CN101490250A - Regulation of protein activity by reversible acetylation - Google Patents

Abstract

This invention discloses the first cellular acetylated substrate protein of SIRT3, Acetyl-CoA synthetase 2 (AceCS2), which is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at lysine 642 (Lys642) in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys642 both in vitro and in vivo. Deacetylation of AceCS2 by SIRT3 activates the acetyl-CoA synthetase activity of AceCS2. Thus, a mammalian sirtuin directly controls the activity of a metabolic enzyme via reversible lysine acetylation. Modulators of the acetylation status or the activity of AceCS2 are useful for the treatment of pathological conditions, such as type II diabetes, hypercholesterolemia, hyperlipidemia, and obesity.

Description

Regulate protein active by reversible acetylize

The cross reference of related application

The application requires the right of priority of the U.S. Provisional Application sequence number 60/813,275 of submission on June 12nd, 2006, and its content mode is by reference included this paper in full in.

Invention field

The present invention discloses the first cell acetylize substrate protein of SIRT3, i.e. Acetyl-CoA synthetase 2 (AceCS2), it is a mitochondrial matrix albumen.Plastosome Sir2-sample albumen (sirtuin) SIRT3 and AceCS2 interact and directly control the activity of this metabolic enzyme by reversible Methionin acetylize.The acetylize state of AceCS2 or active conditioning agent can be used for treatment, for example pathologic state such as type ii diabetes, hypercholesterolemia, hyperlipidaemia and obesity.

Background of invention

Reversible Methionin acetylize is the post translational protein modification that is subjected to altitude mixture control, and it is by protein deacetylase and Transacetylase control (Han and Martinage, 1992, Int J Biochem 24:19-28; Yang, 2004, Bioessays 26:1076-87).Though at first with transcribe relevant with the chromatin dynamics, but reversible Methionin acetylize is as cell function, (Hubbert etc., 2002, the Nature417:455-458 that occur of the instrumentality of cell mobility, immune cynapse formation, apoptosis and protein transportation for example; Kawaguchi etc., 2003, Cell 115:727-38; Serrador etc., 2004, Immunity 20:417-28; Cohen etc., 2004, Science 305:390-2; Cohen etc., 2004, Mol Cell 13:627-38).Know though examine the nonhistones and acetylizad importance of reversible Methionin histone, the effect of protein modification in plastosome that reversible Methionin acetylize causes is still unknown.Yet, the posttranslational modification of mitochondrial protein, for example the importance of phosphorylation and ADP-ribosylation is understood by people gradually, and the reversible acetylize of mitochondrial protein also may play an important role in the adjusting of mitochondrial function.

Niacinamide (NAM) adenine dinucleotide NAD ⁺It is the important life-span instrumentality (Kaeberlein etc. that in yeast saccharomyces cerevisiae (Saccharomy cescerevisiae), Caenorhabditis elegans (Caenorhabditiselegans) and the drosophila melanogaster (Drosophila melanogaster) thermal limit (CR) signal reacted that-dependency deacetylase silent message is regulated albumen 2 (Sir2), 1999, Genes Dev 13:2570-80; Tissenbaum and Guarente, 2001, Nature 410:227-30; Lin etc., 2000, Science 289:2126-8; Rogina and Helfand, 2004, Proc Natl Acad Sci USA 101:15998-6003; Lin etc., 2004, Genes Dev 18:12-6; Lin etc., 2002, Nature 418:344-8; Anderson etc., 2003, Nature 423:181-5).Known 7 kinds of Mammals Sir2 homologues (SIRTI-7) (Frye, 1999, Biochem Biophys Res Commun 260:273-9; Frye, 2000 Biochem Biophys ResCommun 273:793-8; Blander and Guarente, 2004, Annu Rev Biochem 73:417-35; North and Verdin, 2004, Genome Biol 5:224).In plastosome, find SIRT3, SIRT4 and SIRT5 (Shi etc., 2005, J Biol Chem 280:13560-7 recently; Michishita etc., 2005, Mol Biol Cell 16:4623-35; Onyango etc., 2002, Proc Natl Acad Sci USA 99,13653-8; Schwer etc., 2002, J Cell Biol 158:647-57) there is plastosome Sir2-sample protein substrate albumen in prompting.

The present invention discloses the first cell acetylize substrate protein that identifies SIRT3, Acetyl-CoA synthetase 2 (AceCS2), and it is a mitochondrial matrix albumen.

AceCS catalysis acetate links to each other with CoA to produce acetyl-CoA, this is to comprise in the various pathways metabolisms of the synthetic and tricarboxylic acid cycle of lipid acid and cholesterol used basic molecule (summary can be referring to Bremer and Osmundsen, 1984, publish in Fatty Acid Metabolism and ItsRegulation (fatty acid metabolism and adjusting thereof) (Numa, S. compile), 113-154, Ai Ersaiwei scientific publication company (Elsevier Science Publisher), Amsterdam).

The AceCS of various microorganisms and senior biology shows a kind of superfamily (Toh, 1990, ProteinSequences Data Anal 3:517-521; Toh, 1991, Protein Sequences Data Anal4:111-117), comprise Mammals long acyl-CoA synthetic enzyme, ACS1-ACS5 (Fujino and Yamamoto, 1992, J Biochem 111:197-203; Fujino etc., J Biol Chem271:16748-16752; Kang etc., 1997, Proc Natl.Acad Sci USA 94:2880-2884; Suzuki etc., 1990, J Biol Chem 265:8681-8685; Oikawa etc., 1998, J Biochem124:679-685).All enzymes in this superfamily all contain the consensus sequence motif, Ser-Gly-(little wetting ability residue) ₂-Gly-(any residue)-Pro-Lys-Gly (SEQ ID NO:1) and the normal two-step reaction of catalysis: adenylylation substrate and thioesters subsequently form (Toh, 1990, Protein Sequences DataAnal 3:517-521; Toh, 1991, Protein Sequences Data Anal 4:111-117).

Clone and characterized ox and Muridae AceCS cDNA recently.Two kinds of Muridae AceCS that function is different have been described: heart AceCS and liver AceCS.The liver type enzyme that is called AceCS1 is the cytosol enzyme, and the heart type enzyme that is called AceCS2 is arranged in mitochondrial matrix (Fujino etc., 2001, J Biol Chem276:11420-11426).AceCS2 mRNA is induced this discovery prompting AceCS2 to provide to be mainly used in after fasting and is being given birth to the ketone state, for example acetyl-the CoA (Fujino etc., 2001, J Biol Chem 276:11420-11426) of oxidation under hunger and the diabetic disease states.Find that this prompting (Fujino etc., 2001, J Biol Chem 276:11420-11426) is supported in the rising of AceCS2 level in the Zucker diabetes rat.Induce AceCS2 in some cases though Fujino etc. have described, they do not study precise function and the regulating effect (Fujino etc., 2001, J Biol Chem 276:11420-11426) of AceCS2.

For understanding better and treatment is characterised in that the pathologic state that acetate (substrate of AceCS2) level raises, for example give birth to the ketone state, it is most important to illustrate these regulation mechanisms.The present invention provides a kind of like this regulation mechanism by the cell acetylize substrate that Acetyl-CoA synthetase 2 (AceCS2) is accredited as cyclophorase and plastosome Sir2-sample Protein S IRT3.AceCS2 Methionin 642 (Lys642) in the avtive spot of enzyme is located by reversible acetylize.SIRT3 interacts with AceCS2 in vitro and in vivo and makes Lys642 deacetylated.SIRT3 makes the Acetyl-CoA synthetase activity of the deacetylated activation of AceCS2 AceCS2.

Preferably identify level or the active material of deacetylase of activation SIRT3, or regulate level, acetylize state or the active material of AceCS2.These materials are characterised in that the pathologic state that the acetate level raises in treatment, for example give birth to the ketone state and to small part be because of this acetate level due to raising other disease or illness in have therapeutic and use.In this article, the invention provides the method for identifying this material, containing the method for compositions of this material and utilize this material treatment type ii diabetes, hypercholesterolemia, hyperlipidaemia and obesity.

Summary of the invention

The present invention discloses Mammals Sir2-sample Protein S IRT3 and directly controls metabolic enzyme by reversible Methionin acetylize, the activity of Acetyl-CoA synthetase 2 (AceCS2).This paper provides the level that evaluation can improve the SIRT3 polypeptide or method, composition and the test kit of the active material of deacetylase.The present invention also provides method, composition and the test kit of level, acetylize state or active material that evaluation can regulate Acetyl-CoA synthetase 2 (AceCS2) polypeptide.The invention still further relates to these materials and reducing the disease that individual acetate level and treatment are characterised in that the rising of acetate level, for example application in the method for type ii diabetes, hypercholesterolemia, hyperlipidaemia or obesity, composition and the test kit.

More particularly, one aspect of the present invention provides the level that evaluation can improve the SIRT3 polypeptide or the method for the active material of deacetylase.In the present invention's one preferred implementation; this method may further comprise the steps: the cell of expressing SIRT3 polypeptide and AceCS2 polypeptide is contacted with candidate substances; if (b) effect is arranged, then measure the effect of acetylize AceCS2 level in the candidate substances pair cell.Step (b) can comprise the immunological testing that utilizes acetylize AceCS2 specific antibody.This method is optional to comprise the structure of identifying candidate substances or the step of sequence.

Preferred cell is a mammalian cell, more preferably people's cell.In some embodiments, described mammalian cell is selected from down group: heart cell, muscle cell and brain cell.

In another aspect of this invention, evaluation can improve the level of SIRT3 polypeptide or the method for the active material of deacetylase may further comprise the steps: (a) make to comprise NAD ⁺Test mixture in the SIRT3 polypeptide contact with candidate substances with acetylize AceCS2 polypeptide and (b) if effect arranged, then measure the effect of candidate substances to acetylize AceCS2 level in the test mixture.Do not compare with second level of acetylize AceCS2 in the test mixture of handling with candidate substances, first level of acetylize AceCS2 reduces and shows that this material can improve the level or the deacetylase activity of SIRT3 polypeptide in the test mixture.

In a preferred implementation of this method, acetylize AceCS2 polypeptide comprises ¹⁴The ethanoyl of C-mark is by detecting ¹⁴The release implementation step (b) of C-mark ethanoyl.

Another embodiment of the present invention provides the method for level, acetylize state or active material that evaluation can regulate the AceCS2 polypeptide.In a preferred embodiment of the present invention; this method may further comprise the steps: the cell of expressing the AceCS2 polypeptide is contacted with candidate substances; if (b) effect is arranged, then measure in the cell candidate substances to level, acetylize state or the active effect of AceCS2.

In another aspect of this invention; the method that evaluation can be regulated AceCS2 polypeptide level, acetylize state or active material may further comprise the steps: the AceCS2 polypeptide in the test mixture is contacted with candidate substances; if (b) effect is arranged, then measure level, acetylize state or the active effect of candidate substances to AceCS2 in the test mixture.

The present invention provides the biological active agents of identifying by one of screening method of the present invention on the other hand.

Also having on the other hand.The invention provides the method for the acetylize state of regulating the AceCS2 polypeptide.In a preferred embodiment of the present invention; this method comprises will express the cell and the step that contacts according to the obtainable material of the method for the invention of AceCS2 polypeptide, and described method is for example identified level or the method for the active material of deacetylase or the method that evaluation can be regulated level, acetylize state or the active material of AceCS2 polypeptide that can improve the SIRT3 polypeptide.

The present invention also provides the method for the individual pathologic state of treatment, and wherein said pathologic state is characterised in that the acetate level raises.In a preferred embodiment of the present invention, this method comprises that the level that can improve the SIRT3 polypeptide or the active material of deacetylase of will treat significant quantity give individual step, and described polypeptide makes AceCS2 deacetylated.Thereby can treat described pathologic state.

In a preferred embodiment of the present invention, described pathologic state is selected from down group: type ii diabetes, hypercholesterolemia, hyperlipidaemia and obesity.

The individual preferred people or the non-human animal that are treated.

Also having on the other hand, the invention provides the level or the active pharmaceutical composition of deacetylase that can improve SIRT3.In a preferred embodiment of the present invention, pharmaceutical composition comprises (i) according to the obtainable material of the inventive method, described method is for example identified the level that can improve the SIRT3 polypeptide or the method and the (ii) pharmaceutically acceptable carrier of the active material of deacetylase.

The present invention also provides level, acetylize state or the active pharmaceutical composition of regulating the AceCS2 polypeptide.In preferred implementation of the present invention; pharmaceutical composition comprises (i) according to the obtainable material of the method for the invention; described method is for example identified the method and the (ii) pharmaceutically acceptable carrier of level, acetylize state or the active material that can regulate the AceCS2 polypeptide.

The present invention provides test kit in addition on the other hand.Can improve the level of SIRT3 or the active preferred reagent of deacetylase is box-packed has (i) to contain container according to the obtainable material of the inventive method; described method is for example identified the method for the level that can improve the SIRT3 polypeptide or the active material of deacetylase and (ii) with this material and the cells contacting of expressing SITR3 polypeptide and AceCS2 polypeptide with measure the working instructions of the effect (if effective words) of acetylize AceCS2 level in this material pair cell.

The present invention provides level, acetylize state or the active test kit that can regulate the AceCS2 polypeptide in addition on the other hand.In a preferred embodiment of the present invention; this test kit is equipped with (i) and contains container according to the obtainable material of the method for the invention; described method is for example identified the method for level, acetylize state or the active material that can regulate the AceCS2 polypeptide and (ii) with the cells contacting of expressing the AceCS2 polypeptide with measure the working instructions of this material to level, acetylize state or the active effect (if effective words) of AceCS2.

The useful matter that comprises in pharmaceutical composition and the test kit is level, acetylize state or the active material that can improve the level or the active material of deacetylase of SIRT3 polypeptide and can regulate the AceCS2 polypeptide.

Method of the present invention, composition and test kit are supported the details that this paper describes fully.

The accompanying drawing summary

Fig. 1 shows that people's Acetyl-CoA synthetase 2 (AceCS2) is the solubility mitochondrial protein.(A) people AceCS2 ^SignBe transfected into the Hela cell.With anti--sign (Flag) antibody with at the common staining cell of antibody of endogenous plastosome labelled protein MnSOD, analyze by the confocal laser scanning microscopy.Shown AceCS2 in the same focusing section ^Sign(left side) and MnSOD (in) fluorescent signal.The image proof AceCS2 that (right side) merges ^SignBe positioned plastosome.(B) from stably express AceCS2 ^SignHEK293 cell preparation subcellular components.Antibody shown in the utilization carries out immunoblotting and analyzes the cell general extractive (always) of equivalent (30 μ g), plastosome, light film (light membrane) (LM) and cytosol albumen.(C) AceCS2 ^SignInferior plastosome location.Handle stably express AceCS2 by hypotonic swelling with Proteinase K ^SignThe plastosome of HEK293 cell be transformed into line grain corpusculum.Behind the incubation (0 ℃, 20 minutes), by the centrifugal grain of defiber once more corpusculum, with shown in antibody pass through immunoblotting assay.Make immunoblotting witness line plastochondria outer membrane rupture with intermembrane space albuminous cell pigment c (Cyt.C).Make the integrity that immunoblotting confirms mitochondrial inner membrane with stromatin mark SIRT3 and GDH.With anti--sign antibody test AceCS2 ^Sign(D) AceCS2 ^SignIt is soluble protein.From stably express AceCS2 ^SignHEK293 cellular segregation plastosome, it directly is dissolved in the SDS sample buffer (T, overall) or with yellow soda ash (Na ₂CO ₃, pH11.5) extract.Divide film-forming components (P, throw out) with the alkaline extraction thing and contain the component (S, supernatant liquor) of yellow soda ash soluble proteins by ultracentrifugation.Detect whether have labelled protein COX-IV, the MnSOD of abundant sign and the efficient that SIRT3 followed the trail of or monitored alkaline extraction by different components being made immunoblotting.Details is seen embodiment 3.

Fig. 2 has shown the N-end sequence of identifier AceCS2.(A) the N-end of people AceCS2 contains amphipathic alpha-helix.AceCS2 zone (amino-acid residue 3-20 by computer secondary structure analysis prediction formation alpha-helix; SEQ ID NO:2) mapping is the spiral colyliform.R, alkaline residue; T, L, V and A, hydrophobic residue; G and S, neutral residue.(B) analyze plastosome presequence and the cleavage site of measuring people AceCS2 by order-checking of N-terminal protein matter and LC/MS-MS.MPP, the mitochondrial matrix processing peptidase.See embodiment 4 for details.

Fig. 3 shows that conservative property Lys642 undergos mutation among the AceCS2 and causes AceCS2 to lose enzymic activity.(A) Acetyl-CoA synthetase of different plant species (ACS intestines salmonella (S.enterica), SEQ ID NO:3; The ACS2p yeast saccharomyces cerevisiae, SEQ ID NO:4; The ACS Caenorhabditis elegans, SEQ ID NO:5; The ACS drosophila melanogaster, SEQ ID NO:6; AceCS2 brown rat (R.norvegicus) and AceCS2 mouse (Mmusculus), SEQ ID NO:7) with the multisequencing comparison in the avtive spot zone of people AceCS2 (SEQ ID NO:7).Hollow triangle marks avtive spot Methionin.(B) Lys642 is most important for the AceCS2 function.The Lys642 sudden change makes this enzyme-deactivating among the AceCS2.AceCS2 ^HAOr AceCS2-K642Q ^HA(wherein Lys642 is by glutamine (Q) alternate AceCS2 mutant) expresses in the HEK293 cell, and immunoprecipitation is measured Acetyl-CoA synthetase (ACS) specific activity.Data are three independently mean value ± SD of Acetyl-CoA synthetase activity test.(C) AceCS2 of immunoprecipitation ^HAAnd AceCS2-K642Q ^HACoomassie blue stain.Can distinguish AceCS2 by Coomassie blue stain ^HAOr AceCS2-K642Q ^HA, its expression level equates.See embodiment 5 for details.

Fig. 4 shows people AceCS2 acetylize in Lys642 place body.With SIRT3-specific siRNA transfection stably express AceCS2 ^SignThe HEK293 cell, immunoprecipitation AceCS2 after 5 days ^SignBy the sedimentary AceCS2 of SDS-PAGE separating immune ^Sign, its preparation is used for mass spectrum.The tryptic digestion thing is implemented liquid chromatography link coupled tandem mass spectrometry (LC/MS-MS) to identify Methionin acetylize site.AcK, acetylize Methionin.SgacKVoxMR，SEQ?ID?NO：8。See embodiment 6 for details.

Fig. 5 shows the excessive acetylize of people AceCS2 that lacks Sir2-sample PROTEIN C obB gene or proteic escherichia coli expression.(A) remove or suppress the excessive acetylize of avtive spot Methionin that bacterium Sir2-sample PROTEIN C obB causes people AceCS2 in the bacterium.Utilization knocks out the recombinant human AceCS2 (AceCS2:KR) of mutant or the terminal myc-His-mark of wild-type e. coli K-12 expression C-.With ethanoyl-Methionin-specific antibody as immunoblotting analyze equivalent from CobB-or CobB ⁺The AceCS2 (wild-type) of colibacillary purification of recombinant human myc-His-mark or AceCS2-K642R (KR) (Ac-AceCS2 ^Myc-His).Remove trace, detect demonstration AceCS2 (AceCS2 again with α-myc antibody ^Myc-His) aggregate level.(B) anti--ethanoyl-Methionin antibodies specific identification contains the synthetic peptide of AceCS2 of acetylize Lys642 residue (acK642), but not with the peptide that contains non-acetylize Lys642 (K642) cross reaction.Utilize peptide coupling reagent box (NEB) with California Hai Dade city Ai Lemu bio-pharmaceutical company (Elim Biopharmaceuticals, Inc., Hayward, synthetic peptide coupling CA) is in carrier proteins CP39.Used AceCS2 peptide is: NH ₂-CPKTRSG (ac) KVMRRLL-CONH ₂(acK642, SEQ ID NO:9) and NH ₂-CPKTRSGKVMRRLL-CONH ₂(K642, SEQ ID NO:10).For the coupling purpose adds N-terminal cysteine residue.Differentiate 150ng CP39-link coupled K642 or acK642 by SDS-PAGE; utilize not (the Cell SignalingTechnology of interests cell signaling technology company of Massachusetts shellfish; Beverly, polyclone MA) is anti--and ethanoyl-Methionin antibody analyzes as immunoblotting.See embodiment 7 for details.

Fig. 6 shows that suppressing Sir2-sample protein-active with niacinamide (NAM) causes the excessive acetylize of recombinant human AceCS2.(A) suppressing bacterium Sir2-sample albumen with niacinamide during recombinant human AceCS2 protein expression causes people AceCS2 in the excessive acetylize in Lys642 place.Utilize the AceCS2:WT or the AceCS2:KR of bacillus coli DH 5 alpha bacterial expression myc-His-mark.Shown in (5A), analyze the equivalent amount of purified recombinant protein.(B) the tryptic digestion thing of the reorganization AceCS2:WT of the intestinal bacteria purifying handled from niacinamide is implemented the acetylize (acK) that liquid chromatography link coupled tandem mass spectrometry confirms Lys642.The tandem mass spectrum that has shown the doubly charged ion of m/z 368.1948.SgacKVoxMR，SEQ?ID?NO：8。See embodiment 8 for details.

Fig. 7 shows that subtracting reduction endogenous SIRT3 level by striking of siRNA-mediation has improved AceCS2 ^SignThe acetylize level.To be transfected into stably express AceCS2 at the double-stranded siRNA of people SIRT3 (siSIRT3) or Lampyridea luciferase GL3 (si contrast) ^SignThe HEK293 cell.Lysing cell is used the antibody (Ac-AceCS2 at acetylize Methionin ^Sign) analyze the AceCS2 of immunoprecipitation as immunoblotting ^SignAcetylize.Remove the trace on the film, detect total AceCS2 amount (AceCS2 once more ^Sign).Make efficient and the specificity that immunoblotting confirms that siRNA handles with anti--SIRT3 and anti--Actin muscle antibody pair cell general extractive.See embodiment 9 for details.

Fig. 8 is presented at the external SIRT3 of being rather than SIRT5 makes AceCS2 deacetylated.(A) will handle the recombinant human AceCS2 of intestinal bacteria purifying from niacinamide ^Myc-HisWith from the sedimentary sign of HEK293 cellular immunization-mark SIRT3 or SIRT5 incubation.Utilize in the reaction and express pcDNA ^SignAnti--sign the immunoprecipitate (left road) in contrast of HEK293 cell.32 ℃, be with or without niacinamide adenosine dinucleotides (NAD ⁺, 1mM) have the deacetylated reaction of incubation down 3 hours.Institute responds, and all (TSA 500nM) carries out under the existence at the plain A of system hair tinea.Add SDS sample buffer termination reaction, boil and pass through immunoblotting assay.Detect acetylize AceCS2 with ethanoyl-Methionin-specific antibody ^Myc-His(Ac-AceCS2 ^Myc-His).Remove trace, with the aggregate level (AceCS2 of α-myc antibody test AceCS2 ^Myc-His).Come (the figure below that exists of proof mark-mark SIRT3 and SIRT5 as immunoblotting with anti--sign antibody; Input (Sir2-sample albumen)).(B) 32 ℃, be with or without NAD ⁺(1mM) exist down and the recombinant human AceCS2 that has TSA (500nM) to exist down will to handle the intestinal bacteria purifying from niacinamide with the reorganization Sir2-sample albumen incubation of purifying 3 hours, as analysis as described in (A).Utilize anti--myc-antibody test reorganization SIRT3 or SIRT5 albumen (figure below; Input (Sir2-sample albumen)).(C) external, the catalysis deactivation mutant of SIRT3 rather than SIRT3 makes AceCS2 deacetylated.Carry out deacetylation test and analysis as mentioned above.Indicate the place, comprising niacinamide (NAM between incubation period; 10mM).See embodiment 10 for details.

Fig. 9 shows the co-immunoprecipitation of SIRT3 and AceCS2 and makes the AceCS2 in the cell deacetylated.(A) the SIRT3 overexpression reduces the acetylize of the AceCS2 of ectopic expression.Use AceCS2 ^HAAnd pcDNA ^Sign, SIRT3 ^SignOr SIRT5 ^SignIn any cotransfection COS-1 cell.Use the AceCS2 that makes the immunoblotting assay immunoprecipitation at the antibody of acetylize Methionin ^HAAcetylize (Ac-AceCS2 ^HA).Remove the trace on the film, with anti--HA antibody test AceCS2 total amount (AceCS2 ^HA).Confirm SIRT3 in total cell pyrolysis liquid with anti--sign antibody (Sir2-sample albumen (sign)) as immunoblotting ^SignAnd SIRT5 ^SignExpression.(B) AceCS2 and SIRT3 co-immunoprecipitation from cell.Analyze stably express AceCS2 ^SignOr blank sign-control vector (pcDNA ^Sign) the anti--sign immunocomplex of HEK293 clone in whether have endogenous SIRT3.See embodiment 11 for details.

Figure 10 shows the acetyl-CoA synthase activity of the acetylize control people AceCS2 of Lys642.(A) be with or without in the presence of the niacinamide (NAM) express recombinant people AceCS2 in bacterium.With ethanoyl-Methionin specific antibody or anti--myc antibody mediated immunity trace to analyze acetylize level and the total amount of AceCS2.(B) be with or without the specific activity that there is the purifying AceCS2 that expresses down in NAM.Data are mean value ± SD of three independent experiments.(C) be that SIRT3 rather than SIRT3-H248Y make AceCS2 that NAD take place ⁺-dependency is deacetylated.Figure below shows equal amounts of S IRT3 or SIRT3-H248Y is added deacetylated reaction.(D) SIRT3 makes that AceCS2 is deacetylated to activate its Acetyl-CoA synthetase activity.At the AceCS2 that is with or without incubation equivalent in the presence of the SIRT3 mutant (SIRT3-H248Y) of SIRT3 or catalysis deactivation.Indicate the place, adding NAD between incubation period ⁺(1mM).32 ℃, after 3 hours, add niacinamide (NAM; 10mM) stop deacetylated reaction, measure the specific activity of AceCS2.Data are mean value ± SD of three independent determinations of activity.(E) SIRT3 activates the AceCS2 time-dependent manner.Will be from the bacterium that NAM handles the recombinant human AceCS2 and reorganization SIRT3 or SIRT3-H248Y incubation of purifying.Make sample be maintained at 32 ℃, add NAD ⁺(1mM).Take out sample aliquot at the time point of indicating, mix being incorporated in incubation on ice with NAM.Immunoblotting shows it is that SIRT3 rather than SIRT3-H248Y make AceCS2 deacetylation gradually.(F) measure with the time shown in reorganization SIRT3 or the SIRT3-H248Y incubation after the specific activity of AceCS2.Mean value ± the SD that has shown three independent activity tests.See embodiment 12 for details.

Figure 11 is a synoptic diagram of regulating AceCS2 by the reversible Methionin acetylize of Lys642.SIRT3 makes that AceCS2 is deacetylated to activate its Acetyl-CoA synthetase activity.Therefore identity the unknown of the acetyltransferase of a-protein ceCS2 is labeled as acetyltransferase X.See embodiment 13 for details.

Detailed Description Of The Invention

Definition

Unless otherwise defined, all technology used herein and scientific terminology have the skill in the affiliated field of the present invention The conventional connotation of understanding of art personnel. Many terms used herein are provided below with reference to document for the technical staff Generic definition: Singleton etc., Dictionary of Microbiology and Molecular Biology (microorganism and molecular biology dictionary) (the 2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Cambridge technical dictionary) (Walker just, 1988); The Glossary of Genetics (science of heredity glossary), the 5th edition, R.Rieger etc. (volume), SV company (Springer Verlag) (1991); Hale and Marham, The Harper Collins Dictionary of Biology (Harper Corinth biology dictionary) (1991). Unless statement is arranged in addition, following art used herein The connotation of language is as described below.

" AceCS2 " used herein expression acetyl-CoA synzyme 2.

" AceCS2 " used herein or " acetyl-CoA synzyme 2 " represents and comprises mammal The nucleic acid of AceCS2 polypeptide and encoding mammalian AceCS2 polypeptide. In some embodiments, AceCS2 polypeptide or AceCS2 nucleic acid from saccharomycete, fruit bat, zebra fish (zebrafish), trypanosome, C. Elegans Automatic Screening and other species. Exemplary AceCS2 peptide sequence is seen GenBank, for example the people (GenBank accession number Q9NUB1; Mouse (GenBank accession number BAB21612); Ox (GenBank Accession number BAB21611). Exemplary AceCS2 nucleotide sequence can be referring to GenBank, for example the people (GenBank accession number BC039261); Mouse (GenBank accession number AB046742) and ox (GenBank accession number AB046741).

" acetylation state " used herein is illustrated in polypeptide, preferred acetyl-CoA synzyme 2 (AceCS2) Being with or without acetyl group on the polypeptide exists.

" activator " used herein refers to certain material, and this substance is for example induced or to activate the present invention many The expression of peptide, or be combined with polypeptide of the present invention, or stimulate, increase, open, activate, promote or strengthen this The activation of invention polypeptide, or the activity of sensitization or rise polypeptide of the present invention. Activator comprise the coding SIRT3 or The nucleic acid of AceCS2 and natural generation and synthetic compound or material, little chemical molecular etc. Activator Test comprise, for example candidate substances is applied to express SITR3 or AceCS2 cell, measure then merit Can effect. To and not contain with the sample that comprises SIRT3 or AceCS2 or the sample of possible Treatment with activating agent The control sample of activator is made comparisons with the detection effect degree. Control sample (not processing with candidate substances) is specified Be relative activity value 100%. When compared with the control, the polypeptide active value is 110%, optional 130%, 150%, Optional 200%, 300%, 400%, 500% or 1000-3000% or when higher, this polypeptide obtains activation.

" activity of AceCS2 " used herein or " activity of AceCS2 polypeptide " refer to (i) AceCS2 with Certain polypeptide or peptide, preferred SIRT3 polypeptide combination, (ii) AceCS2 and certain polypeptide or peptide, preferred SIRT3 Polypeptide interacts, and (iii) AceCS2 is assembled into the multiprotein complex that comprises the SIRT3 polypeptide, (iv) will AceCS2 is positioned in the mitochondria, or (v) with acetic acid, ATP with CoA enzymatic conversion become acetyl-CoA and AMP (adenosine monophosphate), namely utilize acetic acid, ATP and CoA as substrate with produce acetyl-CoA and AMP.

" activity of SIRT3 " used herein or " activity of SIRT3 polypeptide " refer to that (i) SITR3 and certain are many Peptide or peptide, preferred AceCS2 polypeptide combination, (ii) SIRT3 and certain polypeptide or peptide, preferred AceCS2 polypeptide Interact, (iii) SIRT3 is assembled into the multiprotein complex that comprises the AceCS2 polypeptide, (iv) will SIRT3 is positioned in the mitochondria, (v) makes certain polypeptide or peptide, and preferred AceCS2 polypeptide is deacetylated, or (vi) with certain polypeptide or peptide, preferred AceCS2 polypeptide A DP-ribosylation.

" material " used herein or " candidate substances " can be any chemical compounds, for example big molecule Or little molecule. The molecular weight of described candidate substances can be less than about 10,000 g/mols, less than 5,000 grams / mole, less than 1,000 g/mol, or less than about 500 g/mols. Described candidate substances can be (for example, plant or the animal product) of natural generation, synthetic or the two. Macromolecular example has albumen Matter, protein complex and glycoprotein, nucleic acid, for example DNA, RNA and PNA (peptide nucleic acid). Little The example of molecule has peptide, peptide mimics (for example, intending peptide), amino acid, amino acid analogue, multinuclear glycosides Acid, polynucleotides analog, nucleotides, nucleotide analog, organic or inorganic compound, for example Assorted organic (heteroorganic) or organo-metallic compound. Candidate substances can be by side described herein Unique material of method check. Perhaps, can check material standed for by methods described herein continuously or simultaneously The set of matter.

Candidate substances comprises many chemical classes, normally synthetic, semisynthetic or natural generation inorganic Or organic molecule. Chemical substance can be that molecular weight is greater than 50 but less than about 2,500 daltonian organic littleization Compound. Chemical substance can comprise the functional group, particularly hydrogen bond required with the protein structure interaction, At least one amido, carbonyl, hydroxyl or carboxyl can be comprised, also at least two chemical functional groups can be comprised. Wait The ring-type carbon that selects material to comprise to replace with one or more functional groups or heterocycle structure and/or fragrance or virtue how Family's structure. Also finding in the biomolecule has candidate substances, comprise peptide, sugar, aliphatic acid, steroids, purine, Pyrimidine, their derivative, analogue or combination.

" antagonist " used herein expression reduces, eliminates or disturb the chemicals of the physiological role of polypeptide Matter. Described antagonist can be, for example chemical antagonist, pharmacokinetics antagonist, noncompetitive antagonism Agent or physiology antagonist, biomolecule for example is such as polypeptide. Preferred antagonist can reduce, eliminates or disturb The physiological role of AceCS2 polypeptide.

Specifically, antagonist can be at first polypeptide, and for example SITR3 polypeptide and second polypeptide are for example tied Close the companion, for example work on the level of AceCS2 polypeptide interaction. For example, antagonist can be competed Striving property or noncompetitive (for example, atopy) suppress the combination of first polypeptide and second polypeptide. " medicine is for power Learn antagonist " effectively reduce active medicine in the concentration at its action site place, for example by increasing by first polypeptide Metabolic degradation speed. " competitive antagonist " is to disturb first polypeptide and second polypeptide mutually to do with dystopy With mode directly in conjunction with the molecule of first polypeptide. Noncompetitive antaganist is described antagonist not directly with first Polypeptide is competed mutually with the combination of second polypeptide, but the situation of the signal transduction pathway after the blocking-up combination. Physiology Antagonist is not quite accurately described the interaction of two kinds of materials, and wherein a kind of reaction meeting is in vivo offset Alternative effect. Antagonist can also be the material that reduces or eliminate first expression of polypeptides. Therefore, The AceCS2 antagonist can be for example to reduce or eliminate the material of following effect: (i) encode AceCS2's The expression of gene, (ii) translation of AceCS2 mRNA, (iii) posttranslational modification of AceCS2, or (iv) exist The interaction of AceCS2 and other polypeptide during multiprotein complex forms.

Term used herein " ASON " comprises with the nucleotides of target sequence complete complementary and has Those of one or more mispairing nucleotides are as long as this ASON can be with the target sequence specific hybrid Can. For example, ASON of the present invention is included at least 15 continuous nucleotides to maximum total length orders Show at least 70% or higher, preferred 80% with any nucleotides sequence of AceCS2 gene on the span of row Or higher, more preferably 90% or higher, in addition more preferably 95% or higher homology (be also referred to as the sequence phase The same sex) polynucleotides. For example, ASON of the present invention is included at least 15 continuous nucleotides The multinuclear that has homology with any nucleotide sequence of AceCS2 gene on the span of maximum full length sequences Thuja acid. Can adopt algorithm known in the art to measure homology. In addition, the derivative of ASON or Modified outcome also can be used as ASON of the present invention. The example of this modified outcome comprises low alkyl group Phosphonate ester is modified, for example methyl-phosphonate type or phosphinic acid ethyl ester type, phosphorothioate and phosphoramidic acid Salt (phosphoroamidate) is modified.

" linking to each other " used herein expression contact, interaction or combination.

When referring to material, term used herein " BA " is known in the art, the expression material Certain form, this form is so that the part administered dose of this material or material can be by the object that is given or patient Absorb, take in or be acceptable for the object or the patient that give at physiology.

" biological sample " used herein expression contains the biological tissue of nucleic acid or polypeptide or the sample of liquid. These samples are usually from the people, but comprise from non-human primates or rodent, for example the Mouse and rat branch From tissue. Biological sample also can comprise histotomy, the biological biopsy of for example obtaining for the histology purpose With postmortem sample, freezing microtome section, cerebrospinal fluid, blood, blood plasma, serum, phlegm, ight soil, tear, mucus, Hair, skin etc. Biological sample also comprises explant and available from former generation and/or the transformant of patient tissue Culture. " biological sample " also represents cell or cell mass or a certain amount of tissue or the body fluid of animal. Modal biological sample is taken from animal, but term " biological sample " also can represent the thin of body inner analysis Born of the same parents or tissue namely do not take out from animal. " biological sample " contains the cell of animal usually, but this art Language also represents to can be used for detecting the acellular organism material of polynucleotide sequence or expression of polypeptides level, for example The acellular component of blood, serum, saliva, cerebrospinal fluid or urine. The present invention can utilize polytype biology The product that imitate include but not limited to: tissue biological's biopsy or blood sample. It is used herein that " tissue biological lives Inspection " be expressed as diagnostic purpose and from animal, a certain amount of tissue that takes out among the preferred people. " tissue biological's biopsy " Can represent the biological biopsy of any type, for example biological biopsy of syringe needle, the first-born thing biopsy of fine needle, operation Biological biopsy etc.

Expression obtains to be used for the biological sample of the method for the invention " to provide biological biopsy samples ". The most normal What see is the cell sample that takes out object, but the cell that separates before also can adopting (for example, by other people at it Its time and/or be that other purpose separates), or by implementing in vivo the inventive method. Have the treatment history or The file of achievement history is organized particularly useful.

" CoA " used herein represents coacetylase.

" combinatorial chemistry library " used herein refers to by chemosynthesis or biology synthetic, by making up many chemistry " structure piece ", and for example reagent and the set of all cpds that produces.For example, by one group of chemical building piece (amino acid) being combined into given compound length (that is, the amino acid number in the polypeptide compound) to form linear combination chemistry library, as polypeptide libraries in each possible mode.By the synthetic millions of kinds of chemical compounds of this combined mixing energy of chemical building piece.

The level activity lower and/or gene expression product that the expression gene expression product " is expressed and reduce " to term used herein reduces.Compared with the control, described reduction preferably at least 20%, described reduction more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%, described reduction most preferably at least 100%.

The synonym of term " mensuration " belongs to scope of the present invention, includes but not limited to: detect, measure, check or test certain molecule, whether AceCS2 for example of the present invention, SIRT3, marker or small molecules exist, content or concentration.This term is represented qualitative and quantitative assay.

" mensuration effect " used herein or " measurement function effect " expression check is under indirectly or direct influence, and certain material is to increase or reduce certain parameter, for example functional, enzyme, physics or chemical action.Can detect these effects by any way well known by persons skilled in the art, for example proteinic spectral signature (as fluorescence, absorbancy, specific refractory power), hydromeehanics (for example, shape), chromatogram or dissolubility property change; Detection can be induced marker or gene transcription activation, for example SIRT3 or AceCS2; Detect in conjunction with active, for example SIRT3 and AceCS2's combines; The deacetylated activity of check SIRT3; Measure the acetylize state of AceCS2; Detect cell proliferation; Detect apoptosis; Detect polypeptide, for example Subcellular Localization of SIRT3 or AceCS2; Detect the generation of acetyl-CoA or AMP; Detect the reduction of acetate level, ATP level or CoA level, or the like.Also can adopt test well known by persons skilled in the art, for example in vitro tests is as cell proliferation; Somatomedin or serum dependency; MRNA in the cell and protein expression, and the further feature of cell is measured the function of something confrontation disease, illness, cancer or other pathologic state.Can assess these effects by many modes well known by persons skilled in the art, for example microscopy is quantitative or the qualitative detection morphological feature changes, detecting RNA or protein level changes, detect rna stability, identify downstream or reporter gene expression (CAT, luciferase, β-gal, GFP etc.), for example, by chemoluminescence, fluorescence, colorimetric reaction, antibodies, can induce marker, part in conjunction with test, apoptosis test, detect the generation of acetyl-CoA and AMP, or the like." function " comprises in external, the body and isolated activity.

" illness " used herein, " disease " or " pathologic state " but inclusive ground use, refer to and the normal configuration of any part, organ or the system (or their any combination) of health or any deviation of function.Disease specific shows as distinctive symptom and sign, comprises biology, chemistry and physical change, and disease specific is normal relevant with various other factorses (including but not limited to demography, environment, work, heredity and medical history factor).Can pass through some characteristic sign of the whole bag of tricks quantitative assay, symptom and correlative factor to obtain important diagnostic information.

" significant quantity " used herein, " effective dose ", " sufficient quantity ", " effectively dosage ", " treatment significant quantity " or their grammer equivalents are represented to be enough to produce required result, improvement or are reduced symptom in some way or the dosage of termination or reverse progression of disease.In some embodiments, required result is that SIRT3 locatees increase in plastosome.In other embodiments, required result is that AceCS2 locatees increase in plastosome.In also having other embodiment, required result is deacetylated active the increasing of SIRT3.In another embodiment, required result is the deacetylated state increase of AceCS2 or reduces.In also having other embodiment, required result utilizes acetate, ATP and CoA to produce acetyl-CoA and AMP increase or reduce (referring to Figure 11) as substrate.In also having another embodiment, required result is that the acetate level reduces.No matter give symptom that pharmaceutical composition described herein improves specified disease and refer to give relevant any effect that alleviates with pharmaceutical composition, be permanent or interim, lasting or of short duration.Can interior and external the giving " significant quantity " of body.

" total length " polypeptide or nucleic acid refer to contain polypeptide or polynucleotide sequence or its variant of all elements that normally comprise in one or more natural polynucleotide or the peptide sequence." total length " can be before or after each stage of translation post-treatment or montage (comprise other road montage and signal peptide cutting).

When term " allos " was used to address the each several part of nucleic acid, it showed that this nucleic acid is included in two or more subsequences that occurring in nature does not have identical relation each other.For example, the nucleic acid generation of recombinating usually, make independent basis because of two or more series arrangement constitute new functional nucleic acid, the coding region in a kind of promotor of source and another source for example.Similarly, heterologous protein shows that this protein is included in occurring in nature and does not have two or more subsequences of identical relation (for example, fusion rotein) each other.

" host cell " is n cell or the transformant that contains expression vector and support this expression vector to duplicate or express.Host cell can be cultured cells, explant, cells in vivo etc.Host cell can be a prokaryotic cell prokaryocyte, for example intestinal bacteria, or eukaryotic cell, for example yeast, insect cell, Amphibians cell or mammalian cell, as CHO, 293,3T3, HeLa, or the like (referring to, American Type Culture Collection catalogue for example).

For purposes of the present invention, term " hybridization " or " specific hybrid " are used to represent the ability of two kinds of nucleic acid molecule hybridization under " stringent hybridization condition ".Phrase " stringent hybridization condition " refers to that under this condition nucleic acid molecule can be hybridized with its target sequence usually, but does not detect and other sequence hybridization in the nucleic acid mixture of complexity.Stringent condition is a sequence dependent, and is different in varying environment.Long sequence specific hybrid under comparatively high temps.Tijssen is seen in the detailed guidance of nucleic acid hybridization, Techniques inBiochemistry and Molecular Biology--Hybridization with Nucleic Probes (biological chemistry and Protocols in Molecular Biology-with nucleic acid hybridization), " Overview of principles ofhybridization and the strategy of nucleic acid assays (hybridization principle and nucleic acid testing program summary) ", (1993).Under ionic strength that is limited and pH, the pyrolysis chain temperature (T of the common bit sequencing row of the stringent condition of selection _m) low 5-10 ℃ approximately.T _mBe under ionic strength, pH and the nucleic acid concentration that limits, have during balance 50% the complementary probe of target and target sequence hybridization temperature (excessive because of target sequence, at T _mThere is 50% probe to be occupied during following balance).Also can add destabilizing agent, for example methane amide is to realize stringent condition.For selectivity or specific hybrid, positive signal is the twice at least of background, 10 times of preferred background hybridization.Exemplary stringent hybridization condition can be as follows: 50% methane amide, 5 * SSC, and 1%SDS, 42 ℃ of incubations, or 5 * SSC, 1%SDS, 65 ℃ of incubations, 50 ℃, 0.2 * SSC and 0.1%SDS washing.Antisense oligonucleotide and derivative thereof act on generation in the following manner, the proteic cell of AceCS2 coded by said gene for example: in conjunction with the DNA or the mRNA of coded protein, suppress it and transcribe or translate, impel mRNA degraded and arrestin matter to express, thus arrestin matter function.

Term " hypercholesterolemia " refers to the unusual high state of the cholesterol levels in the object blood flow.

Term " hyperlipidaemia " refers to that the fatty substance level of blood in the object is higher than normally.Fatty substance comprises cholesterol, cholesteryl ester, phosphatide and triglyceride level.

Term " individuality ", " object ", " host " and " patient " are used interchangeably herein, and the expression Mammals includes but not limited to: Muridae, ape, cat, dog, horse, ox, agriculture Mammals, sports Mammals and mammal pet and people.Preferred people.In some embodiments, these terms also comprise XenoPus (Xenopus), zebra fish, trypanosome, beautiful nematode, fruit bat and yeast.

" inhibitor " used herein refers to certain material, its energy for example checks or deactivation expression of polypeptides of the present invention, or combines with polypeptide of the present invention, or reduce, close, deactivation, obstruction or reduce the activation of polypeptide of the present invention, or make the active insensitive of polypeptide of the present invention or reduce its activity.Inhibitor comprises interference, the AceCS2 nucleic acid of expressing for example, and as siRNA, sense-rna and ribozyme, and natural generation and synthetic compound and material, chemical small molecules etc.Also can adopt the test of activator test (referring to above) as inhibitor.Sample that comprises AceCS2 or the sample that to handle with possible inhibitor and the control sample that does not contain inhibitor are made comparisons with the detection effect degree.Control sample (not handling with candidate substances) is appointed as relative reactivity value 100%.When compared with the control, activity value reduces by 10%, optional 20%, optional 30%, optional 40%, optional 50%, 60%, 70%, 80% or during 90-100%, this polypeptide is inhibited.

" external " used herein expression obtains one or more cells, or outside the living organism of isolated cell system.

" in the body " used herein expression obtains one or more cells, or within the living organism of isolated cell system.

Term used herein " is given birth to the ketone state " and is referred to be characterised in that individual liver is released into a large amount of acetate state (Buckley and Williamson, 1977, the Biochem J 166:539-45 of blood flow; Seufert etc., 1974, Biochem Biophys Res Commun 57:901-9; Yamashita etc., 2001, BiochimBiophys Acta 1532:79-87).Living ketone state can be the effect due to long-term fasting or the diabetes.In addition, and the activation under living ketone state of the liver Acetyl-CoA hydrolase of generation acetate (Matsunaga etc., 1985, Eur J Biochem 152,331-6).

" mark " or " but test section " is to pass through the composition that spectrography, photochemistry, biological chemistry, immunochemistry, chemistry or other physical method detect.For example, useful marker comprises ³H, ¹²⁵I, ³²P, ¹⁴C, fluorescence dye, electronics dense dose (electron-dense reagent), enzyme are (for example, commonly used enzyme among the ELISA) but, vitamin H, digitoxigenin or haptens and protein maybe can make other entity of test format, for example the substrate by radio-labeling being mixed SIRT3, AceCS2, SIRT3 (for example, NAD or polypeptide) or substrate (for example, acetate) or the micromolecular compound of AceCS2.Preferred marker is ¹⁴C is preferably in ethanoyl.

The mRNA amount that exists in " level of mRNA " expression cell in the biological sample used herein or the biological sample from genetic transcription.The common encoding function albumen of mRNA is though exist the sudden change that can change or eliminate coded protein function.Need not quantitative assay " level of mRNA ", but can detect simply,, make comparisons with the expectation level of the level of control sample or control sample or do not compare for example by people's subjective visual inspection.Preferred mRNA is SIRT3mRNA or AceCS2 mRNA.

The polypeptide amount that exists in " polypeptide level " expression cell in the biological sample used herein or the biological sample from the mRNA translation.Described polypeptide can have or not have protein active.Need not quantitative assay " polypeptide level ", but can detect simply,, make comparisons with the expectation level of the level of control sample or control sample or do not compare for example by people's subjective visual inspection.Preferred polypeptide is SIRT3 or AceCS2 polypeptide.

" Mammals " used herein or " mammiferous " expression Mammals classification or relevant with it comprises carnivore order (for example, dog and cat), Rodentia (for example, mouse, cavy and rat) and Primates (for example, people, chimpanzee and monkey).

Term " adjusting " or " adjusting " are known in the art, and (that is, activation, stimulation, increase) or certain reaction of downward modulation (that is, suppress, check, reduce or reduce), perhaps these two kinds of forms of combination or independent appearance are raised in expression.

Polypeptide used herein, for example the level of AceCS2 or active " conditioning agent " comprise the activator and/or the inhibitor of this polypeptide, represent to activate or suppress the material of polypeptide expression level or polypeptide active with it.Preferred polypeptide is AceCS2.

" multiprotein complex " used herein refers to that the combination each other of two or more polypeptide is connected with non-covalent.

" natural generation " used herein nucleic acid molecule represents to have the RNA or the dna molecular of the nucleotide sequence that produces at occurring in nature.For example, the nucleic acid molecule codified native protein of natural generation.

" natural generation " used herein polypeptide represents to have the peptide molecule of the aminoacid sequence that produces at occurring in nature.

" nucleic acid " used herein or " oligonucleotide " or " polynucleotide " or their grammer equivalents are represented covalently bound at least two Nucleotide together.Oligonucleotide is about 5,6,7,8,9,10,12,15,25,30,40,50 or more a plurality of Nucleotide usually, is about 100 Nucleotide at most.Nucleic acid and polynucleotide sequence are the polymkeric substance of any length, comprise longer length, for example 200,300,500,1000,2000,3000,5000,7000,10,000 etc.Nucleic acid of the present invention contains phosphodiester bond usually, though in some cases, comprise may have comprise phosphoramidate, thiophosphatephosphorothioate, phosphorodithioate or neighbour-methyl phosphoramidite connecting key for example other skeleton (referring to Eckstein, Oligonucleotides and Analogues:A Practical Approach (oligonucleotide and analogue: practical approach), the Oxford University Press); And the nucleic acid analog of peptide nucleic acid(PNA) skeleton and connecting key.Other nucleic acid analog comprises having positive main chain (positive backbone); The nonionic main chain; Those analogues with non-ribose main chain, comprise U.S. Patent number 5,235,033 and 5,034,506, and Sanghui and Cook volume, ASC Symposium Series (ASC forum book series) 580, Carbohydrate Modifications in Antisense Research (carbohydrate compound research in the antisense research), described in the 6th and 7 chapters.Also comprise the nucleic acid that contains one or more carbocyclic ring sugar in the nucleic acid definition.Can modify ribose-phosphoric acid skeleton for various reasons, for example for increasing stability and the transformation period or as the probe of biochip of this molecule in physiological environment.The mixture that can prepare natural acid and analogue; Perhaps, can prepare the mixture of different IPs acid-like substance, and the mixture of natural acid and analogue.

Various reference have disclosed this nucleic acid analog, comprise, for example phosphoramidate (Beaucage etc., Tetrahedron 49 (10): 1925 (1993) and reference; Letsinger etc., J.Org.Chem.35:3800 (1970); Sprinzl etc., Eur.J.Biochem.81:579 (1977); Letsinger etc., Nucl.AcidsRes.14:3487 (1986); Sawai etc., Chem.Lett.805 (1984); Letsinger etc., J.Am.Chem.Soc.110:4470 (1988); With Pauwels etc., Chemica Scripta 26:14191986)), thiophosphatephosphorothioate (Mag etc., Nucleic Acids Res.19:1437 (1991); With U.S. Patent number 5,644,048), phosphorodithioate (Briu etc., J.Am.Chem.Soc.111:2321 (1989), neighbour-methyl phosphoramidite connecting key are (referring to Eckstein, oligonucleotide and analogue: practical approach, the Oxford University Press) and peptide nucleic acid(PNA) main chain and connecting key (referring to, Egholm, J.Am.Chem.Soc.114:1895 (1992); Meier etc., Chem.Int.Ed.Engl.31:1008 (1992); Nielsen, Nature 365:566 (1993); Carlson etc., Nature 380:207 (1996), all documents are included in by reference).Other similar nucleic acid comprises and contains positive main chain (Denpcy etc., Proc.Natl.Acad.Sci.USA 92:6097 (1995), nonionic main chain (U.S. Patent number 5,386,023,5,637,684,5,602,240,5,216,141 and 4,469,863; Kiedrowshi etc., Angew.Chem.Intl.Ed.English 30:423 (1991); Letsinger etc., J.Am.Chem.Soc.110:4470 (1988); Letsinger etc., Nucleoside ﹠amp; Nucleotide 13:1597 (1994); ASC forum book series 580, " carbohydrate modification in the antisense research ", Y.S.Sanghui and P.Dan Cook compile, the 2nd and 3 chapters; Mesmaeker etc., Bioorganic ﹠amp; Medicinal Chem.Lett.4:395 (1994); Jeffa etc., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett.37:743 (1996)) and those nucleic acid of non-ribose main chain, comprise U.S. Patent number 5,235,033 and 5,034,506, and ASC forum book series 580, " carbohydrate modification in the antisense research ", Y.S.Sanghui and P.Dan Cook compile, those that the 6th and 7 chapters are described.Also comprise the nucleic acid that contains one or more carbocyclic ring sugar (referring to Jenkins etc., the 169th page of Chem.Soc.Rev., 176 (1995)) in the nucleic acid definition.Rawls, C and E News, has described several nucleic acid analogs for the 35th page on June 2nd, 1997.All these reference are clearly included in way of reference.

Other analogue comprises peptide nucleic acid(PNA) (PNA), and it is the peptide nucleic acid(PNA) analogue.Opposite with the phosphodiester bond main chain of highly charged natural acid, these main chains are non-ionic basically under neutrallty condition.This has two kinds of advantages.The first, the PNA main chain shows that hybridization kinetics improves.With regard to the base pair of mispairing and Perfect Matchings, the melting temperature(Tm) (T of PNA _m) bigger change arranged.Do not match for inside, DNA and RNA show T usually _mReduce 2-4 ℃.For non-ionic type PNA main chain, reduce 7-9 ℃.Similarly, because the nonionic character of these main chains, the hybridization of the base that links to each other with them is to the salt concn relative insensitivity.In addition, the cellular enzymes PNA that do not degrade is therefore more stable.

Nucleic acid can be described strand or two strands, perhaps contains the part of two strands or single stranded sequence simultaneously.Those skilled in the art can know, address the sequence that strand also defines complementary strand; Therefore, sequence as herein described also provides the complementary sequence of this sequence.Nucleic acid can be DNA (genomic dna and cDNA), RNA or crossbred, its amplifying nucleic acid can contain the combination of thymus nucleic acid and ribonucleotide, comprises the combination of bases such as uridylic, VITAMIN B4, thymus pyrimidine, cytosine(Cyt), guanine, inosine, xanthine, xanthoglobulin, iso-cytosine (isocytosine), isoguanine." transcript " is often referred to natural RNA, for example preceding-mRNA, hnRNA or mRNA.Term used herein " nucleosides " comprises Nucleotide, nucleosides and nucleotide analog, and the nucleosides of modifying, for example amido modified nucleosides.In addition, " nucleosides " comprises non-natural analogue structure.Therefore, this paper is called nucleosides with the single peptide nucleic acid(PNA) unit that for example respectively contain base.

" pharmaceutically acceptable " used herein showed and given object, during preferred people's object, can tolerate and can not produce usually the composition of irritated or similar untoward reaction on physiology.The management board that term used herein " pharmaceutically acceptable " preferably is expressed as federation or state government ratifies or lists in American Pharmacopeia or other generally accepted pharmacopeia and can be used for animal, more particularly is used for philtrum.

" polypeptide " used herein and " protein " are used interchangeably, the polymkeric substance of expression amino-acid residue.This term is applicable to that also wherein one or more amino-acid residues are aminoacid polymerss of the artificial chemical simulation thing of corresponding natural amino acid, and contains natural amino acid polymkeric substance and the alpha-non-natural amino acid polymkeric substance of modifying residue.Preferred polypeptide is SIRT3 and AceCS2.

Term " promotor " refers to comprise the nucleotide sequence of one or more regulatory regions, and described regulatory region can be controlled SIRT3 gene or AceCS2 genetic transcription.In some embodiments, promotor is people's promotor.

" purifying " or " isolating " polypeptide or protein are gone up other contaminative protein that does not contain cell material or obtain this proteinic cell or tissue source substantially, or are substantially free of other chemical substance of precursor or chemosynthesis." be substantially free of " and represent that protein of interest matter at least 10% is pure in the goods.In one embodiment, contaminative component in the protein articles (for example, non-protein of interest matter, precursor or the like) is less than about 30%, 20%, 10% and, more preferably 5% (with dry weight basis).Produce if protein or its biologic activity partly are reorganization, also preferably be substantially free of substratum, that is, substratum in the volume of protein articles, account for less than about 20%, be more preferably less than about 10%, most preferably less than about 5%.

When term " reorganization " is used to address, for example when cell, nucleic acid, protein or carrier, it shows this cell, nucleic acid, protein or carrier by introducing heterologous nucleic acids or protein or obtaining modifying by changing natural acid or protein, and perhaps this is cell-derived from the cell of so modifying.Therefore, for example, reconstitution cell is expressed the gene of not finding in natural (non-reorganization) form of cell, the natural gene of perhaps express other unconventionality expression, expressing lowly or not expressing.The nucleic acid that its form is not found at occurring in nature represented in the term of this paper " recombinant nucleic acid ", and it utilizes usually, for example polysaccharase and endonuclease operation nucleic acid and at first in external formation.Operability ground connects different sequences in this way.Therefore, for purposes of the present invention, linear isolating nucleic acid or all think recombinant chou at the expression vector of external formation by connecting disjunct dna molecular under the normal circumstances.In case should be understood that to have prepared recombinant nucleic acid and introduced host cell or organism again, its be non-reorganization duplicate, that is, utilize the cells in vivo machine rather than the manipulation in vitro of host cell; Yet for purposes of the present invention, though duplicating subsequently is non-reorganization, in case reorganization has produced these nucleic acid, it still regards recombinant chou as.Similarly, " recombinant protein " is to adopt recombinant technology, promptly by expressing the protein that above-mentioned recombinant nucleic acid produces.

Term used herein " salt " refers to the salt of active compound of the present invention or material, for example SIRT3 activator or AceCS2 conditioning agent, and their prepare with nontoxic relatively acid or alkali according to the specified substituent of finding on compound described herein.If compound of the present invention, material and small molecules contain relative tart functional group, these compounds that can be by making neutral form and the required alkali of q.s (pure or in suitable inert solvent) contact and obtain base addition salt.The example of pharmaceutically acceptable base addition salt comprises sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salt.If compound of the present invention, material and small molecules contain the functional group of relative alkalescence, these compounds that can be by making neutral form and the required acid of q.s (pure or in suitable inert solvent) contact and obtain acid salt.The example of pharmaceutically-acceptable acid addition comprises derived from mineral acid and nontoxic relatively organic acid salt, described mineral acid for example has hydrochloric acid, Hydrogen bromide, nitric acid, carbonic acid, one hydrogen carbonic acid (monohydrogencarbonic), phosphoric acid, one hydrogen phosphoric acid (monohydrogenphosphoric), dihydrogen phosphoric acid (dihydrogenphosphoric), sulfuric acid, one hydrosulphuric acid (monohydrogensulfuric), hydroiodic acid HI or phosphorous acid etc., described organic acid for example has acetate, propionic acid, isopropylformic acid, toxilic acid, propanedioic acid, phenylformic acid, succsinic acid, suberic acid, fumaric acid, amygdalic acid, phthalic acid, Phenylsulfonic acid, right-toluenesulphonic acids, citric acid, tartrate, methylsulfonic acid etc.Also comprise amino acid whose salt, for example arginic acid salt etc., and organic acid, for example the salt of glucuronic acid or galacturonic acid etc. (referring to, Berge etc. for example, 1977, J Pharm Science 66:1-19).Some concrete material of the present invention contains alkalescence and acidic functionality, can change into acid salt again thereby can change into base addition salt.

Can be by with salt and alkali or acid contact and separate parent compound the regenerate material of the present invention and the small molecules of neutral form in a usual manner.The difference of material and micromolecular parent form and various salt forms is some physical property, the solvability in the polar solvent for example, but for purposes of the present invention, these salt and compound, material and micromolecular parent form equivalence.

Term used herein " Sir2-sample albumen " refers to Sir2-sample deacetylase protein zymoprotein family, a member of preferred Sir2 family, (for example comprise yeast Sir2, GenBank accession number P53685), beautiful nematode Sir-2.1 (for example, GenBank accession number NP_01912) and people SIRT1 (for example, GenBank accession number Q96EB6, NP_036370 and AAD40849) and people SIRT2 (for example, GenBank accession number NM_030593, AF083107, AAD40850, CAD43717, ABB72675, NP_085096, AAH03547) protein.Other family member comprises 4 other yeast Sir2-sample genes (homologue of Sir2) that are called " HST gene ", be HST1, HST2, HST3 and HST4, and 5 other people's homologues, be hSIRT3 (for example, GenBank accession number NP_036371, AAH01042, AAD40851 and Q9NTG7), hSIRT4 (for example, GenBank accession number NP_036372, AAI09321, AAI09320, AAD40852 and Q9Y6E7), hSIRT5 (for example, GenBank accession number CAI19838, CAI19837, NP_112534, NP_036373 and AAH001216), hSIRT6 (for example, GenBank accession number NP_057623, AAH05026, AAH04218 and AAH28220) and hSIRT7 is (for example, GenBank accession number NP_057622, AAH17305, AAI01794 and AAI01792) (Brachmann etc., 1995, GenesDev9:2888-902 and Frye, 1999, Biochem Biophys Res Commun 260 (1): 273-279; Frye, 2000, Biochem Biophys Res Commun 273 (2): 793-798).Preferred Sir2-sample albumen is and SIRT3 that more preferably hSIRT3 has those albumen of more similaritys.

Term used herein " SIRT3 " refers to nucleic acid, polypeptide and polymorphism variant, allelotrope, homologue between mutant and species thereof, this paper with its further describe for: the aminoacid sequence that (1) has at least about 25,50,75,100,150,200,250,300,350 or 400 or more a plurality of amino acid whose zone on with GeneBank accession number NP_036371, AAH01042, AAD40851, the SIRT3 sequence of NM_001017524 or Q9NTG7 preservation has the aminoacid sequence homogeny greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferred 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher aminoacid sequence homogeny; (2) can be in conjunction with modifying the antibody of variant or the generation of segmental immunogen, for example polyclonal antibody with the aminoacid sequence or its conservative property that comprise with GeneBank accession number NP_036371, AAH01042, AAD40851, NM_001017524 or Q9NTG7 preservation; (3) combine with the AceCS2 polypeptide; (4) to small part and indirect regulation acetyl-CoA and AMP generation; (5) deacetylated and acetylize AceCS2 polypeptide; (6) under stringent hybridization condition, modify the variant specific hybrid with nucleotide sequence or its conservative property with GenBank accession number NP_036371, AAH01042, AAD40851 and Q9NTG7 preservation; (7) nucleotide sequence that has is preferably at least about 30,50,100,200,500,1000,1,500,2000 or the zone of more a plurality of Nucleotide on have greater than about 90% with nucleotide sequence with GeneBank accession number NM-012239, BC001042 or AF083108 preservation, more preferably greater than about 96%, 97%, 98%, 99% or higher nucleotide sequence homogeny; (8) have with the peptide sequence of GenBank accession number NP_036371, AAH01042, AAD40851 or Q9NTG7 preservation at least 25, normally 50,75,100,150,200,250,300,350 or 400 continuous amino acid residues; And/or have at least 25 with the nucleotide sequence of GenBank accession number NM_012239, BC001042 or AF083108 preservation, normally 50,75,100,150,200,250,300,350,400,500,600,700,800,900,1,000,1,200,1,500,2,000 or more a plurality of continuous nucleotide.

SIRT3 polynucleotide or peptide sequence be usually from the people, but also can be from other Mammals, but be not limited to: non-human primates, rodents, for example rat, mouse or hamster, ox, pig, horse, sheep or other Mammals.In some embodiments, preferably utilize the SIRT3 of yeast, fruit bat, trypanosome, chicken, beautiful nematode or Xenopus laevis." SIRT3 " polypeptide and polynucleotide comprise natural or recombinant forms.Therefore, in some embodiments, SIRT3 polypeptide as herein described and SIRT3 subdomain polypeptide can comprise the sequence corresponding to people SIRT3 peptide sequence.Therefore, exemplary SIRT3 peptide sequence provided herein is known in the art.For example, the GenBank accession number of people SIRT3 polypeptide is NP_036371, AAH01042, AAD40851, NM_001017524 and Q9NTG7.The GenBank accession number of mouse SIRT3 polypeptide is, for example CAJ18608, Q8R104, AAH25878 and NP_071878; That rat SIRT3 is XP_215124; That ox SIRT3 is XP_869883 and XP_879073; That dog SIRT3 is XP_848300, XP_855809 and XP_856096; That zebra fish SIRT3 is XP_684225 and XP690925.Exemplary SIRT3 polynucleotide sequence provided herein is known in the art.For example, the GenBank accession number of people SIRT3 nucleic acid is NM_012239, BC001042, NM_001017524 and AF083108.The GenBank accession number of mouse SIRT3 is, for example CT010402, BC025878 and NM_022433; That rat SIRT3 is XM_215124; That ox SIRT3 is XM_864790 and XM_873980; That dog SIRT3 is XM_843207, XM_850716 and XM_851003; That zebra fish SIRT3 is XM_679133 and XM_685833.

In some embodiments, the SIRT3 polypeptide can be " total length " SIRT3 polypeptide.Term used herein " total length " refers to have at least the polypeptide of natural SIRT3 polypeptide length." total length " SIRT3 polypeptide or its fragment also can comprise other sequence, for example purification tag (for example sign or HA), or other compound (for example, the fluorophore of Xiang Lianing, marker or cofactor) that links to each other.

The isolation of RNA molecule that " siRNA ", " short interfering rna " or " siRNA " expression are worked as the key intermediate that triggers the sequence-specific RNA degraded, preferred length is greater than 10 Nucleotide, more preferably length is most preferably grown 18,19,20,21,22,23,24,25,26,27,28,29 or 30 Nucleotide greater than 15 Nucleotide.19-25 Nucleotide is the most preferably size of siRNA.SiRNA comprises that also wherein two chains of siRNA duplex are included in a short hairpin RNA (shRNA) in the RNA molecule.Double-stranded siRNA is usually by having justice and antisense strand to constitute.Strand siRNA is usually only by constituting with target gene or mRNA complementary antisense strand.SiRNA comprises any type of RNA, preferred dsRNA (the proteolysis cleaved products of big dsRNA, partially purified RNA, pure RNA, synthetic RNA, the RNA that reorganization produces basically) and because of interpolation, disappearance, replacement and/or the change of the one or more Nucleotide modification RNA different with natural RNA.

" small molecules " is molecular weight less than 5,2,1 or the molecule of 0.5kDa.In many embodiments, this small molecules does not comprise peptide bond or phosphodiester bond.For example, they can be non-polymeric.In some embodiments, the molecular weight of described molecule is at least 50,100,200 or 400 dalton.

With regard to protein, for example SIRT3 or AceCS2, term " subdomain ", " structural domain ", " functional domain " or its grammer equivalents refer to this proteinic fragment, the fragment of SIRT3 or AceCS2 for example, it participates in interacting, for example intramolecularly or molecular interaction interact as combination or catalysis.Intramolecular interaction can be specificity binding interactions or enzymatic interaction (for example, interaction can be instantaneous and form or interrupt covalent linkage).Molecular interaction can be between protein and another protein, between protein and another compound, or between proteinic first molecule and second molecule (for example, dimerization interacts).Proteinic biologic activity part and/or functional domain comprise contained aminoacid sequence and the enough homologies of this proteinic aminoacid sequence or derived from the peptide of this proteinic aminoacid sequence, the amino acid of this peptide is less than the natural protein of total length and shows at least a activity of this natural protein.Can be by various technical evaluation biologic activity part/functional domains, these technology comprise brachymemma analysis, site-directed mutagenesis and proteolysis.Can be by the activity of suitable biological chemistry or biology (for example, genetics) experimental examination mutant or proteolytic fragments.In some embodiments, functional domain is independent folding.Biological active fragment comprises usually and has proteinic at least a active structures territory or motif, for example SIRT3 or AceCS2 core catalyst structure domain.Proteinic biologic activity part/functional domain can be that length is, for example 10,25,50,100,200 or more a plurality of amino acid whose polypeptide.Protein, for example the biologic activity part/functional domain of SIRT3 energy (i) (ii) has deacetylated activity in conjunction with the AceCS2 polypeptide, (iii) is assembled into to comprise for example multiprotein complex of AceCS2, or (iv) makes AceCS2 deacetylated.Protein, for example the biologic activity part/functional domain of AceCS2 energy (i) is in conjunction with the SIRT3 polypeptide, (ii) has enzymatic activity, (iii) be assembled into and comprise for example multiprotein complex of SIRT3, (iv) utilize acetate, ATP and CoA to produce acetyl-CoA and AMP, or be positioned plastosome as substrate.

" being correlated with " used herein polypeptide refers to homologue, isotype, straight fusion rotein or fragment or their any combination to homologue, polypeptide.

" SIRT3 homologue " or " AceCS2 homologue " refers to contained aminoacid sequence similar to SIRT3 or AceCS2 respectively, but not necessarily has the polypeptide of or identical function similar to SIRT3 or AceCS2.

" SIRT3 isotype " or " AceCS2 isotype " refers to same genes encoding respectively, but its pI or MW are different or all different SIRT3 or AceCS2 variants.The amino acid of these isotypes is formed can be different (for example, alternately montage or proteolysis limited due to), in addition, can be because of due to the discrepant posttranslational modification (for example, glycosylation, acetylize or phosphorylation).

" SIRT3 is directly to homologue " used herein or " AceCS2 is directly to homologue " refer to non-human polypeptides, (i) its aminoacid sequence that comprises is similar to the aminoacid sequence of people SIRT3 or people AceCS2 respectively, with (ii) its function that has respectively with the functional similarity of people SIRT3 or people AceCS2 or identical.

" SIRT3 fusion rotein " used herein refers to comprise the polypeptide of following aminoacid sequence: (i) the segmental aminoacid sequence of SIRT3, SIRT3 fragment, SIRT3 subdomain polypeptide, SIRT3 related polypeptide or SIRT3 related polypeptide and (ii) heterologous polypeptide, the aminoacid sequence of promptly non--SIRT3, non--SIRT3 fragment or non--SIRT3 related polypeptide.Similarly, " AceCS2 fusion rotein " used herein refers to comprise the polypeptide of following aminoacid sequence: (i) the segmental aminoacid sequence of AceCS2, AceCS2 fragment, AceCS2 subdomain polypeptide, AceCS2 related polypeptide or AceCS2 related polypeptide and (ii) heterologous polypeptide, the aminoacid sequence of promptly non--AceCS2, non--AceCS2 fragment or non--AceCS2 related polypeptide.

Term used herein " solvate " refers to form with solvent The compounds of this invention, material and the small molecules of mixture.Can comprise conventional organic solvent with the solvent of The compounds of this invention, material and small molecules formation solvate, for example alcohols (methyl alcohol, ethanol etc.), ether, acetone, ethyl acetate, halogenated solvent (methylene dichloride, chloroform etc.), hexane and pentane.Other solvent comprises water.If water is double solvents, then this mixture is called " hydrate ".

The people or the non-human animal that will need treatment pathologic state, illness or disease used herein by " object " or " patient " expression of described method treatment pathologic state, illness or disease.Term " non-human animal " comprises all vertebratess, Mammals for example, as non-human primates (particularly senior primates), sheep, dog, rodents (for example, mouse or rat), cavy, goat, pig, cat, rabbit, ox and nonmammalian, as chicken, Amphibians, Reptilia etc.In an embodiment preferred, described to liking the people.In another embodiment, described to as if laboratory animal or be suitable as the animal of disease model.

Term used herein " treatment " comprising: (1) prevention pathologic state, illness or disease, promptly, cause this pathologic state, illness or disease-susceptible humans, but the object that does not experience any symptom of this pathologic state, illness or disease does not as yet produce the clinical symptom of this pathologic state, illness or disease; (2) suppress pathologic state, illness or disease, that is, the development of this pathologic state, illness or disease or its clinical symptom is stagnated or reduction; Or (3) alleviation pathologic state, illness or disease, that is, this pathologic state, illness or disease or its clinical symptom are disappeared.These terms also comprise prevention, diagnosis and treatment and healing.Any way that the treatment expression makes the doing well,improving of pathologic state, illness or disease or changes valuably.The object preferred mammal that needs this treatment, more preferably people.

The evaluation of II.SIRT3 activator and AceCS2 conditioning agent and test

The present invention identifies the AceCS2 as first cell target of SIRT3.In addition, the present invention also discloses SIRT3 and makes AceCS2 deacetylated in vitro and in vivo.In addition, this paper has described the enzymic activity of the acetylize state decision AceCS2 of AceCS2, promptly utilizes acetate, ATP and CoA to produce acetyl-CoA and AMP as substrate.According to discovery as herein described, the inventor has designed and has identified the whole bag of tricks that improves SIRT3 level or active material and regulate AceCS2 level, acetylize state or active material.

Adopt method known in the art and as herein described to identify the material of activation SIRT3 or the material of adjusting AceCS2.Can adopt many different screening schemes to identify and improve SIRT3 level or active or adjusting AceCS2 level or active material.Term " evaluation " and " screening " are used interchangeably in this article.

Preferably energy selectivity raising, selectivity activation or selectivity are regulated level, acetylize state or the active material of described polypeptide.Therefore; in some embodiments, be applicable to that methods of treatment described herein also can improve (i) SIRT3 level or activity or (ii) regulate AceCS2 level, acetylize state or active material is respectively the selectivity movable agent of SIRT3 or the selective modulator of AceCS2.Therefore, be not obvious inhibition or activate other enzyme, comprise for example proteic material of other Sir2-sample, for example at the IC of SIRT3 as the material of the selectivity movable agent of SIRT3 ₅₀On, this material is no more than about 5% to the inhibition of another kind of Sir2-sample protease activity or activation, be no more than about 10% or be no more than about 25%.In addition, be not obvious inhibition or activate other enzyme, comprise that other AMP-for example forms the material of enzyme, for example at the IC of AceCS2 as the material of the selective modulator of AceCS2 ₅₀On, this material forms the inhibition of enzyme or activation to other AMP and is no more than approximately 5%, is no more than about 10% or be no more than about 25%.

In some embodiments of the present invention, screening scheme is in external application.Can be at cell, particularly mammalian cell is used other preferred screening scheme in preferred people's cell.The particularly preferred cell that is used for screening method of the present invention is heart cell, muscle cell or brain cell.

The used described polypeptide of the inventive method can be natural polypeptides or recombinant polypeptide.In some embodiments, described polypeptide is SIRT3 related polypeptide and/or AceCS2 polypeptide.

As used herein, SIRT3 or the AceCS2 polypeptide that is used for described method can include but not limited to from various species: people, mouse or rat.Preferred SIRT3 and AceCS2 polypeptide are people SIRT3 and AceCS2 polypeptide.

As further described herein, SIRT3 or AceCS2 polypeptide can be total length SIRT3 or AceCS2 or its fragment or structural domain.In addition, SIRT3 and AceCS2 related polypeptide, SIRT3 and AceCS2 homologue, SIRT3 and AceCS2 isotype or SIRT3 and AceCS2 directly can be used for implementing the inventive method and composition to homologue.Preferred AceCS2 polypeptide fragment of the present invention comprises sequence NH ₂-KRLPKTRSGKVMRRLLRKII-COOH (SEQ ID NO:7) (Fig. 3 A).Other preferred embodiment in, the AceCS2 polypeptide comprises sequence NH ₂-CPKTRSG (ac) KVMRRLL-COOH (SEQ ID NO:11), it has acetylizad Methionin (acK642), or NH ₂-CPKTRSGKVMRRLL-COOH (SEQ ID NO:12).

The present invention's test generally includes one or more contrasts.Therefore, specimen comprises candidate substances, and control sample has all components except that candidate substances in the specimen.Usually the many parts of test mixtures of the material that contains different concns that run parallel, thus differential responses obtained at various concentration.One of these concentration are usually as negative control, i.e. 0 concentration or be lower than detection level.

Those of ordinary skills should know that except that described polypeptide, shaker test can comprise all ingredients.These reagent comprise the described and known in the art buffer reagent of this paper embodiment, salt, stablizer, stain remover, proteinase inhibitor, nucleic acid inhibitor, biocide etc.

Can adopt various test as herein described to identify, test and verify and described hereinly can improve SIRT3 level or active candidate substances, maybe can regulate AceCS2 level, acetylize or active material.These tests include but not limited to: for example (i) Northern trace test, and (ii) in situ hybridization, (iii) western blotting test, (iv) immunoprecipitation test, (v) immunohistochemical test, (vi) test cell line, (vii) in vivo test, or the like.In vitro tests can utilize the SIRT3 and/or the AceCS2 polypeptide of purifying or comprise SIRT3 and/or the multiprotein complex of AceCS2 polypeptide, as further described herein.

The material that can select the activity of described polypeptide to be improved or be reduced to required degree is done further research, assessment cell availability, cytotoxicity, biocompatibility etc.

The test that employing is known, for example trypan blue dye exclusion assessment candidate substances such as (MTT ([3-(4,5-dimethylthiazole-2-yl)-2,5-phenylbenzene-2H-bromination tetrazolium]) tests) is to testing the issuable any cytotoxicity of used cell.Preferably do not produce Cytotoxic material or do not produce obvious Cytotoxic material.

In case identified candidate substances and it be further used in method of the present invention, composition or the test kit that with one of screening method of the present invention it is commonly referred to material or biological active agents rather than candidate substances.

In some embodiments, interested especially is can (i) to improve the SIRT3 activity, (ii) improve SIRT3 polypeptide level in the cell or (iii) improve the material of SIRT3mRNA level in the cell.Interested especially also have can (i) improve the AceCS2 activity, (ii) improve AceCS2 polypeptide level in the cell or (iii) improve the material of AceCS2mRNA level in the cell.These materials can be used for treating and are characterised in that acetate horizontal abnormality height or the pathologic state that acetyl-the CoA horizontal abnormality is low, for example type ii diabetes, hypercholesterolemia, hyperlipidaemia and obesity.

In some embodiments, interested especially is can (i) to reduce the AceCS2 activity, (ii) reduce AceCS2 polypeptide level in the cell or (iii) reduce the material of AceCS2mRNA level in the cell.These materials can be used for treating the pathologic state that is characterised in that acetyl-the CoA horizontal abnormality is high.

A. The material of activation SIRT3

These screening methods are usually directed to screen various materials can improve SIRT3 level or activity to identify, the active material of deacetylase of preferred SIRT3.Screening method generally includes following steps: the mammalian cell that (a) make candidate substances and (i) SIRT3 and/or AceCS2, (ii) contains the biological sample of SIRT3 and/or AceCS2 or (iii) express SIRT3 and/or AceCS2 contacts; (b) in level that check SIRT3 in the presence of the candidate substances is arranged or level or the activity of activity and/or AceCS2.With appropriate control (for example, there is not the SIRT3 under the candidate substances existence, there is not candidate substances to have the biological sample that contains SIRT3 down, or do not have candidate compound to have the mammalian cell of expressing SIRT3 down) in the activity of SIRT3 compare, detect level or the active activity that shows this candidate substances activation SIRT3 that improves of SIRT3.

On the one hand, screening method comprises that screening of candidate substances can improve SIRT3 level or active material to identify.Term " improves SIRT3 level or activity " and comprises with suitable contrast and comparing, SIRT3 polypeptide, SIRT3 nucleic acid or SIRT3 activity, and for example the active detection level of SIRT3 deacetylase improves.

On the one hand, the invention provides the method that evaluation can improve SIRT3 polypeptide level or the active material of deacetylase.In preferred implementation of the present invention, this method may further comprise the steps: if (a) cell of expressing SIRT3 polypeptide and AceCS2 polypeptide contact with candidate substances and (b) effect, the effect of acetylize AceCS2 level in the mensuration candidate substances pair cell arranged.Identify by this and can improve SIRT3 polypeptide level or the active candidate substances of deacetylase.

In another aspect of this invention; the method that evaluation can improve SIRT3 polypeptide level or the active material of deacetylase may further comprise the steps: if (a) SIRT3 polypeptide and acetylize AceCS2 polypeptide in the test mixture contacted with candidate substances and (b) effect is arranged, measure the effect of candidate substances to acetylize AceCS2 level in the test mixture.Identify by this and can improve SIRT3 polypeptide level or the active candidate substances of deacetylase.Do not compare with second level of acetylize AceCS2 in the test mixture of handling with candidate substances, first level of acetylize AceCS2 reduces and shows that this material can improve the level or the deacetylase activity of SIRT3 polypeptide in the test mixture.

In a preferred embodiment, described test mixture comprises NAD ⁺

Can adopt variety of way to measure the effect of acetylize AceCS2 level in candidate substances pair cell or the test mixture.In a preferred embodiment, this step comprises the immunological testing of the specific antibody that utilizes acetylize AceCS2 described herein.Have candidate substances to exist down, the minimizing of acetylize AceCS2 polypeptide shows this material activation SIRT3.

In another preferred implementation of the present invention, AceCS2 comprises marker, and for example the ethanoyl of mark is preferred ¹⁴The ethanoyl of C-mark.Therefore, in this embodiment, the AceCS2 polypeptide by certification mark discharges ¹⁴C-mark ethanoyl is measured the effect of acetylize AceCS2 level in candidate substances pair cell or the test mixture.Can conventional sense discharge ¹⁴The C mark for example carries out scintillation counting then by chromatography.There is candidate substances to exist down ¹⁴The C marker discharges to increase and shows that this material can activate SIRT3.

Evaluation can improve the optional structure or the sequence of identifying candidate substances that comprise of method of SIRT3 level or active material.

With suitable contrast, preferably do not have candidate substances to exist contrast down to compare, interested material standed for mass-energy with SIRT3 level or active raising at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 500% or higher.

B. Regulate the material of Acetyl-CoA synthetase 2

Adopt method known in the art and as herein described to identify inhibitor and activator, this paper is called level, acetylize state or the active conditioning agent of AceCS2.Can adopt many different screening schemes to identify and to regulate AceCS2 level, acetylize state or active material.Term " adjusting " comprises with suitable contrast to be compared, the detection level of AceCS2, acetylize state or active the raising or reduction.Preferred AceCS2 activator.Yet some embodiment may need to suppress AceCS2.

These screening methods are usually directed to screen various materials can regulate AceCS2 level, acetylize state or active material to identify.This method generally includes following steps: the mammalian cell that (a) makes candidate substances with (i) AceCS2, (ii) contain the biological sample of AceCS2 or (iii) express AceCS2 contacts; (b) in level, acetylize state or activity that check AceCS2 in the presence of the candidate substances is arranged.With appropriate control (for example; there is not the AceCS2 under the candidate substances existence; there is not candidate substances to have the biological sample that contains AceCS2 down; or do not have candidate compound to have the mammalian cell of expressing AceCS2 down) in level, acetylize state or the activity of AceCS2 compare, detect AceCS2 level, acetylize state or active improve or reduce show that this material standed for mass-energy regulates level, acetylize state or the activity of AceCS2.

1. regulate Acetyl-CoA synthetase 2 levels, acetylize state or active material

One aspect of the present invention provides the method for identifying level, acetylize state or the active material that can regulate the AceCS2 polypeptide.In preferred implementation of the present invention, this method may further comprise the steps: if (a) cell of expressing the AceCS2 polypeptide contact with candidate substances and (b) effect, AceCS2 level, acetylize state or active effect in the mensuration candidate substances pair cell arranged.Identify by this and can regulate AceCS2 level, acetylize state or active candidate substances.

In another aspect of this invention; the method that evaluation can be regulated level, acetylize state or the active material of AceCS2 polypeptide may further comprise the steps: if (a) AceCS2 polypeptide in the test mixture is contacted with candidate substances and (b) effect is arranged, measure candidate substances to AceCS2 level, acetylize state or active effect in the test mixture.Identify by this and can regulate AceCS2 polypeptide level, acetylize state or active candidate substances.

In an embodiment preferred, cell or test mixture comprise the substrate of AceCS2 polypeptide.Preferred substrate is the acetate that can mark.In such embodiment, the mensuration candidate substances can comprise AceCS2 synthetic acetyl-CoA and the AMP level of detecting to the step of AceCS2 active function.

The preferred activity of AceCS2 is that enzymatic conversion becomes acetyl-CoA and AMP with CoA with acetate, ATP.Therefore, on the one hand, the invention provides the method that evaluation can be regulated the candidate substances of AceCS2 enzymic activity.One preferred embodiment in, this method may further comprise the steps: (a) having in the presence of the substrate, the AceCS2 polypeptide is contacted with candidate substances and (b) check AceCS2 enzymic activity to identify the candidate substances that can regulate the AceCS2 enzymic activity.Identify by this and can regulate the active candidate substances of AceCS2 polypeptidase.

Preferred substrate is acetate, ATP and/or CoA.This paper has described the test of measuring the AceCS2 enzymic activity.

The material that can regulate the AceCS2 level comprises that (i) for example can regulate the material of AceCS2 mRNA level and (ii) can regulate the material of AceCS2 polypeptide level by activation or inhibition AceCS2 genetic expression.This paper provides the method for measuring mRNA and polypeptide level.

2. with Acetyl-CoA synthetase 2 bonded materials

The present invention also provide evaluation can with the method for Acetyl-CoA synthetase 2 (AceCS2) bonded material.One aspect of the present invention provide evaluation can with the method for AceCS2 polypeptide bonded material.Whether in the present invention's one preferred implementation, this method may further comprise the steps: the cell of expressing the AceCS2 polypeptide is contacted with candidate substances and (b) measure candidate substances to combine with AceCS2.Identifying by this can be in conjunction with the candidate substances of AceCS2 polypeptide.

Whether in another aspect of this invention, evaluation can may further comprise the steps in conjunction with the method for the material of AceCS2 polypeptide: (a) make AceCS2 polypeptide in the test mixture contact with candidate substances and (b) measure candidate substances and combine with AceCS2.Identifying by this can be in conjunction with the candidate substances of AceCS2 polypeptide.

The AceCS2 polypeptide that can be used for implementing the method for the invention is the AceCS2 that natural A ceCS2 polypeptide or reorganization produce.

Can adopt the whole bag of tricks described herein and known in the art to detect the situation that combines of candidate substances and AceCS2 polypeptide.

Can earlier the AceCS2 polypeptide be attached to solid support, contact with candidate substances again.This paper has described the coupling of described polypeptide and solid support, solid support and their application in the method for the invention.

3. regulate interactional material between AceCS2 and the SIRT3

The inventor proves that AceCS2 can interact with SIRT3.Therefore, the present invention also provides evaluation can regulate the method for interactional material between AceCS2 polypeptide and the SIRT3 polypeptide.

One aspect of the present invention provides evaluation can regulate the method for interactional material between AceCS2 polypeptide and the SIRT3 polypeptide.In the present invention one preferred embodiment, this method may further comprise the steps: if (a) cell of expressing SIRT3 polypeptide and AceCS2 polypeptide contact with candidate substances and (b) effect is arranged, the mensuration candidate substances is to AceCS2 and the interactional effect of SIRT3 polypeptide.Identify by this and can regulate interactional candidate substances between AceCS2 polypeptide and the SIRT3 polypeptide.

In another embodiment of the present invention, the method that evaluation can be regulated interactional material between AceCS2 polypeptide and the SIRT3 polypeptide may further comprise the steps: if (a) test mixture that contains AceCS2 polypeptide and SIRT3 polypeptide is contacted with candidate substances and (b) effect arranged, measure candidate substances to AceCS2 polypeptide and the interactional effect of SIRT3 polypeptide.Identify by this and can regulate interactional candidate substances between AceCS2 polypeptide and the SIRT3 polypeptide.

4. regulate the material that Acetyl-CoA synthetase 2 is assembled into multiprotein complex

The inventor proves and forms the multiprotein complex contain SIRT3 by AceCS2 (for example embodiment 11, Fig. 9).Therefore, the present invention also provides evaluation can regulate the method that AceCS2 is assembled into the material of multiprotein complex.Multiprotein complex preferably comprises the SIRT3 polypeptide.

One aspect of the present invention provides evaluation can regulate the method that AceCS2 is assembled into the material of multiprotein complex.In the present invention's one preferred implementation, this method may further comprise the steps: if (a) cell of expressing AceCS2 polypeptide and SIRT3 polypeptide contact with candidate substances and (b) effect is arranged, the mensuration candidate substances is assembled into the effect of the multiprotein complex that contains the SIRT3 polypeptide to AceCS2.Identify by this and can regulate the candidate substances that the AceCS2 polypeptide is assembled into multiprotein complex.

The present invention provides evaluation can regulate the method that the AceCS2 polypeptide is assembled into the material of the multiprotein complex that contains AceCS2 polypeptide and at least a second polypeptide on the other hand.This method may further comprise the steps: if (a) test mixture that contains the AceCS2 polypeptide and second polypeptide is contacted with candidate substances and (b) effect arranged, measure candidate substances is assembled into multiprotein complex to AceCS2 polypeptide in the test mixture effect.Identify by this and can regulate the candidate substances that the AceCS2 polypeptide is assembled into multiprotein complex.

In this method one preferred implementation, described second polypeptide is the SIRT3 polypeptide.

In another preferred implementation of the present invention, AceCS2 comprises epi-position-label, and for example sign, hemagglutinin (HA) or 6 * His-label (SEQ ID NO:13) are as described herein.In other embodiments, the SIRT3 polypeptide comprises the epi-position label.The polypeptide that the detection easily of epi-position label and purifying link to each other with these labels.Utilize anti--sign antibody, anti--HA antibody or anti--6 * His-antibody and ordinary method known in the art to detect and purifying.

Can adopt the whole bag of tricks to measure candidate substances is assembled into multiprotein complex to AceCS2 in the test mixture effect.Preferred method is column chromatography as herein described or immunoprecipitation.In the method, antibody, for example anti--AceCS2 are used for immunoprecipitation AceCS2 and any other polypeptide of bonded with it.Can pass through, for example SDS-PAGE, mass spectroscopy or immunoblotting visual observations combine and therefore form the polypeptide of multiprotein complex with AceCS2.Can utilize the situation that combines of anti--SIRT3 TPPA SIRT3 polypeptide and AceCS2.

Also can adopt evaluation can regulate method that AceCS2 is assembled into the material of multiprotein complex identifies except that SIRT3 and AceCS2 bonded cell protein.For example, can utilize the antibody of the AceCS2 of AceCS2 in the immunoprecipitation cell or epi-position mark to detect and purifying and AceCS2 bonded cell polypeptide.Can pass through, for example SDS-PAGE or mass spectroscopy detect and AceCS2 bonded polypeptide, and these polypeptide also can further be identified by peptide fingerprinting described herein and known in the art.A kind of such polypeptide that can adopt this method to identify is the acetyltransferase polypeptide of acetylize AceCS2 polypeptide.

In preferred embodiment; (i) level, acetylize state or the activity of adjusting AceCS2 polypeptide; (ii) combine, (iii) regulate AceCS2 and be assembled into multiprotein complex, or the material of (iv) regulating the cellular localization of AceCS2 is the SIRT3 polypeptide with the AceCS2 polypeptide.The SIRT3 polypeptide can be natural SIRT3 polypeptide or its fragment.The SIRT3 polypeptide can also be recombinant expressed SIRT3 polypeptide or its fragment.In addition, the SIRT3 polypeptide can be SIRT3 related polypeptide or its fragment.

5. regulate the material of the cellular localization of Acetyl-CoA synthetase 2

The inventor proves that in this article AceCS2 and SIRT3 are positioned plastosome.Because the vital role of SIRT3 in AceCS2 is deacetylated, it is most important for control AceCS2 enzymic activity that adjusting AceCS2 and/or SIRT3 are positioned plastosome.

Therefore, another aspect of the present invention provides evaluation can regulate the method for the material of SIRT3 polypeptide or the cellular localization of AceCS2 polypeptide.In the present invention's one preferred implementation, this method may further comprise the steps: (a) make the cell of expressing SIRT3 polypeptide and/or AceCS2 polypeptide contact and (b) measure the cellular localization of AceCS2 polypeptide and/or SIRT3 polypeptide with candidate substances, thereby identify the material that can regulate SIRT3 polypeptide and/or the cellular localization of AceCS2 polypeptide.

Preferred material is that enhancing SIRT3 polypeptide and/or AceCS2 are positioned mitochondrial material.The location that strengthens SIRT3 polypeptide and/or AceCS2 polypeptide comprises and prolongs SIRT3 polypeptide and/or the AceCS2 polypeptide retention time at required cell position.

Can adopt the whole bag of tricks known in the art to measure the cellular localization of SIRT3 or AceCS2 polypeptide.Preferred method is the immunohistochemical methods method or utilizes anti--SIRT3 antibody and anti--AceCS2 antibody to pass through immunoblotting assay plastosome component (for example, referring to Fig. 1 and respective embodiments).

Can adopt the whole bag of tricks described herein and known in the art to measure the influence of candidate substances: (i) level of AceCS2 polypeptide, acetylize state or activity to following incident; (ii) in conjunction with the AceCS2 polypeptide; (iii) interact between AceCS2 polypeptide and the SIRT3 polypeptide, (iv) AceCS2 is assembled into multiprotein complex or (the v) cellular localization of AceCS2 polypeptide.

Interested candidate substances is and suitable contrast; the preferred candidate material exists contrast down to compare, and can strengthen AceCS2 level, acetylize state or activity at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 500% or higher material.

C. Utilize the test of protein purification or multiprotein complex

Evaluation selectivity SIRT3 activator or selectivity AceCS2 conditioning agent (activator or inhibitor) can carry out based on mode cell or acellular with the test of general activator or inhibitor.For example, test can comprise described polypeptide or described nucleic acid and test substances incubation (or contact) under level that can activate described polypeptide or described nucleic acid or active condition, and monitoring or measure and do not have test substances to exist down to compare, the activation levels under the test substances existence is arranged.

AceCS2 can be natural A ceCS2 or reorganization AceCS2.SIRT3 can be natural SIRT3 or reorganization SIRT3.Can from, for example people or mouse tissue or for example people or mouse cell purifying natural AceCS2 or SIRT3.Can be from any suitable expression system purification of Recombinant AceCS2 known in the art and SIRT3, from host cell, preferred mammal host cell purification of recombinant proteins.

Can AceCS2 and SIRT3 be purified to suitable purity by standard technique, described standard technique for example includes but not limited to: column chromatography, immune purification process, utilize the selective precipitation of ammonium sulfate, or the like.

In some embodiments of the present invention, screening method and testing experiment comprise and utilize multiprotein complex, and it comprises (i) AceCS2 and SIRT3 at least, or the (ii) AceCS2 and second polypeptide, or the (iii) SIRT3 and second polypeptide.For isolating protein, can from, for example people or mouse tissue, people or mouse cell obtain, or the albumen of these multiprotein complexs of reorganization preparation.If the reorganization preparation is assembled into multiprotein complex with described albumen earlier usually, again they are used for one of described method.Can adopt the whole bag of tricks known in the art to carry out.

For in vitro tests, can the described polypeptide of (but not necessarily) purifying.Can adopt the described protein of methods known in the art purifying.Can the partial purification cell or the described polypeptide of host cell, however it is pure preferably to be measured to described polypeptide at least 90% by standard SDS-PAGE.

D. Test cell line

Can adopt test cell line to identify and test activation SIRT3 level or active candidate substances and adjusting AceCS2 level, acetylize state or active material (for example, referring to embodiment 5,6,9 and 11).In addition, can adopt coli test as herein described to identify and/or test candidate substances (referring to embodiment 7,8 and 12).

Test cell line utilizes eukaryotic cell, for example mammalian cell usually.In some embodiments, can utilize yeast cell.Described cell can be from the isolating primary cell of donor biological sample.Perhaps, described cell can be the existing clone that obtains by American Type Culture Collection.

(referring to embodiment) as described herein, cell use expression constructs, and for example AceCS2 expression constructs or SIRT3 expression constructs are instantaneous or the cell of stable transfection.

E. In vivo test

Also can identify and test activation SIRT3 level or active candidate substances in vivo and regulate AceCS2 level, acetylize state or active material.In the method; give animal with certain material; preferred mouse; each time point after giving this material obtains blood sample or the tissue sample of this animal; and test, for example: (i) acetyl-CoA level, (ii) ATP level; the (iii) level of AceCS2, acetylize state or activity, or (iv) level or the activity of SIRT3.

F. Detect mRNA

This paper provides the method for test and check methods described herein compounds identified, material or antagonist, comprises that various accepted tests determine whether given candidate substances or small molecules can be used for implementing the inventive method.The inventive method can be chosen wantonly and comprise detection nucleic acid, for example the step of mRNA or polypeptide.In one embodiment, this method comprises measures or detects mRNA, preferred SIRT3 mRNA or AceCS2mRNA.Also can adopt following method to measure other mRNA of coding polypeptide described herein.Those skilled in the art know the method for the mRNA expression of assessment specific gene, comprise hybridization and amplification test etc.

1. direct cross test

The known employing nucleic acid hybridization technique of those skilled in the art detects and/or the method for quantitative assay genetic transcription thing (mRNA or from the cDNA of its generation) level.For example, the existence of assessment polynucleotide whether or a kind of method of quantity comprise the Northern trace.Also can be by technology known in the art; for example Dot blot, in situ hybridization, RNA enzyme protection, detection DNA microchip array (probing DNA microchiparray) etc. are (for example; referring to Sambrook; J., Fritsch, E.F. and Maniatis; " MolecularCloning A Laboratory Manual (molecular cloning laboratory manual) "; press of cold spring harbor laboratory publishes, and the 2nd edition, 1989) the analyzing gene expression level.

2. amplification test

In another embodiment, adopt the amplification test to detect the expression of gene level.In this test, nucleotide sequence is as the template of amplified reaction (for example polymerase chain reaction or PCR).In quantitative amplification, the amount of amplified production and the template amount in the primary sample are proportional.Making comparisons with suitable contrast is the tolerance of mRNA level in the sample.Those skilled in the art know the method for quantitative amplification.For example (academic press (Academic Press, Inc.), New York) provides the detailed method of quantitative PCR to Innis etc. for (1990) PCR Protocols, A Guide to Methods and Applications (PCR scheme, method and application guide).

An embodiment adopts TaqMan to test quantitative polynucleotide.The TaqMan test utilizes the product fluorescence polynucleotide probes that contains 5 ' fluorescence dye and 3 ' quencher.Probe and the hybridization of PCR product, but self can not extend because of the 3 ' blocker of holding.If amplification PCR product in round subsequently, polymkeric substance then, for example 5 ' of AmpliTaq nuclease can cut the TaqMan probe.This cutting separates 5 ' fluorescence dye and 3 ' quencher, thus cause fluorescence with amplification increase (referring to, Heid etc. for example, 1996, Genome Res 6 (10): 986-94; Morris etc., 1996, J Clin Microbiol 34 (12): 2933-6).

Other suitable amplification method includes but not limited to: ligase chain reaction (LCR) (LCR) (referring to, Wu and Wallace, 1989, Genomics 4:560; Landegren etc., 1988, Science 241:1077; With Barringer etc., 1990, Gene 89:117), transcription amplification (Kwoh etc., 1989, Proc NatlAcad Sci USA 86:1173), self-sustained sequence replication (Guatelli etc., 1990, Proc Nat Acad SciUSA 87:1874), the sub-PCR of spot PCR and dovetail connector (linker adapter PCR) etc.

G. Detect polypeptide

The invention described above method can be chosen wantonly and comprise mensuration or detect polypeptide, for example step of AceCS2 or SIRT3 polypeptide.Also can adopt following method to measure other polypeptide as herein described.

Can measure or detect polypeptide in every way, for example AceCS2, SIRT3 and other polypeptide, described mode includes but not limited to: detection of biological each peptide species in product, cell, organ or the animal (comprising people and non-human animal) that imitates.

Can adopt the whole bag of tricks to measure the polypeptide expression level, include but not limited to: affinity capture, mass spectroscopy, traditional immunoassay and immunoprecipitation test, PAGE, western blotting, RIA or HPLC, (for example, referring to Fig. 1 and 3-10 and respective embodiments) or well known by persons skilled in the art as further described herein.

The detection paradigms that can be used for this purpose comprises optical means, electrochemical method (voltmeter and amperometer technology), atomic force microscopy and radio frequency method, and for example multipole resonance spectrum is learned.Except that microscopy, exemplary optics method (copolymerization burnt and non-copolymerization Jiao) is detection fluorescence, luminous, chemoluminescence, absorbancy, reflectivity, transmissivity and double refraction or specific refractory power (for example, surperficial plasmon resonance, ellipsometry, resonant mirror method, grating coupler waveguide method (grating coupler waveguide method) or interferometric method).

Utilization, for example the adjusting (for example, referring to Fig. 5 B and respective embodiments) of AceCS2 polypeptide acetylize state under the candidate substances existence can be assessed not or be had to antibody as resisting-AceCS2 antibody with as herein described of producing with acetylize AceCS2 peptide.Other antibody as herein described can be used for detecting AceCS2 expression level or SIRT3 expression level.

H. Detect enzymic activity

1. detect SIRT3 deacetylase activity

Now describe various tests and detected the proteic deacetylase activity of Sir2-sample.For example, Imai etc. and Frye reported utilize histone as substrate detect yeast Sir2 and people SIRT2 the active test of NAD-dependency deacetylase (Imai etc., 2000, Nature 403 (6771): 795-800; Frye1999, Biochem Biophys Res Comm 260:273-279).Yet, Imai or the Frye deacetylase activity of all not testing Sir2-sample albumen pair cell polypeptide no matter.

The invention describes the first deacetylated in vitro and in vivo cell SIRT3 target polypeptide, AceCS2 by SIRT3.As further described herein, the AceCS2 polypeptide is relevant with SIRT3.Therefore, in the present invention's one preferred implementation, the enzymic activity of measuring SIRT3 in the presence of the substrate A ceCS2 described herein is being arranged.Can adopt this test assessment SIRT3 deacetylase activity not or when having candidate substances to exist.

Can pass through, for example monitor the deacetylated activity of SIRT3 AceCS2 with the anti--AceCS2 antibody that can detect acetylize and deacetylated AceCS2 and the immunoblotting of acetylize AceCS2 specific antibody.Perhaps, also can monitor the deacetylated activity of SIRT3 by measuring AceCS2 enzymic activity described herein to AceCS2.Also can adopt acetylize state (for example, Fig. 4 and 6 of LC-MS/MS assay determination AceCS2 described herein; Embodiment 6 and 8).

2. detect the AceCS2 enzymic activity

This paper has described the active test of (for example, referring to embodiment 1) test AceCS2 known in the art.Also can measure the AceCS2 enzymic activity, for example measure by isotropic substance or spectrophotometry (Fujino etc., 2001, JBiol Chem 276:11420-11426).

The standard reaction mixture of isotope method contains 100mM Tris-HCl, pH8.5,10mMMgCl ₂, 10mM ATP, 1mM CoA and 10mM[ ¹⁴C] acetate (salt) (940dpm/nmol), cumulative volume 0.2mL.37 ℃ of preincubation added AceCS2 to start this reaction after 1 minute.Behind the incubation 30 minutes, add the ice-cooled Glacial acetic acid of 50 μ l with termination reaction.By point sample a slice chromatography media (ITLC-SG type, the graceful scientific company of gill (Gelman Sciences)) go up with reaction product isolated ([ ¹⁴C] acetyl-CoA), a large amount of washings of water-saturated ethers/formic acid (7:1) come detection of radioactive (Fujino etc., 2001, J Biol Chem 276:11420-11426).

Spectrophotometry is according to utilizing Myokinase, pyruvate kinase and serum lactic dehydrogenase to form AMP.The standard reaction mixture of spectrophotometry contains 100mM Tris-HCl, pH8.5,1mM DTT, 15mM MgCl ₂10mM ATP, 0.25mM phosphoenolpyruvic acid potassium, 1mM acetate (salt), 0.3mM NADH, 80 unit Myokinases (roche molecular biochemicals company (Roche MolecularBiochemicals)), 17 unit acetate desaturases (roche molecular biochemicals company) and 6 unit pyruvate kinases (roche molecular biochemicals company), cumulative volume 1mL.37 ℃ of preincubation added 24 μ L 25mM CoA to start reaction after 1 minute.Utilize the recordable type spectrophotometer to detect the oxidation situation of NADH with 340nm.Form 1 mole of ADP corresponding to 2 moles of NADH of oxidation (Fujino etc., 2001, J BiolChem 276:11420-11426).Other suitable spectrophotometry known in the art (Jones and Lipmann, 1955, Methods in Enzymology (Enzymology method) (academic press, the 1st volume, 585-591 page or leaf; Barak etc., 2004, J Mol Biol 342:383-401).

Another the useful test that detects the AceCS2 enzymic activity be with cell with [ ¹⁴C] acetate (salt) incubation, subsequent analysis ¹⁴C enters CO ₂Situation (Fujiino etc., 2001, J Biol Chem276:11420-11426) with lipid.

I. The double cross test

In another embodiment of screening method of the present invention, can adopt the two-crossing system (" MATCHMAKER Two-Hybrid system (MATCHMAKER pair-crossing system) " that utilizes cell, " Mammalian MATCHMAKER Two-Hybrid Assay Kit (mammalian cell MATCHMAKER pair-the hybrid experiment test kit) ", " MATCHMAKER one-Hybridsystem (MATCHMAKER list-crossbred system) " (clone technology company (Clontech)); " HybriZAP Two-Hybrid Vector System (HybriZAP pair-the hybrid vector system) " (Xstrata genome company (Stratagene)); Also can be referring to Dalton and Treisman, 1992, Cell 68:597-612; Fields and Sternglanz, 1994, Trends Genet 10:286-92).

In two-hybrid system, for example, SIRT3, preferred SIRT3 polypeptide is blended in SRF-land or GAL4-land and expresses in yeast cell.To be blended in VP16 or GAL4 transcriptional activation domain in conjunction with the AceCS2 polypeptide of SIRT3 polypeptide, it is also expressed in yeast cell when having test compounds to exist.Perhaps, the AceCS2 polypeptide in conjunction with the SIRT3 polypeptide can be blended in SRF-land or GAL4-land, the SIRT3 polypeptide is blended in VP16 or GAL4 transcriptional activation domain.Article two, polypeptide is in conjunction with activating reporter gene, thereby can detect positive colony.For reporter gene, except that the HIS3 gene, can use for example Ade2 gene, lacZ gene, CAT gene, luciferase genes etc.

The candidate substances of debond SIRT3 or AceCS2 does not influence the activation of reporter gene.Yet, strengthen SIRT3 and AceCS2 bonded material and can activate reporter gene more strongly.On the contrary, suppressing between SIRT3 and the AceCS2 bonded candidate substances causes activating more weak or does not activate reporter gene.

J. Detect the interaction between two molecules

Also can detect two molecules, fluorescent test is for example adopted in for example interaction between AceCS2 and SIRT3, AceCS2 and candidate substances or SIRT3 and the candidate substances, and wherein at least a molecule has been made fluorescent mark.An example of this test be fluorescence energy transfer (FET or FRET, i.e. FRET (fluorescence resonance energy transfer)) (referring to, Lakowicz etc. for example, U.S. Patent number 5,631,169; Stavrianopoulos etc., U.S. Patent number 4,868,103).Select the fluorophore marker on first " donor " molecule, thereby its fluorescent energy that sends is absorbed by the fluorescent marker on second " receptor " molecule, and then sent fluorescence because of absorbing energy.Perhaps, " donor " protein molecular can utilize the natural fluoresence energy of tryptophan residue simply.Selection can be sent the marker of the light of different wave length, thereby that " receptor " molecular marked compound can be with " donor " is different.Because the efficient that energy shifts between the marker and intermolecular every distance dependent, so can assess spatial relation between the molecule.If in conjunction with occurring between the molecule, then " donor " molecular marked compound emitted fluorescence should be maximum in the test.Can detect the FET binding events easily by standard fluorescence meter detection method well known in the art.

Another example of fluorescent test is fluorescence polarization (FP).For FP, only need a kind of component of mark.The variation that can pass through the molecular size of certification mark component detects binding interactions.Size variation has changed the upset rate of component in the solution, its detect for the change amount of FP (referring to, Nasir etc. for example, 1999, CombChem HTS 2:177-190; Jameson etc., 1995, Methods Enzymol 246:283; Seethala etc., 1998, Anal Biochem 255:257).Can in porous plate, monitor fluorescence polarization, for example utilize Tecan Polarion. ^TM.Reader monitoring (referring to, Parker etc. for example, 2000, J Biomol Screen5:77-88; With Shoeman etc., 1999, Biochemistry 38:16802-16809).

In another embodiment, can adopt real-time biomolecular interaction analysis (BIA) (referring to, for example Sjolander and Urbaniczky, 1991, Anal Chem 63:2338-2345; Szabo etc., 1995, Curr Opin Struct Biol 5:699-705) measures the ability of protein bound target molecule or candidate substances ability in conjunction with described polypeptide." surperficial plasmon resonance " or " BIA " detection of biological specificity in real time interact, and need not any interactant of mark (for example, BIAcore).The quality of mating surface changes the refraction index changing (optics of surperficial plasmon resonance (SPR) now looks like) that (showing combination) causes this near surface light, can be used as the detectable signal that shows real time reaction between the biomolecules thereby produce.

K. The computer test

Also may adopt structure-activity relation (SAR) and principles of structural design to identify and to improve SIRT3 level or active or adjusting AceCS2 level, acetylize state or active material.SAR provides related substances active information at least a correlation test.Has dependency between the constitutional features of substances of interest and the activity.For example, may come one or more required constitutional featuress of identified activity by the SAR of assessment and SIRT3 polypeptide and/or the interactional material of AceCS2 polypeptide family.Can produce material storehouse then, screen these storehouses again with these different characteristicss.Structure design can comprise determines material and target, for example the physical interaction structural models of SIRT3 polypeptide and/or AceCS2 polypeptide.How structural models can show the antagonist of engineered target.This antagonist can be used for changing life-span adjusting (lifespan regulation).

SAR and construction design method all can be used for identifying pharmacophore.Pharmacophore is extremely valuable and be useful concept in drug development and lead drug optimization.Pharmacophore is defined as different three-dimensional (3D) configuration that produces the necessary chemical group of biologic activity.Because pharmaceutically active molecule is wanted effectively, just must in subject, interact with one or more molecular structures, and the required function characteristic of molecule is derived from these and interacts, thereby then this interaction can take place each active compound chemical groups that must contain different configurations.The chemical group that is commonly referred to the description center can be expressed as: (a) atom or atomic group; (b) intend atom, the barycenter at for example Huan center, or molecule; (c) carrier, for example atom pairs, lone electron pair direction (electron lone pairdirections) or planar normal.In case designed pharmacophore, available its retrieval chemical compound database, for example have those databases compatible with this pharmacophore structure (referring to, for example U.S. Patent number 6,343,257; Martin, 1992, J Med Chem 35,2145-54).The database retrieval inquiry is not only according to chemical property information, also according to accurate geometry information.

Computer approach can utilize the template that database retrieval finds to mate (include the Martin of this paper by reference in, 1992, J Med Chem 35:2145-54).The 2-D of retrieval compound and the existing method of 3-D database are suitable for.(American Cyanamid, Pearl River, Lederle N.Y.) have developed shape of molecule retrieval, 3D retrieval and trend-carrier database in pearl river, New York American Cyanamid Company.Retailing merchant and other retrieval are organized retrieval service (MACSS-3D, molecular designing company limited (Molecular Design Ltd.) (holy Leonardo, California) also are provided; CAVEAT, Lauri, G etc., University of California (Berkeley, California); CHEM-X, chemical design company (Chemical Design, Inc.) (agate ten thousand (Mahwah), New Jersey)).Can utilize the retrieval software analysis with obvious chemistry and geometry structure be index the potential drug compound database (for example, Standard Drugs File (standard drug file) (derwent publications ltd. (DerwentPublications Ltd.), London, England), Bielstein database (the smooth database of Bel Si) (Bel Si Tan information firm (Bielstein Information), Frankfort, Germany or Chicago) and Chemical Registry database (chemical registration database) (CAS, Columbus, the Ohio)).

In case identify the compound that mates with pharmacophore, can test its activity, for example combine and/or regulate the biologic activity of polypeptide, as strengthening the enzymic activity of SIRT3 polypeptide and/or AceCS2 polypeptide with polypeptide.

In addition, 3 kinds of III fermentoids have now been measured, be intestinal bacteria CobB (Zhao etc., 2004, J MolBiol 337:731-741) and ancient coccus (A.fulgidus) Sir2-of light sample albumen, Sir2-Afl (Min etc., 2001, Cell 105:269-279) and the structure of Sir2-Af2 (Avalos, 2002, Mol Cell 10:523-535).The crystalline structure information of SIRT5 is by GenBank accession number 2FZQA, 2FZQB, 2B4YA, 2B4YB, 2B4YC and 2B4YD preservation.Can obtain Sir2-P53 peptide-niacinamide (GenBank accession number 1YC5), Sir2af2-NAD-ADP ribose-niacinamide (GenBank accession number 1YC2), NAD ⁺The niacinamide cutting of-dependency Sir2 histone deacetylase and the architecture basics (GenBank accession number 1SZD and 1SZC) that ADP-ribose shifts; The architecture basics of Sir2 enzyme mechanism and adjusting (GenBank accession number 1S7G); Other structured data with people SIRT2 histone deacetylase (1J8F).Finnin etc. (Finnin etc., 2001, Nat Struct Biol 8 (7): 621-5) reported people SIRT2 323 amino acid catalytic cores 1.7

Crystalline structure, it has shown NAD-binding domains (variant that Rossmann is folding) and the less structural domain that is made of spiral module and zinc binding modules.Finnin etc. have also described the conservative property major groove that is in two structural domain interfaces, and pointing out this may be catalytic site.The infall of this major groove is the pocket (pocket) that the spiral module constitutes.Be lined with hydrophobic residue in this pocket, interesting is is conservative property among each comfortable 5 class Sir2 of these residues, is classification specific proteins binding site (Finnin etc., 2001, Nat Struct Biol 8 (7): 621-5) thereby point out this.

See GenBank accession number 1NYH_A with the crystalline structure of the interactional yeast Sir4 of Sir3 coiled coil dimerization motif.In addition, Chang etc. have reported the X-ray structure of the interior coiled coil dimerization motif of C-end of Sir4, the dimerization fragment (residue 464-978) that shows itself and Sir3 forms stable 1:1 complex body and (includes the Chang of this paper etc. by reference in full in, 2003, Structure 11 (6): 637-649).In addition, Murphy etc. provides 2.5 of Sir4 CT fragment (Sir4p 1217-1358)

Resolving power X-ray crystal structure, thus the parallel coiled coil of 72 residue homodimers (GenBank accession number 1PL5S and 1PL5A disclosed; Include the Murphy of this paper etc. by reference in full in, 2003, J Mol Biol334 (4): 769-80).The available group design data of being somebody's turn to do interacts or enhancing SIRT3 polypeptide level or active or adjusting AceCS2 polypeptide level, acetylize state or active material with SIRT3 polypeptide and/or AceCS2 polypeptide in conjunction with SIRT3 polypeptide and/or AceCS2 polypeptide.

Therefore, in one aspect of the invention, can utilize the structure of SIRT3 polypeptide and/or AceCS2 polypeptide and/or demonstration and SIRT3 and/or AceCS2 have the another kind of Sir2-sample albumen of homology or an AceCS2 polypeptide in one or more structural domains structure to identify design and SIRT3 polypeptide and/or the interaction of AceCS2 polypeptide or combine the material of SIRT3 polypeptide and/or AceCS2 polypeptide.

Therefore, the III fermentoid of identifying the structure qualification of enable mode eclipsed provides useful tool for the activator of identifying Mammals Sir2-sample albumen, particularly SIRT3.For example, SIRT3 can with SIRT3 activator cocrystallization, can measure the three-dimensional structure of this complex body.Then can with interact between SIRT3 activator and the SIRT3 amino-acid residue and/or the microcomputer modelling program of importing for information about of the shape of conditioning agent binding site with design more new and SIRT3 activators may be more potent.Those skilled in the art can understand after reading this paper, can design AceCS2 activator and inhibitor by this way.

Therefore; the another kind test that activates SIRT3 polypeptide level or active candidate substances or adjusting AceCS2 polypeptide level, acetylize state or active material comprises the area of computer aided medicinal design, wherein utilizes computer system to produce the three-dimensional structure of SIRT3 or AceCS2 according to its amino acid sequences encoded structural information.In computer program, thereby the aminoacid sequence of input and pre-established algorithm directly and energetically interact and produce proteinic secondary, three grades and quaternary structure model.The model of checking protein structure then can be in conjunction with, the zone of another polypeptide or candidate substances for example to identify in the structure.Utilize these regional evaluations can activate the SIRT3 level then or active activator maybe can be regulated AceCS2 polypeptide level, acetylize state or active conditioning agent.

The protein amino acid sequence of at least 10 amino-acid residues or the nucleotide sequence input computer system of coding SIRT3 or AceCS2 are produced proteinic 3 d structure model.The aminoacid sequence of nucleic acid sequence encoding provided herein has been represented proteinic primary sequence or subsequence, the structural information of its coded protein.From the information of computer keyboard, computer-readable substrate (including but not limited to: electronic storage medium (for example disk, tape, coding tape (cartridge) and chip), optical medium (for example, CD ROM)), the Internet-distributed with by aminoacid sequence (encode 10 amino acid whose nucleotide sequences) the input computer system of RAM with at least 10 residues.Use software well known by persons skilled in the art then, the interaction by aminoacid sequence and computer system produces proteinic 3 d structure model.

Aminoacid sequence representative coding forms the secondary, three grades of proteins of interest matter and the primary structure of quaternary structure information needed.Software checks that some parameter of primary structure coding is to produce structural models.These parameters are called " energy term (energy term) ", mainly comprise electrostatic potential, hydrophobic electromotive force, solvent-accessible surface and hydrogen bond.The secondary energy term comprises Van der Waals gesture (van der Waals potential).Biological molecule forms the structure that reduces energy term in the accumulation mode as far as possible.Therefore, computer program utilize primary structure or amino acid sequences encoded these to produce the secondary structure model.

Energy term according to secondary structure forms the coded tertiary protein structure of secondary structure then.At this moment, the user can import other variable, and for example protein is membrane-bound or soluble, its position and cellular localization thereof in vivo, for example kytoplasm, surface or nuclear.Can utilize these variablees together with the energy term of secondary structure to form the model of tertiary structure.In the modeling process of tertiary structure, computer program is with the hydrophobic surface and similar coupling of secondary structure, with the water-wetted surface and the similar coupling of secondary structure.

In case produced this structure, can identify possible conditioning agent land by computer system.The chemical formula of input aminoacid sequence or nucleotide sequence or compound is to produce the three-dimensional structure of possible conditioning agent, and is as described herein.Then the three-dimensional structure of possible part and SIRT3 or AceCS2 are made comparisons to identify the binding site of SIRT3 or AceCS2.Utilize energy term to determine which kind of conditioning agent binding affinity between protein and the conditioning agent to measure and the combination of proteins probability is higher.

Also utilize homologue between sudden change, polymorphism variant, allelotrope and the species of gene of computer system screening code book invention SIRT3 or AceCS2 polypeptide.These sudden changes can be relevant with morbid state or hereditary feature.In addition, also can adopt GeneChip ^TMAnd homologue between correlation technique screening sudden change, polymorphism variant, allelotrope and species.In case identify variant, can adopt diagnostic test to identify to have patient this sudden change or the allele variant gene, for example diabetes or be in individuality in diabetes, hypercholesterolemia, hyperlipidaemia or the obesity risk.Identify that this SIRT3 and/or AceCS2 gene comprise first aminoacid sequence (or first nucleotide sequence of coding SIRT3 or AceCS2) of accepting input SIRT3 or AceCS2.Sequence is imported aforementioned calculation machine system.Then with first nucleic acid or aminoacid sequence with and substantially the same second nucleic acid or the aminoacid sequence of this first sequence make comparisons.In the above described manner with this second sequence input computer system.In case compared first and second sequences, then can identify Nucleotide or amino acid difference between the sequence.These sequences can be represented the allele variant of various SIRT3 or AceCS2 gene, and the sudden change relevant with morbid state and hereditary feature.

L. High throughput test

One preferred embodiment in, high-throughput screening method comprises provides combinatorial chemistry or the peptide library that contains a large amount of possible treatment compounds (possible conditioning agent or ligand compound).Adopt one or more experiment sievings as herein described this " combinatorial chemistry library " or " ligand library " to show required characteristic active those libraries member (particularly chemical species or subclass) then to identify.Therefore, the material of being identified can be used as routine " leading material " or himself can be used as possibility or actual therapeutic agent.

Those skilled in the art know the preparation and the screening in combinatorial chemistry library.These combinatorial chemistry libraries include but not limited to: peptide library (referring to, for example U.S. Patent number 5,010, and 175; Furka, 1991, Int J PeptProt Res 37:487-493 (1991) and Houghton etc., 1991, Nature 354:84-88).Also can utilize other chemical substance to produce the Chemical Diversity library.These chemical substances include but not limited to: intend peptide (for example, PCT publication No. WO 91/19735), encoded peptide (for example, PCT publication No. WO93/20242), biological at random oligomer (for example, PCT publication No. WO 92/00091), benzodiazepine

Class (for example, U.S. Patent number 5,288,514), various body (diversomer), for example hydantoins, benzodiazepine

Class and dipeptides (Hobbs etc.; 1993; Proc Natl Acad Sci USA90:6909-6913); vinylogous polypeptide (Hagihara etc.; 1992; J Amer Chem Soc 114:6568), the non-peptide peptide mimics (Hirschmann etc., 1992 that contain the glucose support; J Amer Chem Soc114:9217-9218); the little library of compounds of similar organic synthesis (Chen etc., 1994, J AmerChem Soc 116:2661); oligomerization carbamate (Cho etc.; 1993, Science 261:1303), and/or acyltransferase polypeptide phosphonic acid ester (Campbell etc.; 1994; J Org Chem 59:658), nucleic acid library is (referring to Ausubel; Berger and Sambrook), the peptide nucleic acid(PNA) library (referring to; for example U.S. Patent number 5; 539,083), antibody library (referring to; Vaughn etc. for example; 1996, Nature Biotechnology14 (3): 309-314 and PCT/US96/10287), the carbohydrate library (referring to; Liang etc. for example; 1996, Science, 274:1520-1522 and U.S. Patent number 5; 593; 853), the organic molecule library (referring to, benzodiazepine for example

Class, Baum C and EN, on January 18th, 1993, the 33rd page; Isoprenoid, U.S. Patent number 5,569,588; A thiazolidone (thiazolidinone) and a thiazan ketone (metathiazanone), U.S. Patent number 5,549,974; Pyrrolidines, U.S. Patent number 5,525,735 and 5,519,134; Morpholinium compound, U.S. Patent number 5,506,337; Benzodiazepine

Class, U.S. Patent number 5,288,514, or the like).Other example of the method in synthetic molecules known in the art library, DeWitt etc. for example, 1993, Proc Natl Acad Sci USA 90:6909; Erb etc., 1994, ProcNatl Acad Sci USA 91:11422; Zuckemmann etc., 1994, J Med Chem 37:2678; Cho etc., 1993, Science 261:1303; Carrell etc., 1994, Angew Chem Int Ed Engl33:2059; Carell etc., 1994, Angew Chem Int Ed Engl.33:2061; With Gallop etc., 1994, J Med.Chem 37:1233.

The normal high throughput test screening conditioning agent that adopts.Therefore, identifying (i) SIRT3 activator and (ii) in the high throughput test of AceCS2 conditioning agent, may in one day, screen maximum several thousand different candidate substances or part.Specifically, can utilize each hole of microtiter plate to carry out perhaps,, can testing a kind of material in every 5-10 hole if observe concentration or incubation time effect at selected independent experiment that may material.Therefore, the microtiter plate of a standard can check about 100 (for example, 96) to plant material.If utilize 1536 orifice plates, then one flat plate is not difficult to check the about 1500 kinds of different substancess of about 100-.Can check several different flat boards every day; May screen at most with the test of integration system of the present invention about 6,000-20,000 kind of different substances.Developed the microfluidic methods of operation reagent recently.

Can pass through covalently or non-covalently connecting key, for example label directly or indirectly is incorporated into solid components with molecule (s) of interest.Label can be various components.Usually the molecule (label binding substances) with combination tag is fixed in solid support, and interested tagged molecule (for example, SURT3 or AceCS2) is connected in solid support by the interaction of label and label binding substances.

The known molecular of fully describing according to reference interacts, and can utilize many labels and label binding substances.For example, if certain label has natural binding substances, biological example element, A albumen or G albumen can be with itself and suitable label binding substances (the Fc district of avidin, Streptavidin, neutral avidin (neutravidin), immunoglobulin (Ig)) couplings.Can utilize the various natural binding substancess that have, the antibody of the molecule of the label binding substances that biological example is plain and suitable (referring to, SIGMA Immunochemicals 1998 catalogue (Sigma's immune chemical 1998 catalogues), Sigma company (SIGMA), the St. Louis, the Missouri State).

Similarly, any haptens or antigenicity compound can with suitable antibody coupling with form label/label combine right.Thousands of specific antibodies can be buied in commercialization, and reference has been described many other antibody.For example, in a kind of total conformation (common configuration), label is a first antibody, and the label binding substances is the second antibody of this first antibody of identification.Except antibody-AI, receptor-ligand binding also is suitable as label and label-binding substances pairing, for example the agonist of cell-membrane receptor and antagonist are (for example, cell receptor-ligand interaction is as Transferrins,iron complexes, c-kit, virus receptor part, cytokine receptor, Chemokine Receptors, Interferon Receptors, immunoglobulin receptor and antibody, cadherin family, integrin family, selection protein family etc.; Referring to, for example Pigott and Power, TheAdhesion Molecule Facts Book I (the true handbook I of adhesion molecule), (1993)).Similarly, (for example, the effect that it mediates various little parts comprises steroid, Triiodothyronine, retinoid and vitamins D for toxin and venom, virus epitopes, hormone (for example, opium, steroid etc.), intracellular receptor; Peptide), medicine, lectin, sugar, nucleic acid (linear and cyclic polymer conformation), oligosaccharides, protein, phosphatide and antibody can interact with various cell receptors.

Synthetic polymer, for example urethane, polyester, polycarbonate, polyureas, polymeric amide, polymine, poly (arylene sulfide), polysiloxane, polyimide and poly-acetic ester also can form suitable label or label binding substances.The technician knows many other labels/label binding substances pairing in view of this paper content and also can be used for pilot system as herein described.

Conventional joint, for example peptide, polyethers etc. also can be used as label, comprise the peptide sequence between about 5-200 amino acid, for example poly-Gly sequence (SEQ ID NO:14).The known this flexible joint of those skilled in the art.For example, can utilize Huntsville city, Alabama to cut aqueous polymer company (ShearwaterPolymers, Inc., Huntsville, polyoxyethylene glycol joint Ala).These joints are chosen wantonly to have the connection of acyl ammonia, sulfydryl connects or mixes functional group's connection.

Can adopt present known the whole bag of tricks that the label binding substances is fixed on the solid substrate.Usually all or part of chemical reagent that is exposed to solid substrate makes it derivatize or functionalization, and this chemical reagent can will have reactive chemical group with the label binding substances and be fixed in the surface.For example, be fit to be connected in long-chain group partly and comprise amido, hydroxyl, sulfydryl and carboxyl.Aminoalkyl group silicomethane and hydroxyalkyl silicomethane can be used for the various surfaces of functionalization, for example glass surface.Reference fully made up this solid phase biological polymer array (referring to, Merrifield for example, 1965, Endeavour 24:3-7; Merrifield, 1964, Biochemistry 3:1385-90; Merrifield and Stewart, 1965, Nature 207 (996): 522-3; Merrifield, 1965, Science 150 (693): 178-85 (having described for example solid phase synthesis of peptide); Geysen etc., 1987, J Immun Meth 102:259-274 (be described in pin (pin) and go up synthetic solid components); Frank and Doring, 1988, Tetrahedron 44:6031-6040 (having described synthetic various peptide sequences on cellulose discs); Fodor etc., 1991, Science 251:767-777; Sheldon etc., 1993, Clinical Chemistry 39 (4): 718-719; With Kozal etc., 1996, Nature Medicine2 (7): 753-759 (all having described the biopolymer arrays that is fixed in solid substrate).The method non-chemically that the label binding substances is fixed in base material comprises other ordinary method, for example heating by uv irradiating, crosslinked etc.

The invention provides and identify in the high-throughput mode and can improve the in vitro tests that SIRT3 level or active material maybe can be regulated AceCS2 level, acetylize state or active material.Choose the level of detection SIRT3 in the reaction that does not comprise possible activator/conditioning agent or level, acetylize state or the active control reaction of activity or AceCS2 wantonly, because these tests are high unities.These optional control reaction are suitable, have improved the reliability of test.Therefore, in some embodiments, the inventive method comprises this control reaction.For each test form of describing, " no activator " or " no conditioning agent " control reaction that does not comprise the SIRT3 activator or do not comprise the AceCS2 conditioning agent provides in conjunction with active background level.

III.SIRT3 activator and AceCS2 conditioning agent

Screening method of the present invention can utilize any candidate substances, for example cell extract, cell culture supernatant, microbial fermentation product, marine organism extract, plant milk extract, purifying or rough protein, peptide, non-peptide compound, nucleic acid, sugar, lipid, synthetic scintilla compound and natural compounds etc.Also but many methods of coupling combinatorial library retrieval described herein and methods known in the art are to obtain candidate substances of the present invention.Described method comprises (1) biology library, addressable parallel solid phase in (2) space or liquid phase library, and (3) want the synthetic library method of deconvolution, the synthetic library method that (4) " pearl one compound " library method and (5) adopt affinity chromatography to select.The biology library method that adopts affinity chromatography to select is confined to peptide library, and other 4 kinds of methods are applicable to the small molecules library (Lam, 1997, Anticancer Drug Des.12:145) of peptide, non-peptide oligomer or compound.

The example of molecular library synthetic method known in the art (DeWitt etc., 1993, Proc Natl AcadSci USA 90:6909; Erb etc., 1994, Pro.Natl Acad Sci USA 91:11422; Zuckermann etc., 1994, J Med Chem 37:2678; Cho etc., 1993, Science 261:1303; Carell etc., 1994, Angew Chem Int.Ed Engl.33:2059; Carell etc., 1994, AngewChem Int Ed.Engl.33:2061; Gallop etc., 1994, J Med Chem 37:1233).But commercialization buy the preparation combinatorial library device (referring to, 357MPS for example, 390MPS, advanced chemical technology company (Advanced Chem Tech), Louisville Ky., Symphony, Rainin, Wo Ben, the Massachusetts, 433A Applied Biosystems, Inc. (Applied Biosystems), Foster city, California; 9050Plus, Millipore Corp (Millipore), Bedford, Massachusetts).In addition, but many combinatorial library commercialization itself buy (referring to, this company of Kao Muji Nike (ComGenex) for example, Princeton, New Jersey; This company of AsiaSat Nike (Asinex), Moscow, Russia; Bent Bose Corp. (Tripos, Inc.), St. Louis, the Missouri State; Change star company limited (ChemStar, Ltd), Moscow, Russia; 3D drug company (3D Pharmaceuticals), Yi Kexing pauses (Exton), guest's sunset method Ni Zhou; Martek Biosciences Boulder Corp. (Martek Biosciences), Colombia, the Maryland State, etc.).

Library of compounds may reside in the solution (referring to Houghten, 1992, Bio/Techniques13:412) or pearl (Lam, 1991, Nature 354:82), chip (Fodor, 1993, Nature364:555), bacterium (U.S. Patent number 5,223,409), spore (U.S. Patent number 5,571,698; 5,403,484 and 5,223,409), (Scott and Smith, 1990, Science 249:386 on plasmid (Cull etc., 1992, Proc Natl Acad Sci USA 89:1865) or the phage; Devlin, 1990, Science249:404; Cwirla etc., 1990, Proc Natl Acad Sci USA 87:6378; Felici, 1991, J Mol Biol 222 301; U.S. Patent application 20020103360).The candidate substances that contacts with cell or protein according to the inventive method can be single material or combinations of substances.If utilize combinations of substances in the methods of the invention, this material can be linked to each other with cell or protein successively or simultaneously.

The isolating material of screening method of the present invention is to activate SIRT3 level or active drug candidate, or regulates AceCS2 level, acetylize state or active drug candidate.Drug candidate can be used for treatment or prevents pathologic state as herein described, illness or disease.The material that screening method of the present invention obtains comprises that the part-structure of the material that obtains by screening method of the present invention is because of adding, lack and/or the alternative material that changes.Can be in animal model or tested object further screening can effectively activate the ability that SIRT3 level or active material maybe can be regulated AceCS2 level, acetylize state or the treatment of active material or prevention illness, disease or pathologic state.

The material of identifying by any described method as herein described can be used as the biologic activity agent.In a preferred embodiment, the biologic activity agent activates level or the activity of SIRT3 described herein.In another preferred embodiment, level, acetylize state or the activity of AceCS2 described herein regulated in the biologic activity agent.

In addition, the present invention also comprises prodrug.Prodrug is to give any covalent attachment carrier that patient Shi Neng discharges material of the present invention in vivo.General by modifying functional group, thus make the kind modification mode that can produce the parental generation material prepare prodrug by cutting in routine operation or the body.

Can utilize various materials to regulate in the level of AceCS2 described herein, acetylize state or the activity one or more.They comprise that siRNA, sense-rna, ribozyme, small molecules and dominance bears albumen (dominantnegative proteins).Small molecules also can be used as activation SIRT3 level or active material.

As described herein, the conditioning agent of AceCS2 comprises activator and the inhibitor (for example, antagonist) of AceCS2.The negative albumen of siRNA, sense-rna, ribozyme and dominance especially can be used for suppressing the activity of nucleic acid or polypeptide.Therefore, in one embodiment, the invention provides the method that siRNA, sense-rna, ribozyme and dominance are born proteic composition and suppressed AceCS2 nucleic acid or AceCS2 polypeptide with the negative albumen of siRNA, sense-rna, ribozyme and dominance in vitro and in vivo that comprises that suppresses AceCS2 nucleic acid or AceCS2 polypeptide.These method and compositions can be used for treating pathologic state, illness or disease, and disturb the AceCS2 activity.Preferred pathologic state, illness or disease are characterised in that acetyl-CoA level is higher than normal, because of it causes or associated.The negative albumen of siRNA, sense-rna, ribozyme or dominance suffered from the individuality of this pathologic state, illness or disease with after suppressing AceCS2 nucleic acid described herein or AceCS2 polypeptide, the acetyl that this individuality raises-CoA level reduces, and preferably is reduced to normal level.

A. Small molecules

In a preferred embodiment, described material is the small molecules of evaluation as described herein.This paper has described useful small molecules and has comprised their combinatorial library.Useful especially is can activate the SIRT3 level or activity maybe can be regulated AceCS2m level, acetylize state or active small-molecule substance, as described herein.

B. siRNA

In the present invention's one preferred implementation, can regulate that one or more material is short interfering rna (siRNA) in level, acetylize state or the activity of AceCS2 described herein.Referring to, for example PCT application WO0/44895, WO99/32619, WO01/75164, WO01/92513, WO01/29058, WO01/89304, WO02/16620 and WO02/29858 are seen in the description of siRNA technology; With U.S. Patent Publication numbers 20040023390.

In a preferred embodiment, described material is the siRNA at AceCS2 mRNA.

Therefore, the material of the present invention that can be used for implementing the inventive method includes but not limited to: the siRNA of AceCS2.These materials energy (i) usually (ii) disturb the AceCS2mRNA translation or (iii) cause AceCS2 mRNA degraded in conjunction with AceCS2 mRNA.The invention provides and adopt RNA to disturb the composition and the method for regulating the AceCS2 expression.

In many species, introduce double-stranded RNA (dsRNA) (at this paper or be called siRNA (siRNA)) and can induce gene silencing strongly and specifically, a kind ofly be called that RNA disturbs or the phenomenon of RNAi.This phenomenon is nematode, extensively proved in the Caenorhabditis elegans (Fire etc., 1998, Nature 391:806-811), but is dispersed throughout in other biology from the trypanosome to the mouse.Depend on described biology, RNA disturbs and is called " suppressing altogether ", " PTGS ", " having justice to suppress " and " compacting ".RNAi is attractive biotechnology instrument, because it provides the active method of specific gene that knocks out.Formerly think that to knock out genetic expression in the species that are not suitable for genetic analysis or operation particularly useful.

Usually RNAi is described as PTGS (PTGS) phenomenon that dsRNA triggers degradation of homologous mRNA in the kytoplasm.Primary process comprises dsRNA is processed into the shorter unit (be called and lack or siRNA (siRNA)) that instructs homology messenger RNA(mRNA) (mRNA) identification and target cutting.Can many methods in nuclear or kytoplasm, produce the dsRNA that RNAi/PTGS is triggered in the processing back.DsRNA is processed into siRNA, and then degraded mRNA is two step RNA degradation processes.The first step comprises dsRNA endonuclease (III-sample endonuclease; III-sample RNA enzyme) active dsRNA is processed the adopted and sense-rna of having of a growth 21-25 Nucleotide (nt) (that is, siRNA).In fruit bat, this III-sample RNA enzyme is called cuts enzyme (Dicer).In second step, the antisense siRNA of generation be called the different IPs ribonuclease T. complex body combination that RNA-induces silencing complex (RISC) (can cut homology strand mRNA).RISC is roughly cutting mRNA with the middle part in antisense siRNA paired zone, and mRNA subsequently further degrades.The dsRNA of different sources can be processed to produce RNAi/PTGS.

Therefore, in the present invention's one preferred implementation, the used material of the inventive method is the siRNA of AceCS2.Can utilize siRNA to reduce the expression level of AceCS2.The siRNA of AceCS2 and AceCS2mRNA are hybridized, thereby can reduce or suppress the generation of AceCS2 polypeptide.

In designated rna i experiment, need to consider several factors, for example selection of the time length of the character of siRNA, reticent effect and delivery system.For producing the RNAi effect, introduce biological siRNA and should preferably contain exon sequence.Yet, also can select at, the siRNA of exon montage sequence for example.Therefore in addition, the RNAi process depends on homology, must carefully select sequence improving gene specific as far as possible, and reduces homologue as far as possible but not the possibility of cross interference between the gene specific sequence.SiRNA and treat to preferably have more than 90% even 100% homogeny between the sequence of suppressor gene.Efficient is not high basically to be lower than about 80% sequence with the homogeny of target gene.Therefore, the siRNA of AceCS2 and the homology that will suppress between the AceCS2 gene of its expression are high more, and the possibility of the irrelevant genetic expression of influence is low more.

In addition, siRNA's is big or small most important.Present invention relates in general to two strands or the strand siRNA molecule of AceCS2, this molecule comprises the genetic expression that also can regulate AceCS2 at least about 19-25 Nucleotide.In scope of the present invention, the length of siRNA preferably is shorter than 500,200,100,50 or 25 Nucleotide.The length of described siRNA is 19 Nucleotide-Yue 25 Nucleotide more preferably from about.

On the one hand, feature of the present invention is the separation siRNA molecule of at least 19 Nucleotide usually, its at least one chain and AceCS2 at least 10 but to be no more than 30 continuous nucleotides complementary basically, thus can reduce AceCS2 gene or protein expression.

Therefore, the composition that contains siRNA described herein can be used for regulating AceCS2 level, acetylize state or the active method of mammalian cell or treats in the method for pathologic state described herein, illness or disease.

1. select the siRNA target site

The nucleotide sequence of coding AceCS2 has been described in this area, sees GeneBank.For example, people's nucleotide sequence of AceCS2 as seen, GenBank accession number BC039261 for example; Mouse AceCS2 nucleotide sequence as seen, GenBank accession number AB046742 for example; Ox AceCS2 nucleotide sequence as seen, GenBank accession number AB046741 for example.

People's nucleotide sequence of SIRT3 as seen, for example GenBank accession number NM_001017524, NM_012239, BC001042 and AF083108; Mouse SIRT3 nucleotide sequence as seen, for example GenBank accession number CT010402, BC025878 and NM_022433; Rat SIRT3 nucleotide sequence as seen, GenBank accession number XM_215124 for example; Ox SIRT3 nucleotide sequence as seen, for example GenBank accession number XM_864790 and XM_873980; Dog SIRT3 nucleotide sequence as seen, for example GenBank accession number XM_843207, XM_850716 and XM_851003; With zebra fish SIRT3 nucleotide sequence as seen, for example GenBank accession number XM_679133 and XM_685833.

Have these sequences to use, the technician is not difficult to utilize, and for example this paper content identifies that siRNA implements the inventive method and composition and need not too much experiment.

In the present invention's one preferred implementation, at least one chain of siRNA molecule and AceCS2 at least 10 but to be no more than 30 continuous nucleotides complementary basically.Utilize this oligonucleotide can greatly reduce the expression of AceCS2.In other embodiment of the present invention, the n siRNA that inhibition AceCS2 expresses from AceCS2 mRNA comprises the siRNA (referring to GenBank accession number provided herein) that obtains from any AceCS2 sequence described herein.

Will be appreciated that other siRNA that utilizes this paper content to design to regulate the AceCS2 that AceCS2 expresses, and be used to implement the inventive method and need not too much experiment.

The used preferred siRNA of the present invention is shown in following general formula:

5’-[A]-[B]-[A’]-3’

Wherein [A] is the ribonucleoside acid sequence corresponding to AceCS2 gene target sequence; [B] is the ribonucleoside acid sequence that is made of about 23 Nucleotide of about 3-; [A '] be and [A] complementary ribonucleoside acid sequence.The phrase of this paper " target sequence of AceCS2 gene " refers to that if introduce mammalian cell it can effectively suppress or reduce the sequence of AceCS2 mRNA translation.

Except that siRNA disclosed herein, can evaluation as follows can be used for implementing the siRNA of the inventive method.(for example, AceCS2 mRNA) AUG initiator codon begins, and scans the AA dinucleotides sequence of transcript downstream from transcript.Write down the incidence of each AA and adjoin 19 Nucleotide as 3 ' of possible siRNA target site.Do not recommend to design siRNA according to 5 ' and 3 ' non-translational region (UTR) with near the zone (in 75 bases) of initiator codon, because these zones may be abundanter in regulating protein binding site, therefore endonuclease can disturb with complex body according to the siRNA of these zone design that UTR-is protein-bonded to be combined and/or translation initiation complex body (Tuschl etc., 1999, Genes Dev13 (24): 3191-7).Then according to waiting to regulate the species that AceCS2 expresses, possible target site and people's gene database or other Mammals sequence are made comparisons.Show any target sequence of remarkable homology does not consider with other code sequence.Can utilize BLAST to make homology search (Altschul etc., 1997, Nucleic AcidsRes 25:3389-402; Altschul etc., 1990, J Mol Biol 215:403-10).Select qualified target sequence to analyze then.Can visit the length of selecting several preferred target sequences and assessment gene on the website of benefactor department (Ambion) in peace.

Duplex molecule of the present invention comprises sense strand and antisense strand, wherein said sense strand comprises the ribonucleoside acid sequence corresponding to the AceCS2 target sequence, and antisense strand comprises and described sense strand complementary ribonucleoside acid sequence, thereby wherein said sense strand and described antisense strand are hybridized each other and are formed described duplex molecule, wherein said duplex molecule can suppress described genetic expression when introducing the cell of expressing the AceCS2 gene.

Duplex molecule of the present invention can be derived from its primal environment (that is) polynucleotide, the natural surroundings when it is natural molecule, perhaps from its native state through physics or chemically changed, or chemosynthesis.According to the present invention, this duplex molecule comprises those molecules and the derivative thereof that is made of DNA, RNA.DNA can be by suitably constituting such as bases such as A, T, C and G, and T replaces with U in RNA.

Can be from preparing carriers siRNA.Carrier preferably comprises the adjusting sequence in the zone that is adjacent to code book invention duplex molecule, and sequence-directed this molecule of described adjusting is expressed in suitable cell.For example, duplex molecule of the present invention contains by its encoding sequence is cloned into, for example the carrier of the rna plymerase iii transcription unit of small nuclear RNA (snRNA) U6 or people H1 RNA promotor and carry out transcribing in the born of the same parents.

Perhaps, for example target sequence can be cloned into expression vector and produce carrier of the present invention, therefore target sequence is connected in the adjusting sequence (Lee etc., 2002, Nature Biotechnology 20:500-505) of carrier with its mode that can express (transcription DNA molecule) operability.For example, first promotor (for example, the promoter sequence that links to each other with 3 ' of cloned DNA-end) drives the RNA molecule that contains antisense sequences and be transcribed into target sequence, second promotor (for example, the promoter sequence that links to each other with 5 ' of cloned DNA-end) drives to contain has the RNA of adopted sequence molecule to be transcribed into target sequence.The have justice and the antisense strand of expressing are hybridized the construction with generation siRNA in vivo each other, thereby make the gene silencing that contains target sequence.That in addition, can utilize that two kinds of constructions (carrier) produce the siRNA construction respectively has justice and an antisense strand.

For carrier is introduced cell, can utilize the transfection toughener.FuGENE6 (Luo Shi diagnostic companies (RocheDiagnostics)), Lipofectamine 2000 (hero company (Invitrogen)), Oligofectamine (hero company) and Nucleofector (watt pure chemistry product company of section (Wako pure Chemical)) can be used as the transfection toughener.Can adopt the carrier of transfection expression siRNA polynucleotide of the present invention to suppress AceCS2 in the mammalian cell.Therefore, another aspect of the present invention provides duplex molecule (it plays the effect of the siRNA of AceCS2) that comprises sense strand and antisense strand and the carrier of encoding this duplex molecule.

SiRNA also can comprise one or more Nucleotide and change.This change can comprise adding non-nucleotide material, for example adds two ends or inside (at the one or more Nucleotide place of RNA) of 19-25 Nucleotide RNA.In a preferred embodiment, the RNA molecule contains 3 '-hydroxyl.Nucleotide in the RNA molecule of the present invention also can comprise non-standard Nucleotide, comprises non-natural nucleotide or deoxyribonucleotide.Double chain oligonucleotide can contain the main chain of modification, and for example thiophosphatephosphorothioate, phosphorodithioate or known in the art other are modified main chain, perhaps can contain connecting key between non-natural nucleoside.Other modification of siRNA (for example, 2 '-O-methyl ribonucleotides, 2 '-deoxidation-2 '-fluorine ribonucleotide, " universal base " Nucleotide, 5-C-methyl nucleotide, one or more thiophosphatephosphorothioate Nucleotide between connecting key and the non-alkaline residue of upset deoxidation mix (inverted deoxyabasic residue incorporation)) visible Application No. 20040019001 and U.S. Patent number 6,673,611 (including in by reference).The RNA of above-mentioned all this changes is referred to as the siRNA of modification.

RNAi preferably can reduce at least 10%, 20%, 30% or 40% with the expression of AceCS2 in the cell, and more preferably at least 50%, 60% or 70%, most preferably at least 75%, 80%, 90%, 95% or higher.

Can adopt method known in the art and disclosed herein that siRNA is introduced cell, comprise, for example microinjection, electroporation or with the carrier transfection that contains the nucleic acid that can be transcribed into siRNA.Perhaps, can be with the siRNA of AceCS2 directly introducing cell in conjunction with the form of AceCS2 mRNA transcript.For strengthening persistence and film perviousness, siRNA and liposome, poly-L-Lysine, lipid, cholesterol, lipofectin or their derivative can be made up or modify with them.For AceCS2, preferred cholesterol link coupled siRNA (referring to Song etc., Nature Med.9:347-351 (2003)).

For example include the Application No. 20060051815 of this paper by reference in full in and described siRNA and the carrier and preparation and the using method that comprise the siRNA nucleotide sequence.

C. Sense-rna and ribozyme

Can utilize various materials to regulate the level of AceCS2 described herein, acetylize state or activity.For example, can will suppress or the nucleic acid of antagonism AceCS2 genetic expression give cell or object is regulated, preferably suppress AceCS2 and express.Except that above-mentioned siRNA, can utilize the antisense oligonucleotide of destruction AceCS2 genetic expression or expression or the activity that ribozyme is regulated AceCS2.In preferred embodiment, conditioning agent, preferred inhibitor are the sense-rnas of evaluation as described herein.

As mentioned above, can utilize antisense oligonucleotide to reduce this expression of gene level corresponding to any nucleotide sequence of AceCS2 gene.Specifically, antisense oligonucleotide at the AceCS2 gene can be by working in conjunction with any AceCS2 mRNA, thereby suppress AceCS2 gene transcription or translation, promote the protein expression of AceCS2 mRNA degraded and/or inhibition AceCS2 genes encoding, finally suppress the proteinic function of AceCS2.

Also can be according to limiting antisense oligonucleotide of the present invention and siRNA with the mRNA of gene disclosed herein or the ability of cDNA specific hybrid.

Can antisense oligonucleotide and derivative thereof be prepared into external articles by mixing with suitable base-material, for example liniment or application, described base-material is to this derivative non-activity.

Antisense oligonucleotide of the present invention suppresses the protein expression of AceCS2 genes encoding, therefore can be used for suppressing AceCS2 or comprises AceCS2 or the biologic activity of the multiprotein complex of one of SIRT3 polypeptide.

Ribozyme is divided into big ribozyme and little ribozyme usually.Known big ribozyme is the enzyme that can cut the phosphoric acid ester bond of nucleic acid.After the macronucleus enzyme reaction, reaction site is made of 5 '-phosphate group and 3 '-hydroxyl.Big ribozyme also can be divided into (1) I group introne RNA by guanosine catalysis 5 '-splice site place transesterification; (2) by the II group introne RNA of two-step reaction through cover Cable Structure catalysis self-splicing; (3) by the RNA component of hydrolysis at the ribonuclease P of 5 ' site cutting tRNA precursor.On the other hand, compare with big ribozyme, the volume of little ribozyme less (about 40bp) also cuts RNA to produce 5 '-hydroxyl and 2 '-3 ' ring-type phosphoric acid (ester).Little ribozyme comprises hammerhead ribozyme (Koizumi etc., 1988, FEBS Lett.228:225) and hair clip type ribozyme (Buzayan, 1986, Nature 323:349; Kikuchi and Sasaki, 1991, Nucleic Acids Res.19:6751).Design known in the art and make up ribozyme method (referring to Koizumi etc., 1988, FEBSLett.228:225; Koizumi etc., 1989, Nucleic Acids Res.17:7059; Kikuchi and Sasaki, 1991, Nucleic Acids Res.19:6751), can be according to the ordinary method that produces ribozyme, the sequence information of the nucleotide sequence of foundation coding AceCS2 polypeptide makes up the ribozyme that suppresses the AceCS2 expression of polypeptides.

D. Dominance is born albumen

Can utilize all ingredients to suppress level, acetylize state or the activity of AceCS2.In a preferred embodiment, inhibitor be can evaluation as described herein the negative albumen of dominance.

As described herein, the AceCS2 polypeptide is assembled into multiprotein complex.Therefore, one preferred embodiment in, the negative albumen of dominance suppresses level, acetylize state or active or its active fragments of AceCS2 to be regulated, and preferably suppresses the AceCS2 polypeptide and is assembled into the biologic activity multiprotein complex.After suppressing the assembling of biologic activity complex body, can suppress or reduce one or more activity of AceCS2 polypeptide, for example acetate, ATP and CoA be changed into acetyl-CoA and AMP or be positioned mitochondrial enzymic activity.

Can adopt methods known in the art and test disclosed herein to identify the negative albumen of other dominance of AceCS2.

If candidate compound is a protein, then analyze the proteinic aminoacid sequence of gained, according to the synthetic oligo DNA of this sequence, utilize this oligo DNA to screen the cDNA library to obtain this protein DNA of coding as probe.

IV. the usage of material

The material that this paper identifies can be used for the whole bag of tricks, for example can be used for level or the activity of (i) activation SIRT3, (ii) regulates level, acetylize state or the activity of AceCS2, or (iii) treats pathologic state, illness or disease.Can implement these methods in vitro and in vivo.With preferred people of the patient of any treatment in these methods and non-human animal.

A. Improve level or the activity of SIRT3

Utilize screening method evaluation of the present invention or appraisable material to improve, that is, and in level or activity external or body internal stimulus or activation SIRT3.Therefore, one aspect of the present invention provides and improves SIRT3 level or active method.In the present invention's one preferred implementation, this method comprises that wherein the level of SIRT3 or activity are improved with SIRT3 and the step that adopts screening method acquisition of the present invention or obtainable material to contact.

SIRT3 can be at cell, and the preferred mammal cell is more preferably in people's cell.The preferred activity of SIRT3 is the deacetylase activity of SIRT3, preferably makes AceCS2 deacetylated.

Can act in the external or body of check material as described herein.

B. Regulate the horizontal acetylize state or the activity of CoA synthetic enzyme 2

The present invention provides AceCS2 level, acetylize state or the active method of regulating on the other hand.In the present invention's one preferred implementation; this method comprises AceCS2 polypeptide and the step that adopts screening method of the present invention (for example identifying the method that can improve AceCS2 level, acetylize state or active material) acquisition or obtainable material to contact, the wherein level of AceCS2, acetylize state or active adjusted.

AceCS2 can be at cell, and the preferred mammal cell is more preferably in people's cell.The preferred activity of AceCS2 is an enzymic activity, preferred generation acetyl-CoA as described herein.

Can act in the external or body of check material as described herein.

C. The treatment pathologic state

The present invention also provides the method for treatment pathologic state, illness or disease.

Under mammiferous some situation, generation need be by a large amount of acetate of Acetyl-CoA synthetase activatory.For example, giving birth under the ketone situation, as long-term fasting or diabetes, liver is released into blood flow (Buckley and Williamson, 1977, Biochem J 166:539-45 with a large amount of acetate; Seufert etc., 1974, Biochem Biophys Res Commun 57:901-9; Yamashita etc., 2001, Biochim BiophysActa 1532:79-87).In addition, give birth to the liver Acetyl-CoA hydrolase that produces acetate under the ketone situation obtain activation (Matsunaga etc., 1985, Eur J Biochem 152,331-6).Utilize the acetate that discharges in the extrahepatic tissue to need the effect of Acetyl-CoA synthetase.AceCS2 is abundant but be not present in the liver and the true prompting of inductive AceCS2 transforms performance crucial effects (Fujino etc. for giving birth to the acetate that energy produces under the ketone situation under the ketone situation giving birth in heart and skeletal muscle, 2001, J Biol Chem 276,11420-6).Therefore, the medicable preferred pathologic state of the present invention, illness or disease are to give birth to the ketone situation.In addition, with to give birth to the ketone situation relevant or cause the disease of (directly or indirectly) to be suitable for adopting the inventive method to treat by it.

Therefore, on the one hand, the invention provides the method that treatment has the individuality of living ketone situation.In a preferred embodiment; this method comprises that the material with the treatment significant quantity has the step of the individuality of living ketone situation; described material can improve level or the deacetylase activity of SIRT3, and wherein SIRT3 makes AceCS2 deacetylated, thus the individuality that treatment has living ketone situation.Adopt screening method of the present invention to obtain maybe can obtain to improve SIRT3 level or the active material of deacetylase.

In another embodiment of the present invention; the method that treatment has the individuality of living ketone situation comprises that the material with the treatment significant quantity has the step of the individuality of living ketone situation; described material can improve level, acetylize state or the activity of AceCS2, thus the individuality that treatment has living ketone situation.Adopt screening method of the present invention to obtain maybe can obtain to improve AceCS2 level, acetylize state or active material.

In another preferred embodiment, the method for individuality of treatment with living ketone situation comprises and gives this individual step with pharmaceutical composition; Wherein said pharmaceutical composition comprises the obtainable biologically active substance of described method according to the identification of organism active substance, thus the individuality that treatment has living ketone situation.

The ketone situation is given birth in treatment, and particularly reducing the preferred substance that is higher than normal acetate level in the individual blood is to improve the material of AceCS2 to acetate avidity, can reduce K _mMaterial.

Do not want to be confined to theory, but it is believed that the active increase of plastosome AceCS2 can make acetate concentrate oxidized and away from kytoplasm AceCS1, the lipid and the cholesterol that therefore may reduce the AceCS1-mediation are synthetic, give birth to the ketone situation thereby improve, include but not limited to hypercholesterolemia, hyperlipidaemia, obesity and possible type ii diabetes.

The living ketone situation of treatment as described herein can reduce the acetate level in the individual blood flow.Therefore, can be by detecting the treatment of monitoring living ketone situation described herein according to the acetate level in the individual blood of the present invention's treatment.

Compare with the acetate level that individuality raises before treatment, diagnosis have the acetate level that raises in the blood of individuality of living ketone situation preferably reduce at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more.Preferably the acetate level that raises among the patient is reduced to the acetate level in the healthy individual blood.

1.II type diabetes

Type ii diabetes often betides among the grownup, it is characterized in that insulin level is lower than normally in the blood, thereby causes glucose level rising in the blood.Buckley and Williamson have reported the blood acetic acid concentration of diabetes rat also raise (Buckley and Williamson, 1977, Biochem J166:5239-545).In addition, Fujino etc. have reported the AceCS2 mRNA level rising (Fujino etc., 2001, J Biol Chem 276:11420-11426) of Zucker diabetes rat.

Therefore, on the one hand, the invention provides the method for treatment type ii diabetes.In a preferred embodiment; this method comprise with the treatment significant quantity can improve the SIRT3 level or the active material of deacetylase has the step of the individuality of type ii diabetes; wherein SIRT3 makes AceCS2 deacetylated, thereby treatment has the individuality of type ii diabetes.Adopt screening method of the present invention to obtain maybe can obtain to improve SIRT3 level or the active material of deacetylase.

In another embodiment of the present invention; the method of treatment type ii diabetes comprises that can improve AceCS2 level, acetylize state or the active material with the treatment significant quantity has the step of the individuality of type ii diabetes, thereby treatment has the individuality of type ii diabetes.Adopt screening method of the present invention to obtain maybe can obtain to improve AceCS2 level, acetylize state or active material.

The method of treatment diabetes is optional to be comprised existing hypoglycemic drug, and the medicine that can reduce circulating glucose concentration gives individual step.But but can not correct potential biological chemistry defective in this disease though treat the medicine lowering blood glucose level of diabetes B at present, be still ideal for the material of coupling this paper evaluation and some situation of existing hypoglycemic drug.These medicines known in the art, for example comprise (i) sulfonylureas, the (ii) nearest non-sulfonylurea medicine of developing of closing potassium/ATP passage, (iii) be plastosome metabolism supplying bottom medicine (for example, KCl, α-Tong Yijisuan or leucine), insulin sensitizers (for example, thiazolidinediones), hepatic glucose discharge inhibitor (for example, N1,N1-Dimethylbiguanide) or (iv) glucose uptake blocker (for example, acarbose).

2. hypercholesterolemia

Hypercholesterolemia refers to that cholesterol concentration is high singularly in the blood flow.In suffering from the individuality of hypercholesterolemia, high-caliber cholesterol can cause heart trouble, arteriosclerosis, heart attack and apoplexy.For total cholesterol, the above blood cholesterol levels of 240mg/dL can be regarded as unusual high.Blood cholesterol levels between the 200-239mg/dL is regarded critical height as.The following blood cholesterol levels of 200mg/dL is an ideal.

Also can treat individuality with the material that this paper identifies with familial hypercholesterolemia.Affected people has high-caliber low-density lipoprotein (LDL or " bad " cholesterol) all the time, and it causes the too early atherosclerosis of coronary artery.The affected male sex has a heart attack in its 40-50 year usually, and 85% suffers from this sick people can experience heart attack in the time of 60 years old.The incidence of suffering from this sick women's center popular name for outbreak also raises, but takes place in late 10 years than the male sex.

Therefore, on the one hand, the invention provides the method for treatment hypercholesterolemia.In a preferred embodiment; this method comprise with the treatment significant quantity can improve the SIRT3 level or the active material of deacetylase has the step of the individuality of hypercholesterolemia; wherein SIRT3 makes AceCS2 deacetylated, thus the individuality that treatment has hypercholesterolemia.Adopt screening method of the present invention to obtain maybe can obtain to improve SIRT3 level or the active material of deacetylase.

In another embodiment of the present invention; the method of treatment hypercholesterolemia comprises that can improve AceCS2 level, acetylize state or the active material with the treatment significant quantity has the step of the individuality of hypercholesterolemia, thus the individuality that treatment has hypercholesterolemia.Adopt screening method of the present invention to obtain maybe can obtain to improve AceCS2 level, acetylize state or active material.

Treat hypercholesterolemia described herein and can reduce cholesterol levels in the individual blood flow.Preferably with blood cholesterol levels from the level that is reduced to more than the 240mg/dL between the 200-239mg/dL, more preferably be reduced to the following level of 200mg/dL.

3. hyperlipidaemia

Hyperlipidaemia (being also referred to as hyperlipoproteinemia) is to be characterised in that fatty substance in the blood, for example the high lipoid dyscrasias of cholesterol and triglyceride concentration.Atherosclerosis and heart trouble more may take place in the individuality of suffering from hyperlipidaemia.For example, the following triglyceride levels of 150mg/dL is regarded as normally, and the triglyceride levels between the 150-199mg/dL is regarded critical height as, and the triglyceride levels between the 200-499mg/dL is regarded height as, and 500mg/dL or above triglyceride levels are regarded as very high.

Therefore, on the other hand, the invention provides the method for treatment hyperlipidaemia.In a preferred embodiment; this method comprise with the treatment significant quantity can improve the SIRT3 level or the active material of deacetylase has the step of the individuality of hyperlipidaemia; wherein SIRT3 makes AceCS2 deacetylated, thus the individuality that treatment has hyperlipidaemia.Adopt screening method of the present invention to obtain maybe can obtain to improve SIRT3 level or the active material of deacetylase.

In another embodiment of the present invention, the method for treatment hyperlipidaemia comprises that can improve AceCS2 level, acetylize state or the active material with the treatment significant quantity has the step of the individuality of hyperlipidaemia, thus the individuality that treatment has hyperlipidaemia.Adopt screening method of the present invention to obtain maybe can obtain to improve AceCS2 level, acetylize state or active material.

Treat hyperlipidaemia described herein and can reduce triglyceride levels in the individual blood flow.Preferably blood cholesterol levels is reduced to level between the 200-499mg/dL from the level more than the 500mg/dL, more preferably is reduced to the level between the 150-199mg/dL, most preferably be reduced to the following level of 150mg/dL.

4. obesity

Obesity is defined as BMI (weight index) and surpasses 30kg/m ²The patient of BMI between 25-29.9 regards as overweight, but is not obesity.BMI regards as between 18.5-24.9 normally, BMI be 18.5 or the lower body weight of regarding as light partially.American more than half is overweight, and the obesity ratio rises.It is ill and because of the dead danger of diabetes, apoplexy, coronary artery disease, hypertension, hypercholesterolemia and kidney and gallbladder disease that obesity can increase the patient.Obesity can increase the danger of some cancer, and it still produces the Hazard Factor of osteoarthritis and sleep apnea.

Therefore, on the other hand, the invention provides the method for treatment of obesity.In a preferred embodiment; this method comprises can improve the step that SIRT3 level or the active material of deacetylase are diagnosed the individuality of suffering from obesity with the treatment significant quantity; wherein SIRT3 makes AceCS2 deacetylated, thereby the treatment diagnosis suffers from the individuality of obesity.Adopt screening method of the present invention to obtain maybe can obtain to improve SIRT3 level or the active material of deacetylase.

In another embodiment of the present invention, the method for treatment of obesity comprise with the treatment significant quantity can improve the step that AceCS2 level, acetylize state or active material are diagnosed the individuality of suffering from obesity, thereby treatment diagnosis suffers from the individuality of obesity.Adopt screening method of the present invention to obtain maybe can obtain to improve AceCS2 level, acetylize state or active material.

Treat obesity described herein and can reduce BMI.Preferably with BMI from 30kg/m ²Above level is reduced to 25-29.9kg/m ²Between level, more preferably be reduced to 18.5-24.9kg/m ²Between level.

V. pharmaceutical composition

On the one hand, the invention provides pharmaceutical composition or the medicine that comprises the AceCS2 level of to regulate, acetylize state or active at least a material and pharmaceutically acceptable carrier.One preferred embodiment in, described material can improve level or the activity of AceCS2, maybe can reduce the acetylize state of AceCS2.

On the other hand, the invention provides to comprise and to improve SIRT3 level or activity, the pharmaceutical composition or the medicine of preferred active at least a material of deacetylase and pharmaceutically acceptable carrier.

Can give object with treatment, pathologic state for example as herein described or disease with pharmaceutical composition or medicine.

A. Preparation and administration

Described compound, material and the small molecules that available the inventive method compounds identified, material and small molecules pharmaceutical compositions or medicine, described pharmaceutical composition or pharmaceutical pack contain significant quantity together with or be mixed with and be fit to that enteron aisle is used or the vehicle or the carrier of parenteral application.

Improve SIRT3 level or active or (ii) regulate AceCS2 level, acetylize state or active preferred pharmaceutical compositions and comprise (i) screening method described herein and obtain or obtainable material and (ii) pharmaceutically acceptable carrier in order to (i).Can provide the described material of treatment effective dose to be used for methods of treatment described herein.

Can utilize on one or more physiology receivable carrier or vehicle by standard technique preparation pharmaceutical compositions for use of the present invention or medicine.This paper and E.W.Martin " Remington ' sPharmaceutical Sciences " (Lei Mingdun pharmaceutical science) described suitable pharmaceutical carrier.Compound of the present invention, material and small molecules and physiologically acceptable salt thereof and solvate can be mixed with and can comprise through suction, part, nose, mouth, parenteral or rectal administration by any suitable way administration.Therefore, can utilize syringe or other device, give pharmaceutical composition by injection in intradermal, subcutaneous, intravenously, intramuscular, the nose, in the brain, in the tracheae, in the intra-arterial, intraperitoneal, intravesical, pleura, in the coronary artery or in the tumour.Also considered percutaneous dosing, for example sucked or the aerosol administration.But per os, rectum or vagina give tablet or capsule.

For oral administration, pharmaceutical composition or medicine can be taked, for example with tablet or the capsule form of pharmaceutically acceptable vehicle by the ordinary method preparation.Preferably contain active ingredient, be the tablet and the gelatine capsule of micromolecular compound of the present invention and following material: (a) thinner or weighting agent, for example lactose, glucose, sucrose, N.F,USP MANNITOL, sorbyl alcohol, Mierocrystalline cellulose (for example, ethyl cellulose, Microcrystalline Cellulose), glycerine, pectin, polyacrylic ester and/or secondary calcium phosphate, calcium sulfate; (b) lubricant, for example silicon-dioxide, talcum powder, stearic acid, its magnesium salts or calcium salt, Metallic stearates, colloidal silica, hydrogenated vegetable oil, W-Gum, Sodium Benzoate, sodium acetate and/or polyoxyethylene glycol; Also has (c) tackiness agent for tablet, for example neusilin, starch paste, gelatin, tragacanth gum, methylcellulose gum, Xylo-Mucine, polyvinylpyrrolidone and/or Vltra tears; If desired, also have (d) disintegrating agent, for example starch (for example, yam starch or Starch Sodium), glycollate, agar, Lalgine and sodium salt or effervescent mixture; (e) wetting agent, for example Sodium Lauryl Sulphate BP/USP; And/or (f) absorption agent, pigment, seasonings and sweeting agent.

Can carry out film dressing or bag casing to tablet according to methods known in the art.The flowing product of oral administration can be taked, for example solution, syrup or suspension, and perhaps they can be dryed products, thus used water or other suitable carriers re-use after making up.Available pharmaceutically acceptable additive prepares these flowing products by ordinary method, and described additive for example has: suspension agent, as sorbitol syrups, derivatived cellulose or hydrogenation edible-fat; Emulsifying agent is as Yelkin TTS or gum arabic; Non-aqueous carrier is as Prunus amygdalus oil, grease, ethanol or fractionated vegetable oil; And sanitas, as methyl p-hydroxybenzoate or propyl ester, or Sorbic Acid.These goods also can contain suitable buffering salt, seasonings, tinting material and/or sweeting agent.If desired, can suitably prepare the goods of orally give with the controlled release of active ingredient thing.

Compound of the present invention, material and small molecules can be mixed with and be suitable for parenteral admin by injection, for example, by injecting or the continuous infusion administration.The preparation that is used to inject can be a unit dosage, for example places the ampoule that is added with sanitas or the formulation of multi-dose container.Preferred aqueous solution of Injectable composition or suspension preferably prepare suppository with lipomul or suspension.These compositions can and/or contain adjuvant through sterilization, for example the salt and/or the buffer reagent of sanitas, stablizer, wetting agent or emulsifying agent, solution promotor (solution promoter), adjusting osmotic pressure.Perhaps, active ingredient can be to use suitable carriers, for example the powder type that re-uses behind the apirogen water of the sterilization structure.In addition, they also can contain the material that other has therapeutic value.These compositions can contain the 0.1-75% that has an appointment respectively, preferably the active ingredient of about 1-50% respectively according to mixing, granulation or the coating method preparation of routine.

For inhalation, can utilize suitable propelling agent, for example Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas are sent compound, material and small molecules with form routine from compression wrap or atomizer of aerosol spray.With the pressurized aerosol is example, can provide valve to send metered amounts to determine dose unit.Can will be used for sucker or insufflator, for example gelatine capsule and medicine box are mixed with and contain compound and suitable powder binder, for example powdered mixture of lactose or starch.

The compounds of this invention, material and the small molecules and the carrier that comprise significant quantity through the appropriate formulation of skin application.Preferred carrier comprises that absorbable pharmaceutically acceptable solvent is to promote to see through host's skin.For example, transcutaneous device is the form of bandage that comprises holding components, the reservoir form that contains compound, its optional carrier that contains, thereby the optional speed control barrier that comprises can be delivered to host's skin with controlled and predetermined speed with compound in long-time, and the instrument that this device is fixed in skin.Also can use matrix percutaneous preparation (matrixtransdermal formulation).

Topical application for example is used for the preferred aqueous solution well known in the art of appropriate formulation, ointment, emulsifiable paste or the gel of skin and eyes.These preparations can contain solubilizing agent, stablizer, tension-elevating agent, buffer reagent and sanitas.

Also compound, material and small molecules can be mixed with rectal compositions, for example suppository or enema,retention, as contain conventional suppository bases, as the formulation of theobroma oil or other glyceryl ester.

In addition, compound, material and small molecules can be mixed with long-acting goods.Can be by implanting (for example, subcutaneous or intramuscular) or giving these prolonged action preparations by intramuscular injection.Therefore, for example available suitable polymerization or hydrophobic material (emulsion of for example acceptable oil preparation) or ion exchange resin are prepared these compounds, or are mixed with the derivative of slightly soluble (sparingly soluble), as slightly soluble salt.

If desired, these compositions can be present in the packing or sale unit that contains one or more unit dosage, and described unit dosage contains active ingredient.For example, described packing can contain metal or plastic foil, for example Blister Package.Described packing or sale unit can be equipped with the administration working instructions.

In an embodiment of the invention, pharmaceutical composition or pharmaceutical pack (i) as described herein that contain significant quantity can improve SIRT3 level or active or (ii) can regulate AceCS2 level, acetylize state or active material and another kind of therapeutical agent.When using with The compounds of this invention, material or small molecules, these therapeutical agents can be separately, use successively or with one or more other such therapeutical agent coupling (for example, first therapeutical agent, second therapeutical agent and The compounds of this invention).Can give by identical or different route of administration or in same pharmaceutical preparation, give together.

B. Treatment significant quantity and administration

In an embodiment of the invention, will treat effective dose of medicine compositions or medicine and give object, preferred people or non-human animal are to prevent, to treat or control pathologic state described herein or disease.The pharmaceutical composition or the medicine that will be enough to cause effective therapeutic response consumption of object give object.Effectively therapeutic response is to make the symptom of pathologic state, illness or disease or the reaction that complication is stagnated or slowed down to small part.Be enough to realize that the consumption of this purpose is defined as " treatment effective dose ", be also referred to as " treatment significant quantity ".

The dosage of the promoting agent that is given depends on kind, body weight, age, the individual state of warm-blooded animal (Mammals), the surface-area or the volume in zone to be treated, also depends on administering mode.Give specific micromolecular compound in special object with existence, the nature and extent of any side effect also will determine the dosage size.The mammiferous unitary dose of the about 50-70kg of orally give can contain the active ingredient of having an appointment between the 5-500mg.The dosage of active compound of the present invention normally is enough to realize the dosage of required effect.Can be from the intravital drug accumulation observed value of object calculating optimum dosage regimen.Dosage usually can every day, weekly or gave one or many in every month.Those of ordinary skills are not difficult to measure optimal dose, medication and repetition rate.

The dosage of the promoting agent that gives also depends on the character of this promoting agent.For example, the treatment significant quantity of protein or polypeptide (that is, effective dose) is about 0.001-30mg/kg body weight, preferably about 0.01-25mg/kg body weight, 0.1-20mg/kg body weight more preferably from about, even more preferably from about 1-10mg/kg, 2-9mg/kg, 3-8mg/kg, 4-7mg/kg or 5-6mg/kg body weight.Described protein or polypeptide can be at about 1-10 between weeks, and preferred 2-8 is between week, 3-7 between week more preferably from about, even more preferably from about give once weekly in 4,5 or 6 weeks.

Micromolecular exemplary dosage comprises the small molecules/kilogram object or the example weight (for example, about 1 microgram/kilogram-Yue 500 mg/kg, about 100 microgram/kilograms-Yue 5 mg/kg, or about 1 microgram/kilogram-Yue 50 microgram/kilograms) of milligram or microgram amount.It is also understood that with regard to expression to be regulated or activity, micromolecular suitable dose depends on micromolecular intensity.If (for example give animal with these micromolecular one or more; the people) level, acetylize state or level or the activity active or activation SIRT3 polypeptide or nucleic acid to regulate AceCS2 polypeptide or nucleic acid; doctor, animal doctor or investigator can; for example leave prescription earlier, increase dosage subsequently until obtaining suitable reaction than low dosage.In addition, will be appreciated that, for any specific animal target, concrete dosage level depends on various factors, the activity that comprises used particular compound, the age of object, body weight, general health, sex and diet, administration time, route of administration, excretion rate, drug combination, and expression or active regulating degree.

In an embodiment of the invention, dosage every day with each compound of about 1mg/kg object body weight (1mg/kg)-Yue 1g/kg comprises The compounds of this invention, material or micromolecular pharmaceutical composition or medicine in a few days.In another embodiment, every day, dosage was the dosage of the about 500mg/kg of about 5mg/kg-.In also having another embodiment, every day, dosage was the about 250mg/kg of about 10mg/kg-.In another embodiment, every day, dosage was the about 150mg/kg of about 25mg/kg-.Preferred dose is about 10mg/kg.Dosage can give once a day or was divided into sub-doses and gives with multidose every day, for example every day twice, three times or four times.Yet, the technician should be understood that the inventive method compounds identified, material or small molecules can different amounts, give in the different time.The technician it is also understood that some factor can influence required dosage of effective treatment target and delivery time, and these factors include but not limited to: the disease of object or the severity of illness, former treatment, general health and/or age and other disease that exists.In addition, can comprise single therapy with the compounds for treating object for the treatment of significant quantity, or preferably can comprise a series of treatments.

For obtaining required curative effect, can treat effective every day dosage and give compound, material or small molecules many days.Therefore, in object, give compounds for treating pathologic state as herein described or disease in the treatment effectively and need continue 3 days to two weeks or cyclical administration more over a long time.Usually at least for three days on end, often be continuous at least 5 days, more common is continuous at least 10 days, continuous sometimes 20,30,40 or give material during more days.Though successive administration every day is an optimization approach of realizing the treatment effective dose, also can not realize useful curative effect even do not give these materials every day, thereby as long as enough repetitively administereds are continually kept the material of treatment effective concentration in object.For example, can be every other day, gave these materials in per three days, if when perhaps adopting the dosage range of higher object tolerance, then can give once weekly.

These compounds, material and micromolecular optimal dose, toxicity and curative effect are different because of each compound, material or micromolecular relative effectivenes, and measure in cell culture or laboratory animal by standard pharmaceutical procedures, for example by measuring LD ₅₀(lethal dose of 50% colony) and ED ₅₀(medicable dosage in 50% colony).Dosage ratio between toxicity and the curative effect is a therapeutic index, can be expressed as LD ₅₀/ ED ₅₀The ratio.The preferred big material of therapeutic index that shows.Though can utilize the material of toxic side effect, care should be used to designs the delivery system with the site of these material target affected tissue, thereby reduces as far as possible Normocellular potential destruction, and then reduces side effect.

For example can adopt, the data of cell culture test and zooscopy gained are formulated the human dosage range.The dosage of these small-molecule substances is preferably comprising ED ₅₀The circulation composition scope in, toxicity is low or do not have toxicity.According to used formulation and route of administration, dosage changes in this scope.For the used any material of the inventive method, at first can estimate the treatment effective dose from cell culture test.Can in animal model, formulate dosage to realize comprising the IC that measures in the cell cultures ₅₀(realizing the maximum material concentration that suppresses of half of symptom) is in interior circulating plasma concentration range.Can utilize this information to measure the useful dosage of philtrum more accurately.Can pass through, for example high performance liquid chromatography (HPLC) detects blood plasma level.For typical object, the normally about 1ng/kg-100mg/kg of the dose equivalent(DE) of material.

After success is treated, preferably make the object experience keep treatment and treated situation or palindromia to prevent.

C. Food, beverage and feed

In addition, the present invention relates to have food, beverage or the feed of regulating AceCS2 level, acetylize or active activity or having raising SIRT3 level or active ability.Can adopt the universal method of producing F﹠B or feed to produce this food, beverage or feed; comprise in original or the food, beverage or the feed material that boil adding promoting agent, for example can regulate AceCS2 level, acetylize state or activity or SIRT3 level or active material.Can employing and the conventional molded and granulation with food of the present invention, beverage or feed in a like fashion of food, beverage or feed.

In the weight of the food, beverage or the feed that comprise this promoting agent, the preferred 0.001-10% of the concentration of promoting agent, more preferably 0.01-10%, most preferably 0.1-10%.

The concrete food or the beverage that add promoting agent comprise, fruit juice for example, pick-me-up, soup, tea, yogurt drink, milk-product, for example cultured milk prod, frozen product, butter, cheese, sour milk, processing milk and skimmed milk, meat product, for example ham, sausage and hamburger, the flesh of fish, cereal, wheat bran, pastry product, egg food, for example seasoning egg roll and egg curdled milk (egg curd), sweet food, cookies for example, jelly, snacks and chewing gum, bread, noodles, pickles are smoked product, dried fish, the soy sauce seasoning boil food (soy sauce-seasoned boiled food) and seasonings.

Can give to have to regulate AceCS2 level, acetylize or active ability or have the food, beverage or the feed that improve SIRT3 level or active ability and further add nutrition composition (protein, lipid, sugar, VITAMIN and/or mineral substance).

VI. test kit

With regard to above-mentioned diagnosis, research and treatment were used, the present invention also provided test kit.In diagnosis and research application; these test kits can be equipped with following any or all: test reagent; buffer reagent; compound of the present invention; material or small molecules; the SIRT3 polypeptide; AceCS2 polypeptide or any other polypeptide as herein described; SIRT3 nucleic acid; AceCS2 nucleic acid or any other nucleic acid as herein described; anti--SIRT3 antibody; anti--AceCS2 antibody; anti--ethanoyl-Methionin antibody or any other antibody as herein described; detection SIRT3 nucleic acid described herein; the hybridization probe of AceCS2 nucleic acid or any other nucleic acid and/or primer; the SIRT3 expression constructs; AceCS2 expression constructs or any other polypeptide expression construction as herein described; acetate (salt); NAD or any other compound as herein described or composition, or the like.The treatment product can comprise Sterile Saline or another kind of pharmaceutically acceptable emulsion and suspension base mateiral.

Address concrete buffer reagent, substratum, reagent, cell, culture condition etc. or their some subclass are not restrictive, comprise that those of ordinary skills think its interesting or all these associated materials of being worth in existing concrete environment and should be understood to.For example, a kind of buffering system of Chang Keyong or substratum replace another kind of, thereby can be with different but known mode realizes the target identical with adopting described method, material or composition.

All components of test kit are provided in container usually.In preferred implementation of the present invention, improve SIRT3 level or active test kit or regulate AceCS2 level, acetylize state or active test kit the container that comprises described screening method acquisition or obtainable material is housed.

In addition, test kit can be equipped with the operation instruction material that comprises the guidance (being scheme) of implementing the inventive method.The operation instruction material can be present in the described test kit by various forms, can have a or many parts of operation instruction materials in the test kit.Though the operation instruction material comprises the material of writing or printing usually, they are not limited thereto.The present invention includes any media that can store these operation instructions and can link up with the final user.These media include but not limited to: electronic storage medium (for example disk, tape, coding tape (cartridge) and chip), optical medium (for example, CD ROM) etc.These media can comprise the internet address that these operation instruction materials are provided.

In the present invention one preferred embodiment; test kit is equipped with described material is contacted with mammalian cell with raising SIRT3 level or active working instructions, or described material is contacted with mammalian cell to regulate AceCS2 level, acetylize state or active working instructions.One preferred embodiment in, the level of material incentive AceCS2, acetylize state or activity.In another embodiment, material suppresses level, acetylize state or the activity of AceCS2.

Optional comprising in the working instructions about contingent side effect and drug-drug or the interactional warning of medicine-food.

Can prepare all ingredients box and component according to the present invention according to expectation user He this user's of test kit concrete needs.

In the present invention one preferred embodiment, described test kit is a pharmaceutical kit, and the pharmaceutical composition that contains following composition is housed: (i) can improve SIRT3 level or active material and (ii) pharmaceutically acceptable carrier.In another preferred implementation of the present invention, described test kit is a pharmaceutical kit, and the pharmaceutical composition that contains following composition is housed: (i) can regulate AceCS2 level, acetylize state or active material and (ii) pharmaceutically acceptable carrier.Optional being equipped with of pharmaceutical kit explained the working instructions that can maybe pharmaceutical composition should be used for the treatment of pathologic state, illness or disease or any other method as herein described.

Other test kit embodiment of the present invention is equipped with and makes those of ordinary skills can implement any optional feature component of improving one's methods described herein.

Though describe aforementioned invention for purpose clear and that understand in detail by explanation and example, variation, change, improvement and the replacement that those of ordinary skills can make some equivalence in view of instruction of the present invention will be appreciated that and do not break away from design of the present invention and scope.Therefore, embodiment described herein is to be subordinated to various improvement, variation etc., and scope of the present invention is only with reference to claims decision of enclosing.Those skilled in the art are understood that and can change, change or improve various non-key parameters to obtain similar in fact result.

Though each element of the present invention described herein comprises numerous embodiments, unless statement is arranged in addition, otherwise each embodiment of given element of the present invention can with each embodiment coupling of other element of the present invention, each purposes should form different embodiment of the present invention.

Show with way of reference specially and separately as each publication, patent or patent application and to include in that the patent that this paper addresses, patent application and scientific literature comprise that the GenBank database sequence includes this paper in full with way of reference.Be as the criterion with the latter if any any conflict between the concrete instruction of any reference that this paper quotes and this specification sheets.Similarly, be as the criterion with the latter if any any conflict between the definition of the speech of the definition of the speech of this area understanding or phrase and the concrete instruction of this specification sheets or phrase.

Should be understood that from foregoing the present invention has various application.Following examples further illustrate the present invention, and these embodiment are illustrative, do not should be understood to and limit definition of the present invention and scope by any way.

VII. embodiment

Embodiment 1: Universal method

A. cell cultures and transfection

37 ℃, 5%CO ₂HEK293, COS-1 and HeLa cell are cultivated in the DMEM that has added 10% FCS, 2mM L-glutaminate, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates and grown down.Adopt calcium phosphate transfection to come transfection HEK293 cell (Chen and Okayama, 1987, MolCell Biol 7:2745-52).Fugene transfection Hela and Cos-1 cell with Roche Holding Ag (Roche).By in the complete DME growth medium that contains 800 μ g/mL Geneticins (hero company), selecting to produce stably express AceCS2 ^Sign, SIRT3 ^Sign, SIRT3-H248Y ^SignOr contain free ^Sign-control vector pcDNA ^SignHEK293 clone.

B. plasmid and mutagenesis

Adopt standard clone's scheme of PCR-based to produce all expression constructs, verify all expression constructs by dna sequencing.Utilize PCR primer 5 '-CG GAATTCCATGGCGGCGCGCACCCTGGGC-3 ' (SEQ ID NO:15) and 5 '-CG GAATTCCTTAGCAGCAGCCTGCTTGTCCTTGC-3 ' (SEQ ID NO:16) (respectively containing EcoRI restriction site (underscore)) is from (MGC) cDNA (Genbank accession number BC039261 of people's total length mammalian genes preservation institute (Mammalian Gene Collection); Obtain by open Biosys Corp. (Open Biosystems)) pcr amplification people AceCS2 encoding sequence.Then the PCR product cloning is gone into pcDNA3.1+ (hero company)-derivative vector pcDNA ^SignOr pcDNA ^HATo obtain to contain the terminal sign of C--or the AceCS2 of hemagglutinin (HA)-label.According to N-terminal protein matter sequencing result, will be cloned into pTrcHis2C (hero company) corresponding to the open reading frame of ripe AceCS2 (amino-acid residue 38-689).Be encoded into acquaintance SIRT3 (amino-acid residue 102-399 by pcr amplification, (Schwer etc., 2002, J Cell Biol 158:647-57) or the recombinant expression vector of SIRT5 (amino-acid residue 12-310 or amino-acid residue 39-310) and be cloned into pTrcHis2C.Existing (Schwer etc., 2002, the J Cell Biol 158:647-57 of describing of template that is used for pcr amplification SIRT3 and SIRT5 encoding sequence; North etc., 2003, Mol Cell 11437-44).Those, those of ordinary skills can be from the primer of GenBank preservation and corresponding nucleic sequence derivation pcr amplification as herein described except that described herein.

Can adopt site-directed mutagenesis (QuickChange ^TMMutagenesis kit; Xstrata genome company) makes up AceCS2-K642Q-HA, pTrcHis2C-AceCS2-K642R and pTrcHis2C-SIRT3-H248Y (102-399).

C. the expression of recombinant protein and purifying

To use, for example the bacillus coli DH 5 alpha bacterium (hero company) of pTrcHis2C-SIRT3 (102-399), pTrcHis2C-SIRT3-H248Y (102-399), pTrcHis2C-SIRT5 (12-310), pTrcHis2C-SIRT5 (39-310) or pTrcHis2C-AceCS2 (38-689) conversion is cultured to A _600nmInduced 16 hours with 0.5mM sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) for=0.4,25 ℃.4 ℃, under natural condition, use the fast and smart company in Valencia, California city (Qiagen, Valencia, the protein of Ni-NTA agarose purifying 6XHis-mark CA).The protein of dialysis purifying (for example, is preserved damping fluid [50mM Tris-HCl (pH9.0), 4mMMgCl with Sir2-sample albumen ₂, 50mM NaCl, 0.5mM DTT, 5% glycerine (v/v)] or AceCS2 preservation damping fluid [50mM Tris-HCl (pH8.0), 4mM MgCl ₂, 10% glycerine (v/v)]), be adjusted to 0.5g/L ,-80 ℃ of preservations.For inducing the acetylize of reorganization AceCS2, (16 hours, 25 ℃) add niacinamide (50mM) during protein expression.

The CobB-defective type single-gene of parental generation e. coli k-12 BW25113 and same bacterial strain knocks out (KO) mutant strain (JW1106) (Datsenko and Wanner, 2000, Proc Natl Acad Sci U S A97:6640-5) available from the GenoBase preservation e. coli k-12 system knock-out bacterial strain (SystematicKnock Out Strains of E.coli K-12 Collection of GenoBase).37 ℃, non-selectivity ground bacterium colony purifying CobB-defective type KO mutant strain once, check ampicillin sensitive whether to lose Ampicillin Trihydrate resistance (Datsenko and the Wanner that gives the used helper plasmid of single-gene knockout technique then with test, 2000, Proc Natl Acad Sci U S A 97:6640-5).Amplification ampicillin sensitive bacterium colony, make it become chemoreception attitude (Sambrock and Russell, 2001, publish in Molecular Cloning:A Laboratory Manual (molecular cloning: (press of cold spring harbor laboratory laboratory manual), the cold spring port), the 1st volume, the 1.116-1.118 page or leaf) and use the pTrcHis2C-AceCS2 (38-689) or the pTrcHis2C-AceCS2-K642R (38-689) that contain the Ampicillin Trihydrate resistant gene to transform.Express the also protein of purifying 6XHis-mark as mentioned above.

D. antibody

The used antibody of immunoblotting and immunoprecipitation is anti--mtHsp70 (Clone JG1; Affine biological reagent company (Affinity Bioreagents), Na Shanike stand (Neshanic Station), the New Jersey), anti--Hsp90 α (Si Taisi King Company (StressGen)) and anti--manganese superoxide dismutase (MnSOD) (Si Taisijin biotech company (StressGen Biotechnologies), Victoria (Victoria), Canada), anti--cytochrome c oxidase subunit I V (clone 20E8-C12; Molecular probe company (Molecular Probes)), anti--sign M2 or tame rabbit polyclonal resist-indicate (Sigma-aldrich corp (Sigma-Aldrich)), anti--HA (12CA5 and 3F10; The Luo Shi diagnostic companies), acetylize-Methionin polyclonal antibody (signal conduction technique company, Bei Fuli, Massachusetts), anti--Actin muscle C4 (ICN biological medicine company (ICN Biomedicals)), anti--cytochrome c (clone 7H8.2C12; Fa Ma King Company (Pharmingen)), anti--BRG-1 (H88; SC biotech company (Santa Cruz Biotechnology)) and anti--c-myc (9E10; SC biotech company).Produce SIRT3 antiserum(antisera) (Schwer etc., 2002, J Cell Biol 158:647-57) as mentioned above.

E.SDS-PAGE and western blotting

With enhanced chemoluminescence (A Mushanmu Pharmacia Biological Science Co., Ltd (AmershamPharmacia Biosciences)) or West SuperSignal reagent (Pierre Si company (Pierce)) colour developing immunoblotting.Film is nitrocellulose or the (immunity-trace company (Immun-Blot) of poly(vinylidene fluoride) (PVDF) film; Bole laboratory company (Bio-Rad Laboratories)).

F. immunoprecipitation

Ice-cold NP1 damping fluid (1% NP-40,150mM NaCl, 0.5mM EDTA, 50mM Tris-HCl, pH7.4) lysing cell with the protease inhibitor cocktail that contains Roche Holding Ag.4 ℃, with 16, the centrifugal lysate of 200xg 10 minutes adds the anti--sign monoclonal antibody M2 with the agarose covalent coupling.The protein of immunoprecipitation sign-mark is with NP1 damping fluid washing 4 times.

Common-immunoprecipitation experiment uses the NP1 damping fluid that contains 300mM NaCl.With the used immunoprecipitation sign of the NP1 damping fluid washing acetylize test that contains 500mM NaCl-mark Sir2-sample albumen 3 times, with Sir2-sample deacetylase protein enzyme buffer liquid (SDAC) (50mM Tris-HCl (pH9.0), 4mM MgCl ₂, 50mM NaCl, 0.5mM DTT) and washed twice.4 ℃, will indicate-labelled protein wash-out from pearl goes into to contain the SDAC damping fluid of sign-peptide (Sigma company) l hour.

G. confocal microscopy

The HeLa cell is cultivated on cover glass, with Fugene 6 (Roche Holding Ag) transfection, fixing in 3.7% formaldehyde/PBS after 48 hours.Under the room temperature with the penetrating cell of 05% Triton X-100/PBS 5 minutes (Schwer etc., 2002, J Cell Biol 158:647-57).With 1% bovine serum albumin closing cell, with monoclonal anti-sign M2 (1:500) and polyclone anti--MnSOD (1:300) antibody dyes altogether, then with the anti--mouse-Cy2-link coupled that is suitable for the multiple labeling experiment anti--mouse immuning ball protein G and Cy5-link coupled be anti--rabbit immunoglobulin G second antibody (Jackson's immune Research laboratory company (JacksonImmunoResearch Laboratories)) incubation.Cover glass is placed on the slide glass, obtains image with BioRadRadiance 2000 laser scanning microscopes that are equipped with Olympus Bx60 microscope and Olympus PlanApo 60x/1.40 oil-immersion objective.

H. subcellular fractionation and inferior plastosome are located

Carry out subcellular fractionation (Schwer etc., 2002, J Cell Biol 158:647-57 as described; Yang etc., 1997, Science 275:1129-32).Institute carries out at 4 ℃ in steps.In brief, at ice-cold buffer A (250mM sucrose, 10mM KCl, 1.5mM MgCl ₂, 1mM EDTA, 1mMEGTA, 1mM dithiothreitol (DTT) (dithiotreithol), the 0.1mM phenylmethylsulfonyl fluoride, 20mMHepes-KOH, pH7.5) middle with Dounce homogenizer (Hui Dun company (Wheaton)) homogenate cell.Phase contrast microscopy is checked the homogenate situation.With homogenate with centrifugal twice, 5 minute of 960xg to remove all nuclears and broken cell.By centrifugal (7,000xg, 15 minutes), clean twice with buffer A, at last adding the NP1/150 damping fluid of proteinase inhibitor (1% NP-40 (v/v), 150mMNaCl, 0.5mM EDTA, 50mM Tris-HCl, pH7.4) middle cracking is with separate mitochondria.With 100,000xg ultracentrifugation 30 minutes is classified into light membrane component (LM, precipitation) and cytosol component (S-100, supernatant liquor) with PMS.Measure the protein concn (DC protein test, Bole laboratory company) of each component, by each component of immunoblotting assay equivalent.

According to disclosed scheme (Schwer etc., 2002, J Cell Biol 158:647-57; Ryan etc., 2001, publish in Mitochondria (plastosome), Pon and Schon compile (academic press (AcademicPress), the 65th volume, 190-213 page or leaf) and carry out formation of line grain corpusculum and the experiment of proteolytic enzyme accessibility.Handled line grain corpusculum 15 minutes with Proteinase K (150 μ g/mL) for 0 ℃.Add 2mM PMSF and stop protease digestion, come heavy defiber grain corpusculum by centrifugal in sample buffer, cleaning and cracking.

As (Schwer etc., 2002, J Cell Biol 158:647-57; Fujiki etc., 1982, J Cell Biol93:97-102) described by alkaline purification separate mitochondria protein.The mitochondrial pellet of cleaning is resuspended in the 0.1M yellow soda ash (pH11.5) of prepared fresh 0 ℃ of incubation 30 minutes with 250 μ g/mL.Ultracentrifugation (100,000xg, 30 minutes, 4 ℃) precipitation mitochondrial membrane.Throw out is resuspended in the SDS sample buffer, precipitates soluble proteins in the concentrated supernatant, be resuspended in SDS sample buffer (Schwer etc., 2002, J Cell Biol 158:647-57 by trichloroacetate; Fujiki etc., 1982, J Cell Biol93:97-102).

The order-checking of I.N-terminal protein matter

Cracking stably express AceCS2 ^SignThe HEK293 cell, it is resisted-indicates immunoprecipitation.After the cleaning, to the AceCS2 of immunoprecipitation ^SignCarry out SDS-PAGE, be transferred to poly(vinylidene fluoride) (PVDF) film,, downcut, be forwarded to Stamford PAN mechanism carries out the Edman degraded according to standard scheme N-end sequencing by Ponceau S dyeing (Sigma company) visual observations.

J. protein enzyme test

1. external deacetylated test

32 ℃, be with or without NAD ⁺(1mM) exist down; (NAM 10mM) exists down, has the system hair plain A of tinea (500nM) to exist down to be with or without niacinamide; with the purification of Recombinant AceCS2 of equimolar amount and purification of Recombinant Sir2-sample albumen at the deacetylated damping fluid of SDAC (50mM Tris-HCl (pH9.0), 4mM MgCl ₂, 50mM NaCl, 0.5mM DTT) in incubation 3 hours.For the deacetylated experiment of time-histories, shown in time point take out the sample aliquot of deacetylated reaction, mix with the 10mM niacinamide, at incubation on ice until further analysis.By SDS-PAGE and immunoblotting assay reaction.

2. Acetyl-CoA synthetase activity

As (Jones and Lipmann, 1955, publish in Methods in Enzymology (Enzymology method) (academic press, the 1st volume, 585-591 page or leaf; Barak etc., 2004, J Mol Biol 342:383-401) activity of described detection purifying AceCS2.Each reaction (0.5mL) contains 100mM azanol (using the KOH pre-neutralization), 50mM Tris-HCl (pH8.0), 20mM potassium acetate, 10mM MgCl ₂, 10mM ATP, 2mM DTT and 1mM CoA.All chemical (Sigma company) are getable highest purities.To react (system) and add the AceCS2 (6 μ g) of purifying at 35 ℃ of incubations after 5 minutes.Behind the incubation 30 minutes, add 0.5mL stop bath (10% (w/v) FeCl of 2N HCl preparation ₃, 3.3% (w/v) trichoroacetic acid(TCA) solution), will react (system) incubation on ice 2 minutes.With 16, the centrifugal sample of 200xg detected the color that produces to eliminate turbidity in 2 minutes with 540nm.The sample of no AceCS2 forms acetyl hydroximic acid salt (acetylhydroxamate) as standard substance (Barak etc., 2004, J Mol Biol 342:383-401) as blank solution by ethanoyl-phosphoric acid salt (Sigma company).Do not have to detect the Acetyl-CoA synthetase activity under the CoA existence.

K.SiRNA inhibition-consumption SIRT3

According to manufacturer's recommendation, will be with oligomerization transfection amine (oligofectamine) (hero company) at double-stranded siRNA or the Lampyridea luciferase GL3 contrast siRNA (100nM of people SIRT3; Reach agate Kanggong department (Dharmacon); Each siGENOME duplex D-004827-04-0050, people SIRT3 NM_012239) is transfected into HEK293.After the transfection 5 days, lysing cell in the NP1/300 damping fluid that contains proteinase inhibitor, 1 μ M TSA and 10mM niacinamide.Immunoprecipitation AceCS2 ^Sign, and prepare to be used for analytical reagent composition.

The sequence of SIRT3 siRNA duplex is as follows:

Adopted sequence is arranged: 5 '-GGAGUGGCCUGUACAGCAAUU-3 ' (SEQ ID NO:17)

Antisense sequences: 5 '-UUGCUGUACAGGCCACUCCUU-3 ' (SEQ ID NO:18)

The oligonucleotide that uses in the siRNA experiment can be in its 5 ' end phosphorylation.

Elbashir etc. have described luciferase GL3 duplex D-001400-01-20 " Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells. (duplex of 21-Nucleotide RNA mediate rna in the mammalian cell of cultivation disturbs) " among the Nature 411:494-498 (2001).

L. the liquid chromatography-mass spectrometry of Nano grade

By SDS-PAGE separating immune throw out, digest in the gel as (Hojrup, 2004, Methods MolBiol 251:227-44) described carrying out basically.Utilize An Jielun (Agilent) 1100 nanometer streaming systems (An Jielun technology company (Agilent Technologies), Palo Alto, the California), separate the tryptic digestion product by reversed phase chromatography, directly electron spray(ES) goes into to be equipped with the Finnigan LTQ-FT of the 7-tesla mass spectrograph (power ﹠ light company (Thermo Electron) of nanometer electric spray ion source (the celestial Biosys Corp. (Proxeon Biosystems) of Pu Luoke, Odense, Denmark), bremen, Germany) in.ReproSil-Pur C18-AQ 3 μ m resin (doctor Mai Shi limited-liability company (Dr.MaischGmbH) have been loaded, Ah nurse Bach-Antu root (Ammerbuch-Entringen), Germany) 15-cm is from drawing (home-pulled) pyrogenic silica radiator (75 μ m internal diameter) as reversed-phase column.With 500nl/ minute flow velocity the protein digestion product is expelled on the post, uses 5-40%MeCN (preparation of 0.5% acetate) gradient subsequently with 250nl/ minute flow velocity wash-out.For initial analysis, open mass spectrograph as (Olson and Mann, 2004, Proc Natl Acad Sci USA 101:13417-22) is described with the data dependency pattern basically.In brief, by Fourier transform ion cyclotron resonance (FTICR; Resolving power 50,000, m/z 400) obtain mass range at the detection MS of m/z 300-1600 collection of illustrative plates, utilize selection ion monitoring (SIM) scanning among the FTICR to come three kinds of the highest ions of selection intensity to carry out accurate mass measurement, utilize linear ion hydrazine to implement MS/MS and MS/MS/MS simultaneously.

In interested m/z scope, be scheduled to the MS of m/z value successively ²And MS ³Adopt FTICR SIM scanning to select the directed experiment of ionic.Adopt MASCOT search engine (matrix scientific company (MatrixScience), Boston), be that 5ppm and fragment ions tolerance are the centre of form of 0.6Da retrieval Uniprot database and the MS/MS collection of illustrative plates of merging with peptide quality tolerance (peptide mass tolerance).The urea groups of halfcystine methylated is set at fixing the modification, and with the acetylize of N-terminal protein matter, methionine(Met) oxidation and Methionin acetylize as variable modification.Require peptide to be lacked 3 cleavage sites at most by trypsin hydrolyzing.Utilize MS ²And MS ³The peptide of all evaluations of the manual checking of collection of illustrative plates.

Embodiment 2: Evaluation is as the Acetyl-CoA synthetase (AceCS2) of SIRT3 cell target

As the scheme of the Methionin acetylize mitochondrial protein of identifying SIRT3,4 or 5 targets, the human protein of research sequence and the acetylize lysine residue peripheral region sequence similarity of discovery in intestines salmonella (Salmonella enterica) Acetyl-CoA synthetase (ACS).Acetyl-containing-Methionin zone of selecting intestines salmonella ACS is because it is known substrate (Starai etc., 2003, the Genetics163:545-55 of Sir2-sample PROTEIN C obB; Starai etc., 2002, Science 298:2390-2).In second step, utilize MITOPROT II and the analysis of 1.03 editions ubcellular forecasting softwares of PREDOTAR to show plastosome location probability (Small etc., 2004, the Proteomics 4:1581-90 of the human protein of high similarity; Claros and Vincens, 1996, Eur J Biochem 241:779-86).The research obtains to be positioned high people's Acetyl-CoA synthetase albumen (the MITOPROT II:0.997 of mitochondrial probability; PREDOTAR:0.94; The highest scoring=1), itself and mouse Acetyl-CoA synthetase AceCS2 have height sequence similarity (Claros and Vincens, 1996, Eur J Biochem 241:779-86).

Embodiment 3: Identify the plastosome location of AceCS2

For whether surveyor AceCS2 enzyme is mitochondrial protein, the open reading frame of human cloning AceCS2 also is expressed as the protein of sign-mark in the HeLa cell.The confocal laser scanning microscopy shows AceCS2 ^{Mark Will}Plastosome dyeing pattern and observed endogenous mitochondrial matrix albumen manganese superoxide dismutase (MnSOD; Dyeing pattern Figure 1A) is overlapping.

For the plastosome location of further identifier AceCS2, from stably express AceCS2 ^SignHuman embryo kidney 293 (HEK293) cell preparation subcellular components.As estimating, in the plastosome component, detect AceCS2 ^SignTogether with SIRT3 and MnSOD (Figure 1B).Nuclear labelled protein BRG-1 in each component and cytosol labelled protein Hsp90 α are confirmed the purity (Figure 1B) of each component as immunoblotting.

For further determining the inferior plastosome location of AceCS2, from containing AceCS2 ^SignPlastosome prepare line grain corpusculum.Line grain corpusculum goods destroy mitochondrial outer membrane, thereby make Proteinase K can arrive intermembrane space.Handle line grain corpusculum with Proteinase K and cause intermembrane space protein cell pigment c (Cyt.c) forfeiture, though it does not influence mitochondrial matrix albumen glutamate dehydrogenase (GDH) and SIRT3 (Fig. 1 C).Proteinase K is handled line grain corpusculum does not influence AceCS2 yet ^Sign, point out it similar with SIRT3 to GDH, be positioned (Fig. 1 C) in the mitochondrial matrix.Add non-ionic detergent TX-100, Proteinase K complete digestion all proteins (Fig. 1 C between online grain corpusculum and Proteinase K incubation period; Road, a left side).

Whether conformability is connected in the inboard of mitochondrial inner membrane or is dissolved in the mitochondrial matrix usefulness yellow soda ash (pH11.5) extraction plastosome in order to measure AceCS2.This is handled and discharges soluble proteins but not integral protein.Yellow soda ash is handled and is discharged AceCS2 fully, and its distribution pattern is similar to dissolvable matrix Protein S IRT3 and MnSOD (Fig. 1 D).Under used condition, integral protein COX-IV keep linking to each other (Fig. 1 D) with film.

Embodiment 4: The N-terminal order-checking of AceCS2

The plastosome transposase that goes to the nuclear-encoded protein of mitochondrial matrix often to carry adventitia (TOM) complex body is discerned and is gone into the terminal presequence (Rehling etc., 2004, Nat RevMol Cell Biol 5:519-30) of the N-that excises after the matrix in transportation.The terminal presequence of this N-often contains alpha-helix and several alkaline residue.These features see the N-end (Fig. 2 A) of the people AceCS2 that comprises several arginine residues.

By the AceCS2 of Edman degraded to immunoprecipitation ^SignCarry out the N-end sequencing and show that protein has lacked preceding 37 amino-acid residues of open reading frame, this is consistent with the terminal processing of N-of AceCS2 in the mitochondrial matrix.Identify that by liquid chromatography link coupled tandom mass spectrometer (LC-MS/MS) L-Ala 38 is AceCS2 of immunoprecipitation ^Sign-terminal amino acid confirmed protein sequencing result (data not shown).Being close to the terminal upstream of the proteic N-of ripe AceCS2 exists mitochondrial matrix processing peptidase (MPP) R-2 motif prompting AceCS2 to process (Fig. 2 B by MPP in mitochondrial matrix; Ito, 1999, Biochem Biophys ResCommun 265:611-6).There is the human mitochondrion Acetyl-CoA synthetase in these in witness line mitochondrial matrix as a result.

Embodiment 5: The AceCS2 enzymic activity is eliminated in the sudden change of conservative property Lys642 amino-acid residue

According to the conservative property (Fig. 3 A) in the avtive spot zone of the Acetyl-CoA synthetase of intestines salmonella ACS and people and mouse AceCS2 and other species, checked the sudden change of Lys642 whether can influence from the Acetyl-CoA synthetase activity of the sedimentary overexpression people of HEK293 cellular immunization AceCS2.Glutamine with the acetylizad lysine residue of simulation composing type substitutes the AceCS2 albumen (Fig. 3 B) that Lys642 produces complete deactivation, shows that Lys642 is most important for the AceCS2 function.Wild-type AceCS2 similar with the expression level of mutant AceCS2-K642Q (Fig. 3 C).

Embodiment 6: People AceCS2 acetylize in vivo

Whether in vivo for measuring people AceCS2 acetylize, the SIRT3 of siRNA (siRNA)-mediation strikes and subtracts the back from cellular immunization precipitate A ceCS2 ^Sign, and prepare to be used for mass spectroscopy.There is acetylizad lysine residue (acK) in the LC-MS/MS analysis revealed of tryptic digestion product (Fig. 4) 642 of AceCS2.

Embodiment 7: The super second of the people AceCS2 of the expression in escherichia coli of shortage Sir2-sample PROTEIN C obB Acidylate

Similar with the intestines salmonella, intestinal bacteria contain Sir2-sample PROTEIN C obB.Suppose that protein mediated deacetylated of Sir2-sample has the conservative effect of potential in control Acetyl-CoA synthetase albumen develops, then test person AceCS2 expresses whether induce the super acetylize of AceCS2 in the e. coli k-12 bacterial strain that lacks Sir2-sample PROTEIN C obB.Be better than AceCS2 (Fig. 5 A) from the reaction of the people AceCS2 of CobB-knock-out bacterial strain purifying and ethanoyl-Methionin-specific antibody from parental generation CobB wild type strain purifying.This acetylize is special for AceCS2 Lys642, because Lys642 sports reactivity (Fig. 5 A that can not acetylizad arginine have eliminated with anti--ethanoyl-Methionin antibody; Right road).The synthetic peptide that is coupled to carrier proteins is made the specificity (Fig. 5 B) that immunoblotting assay further confirms ethanoyl-Methionin-specific antibody.Ethanoyl-Methionin-specific antibody only with the reactive polypeptide that contains acetylize K642 residue, peptide of acetylize K642 does not react (Fig. 5 B) with containing not for it.

Embodiment 8: Suppress Sir2-sample protein-active by niacinamide and cause the super acetylize of recombinant human AceCS2

Detected and suppressed of the influence of Sir2-sample protein-active people AceCS2 acetylize state.Handle wild-type e. coli bacterial strain (DH5 α with Sir2-sample protein inhibitor niacinamide; Hero company) causes the super acetylize of recombinant human AceCS2 (Fig. 6 A).This acetylize (state) increases special to the Lys642 of AceCS2, does not induce mutant AceCS2 albumen acetylize (Fig. 6 A that carries arginine residues at 642 because niacinamide is handled; Right road).The purification of Recombinant AceCS2 of LC/MS-MS analysis niacinamide processing bacterium confirms the place's Methionin in the AceCS2 avtive spot, Lys642 acetylize (Fig. 6 B).

Embodiment 9: Endogenous SIRT3 level reduces the acetylize that improves AceCS2

For confirm further whether SIRT3 works to acetylize AceCS2 in cell, use siRNA or contrast siRNA to handle expression AceCS2 at SIRT3 ^SignThe HEK293 cell, detect the AceCS2 of immunoprecipitation as immunoblotting with the antibody of acetylize Methionin ^SignAcetylize state (Fig. 7).Strike and subtract AceCS2 behind the endogenous SIRT3 ^SignAcetylize increase, prompting SIRT3 influences AceCS2 in vivo ^SignAcetylize (Fig. 7).

Embodiment 10: SIRT3 makes AceCS2 deacetylated; And SIRT4 and SIRT5 can not

Among the intestines salmonella ACS acetylize of avtive spot Methionin make this enzyme deactivation (Starai etc., 2002, Science298:2390-2).Make the deacetylated intestines salmonella CobB of ACS be and the maximally related III class of people SIRT5 Sir2-sample albumen.SIRT5 is positioned plastosome and processes (Michishita etc., 2005, Mol Biol Cell 16:4623-35) through the N-end.Whether SIRT5 can make AceCS2 deacetylated for test, carries out external deacetylated test.Immunoprecipitation sign-mark Sir2-sample albumen as (North etc., 2003, Mol Cell 11:437-44) described preparation HEK293 cells such as North.Though the sign of immunoprecipitation-mark SIRT5 has low detectable activity (North etc., 2003, Mol Cell 11:437-44) to the acetylizad H4 peptide of chemistry, but it fails to make AceCS2 deacetylated (Fig. 8 A).On the contrary, the sign of immunoprecipitation-mark SIRT3 is with NAD ⁺-dependency mode makes AceCS2 deacetylated (Fig. 8 A).Report does not have the active another kind of plastosome Sir2-sample albumen of deacetylase, and the SIRT4 of sign-mark (North etc., 2003, Mol Cell 11:437-44) does not make AceCS2 deacetylated (data not shown).

For confirming the relevant discovery of SIRT3 and SIRT5, express and purification of Recombinant Sir2-sample albumen.According to SIRT5 is terminal this discovery of brachymemma (Michishita etc., 2005, Mol Biol Cell 16:4623-35) of N-and the terminal MPP R-2 motif that has two kinds of suppositions of its N-, utilizes to lack preceding 11 (Δ 11-SIRT5 respectively ^Myc-His) or 38 (Δ 38-SIRT5 ^Myc-His) two kinds of amino-acid residue different reorganization SIRT5 albumen.Though SIRT3 can make AceCS2 effectively deacetylated, two kinds of reorganization SIRT5 protein are not still accomplished (Fig. 8 B).With estimate the same, SIRT3 makes the deacetylated strictness of AceCS2 depend on NAD ⁺Have (Fig. 8 B).Sir2-sample protein inhibitor niacinamide also stops SIRT3 to make AceCS2 deacetylated fully, and the SIRT3-H248Y mutant of catalysis inactivation (Schwer etc., 2002, J Cell Biol 158:647-57) does not make AceCS2 deacetylated, although there is NAD ⁺(Fig. 8 C).

Embodiment 11: SIRT3 and AceCS2 coimmunoprecipitation also make AceCS2 take off acetyl in cell Change

For determining whether SIRT5 makes AceCS2 deacetylated in cell, coexpression AceCS2 in the COS-1 cell ^HAAnd SIRT3 ^SignOr SIRT5 ^SignImmunoprecipitation AceCS2 ^HA, with the antibody analysis immunocomplex (Fig. 9 A) of acetylize Methionin.The acetylize level of crossing the AceCS2 that expresses the reduction ectopic expression of SIRT3, and SIRT5 does not have influence, although expression level high (Fig. 9 A) far away.

What is interesting is the AceCS2 of endogenous SIRT3 and HEK293 cell ^SignCoimmunoprecipitation (Fig. 9 B).In conjunction with above-mentioned discovery, prompting SIRT3 is the real deacetylase of plastosome AceCS2.

Embodiment 12: The acetylize of AceCS2 reduces its enzymic activity

Significantly improve its this discovery of acetylize level according to handling intestinal bacteria with niacinamide during expressing at people AceCS2, detected AceCS2 and whether controlled its Acetyl-CoA synthetase activity (Figure 10 A) in the acetylize of Lys642 place.From bacterium (with or handle without niacinamide) the specific activity detected value of the AceCS2 of purifying shows the specific activity obviously lower (Figure 10 B) of super acetylizad AceCS2.The acetylize of these observationss prompting AceCS2 suppresses its enzymic activity, this with bacterium in shown consistent (Starai etc., 2002, the Science 298:2390-2) of ACS.

For further checking this hypothesis, NAD is being arranged ⁺The super acetylize AceCS2 and the SIRT3 incubation that have down the intestinal bacteria purifying that will handle from niacinamide.This processing causes AceCS2 deacetylated fully (Figure 10 C).If save NAD in the reaction ⁺, or utilize the SIRT3 mutant of catalysis inactivation, then do not observe deacetylated (Figure 10 C) of AceCS2.

In addition, as mentioned above, the enzymic activity that detects the different AceCS2 of acetylize level shows by the deacetylated remarkable increase with enzymic activity of SIRT3 relevant (Figure 10 D).

In other experiment, find AceCS2 and SIRT3 and NAD ⁺Incubation causes AceCS2 deacetylated gradually in time together, can not observe this phenomenon (Figure 10 E) with the SIRT3-H248Y mutant of inactivation.Deacetylated still relevant (Figure 10 F) of AceCS2 with the increase gradually of enzymic activity.According to data provided herein, can draw the active conclusion of acetylize mode control human mitochondrion AceCS2 of Lys642.

Embodiment 13: Sum up and discuss

In the intestines salmonella, protein acetyltransferase Pat makes this enzyme deactivation with the ACS acetylize, and this enzyme of deacetylated reactivate of Sir2-sample PROTEIN C obB (Starai etc., 2002, Science298:2390-2; Starai etc., 2004, J Mol Biol 340:1005-12).The Acetyl-CoA synthetase reaction that forms acetyl-CoA from acetate (salt), ATP and CoA is carried out in two steps.In the first step, acetate (salt) activates into ethanoyl-adenosine monophosphate (ethanoyl-AMP).In second step, ethanoyl-AMP changes into acetyl-CoA because of the thioester bond of ACS forms activity, and acetyl-CoA and AMP discharge (summary is seen Starai etc., 2004, Cell Mol Life Sci 61:2020-30) successively.The Methionin acetylize specificity inhibition acetate (salt) that has now proved the avtive spot Methionin (Lys609) of bacterium ACS is transformed into ethanoyl-AMP through ATP-dependency adenylylation process; and the unaffected (Starai etc. of second step of ACS reaction; 2002, Science 298:2390-2).As shown in Figure 3A, the Lys642 of AceCS2 is corresponding to the avtive spot Methionin Lys609 of intestines salmonella ACS.Suppose that there is conservative property in the avtive spot zone between ACS and the people AceCS2, the Lys642 that the someone proposes acetylize AceCS2 can disturb and relate to first reactions steps that acetate activates into ethanoyl-AMP.

AceCS2 is subjected to first example of mitochondrial protein of reversible Methionin acetylize control and first target protein of plastosome Sir2-sample deacetylase protein enzyme.Regulate the synoptic diagram of AceCS2 by reversible Methionin acetylize and see Figure 11.Data provided herein support that SIRT3 activation AceCS2 is this model of metabolism controlling mechanism of evolution conservative.Acetyl-the CoA (being called " activatory acetate " in the past) and the exchange of acetate are called " acetate conversion ", and it is present in archeobacteria, eubacterium and the eukaryotic cell, reclaim NAD for cell provides ⁺, produce ATP and additional coenzyme A stock's possibility (summary is seen Wolfe, 2005, MicrobiolMol Biol Rev 69:12-50).Acetate-scavenger enzyme Acetyl-CoA synthetase control acetate conversion.Metabolism approach such as energy generation during for example TCA circulates and cholesterol and fatty acid biological are synthetic need acetyl-CoA as intermediate.

Saccharomycetic growth extremely needs Acetyl-CoA synthetase, but mammalian cell is not like this.Most of acetyl-CoA in the mammalian cell do not produce in relying on the active approach of Acetyl-CoA synthetase.These approach are by pyruvic oxidase and β-Yang Hua pyruvic acid to be changed into acetyl-CoA, thereby form acetyl-CoA as end product.Yet in Mammals, having produced in some cases needs by a large amount of acetates of Acetyl-CoA synthetase activatory.For example, giving birth under the ketone situation, for example long-term fasting or diabetes, liver is released into blood flow (Buckley and Williamson, 1977, Biochem J 166:539-45 with the acetate of a great deal of; Seufert etc., 1974, Biochem Biophys Res Commun57:901-9; Yamashita etc., 2001, Biochim Biophys Acta 1532:79-87).In addition, and the activation under living ketone situation of the liver Acetyl-CoA hydrolase of generation acetate (Matsunaga etc., 1985, EurJ Biochem 152,331-6).The acetate that utilizes extrahepatic tissue to discharge needs the effect of Acetyl-CoA synthetase.AceCS2 is abundant and lack in liver and induce this discovery prompting AceCS2 giving birth to play an important role in the energy-producing acetate conversion under the ketone situation (Fujino etc. under the ketone situation giving birth in heart and skeletal muscle, 2001, J Biol Chem 276,11420-6).According to finding that bacterium Sir2-sample albumen is controlled the Acetyl-CoA synthetase activity and all had Sir2-sample albumen in all three fields in the intestines salmonella, someone proposes between maincenter metabolism and the Sir2-sample albumen general dependency (Starai etc. are arranged, 2003, Genetics163,545-55; Starai etc., 2002, Science 298,2390-2).Discovery as herein described shows can make the AceCS2 inactivation by its avtive spot Methionin of acetylize; plastosome Sir2-sample albumen can make it reactivate; to the Mammals plastosome, these approach have conservative property from bacterium for these discovery these claims of support and proof.

Claims (21)

1. identify the method that improves SIRT3 polypeptide level or the active material of deacetylase for one kind, this method may further comprise the steps:

(a) cell of expressing SIRT3 polypeptide and Acetyl-CoA synthetase 2 (AceCS2) polypeptide is contacted with candidate substances; With

(b), then measure the effect of acetylize AceCS2 level in the candidate substances pair cell if effect is arranged.

2. the method for claim 1 is characterized in that, step (b) comprises the immunological testing of the specific antibody that utilizes acetylize AceCS2.

3. the method for claim 1 is characterized in that, described cell is a mammalian cell.

4. method as claimed in claim 3 is characterized in that, described mammalian cell is selected from down group: heart cell, muscle cell and brain cell.

5. method as claimed in claim 3 is characterized in that, described mammalian cell is people's cell.

6. the method for claim 1 is characterized in that, and is further comprising the steps of:

(c) structure or the sequence of the described candidate substances of evaluation.

7. identify the method that improves SIRT3 polypeptide level or the active material of deacetylase for one kind, this method may further comprise the steps:

(a) make and comprise NAD ⁺Test mixture in the SIRT3 polypeptide contact with candidate substances with acetylizad Acetyl-CoA synthetase 2 (AceCS2) polypeptide and

(b), then measure the effect of candidate substances to acetylize AceCS2 level in the test mixture if effect is arranged;

Wherein with not compare with second level of acetylize AceCS2 in the test mixture of described candidate substances processing, first level of acetylize AceCS2 reduces level or the deacetylase activity that shows this material raising SIRT3 polypeptide in the test mixture.

8. method as claimed in claim 7 is characterized in that, acetylize AceCS2 polypeptide comprises ¹⁴The ethanoyl of C-mark is by detecting ¹⁴The release implementation step (b) of C-mark ethanoyl.

9. identify the method for regulating Acetyl-CoA synthetase 2 (AceCS2) polypeptide level, acetylize state or active material for one kind, this method may further comprise the steps:

(a) cell of expressing the AceCS2 polypeptide is contacted with candidate substances and

(b), then measure level, acetylize state or the active effect of AceCS2 in the candidate substances pair cell if effect is arranged.

10. identify the method for regulating Acetyl-CoA synthetase 2 (AceCS2) polypeptide level, acetylize state or active material for one kind, this method may further comprise the steps:

(a) AceCS2 polypeptide in the test mixture is contacted with candidate substances and

(b), then measure level, acetylize state or the active effect of candidate substances to AceCS2 in the test mixture if effect is arranged.

11. biological active agents of identifying by the described method of claim 1.

12. biological active agents of identifying by the described method of claim 9.

13. comprising, a method of regulating the acetylize state of Acetyl-CoA synthetase 2 (AceCS2) polypeptide, this method make the cell and the step that contacts according to the obtainable material of the described method of claim 1 of expressing the AceCS2 polypeptide.

14. comprising, a method of regulating the acetylize state of Acetyl-CoA synthetase 2 (AceCS2) polypeptide, this method make the cell and the step that contacts according to the obtainable material of the described method of claim 9 of expressing the AceCS2 polypeptide.

15. comprising, a method for the treatment of individual pathologic state, this method give individual step with the raising SIRT3 polypeptide level of treatment significant quantity or the active material of deacetylase;

Wherein SIRT3 makes AceCS2 deacetylated;

Wherein said pathologic state is characterised in that the acetate level raises; With

Wherein said pathologic state obtains medical treatment.

16. method as claimed in claim 15 is characterized in that, described pathologic state is selected from down group: type ii diabetes, hypercholesterolemia, hyperlipidaemia and obesity.

17. method as claimed in claim 15 is characterized in that, described individuality is people or non-human animal.

18. one kind is improved SIRT3 level or the active pharmaceutical composition of deacetylase, it comprises:

(i) according to the obtainable material of the described method of claim 1; With

(ii) pharmaceutically acceptable carrier.

19. regulate Acetyl-CoA synthetase 2 (AceCS2) polypeptide level, acetylize state or active pharmaceutical composition for one kind, it comprises:

(i) according to the obtainable material of the described method of claim 9; With

(ii) pharmaceutically acceptable carrier.

20. one kind is improved SIRT3 level or the active test kit of deacetylase, it is equipped with:

(i) contain container according to the obtainable material of the described method of claim 1; With

(ii) with this material and the cells contacting of expressing SITR3 polypeptide and Acetyl-CoA synthetase 2 (AceCS2) polypeptide and measure the working instructions of acetylize AceCS2 level, acetylize state or active effect in this material pair cell.

21. regulate Acetyl-CoA synthetase 2 (AceCS2) polypeptide level, acetylize state or active test kit for one kind, it is equipped with:

(i) contain container according to the obtainable material of the described method of claim 9; With

(ii) with the cells contacting of expressing the AceCS2 polypeptide and measure the working instructions of AceCS2 level, acetylize state or active effect in this material pair cell.

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