CN102151335A - Regulation of metabolism by protein lysine acetylation modification - Google Patents

Abstract

The invention relates to regulation of metabolism by protein lysine acetylation modification and use thereof. The metabolism of cells can be regulated by regulating the acetylation level of a substrate protein, so that diseases or disorders associated with metabolism can be improved, or relieved or the growth of pathogenic bacteria can be inhibited, and the target product expression level of engineering bacteria is improved.

Description

Protein lysine acetylation modification is to metabolic regulation and control and application thereof

Technical field

The application relates to biological technical field.Specifically, the application relates to by protein (as enzyme) lysine acetylation modification metabolic regulation and control and application thereof.

Background technology

Acetylation modification is the ε-NH that acetyl group is added to the protein interior lysine residue by acetyltransferase (Acetyltransfearases) ₂On, (Deacetylases) removes acetyl group by deacetylase, is a kind of reversible dynamic modification, changed protein active (Yangand Seto 2007) by changing by the electric charge of modifying protein and space structure.From 1958 for the first time since finding acetylation modification on the histone; nearly there are 200 kinds of protein be found and experiment confirm has acetylation modification; comprise many important transcription regulatory factors; as p53, RB, NF-κ B, E2F1 and HIF-1 α etc.; thereby acetylation modification participation regulating cell apoptosis, Subcellular Localization, DNA and numerous important vital movements such as protein interaction, dna replication dna and reparation, DNA transcriptional activity and protein stability, acetylation modification is considered to a kind of important post translational modification gradually and is widely studied.

In recent years; people adopt new technique and Study of New Method Hepar Mus line grain (Kim SC such as mass spectrum and immunoprecipitation; S.R.et al.2006) and the substrate protein of human liver tissue acetylation modification; screening obtains a series of acetylation substrate protein; find that acetylation modification is closely related with the activity regulation of the various metabolic enzymeses in the organism, and test has all proved this point in vitro and in vivo.Equally, in prokaryote, also find acetylation modification active closely related with metabolic enzymes.

Eukaryotic acetylase and deacetylase

The acetylation of eukaryotic protein is found in the histone of yeast (Yeast) isotype biology at first.Acetylizad biological function is confirmed as regulating the structure of histone at first and then influences transcribing of related gene.The regulatory enzyme that participates in Mechanisms of Histone Acetylation Modification be divided into acetyltransferase (Histone acetyltransferase, HATs) and histon deacetylase (HDAC) (Histone Deacetylase, HDACs).Wherein HATs comprises P300, CBP, GCN5 etc.Known HDACs has multiple, in regulating the acetylizad process of groups of cells albumen different functions is arranged.The common trait of HDACs is to be subjected to a class inhibitor TSA (Tricostatin A) and the micromolecular inhibition of analog thereof.

Acetylation was found the albumen of also modifying outside the histone afterwards.P53 is one of found at first nonhistones transcription regulatory factor that is subjected to the acetylation regulation and control.Other a lot of transcription regulatory factor activity of finding were successively regulated and control by acetylation modification also afterwards.The same with histone, transcription regulatory factor also is a nuclear protein, and their acetylation and deacetylation also are the regulation and control that are subjected to HATs and HDACs.

It is found that in to zymic metabolism research process zymic metabolism also is subjected to the regulation and control of acetylation modification.The acetylase that participates in the yeast acetylation modification comprises GCN5 etc., and its deacetylase then is a class and the diverse enzyme of HDACs, and wherein foremost deacetylase is Sir2.The deacylation process that Sir2 participates in needs NAD ⁺With the participation of S-acetyl-coenzyme-A (Acetyl-CoA), Zn ²⁺The ion prothetic group that also is absolutely necessary.The inhibitor of inhibition of histone deacetylases such as TSA can not suppress the enzyme activity of Sir2, and the NAM of one of its deacetylation product (nicotimamide) then is generally used for suppressing the catalysis activity of Sir2.Regulate the Sir2 vigor and can obviously influence the yeast metabolism activity.

In vertebrate cells such as people Mus, find at least 7 proteic analog of Sir2, be named as SirT1-7 respectively.Although whether they all have the deacetylation enzyme activity and also are not determined fully up till now, can affirm that at least they are all relevant with the cellular energy metabolism, their enzyme activity also can both be suppressed by NAM.A lot of NAM molecular structure analog of exploitation not only can suppress SirT at present, can also have specificity to some SirT.

Regulate HATs by inhibitor or activator, HDACs and SirT vigor can be regulated the energy metabolism and the cell division of cell, are used for the treatment of medical purposes such as comprising tumor, life-saving and delaying senility.Be used for clinical SirT1 activator and comprised Resveraltol, be used for the HDACs inhibitor of oncotherapy etc. with practice.

Procaryotic acetylase and deacetylase

Acetyl-CoA synthetase (Acetyl-Coenzyme Synthetases, ACS) be first metabolic enzyme (V.J.Starai 2002) that regulated and control by acetylation modification of finding in the prokaryote, after this in prokaryote, find the chemotactic adjusting PROTEIN C heY (Barak R 2004) of Escherichia coli and chromatin protein A lba (the Stephen D.Bell 2002 of archeobacteria sulfur ore deposit sulfolobus solfataricus P2 again; Victoria L.Marsh 2005) etc. also be regulated and control by the acetylation modification of lysine.Wherein, the most thorough with ACS research to Salmonella enterica, and in Eukaryotic ACS, also obtained confirmation.

In S.enterica; the enzyme of being responsible for the ACS acetylation modification is respectively acetyltransferase Pat (V.J.Starai and Escalante-Semerena 2004) and deacetylase CobB (V.J.Starai 2002); wherein Pat is responsible for ACS is carried out acetylation modification; CobB is responsible for that it is carried out deacetylation and modifies, and this is unique known a pair of acetylation modification enzyme among the present Salmonella enterica.

People such as Starai find that the adenosine that ACS can be effectively blocked in the acetyltransferase effect of Pat turns usefulness into, and when 609 lysines of ACS were acetylation modification, the vigor of ACS just was closed, can not synthesis of acetyl-CoA.CobB then can make 609-lysine deacetylation, by the acetylation state of controlling ACS and then the vigor (V.J.Starai, Jeffrey G.Gardner et al.2005) of regulating and control it.People such as Starai think that further acetylation modification can regulate all AMP-and generate enzyme families; comprise nonribosomal peptide synzyme, luciferase, aryl-CoA and Acetyl-CoA synthetase, and infer that the acetylation modification of lysine is all ubiquitous a kind of regulatory mechanism in eucaryon and prokaryote.Aspect phenotypic assay, when ACS is modified, be in the acetylation inactivated state under the effect of acetyltransferase Pat, can not utilize acetate synthesis of acetyl-CoA, also just can not utilize acetate and can not on the acetate culture medium, grow; And the deacetylation effect of CobB can activate ACS; make it keep certain deacetylation state; recover the vigor of ACS, and then regulate and control the synthetic of acetyl-CoA, impel S.enterica can utilize acetate and propionate to form acetyl-CoA and satisfy the needs of self growing.If knock out deacetylase CobB (cobB-); at this moment just can observe CobB disappearance the S.enterica bacterial strain can not with the low concentration acetate (＜10mM) be grow on the minimal medium of the sole carbon source and the energy (V.J.Starai, Jeffrey G.Gardner et al.2005).

Stephen D.Bell etc. find when research archeobacteria sulfur ore deposit sulfolobus solfataricus P2; the conjugated protein Alba of archaeal dna exists acetylation and deacetylation two states; DNA has been transcribed regulating action; and it can be by the Sir2 homologous protein deacetylation of archeobacteria; as handling the reconstruction in vitro re-recording system, can observe and transcribe the inhibition phenomenon with the Sir2 homologous protein.Further investigation is found; Alba is acetylation on Lys16 and modifies and modulated; that acetylation modification is carried out in this site is acetyltransferase Pat; and the function of pointing out Pat is very conservative; not only in archeobacteria, exist, and in eubacteria, also have (Stephen D.Bell 2002).

People such as Barak R find that the acetylation of CheY is modified by ACS when the chemical chemotactic protein CheY of research E.coli, this acetylation modification enzyme with ACS that has been found that and Alba is all different.In the acetylation modification reaction of ACS, acetate or acetyl-CoA may be the acetylizad substrates of ACS, and find that the acetylation site of CheY concentrates near the C-terminal, mainly comprises 91,92,109,119,122 and 126 lysine.Therefore CheY with the signal protein comparing class of some eucaryons has seemingly experienced proteic phosphorylation and many acetylations equally, is that acetylizad site concentrates on specific zone (Barak R 2004).

The Rim albumen of E.coli is found and can carries out acetylation modification (Yoshikawa A, Isono Set al.1987 to ribosomal protein; Tanaka S, Matsushita Y et al.1989).Have and discover that ribosomal protein S18, S5 and L12 among the E.coli can be comprised RimI, RimJ, RimL etc. by Rim albumen acetylation modification.Other there are some researches show; in E.coli, express Eukaryotic α-1 thymosin; find its modification that is acetylation; α-1 thymosin that the E.coli that RimJ knocks out expresses does not then have acetylation modification; this has illustrated that RimJ can play the acetylation modification effect to Eukaryotic α-1 thymosin; the function that also further illustrates simultaneously acetyltransferase is relatively more conservative (Hongqing Fang, Xu Zhang et al.2009).

Summary of the invention

The inventor finds that the lysine acetylation is a kind of main modification mode for the metabolic enzyme of catalysis intermediate that is arranged in eukaryotic Cytoplasm and mitochondrion with for the metabolic enzyme in bacterial cell participation center.Experiment shows, the metabolic regulation mechanism of a kind of conservative on evolving, response nutrition amount of obtaining and metabolism state has been represented in the lysine acetylation.It is experienced energy state in the born of the same parents in time, regulates the Direction of Reaction, response speed or the stability of overall enzyme neatly, to guarantee the therefore reasonably variation of response external environment of cell.A kind of so instant, flexible, two-way, metabolic mechanism of regulating cell globally, be from the antibacterial to people on evolving conservative a kind of metabolic regulation network.

Therefore, the material that the present invention relates to regulate albumen acetylation level is used for regulating the application of the metabolic material of bio-energy in preparation.

The present invention relates to the application at the material of preparation is used for improving or alleviation is relevant with metabolism disease or imbalance of the material of regulating albumen acetylation level on the other hand.

Other optimal technical scheme of the present invention and advantage will describe in further detail hereinafter in conjunction with the accompanying drawings and embodiments.

Description of drawings

The acetylation modification of Fig. 1 S.enterica center metabolic enzymes.(A) acetylation modification of S.enterica center metabolic enzymes integral level; (B) the SILAC relative quantification result of S.enterica Chinese and Western metabolic enzymes acetylation modification.The enzyme abbreviation is as follows among the figure: AceA:Isocitrate Lyase (isocitrate lyase); AceK:IsocitrateDehydrogenase Kinase/Phosphatase (Isocitrate dehydrogenase kinases/phosphate); ACO:Aconitase (aconitase); ACS:Acetyl-CoA Synthetase (acetyl-CoA synzyme); AE:Aldose1-epimerase (aldose 1-epimerase); ALD:Fructose-bisphosphate Aldolase (fructosediphosphate aldolase); APase:Acylphosphatase (acylphosphatase); CS:Citrate Synthase (citrate synthetase); ENL:Enolase (enolase); FBPase:Fructose-1,6-bisphosphatase (fructose-1); FH:Fumarate Hydratase (fumarate hydratase); GapA:Glyceraldehyde-3-PhosphateDehydrogenase (glyceraldehyde phosphate dehydrogenase); GK:Glucose Kinase (glucokinase); GPI:Phosphohexose Isomerase (oxoisomerase); ICDH:Isocitrate Dehydrogenase (Isocitrate dehydrogenase); MDH:Malate Dehyrogenase (malic dehydrogenase); MS:Malate Synthase (malate synthetase); PDH:Pyruvate Dehydrogenase (pyruvic dehydrogenase); PEPCase:Phosphoenolpyruvate Carboxylase (phosphoenolpyruvate carboxylase); PEPCK:Phosphoenolpyruvate Carboxykinase (phosphoenolpy ruvate carboxy kinase); PFK:Phosphofructokinase (phosphofructokinase); PGK:Phosphoglycerate Kinase (phosphoglyceric kinase); PGM:Phosphoglycerate Mutase (phosphoglyceromutase); PK:Pyruvate Kinase (pyruvate kinase); PTS:Glucose-Specific Phosphotransferase Enzyme IIA Component (glucose specificity phosphotransferase IIA component); SCS:Succinyl-CoA Synthetase (succinic thiokinase); SDH:Succinate Dehydrogenase (succinate dehydrogenase); TPI:Triose Phosphate Isomerase (phosphotriose isomerase); 50S L4:ribosomal protein (ribosomal protein) 50S L4; 50S L11:ribosomal protein (ribosomal protein) 50S L11; 50S L14:ribosomal protein (ribosomal protein) 50S L146; 50S35:ribosomal protein (ribosomal protein) 50S L35.

The growth phenotype of Fig. 2 S.enterica acetyltransferase Pat knockout mutant strain (Δ pat) and deacetylase CobB knockout mutant strain (Δ cobB).(A) wt (◆), Δ pat (▲), Δ cobB (■), the growth phenotype of Δ pat/ Δ cobB (zero) on 5.0mM glucose and citric acid-based basal culture medium; (B) wild type S.enterica contains (▲) or does not have the growth curve of (●) nicotiamide at glucose (redness, upper left) and citric acid (blueness, bottom right) minimal medium; (C) S.enterica exists ¹³Internal metabolism stream on the glucose of C labelling and the citric acid-based basal culture medium.Metabolism distributions in the born of the same parents is measured with 13C labelling and GC-MS, and arrow is represented the direction of clean metabolism stream, and arrow width is big or small corresponding with metabolism stream; (D) Δ pat and Δ cobB metabolism flow ratio value result of variations.Glycolysis (is used v _GapAExpression), glyconeogenesis (is used v _PckAExpression), glyoxylate bypass (is used v _AceAExpression) and TCA circulate and (to use v _ICDHExpression) metabolism stream is all by quantitatively, and calculates v _GapA/ v _PckAAnd v _AceA/ v _ICDHMetabolism flow ratio value, each data all is average results of three independent trialss, and adds standard deviation S D.

Fig. 3 acetylation modification activates EHHADH and MDH.(A) deacetylated enzyme inhibitor improves the acetylation level of EHHADH.The EHHADH of immunoprecipitation heterogenous expression, reuse acetylation lysine antibody carry out the immunoblotting checking.(B) with iTRAQ the EHHADH acetylation modification is carried out quantitative analysis.The peptide section is quantitatively calculated according to iTRAQ label relative density.(C) deacetylated enzyme inhibitor stimulates EHHADH vigor in the born of the same parents.This experiment and subsequent experimental figure as a result derive from reproducible results 3 times.(D) fatty acid-induced EHHADH acetylation modification and vigor.EHHADH detects EHHADH acetylation modification and vigor with HEK293T cell inner expression allos.(E) MDH acetylation modification.MDH at HEK293T cell inner expression band Myc label detects acetylation modification with immunoblotting then.(F) the quantitative mass spectral analysis of MDH.At the MDH of HEK293T cell inner expression band FLAG label, detect with Fourier transform-ion cyclotron resonance mass spectrometry (FTICR) behind the immunoprecipitation.(G) glucose strengthens the MDH acetylation modification.(H) acetylation modification activates MDH.With Actin MDH acetylation modification and the vigor that confidential reference items detect the intracellular endogenous and heterogenous expression of Chang ' s and HEK293T.(I) external deacetylated processing causes the MDH inactivation.Handle the MDH that immunoprecipitation obtains with deacetylase CobB, detect the MDH enzyme activity then.Negative control is not for adding the essential cofactor nicotinamide adenine dinucleotide (NAD) of CobB.(J) glucokinase activator MDH.Experimental design is just handled cultured cells with glucose according to this figure H part.

Fig. 4 acetylation modification (A and B) causes the ASL inactivation.The ASL acetylation modification.In the HEK293T cell, express the ASL or the ASLK288R of FLAG labelling, carry out immunoblotting with acetylation lysine antibody (A) or acetylation K288 antibody (B) behind the immunoprecipitation and detect.(C) exogenous amino acid suppresses the acetylation modification of ASL.The HEK293T cell is handled with variable concentrations aminoacid, obtains ASL by immunoprecipitation then.(D) Trichostatin A and nicotiamide suppress ASL.At the HEK293T cell inner expression wild type ASL that handles through Trichostatin A and nicotiamide and mutant ASL-K288R (vigor be wild type 132%).With the whole protein is with reference to detecting the ASL vigor that immunoprecipitation obtains.(E) amino acid activation ASL depends on the acetylation modification of the 288th lysine of ASL.At HEK293T cell inner expression wild type ASL and the mutant cultivated with variable concentrations aminoacid.(F, G) glucose is to the influence of ASL acetylation modification and enzyme activity.At the HEK293T cell inner expression ASL that cultivates with the variable concentrations glucose, behind immunoprecipitation, detect acetylation modification and the enzyme activity of ASL.(H) external deacetylated modification activates ASL.External deacetylated modification system is similar to Fig. 2 I.

Fig. 5 acetylation modification destroys PEPCK1 stability.(A) glucose induction PEPCK1 acetylation modification level.(B) aminoacid reduces PEPCK1 acetylation modification level.(C) PEPCK1 content descends in the glucose induction born of the same parents.Adopt PEPCK1 content in the PEPCK1 antibody test born of the same parents.(D) Trichostatin A and nicotiamide are handled and have been reduced PEPCK1 content in the born of the same parents.(E) glucose reduces PEPCK1 stability.When experiment is initial, add cycloheximide, suppress the protein translation of HEK293T cell.Detect PEPCK1 protein abundance in the born of the same parents with immunoblotting.(F) suppress deacetylase wild type PEPCK1 unstability is increased, but inoperative to acetylation modification defective mutant.

The activity of Fig. 6 acetylation modification regulation and control center metabolic enzymes.(A) metabolism key enzyme in center is at wt, the difference of the acetylation level of expressing in Δ cobB and the Δ pat bacterial strain.The GapA that has the His label, AceA and AceK albumen cross in Δ cobB and the Δ pat bacterial strain and express and purification respectively at wt.Use every kind of albumen of equivalent and detect the level of acetylation modification.(B) and (C) carry out with Pat that acetylation is handled and CobB carries out deacetylated processing GapA afterwards, the vigor of AceA and AceK.Have His label G apA, AceA and AceK albumen are expressed in the S.enterica of wild type respectively and purification, and carry out at external use Pat that acetylation is handled or CobB carries out deacetylated processing.(B) handle glycolysis and the glyconeogenesis vigor of regulating and control GapA alternately by Pat and CobB.(C) handle the vigor of regulating and control AceA alternately and regulate and control the vigor that AceK controls ICDH by external Pat and CobB.

The specific embodiment

In view of the The selection result of people such as Kim at Mus mitochondrion acetylation substrate protein, the inventor imagines also should exist this acetylation modification phenomenon widely in the S.enterica body.The inventor attempts the isolating high-resolution of highly sensitive and mass spectrum of immunoprecipitation is combined; first the Lys acetylation substrate protein of S.enterica has been carried out scanning on the protein science level; found that 191 proteic 235 peptide sections are acetylation; it is that metabolism is relevant that 50% albumen is wherein arranged approximately; and find the modification that is acetylation of 90% center metabolism protein, also have quite a few albumen relevant with the toxicity of S.enterica.

According to previous studies; the inventor imagines Pat and CobB is present unique known a pair of acetylation modification enzyme; so made up the acetyltransferase Pat knockout mutant strain (pat null mutant) of S.enterica respectively; deacetylase CobB knockout mutant strain (cobB null mutant); the two knockout mutant strains (pat/cobB double null mutant) of acetyltransferase Pat and deacetylase CobB; respectively with the S.enterica of wild type and these bacterium that derives at the LB Nutrient medium; cultivate in glucose and the citric acid-based basal culture medium; and receive bacterium in logarithmic (log) phase mid-term and stable phase respectively; investigation is under different condition of culture, and different endobacillary acetylation modifications have variation how.Found that the lysine of S.enterica-acetylation substrate protein has almost covered all metabolic processes of S.enterica.These albumen or relevant with the center metabolism, or it is relevant with toxicity, or it is relevant with energy metabolism, or synthetic relevant with carbon skeleton, comprise that glycolysis, glyconeogenesis, tricarboxylic acid cycle, glyoxylate cycle, amino acid metabolism, ornithine cycle, electronics oxidation breathe transfer chain, carbohydrate metabolism, lipopolysaccharide biosynthesis, tRNA biosynthesis, nucleotide metabolism, purine metabolism, pyrimidine metabolic, outer membrane protein metabolism, vitamin metabolism and porphyrin metabolism etc.If the deficiency that consideration method itself exists and miss do not have the detected albumen that identifies, the scope of acetylation substrate protein will be well beyond the category of above-mentioned metabolism protein, its coverage extensively beyond imagination.

Especially; the inventor finds that the center metabolizing enzyme class overwhelming majority of S.enterica all is subjected to the acetylation modification regulation and control; albumen as TCA circular correlation has 7; the albumen that glycolysis is relevant has 9; the albumen that glyconeogenesis is relevant has 9; this is to similar with the result of study in the hepatic tissue at the Hepar Mus mitochondrion; thereby proved that further the lysine acetylation modification may be control methods all very conservative from the eukaryote to the prokaryote; and the lysine acetylation modification is a kind of control methods very widely in prokaryote S.enterica, and it may be by to the crucial enzyme " fine setting " of center metabolism and then the realization regulation and control on the integral level.

Above-mentioned test has also proposed a lot of new research directions for the following acetylizad research of inventor develops to hereditism and molecular biology depth direction; such as still only in S.enterica, finding a kind of acetyltransferase Pat and deacetylase CobB up to now; in the face of numerous acetylation substrate protein like this, the inventor has reason to believe acetyltransferase and the deacetylase that also has other in S.enterica certainly.People such as Driscoll in yeast new discovery a kind of and Sir2 be exactly a good example (RobertDriscoll 2007) without any homologous acetyltransferase; People such as Jeffrey G.Gardner find when the acetylation modification of the AcsA of research B.subtilis; the acetylation of AcsA is responsible for modification by acetyltransferase AcuA; AcuC and SrtN are responsible for that then AcsA is carried out deacetylation and keep the synthetic activity of its acetyl-CoA; wherein SrtN then needs NAD+ as coenzyme; and the performance of the activity of AcuC does not need NAD+, and does not have to find to have the homologous protein (Jeffrey G.Gardner and Escalante-Semerena 2009) of AcuC in S.enterica.In order further to confirm the acetylation modification enzyme of metabolic enzyme; the inventor has cloned other the acetylation modification enzyme SpeG that has reported again; RimI; YjgM and YncA; and external to GapA; AceA and AceK have carried out the acetylation modification processing, find that these acetylases can not carry out acetylation modification to metabolic enzymes, up to the present only have the responsible acetylation modification to metabolic enzymes of a pair of acetylation modification enzyme Pat and CobB in the S.enterica body.

According to the result, the inventor thinks that the lysine acetylation is very conservative on evolving between prokaryote S.enterica and the mammalian cell.May have this relation though early existing a lot of experimental fact shows, the inventor has proved that in prokaryote S.enterica acetylation modification is a kind of control methods that extensively exist for the first time.

Phenotype, biochemical test and quantitative RT-PCR analytical test result grow in the cumulated volume description; think that S.enterica is at different carbon sources and different growing stage; acetylation level in the born of the same parents finally is by the common transcriptional level decision of acetyltransferase Pat and deacetylase CobB, thereby has finished the conversion of different metabolic path under different carbon source conditions.Though not clear to the concrete mechanism that Pat and CobB transcribe, the ratio of the level of acetyl-CoA and acetyl-CoA/CoA-SH may be to regulate carbon source and important signal of energy metabolism in the born of the same parents.

One aspect of the invention provides the material of regulating albumen acetylation level to be used for regulating the application of the metabolic material of bio-energy in preparation.

In a preferred embodiment, the material of described regulatory enzyme acetylation level is selected from acetylase or deacetylase, enhancing or suppresses material, the enhancing of acetylase or deacetylase genetic transcription, translation or expression or suppress the material of the enzymatic activity of acetylase or deacetylase.In a preferred embodiment, described acetylase or deacetylase are selected from lysine acetyltransferase, rely on deacetylase, histone deacetylase or the SIRT family deacetylase of NAD.

" strengthen or suppress certain gene and transcribe, translate or express " is technology well known by persons skilled in the art.For example, can adopt the antisense sequences of coding acetylase or deacetylase gene to realize Antisense Suppression.Length for antisensenucleic acids has no particular limits, but the segmental length of antisensenucleic acids is 10-100bp usually, preferably is 15-50bp, more preferably is 20-25bp.Used antisensenucleic acids fragment can be one or more antisensenucleic acids fragments, if use multiple antisensenucleic acids fragment, can not have the zone that overlaps between each antisensenucleic acids fragment, also can have the zone that overlaps.The another kind of material that suppresses acetylase or deacetylase genetic transcription and/or translation can be (little) RNA interfering (siRNA) of gene.It also is a kind of effective ways that seal gene expression that development in recent years is got up that RNA disturbs (RNA interference).The siRNA transfection that it adopts and the homologous 21-23 nucleotide of genes of interest is long is to target cell.The interferential mechanism of RNA comprises two steps---initial step and effect step.In initial step, the dsRNA of importing is digested to the siRNA of 21-23nt size by the RNaseIII family member of specific recognition dsRNA, and the 3 ' end of each siRNA all has 2～3 outstanding nucleotide, 5 ' end band phosphate group.In the effect step, siRNA and nuclease complex (comprise exonuclease, Cobra venom endonuclease, unwindase and class RecA albumen etc.) in conjunction with forming the inductive reticent complex of RNA (RNA induced silence complex, RISC), thereby RISC unwinds into strand with siRNA and activates RISC under the situation that ATP exists, the RISC after the activation degrades mRNA then by the mRNA of base pairing specific recognition genes of interest.SiRNA can be prepared into the form of double-strandednucleic acid, and it contains a positive-sense strand and an antisense strand, and these two chains only form double-stranded under the condition of hybridization.A double-stranded RNA complex can be prepared by positive-sense strand that is separated from each other and antisense strand.Therefore, by way of example, complementary positive-sense strand and antisense strand are chemosynthesis, can produce synthetic double-stranded RNA complex by annealing hybridization thereafter.In addition, the modulin or the cis acting site sequence of regulation and control acetylase or deacetylated expression of enzymes are also contained in the scope of the present invention.

Those skilled in the art also can adopt the material that strengthens or suppress the enzymatic activity of acetylase or deacetylase to realize regulation and control to energy metabolism.These materials are well known by persons skilled in the art, for example histone deacetylase inhibitor Trichostatin A (TSA) or SIRT family deacetylase inhibitors nicotiamide (NAM), or those skilled in the art can determine according to conventional screening experiment.This screening experiment comprises: acetylase or deacetylase are contacted with candidate substances; measure the activity of acetylase or deacetylase then; raise or downward modulation if the enzymatic activity of acetylase or deacetylase has, determine that then this material standed for is for strengthening or suppress the reagent of acetylase or deacetylase enzymatic activity.

In a preferred embodiment, albumen is involved in sugar zymolysis, glyconeogenesis, tricarboxylic acid cycle, ornithine cycle, fatty acid metabolism, the metabolic enzyme of glycogen.In preferred scheme, described enzyme is selected from glyceraldehyde phosphate dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, malic dehydrogenase, argininosuccinase, sour phosphorus enol acetone acid carboxylic kinases, isocitrate lyase or Isocitrate dehydrogenase kinases/phosphate.

The present invention is not only applicable to prokaryote, is applicable to eukaryote yet.In preferred embodiments, biology of the present invention is an antibacterial, for example Salmonella.In another preferred embodiment, biology of the present invention is preferably mammal, comprises people, domestic animal and farm-animals, non-human primates and zoo, sports ground animal, or house pet, as Canis familiaris L., horse, cat, milch cow etc.

In all these acetylation substrate proteins of finding, have a lot relevantly with metabolism, therefore, just can realize the metabolic regulation and control of pair cell by the acetylation level of adjusting substrate protein, and then improvement or alleviation disease or the imbalance relevant with metabolism.

The disease or the imbalance that can improve or alleviate by the present invention for example comprise: not controlled cell proliferative disease such as cancer (because known chronic inflammatory reaction be not a factor of controlled cell proliferative disease), for example including, but not limited to, cancer, lymphoma, germinoblastoma, sarcoma and leukemia.More specific cancer example comprises squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer, the intestines and stomach cancer, pancreas cancer, glioblastoma, cervical cancer, ovarian cancer, hepatocarcinoma, bladder cancer, hepatoma, mastocarcinoma, colon cancer, colorectal carcinoma, carcinoma of endometrium, salivary-gland carcinoma, renal carcinoma, renal carcinoma, carcinoma of prostate, carcinoma vulvae, thyroid carcinoma, hepatocarcinoma and various types of head and neck cancer.

Carbohydate metabolism that can improve or alleviate by the present invention and/or relevant imbalance or the disease of lipid metabolism, for example including, but not limited to, type i diabetes, type ii diabetes, improper glucose tolerance, insulin resistance, hyperglycemia, hyperlipemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia etc.

Can suppress the growth or the dormancy of pathogenic microorganism by the present invention by the metabolism that suppresses pathogenic microorganism, described pathogenic microorganism can be an infectious bacteria in the born of the same parents such as the outer infectious bacteria of born of the same parents such as escherichia coli, leptospira, staphylococcus or tubercule bacillus, Salmonella, Lyme Salmonella, Listerella for example, specifically comprise, but be not limited to staphylococcus (aurococcus, staphylococcus epidermidis) and streptococcus (streptococcus agalactiae, streptococcus faecalis, streptococcus pneumoniae, streptococcus pyogenes); Gram-negative coccus (gonorrhea diplococcus) and gram negative bacilli such as enterobacteria, for example escherichia coli, hemophilus influenza, Salmonella and Shigella; Klebsiella (pneumobacillus), Enterobacter (clostridium perfringen, enterobacter agglomerans), emplastic serratia, Proteus (proteus mirabilis, Proteus rettgeri, proteus vulgaris), this bacillus of Providian, yersinia, acinetobacter calcoaceticus, Rhodopseudomonas (Pseudomonas aeruginosa, Pseudomonas Maltophilia), bacteroides fragilis, peptostreptococcus, clostruidium, Mycoplasma, mycobacteria (mycobacterium tuberculosis), the Lyme Salmonella, the Listerella, leptospira etc.

Can improve laboratory or industrial strain by the metabolism of regulating microorganism by the present invention, for example including, but not limited to antibacterials such as escherichia coli, actinomycetes, bacillus subtilis; Industrial funguses such as aspergillus niger, beer yeast, methanol yeast.

Below in conjunction with concrete experiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Unless description is arranged in addition, enforcement of the present invention will be adopted molecular biology, microbiology, recombinant DNA and immunologic routine techniques, and these all are known to those skilled in the art.These technology have complete description in following document: for example, and Sambrook " molecular cloning experiment guide " the 2nd edition (1989); " dna clone " I and II volume (D.N.Glover edits 1985); " oligonucleotide is synthetic " (M.J.Gait edits, 1984); " nucleic acid hybridization " (B.D.Hames and S.J.Higgins edit .1984); " protein purification: principle and put into practice " the 2nd edition (Springer-Verlag, N.Y.), and " experiment immunization is learned handbook I-IV volume (D.C.Weir and C.C.Blackwell edit 1986).Perhaps, can carry out according to the description that the reagent manufacturer is provided.

One. with the people liver is Study of model work

Utilize mass-spectrometric technique to identify more than 1,300 acetylation small peptide, and corresponding with 1047 different human proteins, do not find acetylizad 703 albumen (experiment 1.1) comprising reporting as yet in the past.Further analyze and find, nearly all enzyme of involved in sugar zymolysis (glycolysis) in the human liver tissue, glyconeogenesis (gluconeogenesis), tricarboxylic acid cycle (tricarboxylic acid (TCA) cycle), ornithine cycle (urea cycle), fatty acid metabolism (fatty acidmetabolism) and glycogen metabolism (glycogen metabolism) all is (experiment 1.2) that is acetylation.And then identified that wherein four approach (fatty acid oxidation, tricarboxylic acid cycle, ornithine cycle and glyconeogenesis) go up each representational enzyme (third part is seen in experiment).Wherein to having that these deacetylase proteinizations play a major role: histone deacetylase (histone deacetylase HDAC I and II) (inhibitor trichostatin A; TSA) and SIRT family deacetylase (SIRT family deacetylases) (inhibitor nicotiamide, NAM) (experiment 1.3).Inventor and then discovery, the acetylation state of the concentration affects metabolic enzyme of metabolism fuel such as glucose, aminoacid and fatty acid (experiment 1.4).

Experiment 1.1: people's liver protein " acetylation group "

In order to study non-nucleoprotein acetylation modification, the inventor has carried out the subcellular fractionation separation to human liver tissue, has obtained nucleus, mitochondrion and three kinds of components of Cytoplasm.Mitochondrion and cytoplasm protein are used trypsin treatment earlier, carry out enrichment and purification with acetylation lysine antibody then, obtain the acetylated peptide section.Subsequently, utilize multi-dimensional chromatograph-tandem mass spectrum connection technology (LC/LC-MS/MS) to identify that the result has identified more than 1300 acetylated peptide section, these peptide sections have been represented 1047 different albumen, and wherein 703 albumen are found the acetylation modification phenomenon first.People such as Kim have identified 195 acetylated proteins in mouse liver, wherein 135 (70%) are also confirmed by inventor's experiment, illustrate that inventor's the coverage rate of protein group experiment is higher.Choudhary etc. have found 1750 acetylizad albumen in human leukemia cell line, but have only in 240 mass spectrometric datas that appear at the inventor.The comparative result of these three groups of data show the albumen acetylation modification in the liver of people and mice comparing class seemingly, but difference is more remarkable in liver and leukaemia.

Experiment 1.2: the enzyme overwhelming majority that participates in intermediate supersession in people's orgotein is acetylation

Acetylated protein and liver holoprotein group have been compared in this experiment, the modification that is acetylation of the pheron of finding more participation intermediate supersession.In fact, all there is acetylation modification in all enzymes of glycolysis, glyconeogenesis, tricarboxylic acid cycle, ornithine cycle, fatty acid metabolism and glycogen metabolism approach.This may be owing to have only the modification (80 cytoribosome albumen have only 7 modifications that are acetylation) that is acetylation of a spot of ribosomal protein; reduced the abundance of ribosomal protein in total acetylation modification albumen, made that other acetylation modification albumen can be identified.These results show not only ubiquity in cell of acetylation modification that the inventor finds, and regulating action is very widely arranged in cellular metabolism probably.Therefore the inventor has studied the acetylation modification effect of some key enzymes on four metabolic pathways.

Experiment 1.3: people liver histone deacetylase histone deacetylase HDAC I and II (inhibitor trichostatin A; TSA) and the deacetylase SIRT family deacetylases of SIRT family (the inhibitor nicotiamide is NAM) to the deacetylated effect of metabolic enzyme

Acyl coenzyme A hydrase/3-hydroxyacyl-CoA dehydrogenase (crotonase, enoyl-Coenzyme Ahydratase/3-hydroxyacyl-Coenzyme A dehydrogenase, EHHADH, EC 1.1.1.35) is acetylation modification.Personnel selection liver histone deacetylase HDAC I and II inhibitor Trichostatin A (trichostatinA; TSA) and the deacetylated enzyme inhibitor nicotiamide (nicotinamide of SIRT family; NAM) behind the processing cell, the acetylation level of EHHADH has increased by 80% (Fig. 3 A).

Trichostatin A and nicotiamide processing can make the 171st the lysine acetylation level of EHHADH be increased to 62%, the 346 lysine acetylation level from 43.5% and be increased to 77.8% (Fig. 3 B) from 46.8%.Corresponding non-acetylated peptide section is owing to the processing of Trichostatin A and nicotiamide reduces simultaneously.

(the acetylation modification lysine residue of four suppositions all is replaced by glutamine to the EHHADH mutant, and acetylation modification level EHHADH4KQ) reduces, and vigor no longer is subjected to the adjusting (Fig. 3 C) of Trichostatin A and nicotiamide.

In addition; Trichostatin A and nicotiamide are handled cell can make malic dehydrogenase (malate dehydrogenase; MDH, acetylation level EC1.1.1.37) is increased to 67.4% from 26.9%, and occurs triple and quadruple acetylation modification phenomenon (Fig. 3 E and 3F).

In the HEK293T cell, Trichostatin A and nicotiamide are handled can activate wild type MDH, but can not activated mutant body (four acetylation modification lysine residues all are replaced by arginine) (Fig. 3 H).

Immunoblotting assay demonstration Trichostatin A and nicotiamide processing make smart ammonia (base) succinic acid lyases, and (EC4.3.2.1) the 288th lysine acetylation level increased by 2.8 times (Fig. 4 B) for argininosuccinate lyase, ASL.Add aminoacid in the culture medium and reduced overall acetylation level of ASL and the 288th lysine acetylation level (Fig. 4 C).Trichostatin A and nicotiamide are handled and are caused the ASL vigor to descend, and add aminoacid its vigor are risen, and show that acetylation modification has suppressed the vigor of ASL (Fig. 4 D and Fig. 4 E).ASL mutant (the 288th lysine residue replaces to arginine) does not then have similar phenomenon (Fig. 4 D and Fig. 4 E).

Trichostatin A and nicotiamide handle HEK293T and the Chang hepatocyte causes sour phosphorus enol acetone acid carboxylic kinases (phosphoenolpyruvate carboxykinase, PEPCK1, EC4.1.1.32) content decline 70% (Fig. 5).

Even do not containing dextrose culture-medium or adding under the aminoacid condition, Trichostatin A and nicotiamide are handled all can cause the PEPCK1 loss of stability.In addition, Trichostatin A and nicotiamide are handled the stability that can not change mutant PEPCK13KR (3 lysine residues all are mutated into arginine).

Experiment 1.4: the relation of metabolic enzyme acetylation and metabolism fuel in the human liver cell

Adding fatty acid in the culture medium can make the acetylation level of EHHADH and vigor increase by 1.7 times and 1.3 times (Fig. 3 D) respectively.Therefore, the acetylation level of EHHADH can be illustrated that acetylation modification has the effect of regulating EHHADH and fatty acid metabolism aspect physiology by the extracellular fuel adjusting.

The glucose of high concentration improves about 60% (Fig. 3 G) of acetylation level of MDH in the cell.

Therefore exogenous amino acid may activate ASL by the deacetylated modification of lysine residue to the 288th.

The inventor has detected the influence of glucose to ASL vigor and acetylation level, finds that glucose makes ASL acetylation level increase by 2.7 times (Fig. 4 F), makes its vigor reduce by 50% (Fig. 4 G) simultaneously.

ASL is subjected to aminoacid and glucose to regulate phenomenon jointly and illustrates that acetylation modification has important effect in different metabolic approach coordination process.

The glucose of high concentration improves cell PEPCK1 acetylation level (Fig. 5 A), and interpolation aminoacid then reduces PEPCK1 acetylation level (Fig. 5 B) in the dextrose culture-medium but do not contain.

PEPCK1 is more stable in the cell that does not contain the dextrose culture-medium growth, but in high glucose medium unstable (Fig. 5 E).

Two. with the Salmonella typhimurium is Study of model work

Utilize mass-spectrometric technique to identify 235 acetylation small peptides; and the Salmonella albumen different with 191 corresponding (table 2 is only wanted declaratives) inventor's result shows that the center metabolic enzyme of Salmonella typhimurium is by acetylation widely (90%) ([experiment 2.1]).Further physiology and genetic research proof is in Salmonella; what the center metabolic enzyme was played acetylation and deacetylated modification is the deacetylase (CobB) [experiment 2.2] that a pair of acetylase (Pat) and NAD rely on; its physiological function is the adaptation [experiment 2.3] of coordinated regulation antibacterial to different carbon source conversions, and the result of these regulation and control obtains the evidence [experiment 2.4] of cellular metabolism stream experiment.In a word; respond different carbon source (fermented type carbon source glucose through zymolysis (glycolysis) with respect to oxidized form carbon source citric acid through glyconeogenesis (gluconeogenesis)); the state of the metabolism of center metabolite stream in the acetylation of bacterial cell central metabolic pathway enzyme, the growth of cell and the cell, this three shows some consistent differences again.Further also explanation of experiment, transcribing of Pat and CobB have different characteristic (Pattern) [experiment 2.5] on different carbon sources, illustrate that its expression might be regulated and control by the difference of carbon source.

For obtaining the positive evidence of acetylation regulation and control, the inventor has further identified the key enzyme (glyceraldehyde phosphate dehydrogenase-glyceraldehyde phosphate dehydrogenase (GapA)) of wherein controlling glycolysis and glyconeogenesis direction and two key enzymes (isocitrate lyase-isocitrate lyase (AceA) and Isocitrate dehydrogenase kinases/phosphate-isocitrate dehydrogenase (ICDH) kinase/phosphatase (AceK)) that determine tricarboxylic acid cycle and glyoxylate bypass bifurcation direction.

In a word, the reversible acetylation of metabolic enzyme has guaranteed the energy state that cell can be by receptor cell inside in time and has changed response speed neatly or variation that direction comes the response external environment.It has represented a kind of metabolic regulation mechanism of all guarding from the antibacterial to the mankind.

Test the protein " acetylation group " of 2.1 Salmonellas under growth conditions on the different carbon sources:

The lysine acetylation modification is to metabolic whole Regulation Mechanism in the prokaryote in order to study, and the inventor has detected the total acetylation state of S.enterica albumen respectively under fermentable carbon source glucose and two kinds of growth conditions of non-fermentable carbon source citric acid.The peptide section of utilizing acetylation lysine specific antibody to come immunoprecipitation to separate to be acetylation and to modify, and in conjunction with high-resolution mass-spectrometric technique identifies among the S.enterica modification that is acetylation of 191 proteic 235 peptide sections altogether.It is that various metabolism are relevant that 50% acetylated protein is wherein arranged, and finds that the enzyme of center metabolism 90% or more is be acetylation modification (Figure 1A).Utilize the SILAC quantitative analysis tech to find that change has taken place 15 center metabolizing enzymes their acetylation state when the different carbon source of response; these results show that it is carbon source (Figure 1B) that S.enterica its acetylation level when being carbon source with the glucose will be higher than with the citric acid, and this point is consistent with the acetylated peptide section result that inventor's count detection arrives.

Testing 2.2 physiology and genetic experiment proof Pat and CobB is a pair of enzyme of being responsible in the Salmonella metabolic enzyme acetylation and deacetylated modification:

When being carbon source with the glucose; S.enterica with respect to wild type; the acetylation level of Δ pat (acetyltransferase knockout mutant strain) bacterial strain center metabolic enzymes reduces, and the acetylation level of Δ cobB (deacetylase knockout mutant strain) bacterial strain center metabolic enzymes then is (Figure 1B) that rises.When being carbon source with the citric acid, the acetylation level of Δ cobB bacterial strain center metabolic enzymes is also than wild type height (Figure 1B).On the other hand; the acetylation level of the ribosomal protein of Δ cobB and Δ pat bacterial strain does not then change, and this has supported a notion---and Pat and CobB are the responsible a pair of main modification enzymes that the center metabolic enzyme of S.enterica is carried out reversible acetylation and deacetylated modification.

Test the physiological function of 2.3 Pat and CobB: the conversion-growth experiment of accommodate carbon source

By comparing wild type, Δ pat and the different growth conditions of Δ cobB bacterial strain under different carbon sources have disclosed the physiological function (Fig. 2) of acetylation modification enzyme when regulating cell adapts to different carbon source.In the glucosyl group basal culture medium; the growth of Δ cobB bacterial strain (high acetylation level) is faster than wild type; in the citric acid-based basal culture medium but than wild type look slowly (Fig. 2 A); and Δ pat bacterial strain (low acetylation level) has opposite growth conditions (Fig. 2 A); the growth phenotype of Δ pat/ Δ cobB double-mutant strain is more similar to Δ pat bacterial strain, and this has illustrated that deacetylase CobB can not modify (Fig. 2 A) to not carried out deacetylation by the target protein of Pat acetylation modification.Inhibitor (6) NAM (nicotine) with deacetylase CobB handles wild-type strain; this is similar to deacetylated enzyme mutant; find that the growth inhibited of bacterial strain on the citric acid-based basal culture medium is apparent in view; growth on the glucosyl group basal culture medium is subjected to suppress then to want much slight (Fig. 2 B), this conclusion consistent (Fig. 1) when S.enterica acetylation level of internal metabolism enzyme when glucose is carbon source will be higher than citric acid and is carbon source.

Test the physiological function of 2.4 Pat and CobB: the conversion of accommodate carbon source-metabolism stream experiment

The inventor with ¹³The glucose of C labelling or citric acid are carbon source, have detected Pat and the CobB physiological function to the intravital metabolism distributions regulation and control of S.enterica in conjunction with the GC-MS technology.The inventor finds that S.enterica has different metabolism distributions patterns when glucose and citric acid are carbon source.When citric acid was carbon source, glyoxylate bypass was activated, and the glyconeogenesis approach is more active.The inventor also Quantitative Comparison wild type, the glycolysis (vGapA) of Δ pat and Δ cobB bacterial strain, glyconeogenesis (vPckA), tricarboxylic acid cycle (vICDH) is estimated different metabolism stream and acetylation state with the metabolism stream of glyoxylate bypass (vAceA).When discovery is carbon source at glucose, the glycolysis of Δ cobB bacterial strain/glyconeogenesis metabolism flow ratio value exceeds 2.07 times (Fig. 2 D) than Δ pat bacterial strain, and the metabolism flow ratio value of the glycolysis/glyconeogenesis of wild-type strain is lower by 47% than Δ cobB bacterial strain, but than Δ pat bacterial strain high by 40.7% (Fig. 2 D).With the citric acid is carbon source, the metabolism flow ratio value of the glyoxylate bypass/tricarboxylic acid cycle of Δ pat bacterial strain exceeds 2.21 times (Fig. 2 D) than Δ cobB bacterial strain, and the metabolism flow ratio value of the glyoxylate bypass/tricarboxylic acid cycle of wild-type strain is lower by 55% than Δ pat bacterial strain, but than Δ cobB bacterial strain high by 43% (Fig. 2 D).These results add that above-mentioned glucose has illustrated that to the result (Fig 1) of center metabolic enzymes acetylation level affects the relevant acetylation modification of carbon source can influence the vigor of various metabolic enzymes, and then regulation and control metabolism distributions.

Test transcribing of 2.5 Pat and CobB and on different carbon sources, have different characteristic (Pattern)

The inventor has also studied the expression of pat and cobB under different carbon source situations.By real-time reverse transcriptional PCR analysis, be confidential reference items with 16S rRNA, the inventor finds pat and two genes of cobB no matter be that they are transcribed than other growth period active (Fig 4A) logarithmic (log) phase when glucose or citric acid are carbon source.Equally, the protein level of Pat and CobB logarithmic (log) phase also be rise (Fig. 4 B, fig.S6).Under the situation of stable phase, the ratio of the pat mRNA/cobB mRNA of S.enterica when glucose is carbon source with citric acid is similar.When the bacterium that rich medium is cultivated changes the glucosyl group basal culture medium over to; the acetylation level reaches peak (Fig. 4 A) at mid-log phase; and when changing the bacterium that rich medium is cultivated over to the citric acid-based basal culture medium; acetylation logarithm early stage and early logarithmic (log) phase all descends rise and reach stable (Fig. 4 A) gradually then in logarithm later stage and stable phase.This illustrates that it is favourable that low-level acetylation pair cell efficiently utilizes citric acid.Though the transcriptional control mechanism of its mechanism Pat and CobB treats that still the inventor probes into, these results show, protein expression by varying level of Pat and the CobB vigor of total cell (or with respect to) responds different carbon sources, with conversion different metabolic path.

Three. the effect in metabolic regulation of acetylase and deacetylase and the functional verification of expression regulation of himself and acetylation target spot enzyme

Previous experiments has proved for the reversible acetylation of middle (center) metabolic enzyme and has been finished by acetylase and deacetylase; therefore they also just might become the metabolic important manipulation target spot of control, comprise modulin or the cis acting site sequence of regulating and control these expression of enzymes.The inhibitor of these enzymes is existing public reagent, still, has also illustrated to be special role, can design special inhibitor.

The acetylation relevant enzymes:

Lysine acetyltransferase (Pat)

Rely on the deacetylase (CobB) of NAD: Sir2 family enzyme.

Histone deacetylase HDAC I﹠amp; II (inhibitor trichostatin A, TSA)-histone deacetylase

SIRT family deacetylases (the inhibitor nicotiamide, NAM)-SIRT family deacetylase

2. target spot enzyme (be acetylation and by deacetylated metabolic enzyme) and acetylation site, they directly play a role in metabolism:

1). three important enzymes of antibacterial:

Antibacterial glycolysis/glyconeogenesis: [experiment 3.2.1.1]

Glyceraldehyde phosphate dehydrogenase (GapA)-glyceraldehyde phosphate dehydrogenase (K331), the enzyme reaction direction is adjusted in acetylation: glycolysis, deacetylated reinforcement glyconeogenesis are strengthened in acetylation.

The inventor expresses GapA and purification in Salmonella pat, cobB mutant strain, and carry out immunoblotting at external use lysine acetylation modification polyclonal antibody and detect, and with the destination protein of expressing in the Salmonella wild-type strain in contrast.The result shows that the acetylation level of GapA in the cobB mutant strain is the highest, wild type secondly, minimum in the pat mutant.

The GapA albumen that utilization is expressed in the Salmonella wild-type strain; the inventor carries out acetylation and deacetylation processing with acetyltransferase Pat and deacetylase CobB respectively external, to investigate the variation owing to the caused enzyme activity of acetylation modification degree change.The result shows that when improving the acetylation level of GapA with Pat, the enzymatic activity of the glycolysis direction of GapA has increased by 100%, and the enzymatic activity of glyconeogenesis direction has reduced by 30%.After with CobB GapA being carried out deacetylation, the enzymatic activity of the glyconeogenesis direction of GapA has increased by 30%, and the enzymatic activity of glycolysis direction has reduced by 27%.

In addition, the inventor carries out point mutation in conjunction with mass spectral result to the acetylation site that GapA is possible, and investigates the influence to enzyme activity.Consider the cause of space structure; because the space structure of glutamine Q is similar to acetylation modification when Mass Spectrometer Method; so the inventor is Q with lysine K site mutation; the acetylation modification of simulated albumin in vivo; and when the K site mutation is arginine R, then this site modification that can not be acetylation.The result shows, when the K331 of GapA site mutation is Q during with the simulation acetylation modification, the enzyme activity of the glycolysis direction of GapA rises.

Antibacterial glyconeogenesis: [experiment 3.2.1.2]

Isocitrate lyase (AceA)-isocitrate lyase (K308) acetylation suppresses the glyoxalic acid approach

The inventor expresses AceA and purification in Salmonella pat, cobB mutant strain, and carry out immunoblotting at external use lysine acetylation modification polyclonal antibody and detect, and with the destination protein of expressing in the Salmonella wild-type strain in contrast.The result shows that the acetylation level of AceA in the cobB mutant strain is the highest, wild type secondly, minimum in the pat mutant.And, comparing the situation that thalline is grown on glucose or citric acid culture medium, the former AceA acetylation level is than the latter's height.

The AceA albumen that utilization is expressed in the Salmonella wild-type strain; the inventor carries out acetylation and deacetylation processing with acetyltransferase Pat and deacetylase CobB respectively external, to investigate the variation owing to the caused enzyme activity of acetylation modification degree change.The result shows that when improving the acetylation level of AceA with Pat, the enzymatic activity of AceA has reduced by 30%.After with CobB AceA being carried out deacetylation, the enzymatic activity of AceA has increased by 24%.

In addition, the inventor carries out point mutation in conjunction with mass spectral result to the acetylation site that AceA is possible, and investigates the influence to enzyme activity.Examine the result and show,, cause the enzyme activity of AceA to descend when the K308 of AceA site mutation is Q during with the simulation acetylation modification.

Antibacterial tricarboxylic acid cycle: [experiment 3.2.1.3]

Isocitrate dehydrogenase (ICDH) kinase/phosphatase (AceK)-Isocitrate dehydrogenase kinases/phosphate (K3) acetylation suppresses kinases, activates ICDH

The inventor expresses AceK and purification in Salmonella pat, cobB mutant strain, and carry out immunoblotting at external use lysine acetylation modification polyclonal antibody and detect, and with the destination protein of expressing in the Salmonella wild-type strain in contrast.The result shows that the acetylation level of AceK in the cobB mutant strain is the highest, wild type secondly, minimum in the pat mutant.And when thalline was grown on glucose or citric acid culture medium, the former AceK acetylation level was than the latter's height.

The AceK albumen that utilization is expressed in the Salmonella wild-type strain; the inventor carries out acetylation and deacetylation processing with acetyltransferase Pat and deacetylase CobB respectively external, to investigate the variation owing to the caused enzyme activity of acetylation modification degree change.The enzyme activity of AceK measures the IDH inactivation by it to the phosphorylation of Isocitrate dehydrogenase IDH, and IDH is a key enzyme in the TCA circulation, and the catalysis 1-Hydroxy-1,2,3-propanetricarboxylic acid. generates the alpha ketoglutaric acid.The result shows that when improving the acetylation level of AceK with Pat, AceK descends 60% to the deactivation of IDH.And AceK is carried out deacetylation with CobB, strengthened the deactivation of AceK to IDH.

2). four important enzymes of people liver:

Fatty acid oxidation:

Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (crotonase) (K165,171,346,584) acetylation activates

[experiment 3.2.2.1]

Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (crotonase; enoyl-Coenzyme Ahydratase/3-hydroxyacyl-Coenzyme A dehydrogenase; EHHADH; EC 1.1.1.35) 2 step reaction of catalysis in the fatty acid oxidation process, the defective of this enzyme causes fatty acid metabolism unusual.The inventor has found the modification that is acetylation of 4 lysine residues in EHHADH, behind the band FLAG label E HHADH of immunoprecipitation cell inner expression, utilize immunoblotting to verify this 4 acetylation modification sites.Personnel selection liver histone deacetylase HDAC I and II inhibitor Trichostatin A (trichostatin A; TSA) and the deacetylated enzyme inhibitor nicotiamide (nicotinamide of SIRT family; NAM) behind the processing cell, the acetylation level of EHHADH has increased by 80% (Fig. 3 A).In order to reaffirm the acetylation modification of EHHADH, the inventor uses the EHHADH that isotope label technique binding immunoassay intermediate processing has obtained expression in the born of the same parents instead, and has carried out relative quantification and absolute quantitation analysis by mass spectrum (iTRAQ).The result shows that Trichostatin A and nicotiamide processing can make the 171st the lysine acetylation level of EHHADH be increased to 62%, the 346 lysine acetylation level from 43.5% and be increased to 77.8% (Fig. 3 B) from 46.8%.Corresponding non-acetylated peptide section is owing to the processing of Trichostatin A and nicotiamide reduces simultaneously.These results show that there is acetylation modification in the EHHADH of considerable part, and this kind modification is dynamic change in vivo.

In order to study the relation of EHHADH acetylation modification and its enzyme activity, the inventor handles the Chang hepatocyte with Trichostatin A and nicotiamide, finds that the vigor of EHHADH in the born of the same parents has increased by 2 times (Fig. 3 C).Use the HEK293T cell instead and also obtained similar result (Fig. 3 C right side).(the acetylation modification lysine residue of four suppositions all is replaced by glutamine to the EHHADH mutant, and acetylation modification level EHHADH4KQ) reduces, and its vigor no longer is subjected to the adjusting (Fig. 3 C) of Trichostatin A and nicotiamide.Adding fatty acid in the culture medium can make the acetylation level of EHHADH and vigor increase by 1.7 times and 1.3 times of (Fig. 3 D respectively.Therefore, the acetylation level of EHHADH can be illustrated that acetylation modification has the effect of regulating EHHADH and fatty acid metabolism aspect physiology by the extracellular fuel adjusting.

Tricarboxylic acid cycle:

Malic dehydrogenase (K185, K301, K307 and K314), acetylation activates

[experiment 3.2.2.2]

The modification that all is acetylation of whole 7 enzymes in the tricarboxylic acid cycle; wherein at malic dehydrogenase (malatedehydrogenase; MDH, 4 lysine residues (the 185th, 301,307 and 314, table S2 and table 3) are arranged in EC1.1.1.37), and modification all is acetylation.The MDH that expresses external source in the cell modifications that be acetylation equally, and with causing 2.4 times (Fig. 3 E) of its acetylation level rising after Trichostatin A and the nicotiamide processing.In order to verify the acetylation modification of MDH, the inventor further analyzes MDH with Fourier transform-ion cyclotron resonance mass spectrometry (FTICR).Found that the MDH peak except the total length of unmodified also has 2 extra peaks, these two extra peak quality have increased progressively 42.01Da respectively, show that MDH exists corresponding monoacylated modification and diacetyl to modify (Fig. 3 F).Trichostatin A and nicotiamide are handled acetylation level that cell can make MDH and are increased to 67.4% from 26.9%, and occur triple and quadruple acetylation modification phenomenon.Second order ms analysis confirmation subsequently 3 in 4 acetylation modification lysine residues of MDH.These data show that acetylation modification is the topmost modification of MDH, and the modification that can be acetylation in cell of most of MDH protein molecular.

The glucose of high concentration improves about 60% (Fig. 3 G) of acetylation level of MDH in the cell.Suppress the deacetylated enzyme activity of hepatocyte, cause the interior MDH vigor of born of the same parents to increase by 2 times.In the HEK293T cell, Trichostatin A and nicotiamide are handled can activate wild type MDH, but can not activated mutant body (four acetylation modification lysine residues all are replaced by arginine) (Fig. 3 H).The MDH that handles the immunoprecipitation acquisition at external use deacetylase (CobB) causes its MDH vigor to descend (Fig. 3 I), shows that acetylation modification directly activates MDH.High concentration glucose stimulates the wild type MDH vigor of abduction delivering in endogenous and the born of the same parents in addition, but to but not effect (Fig. 3 J) of MDH mutant (MDH4KR, 4 acetylation modification lysine residues all are mutated into arginine).These experimental results show that glucokinase activator MDH partly cause is by carrying out the MDH acetylation modification.

Ornithine cycle:

Smart ammonia (base) succinic acid lyases (K69 and K288), acetylation suppresses

[experiment 3.2.2.3]

Ornithine cycle is the essential approach of detoxifcation amino acid metabolites ammonia.(argininosuccinate lyase, ASL EC4.3.2.1) undergo mutation and cause the ornithine cycle fault, and then make the generation of smart ammonia [base] succinum aciduria get muddled smart ammonia (base) succinic acid lyases.The inventor has identified 2 acetylation modification sites (the 69th and 288) of ASL, and has carried out verifying (Fig. 4 A) by expressing external source ASL in the born of the same parents.Immunoblotting assay shows that Trichostatin A and nicotiamide processing make the 288th lysine acetylation level of ASL increase by 2.8 times (Fig. 4 B).Add aminoacid in the culture medium and reduced overall acetylation level of ASL and the 288th lysine acetylation level (Fig. 4 C).Trichostatin A and nicotiamide are handled and are caused the ASL vigor to descend, and add aminoacid its vigor are risen, and show that acetylation modification has suppressed the vigor of ASL (Fig. 4 D and Fig. 4 E).ASL mutant K288R (the 288th lysine residue replaces to arginine) does not then have similar phenomenon (Fig. 4 D and Fig. 4 E).Limited proteolysis and circular dichroism spectra the analysis showed that ASL mutant K288R does not change the ASL protein structure, so exogenous amino acid may activate ASL (Fig. 4 E) by the deacetylated modification of lysine residue to the 288th of ASL.

The fumarate that ornithine cycle produces can enter tricarboxylic acid cycle and then produce power or carry out glyconeogenesis (Ref8).The inventor has detected the influence of glucose to ASL vigor and acetylation level, finds that glucose makes ASL acetylation level increase by 2.7 times (Fig. 4 F), makes its vigor reduce by 50% (Fig. 4 G) simultaneously.The ASL that external use deacetylase CobB handles mammalian cell expression can cause its vigor to raise, and shows that acetylation modification directly makes the ASL inactivation.ASL is subjected to aminoacid and glucose to regulate phenomenon jointly and illustrates that acetylation modification has important effect in different metabolic approach coordination process.When glucose supplies is abundant, the amino acid metabolism that produces for energy closes the glyconeogenesis approach and then is suppressed.Under the situation that the in liberal supply and glucose of aminoacid lacks, cell will utilize aminoacid to come produce power by strengthening the ornithine cycle approach.Cell can utilize acetylation modification to coordinate various metabolic pathways to obtain in the adaptation aspect the metabolism.

Glyconeogenesis:

Acid phosphorus enol acetone acid carboxylic kinases (K70, K71 and K594). acetylation reduces enzyme stability

[experiment 3.2.2.4]

(phosphoenolpyruvate carboxykinase, PEPCK 1, EC4.1.1.32) is the enzyme of being regulated crucial in the glyconeogenesis approach for acid phosphorus enol acetone acid carboxylic kinases.By mass spectral analysis, the inventor finds that PEPCK1 has the modification (being respectively the 70th, 71 and 594) that is acetylation of 3 lysine residues.The glucose of high concentration improves cell PEPCK1 acetylation level (Fig. 5 A), and interpolation aminoacid then reduces PEPCK1 acetylation level (Fig. 5 B) in the dextrose culture-medium but do not contain.These results show that cell may regulate the glyconeogenesis approach by regulating the PEPCK1 acetylation modification according to the situation of born of the same parents' outer fuel.

The inventor finds that the expression of endogenous PEPCK1 is suppressed by the outer high concentration glucose of born of the same parents.This external Trichostatin A and nicotiamide handle HEK293T and the Chang hepatocyte causes PEPCK1 content decline 70% (Fig. 5 D).PEPCK1 is more stable in the cell that does not contain the dextrose culture-medium growth, but in high glucose medium unstable (Fig. 5 E).Even do not containing dextrose culture-medium or adding under the aminoacid condition, Trichostatin A and nicotiamide are handled all can cause the PEPCK1 loss of stability.These results show that acetylation modification can regulate the stability of PEPCK1.The lysine residue in 3 acetylation modification sites that the inventor identifies mass spectrum respectively all is mutated into arginine (PEPCK13KR) or glutamine (PEPCK13KQ), removes or imitate acetylation modification with this.The result shows that mutant PEPCK13KR stability is higher than wild type PEPCK1, and mutant PEPCK13KQ still unstable (Fig. 5 F).In addition, Trichostatin A and nicotiamide are handled the stability that can not change mutant PEPCK13KR.

Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.It is for referencial use that this paper is all included in all publications, patent and the patent application that this paper quotes in.

List of references:

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Claims (12)

1. the material of regulating albumen acetylation level is used for regulating the application of the metabolic material of bio-energy in preparation.

2. application as claimed in claim 1; it is characterized in that the material of described regulatory enzyme acetylation level is selected from material, the enhancing of acetylase or deacetylase, enhancing or inhibition acetylase or deacetylase genetic transcription, translation or expression or suppresses the material of the enzymatic activity of acetylase or deacetylase.

3. application as claimed in claim 2 is characterized in that, described acetylase or deacetylase are selected from lysine acetyltransferase, rely on deacetylase, histone deacetylase or the SIRT family deacetylase of NAD.

4. application as claimed in claim 1 is characterized in that, described albumen is involved in sugar zymolysis, glyconeogenesis, tricarboxylic acid cycle, ornithine cycle, fatty acid metabolism, the metabolic enzyme of glycogen.

5. application as claimed in claim 4; it is characterized in that described enzyme is selected from glyceraldehyde phosphate dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, malic dehydrogenase, argininosuccinase, sour phosphorus enol acetone acid carboxylic kinases, isocitrate lyase or Isocitrate dehydrogenase kinases/phosphate.

6. application as claimed in claim 1 is characterized in that, described biology is antibacterial or mammal.

7. the material of regulating albumen acetylation level is in the application of the material of preparation is used for improving or alleviation is relevant with metabolism disease or imbalance.

8. application as claimed in claim 7 is characterized in that, described disease relevant or imbalance with metabolism be not the controlled cell proliferative disease or with carbohydate metabolism and/or relevant imbalance or the disease of lipid metabolism.

9. the material of regulating albumen acetylation level is used for suppressing the application of the material of the growth of pathogenic microorganism or dormancy in preparation.

10. application as claimed in claim 9 is characterized in that, described pathogenic microorganism is selected from infectious bacteria in outer infectious bacteria of born of the same parents or the born of the same parents.

11. the material of regulating albumen acetylation level is used to improve the application of laboratory or industrial strain.

12. application as claimed in claim 11 is characterized in that, described laboratory or industrial strain are selected from industrial antibacterial or industrial fungus.

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