CN101580871A - Method for screening new drug for lowering cholesterol based on analysis of change of NPC1L1 protein subcellular localization - Google Patents

Abstract

The invention discloses a method for screening a potential substance for lowering cholesterol, which comprises the step of: in the presence or absence of the candidate substance, screening according to difference among endocytic NPC1L1 proteins; or screening according to activity difference among proteins interacting with NPC1L1 protein; or screening according to combination difference of cholesterol and NH2 end structural domain of the NPC1L1 protein. The invention also provides a substance obtained by the method for lowering cholesterol.

Description

Based on analyzing the method that NPC1L1 protein subcellular location changes the screening new drug for lowering cholesterol

Technical field

The invention belongs to cytobiology and field of pharmacology, more specifically, the present invention relates to based on analyzing the method that NPC1L1 protein subcellular location changes the screening new drug for lowering cholesterol; Simultaneously, the present invention has identified in the NPC1L1 albumen structural domain in conjunction with cholesterol, thereby also can screen the be inhibited material of cholesterol absorption of compound in conjunction with this structural domain.

Background technology

Cholesterol is biomembranous important composition composition, also is the precursor of synthetic sterols hormone and bile acide.The absorption of hypercholesterolia can cause a lot of diseases, and wherein common and the most serious is atherosclerosis and coronary heart disease.The approach that Mammals obtains cholesterol mainly contains two: de novo synthesis and absorbing from food.The biosynthetic main molecules mechanism of cholesterol is illustrated, and still for the absorption of cholesterol, the research of cellular and molecular level is also few.

Nieman-Pick C1 Like 1 (NPC1L1) is reported in (Altmann that plays an important role in the cholesterol absorption process recently, S.W. etc. (2004), Niemann-Pick C1 Like 1 protein is critical for intestinalcholesterol absorption.Science 303,1201-1204).NPC1L1 has very high expression in the small intestine of people and mouse, in the people, liver also has very high expression level.The mouse that NPC1L1 knocks out absorbs cholesterol from food ability descends greatly.Except mediation small intestine cholesterol absorption, NPC1L1 also mediates the liver cholesterol and heavily absorbs, and liver is crossed bile and cholesterol in the ight soil decline of the transgenic mice of expression NPC1L1 than normal mouse.

People NPC1L1 albumen contains 1332 amino acid, constitute 13 TMD (Wang, J. etc. (2009), Membrane topology of human NPC1L1, a key protein in enterohepatic cholesterolabsorption.J Lipid Res, Epub ahead of print).The 3-7 section of NPC1L1 contains sterol and experiences structural domain, also contains this structural domain in the NPC1, the HMGCR that regulated by sterol and SCAP albumen.There is dispute about the proteic Subcellular Localization of NPC1L1 always.

Ezetimibe (market name Zetia) can reduce cholesterol absorption, and is used to the clinical treatment hypercholesterolemia.But the drug action mechanism of the absorption of the protein mediated cholesterol of Ezetimibe inhibition NPC1L1 is unclear always.

Therefore, although NPC1L1 albumen known in the art is relevant with the absorption of cholesterol, yet and do not know the mechanism of the cholesterol absorption that NPC1L1 is protein mediated, the research and development of related drugs is restricted.

Summary of the invention

The object of the present invention is to provide a kind of method of screening the decreasing cholesterol material.

The object of the present invention is to provide the material of the decreasing cholesterol that obtains by described screening method.

In a first aspect of the present invention, a kind of method of screening potential decreasing cholesterol material is provided, described method comprises:

(1) handle the proteic cell of expression NPC1L1 with cholesterol, and definite cell vesicle (generally being called the film bubble again) proteic degree of endocytosis NPC1L1;

(2) in the presence of candidate substances, express the proteic cell of NPC1Ll as processing as described in (1), determine the proteic degree of vesica endocytosis NPC1L1 once more; With

(3) compare proteic difference of vesica endocytosis NPC1L1 in (1) and (2);

Wherein, (preferably significantly be lower than,, preferably hang down 40% if the proteic degree of vesica endocytosis NPC1L1 is lower than statistically in (2) as low by 20%; More preferably hang down 60% or lower) (1) middle proteic degree of vesica endocytosis NPC1L1, then described candidate substances is a potential decreasing cholesterol material.

In a preference, described vesica endocytosis mainly is the endocytosis of cage modle albumen (clathrin) mediation.

In another preference, described NPC1L1 albumen contains the aminoacid sequence of 18-260 position among the GenBank accession number FJ481111, and this zone is the binding domains of cholesterol.

In another preference, in (1) or (2), the proteic cell of described expression NPC1L1 is handled through decreasing cholesterol before cholesterol is handled in advance.

In another preference, it is to instigate the amount of cell inner cholesterol to be lower than normal level that described reducing cholesterol is handled, thereby NPC1L1 albumen is transported to the cytolemma zone.

In another preference, in (1) or (2), also comprise: determine cage modle albumen and the proteic combination degree of NPC1L1 in the cell; And, in (3), also comprise: compare clathrin and the proteic combination degree of NPC1L1 in (1) and (2);

Wherein, (preferably significantly be lower than,, preferably hang down 40% if (2) middle cage modle albumen and the proteic combination degree of NPC1L1 are lower than statistically as low by 20%; More preferably hang down 60% or lower) (1) middle cage modle albumen and the proteic combination degree of NPC1L1, then described candidate substances is a potential decreasing cholesterol material.

In another preference, in (1) or (2), also comprise: determine AP2 complex body associated protein and the proteic combination degree of NPC1L1 in the cell; And, in (3), also comprise: compare AP2 complex body associated protein and the proteic combination degree of NPC1L1 in (1) and (2);

Wherein, (preferably significantly be lower than,, preferably hang down 40% if (2) middle AP2 complex body associated protein and the proteic combination degree of NPC1L1 are lower than statistically as low by 20%; More preferably hang down 60% or lower) (1) middle AP2 complex body associated protein and the proteic combination degree of NPC1L1, then described candidate substances is a potential decreasing cholesterol material.

In another preference, described AP2 complex body associated protein is selected from: μ 2, σ 2, β 2 or α subunit.Better, described AP2 complex body associated protein is μ 2 subunits.

In another preference, described cell is a reconstitution cell, contains expression cassette in its genome, and described expression cassette contains: proteic encoding gene of NPC1L1 and reporter gene that operability connects.

In another preference, described reporter gene is GFP or EGFP.

In another preference, described cell is an eukaryotic cell.

In another preference, determine the proteic degree of cell vesicle endocytosis NPC1L1 by quantity, the NPC1L1 albumen of endocytosis and/or the amount of cholesterol of metering endocytosis vesicle; Perhaps, determine the proteic degree of cell vesicle endocytosis NPC1L1 by the amount (as endocytosis has taken place, then the amount of NPC1L1 albumen and/or cholesterol reduces on the cytolemma) of NPC1L1 albumen and/or cholesterol on the metering cytolemma.

In another preference, position or quantitatively by fluorescent mark or painted method.

In another preference, described NPC1L1 albumen is a kind of fusion rotein, and it comprises NPC1L1 albumen and label protein, and described label protein is positioned at the proteic extracellular region of NPC1L1 territory (or vesica inboard).

In another preference, described label protein is striden film district and the 9th at NPC1L1 the proteic the 8th and is striden between the film district.

In another preference, NPC1L1 albumen is connected by peptide bond with label protein.

In another preference, the people NPC1L1 albumen that described NPC1L1 albumen is total length, and described label protein is positioned on the NPC1L1 albumen between the 986th amino acids (Ser) and the 987th amino acids (Leu).

In another preference, the proteic aminoacid sequence of described NPC1L1 is shown in GenBank accession number FJ481111.

In another preference, described label protein is selected from (but being not limited to): one or several Myc label proteins (preferred 3 * Myc), one or several Flag label proteins, one or several His6 label proteins, one or several T7 label proteins, one or several V5 label proteins, one or several HA label proteins, one or several GST label proteins, or the mixing of several label proteins is used.

In another preference, described label protein is selected from (but being not limited to): luciferase, β-gal enzyme.

In another preference, the proteic location of NPC1L1 is determined in the location that is tested and appraised the cell label protein, and then the proteic degree of definite cell vesicle endocytosis NPC1L1.

In another preference, when the label protein major part is positioned on the cytolemma, then NPC1L1 albumen is also located on the cytolemma in a large number, and the proteic degree of cell vesicle endocytosis NPC1L1 is low; When the label protein small part is positioned on the cytolemma, then NPC1L1 albumen is positioned in the cell in a large number, the proteic degree height of cell vesicle endocytosis NPC1L1.

In another preference, the proteic location of NPC1L1 is determined in the described location that is tested and appraised the cell label protein, and then the proteic degree methods of definite cell vesicle endocytosis NPC1L1 comprises:

(1) under the situation of penetrating cell not, mark (preferably utilizing the method for immunohistochemical methods) is positioned at the label protein on the cytolemma;

(2) under the situation of penetrating cell, the label protein of the full cell of mark (preferably utilizing the method for immunohistochemical methods);

(3) analyze or calculate in (1) per-cent that label protein on cytolemma accounts for full cell label protein in (2); (preferred obviously increase is more than 20% in obviously increase statistically as the shared per-cent of the label protein on the cytolemma, more preferably obviously increase more than 40%, further preferred obviously increase is more than 60%), then the ratio that is positioned on the cytolemma of NPC1L1 albumen obviously increases, and then shows that the proteic degree of cell vesicle endocytosis NPC1L1 obviously reduces.

In another preference, the observation of label protein or full cell label protein or analysis quantitatively can utilize fluorescent microscope or utilize flow cytometer on the cytolemma.

In another preference, adopt washing agent to handle impermeable cell, form penetrating cell.

In another preference, described method also comprises: the potential material that obtains is carried out further cell experiment and/or animal experiment, selection and definite for the useful material of decreasing cholesterol.

In a second aspect of the present invention, a kind of method of screening potential decreasing cholesterol material is provided, described method comprises:

(a) handle the proteic cell of expressing mediation NPC1L1 endocytosis with candidate substances;

(b) detect proteic expression or activity described in the described cell;

Wherein, if described candidate substances can reduce described proteic expression or activity, show that then this candidate substances is a potential decreasing cholesterol material.

In another preference, step (a) comprising: in test group, candidate substances is joined in the proteic cell of expressing mediation NPC1L1 endocytosis; And/or

Step (b) comprising: detect proteic expression or activity described in the cell of test group, and with control group relatively, wherein said control group is the described proteic cell of expression that does not add described candidate substances;

(preferably significantly be lower than,, preferably hang down 40% if proteic expression or activity are lower than statistically described in the test group as low by 20%; More preferably hang down 60% or lower) control group, just show that this material standed for is a potential decreasing cholesterol material.

In another preference, the albumen of described mediation NPC1L1 endocytosis is clathrin and/or AP2 complex body.

In a third aspect of the present invention, provide a kind of purposes of material in the preparation anticholesteremic agent of the NPC1L1 of inhibition albumen endocytosis.

In another preference, the material of described inhibition NPC1L1 albumen endocytosis is the material that suppresses clathrin and/or AP2 complex body and NPC1L1 protein-interacting.

In another preference, described material is β, beta-dimethyl-acry-lalkannin or 25-oxycholesterol.

On the other hand, also provide a kind of method that reduces the cell inner cholesterol, described method comprises: suppress (or interference) clathrin and/or AP2 complex body and NPC1L1 protein-interacting.

On the other hand, also provide a kind of method of screening potential decreasing cholesterol material, described method comprises:

(1) with cholesterol and the proteic NH of NPC1L1 ₂The contact of end structure territory, the proteic NH of cholesterol detection and NPC1L1 ₂The end structure territory in conjunction with situation;

(2) with cholesterol and the proteic NH of NPC1L1 ₂The contact of end structure territory, and add candidate substances, the proteic NH of cholesterol detection and NPC1L1 ₂The end structure territory in conjunction with situation;

Wherein, as cholesterol and the proteic NH of NPC1L1 in (2) ₂The bonding strength in end structure territory is significantly smaller than that (preferred combination intensity is obviously weak more than 20%, more preferably obviously weak more than 40%, further preferred obviously weak more than 60%) or binding capacity significantly is lower than, and (the preferred combination amount is obviously low more than 20%, more preferably obviously hang down more than 40%, further preferred obviously low more than 60%) (1) middle cholesterol and the proteic NH of NPC1L1 ₂The bonding strength in end structure territory or binding capacity, then described candidate substances are potential decreasing cholesterol materials.Also promptly, candidate substances can be competed cholesterol and the proteic NH of NPC1L1 as described ₂The combination in end structure territory, then described candidate substances are potential decreasing cholesterol materials.

On the other hand, also provide a kind of method of screening potential decreasing cholesterol material, described method comprises: with candidate substances and the proteic NH of NPC1L1 ₂The contact of end structure territory detects candidate substances and the proteic NH of NPC1L1 ₂The end structure territory in conjunction with situation; As candidate substances and the proteic NH of NPC1L1 ₂Specific combination takes place in the end structure territory, and then described candidate substances is a potential decreasing cholesterol material.

In another preference, the proteic NH of described NPC1L1 ₂18-260 amino acids in the sequence is contained shown in the GenBank accession number FJ481111 in the end structure territory.

On the other hand, provide the employing material that screening method screening as described obtains.

In another preference, described material is 25-oxycholesterol or 27-oxycholesterol.

On the other hand, also provide a kind of fusion rotein, described albumen comprises NPC1L1 albumen and label protein, and described label protein is positioned at the proteic extracellular region of NPC1L1 territory (or vesica inboard).

In another preference, label protein is striden film district and the 9th at NPC1L1 the proteic the 8th and is striden between the film district.

On the other hand, also provide a kind of nucleotide sequence, the described fusion rotein of described nucleic acid sequence encoding.

On the other hand, also provide a kind of recombinant vectors, described recombinant vectors contains described nucleotide sequence.

In another preference, described carrier wherein contains described nucleotide sequence based on the pEGFP-N1 carrier.

On the other hand, also provide a kind of host cell, described host cell contains described recombinant vectors, or is integrated with described nucleotide sequence in its genome.

In another preference, described host cell is a rat hepatoma cell.

On the other hand, also provide the purposes of described host cell, be used to analyze the inferior location of the proteic cell of NPC1L1.

Description of drawings

Fig. 1. intracellular cholesterol levels is regulated the circulation transhipment of NPC1L1 albumen between cytoplasmic membrane and endocytosis loop body.

A. handle the schematic flow sheet of cell: at-60 minutes, (CDX) reduced intracellular cholesterol with cyclodextrin; And then the cholesterol of passing cell CDX bag quilt at 0 minute.

The B.CRL-1601/NPC1L1-EGFP cell is done the processing shown in the A, fixes at different time points, and observes the fluorescence location with Laser Scanning Confocal Microscope.Bar，10μm。

C. to the amount of the localized NPC1L1 of the quantitative plasma membrane of the cell fluorescence among the B.Error line: standard deviation.

D and E.CRL-1601/NPC1L1-EGFP (D) and L02 cell (E) are shown in the A and are handled, and at different time point labeled cell plasmalemma proteins, SDS-PAGE analyzes.TnR, TfR.

Fig. 2 .NPC1L1 albumen is that cell absorption cholesterol institute is essential.

A.CRL-1601 and CRL-1601/NPC1L1-EGFP cell are done the processing shown in Figure 1A, fix at different time points, and Filipin dyeing, and observe fluorescence with two-photon copolymerization Jiao and locate.Bar，10μm。

B. to the amount of the localized NPC1L1 of the quantitative plasma membrane of the cell fluorescence among the A.Error line: standard deviation.

C. in the L02 cell, reduce the expression of NPC1L1 with the RNAi of retrovirus mediation.Be illustrated as westernblot result.

D.CDX handled the L02 cell 60 minutes, handled cell with the cholesterol of 15 μ g/ml CDX bag quilt.Fix at different time points, Filipin dyes, and compares the cell of NPC1L1 reduction and the fluorescence intensity of control cells with the two-photon Laser Scanning Confocal Microscope.Bar，10μm。DIC represents the light field visual field.

E. to the amount of the localized NPC1L1 of the quantitative plasma membrane of the cell fluorescence among the D.Error line: standard deviation.

Fig. 3. overexpression NPC1L1 albumen increases cell to cholesterol absorption.

After A.CDX handled cell, the cholesterol of passing CRL1601 cell and CRL1601/NPC1L1-EGFP cell different concns was after 1 hour, mounting, and Filipin dyeing, the two-photon Laser Scanning Confocal Microscope is observed; Bar, 10 μ m.

Cell fluorescence is quantitative among the B.A; Error line: standard deviation.

C.HEK293T cell transfecting NPC1L1-EGFP plasmid.Handle cell with method among the A after 24 hours, fixing, mounting, dyeing.Bar，10μm。

μ 2 subunits are as the protein-bonded evaluation of NPC1L1 in Fig. 4 .AP2 complex body.

A. do co-immunoprecipitation with the agarose of the anti-EGFP antibody of coupling, the albumen that obtains separates with SDS-PAGE, and silver dyes;

B. co-immunoprecipitation (CoIP) checking NPC1L1 and μ's 2 combines.

Fig. 5. disappearance clathrin/AP2 reduces proteic endocytosis of NPC1L1 and cholesterol picked-up.

A.Western Blot checking RNAi efficient.

The B.CRL-1601/NPC1L1-EGFP cell is done the processing shown in Fig. 6 A, fixes, dyes and locate with the burnt fluorescence of observing of two-photon copolymerization at different time points.Bar，10μm。

C. to the amount of localized NPC1L1 and cholesterol in the quantitative born of the same parents of the cell fluorescence among the B.Error line: standard deviation.

The cage modle albumen of Fig. 6 .siRNA mediation or the gene of μ 2 subunits disturb the cell that suppresses proteic endocytosis of NPC1L1 and cholesterol to absorb, and are the cholesterol concentration dependency.

A. handle the synoptic diagram of cell.

B. detect 2 pairs of Transferrins,iron complexes endocytosis influences of RNAi CHC and μ, show RNAi efficient.

C. biotin labeling epicyte protein experimental verification cage modle albumen is to the necessity of NPC1L1 albumen endocytosis.

D. cage modle albumen and μ 2 subunits are necessary to the protein mediated cholesterol absorption of NPC1L1, and are the cholesterol concentration dependency.

The fluorescent quantitation result of cell among the E.D; Error line: standard deviation.

Fig. 7 .RNAi is reticent, and caveolin-1 does not influence NPC1L1 albumen endocytosis and cholesterol absorption.

A.Western Blot shows RNAi efficient.

B. pair cell is among Fig. 6 A and is handled; Bar, 10 μ m.

The fluorescent quantitation result of cell among the C.B; Error line: standard deviation.

Fig. 8 .Ezetimibe is by influencing vesicle inhibition NPC1L1 albumen and the cholesterol endocytosis that NPC1L1 albumen enters clathrin bag quilt.

A. handle the schematic flow sheet of cell: cell is handled 60min in containing the substratum of cyclodextrin.Add Ezetimibe in culture medium A (containing 5%LPDS, 10 μ M compactin, 50 μ M mevalonic acid) then.Behind the 60min, the CDX bag is directly added by the cholesterol of cholesterol.Fix dyeing at different time points.

The B.CRL-1601/NPC1L1-EGFP cell is done the processing shown in the A, fixing after adding cholesterol 60min, dyeing, and observe fluorescence with the two-photon Laser Scanning Confocal Microscope and locate.Bar，10μm。

C. to the amount of localized NPC1L1 albumen and cholesterol in the quantitative born of the same parents of the cell fluorescence among the B.Error line: standard deviation.

D does the processing shown in the A with F.CRL-1601/NPC1L1-EGFP (D) or L02 cell (F). carry out the test of biotin labeling cytoplasmic membrane at different time points.

The E.CRL-1601/NPC1L1-EGFP cell is done the processing shown in the A, carries out the co-immunoprecipitation test at different time points.

The G.L02 cell is handled 60min in the substratum that contains cyclodextrin and Ezetimibe.The CDX bag is directly added 60min by the cholesterol of cholesterol.Fixing, dyeing.With the burnt fluorescence location of observing of two-photon copolymerization.Bar，10μm。

H. to the amount of localized NPC1L1 albumen and cholesterol in the quantitative born of the same parents of the cell fluorescence among the G.Error line: standard deviation.

Fig. 9 .NPC1L1-NH ₂End 18-260 amino acid is that cholesterol is essential in conjunction with NPC1L1 albumen institute, and is most important to cholesterol absorption.

A. the proteic cell of stably express people NPC1L1 is done immunoprecipitation with the agarose of the anti-EGFP antibody of coupling, and adds 10-160nM[in the albumen that obtains ³H] the external combination of cholesterol, 4 ℃, survey liquid after 4 hours and dodge and detect combining of NPC1L1 and cholesterol.Non-specific band is not for expressing the proteic cell repetitive operation of NPC1L1.Error line: standard deviation.

B. stably express people NPC1L1 albumen or NH ₂The proteic cell of NPC1L1 of-end 18-260 aminoacid deletion is done immunoprecipitation with the agarose of the anti-EGFP antibody of coupling, and adds 80nM[in the albumen that obtains ³H] the external combination of cholesterol, 4 ℃, survey liquid after 4 hours and dodge and detect combining of this albumen and cholesterol.Non-specific band is for adding the repetitive operation of the unmarked cholesterol competition of 10 μ M.NPC1L1 (Δ 18-260aa): NH ₂The NPC1L1 albumen of-end 18-260 aminoacid deletion.

C. cell transient expression people NPC1L1 albumen or NH ₂The amino acid whose NPC1L1 albumen of-end disappearance 18-260 is handled cell with CDX after 24 hours, and is passed cellular cholesterol once more, and 30 minutes, 60 minutes difference mountings, Filipin dyeing, the two-photon Laser Scanning Confocal Microscope is observed; Localized NPC1L1 albumen carries out fluorescent quantitation in the content of pair cell inner cholesterol and the cell.Error line: standard deviation.

Figure 10. compound 25-oxycholesterol suppresses NPC1L1 albumen and cholesterol combination, reduces the absorption of cholesterol.

A. cholesterol, the structural representation of 25-oxycholesterol.

B. the proteic cell of stably express people NPC1L1 is done immunoprecipitation with the agarose of the anti-EGFP antibody of coupling, and adds 80nM[in the albumen that obtains ³H] cholesterol, and the unlabelled 25-oxycholesterol of 250nM carries out external combination-competition, 4 ℃, survey after 4 hours liquid dodge detect NPC1L1 with [ ³H] combination of cholesterol.Non-specific band is not for expressing the proteic cell repetitive operation of NPC1L1.Error line: standard deviation.

C. the proteic clone of stably express people NPC1L1 is used 25-oxycholesterol pre-treatment 1 hour, and CDX handles cell afterwards, and passs cellular cholesterol once more, mounting after 1 hour, and Filipin dyeing, the two-photon Laser Scanning Confocal Microscope is observed; Localized NPC1L1 albumen carries out fluorescent quantitation in the content of pair cell inner cholesterol and the cell.Error line: standard deviation.

Figure 11. 986 to 987 at the NPC1L1 aminoacid sequence are inserted the normal function that 3 * Myc does not influence NPC1L1.

The site of the topological framework mode chart of A.NPC1L1 and S986 amino acids residue.

B. to expressing after the proteic cell of NPC1L1/S986-3 * Myc-L987 removes cholesterol, send cholesterol to cell again and handle in conjunction with Ezetimibe, Filipin dyeing, observation of cell is to the absorbing state of cholesterol.

C. to intracellular NPC1L1/S986-3 * Myc-L987 albumen and cholesterol among the B quantitatively.Error line: standard deviation.

Figure 12. analyze the proteic Subcellular Localization of NPC1L1 quickly and easily with immunohistochemical methods or fluidic cell.

A. handle the schematic flow sheet of cell: at-60 minutes, (CDX) reduced intracellular cholesterol with cyclodextrin; And then the cholesterol of passing cell CDX bag quilt at 0 minute.

B. transient transfection NPC1L1/S986-3 * Myc-L987 under two kinds of conditions of penetrating and not penetrating cell, carries out fluorescence microscope behind the immunostaining with Myc antibody pair cell.

C, D. detects the NPC1L1 cellular localization with flow cytometer.After 1.5% cyclodextrin (CDX) is handled 1hr, under 4 ℃ of conditions,, educated 30 minutes the flow cytometer detection then with fluorescence two temperature resistances of coupling Alexa555 with antibody (9E10) incubation of anti-Myc 30 minutes.

Figure 13. screening suppresses the compound of NPC1L1 albumen endocytosis.

A. β, the structural formula of beta-dimethyl-acry-lalkannin (C6).

B. with CDX with treat that screening of medicaments (C1-C187) handles the CRL1601/NPC1L1-EGFP cell, make NPC1L1 albumen be positioned cytolemma.Take pictures, see the CDX group among the figure.Remove CDX, remain the aglucon of screening of medicaments and cholesterol and handled cell one hour with containing.Take pictures, see chol group among the figure.Have that the compound (C6) of obvious inhibition NPC1L1 albumen endocytosis is selected to come out.Ez：Ezetimibe。

C. with CDX with treat that screening of medicaments (C6) handles the CRL1601/NPC1L1-EGFP cell, make NPC1L1 albumen be positioned cytolemma, fixing, see the CDX group among the figure.Remove CDX, remain the aglucon of screening of medicaments and cholesterol and handle cell with containing, fixing, see chol group among the figure.With Filipin dyeing, take pictures with the two-photon microscope.

D. the intracellular NPC1L1 and the cholesterol of fluorescent quantitation chol group.

Embodiment

The inventor is through deep research, the protein mediated cholesterol absorption of discovery NPC1L1 need be passed through vesica endocytosis process, wherein clathrin (Clathrin) and AP2 complex body are identified out in NPC1L1 albumen endocytosis process and are played important effect, and the vesica endocytosis NPC1L1 albumen (promptly suppress vesica that NPC1L1 albumen enters clathrin bag quilt, influence its endocytosis process) that suppresses the clathrin mediation can significantly suppress the absorption of cell to cholesterol.Therefore, can screen for the useful medicine of reducing cholesterol based on this mechanism.

NPC1L1 albumen is a repeatedly transmembrane protein, plays an important role in cholesterol absorption.The inventor discovers the proteic endocytosis of promotion NPC1L1 that cholesterol can be special, can regulate NPC1L1 albumen at plasma membrane and endocytosis loop body (Endocytic Recycling Compartment, ERC) the circulation transhipment between, when cholesterol reduces, NPC1L1 albumen is positioned at cytoplasmic membrane, and the supply cellular cholesterol can make to annex in the NPC1L1 albumen and be transported to the endocytosis loop body.This process need clathrin/AP2 complex body.The proteic endocytosis of NPC1L1 of blocking-up clathrin mediation can reduce the absorption of cholesterol, shows that NPC1L1 albumen transports the transportation that mediates cholesterol by the vesica endocytosis.Ezetimibe suppresses vesica that NPC1L1 albumen enters clathrin bag quilt, block its endocytosis process and then suppress the absorption of cholesterol.In a word, the inventor studies show that NPC1L1 albumen carries cholesterol and enters cell by the vesica endocytosis that clathrin mediates, thereby provides novel targets for screening novel cholesterol absorption inhibitor.

It is by the amoeboid movement of plasma membrane ECM to be transported into intracellular process (endocytosis) that cell absorbs the outer material of born of the same parents.The mode of its absorption is diversified, endocytosis can be divided into phagolysis, pinosome, receptor mediated endocytosis etc. according to the difference of going into born of the same parents' mechanism.Clathrin for example, caveolin, flotillin etc. all can mediate some materials and enter in the cell.Yet for different materials, the mechanism of going into born of the same parents may be different, and more complicated factors is relevant with the characteristic of the contained albumen of structure, the cell of cell itself, described material itself and some for this.In order to identify the protein factor that participates in NPC1L1 albumen endocytosis process, the inventor separates the complex body that has obtained participating in NPC1L1 albumen endocytosis with co-immunoprecipitation, with the albumen of tandem mass spectrum evaluation and NPC1L1 albumen specific combination.One of them albumen is μ 2 subunits in the AP2 complex body.μ 2 subunits can be discerned the albumen that endocytosis will take place in the endocytosis process, with it in conjunction with and raise other subunits of AP-2.Can recruit clathrin after the AP-2 complex body forms, endocytosis takes place thereupon.In order to verify above result, the inventor disturbs μ 2 subunits that reduce in endogenous clathrin or the AP-2 complex body with the RNA of siRNA mediation, and finding to understand after two genes reduce influence the absorption to cholesterol of the proteic endocytosis of NPC1L1 and cell.Illustrate that NPC1L1 albumen carries cholesterol and enters cell by the vesica endocytosis that clathrin mediates.

The method of the material of screening decreasing cholesterol

Getting this approach of vesica endocytosis that the protein mediated cholesterol absorption of cicada NPC1L1 need mediate NPC1L1 by clathrin, after the proteic vesica endocytosis of inhibition NPC1L1 can significantly suppress cell this mechanism of absorption to cholesterol, can reduce the potential material of cell based on this mechanism screening to the absorption of cholesterol.Thereby, can from described potential material, find for prevention or the useful material of treatment hypercholesterolemia relative disease.

Therefore, the invention provides a kind of method of screening potential decreasing cholesterol material, described method comprises:

(1) handle the proteic cell of expression NPC1L1 with cholesterol, and the proteic degree of vesica endocytosis NPC1L1 in definite cell;

(2) in the presence of candidate substances, express the proteic cell of NPC1L1 as processing as described in (1), determine the proteic degree of NPC1L1 of vesica endocytosis in the cell once more; With

(3) the proteic difference of NPC1L1 of vesica endocytosis in the comparison (1) and (2);

Wherein, if the proteic degree of NPC1L1 of vesica endocytosis is lower than in (1) statistically in (2), then described candidate substances is a potential decreasing cholesterol material.

As optimal way of the present invention, in step (1) or (2), the proteic cell of described expression NPC1L1 is handled through decreasing cholesterol in advance.The inventor finds that under study for action pair cell can make NPC1L1 albumen be transported to the cytolemma zone from endocytosis loop body (ERC) zone in the born of the same parents after carrying out the decreasing cholesterol processing in advance, prepares for absorbing cholesterol; And the cholesterol processing can promote the proteic endocytosis of NPC1L1 specifically.Therefore, during drug screening, pair cell carries out the decreasing cholesterol processing in advance, gives cellular cholesterol afterwards again, and the vesica endocytosis NPC1L1 albumen of observing the clathrin mediation during for drug screening is favourable.

As optimal way of the present invention, in step (1) or (2), also comprise: determine cage modle albumen and/or AP2 complex body associated protein and the proteic combination degree of NPC1L1 in the cell; And in (3), if (2) middle cage modle albumen and/or AP2 complex body associated protein and the proteic combination degree of NPC1L1 are lower than in (1) statistically, then described candidate substances is a potential decreasing cholesterol material.The AP2 complex body is by α, β 2, subunits such as μ 2 and σ 2 are formed, its function is to participate in endocytosis in the conjugated protein vesicle that enters clathrin (comprising heavy chain and light chain) bag quilt, therefore observes cage modle albumen and/or AP2 complex body or its associated protein and can reflect also that with the proteic combination degree of NPC1L1 NPC1L1 albumen enters the degree of the vesica of clathrin bag quilt (or measuring).

Determine to exist or do not exist the difference of endocytosis under the situation of candidate substances or combination degree to realize with the increase and decrease of the amount of the NPC1L1 albumen of the quantity of metering endocytosis vesicle, endocytosis and/or cholesterol; Perhaps also can realize (as endocytosis has taken place, then the amount of NPC1L1 albumen and/or cholesterol reduces on the cytolemma) by the increase and decrease of the amount of NPC1L1 albumen and/or cholesterol on the metering cytolemma.Concrete example be as positioning or quantitatively by fluorescent mark or painted method, and the immunofluorescence label cell surface protein is the effectively quantitative localized method of NPC1L1 albuminous cell film in conjunction with flow cytometer in addition.Also can be in 96 orifice plates with the cell kind, make related drugs and handle the back localized NPC1L1 albumen of fluorescence labeled cell plasma membrane, use the microplate reader reading then, this medicine can suppress NPC1L1 albumen endocytosis if reading is higher than predetermined threshold value, may be as the potential anticholesteremic agent.

If endocytosis or bonded difference are significant or have surpassed certain threshold value that then candidate agent may be effective for decreasing cholesterol.If difference is not remarkable, then can screen with another candidate substances repeating said steps.Usually, those skilled in the art can test multiple candidate substances simultaneously, for example by using porous plate or other high throughput method.

The invention provides a kind of preferred mode of determining NPC1L1 albumen endocytosis situation, with the label protein coupling or be connected in position suitable on the NPC1L1 protein sequence (preferred described label protein is striden film district and the 9th at NPC1L1 the proteic the 8th and striden between the film district), be tested and appraised on the cytoplasmic membrane the proteic location of label and determine the proteic location of NPC1L1, and then the proteic degree of definite cell vesicle endocytosis NPC1L1.When the label protein major part is positioned on the cytolemma, show that then NPC1L1 albumen locatees on the cytoplasmic membrane in a large number, the proteic degree of cell vesicle endocytosis NPC1L1 is little; Be positioned on the cytoplasmic membrane when label protein is a spot of, show that then NPC1L1 albumen major part is positioned in the cell, the proteic degree of cell vesicle endocytosis NPC1L1 is big.

With label protein coupling or the method that is connected on the NPC1L1 protein sequence is that those skilled in the art are known.Preferably, NPC1L1 albumen is connected by peptide bond with label protein.More preferably, described label protein is positioned on the NPC1L1 albumen between the 986th amino acids (Ser) and the 987th amino acids (Leu).

The selection of label protein is that those skilled in the art are known, and when being used for when of the present invention, length is preferred at 4-300 amino acid whose label protein.Described label protein is selected from (but being not limited to): one or several Myc label proteins, one or several Flag label proteins, one or several His6 label proteins, one or several T7 label proteins, one or several V5 label proteins, one or several HA label proteins, one or several GST label proteins, and the mixing of several labels is used.Perhaps, described label protein is selected from some enzyme (but being not limited to): luciferase, β-gal enzyme.Most preferred, described label protein is a Myc label protein (particularly 3 * Myc).

When NPC1L1 albumen is connected by peptide bond with label protein, constituted a kind of fusion rotein.The nucleotide sequence of encoding said fusion protein, the recombinant vectors and the host cell that contain described nucleotide sequence include in the present invention, and they can be used as analyzes NPC1L1 albumen at the localized material in the cell Central Asia.

Among the present invention, have no particular limits for used cell, (for example it needs cholesterol as biomembranous moiety as long as need to absorb cholesterol in growth or metabolic process; Or need cholesterol to synthesize the precursor of sterols hormone or bile acide); And it contains NPC1L1 albumen and clathrin and/or AP2 complex body.Preferable, described cell is a kind of eukaryotic cell.In embodiments of the present invention, described cell is selected from: CRL-1601 (McArdle RH7777 rat hepatoma cell), and L02 (human liver cell system), HuH7 (Bel7402), or the HEK293 cell etc.

As optimal way of the present invention, described cell is a reconstitution cell, contains expression cassette in its genome, and described expression cassette contains: proteic encoding gene of NPC1L1 and reporter gene that operability connects.Described reporter gene for example is green fluorescent protein (GFP) or synergic green fluorescent protein (EGFP), their endogenous fluorophor is being subjected to UV-light or can efficiently launching apparent green glow when blue-light excited, thereby can be intuitively and be used for the proteic location of NPC1L1 and quantitatively exactly.

Based on the inventor's new discovery, the method for another kind of screening potential decreasing cholesterol material comprises:

(a) handle the cell of expressing clathrin and/or AP2 complex body with candidate substances;

(b) expression or the activity of clathrin and/or AP2 complex body in the described cell of detection;

Wherein, if described candidate substances can reduce the expression or the activity of clathrin and/or AP2 complex body, show that then this candidate substances is a potential decreasing cholesterol material.

As a kind of preferred mode, when screening, in order to be easier to observe clathrin and/or expression of AP2 complex body or active change, also control group can be set, described control group can be not add the expression clathrin of described candidate substances and/or the cell of AP2 complex body.

As a kind of preferred mode, described method also comprises: the potential material that obtains is carried out further cell experiment and/or animal experiment, selection and definite for the useful material of decreasing cholesterol.

The potential decreasing cholesterol material that adopts described screening method to obtain is also included within the present invention.

The material of decreasing cholesterol

Based on the inventor's new discovery, the present invention also provides a kind of inhibition or the proteic material of interference cell vesica endocytosis NPC1L1, and described material can be used for preparing for the useful medicine of decreasing cholesterol.Any suppress or the proteic material of interference cell vesica endocytosis NPC1L1 all can be used for the present invention is as potential decreasing cholesterol material.

Based on the inventor's new discovery, the present invention also provides a kind of inhibition (or interference) clathrin and/or AP2 complex body and the NPC1L1 protein-interacting material of (as combining), and described material can be used for preparing for the useful medicine of decreasing cholesterol.Anyly suppress or disturb clathrin and/or AP2 complex body and NPC1L1 protein-interacting, reduce the stability of clathrin and/or AP2 complex body and NPC1L1 protein-interacting, suppress the expression of clathrin and/or AP2 complex body, or the material of transcribing or translating that suppresses clathrin and/or AP2 complex body genes involved all can be used for the present invention, as potential decreasing cholesterol material.

The material that these preliminary screening go out can constitute a screening storehouse, can or treat the material that the hypercholesterolemia relative disease defines usefulness for prevention so that people finally can therefrom filter out.

Utilize screening method of the present invention, the inventor has obtained a kind of compound that can suppress NPC1L1 albumen endocytosis--β, beta-dimethyl-acry-lalkannin.Therefore, the present invention also provides β, the purposes of beta-dimethyl-acry-lalkannin in the preparation anticholesteremic agent.

In addition, the present invention also provides a kind of method that reduces the cell inner cholesterol, and described method comprises: suppress (or interference) clathrin and/or AP2 complex body and NPC1L1 protein-interacting (as endocytosis, combine or combination), thus the absorption of prevention cholesterol.

Major advantage of the present invention is:

Find first that (1) the protein mediated cholesterol absorption of NPC1L1 need be by this approach of vesica endocytosis NPC1L1 albumen of clathrin mediation, the vesica endocytosis NPC1L1 albumen that suppresses the clathrin mediation can significantly suppress the absorption of cell to cholesterol.

(2) solved not know in the prior art NPC1L1 albumen by what kind of approach is transported cholesterol, is difficult to develop the defective of more more active drugs in the cell, thereby provide new fast way for screening anticholesteremic agent.

(3) develop the localized method of fast and convenient analysis NPC1L1 protein subcellular, can be used for the high-throughput screening compound.

(4) identify the structural domain of NPC1L1 protein binding cholesterol and illustrate its function, thereby can screen compound in conjunction with this structural domain active material of cholesterol absorption that is inhibited.

Below in conjunction with specific embodiment, 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.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.

Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.

Test materials and method

Material and plasmid

Anti-mouse of horseradish peroxidase bonded donkey and anti-rabbit 1gG: available from Jackson immune Research laboratory.

Filipin (Filipin) is available from Sigma.

Sulfosuccinimidyl 6-(vitamin H amino) hexanoate and NeutrAvidin-agarose: available from Pierce.

Methyl-β-cyclodextrin (CDX): available from Cyclodextrin Technologies Development Inc..

The cholesterol purifying is to be higher than 99% purity.

Degrease serum (LPDS, d＞1.215g/ml): from newborn calf serum by super centrifugal acquisition.

The proteic coding region of NPC1L1, people source from 5 '-actggatccatggcggaggccggcctgagg-3 ' (SEQ IDNO:1) and 5 '-actggatccgaactgccgcccattgttggg-3 ' (SEQ ID NO:2) as primer, pcr amplification people liver cDNA obtains, and inserts carrier pEGFP-N1 (available from Clontech) by bamHI single endonuclease digestion site clone.As primer, and insert carrier pDsRed-monomer-C1 (available from Clontech) with 5 '-actgaattctatgggcacccgcgacgacga-3 ' (SEQ ID NO:3) and 5 '-actgaattcttagatgttctgacagcact-3 ' (SEQ ID NO:4) by EcoRI single endonuclease digestion site clone.

Cell cultures

CRL-1601 (McArdle RH7777 rat hepatoma cell), L02 (human liver cell system) and HuH7 (Bel7402) (all available from ATCC) monolayer are at 37 ℃ and 5%CO ₂In, cell is grown in culture medium A (Dulbecco ' Eagle ' the s substratum of s improvement, contain 100 units/ml penicillin and 100 μ g/ml Streptomycin sulphates) and adds 10%FBS.Cholesterol-defective (depleting) substratum is that culture medium A adds 5% degrease serum (LPDS), 10 μ M mevastatins (compactin), 50 μ M mevalonic acid (mevalonate) and 1.5% cyclodextrin (CDX).Cholesterol-additional (replenishing) substratum contains culture medium A and adds 5%LPDS, 10 μ M mevastatins, the cholesterol of the CDX bag quilt of 50 μ M mevalonic acid and different concns.The CDX bag is seen existing report (Brown by the method for sterol, A.J etc. (2002), Cholesterol addition to ER membranes alters conformation of SCAP, the SREBPescort protein that regulates cholesterol metabolism.Mol.Cell 10,237-245).

Biotin labeling cytoplasmic membrane albumen

Wash cell twice with PBS, add 4 ℃ of mark 40min of vitamin H (sulfosuccinimidyl 6-(vitamin H amino) hexanoate) of 1mg/ml.Buffer A (20mM Tris-HCl (pH8.0) and 150mM NaCl) is washed 2 times cells and then cell is placed buffer A 15min.With lysis in buffer B (10mM Tris-HCl (pH8.0), 150mM NaCl and 1%NP-40).Each sample adds 100 μ l 50% (v/v) NeutrAvidin-agaroses, and 4 ℃ of combinations are spent the night.Sepharose 4B is washed 3 times with buffer B.Then sepharose 4B temperature in 37 ℃ of sample-loading buffers is bathed 30min.The centrifuging and taking supernatant is walked SDS-PAGE.

RNA disturbs

Double-stranded siRNA is synthetic by Genepharma.The siRNA of target rat clathrin heavy chain (CHC) is referring to Radulescu, A.E. etc. (2007), A role for clathrin in reassembly of the Golgi apparatus.Mol.Biol.Cell 18,94-105.The siRNA sequence of target rat AP2-μ 2 subunits and Caveolin-1 is respectively 5 '-aaggcatgaaggaatcacaga-3 ' (SEQ ID NO:5) and 5 '-aagcaagtgtacgacgcgcac-3 ' (SEQ IDNO:6).The siRNA of target VSV-G is referring to Song, B.L. (2007) .Ufd1 is a cofactor of gp 78 and playsa key role in cholesterol metabolism by regulating the stability of HMG-CoA reductase.Cell Metab 6,115-128 is with comparing (Control siRNA).Sever is seen in the siRNA transfection, N. etc. (2003), Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.J.Biol.Chem.278,52479-52490.

Filipin dyeing

Prepare fresh 5mg/ml Filipin liquid storage.After the cell fixation with 50 μ g/ml Filipin dyeing 30min.The Filipin signal is observed exciting light 720nm with Zeiss LSM 510 confocal microscope two-photons.

Co-immunoprecipitation

After cell carried out relevant treatment, cracking in the IP damping fluid (PBS, 5mM EGTA, 5mMEDTA, 0.5%Digitonin).The cleer and peaceful sepharose 4B that contains GFP antibody mixes on the centrifuging and taking, and 4 ℃ in conjunction with 2 hours.Remove supernatant, wash sepharose 4B 5 times with the IP damping fluid.Acetic acid/glycine elutriant wash-out with PH2.8.-70 ℃ of acetone precipitations spend the night, the centrifugal supernatant of abandoning.Precipitation is dissolved in sample-loading buffer, and SDS-PAGE analyzes.

Fluorescent quantitation

In each experimental group in each experiment, select 100 cells arbitrarily as quantitative object.With Image ProPlus 5.01 software fluorescent quantitations.The total fluorescence intensity quantitative methods of cell is to select this cell with AOI, cuts off background after the calculating fluorescence intensity.Intracellular fluorescence intensity quantitative methods is to be close to circle of picture under the cytolemma with AOI, quantitatively the fluorescence intensity in the circle.What show among the figure is the total fluorescence intensity of fluorescence intensity ratio cell in the cell.

[ ³H] cholesterol is in conjunction with experiment

After cell carried out relevant treatment, cracking is (PBS, 1%NP-40,5mM EGTA, 5mM EDTA) in conjunction with the IP damping fluid.The cleer and peaceful sepharose 4B that contains EGFP antibody mixes on the centrifuging and taking, and 4 ℃ in conjunction with 2 hours, carries out immunoprecipitation.Wash sepharose 4B 5 times with binding substances IP damping fluid, add respectively suitable concn [ ³H] cholesterol of mark, final [ ³H] cholesterol concentration of mark is 10-400nM, final volume is 100 μ l, be dissolved in binding buffer liquid (PBS, 0.001%NP-40) in.4 ℃ in conjunction with 4 hours, washes sepharose 4B 5 times with binding buffer liquid, with acetic acid/glycine elutriant wash-out of PH2.8.Bonded [ ³H] cholesterol of mark surveys liquid flashing counting with scintillation solution.For competitive assay, dissolve [ ³H] in the binding buffer liquid of cholesterol of mark, adding final concentration respectively is the nonisotopically labelled sterols of 10 μ M.Wherein, [ ³H] cholesterol of mark is available from PerkinElmer company.

Fluidic cell detects

Cell is carried out relevant treatment, after the trysinization, dispel into unicellular.Hatched on ice 30 minutes with Anti-Myc monoclonal antibody IgG-9E10 then, PBS washes twice.Anti-mouse IgG fluorescence two is anti-hatch 30 minutes after, PBS washes twice.With BD LSR II SORP flow cytometry analysis.Wherein, Anti-Myc monoclonal antibody IgG-9E10 is available from Roche company, and Anti-mouse IgG fluorescence two is anti-available from invitrogen company.

Embodiment 1 transhipment of cholesterol regulation NPC1L1 albumen between endocytosis loop body (ERC) and cytoplasmic membrane

The hepatic cell line CRL1601 that will contain the pEGFP-N1 carrier transfection rat of NPC1L1 protein-coding region obtains the stably express strain of stably express NPC1L1-EGFP, called after CRL1601/NPC1L1-EGFP.

Conventional Western Blot experimental result shows that the proteic expression amount of endogenous NPC1L1 is similar in the expression amount of NPC1L1-EGFP in the stably express strain and some hepatic cell lines (HepG2, huh7 and L02).Therefore the inventor studies the proteic location of NPC1L1 with this cell strain.

Handle the flow process of CRL1601/NPC1L1-EGFP cell and see Figure 1A ,-60min cholesterol (Chol)-defective culture medium culturing cell, remove aforementioned substratum at 0min, use cholesterol (Chol)-supplemental medium culturing cell instead, until 120min.Also promptly earlier reduce intracellular cholesterol, and then pass the cholesterol of cell CDX bag quilt with CDX.

In the process of aforementioned processing cell,, and observe fluorescence with copolymerization Jiao and locate at different time point fixed cells.Found that NPC1L1 albumen mainly is positioned at the other zone of nuclear under normal operation.NPC1L1 albumen is transported to cytoplasmic membrane (Figure 1B, time from-60 to 0min) when cholesterol reduces.When passing cholesterol again.NPC1L1 albumen again endocytosis in the cell and finally navigate to ERC (Figure 1B, time from 30 to 120min).Fig. 1 C is seen in the location of NPC1L1 albumen on cytoplasmic membrane.

The CRL-1601/NPC1L1-EGFP cell is done to handle shown in Figure 1A, at different time point labeled cell plasmalemma proteins.Thereby affirmed above phenomenon with the method for biotin labeling plasmalemma protein, seen Fig. 1 D.

In order to get rid of the influence between the cell clone, inventor's method with the biotin labeling plasmalemma protein in the L02 cell has detected the proteic location of endogenous NPC1L1 under the different condition.The result shows that endogenous NPC1L1 albumen is subjected to the regulation and control of cholesterol levels too, sees Fig. 1 E.The NPC1L1 albumen of this explanation CRL1601/NPC1L1-EGFP stably express can be simulated the proteic characteristic of endogenous NPC1L1.

Embodiment 2NPC1L1 albumen is necessary to the absorption of free cholesterol

The cholesterol that cell absorbs in the low-density lipoprotein (LDL) is to realize by the endocytosis of ldl receptor.The proteic endocytosis prompting of NPC1L1 NPC1L1 albumen may be by the absorption of similar process mediation cholesterol.In order to verify this idea, the inventor comes cholesterol in the transfect cell with Filipin.CRL-1601 and CRL-1601/NPC1L1-EGFP cell are done the processing shown in Figure 1A, fix at different time points, and Filipin dyeing, and observe fluorescence with two-photon copolymerization Jiao and locate.The result after handling cell and come reducing cholesterol with cyclodextrin (CDX), almost can't see the signal of Filipin shown in Fig. 2 A, illustrate that the amount of cell inner cholesterol is very low; Meanwhile NPC1L1 albumen also is positioned at cytoplasmic membrane (time point is 0min).Handle cell with the substratum of passing cholesterol and can see that the Filipin signal strengthens gradually in the cell, the prompting cholesterol increases.And the CRL1601/NPC1L1-EGFP cell is to the absorption of the cholesterol CRL-1601 cell (time point is from 30-120min) more than contrast.Along with the proteic endocytosis of NPC1L1, endocytosis also takes place in cholesterol.The vesica that contains proteic vesica of NPC1L1 and cholesterol has well location altogether.Fluorescent quantitation shows that the cholesterol of CRL1601/NPC1L1-EGFP cellular uptake is the twice (Fig. 2 B, time point is from 30-120min) of control cells.Pass cell with the cholesterol of different concns (7.5 μ g/ml, 15 μ g/ml, 30 μ g/ml) and also obtained similar result (Fig. 3 A and B).

In order further to confirm above phenomenon, the inventor will be contained the pEGFP-N1 carrier transfection HEK293T cell of NPC1L1 protein-coding region, transient expression NPC1L1-EGFP in the HEK293T cell.Find that the location of NPC1L1-EGFP is also regulated and control, and the cell of crossing the cell comparison photograph of expressing NPC1L1-EGFP absorbs more cholesterol (Fig. 3 C).

Next the inventor reduces the proteic expression of the endogenous NPC1L1 of L02 cell with the RNAi of retrovirus mediation, and by Fig. 2 C as can be known, RNAi can effectively lower the proteic expression of NPC1L1 in the cell.Find that the picked-up of cholesterol has also reduced about 60% (Fig. 2 D-E) after the NPC1L1 protein expression reduces.In the Huh7 cell, also obtained similar effects.

In addition, the inventor has also observed effluxing of cholesterol, finds that NPC1L1 albumen does not influence cholesterol and effluxes.

Embodiment 3 disappearance clathrin/AP2 reduce proteic endocytosis of NPC1L1 and cholesterol picked-up

In order to seek the factor that participates in NPC1L1 albumen endocytosis, the inventor has carried out a large amount of co-immunoprecipitations.Band for the NPC1L1 protein-specific is identified with tandem mass spectrum.One of them albumen is μ 2 subunits of AP2 complex body.The AP2 complex body is by α, and β 2, compositions such as μ 2 and σ 2, and known its function is to participate in endocytosis in the conjugated protein vesicle that enters clathrin bag quilt.Co-immunoprecipitation has been verified mass spectral result, and NPC1L1-EGFP is described, μ 2 and CHC see Fig. 4 in same complex body.

Mass spectrum evaluation to all NPCs 1L1 protein-binding protein (Band1-7) among Fig. 4 A sees Table 1, and wherein Band5 is μ 2 subunits of AP2 complex body.

Table 1

Next the inventor reduces the expression of endogenous μ 2 and CHC with the RNAi method, and Western blot verifies RNAi jamming effectiveness (Fig. 5 A).RNAi μ 2 and CHC have suppressed Transferrins,iron complexes, and (Tranferrin, endocytosis TnR) illustrate RNAi effectively (Fig. 6 B).

With the processing shown in the CRL-1601/NPC1L1-EGFP cell mapping 6A, fix, dye and locate with the burnt fluorescence of observing of two-photon copolymerization at different time points.The analysis of NPC1L1 albumen endocytosis is found that the proteic endocytosis of NPC1L1 also depends on this approach, and RNAi μ 2 or CHC can suppress the picked-up (Fig. 5 B) of proteic endocytosis of 50%NPC1L1 and cholesterol.Experiment with the biotin labeling cytoplasmic membrane has also proved above conclusion (Fig. 5 C).The experiment of biotin labeling epicyte protein is proof also, and cage modle albumen is necessary (Fig. 6 C) to NPC1L1 albumen endocytosis.

Further test is also found, when passing the cholesterol of cell different concns (7.5 μ g/ml, 15 μ g/ml, 30 μ g/ml), RNAi μ 2 or CHC also can suppress proteic endocytosis of NPC1L1 and cholesterol picked-up (Fig. 6 D-E).

In addition, the reticent expression that reduces endogenous Caveolin-1 of RNAi does not influence the endocytosis (Fig. 7) of NPC1L1 albumen and cholesterol.

Embodiment 4Ezetimibe suppresses NPC1L1 albumen and cholesterol endocytosis

Ezetimibe is the effective inhibitors of cholesterol absorption, and has been approved the clinical treatment hypercholesterolemia.And known NPC1L1 protein binding Ezetimibe.It is also indeterminate but how Ezetimibe suppresses the absorption of the protein mediated cholesterol of NPC1L1.

The inventor reduces intracellular cholesterol with CDX makes NPC1L1 albumen be positioned at cytoplasmic membrane, sends cholesterol (Fig. 8 A) to cell again behind medicine (Ezetimibe or U18666A) the processing cell with different concns.Discovery is along with the raising of Ezetimibe concentration, and the proteic endocytosis of NPC1L1 is suppressed, and the picked-up of cholesterol also reduces (Fig. 8 B, C).U18666A is the medicine that influences the cholesterol transportation in LDL source, and its target spot may be NPC1, and it does not influence the proteic endocytosis of NPC1L1.

Method with the biotin labeling plasmalemma protein has also proved above phenomenon (Fig. 8 D), and Ezetimibe does not influence the endocytosis of TfR.The effect that Ezetimibe is described is special.

The test of ensuing co-immunoprecipitation shows, the proteic endocytosis of Ezetimibe cholesterol inductive NPC1L1 is by suppressing (Fig. 8 E) that vesicle that NPC1L1 albumen enters clathrin bag quilt is realized.The picked-up of proteic endocytosis of endogenous NPC1L1 and cholesterol also is subjected to the inhibition (Fig. 8 F, G, H) of Ezetimibe in the L02 cell.

In a word, Ezetimibe suppresses the picked-up of proteic endocytosis of NPC1L1 and cholesterol by the vesicle that suppresses NPC1L1 albumen and enter clathrin bag quilt.

The proteic NH of embodiment 5NPC1L1 ₂-structural domain (18-260 amino acid) is in conjunction with cholesterol, and is most important to cholesterol absorption

In order whether to detect NPC1L1 albumen in conjunction with cholesterol, the contriver utilize the method for immunoprecipitation from the CRL1601/NPC1L1-EGFP cell, be purified into its carboxyl terminal have people's total length of EGFP NPC1L1 albumen (the GenBank accession number: FJ481111), add again [ ³H] after the cholesterol of mark, detect NPC1L1 albumen can in conjunction with [ ³H] cholesterol, shown in Fig. 9 A, along with [ ³H] increase of cholesterol amount, NPC1L1 albumen with [ ³H] combination of cholesterol is saturation curve, and this result shows that the NPC1L1 protein-specific is in conjunction with the cholesterol molecule.

And then the inventor adopts ordinary method with NPC1L1 albumen NH ₂End regions (18-260 amino acid) lacks, and finds the ability of disappearance amino acid/11 8-260 remarkably influenced NPC1L1 protein binding cholesterol, and this shows NPC1L1 albumen NH ₂The 18-260 amino acid of end regions is cholesterol binding domains (Fig. 9 B).

In order to study NPC1L1 albumen NH ₂Hold of the influence of 18-260 amino acid structure territory to cholesterol absorption, in rat hepatocytes CRL-1601, transient transfection expressing human NPC1L1-EGFP albumen, or NH respectively ₂The people NPC1L1-EGFP albumen of-end 18-260 aminoacid deletion is fixed at different time points, and filipin dyes, and observes with the two-photon Laser Scanning Confocal Microscope.By to each time point cell inner cholesterol content, and NPC1L1 albumen carries out quantitative analysis in the born of the same parents, finds that total length NPC1L1 albumen gets back in the cell gradually, absorbs a large amount of cholesterol simultaneously and enters cell; And expression NH ₂-end 18-260 aminoacid deletion NPC1L1 albumen gets back in the cell and the cholesterol absorption of mediation all significantly reduces (Fig. 9 C).Show people NPC1L1 albumen NH ₂-end structure territory (18-260 amino acid) can be in conjunction with the cholesterol molecule, and most important for cholesterol absorption.

Embodiment 6 compound 25-oxycholesterols suppress NPC1L1 albumen and cholesterol combination, and then reduce the absorption of cholesterol

Because NPC1L1 albumen can combine with the cholesterol specificity, inventor's imagination if find combining of a kind of compound competition NPC1L1 albumen and cholesterol, just can play the effect of inhibition cholesterol absorption.According to this hypothesis, the contriver finds 25-oxycholesterol (structure is seen Figure 10 A), can compete [ ³H] cholesterol of mark combines (Figure 10 B) with NPC1L1 is proteic.

Further, handle the CRL-1601/NPC1L1-EGFP cell with the 25-oxycholesterol, fix, dye and observe fluorescence with the two-photon Laser Scanning Confocal Microscope and locate at different time points, carry out quantitative analysis in proteic location by NPC1L1 in pair cell inner cholesterol content and the cell, find that the 25-oxycholesterol can significantly suppress the absorption and the proteic endocytosis of NPC1L1 (Figure 10 C) of cholesterol.

In sum, compound 25-oxycholesterol can be by the combining of competition cholesterol and NPC1L1, thereby the inhibition cell is to the picked-up of cholesterol.Therefore, the proteic NH of NPC1L1 ₂-structural domain (18-260 amino acid) can be used as the target spot of screening cholesterol absorption inhibitor; 25-oxycholesterol and analogue thereof may be as new cholesterol absorption inhibitors.

Embodiment 7 is behind 986 and 987 insertion 3 * Myc of NPC1L1 aminoacid sequence, and NPC1L1 still has normal physiological function

For further topological framework and the function of research NPC1L1, the inventor explores through a large amount of, identifies to insert behind proteic the 986th amino acids residue of NPC1L1 that 3 * Myc label does not influence the transhipment of NPC1L1 and to the absorption (Figure 11,12) of cholesterol.

Under the normal culture condition, NPC1L1/S986-3 * Myc-L987 mainly be positioned at the other zone of nucleus (Figure 11 B ,-60min).(Figure 12 B 0min), reduces cellular cholesterol levels, and NPC1L1/S986-3 * Myc-L987 albumen is transported to cytoplasmic membrane when the cyclodextrin with 1.5% was handled cell 1 hour.When the cholesterol of sending cyclodextrin bag quilt to cell again after 1 hour, NPC1L1/S986-3 * Myc-L987 again endocytosis in tenuigenin and navigate to ERC (Figure 12 B, 60min).This circulation location is consistent with the NPC1L1 of wild-type.

In order to verify further whether NPC1L1/S986-3 * Myc-L987 albumen is normal to the function of cholesterol absorption, the inventor comes quantitative intracellular cholesterol with the painted method of Filipin.At first remove the cholesterol of cytolemma, and then send cholesterol, observe the absorbing state of cholesterol with Filipin dyeing with cyclodextrin.As Figure 11 B, shown in the 11C, under the condition that does not have Ezetimibe to exist, NPC1L1/S986-3 * Myc-L987 can carry a large amount of cholesterol and enter cell.Under the condition that 10 μ M Ezetimibe handle, NPC1L1/S986-3 * Myc-L987 part is advanced tenuigenin by endocytosis, and cholesterol also part is absorbed.Yet when handling cell with 40 μ M Ezetimibe, NPC1L1/S986-3 * Myc-L987 is suppressed on the cytoplasmic membrane fully, and cholesterol can not be entered cell by endocytosis simultaneously.

Therefore, NPC1L1/S986-3 * Myc-L987 shows identical characteristic with wild-type NPC1L1, be that intracellular cholesterol levels is regulated the circulation transhipment of NPC1L1 albumen between cytoplasmic membrane and endocytosis loop body, this albumen can mediate the absorption of cholesterol, and absorption process can be suppressed by the Ezetimibe specificity.

The used cell of present embodiment is CRL1601, is transfected into the pEGFP-N1 plasmid that contains NPC1L1/S986-3 * Myc-L987 expression cassette with the method for transient transfection.

The methods analyst NPC1L1 that embodiment 8 usefulness immunohistochemical methodss, fluidic cell detect is in intracellular location

To the NPC1L1 The study of topology, the contriver thinks that when NPC1L1/S986-3 * Myc-L987 protein transport was to cytoplasmic membrane, 3 * Myc label will be exposed to extracellular matrix according to the contriver.If impermeable cell (not penetratingly is meant not penetrating cytolemma, with this understanding, extraneous small molecules (as antibody) can not enter in the cell, the NPC1L1 on an antibody labeled cell film surface, NPC1L1 in can not labeled cell) with the antibody incubation of anti-Myc, have only the NPC1L1/S986-3 * Myc-L987 on the cytolemma can be, therefore can utilize these characteristics to come NPC1L1 on the labeled cell film by the Myc antibody test.

Shown in Figure 12 B, in not having penetrating cell, add Myc antibody, then with fluorescently-labeled two anti-detections, has only detected (Figure 12 B of NPC1L1/S986-3 * Myc-L987 on the cytolemma, 0min), intracellular NPC1L1/S986-3 * Myc-L987 then can not be detected (Figure 12 B ,-60min and 60min).(penetrating being meant punched on cytolemma and at complete penetrating cell, Wai Jie small molecules (as antibody) just can enter cytolemma like this, therefore in can labeled cell and the NPC1L1 albumen of surface of cell membrane) in, inside and outside NPC1L1/S986-3 * the Myc-L987 of cell can be by anti-Myc antibody test to (Figure 12 B, penetrating cell dyeing).This method can be analyzed the Subcellular Localization of NPC1L1 quickly and easily.

For more further investigation of albumen location to NPC1L1, the contriver will be contained the hepatic cell line CRL1601 of the pEGFP-N1 carrier transfection rat of NPC1L1/S986-3 * Myc-L987 encoding histone frame, obtain the cell strain of stably express NPC1L1/S986-3 * Myc-L987-EGFP, called after CRL1601/NPC1L1/S986-3 * Myc-L987-EGFP.Utilize CRL1601/NPC1L1/S986-3 * Myc-L987-EGFP cell, the contriver has used flow cytometry analysis before and after cyclodextrin (CDX) extracting cholesterol, and the inferior location of the proteic cell of NPC1L1/S986-3 * Myc-L987-EGFP changes.Discovery that can be clearly, after CDX handles, the amount of the NPC1L1/S986-3 * Myc-L987-EGFP on the cytolemma obviously increase (Figure 12 C, D).This is again a kind of localized method of NPC1L1 protein subcellular of analyzing quickly and easily.

The preparation method of penetrating cell is known in the art, in the present embodiment, adopts the Triton X 100 of 0.1-0.2% to handle cell 5 minutes, obtains penetrating cell.

Embodiment 7 to embodiment 8 shows that the contriver has made up a new NPC1L1 protein expressing plasmid that has 3 * Myc label, and when the NPC1L1 that expresses was positioned on the cytolemma, 3 * Myc label can be exposed to extracellular matrix.Utilize this characteristics, the contriver uses the NPC1L1 albumen on the method label film of immunohistochemical methods, and utilizes fluidic cell to detect the Subcellular Localization of evaluation NPC1L1 that also can be easy.Therefore, the inventor has successfully created a kind of method of new analysis NPC1L1 Subcellular Localization, utilizes this method, can identify NPC1L1 quickly and easily in intracellular location.The foundation of this method is for high throughput analysis NPC1L1 lays a good foundation in intracellular location.

Embodiment 9 drug screening methods

One. screening method is as follows:

1. primary dcreening operation

Be once screening with about 20 kinds of compounds, will express the cell kind of NPC1L1-EGFP in 12 orifice plates;

Handle cell with CDX after 48 hours, reduce intracellular cholesterol, make NPC1L1 albumen be positioned cytolemma;

Handled cell one hour with candidate drug;

To contain the cholesterol that adds CDX bag quilt in the substratum of candidate drug;

Observe the proteic location of NPC1L1 after one hour, finding out influences the localized medicine of NPC1L1 albumen.

As a result, the inventor has found the compound that can suppress NPC1L1 albumen endocytosis a: β, beta-dimethyl-acry-lalkannin (β, β-DIMETHYLACRYL ALKANNIN, C6) (Figure 13 A-B) in more than 180 kind of compound of initial screening.

2. dusting cover

Select the positive drug in the primary dcreening operation, handle cell with aforementioned similar method;

Fix Filipin dyeing and mounting at different time points;

Use the two-photon microscopic examination, take pictures, intracellular NPC1L1 of fluorescent quantitation and cholesterol (Figure 13 C-D).As seen, β, beta-dimethyl-acry-lalkannin (C6) can suppress the endocytosis of general about 50% NPC1L1 and the absorption of cholesterol.So β, beta-dimethyl-acry-lalkannin can be used as the compound that a potential suppresses cholesterol absorption.

Two. screening method is as follows:

The cell that this screening method adopts is CRL1601, wherein is transfected into and contains the proteic NH of NPC1L1 ₂The pEGFP-N1 plasmid of-structural domain (18-260 amino acid) expression cassette, thus make cytotostatic express the proteic NH of NPC1L1 ₂-structural domain (18-260 amino acid)-EGFP, described cell called after CRL-1601/NPC1L1 (18-260)-EGFP after the lysis, utilizes anti-EGFP antibody mediated immunity purifying to obtain NPC1L1 (18-260)-EGFP albumen, and in albumen, add [ ³H] cholesterol of mark, the above-mentioned biased sample that makes is divided into two groups:

Test group: NPC1L1 (18-260)-EGFP albumen wherein adds candidate substances;

Control group: NPC1L1 (18-260)-EGFP albumen does not wherein add candidate substances.

Detect in two groups [ ³H] cholesterol and the proteic NH of NPC1L1 of mark ₂End structure territory (18-260 amino acid) in conjunction with situation, as cholesterol in the test group and the proteic NH of NPC1L1 ₂The binding capacity in end structure territory significantly be lower than in the control group [ ³H] cholesterol and the proteic NH of NPC1L1 of mark ₂The binding capacity in end structure territory (low more than 30%), then described candidate substances is the decreasing cholesterol material.

As candidate substances, found that with 25-oxycholesterol or 27-oxycholesterol they all can be competitively and the proteic NH of NPC1L1 ₂-structural domain (18-260 amino acid) combination, thus cholesterol and the proteic NH of NPC1L1 suppressed ₂The combination of-structural domain (18-260 amino acid).

Discuss

The inventor's data show that Ezetimibe is by suppressing the absorption that the proteic endocytosis of NPC1L1 suppresses cholesterol.Its molecular mechanism is that Ezetimibe influences the vesicle that NPC1L1 albumen enters clathrin bag quilt.What is interesting is that Ezetimibe does not influence the endocytosis of Transferrins,iron complexes and LDL.These results show that all Ezetimibe is the effective and special inhibitor of NPC1L1 albumen.But how Ezetimibe influences the vesicle that NPC1L1 albumen enters clathrin bag quilt also do not know.Illustrate this problem, the inventor started with from the starting stage of NPC1L1 albumen endocytosis.NPC1L1 albumen contains sterol and experiences structural domain.There are 6 albumen to contain this structural domain: HMGCR in the people's gene group, SCAP, NPC1, Patched, TRC8, and NPC1L1.Can infer that the proteic sterol receptive field of NPC1L1 may be directly in conjunction with cholesterol, cause endocytosis thereby combine with clathrin/AP2 complex body by conformational change.Ezetimibe may compete cholesterol and combine with NPC1L1 is proteic.Another kind may be that Ezetimib can influence the proteic conformation of NPC1L1 and causes cholesterol can not promote NPC1L1 albumen to combine with clathrin/AP2 complex body.The distribution that Ezetimibe also may influence the cholesterol around the NPC1L1 albumen causes preventing of NPC1L1 albumen endocytosis.

The people obtains cholesterol by de novo synthesis and food absorption.Compare with suppressing cholesterol synthetic medicine (as: Statins), Ezetimibe is the medicine of inhibition cholesterol absorption unique on the market.Because different Id difference causes the sensitivity difference of different people to Ezetimibe.Therefore, seek more cholesterol absorption inhibitor and seem particularly important.The inventor's work has disclosed the molecular mechanism of cholesterol absorption, can establish Basic of Biology for new medicament screen.Simultaneously, the cell levels that the inventor sets up is observed the inhibitor that the localized system of NPC1L1 protein subcellular can be used to screen NPC1L1 albumen endocytosis, and these inhibitor have the possibility that suppresses the cholesterol absorption medicine.

Cell is main by four kinds of modes from the external substance exchange: freely spread, assist diffusion, active transport and endocytosis and exocytosis.Wherein freely spreading is that hydrophobic and simple small molecules directly passes through the cytoplasmic membrane phospholipid bilayer, and this process does not need albumen to participate in; Assist diffusion be the small molecules of the channel protein mediation on the cytoplasmic membrane from the high side of concentration to a low side, as ionic channel etc.; Active transport requires the expenditure of energy, and it also is protein mediated, as ionic pump etc.; Endocytosis is by the exchange of substance of the mediation of caving in of cytoplasmic membrane, comprises phagolysis and pinocytosis, and pinocytosis comprises the endocytosis of clathrin mediation again, the endocytosis of caveolin mediation and the endocytosis of other unknown forms.

Assisting the mediation albumen of diffusion and active transport is channel protein, is characterized in that this proteinoid is that monomer or polymer form passage at cytolemma, allows material directly pass in and out cytolemma, and these materials mostly are the little material of unit molecule such as water, glucose and amino acid etc.Endocytosis is receptor-mediated by on the cytolemma.

Ldl receptor is cholesterol transportation and the common molecular receptor of endocytosis, it is characterized in that once striding the membranin of film.Its extracellular region territory can be raised the relevant albumen of endocytosis by some special motif in the kytoplasm and carry out endocytosis then in conjunction with LDL or VLDL.And for NPC1L1, it contains general 13 times and strides diaphragm area, and NPC1L1 promoted be the absorption of unit molecule material cholesterol, therefore no matter be aspect topological framework and part, NPC1L1 and ldl receptor all differ widely, and therefore people can not think that the function of NPC1L1 and ldl receptor are similar usually.Ironically participate in the bimolecular complex body that albumin A BCG5/8 that cholesterol effluxes is made up of two albumen of striding film for six times, its topological framework and NPC1L1 have similar part.Have prediction ABCG5/8 to efflux cholesterol by the mode that is similar to pump by hydrolysising ATP, ABCG1 and the ABCA1 very similar to ABCG5/8 also are presumably molecular pump.NPC1 is the albumen (homology 50%) very high with the NPC1L1 homology, and its topological framework and NPC1L1 are closely similar.NPC1 outwards plays a key effect (endoplasmic reticulum and plasma membrane) transportation from lysosome in cholesterol.There is report to show that the NPC1 albumen of vivoexpression has the ability of pump lipid acid, but can not the pump cholesterol.In conjunction with ABCG5/8 and NPC1 research, those skilled in the art are very natural usually can to infer that NPC1L1 may be a molecular pump, promotes the absorption of cholesterol by pump cholesterol in cell.

And the inventor's experimental result is exactly unexpected.At first be if NPC1L1 is a molecular pump, so no matter in human body or in the cell cultures, free unit molecule cholesterol will be the most effective substrate.But cholesterol will form micelle with bile acide and could effectively be absorbed in human body, and is absorbed by NPC1L1 with the cholesterol of the cholesterol specific ionization of CDX bag quilt is easier in cell cultures; Secondly,, when to cellular cholesterol, should see that cholesterol is introduced into cell so, but the inventor sees is that NPC1L1 and cholesterol enter in the cell simultaneously if NPC1L1 is a molecular pump; At last, if NPC1L1 is a molecular pump, still be that reticent clathrin reduces the NPC1L1 endocytosis no matter with the microfilament inhibitor, all will increase the absorption of cholesterol, but the inventor sees is that cholesterol absorption also has been suppressed.These results do not support that not only NPC1L1 is the hypothesis of molecular pump, have proved that on the contrary the absorption of the cholesterol of NPC1L1 mediation is the mode that is similar to the ldl receptor endocytosis.Be not difficult to find out in the report of its tangible NPC1 that molecular pump substrate in the experiment in vitro of NPC1 is the substrate cholesterol under lipid acid rather than its physiological condition, the mode that this explanation NPC1 transports cholesterol in vivo similarly is not a molecular pump, moreover free unit molecule cholesterol pair cell is toxic, the unit molecule cholesterol pumps in the tenuigenin if NPC1 or NPC1L1 directly will dissociate, and pair cell health is very unfavorable.

More than explanation NPC1L1 is similar to the acceptor of cholesterol, and NPC1L1 absorbs cholesterol by the mode of vesica endocytosis.Illustrate that without any evidence NPC1L1 is the acceptor rather than the cholesterol passage of cholesterol in the prior art at present, also explanation is not how to absorb cholesterol when NPC1L1 albumen is positioned cytolemma.And concrete NPC1L1 albumen is screened anticholesteremic agent as target spot scheme is not proposed also in the prior art.The present invention has disclosed for the first time to start with from the proteic function of NPC1L1 and has screened potential decreasing cholesterol material, and this is more reliable in conjunction with experiment than external.Absorb cholesterol just because of having disclosed NPC1L1 albumen first by the vesica endocytic pathway,, promptly screen potential decreasing cholesterol material, and screening has obtained a kind of active compound by analyzing NPC1L1 albumen endocytosis so the talent of the present invention proposes this invention.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

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＜110〉Shanghai Inst. of Life Science, CAS

＜120〉based on analyzing the method that NPC1L1 protein subcellular location changes the screening new drug for lowering cholesterol

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Claims (26)

1. a method of screening potential decreasing cholesterol material is characterized in that, described method comprises:

(1) handle the proteic cell of expression NPC1L1 with cholesterol, and the proteic degree of definite cell vesicle endocytosis NPC1L1;

(2) in the presence of candidate substances, express the proteic cell of NPC1L1 as processing as described in (1), determine the proteic degree of vesica endocytosis NPC1L1 once more; With

(3) compare proteic difference of vesica endocytosis NPC1L1 in (1) and (2);

Wherein, if the proteic degree of vesica endocytosis NPC1L1 is lower than the proteic degree of vesica endocytosis NPC1L1 in (1) statistically in (2), then described candidate substances is a potential decreasing cholesterol material.

2. the method for claim 1 is characterized in that, in (1) or (2), the proteic cell of described expression NPC1L1 is handled through decreasing cholesterol before cholesterol is handled in advance.

3. the method for claim 1 is characterized in that, in (1) or (2), also comprises: determine cage modle albumen and the proteic combination degree of NPC1L1 in the cell; And,

(3) in, also comprise: compare clathrin and the proteic combination degree of NPC1L1 in (1) and (2);

Wherein, if (2) middle cage modle albumen and the proteic combination degree of NPC1L1 are lower than cage modle albumen and the proteic combination degree of NPC1L1 in (1) statistically, then described candidate substances is a potential decreasing cholesterol material.

4. the method for claim 1 is characterized in that, in (1) or (2), also comprises: determine AP2 complex body associated protein and the proteic combination degree of NPC1L1 in the cell; And,

(3) in, also comprise: compare AP2 complex body associated protein and the proteic combination degree of NPC1L1 in (1) and (2);

Wherein, if (2) middle AP2 complex body associated protein and the proteic combination degree of NPC1L1 are lower than AP2 complex body associated protein and the proteic combination degree of NPC1L1 in (1) statistically, then described candidate substances is a potential decreasing cholesterol material.

5. the method for claim 1 is characterized in that, described cell is a reconstitution cell, contains expression cassette in its genome, and described expression cassette contains: proteic encoding gene of NPC1L1 and reporter gene that operability connects.

6. the method for claim 1 is characterized in that, described NPC1L1 albumen is a kind of fusion rotein, and it comprises NPC1L1 albumen and label protein, and described label protein is positioned at the proteic extracellular region of NPC1L1 territory.

7. method as claimed in claim 6 is characterized in that, described label protein is striden film district and the 9th at NPC1L1 the proteic the 8th and striden between the film district.

8. method as claimed in claim 7 is characterized in that, described label protein is between the 986th amino acids on the NPC1L1 albumen and the 987th amino acids.

9. method as claimed in claim 8 is characterized in that, the proteic location of NPC1L1 is determined in the location that is tested and appraised the cell label protein, and then the proteic degree of definite cell vesicle endocytosis NPC1L1.

10. method as claimed in claim 9 is characterized in that, the proteic location of NPC1L1 is determined in the described location that is tested and appraised the cell label protein, and then the proteic degree methods of definite cell vesicle endocytosis NPC1L1 comprises:

(1) under the situation of penetrating cell not, mark is positioned at the label protein on the cytolemma;

(2) under the situation of penetrating cell, the label protein of the full cell of mark;

(3) analyze or calculate in (1) per-cent that label protein on cytolemma accounts for full cell label protein in (2); Obviously increase statistically as the shared per-cent of the label protein on the cytolemma, then the ratio that is positioned on the cytolemma of NPC1L1 albumen obviously increases, and then shows that the proteic degree of cell vesicle endocytosis NPC1L1 obviously reduces.

11. the method for claim 1 is characterized in that, described method also comprises: the potential material that obtains is carried out further cell experiment and/or animal experiment, selection and definite for the useful material of decreasing cholesterol.

12. a method of screening potential decreasing cholesterol material is characterized in that, described method comprises:

(a) handle the proteic cell of expressing mediation NPC1L1 endocytosis with candidate substances;

(b) detect proteic expression or activity described in the described cell;

Wherein, if described candidate substances can reduce described proteic expression or activity, show that then this candidate substances is a potential decreasing cholesterol material.

13. method as claimed in claim 12 is characterized in that,

Step (a) comprising: in test group, candidate substances is joined in the proteic cell of expressing mediation NPC1L1 endocytosis; And/or

Step (b) comprising: detect proteic expression or activity described in the cell of test group, and with control group relatively, wherein said control group is the described proteic cell of expression that does not add described candidate substances;

If proteic expression or activity are lower than control group statistically described in the test group, just show that this material standed for is a potential decreasing cholesterol material.

14., it is characterized in that the albumen of described mediation NPC1L1 endocytosis is clathrin and/or AP2 complex body as claim 12 or 13 described methods.

15. the purposes of material in the preparation anticholesteremic agent that suppresses NPC1L1 albumen endocytosis.

16. purposes as claimed in claim 15 is characterized in that, the material of described inhibition NPC1L1 albumen endocytosis is the material that suppresses clathrin and/or AP2 complex body and NPC1L1 protein-interacting.

17., it is characterized in that the material of described inhibition NPC1L1 albumen endocytosis is β, beta-dimethyl-acry-lalkannin or 25-oxycholesterol as claim 15 or 16 described purposes.

18. a method of screening potential decreasing cholesterol material is characterized in that, described method comprises:

(1) with cholesterol and the proteic NH of NPC1L1 ₂The contact of end structure territory, the proteic NH of cholesterol detection and NPC1L1 ₂The end structure territory in conjunction with situation;

(2) with cholesterol and the proteic NH of NPC1L1 ₂The contact of end structure territory, and add candidate substances, the proteic NH of cholesterol detection and NPC1L1 ₂The end structure territory in conjunction with situation;

Wherein, as cholesterol and the proteic NH of NPC1L1 in (2) ₂The bonding strength in end structure territory is significantly smaller than or binding capacity significantly is lower than cholesterol and the proteic NH of NPC1L1 in (1) ₂The bonding strength in end structure territory or binding capacity, then described candidate substances are potential decreasing cholesterol materials.

19. a method of screening potential decreasing cholesterol material is characterized in that, described method comprises: with candidate substances and the proteic NH of NPC1L1 ₂The contact of end structure territory detects candidate substances and the proteic NH of NPC1L1 ₂The end structure territory in conjunction with situation; As candidate substances and the proteic NH of NPC1L1 ₂Specific combination takes place in the end structure territory, and then described candidate substances is a potential decreasing cholesterol material.

20., it is characterized in that the proteic NH of described NPC1L1 as claim 18 or 19 described methods ₂18-260 amino acids in the sequence is contained shown in the GenBank accession number FJ481111 in the end structure territory.

21. adopt material as claim 18 or 19 described method screening acquisitions.

22. a fusion rotein is characterized in that described albumen comprises NPC1L1 albumen and label protein, and described label protein is positioned at the proteic extracellular region of NPC1L1 territory.

23. a nucleotide sequence is characterized in that, the described fusion rotein of described nucleic acid sequence encoding claim 22.

24. a recombinant vectors is characterized in that, described recombinant vectors contains the described nucleotide sequence of claim 23.

25. a host cell is characterized in that, described host cell contains the described recombinant vectors of claim 24, or is integrated with the described nucleotide sequence of claim 23 in its genome.

26. the purposes of the described host cell of claim 25 is characterized in that, is used to analyze the inferior location of the proteic cell of NPC1L1.

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