CN101580871B

CN101580871B - Method for screening new drug for lowering cholesterol based on analysis of change of NPC1L1 protein subcellular localization

Info

Publication number: CN101580871B
Application number: CN2009101414268A
Authority: CN
Inventors: 宋保亮; 葛亮; 王婧; 李培山; 曲玉秀; 缪红华; 戚炜; 王江; 张锦辉
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Center for excellence and innovation of molecular cell science, Chinese Academy of Sciences
Priority date: 2008-05-13
Filing date: 2009-05-13
Publication date: 2013-06-05
Anticipated expiration: 2029-05-13
Also published as: CN101580871A; WO2009138035A1

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 the method for analyzing NPC1L1 protein subcellular location variation 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 the method for analyzing NPC1L1 protein subcellular location variation screening new drug for lowering cholesterol; Simultaneously, the present invention has identified in NPC1L1 albumen the 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, is also 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 main molecules mechanism of Biosynthesis 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 the transgenic mice of expression NPC1L1 than bile and the decline of the cholesterol in ight soil of normal mouse.

People NPC1L1 albumen contains 1332 amino acid, consist of 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 in Subcellular Localization about NPC1L1 albumen 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 always unclear.

Therefore, although NPC1L1 albumen known in the art is relevant to 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) process the cell of expressing NPC1L1 albumen with cholesterol, and the degree of definite cell vesicle (generally being called again the film bubble) endocytosis NPC1L1 albumen;

(2) under candidate substances exists, express the cell of NPC1L1 albumen as processing as described in (1), again determine the degree of vesica endocytosis NPC1L1 albumen; With

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

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

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

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

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

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

In another preference, in (1) or (2), also comprise: the combination degree of determining cage modle albumen and NPC1L1 albumen in cell; And, in (3), also comprise: the combination degree that compares (1) and (2) middle clathrin and NPC1L1 albumen;

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

In another preference, in (1) or (2), also comprise: the combination degree of determining the relevant albumen of AP2 complex body in cell and NPC1L1 albumen; And, in (3), also comprise: the combination degree that compares (1) and (2) middle relevant albumen of AP2 complex body and NPC1L1 albumen;

Wherein, if in (2) combination degree of the relevant albumen of AP2 complex body and NPC1L1 albumen statistically lower than (preferably significantly lower than, as low by 20%, preferably hang down 40%; More preferably low by 60% or lower) combination degree of the relevant albumen of AP2 complex body and NPC1L1 albumen in (1), described candidate substances is potential decreasing cholesterol material.

In another preference, the relevant albumen of described AP2 complex body is selected from: μ 2, σ 2, β 2 or α subunit.Better, the relevant albumen of described AP2 complex body is μ 2 subunits.

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

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

In another preference, described cell is eukaryotic cell.

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

In another preference, the method by fluorescent mark or dyeing positions or quantitatively.

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 extracellular region territory (or vesica is inboard) of NPC1L1 albumen.

In another preference, described label protein is between the 8th cross-film district of NPC1L1 albumen and the 9th cross-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 NPC1L1 albumen between the 986th amino acids (Ser) and the 987th amino acids (Leu).

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

In another preference, when the label protein major part is positioned on cytolemma, also on a large amount of positioning cells films, the degree of cell vesicle endocytosis NPC1L1 albumen is low for NPC1L1 albumen; When the label protein small part is positioned on cytolemma, NPC1L1 albumen is positioned in cell in a large number, and the degree of cell vesicle endocytosis NPC1L1 albumen is high.

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

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

(2) in the situation that 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); Obviously increasing statistically as the label protein percentage on cytolemma (preferably obviously increases more than 20%, more preferably obviously increase more than 40%, further preferred obviously increase is more than 60%), the ratio of NPC1L1 protein localization on cytolemma obviously increases, and then shows that the degree of cell vesicle endocytosis NPC1L1 albumen obviously reduces.

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

In another preference, adopt washing agent to process 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, select and determine 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) process the cell of the albumen of expressing mediation NPC1L1 endocytosis with candidate substances;

(b) detect protein expression or activity described in described cell;

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

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

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

If protein expression described in test group or activity statistically lower than (preferably significantly lower than, as low by 20%, preferably low 40%; More preferably hang down 60% or lower) control group, just show that this material standed for is 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) use the NH of cholesterol and NPC1L1 albumen ₂The contact of end structure territory, the NH of cholesterol detection and NPC1L1 albumen ₂The end structure territory in conjunction with situation;

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

Wherein, the NH of cholesterol as middle in (2) and NPC1L1 albumen 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 lower than (the preferred combination amount is obviously low more than 20%, more preferably obviously low more than 40%, further preferred obviously low more than 60%) NH of cholesterol and NPC1L1 albumen in (1) ₂The bonding strength in end structure territory or binding capacity, described candidate substances is potential decreasing cholesterol material.Also namely, candidate substances can be competed the NH of cholesterol and NPC1L1 albumen as described ₂The combination in end structure territory, described candidate substances is potential decreasing cholesterol material.

On the other hand, also provide a kind of method of screening potential decreasing cholesterol material, described method comprises: with the NH of candidate substances and NPC1L1 albumen ₂The contact of end structure territory, the NH of detection candidate substances and NPC1L1 albumen ₂The end structure territory in conjunction with situation; NH as candidate substances and NPC1L1 albumen ₂Specific combination occurs in the end structure territory, and described candidate substances is potential decreasing cholesterol material.

In another preference, the NH of described NPC1L1 albumen ₂18-260 amino acids in sequence is contained shown in 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 extracellular region territory (or vesica is inboard) of NPC1L1 albumen.

In another preference, label protein is between the 8th cross-film district of NPC1L1 albumen and the 9th cross-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 take the pEGFP-N1 carrier as the basis.

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 rat hepatoma cell.

On the other hand, also provide the purposes of described host cell, be used for analyzing the Subcellar location of NPC1L1 albumen.

Description of drawings

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

A. process the schematic flow sheet of cell: at-60 minutes, (CDX) reduced intracellular cholesterol with cyclodextrin; And then passed the coated cholesterol of cell CDX at 0 minute.

The B.CRL-1601/NPC1L1-EGFP cell is done the processing shown in A, fixes at different time points, and uses the confocal microscopy fluorescence localization.Bar，10μm。

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

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

Fig. 2 .NPC1L1 albumen is that Cell uptake 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 localization with two-photon copolymerization Jiao.Bar，10μm。

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

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

D.CDX processed the L02 cell 60 minutes, processed cell with the cholesterol that 15 μ g/ml CDX are coated.Fix at different time points, Filipin dyeing, and compare 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 NPC1L1 of the quantitative plasma membrane of the cell fluorescence in D location.Error line: standard deviation.

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

A.CDX passs the cholesterol of CRL1601 cell and CRL1601/NPC1L1-EGFP cell different concns after 1 hour after processing cell, mounting, Filipin dyeing, two-photon confocal microscopy; Bar, 10 μ m.

In B.A, cell fluorescence is quantitative; Error line: standard deviation.

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

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

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. the combination of co-immunoprecipitation (CoIP) checking NPC1L1 and μ 2.

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

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 use the burnt fluorescence localization of observing of two-photon copolymerization at different time points.Bar，10μm。

C. to the NPC1L1 of the quantitative inner cellular localization of cell fluorescence in B and the amount of cholesterol.Error line: standard deviation.

The cage modle albumen of Fig. 6 .siRNA mediation or the gene of μ 2 subunits disturb the endocytosis of inhibition NPC1L1 albumen and the Cell uptake of cholesterol, and are the cholesterol concentration dependency.

A. process the schematic diagram of cell.

B. detect RNAi CHC and μ 2 to the impact of Transferrins,iron complexes endocytosis, show RNAi efficient.

C. the necessity of biotin labeling epicyte protein experimental verification cage modle albumen to 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 in E.D; Error line: standard deviation.

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

A.Western Blot shows RNAi efficient.

B. cell is in Fig. 6 A and is processed; Bar, 10 μ m.

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

Fig. 8 .Ezetimibe enters clathrin coated vesicle inhibition NPC1L1 albumen and cholesterol endocytosis by affecting NPC1L1 albumen.

A. process the schematic flow sheet of cell: cell is processed 60min in containing the substratum of cyclodextrin.Then add Ezetimibe in culture medium A (containing 5%LPDS, 10 μ M compactin, 50 μ M mevalonic acid).After 60min, the cholesterol of the coated cholesterol of CDX directly adds.Fix dyeing at different time points.

The B.CRL-1601/NPC1L1-EGFP cell is done the processing shown in A, fixing after adding cholesterol 60min, dyeing, and with two-photon confocal microscopy fluorescence localization.Bar，10μm。

C. to the NPC1L1 albumen of the quantitative inner cellular localization of cell fluorescence in B and the amount of cholesterol.Error line: standard deviation.

D and F.CRL-1601/NPC1L1-EGFP (D) or L02 cell (F) are done the processing shown in A and are carried 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 A, carries out the co-immunoprecipitation test at different time points.

The G.L02 cell is processed 60min in the substratum that contains cyclodextrin and Ezetimibe.The cholesterol of the coated cholesterol of CDX directly adds 60min.Fixing, dyeing.With the burnt fluorescence localization of observing of two-photon copolymerization.Bar，10μm。

H. to the NPC1L1 albumen of the quantitative inner cellular localization of cell fluorescence in G and the amount of cholesterol.Error line: standard deviation.

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

A. the cell of stably express people NPC1L1 albumen 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 the combination that detects NPC1L1 and cholesterol.Non-specific band is not for expressing the cell repetitive operation of NPC1L1 albumen.Error line: standard deviation.

B. stably express people NPC1L1 albumen or NH ₂The cell of the NPC1L1 albumen 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 the combination that detects this albumen and cholesterol.Non-specific band is the repetitive operation that adds the 10 unmarked cholesterol competitions of 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 processed cell with CDX after 24 hours, and is again passed cellular cholesterol, and 30 minutes, 60 minutes difference mountings, Filipin dyeing, two-photon confocal microscopy; The content of cell inner cholesterol and the NPC1L1 albumen of thin inner cellular localization are carried out fluorescent quantitation.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 cell of stably express people NPC1L1 albumen 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 cell repetitive operation of NPC1L1 albumen.Error line: standard deviation.

C. the clone of stably express people NPC1L1 albumen is used 25-oxycholesterol pre-treatment 1 hour, and CDX processes cell afterwards, and again passs cellular cholesterol, mounting after 1 hour, Filipin dyeing, two-photon confocal microscopy; The content of cell inner cholesterol and the NPC1L1 albumen of thin inner cellular localization are carried out fluorescent quantitation.Error line: standard deviation.

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

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

B. send cholesterol and process in conjunction with Ezetimibe, Filipin dyeing, the absorbing state of observation of cell to cholesterol to after the cell Cholesterol removal of expressing NPC1L1/S986-3 * Myc-L987 albumen, then to cell.

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

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

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

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

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

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

B. use CDX and medicine to be screened (C1-C187) to process the CRL1601/NPC1L1-EGFP cell, make the NPC1L1 protein localization in cytolemma.Take pictures, see the CDX group in figure.Remove CDX, processed cell one hour with the aglucon that contains medicine to be screened and cholesterol.Take pictures, see chol group in figure.There is the compound (C6) of obvious inhibition NPC1L1 albumen endocytosis out selected.Ez：Ezetimibe。

C. use CDX and medicine to be screened (C6) to process the CRL1601/NPC1L1-EGFP cell, make the NPC1L1 protein localization in cytolemma, fixing, see the CDX group in figure.Remove CDX, process cell with the aglucon that contains medicine to be screened and cholesterol, fixing, see chol group in figure.With Filipin dyeing, take pictures with Two Photon Fluorescence.

D. 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 to be passed through vesica endocytosis process, wherein clathrin (Clathrin) and AP2 complex body are identified out plays important effect in NPC1L1 albumen endocytosis process, the vesica endocytosis NPC1L1 albumen (namely suppressing NPC1L1 albumen enters the coated vesica of clathrin, affects its endocytosis process) that suppresses the clathrin mediation can significantly suppress cell to the absorption of cholesterol.Therefore, can screen for reducing the useful medicine of cholesterol based on this mechanism.

NPC1L1 albumen is a repeatedly transmembrane protein, plays an important role in cholesterol absorption.The inventor studies the endocytosis of the promotion NPC1L1 albumen of finding 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, the NPC1L1 protein localization is at cytoplasmic membrane, and the supply cellular cholesterol can make to annex in NPC1L1 albumen and be transported to the endocytosis loop body.This process need clathrin/AP2 complex body.The endocytosis of the NPC1L1 albumen of blocking-up clathrin mediation can reduce the absorption of cholesterol, shows that NPC1L1 albumen transports to mediate the transportation of cholesterol by the vesica endocytosis.Ezetimibe suppresses the absorption that NPC1L1 albumen enters the coated vesica of clathrin, blocks its endocytosis process and then inhibition 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.

The outer material of Cell uptake born of the same parents is by the amoeboid movement of plasma membrane, ECM to be transported into intracellular process (endocytosis).The mode of its absorption is diversified, endocytosis can be divided into phagolysis, pinosome, receptor mediated endocytosis etc. according to the difference that enters born of the same parents' mechanism.Clathrin for example, caveolin, flotillin etc. all can mediate some materials and enter in cell.Yet for different materials, the mechanism that enters born of the same parents may be different, albumen, the characteristic of described material itself and the factor analysis that some are more complicated that structure, the cell of this and cell itself is contained.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 identified the albumen that endocytosis will occur 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 occurs thereupon.In order to verify above result, the inventor disturbs to reduce μ 2 subunits in endogenous clathrin or AP-2 complex body with the RNA of siRNA mediation, finds to understand after two genes reduce the endocytosis that affect NPC1L1 albumen and cell to the absorption of cholesterol.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

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

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

(1) process the cell of expressing NPC1L1 albumen with cholesterol, and the degree of the vesica endocytosis NPC1L1 albumen in definite cell;

(2) under candidate substances exists, express the cell of NPC1L1 albumen as processing as described in (1), again determine the degree of the NPC1L1 albumen of vesica endocytosis in cell; With

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

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

As optimal way of the present invention, in step (1) or (2), the cell of described expression NPC1L1 albumen is processed through decreasing cholesterol in advance.The inventor finds under study for action, and cell is carried out after decreasing cholesterol processes in advance, can make endocytosis loop body (ERC) zone of NPC1L1 albumen in the born of the same parents be transported to the cytolemma zone, prepares for absorbing cholesterol; And cholesterol is processed the endocytosis that can promote specifically NPC1L1 albumen.Therefore, during drug screening, in advance cell is carried out decreasing cholesterol and process, give afterwards cellular cholesterol again, 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: the combination degree of determining the relevant albumen of cage modle albumen in cell and/or AP2 complex body and NPC1L1 albumen; And in (3), the combination degree of albumen and NPC1L1 albumen is statistically lower than in (1) if (2) middle cage modle albumen and/or AP2 complex body are correlated with, and described candidate substances is potential decreasing cholesterol material.The AP2 complex body is by α, β 2, the subunits such as μ 2 and σ 2 form, its function is to enter in the coated vesicle of clathrin (comprising heavy chain and light chain) in conjunction with albumen to participate in endocytosis, and the combination degree of therefore observing cage modle albumen and/or AP2 complex body or its associated protein and NPC1L1 albumen can reflect that also NPC1L1 albumen enters the degree of the coated vesica of clathrin (or amount).

Determine to exist or do not exist the difference of endocytosis in 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 by the increase and decrease of the amount of NPC1L1 albumen and/or cholesterol on the metering cytolemma (as endocytosis has occured, on cytolemma, the amount of NPC1L1 albumen and/or cholesterol reduces).Concrete example be as positioning by the method for fluorescent mark or dyeing or quantitatively, and the immunofluorescence label cell surface protein is the effectively quantitative method of NPC1L1 albuminous cell film location in conjunction with flow cytometer in addition.Also can be with the cell kind in 96 orifice plates, make to use after related drugs is processed the NPC1L1 albumen of fluorescence labeled cell plasma membrane location, then use the microplate reader reading, if reading higher than predetermined threshold value this medicine can suppress NPC1L1 albumen endocytosis, may be as potential anticholesteremic agent.

If the difference of endocytosis or combination is significant or surpassed certain threshold value, candidate agent may be effective for decreasing cholesterol.If difference is not remarkable, 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 between the 8th cross-film district of NPC1L1 albumen and the 9th cross-film district), the location of NPC1L1 albumen is determined in the location that is tested and appraised label protein on cytoplasmic membrane, and then the degree of definite cell vesicle endocytosis NPC1L1 albumen.When the label protein major part is positioned on cytolemma, show that on a large amount of positioning cells plasma membranes of NPC1L1 albumen, the degree of cell vesicle endocytosis NPC1L1 albumen is little; When a small amount of being positioned on cytoplasmic membrane of label protein, show that NPC1L1 albumen major part is positioned in cell, the degree of cell vesicle endocytosis NPC1L1 albumen is large.

That those skilled in the art are known with label protein coupling or the method that is connected on the NPC1L1 protein sequence.Preferably, NPC1L1 albumen is connected by peptide bond with label protein.More preferably, described label protein is positioned on 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 Myc label protein (particularly 3 * Myc).

When NPC1L1 albumen is connected by peptide bond with label protein, consisted of 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 material of location, the cell Central Asia.

In the present invention, have no particular limits for cell used, (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.Better, 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 reconstitution cell, contains expression cassette in its genome, and described expression cassette contains: encoding gene and the reporter gene of the NPC1L1 albumen that operability connects.Described reporter gene is for example the green fluorescent protein (EGFP) of green fluorescent protein (GFP) or synergy, their endogenous fluorophor is being subject to UV-light or can efficiently launching apparent green glow when blue-light excited, thereby can intuitively and be used for exactly the location of NPC1L1 albumen and quantitatively.

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

(a) process 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 expression or the activity of clathrin and/or AP2 complex body, show that this candidate substances is potential decreasing cholesterol material.

As a kind of preferred mode, when screening, in order to be easier to observe clathrin and/or the 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, select and determine 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 the material of a kind of inhibition or interference cell vesica endocytosis NPC1L1 albumen, and described material can be used for preparing for the useful medicine of decreasing cholesterol.Any suppress or the material of interference cell vesica endocytosis NPC1L1 albumen all can be used for the present invention is as potential decreasing cholesterol material.

New discovery based on the inventor, the present invention also provides the material of a kind of inhibition (or interference) clathrin and/or AP2 complex body and NPC1L1 protein-interacting (as combination), 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 composite bulk phase correlation gene all can be used for the present invention, as potential decreasing cholesterol material.

The material that these preliminary screening go out can consist of one and screen the storehouse, can or treat the material that the hypercholesterolemia relative disease defines use 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, described method comprises: suppress (or interference) clathrin and/or AP2 complex body and NPC1L1 protein-interacting (as endocytosis, combination or combination), thereby stop the absorption of cholesterol.

Major advantage of the present invention is:

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

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

(3) develop the method for fast and convenient analysis NPC1L1 protein subcellular location, 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 the be inhibited material of cholesterol absorption activity of compound in conjunction with this structural domain.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention 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 all specialties and scientific words and the one skilled in the art who uses in literary composition is familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.

Test materials and method

Material and plasmid

The anti-mouse of the donkey of horseradish peroxidase combination and anti-rabbit igg: available from Jackson immune Research laboratory.

Filipin (Filipin) is available from Sigma.

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

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

The cholesterol purifying is the purity higher than 99%.

Degrease serum (LPDS, d＞1.215g/ml): obtain by ultracentrifugation from newborn calf serum.

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

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, Growth of Cells is added 10%FBS in culture medium A (Dulbecco ' Eagle ' the s substratum of s improvement, contain 100 units/ml penicillin and 100 μ g/ml Streptomycin sulphates).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 that the CDX of 50 μ M mevalonic acid and different concns is coated.The method of the coated sterol of CDX is seen and is had been reported (Brown, 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 is amino) hexanoate) of 1mg/ml.Buffer A (20mM Tris-HCl (pH 8.0) and 150mM NaCl) is washed 2 times cells and then cell is placed in 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 gp78 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.Cell is fixing rear with 50 μ g/ml Filipin dyeing 30min.The Filipin signal is observed with Zeiss LSM 510 confocal microscope two-photons, exciting light 720nm.

Co-immunoprecipitation

After cell was carried out relevant treatment, cracking is (PBS, 5mM EGTA, 5mMEDTA, 0.5%Digitonin) in the IP damping fluid.On centrifuging and taking, the cleer and peaceful sepharose 4B that contains GFP antibody mixes, and 4 ℃ in conjunction with 2 hours.Remove supernatant, wash sepharose 4B 5 times with the IP damping fluid.With the acetic acid of PH2.8/glycine elutriant wash-out.-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 arbitrarily 100 cells as quantitative object.With Image ProPlus 5.01 software fluorescent quantitations.The total quantitative method of fluorescence intensity of cell is to select this cell with AOI, cuts off background after the calculating fluorescence intensity.The quantitative method of intracellular fluorescence intensity is to be close to AOI to draw a circle under cytolemma, quantitatively the fluorescence intensity in circle.What show in figure is the total fluorescence intensity of fluorescence intensity ratio cell in cell.

[ ³H] cholesterol is in conjunction with experiment

After cell was carried out relevant treatment, cracking is (PBS, 1%NP-40,5mM EGTA, 5mM EDTA) in conjunction with the IP damping fluid.On centrifuging and taking, the cleer and peaceful sepharose 4B that contains EGFP antibody mixes, 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, is dissolved in binding buffer liquid (PBS, 0.001%NP-40).4 ℃ in conjunction with 4 hours, washes sepharose 4B 5 times with binding buffer liquid, with the acetic acid of PH2.8/glycine elutriant wash-out.In conjunction with [ 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 respectively final concentration is the 10 nonisotopically labelled sterols of μ M.Wherein, [ ³H] cholesterol of mark is available from PerkinElmer company.

Fluidic cell detects

Cell is carried out relevant treatment, after trysinization, dispel into unicellular.Then hatched on ice 30 minutes with Anti-Myc monoclonal antibody IgG-9E10, 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, in the expression amount of NPC1L1-EGFP in the stably express strain and some hepatic cell lines (HepG2, huh7 and L02), endogenous NPC1L1 protein expression amount is similar.Therefore the inventor studies the location of NPC1L1 albumen with this cell strain.

Process 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 namely first reduce intracellular cholesterol with CDX, and then pass the coated cholesterol of cell CDX.

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

The CRL-1601/NPC1L1-EGFP cell is done to process 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 impact between cell clone, inventor's method with the biotin labeling plasmalemma protein in the L02 cell has detected the location of endogenous NPC1L1 albumen under the different condition.Result shows that endogenous NPC1L1 albumen is subject 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 characteristic of endogenous NPC1L1 albumen.

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

Cholesterol in Cell uptake low-density lipoprotein (LDL) is to realize by the endocytosis of ldl receptor.The endocytosis prompting NPC1L1 albumen of NPC1L1 albumen may be by the absorption of similar process mediation cholesterol.In order to verify this idea, the inventor comes cholesterol in 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 localization with two-photon copolymerization Jiao.Result after reducing cholesterol with cyclodextrin (CDX) processing cell, almost be can't see the signal of Filipin as 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).Can see that with the medium treatment cell of passing cholesterol in cell, the Filipin signal strengthens gradually, 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 endocytosis of NPC1L1 albumen, endocytosis also occurs in cholesterol.Contain the vesica of NPC1L1 albumen and the vesica of cholesterol location is altogether arranged well.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.The location of finding NPC1L1-EGFP is is also regulated and controled, and crosses the more cholesterol of Cell uptake (Fig. 3 C) of the cell ratio contrast of expressing NPC1L1-EGFP.

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

In addition, the inventor has also observed effluxing of cholesterol, finds that NPC1L1 albumen does not affect cholesterol efflux.

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

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, the compositions such as μ 2 and σ 2, and known its function is to enter in the coated vesicle of clathrin in conjunction with albumen to participate in endocytosis.Co-immunoprecipitation has been verified mass spectral:mass spectrographic result, and NPC1L1-EGFP is described, μ 2 and CHC see Fig. 4 in same complex body.

Mass Spectrometric Identification to all NPCs 1L1 protein-binding protein (Band1-7) in 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 the endocytosis of Transferrins,iron complexes (Tranferrin, TnR), and RNAi effectively (Fig. 6 B) is described.

With the processing shown in CRL-1601/NPC1L1-EGFP cell mapping 6A, fix, dye and use the burnt fluorescence localization of observing of two-photon copolymerization at different time points.The endocytosis of the analysis of NPC1L1 albumen endocytosis being found NPC1L1 albumen also depends on this approach, and RNAi μ 2 or CHC can suppress the endocytosis of 50%NPC1L1 albumen and the picked-up of cholesterol (Fig. 5 B).Experiment with the biotin labeling cytoplasmic membrane has also proved conclusions (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 endocytosis and the cholesterol picked-up (Fig. 6 D-E) of NPC1L1 albumen.

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

Embodiment 4Ezetimibe suppresses NPC1L1 albumen and cholesterol endocytosis

Ezetimibe is the effective inhibitor of cholesterol absorption, and is arrived the clinical treatment hypercholesterolemia by approval.And known NPC1L1 protein binding Ezetimibe.It is also indefinite but how Ezetimibe suppresses the absorption of the protein mediated cholesterol of NPC1L1.

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

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

Ensuing co-immunoprecipitation is tested and is shown, the endocytosis of the NPC1L1 albumen that the Ezetimibe cholesterol is induced is to enter by suppressing NPC1L1 albumen (Fig. 8 E) that the coated vesicle of clathrin is realized.The picked-up of the endocytosis of endogenous NPC1L1 albumen and cholesterol also is subject to the inhibition (Fig. 8 F, G, H) of Ezetimibe in the L02 cell.

In a word, Ezetimibe enters the coated vesicle of clathrin and suppresses the endocytosis of NPC1L1 albumen and the picked-up of cholesterol by suppressing NPC1L1 albumen.

The NH of embodiment 5NPC1L1 albumen 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 be purified into from the CRL1601/NPC1L1-EGFP cell its carboxyl terminal with the NPC1L1 albumen of people's total length of EGFP (the GenBank accession number: FJ481111), then add [ ³H] after the cholesterol of mark, detect NPC1L1 albumen can in conjunction with [ ³H] cholesterol, as 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 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 ₂The impact of end 18-260 amino acid structure territory on cholesterol absorption, in rat hepatocytes CRL-1601, transient transfection is expressed people NPC1L1-EGFP albumen respectively, or NH ₂The people NPC1L1-EGFP albumen of-end 18-260 aminoacid deletion is fixed at different time points, filipin dyeing, and use the two-photon confocal microscopy.By to each time point cell inner cholesterol content, and in born of the same parents, NPC1L1 albumen carries out quantitative analysis, finds that total length NPC1L1 albumen gets back in cell gradually, absorbs simultaneously a large amount of cholesterol and enters cell; And expression NH ₂-end 18-260 aminoacid deletion NPC1L1 albumen gets back in 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 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

Due to NPC1L1 albumen can with the cholesterol specific binding, inventor imagination if find the combination 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 and the combination of NPC1L1 albumen (Figure 10 B).

Further, process the CRL-1601/NPC1L1-EGFP cell with the 25-oxycholesterol, fix, dye and use two-photon confocal microscopy fluorescence localization at different time points, by quantitative analysis is carried out in the location of NPC1L1 albumen in cell inner cholesterol content and cell, find that the 25-oxycholesterol can significantly suppress the absorption of cholesterol and the endocytosis of NPC1L1 albumen (Figure 10 C).

In sum, compound 25-oxycholesterol can pass through the combination of competition cholesterol and NPC1L1, thereby suppresses cell to the picked-up of cholesterol.Therefore, the NH of NPC1L1 albumen 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 after 986 and 987 insertion 3 * Myc of NPC1L1 aminoacid sequence, and NPC1L1 still has normal physiological function

In order further to study topological framework and the function of NPC1L1, the inventor explores through a large amount of, identify and insert after the 986th amino acids residue of NPC1L1 albumen that 3 * Myc label does not affect the transhipment of NPC1L1 and to the absorption (Figure 11,12) of cholesterol.

Under normal culture condition, NPC1L1/S986-3 * Myc-L987 mainly be positioned at nucleus other zone (Figure 11 B ,-60min).Process cell 1 hour (Figure 12 B, 0min) when the cyclodextrin with 1.5%, cellular cholesterol levels is reduced, NPC1L1/S986-3 * Myc-L987 albumen is transported to cytoplasmic membrane.When sending the coated cholesterol of cyclodextrin after 1 hour to cell again, NPC1L1/S986-3 * Myc-L987 endocytosis again is interior and navigate to ERC (Figure 12 B, 60min) to tenuigenin.This circulation is located 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 method for Filipin dyeing.At first remove the cholesterol of cytolemma with cyclodextrin, and then send cholesterol, observe the absorbing state of cholesterol with Filipin dyeing.As Figure 11 B, shown in 11C, do not having under the condition of Ezetimibe, NPC1L1/S986-3 * Myc-L987 can carry a large amount of cholesterol and enter cell.Under the condition that 10 μ M Ezetimibe process, NPC1L1/S986-3 * Myc-L987 part is advanced tenuigenin by endocytosis, and cholesterol also part is absorbed.Yet when processing cell with 40 μ M Ezetimibe, NPC1L1/S986-3 * Myc-L987 is suppressed on 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 present embodiment cell used is CRL1601, is transfected into the method for transient transfection the pEGFP-N1 plasmid that contains NPC1L1/S986-3 * Myc-L987 expression cassette.

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

According to the research of contriver to the NPC1L1 topological framework, the contriver thinks, when on NPC1L1/S986-3 * Myc-L987 albumen transporte to cells plasma membrane, 3 * Myc label will be exposed to extracellular matrix.If impermeable cell (not penetratingly refers to not penetrating cytolemma, with this understanding, extraneous small molecules (as antibody) can not enter in cell, the NPC1L1 on antibody labeled cell film surface, NPC1L1 in can not labeled cell) with the antibody incubation of anti-Myc, only have the NPC1L1/S986-3 * Myc-L987 on cytolemma can be by the Myc antibody test, therefore can utilize these characteristics to come NPC1L1 on the labeled cell film.

As shown in Figure 12 B, add Myc antibody in there is no penetrating cell, then use fluorescently-labeled two anti-detections, only has detected (Figure 12 B of NPC1L1/S986-3 * Myc-L987 on cytolemma, 0min), intracellular NPC1L1/S986-3 * Myc-L987 can not be detected (Figure 12 B ,-60min and 60min).(penetrating referring to punched on cytolemma and at complete penetrating cell, extraneous 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).The method can be analyzed the Subcellular Localization of NPC1L1 quickly and easily.

For the protein localization to NPC1L1 is more furtherd investigate, 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 Subcellar location of NPC1L1/S986-3 * Myc-L987-EGFP albumen changes.Discovery that can be clearly, after CDX processed, the amount of the NPC1L1/S986-3 * Myc-L987-EGFP on cytolemma obviously increased (Figure 12 C, D).This is again a kind of method of analyzing quickly and easily NPC1L1 protein subcellular location.

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 process cell 5 minutes, obtains penetrating cell.

Embodiment 7 to embodiment 8 shows, the contriver has built a new NPC1L1 protein expressing plasmid with 3 * Myc label, and when the NPC1L1 that expresses was positioned on 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 quickly and easily NPC1L1 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

Take about 20 kinds of compounds as primary screening, will express the cell kind of NPC1L1-EGFP in 12 orifice plates;

Process cell with CDX after 48 hours, reduce intracellular cholesterol, make the NPC1L1 protein localization in cytolemma;

Processed cell one hour with candidate drug;

To contain the cholesterol that adds CDX to be coated with in the substratum of candidate drug;

Observe the location of NPC1L1 albumen after one hour, find out the medicine that affects the NPC1L1 protein localization.

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

2. dusting cover

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

Fix Filipin dyeing and mounting at different time points;

Observe with Two Photon Fluorescence, take pictures, the intracellular NPC1L1 of fluorescent quantitation and cholesterol (Figure 13 C-D).As seen, β, beta-dimethyl-acry-lalkannin (C6) can suppress the endocytosis of NPC1L1 of general 50% left and right and the absorption of cholesterol.So β, beta-dimethyl-acry-lalkannin can be used as the compound of a potential inhibition cholesterol absorption.

Two. screening method is as follows:

The cell that this screening method adopts is CRL1601, wherein is transfected into the NH that contains NPC1L1 albumen ₂The pEGFP-N1 plasmid of-structural domain (18-260 amino acid) expression cassette, thus make cytotostatic express the NH of NPC1L1 albumen ₂-structural domain (18-260 amino acid)-EGFP, described cell called after CRL-1601/NPC1L1 (18-260)-EGFP, after lysis, utilize anti-EGFP antibody mediated immunity purifying to obtain NPC1L1 (18-260)-EGFP albumen, and add in albumen [ 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 of mark and the NH of NPC1L1 albumen ₂End structure territory (18-260 amino acid) in conjunction with situation, as the NH of cholesterol in test group and NPC1L1 albumen ₂The binding capacity in end structure territory significantly lower than in control group [ ³H] cholesterol of mark and the NH of NPC1L1 albumen ₂The binding capacity in end structure territory (low more than 30%), described candidate substances is the decreasing cholesterol material.

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

Discuss

The inventor's data show that Ezetimibe suppresses the absorption of cholesterol by the endocytosis that suppresses NPC1L1 albumen.Its molecular mechanism is that Ezetimibe affects NPC1L1 albumen and enters the coated vesicle of clathrin.What is interesting is that Ezetimibe does not affect 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 affects NPC1L1 albumen enter the coated vesicle of clathrin and 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 human genome, SCAP, NPC1, Patched, TRC8, and NPC1L1.The sterol receptive field that can infer NPC1L1 albumen may directly in conjunction with cholesterol, cause endocytosis thereby be combined with clathrin/AP2 complex body by conformational change.Ezetimibe may compete the combination of cholesterol and NPC1L1 albumen.Another kind may be that the conformation that Ezetimib can affect NPC1L1 albumen causes cholesterol can not promote NPC1L1 albumen to be combined with clathrin/AP2 complex body.The distribution that Ezetimibe also may affect the cholesterol around NPC1L1 albumen causes preventing of NPC1L1 albumen endocytosis.

The people obtains cholesterol by de novo synthesis and food absorption.Compare with the medicine (as: Statins) that suppresses cholesterol biosynthesis, Ezetimibe is the medicine of inhibition cholesterol absorption unique on market.Because different Id difference causes different people different to the sensitivity of 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 system that the cell levels observation NPC1L1 protein subcellular that the inventor sets up is located can be used for the inhibitor of screening 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: free diffusing, assistance diffusion, active transport and endocytosis and exocytosis.Wherein free diffusing 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; Assisting diffusion is that the small molecules of the channel protein mediation on cytoplasmic membrane is from the high side of concentration to a low side, as ionic channel etc.; Active transport requires the expenditure of energy, and it is also 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 again clathrin-mediated endocytosis, the endocytosis of caveolin mediation and the endocytosis of other unknown forms.

Assisting the mediating proteins 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 cytolemma.

Ldl receptor is cholesterol transportation and the common molecular receptor of endocytosis, it is characterized in that the membranin of a cross-film.Its extracellular region territory can in conjunction with LDL or VLDL, then be raised the relevant albumen of endocytosis by some special motif in kytoplasm and carry out endocytosis.And for NPC1L1, it contains general 13 cross-film zones, and the NPC1L1 promotion is 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.The bimolecular complex body that the albumin A BCG5/8 that ironically participates in cholesterol efflux is comprised of the albumen of two six cross-films, 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 usually very natural can 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 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 coated cholesterol specific ionization of CDX is easier in cell cultures; Secondly, if NPC1L1 is a molecular pump, when to cellular cholesterol, should see that cholesterol is introduced into cell so, but the inventor sees is that NPC1L1 and cholesterol enter in cell simultaneously; At last, if NPC1L1 is a molecular pump, no matter reduce the NPC1L1 endocytosis with actin inhibitor or reticent clathrin, all will increase the absorption of cholesterol, but the inventor sees is also suppressed lived of cholesterol absorption.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 molecular pump, moreover free unit molecule cholesterol is toxic to cell, the unit molecule cholesterol pumps in tenuigenin if NPC1 or NPC1L1 directly will dissociate, and is very unfavorable to cell health.

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

All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.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 limited range equally.

Sequence table

＜110〉Shanghai Inst. of Life Science, CAS

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

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Claims

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

(1) process the cell of expressing NPC1L1 albumen with cholesterol, and the degree of definite cell vesicle endocytosis NPC1L1 albumen;

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

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

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

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

(3) in, also comprise: the combination degree that compares (1) and (2) middle clathrin and NPC1L1 albumen;

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

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

(3) in, also comprise: the combination degree that compares (1) and (2) middle relevant albumen of AP2 complex body and NPC1L1 albumen;

Wherein, if in (2), the combination degree of the relevant albumen of AP2 complex body and NPC1L1 albumen is statistically lower than the combination degree of (1) middle relevant albumen of AP2 complex body and NPC1L1 albumen, described candidate substances is potential decreasing cholesterol material.

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

6. the method for claim 1, is characterized in that, described NPC1L1 albumen also connects label protein, and described label protein is positioned at the extracellular region territory of NPC1L1 albumen.

7. method as claimed in claim 6, is characterized in that, described label protein is between the 8th cross-film district of NPC1L1 albumen and the 9th cross-film district.

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

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

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

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

(2) in the situation that 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 label protein percentage on cytolemma, the ratio of NPC1L1 protein localization on cytolemma obviously increases, and then shows that the degree of cell vesicle endocytosis NPC1L1 albumen 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, select and determine for the useful material of decreasing cholesterol.

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

(b) detect protein expression or activity described in described cell;

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

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

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

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

15. the purposes of material in the preparation anticholesteremic agent that suppresses NPC1L1 albumen endocytosis, the material of described inhibition NPC1L1 albumen endocytosis is β, beta-dimethyl-acry-lalkannin or 25-oxycholesterol.

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

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

17. a method of screening potential decreasing cholesterol material is characterized in that described method comprises: with the NH of candidate substances and NPC1L1 albumen ₂The contact of end structure territory, the NH of detection candidate substances and NPC1L1 albumen ₂The end structure territory in conjunction with situation; NH as candidate substances and NPC1L1 albumen ₂Specific combination occurs in the end structure territory, and described candidate substances is potential decreasing cholesterol material.

18. method as described in claim 16 or 17 is characterized in that, the NH of described NPC1L1 albumen ₂18-260 amino acids in sequence is contained shown in GenBank accession number FJ481111 in the end structure territory.

19. a fusion rotein is characterized in that described albumen comprises NPC1L1 albumen and label protein, and described label protein is positioned at the extracellular region territory of NPC1L1 albumen; The aminoacid sequence of described NPC1L1 albumen is as shown in GenBank accession number FJ481111; The people NPC1L1 albumen that described NPC1L1 albumen is total length, and described label protein is between the 986th amino acids on NPC1L1 albumen and the 987th amino acids; Described label protein is the Myc label protein.

20. a nucleotide sequence is characterized in that, the described fusion rotein of described nucleic acid sequence encoding claim 19.

21. a recombinant vectors is characterized in that, described recombinant vectors contains the described nucleotide sequence of claim 20.

22. a host cell is characterized in that, described host cell contains the described recombinant vectors of claim 21, or is integrated with the described nucleotide sequence of claim 23 in its genome.

23. the purposes of the described host cell of claim 22 is characterized in that, is used for analyzing the Subcellar location of NPC1L1 albumen.