国家人类基因组北方研究中心.ppt

单击此处编辑母版标题样式,*,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,Protein Complex and Protein-protein Interaction,彭鲲鹏,国家人类基因组北方研究中心,Email:pengkp,Central dogma:the story of life,RNA,DNA,Protein,Protein is the final player in cell life,Proteins function in association with other proteins or biomolecules,but not in isolation,Introduction to Proteomics,the analysis of genomic complements of proteins,dynamic,systematic,discovery-driven,Goals of Proteomics,to discover,protein function,to understand,cellular processes,to understand,disease states,to discover,drug target,to identify,biomarker,Types of Proteomics,ExpressionProteomics,Quantitative study of protein expression,and their changes,between samples that differs by some variable,Functional Proteomics,To study,protein-protein interaction,3-D structures,cellular localization and PTM,s,in order to understand the physiological function of the whole set of proteome.,Blue Native PAGE,detergent,CBB,6-,ACA,_,+,Blue Native PAGE,Sample Preparation,Blue Native PAGE,SDS-PAGE,Solubilization with nonionic,detergent(laurylmaltoside,TX-100,CHAPS,Mega 9,octylglucoside,Brij 35,etc),supplemented with,6-aminocaproic acid,Separation gel:6-13%gradient,Cathode buffer contains0.02%Coomassie blue G250,Separation of members of,multiprotein complex,Blue Native PAGE of chloroplast thylakoid membranes,BBRC 1999,259:569-575,BN-PAGE of solubilized chloroplast thylakoid membranes(a),followed by SDSPAGE in the second dimension(b).,CF,0,F,1,ATP synthase was indicated.,Blue Native PAGE of chloroplast thylakoid membranes,BBRC 1999,259:569-575,lane 1:LMW marker,lane 2:CF,0,F,1,ATP synthase,purified by density gradient centrifugation,lane 3:electroeluted protein from the intense band(Rf=,0.38)in BN-PAGE(a).,Blue Native PAGE of multiprotein complex from whole cellular lysate,MCP 3:176-182,2004,Dialysis permits the analysis of multiprotein complexes of whole cellular lysates by BN-PAGE.,Identification and analysis of distinct proteasomes by WCL 2D BN/SDS-PAGE,A,WCL of HEK293 cells was separated by 2D BN/SDS-PAGE(5.514 and 10%,respectively),and immunoblotting was performed with specific antibodies recognizing either subunits of the 20S core complex(Mcp21 and 2),or a subunit of the 19S cap of the 26S proteasome(S4 ATPase),or a subunit of the PA28 regulatory subunit(PA28).,B,An identical sample was boiled in 1%SDS,resolved by 2D BN/SDS-PAGE,and immunoblotted as described in,A,.,MCP 3:176-182,2004,Blue Native PAGE,Visualization of MPCs on a 2D WCL BN/SDS gel,B,WCL of HEK293 cells was boiled with 1%SDS before separation and staining.,A,WCL of HEK293 cells was prepared using Triton X-100 and separated by 2D BN/SDS-PAGE(5.517 and 10%,respectively).,MCP 3:176-182,2004,Blue Native PAGE,Far Western,Max:functional cloning of a Myc-binding protein,A,.,CKII,casein kinase II phosphorylation site;BR,basic region;HLH,helix-loop-helix;LZ,leucine zipper.,B,.Plaques that express beta-galactosidase fusion prteins were screened for their ability to react with,125,I-labeld GST-MycC92.,Top left,secondary plating of five putative positive demonstrates the reactivity of two of the primary plaques,Max11 and Max14.,Top right,as a negative control,GST was labeled to a similar specific activity and compared with GST-MycC92 for bidning to Max14 plaques.,Bottom,binding of GST-MycC92 to Mzx14 plaques was assayed with or without affinity purified carboxyl terminal-specific anti-Myc(Ab)or peptide immunogen(peptide).,MycC92,Science 251:1211-7,1991,Far Western,Association of Rb with HIP1,HeLa nulear extract(100 ug)(lane 1,2)and HIP1(200 ng)purified from HeLa(lane 3,4)were electrophoresed,blotted,and renatured in situ.Adjacent strips were cut from the filters and probed with,32,P-GST-RB(379-928)(lane 1,3)or,32,P-GST-RB(379-928;706F)(lane 2,4),Cell 70:351-364,1992,Far Western,GST Pulldown,Interactions of Cellular Polypeptides with the Cytoplasmic Domain of the Mouse Fas Antigen,GST Pulldown,JBC 271:8627-32,1996,Fas:45-kilodalton transmembrane receptor that initiates apoptosis;,The biochemical mechanisms responsible for Fas action are incompletely understood;,the cytoplasmic domain is clearly necessary for Fas to function as a receptor;,The cytoplasmic domain does not display any known enzymatic activities but is capable of interacting with a number of proteins.,GST-mFas fusion proteins,GST Pulldown,149,166,204,293,306,1,306,194,194,194,194,194,194,292,283,276,268,221,194,306,306,221,GST-mFas-associated polypeptides from,32,S-labeled HeLa,L929,and Jurkat cell lysates,GST Pulldown,Preclearation,:25 ug GST/50 ul GSH-Seph.,Incubation,:10 ug GST/GST-mFas-(194-306),Wash,:0.5%NP-40,20 mM Tris,pH 8.0,200 mM NaCl,Elution,:50 ul 20 mM GSH in 50 mM Tris,GST-mFas-associated polypeptides are stable to high salt concentrations,GST Pulldown,HeLa cell lysates were screened with either GST or GST-mFas-(194306)as described above except that the Sepharose-protein complexes were washed with Lysis Buffer containing different salt concentrations(as indicated).The eluted material was subjected to 12%SDS-PAGE and fluorography.,Association is blocked by preincubation with a polyclonal antibody against GST-mFas,GST Pulldown,A,.the antibody recognized the Fas intracellular domain;,B,.association of proteins from HeLa lysate with GST-mFas was blocked by anti-GST-mFas IgG;,C,.anti-GST antibody had no effect up to 100,u,g of IgG.,Differential association with mutant forms of GST-mFas,GST Pulldown,HeLa,L929,292,283,276,268,221,Schematic representation of the mouse Fas antigen and its binding proteins,GST Pulldown,Epitope tagging,1,2,3,4,5,6-9,GST Pulldown,Co-Immunoprecipitation,In the intact cell,protein X is present in a complex with protein Y.This complex is preserved after cell lysis and allows protein Y to be coimmunoprecipitated with protein X(complex 1).However,the disruption of subcellular compartmentalization could allow artifactual interactions to occur between some proteins,for example,protein X and protein B(complex 2).Furthermore,the antibody that is used for the immunoprecipitation may cross-react nonspecifically with other proteins,for example,protein A(complex 3).The key to identification of protein:protein interactions by coimmunoprecipitation is to perform the proper controls so as to identify protein Y but not protein A and B.,Co-Immunoprecipitation,Antibody Identification,The protein against which the antibody was raised should be precipitated from cell lysate.,(1)Independent antibodies raised against the same protein recognize the same polypeptide;,(2)Target protein should not be identified with antibodies from cell lines without target protein;,False positive and control,Co-Immunoprecipitation,1.Antibody control,Monoclonal Ab:another MoAb against similar protein,Antiserum:serum before immunization from the same animal,Polyclonal Ab:purified PoAb against another protein,2.Multiple antibodies,different Abs against different epitopes;,the epitope may be the site for association with other proteins;,3.Cell lines depleted of target protein,Control experiment should be practised in depleted cell lines,4.Inactive biological mutant,5.Interaction verification before and after cell lysis,unphysiological interaction,Reduction of nonspecific protein background,Co-Immunoprecipitation,1.,to increase ionic strength in wash buffer;,2.to reduce the amount of primary Ab;,3.to preclear cell lysate with control Ab.,Binding of pVHL to Elongin B and C,Co-Immunoprecipitation,1.von Hippel-Lindau disease is a hereditary cancer syndrome characterized by the development of multiple tumors;,2.VHL susceptibility gene,mutated in the majority of VHL kindreds,is a tumor suppressor;,3.to elucidate the biochemical mechanisms underlying tumor suppression by pVHL,search for cellular proteins that bound to wt pVHL,but not to tumor-derived pVHL mutants.,Science 269:1444-6,1995,Identification of VHL-associated proteins,Co-Immunoprecipitation,Lysates from 786-O renal carcinoma cells,transfected with the indicated pVHL constructs,were immunoprecipitated with anti-HA(A and B)or with anti-VHL(C).,Detection by autoradiography(A,C)or by immunoblotting(B).,open arrows:exo pVHL,closed arrows:VHL-AP,pVHL(1-115),:without residues frequently altered by naturally occurring VHL mutations and,unlike pVHL(wt),does not suppress tumor formation in vivo.,pVHL(167W),:the predicted product of a mutant VHL allele that is common in VHL families.,anti-VHL,Mapping the p14 and p18 binding site on pVHL,Co-Immunoprecipitation,a-HA,A,.786-O cells producing HA-VHL(wt)or HA-VHL(1-115)were labeled with,35,S-methione,lysed,and immunoprecipitated with anti-HA.Parental 786-O cells were similarly labeled,lysed,and incubated with GSH Sepharose preloaded with GST-VHL(117-213)or GST alone.,B and C,.786-O cells were labeled,lysed,and incubated with GSH Sephorase preloaded with the indicated GST-VHL fusion protein.In(C),the indicated peptides(final conc.0.1,1,or 10 uM)were added to the GST-VHL fusion protein before incubation with the radiolabeled extract.The wt peptide is,TL,KE,RC,L,QWR,SLVKP(underlined residues are sites of germ-line missense mutations).The mutant peptide is TLKER,F,LQWRSLVKP.,the binding site for Elongin B and C in pVHL,Co-Immunoprecipitation,Distribution of germ-line VHL mutations.The shaded region represents the bidning site for Elongin B and C.,Binding of pVHL to Elongin B and Elongin C in vivo,Co-Immunoprecipitation,A,.ACHN(VHL+/+),CAKI-1(VHL+/+),786-O(VHL-/-),and 293(VHL+/+)cells were labeled with 35S-methione,lysed,and immunoprecipitated with anti-VHL or a control antibody.The immunoprecipitaes were washed under high-salt conditions.The identification of pVHL(wt)(open arrow)was confirmed by anti-pVHL immunoblot analysis.The 19 kD protein immediately above p18(*)in the ACHN,CAKI-1,and 293 cell anti-VHL immunoprecipitates reacts with a polyclonal antibody to VHL.,B.Comparison of peptides generated by partial proteolysis of Elongin B and C,translated in vitro,with p18 and p14.,TAP:tandem affinity purification,Sequence and structure of the TAP tag,CBP,TEV,Ig BD,bait,TAP,Overview of the TAP procedure,TAP,Schematic representation of the split TAP tag strategy,TAP,Schematic representation of the substraction strategy,TAPProtein composition of TAP-purified U1 snRNP,TAP,Step-by-step analysis of the TAP strategy,TAP,Proteins present in the final TAP fraction(lanes 7 and 8),or present after each of the single affinity purification steps(lanes 14),were analyzed.Snu71-TAP(lanes 1,3,and 7)or wild-type extracts(lanes 2,4,and 8)were used.Lane 5:molecular weight marker.Lane 6:an amount of TEV protease identical to the amount used to elute proteins bound to IgG beads(lanes 2,3,7,and 8).Right arrows indicate the U1 snRNP-specific proteins including the tagged Snu71p after TEV cleavage;the arrow on the left indicates the Snu71p protein fused to the TAP tag before TEV cleavage.,TAP in higher eucaryotes,TAP,Questions:,overexpression,endogenous expression,Solutions:,RNA interference,Knockin technique,Strengths and weaknesses of commonly used affinity approaches for the retrieval of protein complexes,FRET:fluorescence resonance energy transfer,When will FRET occur?,1)Spectral overlap,Donor emission spectrum must,significantly overlap the absorption,spectrum of the acceptor(30%),2)Distance between the donor and,acceptor is between 2-10 nm,3)Favorable orientation of fluorophores,2 10 nm,Donor,emission,Acceptor,absorption,FRET,E:energy transfer efficiency,R,0,:intermolecular distance when half of energy is transfered,r:distance between fluorophores,E=R,0,6,/(R,0,6,+r,6,),when r=2R,0,E=1/65,R,0,=4.9 nm,Imaging protein phosphorylation by FRET,target,GFP,Fab,Cy3,transfection,microinjection,or incubation,target,GFP,Fab,Cy3,activator,laser,FRET,Detection of protein interaction by FRET,target,GFP,Fab,Cy3,Protein 1,CFP,Protein 2,YFP,FRET,Protein 1,Cy3,Protein 2,FITC,in vitro,phosphorylation,in vivo,FRET reveals interleukin(IL)-1-dependent aggregation of IL-1 type I receptors that correlates with receptor activation,FRET,JBC 270:27562-8,1995,Abbreviation,FRET,IL-1:interleukin 1,IL-1 RI:IL-1 type I receptor,IL-1ra:IL-1 receptor antagnist,CHO-mu1c:CHO-K1 cells stably transfected with wild-type IL-1 receptor,CHO-extn:CHO-K1 cells stably transfected with cytoplasmic tail-truncated IL-1 receptor,M5:noncompetitive anti-IL1 RI monoclonal antibody,FITC-M5:M5 labeled with a donor probe,FITC,Cy3-M5:M5 labeled with a acceptor probe,Cy3,IL-1,a,-dependent FRET between donor FITC-M5 and acceptor Cy3-M5 bound to IL-1 RI on the surface of CHO-mu1c cells,FRET,A,a mixture of 5 nM FITC-M5 and 5 nM Cy3-M5 was incubated with CHO-mu1c cells(3 X 10,6,cells/ml)containing wild-type transfected receptors for 50 min at 22 C.IL-1a or IL-1ra was added at a final concentration of 30 nM immediately after the time point at t=0 min(arrow),and changes in the ratio of Cy3-M5 fluorescence to FITC-M5 fluorescence were monitored over time.Changes in this ratio were also monitored for the control sample to which no ligand was added.,B,normalized fluorescence ratio for cells with added IL-1a or IL-1ra calculated from data in A.,IL-1a,IL-1ra,control,IL-1a,IL-1ra,IL-1a but not IL-1ra causes aggregation between IL-1 RI-labeled with FITC and Cy3 Fab fragments of M5 as detected by FRET,FRET,A mixture of 20 nM FITC-M5-Fab and 20 nM Cy3-M5-Fab was added to CHO-mu1c cells transfected with wild-type receptors and incubated at 22 C for 50 min.IL-1a or IL-1ra was added to a final concentration of 10 nM immediately after the time point at 0 min.Changes in the normalized ratio of Cy3-M5 Fab fluorescence to FITC-M5 Fab fluorescence were monitored over time at 22 C.,IL-1a,IL-1ra,IL,-1-dependent energy transfer between IL-1 RI is temperature,FRET,A mixture of 20 nM FITC-M5 Fab and 12 nM Cy3-M5 Fab was added to CHO-mu1c cells(3 X 106,cells/ml)with transfected wild-type IL-1 RI and preincubated at either 4 C(A)or 22 C(B)for 50 min.Immediately after the base-line data point at t=0 min,IL-1a was added(arrow)at a final concentration of 10 nM to both samples.Changes in the normalized ratio of Cy3-M5 Fab fluorescence to FITC-M5 Fab fluorescence was monitored over time at the corresponding preincubation temperature.At t=85 min,the temperature for sample(A)was changed from 4 to 22 C,and the temperature for sample(B)was changed from 22 to 4 C.Changes in the normalized fluorescence ratio continued to be monitored until t=180 min.A,B,IL-1a-dependent FRET can be detected between FITC-M5 Fab and Cy3-M5 Fab bound to the cytoplasmic tail deleted mutant IL-1 RI on CHO-extn cells,FRET,A mixture of 20 nM FITC-M5 Fab and 12 nM Cy3-M5 Fab was added to wild-type transfected receptors on CHO-mu1c cells and incubated at 22 C for 50 minA).A mixture of 20 nM FITC-M5 Fab and 12 nM Cy3-M5 Fab was added to CHO-extn cells(cytoplasmic tail deleted mutant IL-1 RI)and incubated at 22 C for 50 min(B).IL-1a was added to a final concentration of 20 nM at the arrow,and changes in the normalized ratio of Cy3-M5 Fab fluorescence to FITC-M5 Fab