Protein Complex and Protein-protein Interaction 彭鲲鹏 国家人类基因组北方研究中心 Email: firstname.lastname@example.org
Central dogma: the story of life Protein is the final player in cell life DNA RNA Protein
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 drug target to understand cellular processes to discover protein function to identify biomarker to understand disease states
Types of Proteomics • Expression Proteomics • 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 PTMs in order to understand the physiological function of the whole set of proteome.
Approaches Genetic: yeast two-hybrid phage display Biophysical: Mass Spectrometry SPR FRET Biochemical: Blue native PAGE Far Western Pull-down Coimmunoprecipitation TAP Crosslinking Bioinformatic: Co-occurrence Neighborhood Surface patch
Blue Native PAGE • separation of native proteins in complex. • Coomassie Blue G: stable and negatively charge multiprotein complex. • 6-aminocaproic acid: solubilize membrane protein complex instead of salts. • the resolution is not so high that the prepurification is needed. Anal Biochem 1991, 199:223-231
Blue Native PAGE _ detergent CBB 6-ACA +
Blue Native PAGE Solubilization with nonionic detergent (laurylmaltoside, TX-100, CHAPS, Mega 9, octylglucoside, Brij 35, etc), supplemented with 6-aminocaproic acid Sample Preparation Blue Native PAGE Separation gel: 6-13% gradient Cathode buffer contains 0.02% Coomassie blue G250 SDS-PAGE Separation of members of multiprotein complex
Blue Native PAGE of chloroplast thylakoid membranes BN-PAGE of solubilized chloroplast thylakoid membranes (a) followed by SDS–PAGE in the second dimension (b). CF0F1 ATP synthase was indicated. BBRC 1999, 259:569-575
Blue Native PAGE of chloroplast thylakoid membranes lane 1: LMW marker lane 2: CF0F1 ATP synthase, purified by density gradient centrifugation lane 3: electroeluted protein from the intense band (Rf=0.38) in BN-PAGE (a). BBRC 1999, 259:569-575
Blue Native PAGE of multiprotein complex from whole cellular lysate Dialysis permits the analysis of multiprotein complexes of whole cellular lysates by BN-PAGE. MCP 3:176-182, 2004
Blue Native 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 (5.5–14 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 A, WCL of HEK293 cells was prepared using Triton X-100 and separated by 2D BN/SDS-PAGE (5.5–17 and 10%, respectively). B, WCL of HEK293 cells was boiled with 1% SDS before separation and staining. MCP 3:176-182, 2004
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 125I-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 32P-GST-RB(379-928) (lane 1, 3) or 32P-GST-RB(379-928;706F) (lane 2, 4) Cell 70:351-364, 1992
GST Pulldown Interactions of Cellular Polypeptides with the Cytoplasmic Domain of the Mouse Fas Antigen 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. JBC 271:8627-32, 1996
GST Pulldown 1 149 166 204 293 306 194 306 194 292 194 283 194 276 194 268 194 221 194 306 221 306 GST-mFas fusion proteins
GST Pulldown GST-mFas-associated polypeptides from 32S-labeled HeLa, L929, and Jurkat cell lysates 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 Pulldown GST-mFas-associated polypeptides are stable to high salt concentrations HeLa cell lysates were screened with either GST or GST-mFas-(194–306) 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.
GST Pulldown Association is blocked by preincubation with a polyclonal antibody against GST-mFas 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 ug of IgG.
GST Pulldown HeLa 292 268 221 283 276 L929 Differential association with mutant forms of GST-mFas
GST Pulldown Schematic representation of the mouse Fas antigen and its binding proteins
GST Pulldown 1 2 3 4 5 6-9 Epitope tagging
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;
Co-Immunoprecipitation False positive and control 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
Co-Immunoprecipitation Reduction of nonspecific protein background 1. to increase ionic strength in wash buffer; 2. to reduce the amount of primary Ab; 3. to preclear cell lysate with control Ab.
Co-Immunoprecipitation Binding of pVHL to Elongin B and C 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
Co-Immunoprecipitation anti-VHL Identification of VHL-associated proteins 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.
Co-Immunoprecipitation a-HA Mapping the p14 and p18 binding site on pVHL A. 786-O cells producing HA-VHL(wt) or HA-VHL(1-115) were labeled with 35S-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 TLKERCLQWRSLVKP (underlined residues are sites of germ-line missense mutations). The mutant peptide is TLKERFLQWRSLVKP.
Co-Immunoprecipitation the binding site for Elongin B and C in pVHL Distribution of germ-line VHL mutations. The shaded region represents the bidning site for Elongin B and C.
Co-Immunoprecipitation Binding of pVHL to Elongin B and Elongin C in vivo 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 bait CBP TEV Ig BD Sequence and structure of the TAP tag
TAP Overview of the TAP procedure
TAP Schematic representation of the split TAP tag strategy
TAP Schematic representation of the substraction strategy
TAP Protein composition of TAP-purified U1 snRNP
TAP Step-by-step analysis of the TAP strategy Proteins present in the final TAP fraction (lanes 7 and 8), or present after each of the single affinity purification steps (lanes 1–4), 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 TAP in higher eucaryotes Questions: overexpression endogenous expression Solutions: RNA interference Knockin technique
Strengths and weaknesses of commonly used affinity approaches for the retrieval of protein complexes
When will FRET occur? Acceptor absorption Donor emission 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 2 ~ 10 nm 3) Favorable orientation of fluorophores FRET: fluorescence resonance energy transfer
R0 = 4.9 nm FRET E: energy transfer efficiency R0: intermolecular distance when half of energy is transfered r: distance between fluorophores E = R06/(R06 + r6) when r = 2R0, E = 1/65
FRET Fab Fab Cy3 Cy3 target GFP microinjection or incubation transfection laser target GFP activator Imaging protein phosphorylation by FRET
FRET Fab Cy3 target GFP Protein 2 FITC Protein 2 YFP Protein 1 CFP Protein 1 Cy3 Detection of protein interaction by FRET in vitro phosphorylation in vivo
FRET FRET reveals interleukin (IL)-1-dependent aggregation of IL-1 type I receptors that correlates with receptor activation JBC 270:27562-8, 1995