Protein Complex and Protein protein Interaction

  

Protein Complex and

Protein Complex and

  

Protein-protein Interaction

Protein-protein Interaction

  彭彭彭 国家人类基因组北方研究中心 Email: pengkp@yahoo.com.cn

  

Protein is the final player in cell life

Protein is the final player in cell life

  Central dogma: Central dogma: the story of life the story of life DNA RNA Protein

  

Proteins function in association with

Proteins function in association with

other proteins or biomolecules, but

other proteins or biomolecules, but

not in isolation

not in isolation

  Introduction to Proteomics Introduction to Proteomics

   the analysis of genomic complements of proteins

   dynamic

   systematic

   discovery-driven

Goals of Proteomics

  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

Types of Proteomics

   Expression Proteomics

• – their changes between samples that differs by some variable

  Quantitative study of protein expression and

  

Functional Proteomics

• – structures, cellular localization and PTMs in order to understand the physiological function of the whole set of proteome.

  To study protein-protein interaction , 3-D

  

Approaches

Approaches

  Genetic: Biophysical: yeast two-hybrid Mass Spectrometry phage display SPR

  FRET Biochemical: Blue native PAGE Far Western

Bioinformatic:

  Pull-down Co-occurrence Coimmunoprecipitation Neighborhood TAP Surface patch Crosslinking

  Blue Native PAGE 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.

  

Blue Native PAGE

Blue Native PAGE

  _

  detergent CBB 6-ACA

Blue Native PAGE

Blue Native PAGE

  Solubilization with nonionic detergent (laurylmaltoside, TX-100, Sample Preparation CHAPS, Mega 9, octylglucoside,

  Brij 35, etc), supplemented with 6-aminocaproic acid 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 Blue Native PAGE of chloroplast thylakoid membranes thylakoid membranes

  BN-PAGE of solubilized chloroplast thylakoid membranes (a) followed by SDS–PAGE in the second dimension (b). CF F ATP synthase was ₁ indicated. Blue Native PAGE of chloroplast Blue Native PAGE of chloroplast thylakoid membranes thylakoid membranes

  lane 1: LMW marker lane 2: CF F ATP synthase, ₁ 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 Blue Native PAGE of multiprotein complex from whole cellular lysate complex from whole cellular lysate

  Dialysis permits the analysis of multiprotein complexes of whole cellular lysates by BN- PAGE. Blue Native PAGE Blue Native PAGE

  Identification and analysis of distinct proteasomes by WCL 2D BN/SDS-PAGE

  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. Blue Native PAGE Blue Native PAGE

Visualization of MPCs on a 2D WCL BN/ SDS gel

  A, WCL of HEK293 cells was prepared

  B, WCL of HEK293 cells was using Triton X-100 and separated by boiled with 1% SDS before

  2D BN/SDS-PAGE (5.5–17 and 10%, separation and staining. respectively).

  

Far Western

Far Western Far Western Far Western

  Max: functional cloning of a Myc-binding protein Max: functional cloning of a Myc-binding protein A. _MycC9 CKII, casein kinase II phosphorylation ₂ site; BR, basic region; HLH, helix-loop- helix; LZ, leucine zipper.

  B. Plaques that express beta-galactosidase fusion prteins were screened for their ability ₁₂₅ to react with 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). Far Western Far Western

  Association of Rb with HIP1

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 ₃₂ with P-GST-RB(379-928) (lane 1, ₃₂

  3) or P-GST-RB(379-928;706F) (lane 2, 4)

  

GST Pulldown

GST Pulldown GST Pulldown 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. GST Pulldown GST Pulldown _{1 149 166 204 293 306} GST-mFas fusion proteins _{194 292} 194 306 ₁₉₄ 194 283 _{276 194 221}

194 268

_{221 306} 194 ₃₀₆

  GST Pulldown GST Pulldown

  32 GST-mFas-associated polypeptides from S- 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 Pulldown

  GST-mFas-associated polypeptides GST-mFas-associated polypeptides are stable to high salt concentrations 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 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

  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

  GST Pulldown

  GST Pulldown GST Pulldown

  Epitope tagging Epitope tagging ₁

  2

  3 _6-9

  4 ⁵ Co-Immunoprecipitation

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 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 Co-Immunoprecipitation

  False positive and control

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 Co-Immunoprecipitation

  Reduction of nonspecific protein Reduction of nonspecific protein background 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. Binding of pVHL to Elongin B and C Binding of pVHL to Elongin B and C

  Co-Immunoprecipitation Co-Immunoprecipitation

  1. von Hippel-Lindau disease is a hereditary cancer

  1. von Hippel-Lindau disease is a hereditary cancer

  syndrome characterized by the development of multiple

  syndrome characterized by the development of multiple

  tumors;

  tumors;

  2. VHL susceptibility gene, mutated in the majority of

  VHL kindreds, is a tumor suppressor;

  VHL kindreds, is a tumor suppressor;

  3. to elucidate the biochemical mechanisms underlying

  3. to elucidate the biochemical mechanisms underlying

  tumor suppression by pVHL, search for cellular proteins

  tumor suppression by pVHL, search for cellular proteins

  

that bound to wt pVHL, but not to tumor-derived pVHL

  that bound to wt pVHL, but not to tumor-derived pVHL mutants. mutants. Co-Immunoprecipitation Co-Immunoprecipitation

  Identification of VHL-associated proteins

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). _anti-VHL 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 Co-Immunoprecipitation

  

Mapping the p14 and p18 binding

Mapping the p14 and p18 binding

site on pVHL

site on pVHL

  A. 786-O cells producing HA-VHL(wt) or HA- ₃₅ VHL(1-115) were labeled with 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) _a-HA 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 Co-Immunoprecipitation the binding site for Elongin B and C the binding site for Elongin B and C in pVHL in pVHL Distribution of germ-line VHL mutations.

  The shaded region represents the bidning site for Elongin B and C. Co-Immunoprecipitation Co-Immunoprecipitation

  

Binding of pVHL to Elongin B and

Binding of pVHL to Elongin B and

  

Elongin C in vivo

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: tandem affinity purification

TAP: tandem affinity purification Sequence and structure of the TAP tag Sequence and structure of the TAP tag

  CBP TEV

  Ig BD bait TAP

  TAP

  Overview of the TAP procedure Overview of the TAP procedure

  TAP TAP

  Schematic representation of the split TAP tag strategy TAP

  TAP

  Schematic representation of the substraction strategy TAP

  TAP

  Protein composition of TAP- purified U1 snRNP TAP

  TAP

  TAP 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

  TAP in higher eucaryotes 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

  

FRET:

FRET:

fluorescence resonance energy transfer

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 ^{Donor 2 ~ 10 nm emission Acceptor absorption} FRET FRET

  R = 4.9 nm _{6 6 6} E: energy transfer efficiency E = R /(R + r )

  R : intermolecular distance when half of energy is transfered r: distance between fluorophores when r = 2R , E = 1/65 FRET FRET

  Imaging protein phosphorylation by FRET Imaging protein phosphorylation by FRET _{target GFP Fab} Cy3 microinjection transfection or incubation laser _{target GFP Fab}

Cy3 activator FRET FRET

  Detection of protein interaction by FRET Detection of protein interaction by FRET _{Protein 2 target GFP Protein 2 YFP} FITC _{Protein 1 Fab Cy3 Cy3} Protein 1 CFP in vitro phosphorylation in vivo

  

FRET reveals interleukin (IL)-1-

FRET reveals interleukin (IL)-1-

dependent aggregation of IL-1 type I

dependent aggregation of IL-1 type I

receptors that correlates with

receptors that correlates with

receptor activation

receptor activation

  FRET FRET FRET FRET

  Abbreviation

Abbreviation

  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 FRET FRET

  IL-1a -dependent FRET between donor

  IL-1 -dependent FRET between donor FITC-M5 and acceptor Cy3-M5 bound to

  FITC-M5 and acceptor Cy3-M5 bound to

  

IL-1 RI on the surface of CHO-mu1c cells

  

IL-1 RI on the surface of CHO-mu1c cells

_IL-1a

  A, a mixture of 5 nM FITC-M5 and 5 nM Cy3-M5 _{6 IL-1ra ml) containing wild-type transfected receptors for} was incubated with CHO-mu1c cells (3 X 10 cells/ _control 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 _IL-1a fluorescence were monitored over time. Changes in this ratio were also monitored for the control sample to which no ligand was added. B, _IL-1ra normalized fluorescence ratio for cells with added IL-1a or IL-1ra calculated from data in A. FRET FRET

  IL-1a but not IL-1ra causes aggregation

  IL-1a but not IL-1ra causes aggregation between IL-1 RI-labeled with FITC and Cy3 between IL-1 RI-labeled with FITC and Cy3

  

Fab fragments of M5 as detected by FRET

Fab fragments of M5 as detected by FRET

  A mixture of 20 nM FITC-M5-Fab and 20 nM Cy3-M5-Fab was added to _IL-1a CHO-mu1c cells transfected with wild-type receptors and incubated at _IL-1ra 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. FRET FRET

  IL-1-dependent energy transfer between IL-1 RI is temperature

  A mixture of 20 nM FITC-M5 Fab and 12 nM Cy3-M5 Fab was added to CHO-mu1c cells (3 _{6 B} X 10 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) _A 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. FRET FRET

  

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

  A mixture of 20 nM FITC-M5 Fab and 12 nM Cy3-M5 Fab was added to wild-type _{A and incubated at 22 °C for 50 min (A). A} transfected receptors on CHO-mu1c cells _B 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 fluorescence were monitored over time at 22 °C.

  

SPR: Surface Plasma Resonance

SPR: Surface Plasma Resonance

  Diagram of BIAcore Diagram of BIAcore

  SPR SPR

  Interactions between lectins and immobilized glycoproteins SPR

  SPR

  SPR SPR

Interactions between lectins and immobilized glycoproteins

  An overlay plot of binding curves showing the interaction between lectins and immobilized thyroglobulin.

  Lectin solutions (50 µg/ml in 10 mM HEPES, 0.5 mM MnCl2 , 0.5 M CaCl2 and 0.05% surfactant, pH 7.4) were injected. Bound lectin was dissociated by 100 mM HCl (15 µl, 5 µl/min). SPR SPR

Summary of the interaction of seven lectins of different nominal specificities with immobilized glycoproteins

  Binding of lectin to the glycoprotein is indicated by “+” and lack of binding by “-” in the above table. As control experiments, the lectins were injected over (i) an immobilized non-glycosylated protein (recombinant HIV-1 reverse transcriptase expressed in E. coli) and (ii) a blank surface which was subjected to immobilizationchemistry in absence of a protein. The lectins did not show any binding in the control experiments. SPR SPR

  SPR-MS: Ligand Fishing with SPR-MS:

  Biacore 3000

  Detection Identification

  Selective binding, recovery and identification by MALDI MS of a specific interaction partner

  

Other important techniques in

Other important techniques in

protein interaction research

protein interaction research

  Mass Spectrometry Cross-linking Ultracentrifuge ChIP (Chromatin immunoprecipitation)

  Mass Spectrometry

  Mass spectrometry is indispensable for protein identification

  

Mass spectrometry is indispensable for protein identification

and will be in the center of proteomics research. and will be in the center of proteomics research.

  High sensitivity High resolution High throughput

  

Reference data bases

Reference data bases

  

   Prediction server

Interactions

• – Predictome (Boston U)
• – MIPS
• – Plex (UTexas)
• – DIP
• – STRING (EMBL)
• – YPD
• – Intact (EBI)
• – BIND/ Blueprint

  

Protein complexes

• – GRID
• – MIPS
• – MINT
• – YPD

  

From defining the proteome

to understanding function

Thanks!