Protein Complex and Protein protein Interaction

(1)

Protein Complex and

Protein-protein Interaction

彭彭彭

国家人类基因组北方研究中心

(2)

Central dogma:

the story of life

RNA DNA

Protein

Protein is the final player in cell life

(3)

Proteins function in association with

other proteins or biomolecules, but

(4)

Introduction to Proteomics



the analysis of genomic complements

of proteins



dynamic



systematic

(5)

Goals of Proteomics

to discover

protein function

to understand

cellular processes

to understand

disease states

to discover

drug target

(6)

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

(7)

Approaches

Genetic:

yeast two-hybrid phage display

Biochemical:

(8)

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.

(9)

Blue Native PAGE

detergent

CBB

6-ACA

_

(10)

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 contains

0.02% Coomassie blue G250

(11)

Blue Native PAGE of chloroplast

thylakoid membranes

BBRC 1999, 259:569-575

BN-PAGE of solubilized

chloroplast thylakoid

membranes (a)

followed by SDS–PAGE in

the second dimension (b).

CF

₀

F

₁

ATP synthase was

(12)

Blue Native PAGE of chloroplast

thylakoid membranes

BBRC 1999, 259:569-575

lane 1: LMW marker

lane 2: CF₀F₁ATP synthase, purified by density gradient centrifugation

(13)

Blue Native PAGE of multiprotein

complex from whole cellular lysate

MCP 3:176-182, 2004

Dialysis permits

(14)

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

(15)

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 (5.5–17 and 10%, respectively).

MCP 3:176-182, 2004

Blue Native PAGE

(16)

Far Western

(17)

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).

MycC9 2

Science 251:1211-7, 1991

(18)

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

(19)

GST Pulldown

(20)

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

(21)

GST-mFas fusion proteins

GST Pulldown

149 166 204 293 306

(22)

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

(23)

GST-mFas-associated polypeptides

are stable to high salt concentrations

GST Pulldown

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%

(24)

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

(25)

Differential association with mutant

forms of GST-mFas

GST Pulldown

HeLa

L929

(26)

Schematic representation of the mouse Fas

antigen and its binding proteins

(27)

Epitope tagging

1

2

3

4 5 6-9

(28)

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

(29)

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;

(30)

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

(31)

Reduction of nonspecific protein

background

1. to increase ionic strength in wash buffer;

2. to reduce the amount of primary Ab;

(32)

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.

(33)

Identification of VHL-associated proteins

Lysates from 786-O renal

carcinoma cells, transfected with the indicated pVHL constructs, were immunoprecipitated with 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.

(34)

Mapping the p14 and p18 binding

site on pVHL

a-HA

A. 786-O cells producing 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 TLKERCLQWRSLVKP (underlined residues are sites of germ-line missense

(35)

the binding site for Elongin B and C

in pVHL

(36)

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.

(37)

TAP: tandem affinity purification

(38)

Sequence and structure of the TAP tag

CBP

TEV

Ig BD

bait

TAP

(39)

Overview of the TAP procedure

(40)

Schematic representation of the

split TAP tag strategy

(41)

Schematic representation of the

substraction strategy

(42)

Protein composition of

TAP-purified U1 snRNP

(43)

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 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

(44)

TAP in higher eucaryotes

TAP

Questions:

overexpression

endogenous expression

Solutions:

RNA interference

(45)

Strengths and weaknesses of commonly

used affinity approaches for the retrieval

(46)

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

(47)

FRET

E: energy transfer efficiency

R₀: intermolecular distance when half of energy is transfered r: distance between fluorophores

E = R

₀6

/(R

06

+ r

6

)

when r = 2R₀, E = 1/65

(48)

Imaging protein phosphorylation by FRET

target GFP Fab Cy3

transfection microinjection or incubation

target GFP Fab Cy3

activator

laser

(49)

Detection of protein interaction by FRET

target GFP

Fab Cy3 Protein 1 CFP

Protein 2 YFP

FRET

Protein 1 Cy3

Protein 2 FITC

(50)

FRET reveals interleukin

(IL)-1-dependent aggregation of IL-1 type I

dependent aggregation of IL-1 type I

receptors that correlates with

receptor activation

FRET

(51)

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

(52)

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 106_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

(53)

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

(54)

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

(55)

IL-1a-dependent FRET can be detected between

FITC-M5 Fab and Cy3-FITC-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 min (A). 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 fluorescence were monitored over time at 22 °C.

A

(56)

(57)

Diagram of BIAcore

SPR

(58)

Interactions between

lectins and immobilized glycoproteins

SPR

(59)

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

(60)

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

(61)

SPR-MS:

SPR-MS: Ligand Fishing with

Biacore 3000

SPR

Selective binding, recovery and

identification by MALDI MS of a specific

interaction partner

SPR

MS

(62)

Other important techniques in

protein interaction research

Mass Spectrometry

Cross-linking

Ultracentrifuge

(63)

Mass spectrometry is indispensable for protein identification

and will be in the center of proteomics research.

Mass Spectrometry

(64)

Reference data bases



Interactions

–

MIPS

–

DIP

–

YPD

–

Intact (EBI)

–

BIND/ Blueprint

–

GRID

–

MINT



Prediction server

–

Predictome (Boston U)

–

Plex (UTexas)

–

STRING (EMBL)



Protein complexes

(65)

From defining the proteome

to understanding function