Principle of in vivo Proximity-Utilizing Biotinylation

Ligase: humanized Biotin ligase – humBirABAP-Biotin Accepting PeptideB: Biotin

Biotin pulse (B)

BAP В

Bir

A

А Interaction (proximity)

Bir

A

А

BAP В

Bir

A

А

B

BAP В

А

В

B

Bir

A

BAP

Detection by Western BlottingConfocal microscopyStreptavidin pulldownLC-MS/MS

A (B) – HP1a, HP1b, HP1g, Tap54a, Tap54b, wtKap1, mutKap1, H2A, H2Az, mH2A, H2A.BBD, H3.1, CenpA, PCNA, GFP, Rad18

Protein BirA-A << Protein BAP-B

Vector design

Kozak His-tag XhoI NotI

His-tag BAP/MS BiotinLC-MS/MS

B

Kinetics of biotinylation of different BAP. Detection on LC-MS/MS

Strept

a-His

Old BAP BAP1070 BAP11085

min

30

min

3 h

24 h

5 m

in

30

min

3 h

24 h

5 m

in

30

min

3 h

24 h

0 200 400 600 800 1000 1200 1400 16000

102030405060708090

100

OldBAP BAP1070

Time, min

Bio

tin

yla

tio

n, %

Old BAP - MAGLNDIFEAQKIEWHE (Half-saturation time 15min)BAP1070 - ---------- ILEAQKIVR ---- (Half-saturation time 680 min)BAP1108 - ---------- ILEAQKIHR ---- (Half-saturation time 700 min)

Cell lysate / gel slices

Propionic anhydride

Trypsin

LC-MS/MS

Detection of the ubiquitination of the protein of interest

Biotin pulse (B)

BAP В

Bir

A

А Interaction (proximity)

Bir

AА

BAP В

Bir

A

А

B

BAP В

А

В

B

Bir

A

BAP

Easy detection by Western Blotting

Ub

BAP В

Bir

A

А

BAP В

А

В

B

Bir

A

BAP

Biotin pulse (B)

Interaction (proximity) B

BAP В

Ub

Ub

Ub

E1,

E2,

E3

– li

ga

se +

U

biq

uit

in

M 0 1 2

M – Marker of molecular weight0 – nontransfected cell1 – BirA-GFP+BAP-B (control)2 – BirA-A+BAP-B

Bendogenous

b-BAP-B

b-Ub-BAP-B

8.5

kDa

Ub

iqu

itin

3.0

kDa

BA

P1

070

Application of PUB for study of PTM (e.g. ubiquitination, acetylation)

Protein B: PCNA, H2A, H2Az, mH2A, H3.1A - protein of interest (PolH, Rad18, GFP as control)

Endogenous protein B

Ub

iqu

itin

atio

n le

vel

Fractions of BAP-B fusions

Biotinylated b-BAP-B subfraction

Protein B

Ub

iqu

itin

atio

n le

vel Bir

A

А

+Protein B

Bir

A

GFP

+

Ub

iqu

itin

atio

n le

vel

Human Polymeraze PolH - Ubiquitin interacting protein

c | Crystal structure of the human Y‑family DNA polymerase Pol η in a ternary complex with a cyclobutane pyrimidine dimer (CPD). In this view, the 3′T of the CPD is in the active site and is correctly paired with incoming dATP (PDB code 3MR3)42. The template strand is shown in rust colour, and the primer is olive green. The incoming dNTP is shown in yellow. The burgundy stick represents the position of the CPD. The small blue spheres represent the metal ions. The protein backbone is represented by ribbon surrounded by semi-transparent solvent accessible surface.

Specific recruitment of PolH to sites of DNA damage

POLH

mutation of UbZ

POLH

mutation of UbZ

POLH

UbZmutation of PCNA-binding site or PIP

Ubi

quiti

n

Human Polymeraze PolH - Ubiquitin interacting protein

DNA polymerase is specifically involved in DNA repair. Plays an important role in translesion synthesis, where the normal high fidelity DNA polymerases cannot proceed and DNA synthesis stalls. Plays an important role in the repair of UV-induced pyrimidine dimers. Mass - 78,4 kDa

POLH

UbZ PCNA-binding site or PIP

POLH interacts with ubiquitinated PCNA, but also has domain interacting with nonubiquitinated PCNA

Ubi

quiti

n

Ubi

quiti

n

PCNA

PCNA-binding site or PIP

mutation of PCNA-binding site or PIP

POLH-PIP mutant

POLH-UbZ mutant

POLH-DD mutant

Wilde type POLH

Detection of POLH interaction with Ub-BAP-PCNA

wt POLH

BirA

Biotin pulse (B)

Ub

PCNABAP

Interaction (proximity)

UbZ PCNA-binding site or PIP

PCNA

BirA

wt POLH

BAP

wt POLHUb

BirA

B

Ub

PCNABAP

E1, E2, E3 ligase

Ub Ub

Ub

BirA - humanized Biotin ligase BAP - Biotin Accepting PeptideB - BiotinPCNA – Proliferating Cell Nuclear AntigenwtPOLH – wild type human DNA PolymeraseUb - Ubiquitin

Analysis of post-translational modifications of a specific protein fraction using Proximity Utilizing Biotinylation (PUB)

B. Ubiquitination status of POLH-proximal PCNA. Western blot with α-PCNA antibodies.(1) untrasfected cells. (2, 4) BAP-PCNA cotransfected with BirA-GFP fusion. (3, 5) BAP-PCNA cotransfected with Bir-POLH fusion. (2, 3) Flowthrough fraction. (4, 5) Eluate. Note that the BAP-PCNA from the flowthrough fraction was further purified via Ni-NTA chromatography in order to decrease the signal from endogenous PCNA. The endogenous PCNA, the BAP-PCNA fusion and the ubiquitinated BAP-PCNA are indicated by arrows.

6 hr48 hr Streptavidin

Biotin pulse5 min

pulldown

Elu

FT

UVС 20

J/m2

CMV.BAP.PCNA CMV.BirA.PolH

B

Detection of ubiquitinated proteins interacting with aBirA-fusion of the protein of interest

BirA

А

ВBiotin pulse (B)

Interaction (proximity)

E1,

E2,

E3

– li

ga

se +

BA

P-

Ub

iqu

itin

C

D

CUb

BA

P

ВUb

BA

PInteracting partners of protein A

Noniteracting partners of protein A

Ub

BA

P

ВUb

BA

P

CUb

BA

P

B

B

ВUb

BA

P

BirA

А

CUb

BA

P

BirA

А

Ub

BA

PD

D

No interaction

Western blotting

Streptavidin pulldown, SDS PAGE, LC-MS/MS

Data analysis LC-MS/MS

Detection of POLH interaction with BAP-Ub-PCNA

wt POLH

BirA

Biotin pulse (B)

PCNA

Interaction (proximity)

UbZ PCNA-binding site or PIP

PCNA

BirA

wt POLH

wt POLHUb

BirA

B

Ub

PCNA

BAP

E1, E2, E3 ligase

Ub Ub

Ub

BAP Ub

BirA - humanized Biotin ligase BAP - Biotin Accepting PeptideB - BiotinPCNA – Proliferating Cell Nuclear AntigenwtPOLH – wild type human DNA PolymeraseUb - Ubiquitin

PCNA

UV + + + + +55

36

Str

epta

vid

in-H

RP

BA

P-U

b-P

CN

A

55

36

0 1 2 3 4 5

Ub

-PC

NAX

aP

CN

A

BAP-Ub + + + + +BirA-GFP + BirA-wtPolH + BirA-UbZmutPolH + BirA-PIPmutPolH + BirA-DDPolH +

Detection of ubiquitinated proteins interacting with BirA-POLH

BAP-ubiquitinated PCNA is preferentially biotinylated via its interaction with BirA-POLH.

Left top - Western with streptavidin-HRP,

Left bottom - Western with anti-PCNA antibodies.

The positions of PCNA, Ub-PCNA and BAP-Ub-PCNA are indicated. The position of an unknown ubiquitinated protein, induced by UV treatment, is indicated by X.

6 hr

Biotin pulse15 min

48 hr

CMV.BAP.Ub CMV.BirA.PolH

UVС 20

J/m2

Western blotting

Streptavidin pulldown, SDS PAGE, LC-MS/MS

6 hr

Biotin pulse15 min

48 hr

48 hr

CMV.BAP.Ub CMV.BirA.GFP

C13, N15

C12, N14

C13, N15

C12, N14

UVС 20

J/m2

6 hr

C13, N15

C12, N14

Biotin pulse15 min

CMV.BAP.Ub CMV.BirA.PolH

UVС 20

J/m2

Identification of ubiquitinated partners of POLH using SILAC

HEAVY

LIGHT

MS/MS of PCNA

derived peptide

15

CONCLUSIONS

Validation of our approach using well characterized interaction between the translesion DNA polymerase POLH and ubiquitinated PCNA.

Purification of the biotinylated fraction of a BAP-fusion of the protein of interest allowed us to study PTMs specific for the particular fraction of this protein located in proximity to another protein of interest fused to BirA.

Whereas in the case of ubiquitination the PTM analysis could be accomplished by Western blot analysis of cellular extracts with streptavidin-HRP, in most other cases it will require biochemical purification of the biotinylated fraction of the protein.

Preferential biotinylation of BAP-Ub-PCNA by the BirA-POLH fusion depends on the integrity of the UBZ and PCNA-binding domains of POLH protein, i.e., on the binding between the two proteins.

Further, we demonstrated that the approach can be used for mass-spectrometry identification of the ubiquitinated proteins interacting with the BirA fusion of interest.The intensity of the biotinylation signal in the ‘BAP-Ub + BirA-POLH’ samples depends on the interaction between BirA-POLHwt and ubiquitinated PCNA, demonstrating that PUB allows us to preferentially label ubiquitinated proteins interacting with the protein of interest.

16

KAZAKHSTANI-FRENCH COOPERATION PROJECT

Project 05.01.06.H24. Development of new approaches for diagnostics and therapy of chronic diseases by proteomics methods

17

Group MembersVasily OgryzkoMuhammad ShoaibEvelyne SaadeEmilie CochetMartine ComissoChloé RobinAnamarija JurisicIrina PirozhkovaRakhan Aimbetov

UMR8126Joelle Wiels and Marc Lipinski

ACKNOWLEDGEMENTS

UMR8200Patricia Kannouche

18

THANK YOU