Combining isothermal titration calorimetry, fluorescence ... · Combining isothermal titration...

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Combining isothermal titration calorimetry, fluorescence anisotropy and kinetic approaches to decipher the molecular mechanisms of the redox activation of the Yap1 transcription factor in S. cerevisiae UMR 7365 IMOPA Structural and Molecular Enzymology Nancy, France Antoine Bersweiler Alexandre Kriznik Hortense Mazon Dr B. D’Autréaux Dr A. Delaunay-Moisan Dr G. Belli Dr M. Toledano LSOC CEA Saclay France Dr. S. Rahuel-Clermont

Transcript of Combining isothermal titration calorimetry, fluorescence ... · Combining isothermal titration...

Combining isothermal titration calorimetry, fluorescence anisotropy and kinetic approaches to decipher the molecular mechanisms of the

redox activation of the Yap1 transcription factor in S. cerevisiae

UMR 7365 IMOPA

Structural and Molecular

Enzymology

Nancy, France

Antoine Bersweiler

Alexandre Kriznik

Hortense Mazon

Dr B. D’Autréaux

Dr A. Delaunay-Moisan

Dr G. Belli

Dr M. Toledano

LSOC

CEA Saclay

France

Dr. S. Rahuel-Clermont

H2O2Reactive oxygen species = oxidant

ToxicCellular messenger

Alteration of biomolecular

function

Stress proteins ChaperonesEnzymes/transcription

factors activation

Normal physiological conditions

Oxidative stress

H2O2 local concentration

H2O2 as a signaling molecule

Stimuli UV Growth factor deprivation

p66shc H2O2

Stimuli Growth factors Cytokines

NOX

H2O2

H2O2 as a signaling molecule: issues

Specificity

Concentration tightly regulated by cellular antioxidant systems low in vivo concentrations

Signal Transduction ?

Molecular mechanisms that generate specificity in the transmission of oxidative

stress signals or redox signals ?

Yap1reduced

Yap1oxidized

H2O2

Delaunay et al., EMBO J 2000, Delaunay et al. Cell 2002Veal et al. JBC 2003

Yap1: redox activation of transcription factor by thiol peroxidase

H2O2

75 Kda

Yap1reduced

Yap1oxidized

Orp1

H2O2

Delaunay et al., EMBO J 2000, Delaunay et al. Cell 2002Veal et al. JBC 2003

= thiol peroxidase

Yap1: redox activation of transcription factor by thiol peroxidase

H2O2

H2O2

Ybp1

Orp1

79 kda18 kda

75 Kda

-Yap1S

598

S303

Orp1SH

SH

36

82

Orp1SOH

SH

36

82

H2O2

H2O

H2O

-Yap1SH

598

SH303

-Yap1S

598

SH303

Orp1HS

S36

82

Based on D’Autréaux et Toledano, Nature Reviews, 2007

Yap1: the redox relay 1st step of activation pathway

Relay of oxidative equivalents

Oxidative activation of Yap1 is sequential

Based on D’Autréaux et Toledano, Nature Reviews, 2007

-Yap1S

598

S303

Orp1SH

SH

36

82

Orp1SOH

SH

36

82

Orp1S

S

36

82

H2O

H2O2

H2O

H2O

-Yap1SH

598

SH303

Trx

-Yap1S

598

SH303

Orp1HS

S36

82

Yap1: Specificity?

Competition peroxidase/Yap1

Based on D’Autréaux et Toledano, Nature Reviews, 2007

-Yap1S

598

S303

Orp1SH

SH

36

82

Orp1SOH

SH

36

82

Orp1S

S

36

82

H2O

H2O2

H2O

H2O

-Yap1SH

598

SH303

Trx

Competition peroxidase/Yap1

-Yap1S

598

SH303

Orp1HS

S36

82

Yap1: Specificity?

Functional coupling betweenOrp1 and Yap1?

Direct recognition Orp1/Yap1?

Based on D’Autréaux et Toledano, Nature Reviews, 2007

-Yap1S

598

S303

Orp1SH

SH

36

82

Orp1SOH

SH

36

82

Orp1S

S

36

82

H2O

H2O2

H2O

H2O

-Yap1SH

598

SH303

Ybp1 ?

Trx

Competition peroxidase/Yap1

-Yap1S

598

SH303

Orp1HS

S36

82

Yap1: Specificity?

Functional coupling betweenOrp1 and Yap1?

Direct recognition Orp1/Yap1?Role of Ybp1 ?

Mechanism of Ybp1 action? Protein-protein interactions

Orp1SH

SH

36

82

-Yap1SH

598

SH303

Ybp1

Molecular recognition betweenthe redox relay partners?

E

polarizedligth

excitation

rapid rotation hν’ stronglydepolarized lightweak anisotropy

slow rotationhν’ weakly

depolarized lightstrong anisotropy

L

fluorescent probe: Alexa Fluor (AF) 488 5-SDP Ester

RL

absorption emission

Fluorescence anisotropy

Mechanism of Ybp1 action? Protein-protein interactions

Orp1SH

SH

36

82

-Yap1SH

598

SH303

Ybp1

Molecular recognition betweenthe redox relay partners?

Mechanism of Ybp1 action? Protein-protein interactions

Orp1SH

SH

36

82

-Yap1SH

598

SH303

Ybp1

Molecular recognition betweenthe redox relay partners?

Ybp1KD = 1,3 μM

Ybp1+-HS

HS

598

303

Yap1 -

598

303

Yap1SH

SH

Yap1·Ybp1 1·1

stoichiometry = 1:1

%

m/z

Ybp1

MW = 79780 Da

MW = 154296 Da

Ybp1

-598

303

Yap1SH

SH

Ybp1

-Yap1SH

SH

Collaboration S. Cianférani, Strasbourg

Native mass spectrometry

Ybp1 recruits Orp1 and Yap1 into a ternary complex

Orp1SH

SH

36

82

-Yap1SH

598

SH303

Ybp1

-Yap1SH

598

SH303

Ybp1

ITC titration of Yap1, Ybp1 and Yap1-Ybp1 by Orp1

kcal

mol

-1 O

rp1

inje

cted

time (min)

μca

l/sec

molar ratio

Orp1 and Yap1 do not interact

-Yap1HS

HSOrp1

SH

SH+

Orp1SH

SH

kcal

mo

l-1 O

rp1

inje

cted

time (min)

μca

l/sec

molar ratio

μca

l/sec

time (min)

kcal

mo

l-1 O

rp1

inje

cted

molar ratio

-Yap1HS

HSOrp1

SH

SH+ Orp1

SH

SHYbp1 +

Ybp1

KD = 0,8 μMN = 1

Ybp1 and Orp1 form a 1·1 complex

μca

l/sec

kcal

mol

-1 O

rp1

inje

cted

time (min)

molar ratio

Orp1 interacts with Ybp1·Yap1

Two hypotheses

μca

l/sec

kcal

mol

-1 O

rp1

inje

cted

time (min)

molar ratio

Orp1 interacts with Ybp1·Yap1

Two hypotheses

KD = 0,7 μMN = 1

Orp1 does not interact with Yap1Orp1 interacts with Ybp1

Orp1SH

SH

36

82

-Yap1SH

598

SH303

Ybp1

-Yap1SH

598

SH303

Ybp1

X

Ybp1 recruits Orp1 and Yap1 into a ternary complex

-Yap1S

598

S303

Orp1SOH

SH

36

82

H2O

-Yap1SH

598

SH303

-Yap1S

598

SH303

Orp1HS

S36

82

Ybp1 ?

kYap

H2O2

Orp1SH

SH

36

82

X

X

Rate of the first step of Yap1 activation pathway

H2O

X

Orp1S

S

36

82

Role of ternary complex in Yap1 oxidation mechanism?

Orp1SOH

SH

36

82

H2O

-Yap1SH

598

SH303

-Yap1S

598

SH303

Orp1HS

S36

82

Ybp1 ?

kYap

H2O2

Orp1SH

SH

36

82

XH2O

Reconstitution of first step of activation pathway in vitro

ms to min incubations Reverse phase chromatographic

analysis (C8)

Elution time (min)

abso

rban

ce (2

15 n

m)

Orp1SH

SH

-Yap1HS

HS

→Ybp1

kYap determination

Reactiontime (s)

S

HSOrp1Orp1

SH

S

S

-Yap1S

HSOrp1

SH

Orp1SH

SH

-Yap1HS

HS

Orp1SH

SNH

Elution time (min)

abso

rban

ce (2

15 n

m)

Orp1SH

SNH

→Ybp1

kYap determination

Reactiontime (s)

Elution time (min)

abso

rban

ce (2

15 n

m)

S

-Yap1S

HSOrp1

SH

Orp1SH

SH

-Yap1HS

HS

Orp1SH

SNH

S

HSOrp1Orp1

SH

S

→Ybp1

kYap determination

Reactiontime (s)

Elution time (min)

abso

rban

ce (2

15 n

m)

S

-Yap1S

HSOrp1

SH

Orp1SH

SH

-Yap1HS

HS

Orp1SH

SNH

S

HSOrp1Orp1

SH

S

→Ybp1

kYap determination

Reactiontime (s)

Elution time (min)

abso

rban

ce (2

15 n

m)

S

-Yap1S

HSOrp1

SH

Orp1SH

SH

-Yap1HS

HS

Orp1SH

SNH

S

HSOrp1Orp1

SH

S

→Ybp1

kYap determination

Reactiontime (s)

Elution time (min)

abso

rban

ce (2

15 n

m)

observed rate constant = 0.3 s-1

observed rate constant = 2.6 s-1

S

-Yap1S

HSOrp1

SH

Orp1SH

SH

-Yap1HS

HS

Orp1SH

SNH

S

HSOrp1Orp1

SH

S

Ybp1

Ybp1

fraction of total

time (s)

Ybp1

Ybp1

S

-Yap1S

HSOrp1

SH

Ybp1 accelerates the reaction Orp1-SOH + Yap1

Reactiontime (s)

Elution time (min)

abso

rban

ce (2

15 n

m)

observed rate constant = 0.3 s-1

observed rate constant = 2.6 s-1

Wild type Orp1 = 5 s-1

S

-Yap1S

HSOrp1

SH

Orp1SH

SH

-Yap1HS

HS

Ybp1

Ybp1

fraction of total

time (s)

Ybp1

S

-Yap1S

HSOrp1

SH

Ybp1 accelerates the reaction Orp1-SOH + Yap1

Reactiontime (s)

Orp1S

S

kYap

Ybp1

Orp1

In vivo Orp1 concentration = 0,5 µMExperiments @ 50 µM

Real effect of complex formation = 600 fold

Role of ternary complex in Yap1 oxidation mechanism?

kYap = Cste suggests that the reaction occurs within the ternary complex

Simulation of kYap = f(Orp1) based on KD of 0,7 µM

In the absence of Ybp1 the reaction is bimolecular

Orp1SH

SH

36

82

Orp1SOH

SH

36

82

Orp1S

S

36

82

H2O

H2O2

H2O

kSS = 500 s-1

Intramolecular disulfide formation in Orp1 is very fast

Ma et al. JBC 2007

Orp1SH

SH

36

82

Orp1SOH

SH

36

82

Orp1S

S

36

82

H2O

H2O2

H2O

kSS

Ybp1

Trx

Does Ybp1+Yap1 inhibit formation?

Within

kss decreased from 500 to 6 s-1

Reduced competition with Yap1

Reaction between Orp1 and Yap1 is possible

time (min)

kcal

mol

-1 O

rp1

inje

cted

μca

l/sec

time (min)

kcal

mol

-1 O

rp1

inje

cted

μca

l/sec

molar ratio

The disulfide form of Orp1 doesn’t interact with Ybp1 and Yap1·Ybp1

-Yap1HS

HS+ Ybp1 + +

molar ratio

μca

l/sec

kcal

mol

-1 O

rp1

inje

cted

time (min)

molar ratio

Orp1S

SOrp1

S

SOrp1

S

SYbp1

-Yap1SH

SH

time (min)

kcal

mol

-1 O

rp1

inje

cted

μca

l/sec

time (min)

kcal

mol

-1 O

rp1

inje

cted

μca

l/sec

molar ratio

The disulfide form of Orp1 doesn’t interact with Ybp1 and Yap1·Ybp1

-Yap1HS

HS+ Ybp1 + +

molar ratio

μca

l/sec

kcal

mol

-1 O

rp1

inje

cted

time (min)

molar ratio

Orp1S

SOrp1

S

SOrp1

S

SYbp1

-Yap1SH

SH

-Yap1SH

598

SH303

Orp1SH

SH

36

82Ybp1

-Yap1SH

598

SH303

Ybp1 recruits Orp1 (ITC)

Bersweiler et al., in revision

Yap1 activation mechanism

Orp1SH

SH

36

82Ybp1

-Yap1SH

598

SH303

Ybp1

-Yap1SH

598

SH303

Orp1SOH

SH

36

82

-Yap1SH

598

SH303

Ybp1 kYap1

Ybp1 recruits Orp1 (ITC)Orp1 disulfide formation is decreased within the ternary complex

Bersweiler et al., in revision

Yap1 activation mechanism

Orp1SH

SH

36

82Ybp1

-Yap1SH

598

SH303

Ybp1

-Yap1SH

598

SH303

Orp1SOH

SH

36

82

-Yap1SH

598

SH303

Ybp1 kYap1

Ybp1 recruits Orp1 (ITC)Orp1 disulfide formation is decreased within the ternary complex

Bersweiler et al., in preparation

Yap1 activation mechanism

Orp1SH

SH

36

82Ybp1

-Yap1SH

598

SH303

Ybp1

Orp1SOH

SH

36

82

-Yap1SH

598

SH303

Ybp1 kYap1

kSS

Ybp1 recruits Orp1 (ITC)Orp1 disulfide formation is decreased within the ternary complex

Bersweiler et al., in preparation

Yap1 activation mechanism

Orp1SH

SH

36

82Ybp1

-Yap1SH

598

SH303

Ybp1

-Yap1SH

598

SH303

signal

Sensor/receptor

modified sensor

sensing

transducer

transduction

effector

Case of redox signaling

Oxidant

sensing

transducer

transduction

effector

Signaling pathway

Cellular signaling pathways

proteinS-

proteinSox

Molecular recognition

Oxidation

Molecular recognition

Thank you for your attention

In vitro reconstitution of the redox relayRecombinant purified proteins

Collaboration M. Toledano

Ybp1

High H2O2

Low H2O2

Conclusion: requirement of both Orp1 and Ybp1 in efficient and sensitive Yap1 oxidation by H2O2

H2O2

proliferation growth arrest apoptose10-8 10-6 10-4 local

concentration (M)

proteinSox

cellular response

effector

proteinS-

sensorSox

sensorS-

H2O2

relay

sensing

lower reactivity in most cases

high reactivity with H2O2

OxyR

signaling

Prx2/Stat3

Sobotta et al., Nature Chemical Biology, 2015

oxidative stress

Direct/indirect sensing of the H2O2 signal

sensorS-

= Enzyme family Thiol peroxidase

Table 1. Binding and thermodynamic parameters of Orp1 binding to Ybp1 and the Yap1SSS SSS·Ybp1

complex deduced from ITC titrationsa

Dissociation constant (µM)

Stoichiometry ΔH cal mol-1

-TΔS cal mol-1

Ybp1 0.8 ± 0.1 0.8 ± 0.1 - 4890 ± 400 - 3430

Yap1SSS SSS·Ybp1 0.7 ± 0.2 0.8 ± 0.1 - 4210 ± 240 - 4235 a Parameters deduced from the analysis of binding isotherms shown Fig. 3C

Orp1SH

SH

36

82

Orp1SOH

SH

36

82

Orp1S

S

36

82

H2O

H2O2

H2O

kSS

0 1 2 3 4 51.5

2.0

2.5

3.0

3.5

H2O

2 5 to 800 µM

Flu

ore

scen

ce in

ten

sity

Time (s)

0.00 0.01 0.02 0.03 0.04 0.051.5

2.0

2.5

3.0

3.5B

Flu

ore

scen

ce in

ten

sity

Time (s)

A

H2O

2 1 to 20 mM

kSOH = 1.5 105 M-1s-1

kSS = 500 s-1

Intramolecular disulfide formation in Orp1 is very fast

Ybp is required for the reaction WT Orp1-SOH + Yap1

Orp1 model

Ma et al. JBC 2007

Non reducing SDS-PAGE

Wood et al., Letters to Nature 2003

N-ter Cys richdomain

C-ter Cys richdomain / NES

Ybp1 required in vivo for the first step of Yap1 activation

Mechanism of Ybp1 action?

DYbp DYbp

- + - + - + - +

Yap1 C303S

X

Collaboration M. Toledano