Redox regulated transcription factorsgenomics.unl.edu/RBC_2015/COURSE_FILES/tue2.pdf · 2015. 5. 19. · Redox regulated transcription factors Katarina Johansson, PhD Division of

Redox regulated transcription factors

Katarina Johansson, PhD Division of Biochemistry

Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden

[email protected]

19 maj 2015 [email protected]

Outline

§  General introduction §  How is a redox signal sensed and transmitted?

à OxyR – E. coli à Yap1 – S. cerevisiae

§  Redox regulated transcription factors in mammals §  How we study redox regulated transcription factors


Redox regulation

²  Cellular regulation via reversible reduction/oxidation

²  Difficult to study because of fast, transient and complex reactions

²  Key factors in redox regulation; Trx- and GSH- systems


PrxASK-1RNR Ref-1Etc.

GPxGrxGSTEtc.

(Or TRP14)

Cysteine biochemistry allows redox- dependent signaling

§  Signaling molecules à H2O2

à NO à Lipidhydroperoxides à H2S

§  Cysteine biochemistry à Sulfenic form (RSOH) à Disulfides (RSSR) à Glutathionylated cysteines (RSSG) à Nitrosylated cysteines (RSNO)

§  Reducing systems à Trx system à GSH system


Finkel T - J. Cell Biol. (2011)

How is the redox signal transferred and how is specificity achieved?


Principles of redox signaling

à 1) Thermodynamic model.

à 2) Direct targeting.

à 3) Facilitated targeting.


Winterbourn CC and Hampton MB, 2008, FRBM

OxyR – a well studied redox regulated transcription factor in E. coli - Direct targeting


Figure 4 Model for OxyR activation and deactivation.

M Zheng et al. Science 1998;279:1718-1722

§  Direct targeting

§  OxyR is activated by ROS and induces a number of protective enzymes, including Catalase, Grx1, and Glutathione reductase (GSSG→2GSH)


(Brigelius-Flohé and Flohé, ARS, 2011)

Yap1 – a well studied redox regulated transcription factor in S. cerevisiae - Facilitated targeting

•  The H2O2 signal is sensed by a Cys residue in Orp1, which oxidizes to sulfenic acid (Cys-SOH)

•  Orp1 transduces this signal to Yap1 by formation of an intermolecular disulfide bond

•  Thereafter, intramolecular disulfide bonds are formed in activated Yap1

•  Activated Yap1 may finally induce several antioxidant genes, including proteins in the thioredoxin and GSH systems

Localisation of the oxidant may increase the specificity of the redox signaling

§  Growth factors

à  Induce NOX that produce superoxide

§  Extracellular H2O2 is channelled back through aquaporins

§  Tyrosine kinases (Src) à  Inactivate PrxI à  Accumulation of

H2O2

§  PTP with low pKa thiol à  Oxidized and

inactivated


Finkel, 2011, JCB

PTP – Protein Tyrosine PhosphatasesPrxI – Peroxiredoxin INOX – NADPH-dependent oxidases

BUT… PTPs react poorly with H2O2 – likely oxidation mediated by another thiol protein e.g. Prx

Redox regulated transcription factors in mammals

§  Many different transcription factors are redox regulated à  Nrf2, NFkB, HIF, p53, Oct-4, AP-1, FOXO, Notch, β-Catenin etc

§  Highly important for development à  Embryonic lethal -  NFkB (p65), AP-1 (c-Jun, JunB), HIF (HIF1a, HIF1b, ARNT), Ref-1

§  Redox regulated transcription factors have been implicated to be involved in the regulation of self-renewal and differentiation of stem cells à  Nrf2, NFkB, HIF, p53, Oct-4, AP-1, FOXO, Notch, β-Catenin

§  Redox regulated transcription factors are involved in many different diseases such as cancer, chronic inflammation, cardiovascular-, neurological-, and pulmonary diseases

§  Knowledge about the mechanism of redox regulated transcription factors is limited due to complex, transient and fast reactions


Many levels of redox regulation of transcription factors in cells


Ufer et al., ARS, 2010

p53 is redox regulated by the GSH- and Trx-systems


§  Tumor suppressor protein that activates genes involved in cell cycle arrest, apoptosis, and DNA repair

§  Activates upon stress –such as hypoxia, heat shock, DNA damage etc

§  p53 activates many different redox-controlling genes, including Gpx and Grx3

Maillet, A and Pervaiz, S, ARS, 2012


Maillet, A and Pervaiz, ARS, 2012

Crosslinks between p53 and ROS/RNS pathways

Nrf2 – nuclear factor erythroid 2 (NF-E2)-related transcription factor

§  Redox regulated transcription factor that induce proteins that protect against oxidative stress and xenobiotics (including the GSH- and Trx systems)

§  An induction of Nrf2 is beneficial for patients with degenerative-

and autoimmune diseases like MS, atherosclerosis, Parkinson, arthritis

§  An induction of Nrf2 in cancer make the cancer cells more viable and resistant to anticancer drugs


Nrf2 – a redox regulated transcription factor that protect cells from ROS


•  In normal conditions Keap1 targets Nrf2 for proteosomal degradation

•  Upon oxidative stress Keap1 are oxidized – releases Nrf2 that can translocate to the nucleus

Surh, YJ, Nature Reviews Cancer, 2003

Hypoxia Inducing Factor (HIF)

§  HIF regulates more than 70 genes coding for proteins involved in angiogenesis, erythropoiesis, energy metabolism, cell survival.

§  HIF is a heterodimeric transcription factor consisting of one HIFα (HIF1α, HIF2α or HIF3α) and one HIF1β (ARNT) subunit.

§  The oxygen sensors are, Prolyl-4-hydorxylasese domain 1-3 (PHD1, PHD2, and PHD3) and a second oxygen sensor FIH (Factor inhibiting HIF)

§  HIF are important in stem cells, for development but have also

been highly linked to metastatic cancer.


Redox Regulation of HIF


•  Oxidation of Fe(II) to Fe(III) in PHDs induces HIF

•  Direct oxidation of HIF1α

•  Reduction of Cys800 in HIF by APE1/Ref-1 or Trx system – facilitates binding to DNA


Different levels of redox regulation of HIF-1 in cells

Ufer et al., ARS, 2010

NFκB, a key regulator of the immune response

§  NFkB is a protein complex made up from homo- or heterodimers

à  p50, p52, p65, c-Rel and RelB. à Most common is the p50/p65 complex

§  Incorrect regulation of NF-κB has been linked to cancer,

inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development.


Enzymes

Growth factors

Stress response genes

Early response genes

Viruses

Apoptosis Regulators

Cytokines/Chemokines

Cell adhesion molecules

Miscellaneous

Transcription factors/Regulators

Cell surface receptors

Immunoreceptors

Antigen presentation

Acute phase proteins


NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells)

NFkB can be activated by many different stimuli

19 maj 2015 [email protected] (Brigelius-Flohé and Flohé, ARS, 2011)

(Brigelius-Flohé and Flohé, ARS, 2011)

Redox regulation of NFκB via the classical pathway

§  Redox regulation of NFκB occurs at different levels à  By Trx and other redoxins (Grx,

TRP14, Prx) – preventing activation of NFκB

à  A complex cascade of redox sensitive phosphorylation and de-phosphorylation

à  On the other hand, Trx system and APE1/Ref-1 have been shown to be crucial for NFκB binding to the DNA.


Simultaneous assessment of Nrf2, NFκB and HIF activation at Single-Cell Resolution


Redox regulated transcription factors – Nrf2, NFκB and HIF


HIF1α&

Hypoxia&

Normoxia&

Cytosol&

Nucleus&

NFkB&

S& S&

SH& SH&

NFkB&Nrf2&

HIF1α&

red&

KEAP1&

S& S&

NFkB&&&&IkB&

&&&IkB&

HIF1α&

OH&OH&pVHL&

ARE& AnHoxidant&genes& NFkB& HRE& Target&genes&Immune&response&gene&

ub&ub&

ub&ub&

ub&ub&

ub&ub&

Nrf2&ub&

ub&ub&

ub&

KEAP1&

SH& SH&

AnHoxidant&enzymes,&detoxifying&enzymes,&Trx=&and&GSH&systems&

Chemokines,&adhesion&molecules&and&enzymes&(IL=8&etc)&

Glycolysis,&angiogenesis&(VEGF),&&survival&and&invasion&factors&&

Nrf2&

Inflamma*on$

Redox regulated transcription factors – Nrf2, NFκB and HIF


Luciferase assays to study the activation of Nrf2, NFκB and HIF

With luciferase assays it is only possible to characterize è  one transcription factor response at a timeè  the whole cell culture response (not individual cells)

HIF Luciferase

HIF

NFκB Luciferase

NFkB

Nrf2 Luciferase

Nrf2


New tool: pTRAF – Plasmid for Transcription factor Reporter Activities based on Fluorescence

≈ 8 kbp

²  Nrf2, HIF and NFκB response elements coupled to three different fluorescent proteins


Overlay pictures of HEK293 cells transfected with pTRAF – 24h exposure in normoxia or hypoxia


Control - 21% O2 TNF - 21% O2 Auranofin - 21% O2 TNF+Auranofin - 21% O2

Control - 1% O2 TNF - 1% O2 Auranofin - 1% O2 TNF+Auranofin - 1% O2

NFκB Nrf2 HIF1α

Operetta® High Content Imaging System

² Fluorescent Microscope ² High-throughput screens ² 96 - 384 well plates x 48 ²  Incubator ² Live or fixed cells


Operetta® - High Content Imaging System


Hypoxia Total I /cellI/c

ell

CtrlEtO

HAur

SulphorAHQ

BHQ

tBuOOH

Cobalt

CDNBDox

Cisplat

in

Etoposid

e

CuOOHGSNO

TNF

TNF+

Aur

TNF+

Sulpho

r

TNF+AHQ

TNF+Cobalt

TNF+

CDNB

TNF+

Dox

TNF+

Cisplai

n

TNF+Eto

poside

Untransfe

cted

0

20000

40000

60000

80000

mCherryYPetCFP

Hypoxia Total I /cell

I/cel

l

CtrlEtO

HAur

SulphorAHQ

BHQ

tBuOOH

Cobalt

CDNBDox

Cisplat

in

Etoposide

CuOOHGSNO

TNF

TNF+Aur

TNF+Sulphor

TNF+AHQ

TNF+Cobalt

TNF+CDNB

TNF+Dox

TNF+Cisp

lain

TNF+Etoposide

Untransfe

cted

0

20000

40000

60000

80000

mCherryYPetCFP

Normoxia Total I / cellI/c

ell

CtrlEtO

HAur

SulphorAHQ

BHQ

tBuOOH

Cobalt

CDNBDox

Cisplat

in

Etoposide

CuOOHGSNO

TNF

TNF+Aur

TNF+Sulphor

TNF+AHQ

TNF+Cobalt

TNF+CDNB

TNF+Dox

TNF+Cisp

lain

TNF+Etoposide

Untransfe

cted

0

50000

100000

150000

200000

250000

Nrf2HIFNFkB

Normoxia Total I / cell

I/cel

l

CtrlEtO

HAur

SulphorAHQ

BHQ

tBuOOH

Cobalt

CDNBDox

Cisplat

in

Etoposide

CuOOHGSNO

TNF

TNF+Aur

TNF+Sulphor

TNF+AHQ

TNF+Cobalt

TNF+CDNB

TNF+Dox

TNF+Cisp

lain

TNF+Etoposide

Untransfe

cted

0

50000

100000

150000

200000

250000

Nrf2HIFNFkB

pTRAF transfected HEK293 cells analysed with the Operetta

21% O2

1% O2


pTRAF transfected HEK293 cells analysed with Operetta in 21 % O2

pTRAF transfected cells in 21% O2

Ctrl TNF Auranofin TNF+Aur0

20000

40000

60000

80000

100000

I/cell

NFκBNrf2 HIF


pTRAF transfected HEK293 cells analysed with Operetta at 1% O2

pTRAF transfected cells in 1% O2

Ctrl TNF Auranofin TNF+Aur0

10000

20000

30000

40000

50000

I/cell

NFκBNrf2 HIF


Nrf2 activity in HEK293 cells transfected with pTRAF exposed for 24h in 21% O2

Nrf2 - 21% O2

I/Cell

EtOH

Auranofin

Sulforophane

AHQ

Doxorubicin

Cisplatin

TNF

TNF+Aur

TNF+Sulf

TNF+AHQ

TNF+Dox

TNF+Cisp

0

20000

40000

60000

80000

100000


HIF - 21% O2

EtOH

Auranofin

Sulforophane

AHQ

Doxorubicin

Cisplatin

TNF

TNF+Aur

TNF+Sulf

TNF+AHQ

TNF+Dox

TNF+Cisp

0

5000

10000

15000

20000

I/Cell

HIF activity in HEK293 cells transfected with pTRAF exposed for 24h in 21% O2

NFκB activity in HEK293 cells transfected with pTRAF exposed for 24h in 21% O2

NFkB - 21% O2

I/Cell

EtOH

Auranofin

Sulforophane

AHQ

Doxorubicin

Cisplatin

TNF

TNF+Aur

TNF+Sulf

TNF+AHQ

TNF+Dox

TNF+Cisp

0

50000

100000

150000

200000

How are Nrf2, HIF and NFκB redox regulated?


GSSG

2GSH

TrxR

NADPH + H+

NADP+

GR

Trx-(SH)2

Trx-S2

GSH-systemTrx-system


GPxGrxGSTEtc.

(Or TRP14)

Se

SeTrxR1 activity in HEK293 cells

Contro

l

5nM Se

25nM

Se

500n

M Se0.00

0.05

0.10

0.15

0.20

0.25

Abso

rban

ce

NFκB

Nrf2

HIF

Ctrl Aur TNF TNF+Aur0

5000

10000

15000

I/cell

-Selenite+Selenite


20000

40000

60000

80000

I/cell

-Selenite+Selenite


20000

40000

60000

80000

100000

I/cell

-Selenite+Selenite

HIF response in 1% O2


20000

40000

60000

80000

100000

I/cel

l

-Selenite+Selenite (25nM)


50000

100000

150000

200000

I/cell


Nrf2- response in 1% O2


20000

40000

60000

80000

I/cel

l


How are Nrf2, HIF and NFκB redox regulated?


GSSG

2GSH

TrxR

NADPH + H+

NADP+

GR

Trx-(SH)2

Trx-S2

GSH-systemTrx-system


GPxGrxGSTEtc.

(Or TRP14)

Se

Se

NFκB

Nrf2

HIF


5000

10000

15000

I/cell

-Selenite+Selenite


20000

40000

60000

80000

I/cell

-Selenite+Selenite


20000

40000

60000

80000

100000

I/cell

-Selenite+Selenite

HIF response in 1% O2


20000

40000

60000

80000

100000

I/cel

l



50000

100000

150000

200000

I/cell


Nrf2- response in 1% O2


20000

40000

60000

80000

I/cel

l


TrxR1 activity in HEK293 cells

Contro

l

5nM Se

25nM

Se

500n

M Se0.00

0.05

0.10

0.15

0.20

0.25

Abso

rban

cePader et al, 2014, PNAS

Control 21%O2 TNF 21%O2 Auranofin 21%O2 Auranofin+TNF 21%O2

Control 1%O2 TNF 1%O2 Auranofin 1%O2 Auranofin+TNF 1%O2

HEK293 cells stably expressing pTRAF - Exposed for 24 in 21% O2 and 1% O2

NFκBNrf2 HIF


How are the heterogenic induction pattern Nrf2, HIF and NFκB regulated?


HEK293 cells that stably express pTRAF induced with Auranofin and TNFα for 24h in 21%O2

Sort with FACS

Nrf2

NFκB

HIF Why?RT-PCRGene arrays

On going and future…

²  Mechanistic studies with regards to the Trx- and GSH systems

²  Further studies of stochastic responses ²  Develop new pTRAF variants for studies of other transcription

factors (p53, AP-1, Oct-4, c-Myc, β-catenin (wnt)…) ²  Cancer stem cells

²  Mechanism of clinically used drugs as well as new potential drug leads to treat cancer, MS, Parkinson´s, atherosclerosis


Conclusions

§  Redox regulation is important for control of many different signaling pathways

§  Knowledge on how redox regulated transcription factors is controlled is limited due to complex, transient and fast reactions

§  Redox regulation involves specific oxidation or reduction of regulatory target proteins, often via uniquely reactive Cys residue(s) as sensors

§  Compartmentalization, direct targeting and facilitated targeting are likely to be highly important in redox regulation of transcription factors in mammals


Reference list:

§  Brigelius-Flohe R., and Flohe L., Basic Principles and Emerging concepts in the Redox Control of Transcription Factors, 2011, Antioxiants & Redox Signaling, 15(8); 2335-2381

§  Winterbourn CC., and Hampton MB., Thiol chemisty and specificity in redox signaling, 2008, FRBM, 45;549-561

§  Arner ESJ., and Holmgren A., Physiological functions of thioredoxin and thioredoxin reductase, Eur J. Biochem. 2000, 267;6102-6109

§  Ufer et al, Redox control in Mammalian Embryo Develoment, 2010, Antioxidants & Redox Signaling, 13;6, 833-875

§  Finkel, T, Signal transduction by reactive oxygen species, 2011, J. Cell Biol. 194;(1) 7-15

§  Marinho H.S et al, Hydrogen peroxide sensing, signaling and regulation of transcription factors, 2014, Redox biology 2, 535-562


Documents

Redox regulated transcription factorsgenomics.unl.edu/RBC_2015/COURSE_FILES/tue2.pdf · 2015. 5. 19. · Redox regulated transcription factors Katarina Johansson, PhD Division of