Redox regulated transcription factorsgenomics.unl.edu/RBC_2015/COURSE_FILES/tue2.pdf · 2015. 5....

Redox regulated transcription factors

Katarina Johansson, PhD Division of Biochemistry

Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden

Katarina.Johansson@ki.se

19 maj 2015 Katarina.Johansson@ki.se

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

§  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 Katarina.Johansson@ki.se (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&

SH& SH&

NFkB&Nrf2&

HIF1α&

KEAP1&

NFkB&&&&IkB&

&&&IkB&

HIF1α&

OH&OH&pVHL&

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

ub&ub&

Nrf2&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&&

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

NFκB Luciferase

Nrf2 Luciferase

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

CtrlEtO

SulphorAHQ

tBuOOH

Cobalt

CDNBDox

Cisplat

Etoposid

CuOOHGSNO

Sulpho

TNF+AHQ

TNF+Cobalt

Cisplai

TNF+Eto

poside

Untransfe

mCherryYPetCFP

Hypoxia Total I /cell

CtrlEtO

SulphorAHQ

tBuOOH

Cobalt

CDNBDox

Cisplat

Etoposide

CuOOHGSNO

TNF+Aur

TNF+Sulphor

TNF+AHQ

TNF+Cobalt

TNF+CDNB

TNF+Dox

TNF+Cisp

TNF+Etoposide

Untransfe

mCherryYPetCFP

Normoxia Total I / cellI/c

CtrlEtO

SulphorAHQ

tBuOOH

Cobalt

CDNBDox

Cisplat

Etoposide

CuOOHGSNO

TNF+Aur

TNF+Sulphor

TNF+AHQ

TNF+Cobalt

TNF+CDNB

TNF+Dox

TNF+Cisp

TNF+Etoposide

Untransfe

100000

150000

200000

250000

Nrf2HIFNFkB

Normoxia Total I / cell

CtrlEtO

SulphorAHQ

tBuOOH

Cobalt

CDNBDox

Cisplat

Etoposide

CuOOHGSNO

TNF+Aur

TNF+Sulphor

TNF+AHQ

TNF+Cobalt

TNF+CDNB

TNF+Dox

TNF+Cisp

TNF+Etoposide

Untransfe

100000

150000

200000

250000

Nrf2HIFNFkB

pTRAF transfected HEK293 cells analysed with the Operetta

21% O2

pTRAF transfected HEK293 cells analysed with Operetta in 21 % O2

pTRAF transfected cells in 21% O2

Ctrl TNF Auranofin TNF+Aur0

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

I/cell

NFκBNrf2 HIF

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

Nrf2 - 21% O2

I/Cell

Auranofin

Sulforophane

Doxorubicin

Cisplatin

TNF+Aur

TNF+Sulf

TNF+AHQ

TNF+Dox

TNF+Cisp

100000

HIF - 21% O2

Auranofin

Sulforophane

Doxorubicin

Cisplatin

TNF+Aur

TNF+Sulf

TNF+AHQ

TNF+Dox

TNF+Cisp

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

Auranofin

Sulforophane

Doxorubicin

Cisplatin

TNF+Aur

TNF+Sulf

TNF+AHQ

TNF+Dox

TNF+Cisp

100000

150000

200000

How are Nrf2, HIF and NFκB redox regulated?

NADPH + H+

Trx-(SH)2

Trx-S2

GSH-systemTrx-system

GPxGrxGSTEtc.

(Or TRP14)

SeTrxR1 activity in HEK293 cells

Contro

5nM Se

M Se0.00

Ctrl Aur TNF TNF+Aur0

I/cell

-Selenite+Selenite

I/cell

-Selenite+Selenite

100000

I/cell

-Selenite+Selenite

HIF response in 1% O2

100000

-Selenite+Selenite (25nM)

100000

150000

200000

I/cell

Nrf2- response in 1% O2

How are Nrf2, HIF and NFκB redox regulated?

NADPH + H+

Trx-(SH)2

Trx-S2

GSH-systemTrx-system

GPxGrxGSTEtc.

(Or TRP14)

I/cell

-Selenite+Selenite

I/cell

-Selenite+Selenite

100000

I/cell

-Selenite+Selenite

HIF response in 1% O2

100000

150000

200000

I/cell

Nrf2- response in 1% O2

TrxR1 activity in HEK293 cells

Contro

5nM Se

M Se0.00

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

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

Redox regulated transcription factorsgenomics.unl.edu/RBC_2015/COURSE_FILES/tue2.pdf · 2015. 5....

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