”Redox regulated transcription factors”

June 10, 2011 Elias Arnér

”Redox regulated transcription factors”

Elias Arnér, MD PhD

Division of Biochemistry

Medical Biochemistry and Biophysics

Karolinska Institutet

Stockholm, Sweden

[email protected]


Redox regulation

— A process of regulated activation or

inhibition of cellular signaling pathways

through redox control


Redox regulation

- examples of chemical reactions

R S

S

2 e- + 2 H+

R SH

SH

(A dithiol) (A disulfide)

(two-electron transfer)

(Active, or Inactive)

R SH

(Glutathionylation)

GSSG GSH

R SSG


R SH

(Nitrosylation)

GSNO GSH

R SNO


Grx’s

Trx’s

(?)


Many levels of redox regulation in cells

- there is very much yet to discover

Arnér ES, Holmgren A. (2000) Eur J Biochem. 267:6102-6109


Redox regulation

- a few examples of regulated proteins

• Oxidation of Keap-1 releases and activates the transcription factor Nrf2,

which induces transcription of a number of predominantly GSH-

dependent antioxidant enzyme systems

• Several protein tyrosine phosphatases (PTP’s) are transiently inactivated

by oxidation (probably due to local NOX activity) as a step in protein

phosphorylation cascades, thus activating protein phosphorylation

cascades

• Many proteins have been identified to be reversibly glutathionylated, the

significance of which is yet largely unknown

• Nitrosylation of caspase-3 may inactivate the caspase and prevent

apoptosis (this nitrosylation can be removed by the thioredoxin system)

Many proteins may be “redox regulated”


Fomenko et al., Science 315, 387-389 (2007)

Many proteins may be “redox regulated”


Functions of Cys residues in proteins

1. Redox catalysis (e.g., thiol oxidoreductases)

2. Regulatory cysteines (e.g., kinases, phosphatases)

3. Structural disulfides (e.g., secretory proteins)

4. Metal-coordinating cysteines (e.g., zinc fingers)

5. Non-redox catalysis (e.g., certain proteases)

6. Sites of posttranslational modifications (e.g., membrane targeting)

7. Other functions

Any potential targeting of Cys can result in “redox regulation”

What is a “genuine” redox regulatory step?

- Has to be determined experimentally on a case-by-case basis!

A few examples of known mechanisms in

redox regulation…



OxyR is a transcription factor in bacteria activated by

ROS and inducing a number of protective enzymes:

•Catalase

•Alkyl hydroperoxide reductase

•Glutathione reductase (GSSG→2GSH)

•Dps, Protein implicated in protection of DNA from

oxidants

•OxyS, Small nontranslated regulatory RNA involved

in DNA repair

•…

OxyR – a well studied redox regulated

transcription factor in E. coli

OxyR – a well known redox regulated

transcription factor in E. coli


Yap1 – a well studied redox regulated

transcription factor in S. cerevisiae


From: ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis

Benoît D'Autréaux & Michel B. Toledano

Nature Reviews Molecular Cell Biology 8, 813-824 (October 2007)

• The H2O2 signal is sensed by a Cys

residue in Orp1, which oxidizes to sulfenic

acid (Cys-SOH) and subsequently 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


The human Keap-1/Nrf2 axis

Keap-1: Kelch-like ECH-associated protein 1

624 amino acids long, has 27 Cys residues.

Contains BTB and C-terminal KELCH-1-like

(BACK) domains.

Cytosolic repressor of the transcription

factor Nfr2 under reductive conditions.

Nrf2: Nuclear factor (erythroid-derived 2)-like 2 (also known as NFE2L2)

bZIP transcription factor which regulates

antioxidant response by recognizing AREs

(Antioxidant response elements).

AREs activated by Nrf2: HO1, NQO1,

GCL, GST’s, TXNRD1,…


The human Keap-1/Nrf2 axis

Highly simplified scheme:

From:

Cancer chemoprevention with dietary phytochemicals

Young-Joon Surh

Nature Reviews Cancer 3, 768-780 (October 2003)

The inhibitory

Keap1/Nrf2 complex

requires free and

reduced Cys residue(s)

in Keap1, and is

thereby induced by

either oxidation or by

electrophilic agents

Protein phosphorylation cascades


From: Robert M Carey. Pathophysiology of Primary Hypertension. Compr

Physiol 2011, Supplement 9: Handbook of Physiology, The Cardiovascular

System, Microcirculation: 794-895. First published in print 2008. doi:

10.1002/cphy.cp020418

Oxidation and thereby

inactivation of PTP’s

allows for efficient

protein phosphorylation

cascades to occur

Compartmentalization?

Mediator of H2O2 signal?

Reactivity with H2O2 and amount of target

are key determinants for redox regulation –

what actually reacts?


Reactivity with H2O2 and amount of target

are key determinants for redox regulation –

what actually reacts?


Are highly reactive oxidant “sensors”,

e.g. Prx’s, intermediates for oxidant

signaling to target proteins?

Yes, most likely!

Peroxidases may perhaps

mediate signaling events?


Fomenko D E et al. PNAS 2011;108:2729-2734

Deletion of all eight

peroxidases in S.

cerevisiae does not lead

to severely impaired

defense against H2O2,

but abolishes the

triggering of signaling

events by H2O2

Mechanism by which peroxidases

may mediate signaling events


Peroxiredoxins as

antioxidant enzymes,

removing H2O2 using

the Trx system

Peroxiredoxins in

signaling, transfering

oxidative equivalents

from H2O2 to specific

target proteins through

protein-protein

interactions

From:

The Dual Functions of Thiol-

Based Peroxidases in

H2O2 Scavenging and Signaling

Fourquet, S. et al, 2008 Antioxid.

Redox Signal. 10, 1565–1575

Redox regulation may require

oxidation in one compartment but

reduction in another


From:

Dr. Katarina Johansson


Conclusions

Redox regulation involves specific oxidation or reduction of target proteins

with regulation of activity, most often occurring through uniquely reactive

Cys residue(s) in “sensor” proteins

Redox regulation is likely to be important for control of many cellular

signaling pathways and seem to be highly complex and transient

Compartmentalisation as well as protein-protein interactions are likely to

be important in redox regulation, in addition to mere reduction/oxidation

reactions

A thorough knowledge of the chemistry of redox reactions as well as the

biological context of redox active proteins is therefore essential for a

better understanding of redox biology

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”Redox regulated transcription factors”