Redox regulated transcription factors, MD PhD Division of Biochemistry Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden Elias.Arner@ki.se
Redox regulation A process of regulated activation or inhibition of cellular signaling pathways through redox control
Redox regulation - examples of chemical reactions (A disulfide) (A dithiol) R S S (Active, or Inactive) 2 e - + 2 H + R SH (two-electron transfer) SH (Nitrosylation) R SH GSSG Grx s (Glutathionylation) GSH GSNO R SH R SSG (Active, or Inactive) (?) Trx s GSH R SNO (Active, or Inactive)
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 GSHdependent 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 a well studied redox regulated transcription factor in E. coli 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 known redox regulated transcription factor in E. coli
Yap1 a well studied redox regulated transcription factor in S. cerevisiae The H 2 O 2 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 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 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: 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 From: Cancer chemoprevention with dietary phytochemicals Young-Joon Surh Nature Reviews Cancer 3, 768-780 (October 2003)
Protein phosphorylation cascades Oxidation and thereby inactivation of PTP s allows for efficient protein phosphorylation cascades to occur Compartmentalization? Mediator of H 2 O 2 signal? 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
Reactivity with H 2 O 2 and amount of target are key determinants for redox regulation what actually reacts?
Reactivity with H 2 O 2 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? 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 Fomenko D E et al. PNAS 2011;108:2729-2734
Mechanism by which peroxidases may mediate signaling events Peroxiredoxins as antioxidant enzymes, removing H2O2 using the Trx system From: The Dual Functions of Thiol- Based Peroxidases in H2O2 Scavenging and Signaling Fourquet, S. et al, 2008 Antioxid. Redox Signal. 10, 1565 1575 Peroxiredoxins in signaling, transfering oxidative equivalents from H2O2 to specific target proteins through protein-protein interactions
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