Course in Redox regulation, oxidative stress and ...genomics.unl.edu/RBC_2012/COURSE_FILES/S3.pdfOmi/HtrA2 mature form In vitro activation of serine Not necessary 34 protease activity

Redox Regulators

Group members: Fernando Toshio Ogata

Ryan Grove Dragana Celojevic

Urmi Basu

Course in Redox regulation, oxidative stress and selenoproteins at Karolinska Institutet 2012

Redox Regulators - 1 - OxyR

OxyR: •  First discovered in Salmonella typhimurium;

•  Highly conserved among bacteria; •  Member of LysR family;

•  DNA-binding protein

Describe the mechanism by which OxyR responds to hydrogen peroxide and regulates gene expression


Structure of OxyR


A few genes regulated by OxyR: •  hydroperoxidase I (katG),

•  alkyl hydroperoxide reductase (ahpCF)

•  a small regulatory RNA (oxyS),

•  glutathione reductase (gorA)

•  glutaredoxin I (grxA)

•  a nonspecific DNA-‐binding protein (dps)

Redox Regulators - 2 - Yap1p

Describe the mechanism by which Yap1p is involved in the response to

oxidative stress in S. cerevisiae

Yeast AP-‐1 Protein (Yap1)

•  AP-‐1 is a class of dimeric TFs – Basic leucine zipper – Respond to external sLmuli

•  Yeast AP-‐1 – Response to oxidaLve stress

•  Nuclear locaizaLon –  Induces expression of many redox response genes in various pathways

Glover, JN, Harrison, SC. Nature 1995 373: 257-‐261 Wood, MJ et al. Nature 2004 430: 917-‐921


Nuclear LocalizaLon of Yap1

Yap1-‐SH Yap1-‐SH

Yap1-‐SH Yap1-‐SH Crm1

Cytosol

Nucleus

Wood, MJ et al. Nature 2004 430: 917-‐921


Nuclear LocalizaLon of Yap1

Yap1-‐SH Yap1-‐SH

Yap1-‐SH Yap1-‐SH Crm1

Cytosol

Nucleus

Yap1-‐SS

H2O2

Yap1-‐SS

Yap1-‐SS Crm1

X

Yap1-‐SS

Trx2

Redox Gene

Toone, WM et al. Oncogene 2001 20: 2336-‐2346


Genes Induced

Herrero et al. Biochimica et Biophysica Acta 2008 1780: 1217-‐1235 Toone, WM and Jones, N. Curr Opin Gen Dev 1999 9: 55-‐61


Redox Regulators - 3 - Floodgate hypothesis

Prxs reduce hydroperoxides by using the peroxide reactivity of the cysteine sulfur atom and thioredoxin as the hydrogen-donor pathway

Prxs as peroxide floodgate: low concentration of hydroperoxides - closed floodgate – Prxs keep peroxides away from susceptible targets high concentrations - opened floodgate - Prxs are temporarily inactivated during H2O2 signaling

What is the floodgate hypothesis in mammalian redox signaling, involving peroxiredoxins?

Wood et al. Science 2003


What are the arguments for and against this signaling mechanism?

Ø  altered expression of Prxs affect intracellular levels of hydrogen peroxide

Ø  reversible inactivation of Prxs provides a redox-switch mechanism

that regulates these signaling functions relative to the levels of H2O2 Ø  high reactivity for H2O2 - ideal for H2O2 sensing and signaling Ø  posttranslational modifications of Prxs (altered peroxidase function)

suggests an extended set of regulatory functions Ø  Prx transfer oxidative signals from peroxides to target proteins due to

ability to oxidize proteins-thiols - allowing the oxidation of regulatory proteins that are otherwise nonreactive to peroxide.

addition, a multitude of studies have shown that overex-pressed Prxs, as much as SeGPxs, can interfere with cell death(48, 70, 112, 114), which is at least another demonstration ofthe important signaling role of H2O2 in these death pathways.

Prxs can also affect cell signaling by virtue of an interac-tion with and modulation of regulatory proteins in a man-ner independent of their peroxidase function. IndividualPrxs have indeed been shown to interact specifically with amultitude of regulatory proteins (listed in Table 2), and, insome instances, to alter their regulatory functions, as in theinteraction of Prx1 with the c-Abl protooncogene (101), thec-MYC protooncogene (65), the JNK-GST! protein complex(49), and with the androgen receptor (75). Establishing thein vivo biologic relevance of these interactions will requirefurther studies.

Summary and Perspectives

The thiol peroxidases, Prxs and CysGPxs, are fascinatingperoxide-metabolizing enzymes that clearly carry biologicfunctions beyond those of simple peroxide-protective en-zymes. Their biochemical and enzymatic attributes specify

both distinctive peroxide-scavenging functions and the abil-ity to signal H2O2. Their high reactivity for peroxide togetherwith their relative overall catalytic inefficiency and theirproperty to undergo reversible substrate-mediated inactiva-tion provide the unique ability to scavenge the low levels ofendogenously produced peroxide, also restricting scaveng-ing to these low concentrations. Such an enzymatic charac-teristic confers the quasi-exclusive function of protecting thegenome against mutations that would otherwise arise dur-ing normal cell metabolism, as shown in E. coli and in S. cere-visiae (36, 88). Regarding this function, selenothiol-basedGPxs, which do not exist in prokaryotes and fungi, also mustbe considered as important in mammals.

Reactivity and reversible inactivation are also attributes ex-ploited by Prxs for operating H2O2 signaling, which generallyoccurs under low to very low concentrations of the oxidant.Whereas prokaryotic thiol peroxidases primarily operate asperoxide-protective enzymes, eukaryotic ones are also used inthe regulation of H2O2 signaling. Model fungi have revealed,for both Prxs and CysGPxs, the unique regulatory function ofdetecting and propagating H2O2 signals. This function is againdependent on the unique peroxide reactivity of these enzymes

SIGNALING FUNCTIONS OF THIOL-BASED PEROXIDASES 1571

TABLE 2. PROTEIN-PROTEIN INTERACTIONS INVOLVING MAMMALIAN PEROXIREDOXINS

Peroxiredoxin Interaction partner Significance CysP Reference

Prx1 JNK-GST! complex Prevents JNK activation by IR Not necessary 49c-Abl Abl tyrosine kinase activity inhibition 101eEF1A-2 Co-transfection of both proteins 16

protects cells from H2O2-inducedapoptosis

Omi/HtrA2 mature form In vitro activation of serine Not necessary 34protease activity

Androgen receptor, on Prx1 increases AR activation by H/R Not necessary 75hypoxia/reoxygenation and synthetic androgen R1881

c-Myc (Myc Box II domain) Prx1 inhibits tumorigenicity 65induced by myc

MIF Decreased D-dopachrome Not necessary 45tautomerase activity of MIF

Presenilin-1 PS-1 overexpression prevents Prx1 115overexpression-induced cell deathin SCG primary neurons culture

Syk Not assessed 113Prx2 PDGFR", on PDGF Prevents PDGFR" Tyr857 20

phosphorylation andCdk5 Prx2 phosphorylation 83PLD1 on PMA Inhibits H2O2-induced 109

PLD1 activityStomatin (7.2b) Could be linked to a role of Prx2 64

in Ca2!-dependent K! transportPrx3 LZK Enhances IKK and NF-#B activation Not necessary 61

for interactionAbrin A chain Prx3 blocks Abrin-induced apoptosis 89RPK118 RPK118 could be a transporter 56

of Prx3 to mitochondriaFANCG Protection against Prx3 cleavage 66

and mitochondrial oxidative stressPrx4 Thromboxane A2 TP" relocalization to ER and TP" 31

receptor, isoform " degradation on H2O2Prx6 p67phox Linked to Prx6 phospholipase 50

A2 activitySurfactant protein A Inhibition of Prx6 PLA2 activity 108Saitohin Possible link to Pick disease 30

PROTEIN-‐PROTEIN INTERACTIONS INVOLVING MAMMALIAN PEROXIREDOXINS

Fourquet et al. An9oxid Redox Signal 2008


Redox Regulators - 4 - Keap1-Nrf2-ARE signaling pathway

Keap1-Nrf2-ARE signaling pathway


Ø  Nuclear factor erythroid 2-related factor 2 (Nrf2), a member of “Cap n Collar” subfamily of the bZIP transcription factors

Ø  Binds to Antioxidant response element (ARE) DNA sequence to induce transcription of detoxifying and antioxidant enzymes to counteract electrophilic and oxidative stress

Ø  Controls both inducible and constitutive gene expression mediated by ARE

Ø  Activity is repressed by an actin associated protein, Kelch-like ECH-associated protein1 (Keap1).

World J Cardiol. 2011

How does it help cells respond to oxidative stress


What genes are regulated by this system?

Genes encoding phase II detoxification enzymes as well as antioxidant proteins including: Ø  Glutathione-S-transferases

Ø  NAD(P)H:quinone oxidoreductase-1

Ø  Glutathione peroxidase

Ø  Gamma-glutamylcysteine synthase

Ø  Ferritin

Ø  Heme oxygenase-1


Documents

Course in Redox regulation, oxidative stress and ...genomics.unl.edu/RBC_2012/COURSE_FILES/S3.pdfOmi/HtrA2 mature form In vitro activation of serine Not necessary 34 protease activity