Functions of the actin oxidation cycle

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The highly dynamic nature and organization of the actin cytoskeleton in vivo underlies its functional ability to respond rapidly to internal and external cellular signals. Actin re-organization is essential in almost all cell physiological processes such as chemotaxis, cell growth and division, neurite extension, and phagocytosis. The complexity of these events necessitates multiple actin regulating factors. Examples of well known regulators are actin binding proteins such as profilin, gelsolin and cofilin, as well as lipid kinases/phosphatases, and signal transduction factors such as the Rac and Rho family of small G-proteins. Recently, the actin oxidation cycle has emerged as a powerful regulatory mechanism with hydrogen peroxide (H2O2) as the major player. Reactive oxygen species such as H2O2 are produced by a variety of extracellular signaling events (e.g., cytokines, growth factors) which activate NADPH oxidase and lipoxygenase (1). In vivo, actin cycles between reduced (actR) and oxidized (actO) forms (2; see Figure 1), with reactions centered at two main redox sensitive cysteine amino acid residues, Cys272 and Cys374 in b-actin (3). Cys272 is oxidized to sulfenic (Cys-SOH), sulfinic (Cys-SO2H) or sulfonic (Cys-SO3H) acid residues in the presence of H2O2. The Cys374 amino acid is oxidized either in the same way or by forming a disulfide bridge which creates actin dimers (4). These modifications are reversible to the reduced form by reductive cellular pathways involving thioredoxin/thioreductase/NADPH (TRX) or glutathione/glutathione reductase/NADPH (GLT).

Interestingly, these two oxidized cysteines confer important biological roles for oxidized actin. For example, actO has approximately a 10 fold reduced affinity for profilin (Cys374 oxidation; 3) and production of actO in vitro causes depolymerization of preformed filaments which is reversible by addition of reducing agents (3). These two characteristics of actO could hypothetically facilitate the formation of many short dynamic filaments in a localized oxidative environment. Additionally, in vitro oxidation of Cys374 by H2O2 induces formation of actin dimers that can incorporate into filaments to create a nucleating center for branched filament formation (4). The Arp2/3 protein may be involved in stabilizing these

structures and forming daughter filaments (3). Finally, the sulfenic product of Cys272 oxidation is a sensor for the redox state which determines the fate of cells by apoptosis (5).

Another interesting aspect of the actin oxidative cycle is that the small G-protein Rac possibly has another role in actin regulation, apart from the well-studied Rac/effector-mediated control of growth cone dynamics (6). Rac could be involved in the signaling cascade that leads to amplification of the H2O2 concentration (7, 8). Down-regulation of lipid or protein tyrosine kinase phosphatases (e.g., PTEN) by H2O2 increases the amount of PIP2 and PIP3 (9). These phosphoinositides target guanine exchange factors that activate Rac (10, 11), which can activate NADPH oxidase (12, 13), resulting in increased production of H2O2 (9). Actin could then be oxidized by this amplified pool of H2O2 at the cell periphery in a temporally and spatially relevant manner to regulate actin dynamics at the cell periphery. Later in the progression of actin towards the cell lumen by treadmilling, actO could be converted to actR by the reductive systems TRX and GLT.

Figure 1: Potential functions of actO in vivo. Actin cycles between oxidized (actO) and reduced (actR) forms in vivo with actO having possible roles in dimer formation and short filament formation. ActO is also a sensor for cellular redox potential which in the oxidized state can lead to apoptosis. TRX, thioredoxin/thioreductase/NADPH; GLT, glutathione/glutathione reductase/NADPH.

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References

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Importantly, actin oxidation research has clinical relevance as two intriguing models for the role of actO in human diseases are emerging. In in vitro models of inflammatory bowel syndrome, actO plays a key role in compromising the intestinal barrier integrity of colonic Caco-2 cells (14). Also, in ischemia, actO contributes to apoptosis and death of cardiac cells (15).

1. Sundaresan M, Yu ZX, Ferrans VJ, Irani K, and Finkel T. 1995. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 270, 296-299.

2. Fiaschi T, Cozzi G, Raugei G, Formigli L, Ramponi G, and Chiarugi P. 2006. Redox regulation of beta-actin during integrin-mediated cell adhesion. J. Biol. Chem. 281, 22983–22991.

3. Lassing I, Schmitzberger F, Bjornstedt M, Holmgren A, Nordlund P, Schutt CE, and Lindberg U. 2007. Molecular and structural basis for redox regulation of b-actin. J. Mol. Biol. 370, 331-348.

4. Tang JX, Janmey PA, Stossel TP, and Ito T. 1999. Thiol oxidation of actin produces dimers that enhance the elasticity of the F-actin network. Biophys. J. 76, 2208–2215.

5. Farah ME and Amberg DC. 2007. Conserved actin cysteine residues are oxidative stress sensors that can regulate cell death in yeast. Mol. Biol. Cell. 18, 1359-1365.

6. Hall A. 1998. Rho GTPases and the actin cytoskeleton. Science. 279, 509-514.

7. Moldovan L, Irani K, Moldovan NI, Finkel T, and Goldschmidt-Clermont PJ. 1999. The actin cytoskeleton reorganization induced by Rac1 requires the production of superoxide. Antioxid. Redox Signal. 1, 29-43.

8. Rhee SG, Bae YS, Lee SR, and Kwon J. 2000. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Sci. STKE. 2000, PE1.

9. Rhee SG, Kang SW, Jeong W, Chang TS, Yang KS, and Woo HA. 2005. Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins. Curr. Opin. Cell Biol. 17, 183-189.

10. Hawkins PT, Eguinoa A, Qiu R-G, Stokoe D, Cooke FT, Walters R, Wennstrom S, Claesson-Welsh L, Evans T, Symons M, and Stephens L. 1995. PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr. Biol. 5, 393-403.

11. Han J, Luby-Phelps K, Das B, Shu X, Xia Y, Mosteller RD, Krishna UM, Falck JR, White MA, and Broek D. 1998. Role of substrates and products of PI3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science. 279, 558-560.

12. Abo A, Pick E, Hall A, Totty N, Teahan CG, and Segal AW. 1991. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 353, 668–670.

13. Moldovan L, Mythreye K, Goldschmidt-Clermont PJ, and Satterwhite LL. 2006. Reactive oxygen species in vascular endothelial cell motility. Roles of NAD(P)H oxidase and Rac1. Cardiovasc. Res. 71, 236–246.

14. Banan A, Zhang Y, Losurdo J, and Keshavarzian A. 2012. Carbonylation and disassembly of the F-actin cytoskeleton in oxidant induced barrier dysfunction and its prevention by epidermal growth factor and transforming growth factor a in a human colonic cell line. Gut. 46, 830-837.

15. Canton M, Neverova I, Menabo R, Van Eyk J, and Di Lisa F. 2004. Evidence of myofibrillar protein oxidation induced by postischemic reperfusion in isolated rat hearts. Am. J. Physiol. Heart Circ. Physiol. 286, H870–H877.

Actin Biochem Kits™ Cat. # Amount

Actin Polymerization Biochem Kit™ BK003 30-100 assays

G-actin/F-actin in vivo Biochem Kit™ BK037 30-100 assays

Actin Binding Protein Spin-Down Assay Biochem Kit™ Muscle

BK001 30-100 assays

Actin Binding Protein Spin-Down Assay Biochem Kit™Non-muscle

BK013 30-100 assays

F-actin Visualization Biochem Kit™ BK005 300 assays

Antibodies Antigen Host Grade Cat. # AmountActin Antibody C-terminal of

actinRabbit Affinity

PurifiedAAN01-AAAN01-B

1 x 100 µg3 x 100 µg

Cofilin Antibody N-terminal of human cofilin1

Rabbit Affinity Purified

ACFL02-AACFL02-B

1 x 50 µg3 x 50 µg

Fibrillarin Antibody 72B9 (IgG2a) Mouse Ascites AFB01-AAFB01-B

1 x 100 µg3 x 100 µg

Profilin Antibody Purified human profilin

Rabbit Affinity Purified

APUF01-AAPUF01-B

1 x 50 µg3 x 50 µg

Phalloidin Excitation Emission Signal stability *(T1/2 in secs)

Cat. # Amount

Acti-stain™ 488 phalloidin

480 nm 535 nm 57 PHDG1-A 300 Slides

Acti-stain™ 535 phalloidin (Rhodamine Phalloidin)

535 nm 585 nm 27 PHDR1 300 Slides

Acti-stain™ 555 phalloidin

535 nm 585 nm 46 PHDH1-A 300 Slides

Acti-stain™ 670 phalloidin

640 nm 670 nm 8 PHDN1-A 300 Slides

* Stability measured without antifade. For comparison, fluorescein phalloidin has a T1/2 of 6 secs.** One slide equals enough phalloidin to stain a 25 mm2 coverslip

Actin Binding Proteins Source Purity Cat. # Amounta-Actinin Protein Rabbit skeletal muscle >90% AT01-A

AT01-C2 x 50 µg10 x 50 µg

Arp2/3 Protein Complex Bovine brain >95% RP01-ARP01-B

2 x 50 µg6 x 50 µg

Cofilin Protein Recombinant human cofilin 1

95% CF01-ACF01-C

1 x 100 µg4 x 100 µg

Gelsolin Protein Recombinant human, plasma isoform

>95% HPG6-AHPG6-B

4 x 20 µg20 x 20 µg

Myosin Cardiac Protein Bovine cardiac muscle 95% MY03-AMY03-B

1 x 1 mg5 x 1 mg

Myosin: Heavy Meromyosin Protein

Chymotrypsin digest of rabbit skeletal muscle myosin II

70% MH01-AMH01-B

4 x 50 µg20 x 50 µg

Myosin II Protein Rabbit skeletal muscle 95% MY02-AMY02-B

5 x 1 mg20 x 1 mg

Profilin Protein Recombinant human profilin 1

>95% PR01-APR01-C

1 x 50 µg4 x 50 µg

WASP protein VCA Domain: GST taggedBinds & activates Arp2/3

Recombinant human >95% VCG03-AVCG03-B

1 x 500 µg5 x 500 µg

Rac1 Activation Assay & Activator Cat. # Amount

Rac1 G-LISA® Activation Assay, colorimetric BK128 96 assays

Rho/Rac/Cdc42 Activator IDeamidation of Rho Gln-63 & Rac/Cdc42 Gln-61

CN04-ACN04-B

3 x 20 µg9 x 20 µg