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J. Biol. Chem., Vol. 282, Issue 11, 8175-8187, March 16, 2007

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Oxidation and Nitrosylation of Cysteines Proximal to the Intermediate Filament (IF)-binding Site of Plectin

EFFECTS ON STRUCTURE AND VIMENTIN BINDING AND INVOLVEMENT IN IF COLLAPSE^*

Radovan Spurny, Kamaran Abdoulrahman¹, Lubomir Janda, Dominik Rünzler, Gottfried Köhler, Maria J. Castañón, and Gerhard Wiche²

From the Departments of Molecular Cell Biology and Biomolecular Structural Chemistry, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria

As an intermediate filament (IF)-based cytolinker protein, plectin plays a key role in the maintenance of cellular cytoarchitecture and serves at the same time as a scaffolding platform for signaling cascades. Consisting of six structural repeats (R1–6) and harboring binding sites for different IF proteins and proteins involved in signaling, the plectin C-terminal domain is of strategic functional importance. Depending on the species, it contains at least 13 cysteines, 4 of which reside in the R5 domain. To investigate the structural and biological functions of R5 cysteines, we used cysteine-to-serine mutagenesis and spectroscopic, biochemical, and functional analyses. Urea-induced unfolding experiments indicated that wild-type R5 in the oxidized, disulfide bond-mediated conformation was more stable than its cysteine-free mutant derivative. The binding affinity of R5 for vimentin was significantly higher, however, when the protein was in the reduced, more relaxed conformation. Of the four R5 cysteines, one (Cys4) was particularly reactive as reflected by its ability to form disulfide bridges with R5 Cys1 and to serve as a target for nitrosylation in vitro. Using immortalized endothelial cell cultures from mice, we show that endogenous plectin is nitrosylated in vivo, and we found that NO donor-induced IF collapse proceeds dramatically faster in plectin-deficient compared with wild-type cells. Our data suggest an antagonistic role of plectin in nitrosylation (oxidative stress)-mediated alterations of IF cytoarchitecture and a possible role of R5 Cys4 as a regulatory switch.

Received for publication, September 5, 2006 , and in revised form, December 20, 2006.

^* This work was supported by Grant 717862-B09 from the Austrian Science Research Fund. Thecostsofpublicationofthisarticleweredefrayedinpartbythe payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental "Experimental Procedures," Equations 1 and 2, Refs. 1–6, and Figs. S1 and S2.

¹ Present address: College of Pharmacy, Salahaddin University, Erbil-Kurdistan Region, Federal Republic of Iraq.

² To whom correspondence should be addressed: Dept. of Molecular Cell Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria. Tel.: 43-1-4277-52852; Fax: 43-1-4277-52854; E-mail: gerhard.wiche{at}univie.ac.at.

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