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

Abstract

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 donorinduced 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.

Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle

“…also supports our concept that IFs that are not anchored via plectin at subcellular docking sites become destabilized, more mobile, and presumably more accessible to posttranslational modifications, which in turn might make them more prone to aggregation. Such a mechanism would explain previous results showing that hyperphosphorylation-and oxidative stress-induced (nitrosylationinduced) disruption and collapse of IF networks in plectin-deficient keratinocyte and endothelial cells, respectively, occurred more readily in plectin-deficient cells compared with wild-type cells (20,21). The fact that plectin deficiency concurred with spontaneous protein aggregate formation in myotubes, but not in the other previously tested cell systems, probably reflects the higher mechanical load of these cells due to their spontaneous contraction.…”

The Structure of the Plakin Domain of Plectin Reveals a Non-canonical SH3 Domain Interacting with Its Fourth Spectrin Repeat

“…First, it might affect the affinity of plectin for some ligands due to a reduction in the conformational adaptability of this region. Similarly, oxidation of a Cys near the intermediate filament-binding site located in the C-terminal region of plectin reduces its affinity for vimentin (62). Second, disulfide crosslinking might reduce the flexibility of this segment of the plakin domain, increasing the stability of the plakin domain.…”

Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody

“…Given that vimentin is the target of various posttranslational modifications that include oxidation (46,47), we thus examined whether these oxidative adducts were associated with vimentin exposed on the surface of senescent cells. To that end, we performed a combinatorial vimentin IP and time-course experiment.…”