Vimentin disruption by lipoxidation and electrophiles: Role of the cysteine residue and filament dynamics

Abstract

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress, which induce extensive reorganization of the vimentin cytoskeletal network. Here, we have investigated the mechanisms underlying these effects. In vitro, electrophilic lipids, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and 4-hydroxynonenal (HNE), directly bind to vimentin, whereas the oxidant diamide induces disulfide bond formation. Mutation of the single vimentin cysteine residue (Cys328) blunts disulfide formation and reduces lipoxidation by 15d-PGJ2, but not HNE. Preincubation with these agents differentially hinders NaCl-induced filament formation by wild-type vimentin, with effects ranging from delayed elongation and increased filament diameter to severe impairment of assembly or aggregation. Conversely, the morphology of vimentin Cys328Ser filaments is mildly or not affected. Interestingly, preformed vimentin filaments are more resistant to electrophile-induced disruption, although chemical modification is not diminished, showing that vimentin (lip)oxidation prior to assembly is more deleterious. In cells, electrophiles, particularly diamide, induce a fast and drastic disruption of existing filaments, which requires the presence of Cys328. As the cellular vimentin network is under continuous remodeling, we hypothesized that vimentin exchange on filaments would be necessary for diamide-induced disruption. We confirmed that strategies reducing vimentin dynamics, as monitored by FRAP, including cysteine crosslinking and ATP synthesis inhibition, prevent diamide effect. In turn, phosphorylation may promote vimentin disassembly. Indeed, treatment with the phosphatase inhibitor calyculin A to prevent dephosphorylation intensifies electrophile-induced wild-type vimentin filament disruption. However, whereas a phosphorylation-deficient vimentin mutant is only partially protected from disorganization, Cys328Ser vimentin is virtually resistant, even in the presence of calyculin A. Together, these results indicate that modification of Cys328 and vimentin exchange are critical for electrophile-induced network disruption.

Proteome Oxidative Modifications and Impairment of Specific Metabolic Pathways During Cellular Senescence and Aging

“…For instance, in fibroblasts, vimentin is particularly affected during senescence as it simultaneously suffers from direct carbonylation, as well as HNE and AGE adductions. Vimentin has been shown to be prone to binding of HNE and it has been observed that senescent primary fibroblasts express on their cell surface vimentin presenting lipid peroxidation adducts . Indeed vimentin is subject to various nonenzymatic modifications, mostly oxidative in nature, which may be involved in the regulation of the vimentin network under stress conditions.…”

Zinc Differentially Modulates the Assembly of Soluble and Polymerized Vimentin

“…Vimentin plays key roles in essential cell functions such as division and migration and contributes to cellular structural support and plasticity and organelle positioning. In addition, recent reports have unveiled its complex interplay with other cytoskeletal systems [1,2] and its involvement in redox sensing [3,4], regulation of gene expression, [5] or protection of the nucleus and DNA from damage [6]. Moreover, biophysical techniques are providing high resolution information on the mechanics and dynamic performance of the vimentin network, with a clear impact on the physical properties of cells [6,7].…”

“…In cells, vimentin is subjected to an exquisite regulation, both by transcriptional and posttranscriptional mechanisms relying on a plethora of posttranslational modifications, including phosphorylation, glycosylation, SUMOylation, as well as non-enzymatic processes, like oxidative modifications [4,16]. In particular, modifications of the vimentin single cysteine residue, C328 in human vimentin, play a key role in filament assembly in vitro and network remodeling in cells [3,4,17]. Oxidative modifications of C328 disrupt in vitro filament formation in a manner dependent on the structure of the modifying moiety [4].…”

“…In particular, modifications of the vimentin single cysteine residue, C328 in human vimentin, play a key role in filament assembly in vitro and network remodeling in cells [3,4,17]. Oxidative modifications of C328 disrupt in vitro filament formation in a manner dependent on the structure of the modifying moiety [4]. Moreover, C328 appears to occupy a key position in assembled filaments in such a way that modifications or substitutions of this residue by other amino acids affect vimentin performance in cellular functions.…”

“…Thus, C328S vimentin is less efficient than the wild-type (wt) protein at forming extended networks and supporting organelle positioning in cells [3]. Moreover, cysteine-deficient vimentin displays an attenuated response against several types of stress [3,4]. Although knowledge of the regulation of vimentin is rapidly expanding, there are still several intriguing aspects like its potential interaction with regulatory or associated proteins and the mechanisms controlling assembly in the cellular context.…”