Abstract
The intermediate filament protein vimentin is involved in essential cellular processes, including cell division and stress responses, as well as in the pathophysiology of cancer, pathogen infection, and autoimmunity. The vimentin network undergoes marked reorganizations in response to oxidative stress, in which modifications of vimentin single cysteine residue, Cys328, play an important role, and is modulated by zinc availability. However, the molecular basis for this regulation is not fully understood. Here, we show that Cys328 displays a low pKa, supporting its reactivity, and is readily alkylated and oxidized in vitro. Moreover, combined oxidation and crosslinking assays and molecular dynamics simulations support that zinc ions interact with Cys328 in its thiolate form, whereas Glu329 and Asp331 stabilize zinc coordination. Vimentin oxidation can induce disulfide crosslinking, implying the close proximity of Cys328 from neighboring dimers in certain vimentin conformations, supported by our computational models. Notably, micromolar zinc concentrations prevent Cys328 alkylation, lipoxidation, and disulfide formation. Moreover, zinc selectively protects vimentin from crosslinking using short-spacer cysteine-reactive but not amine-reactive agents. These effects are not mimicked by magnesium, consistent with a lower number of magnesium ions hosted at the cysteine region, according to molecular dynamics simulations. Importantly, the region surrounding Cys328 is involved in interaction with several drugs targeting vimentin and is conserved in type III intermediate filaments, which include glial fibrillary acidic protein and desmin. Altogether, our results identify this region as a hot spot for zinc binding, which modulates Cys328 reactivity. Moreover, they provide a molecular standpoint for vimentin regulation through the interplay between cysteine modifications and zinc availability.
Highlights
Introduction published maps and institutional affilVimentin is a type III intermediate filament protein that forms robust filaments in mesenchymal cells
We have previously shown that a Cys328Ser mutant is competent for filament formation in vitro and confers resistance to the alterations of vimentin filaments induced by oxidants and electrophilic agents [10], it shows subtle but significant differences in polymerization features and response to zinc supplementation compared to vimentin wt [45]
We have previously shown that the vimentin network is thinner and more susceptible to oxidative damage in fibroblasts from patients with genetic zinc deficiency, i.e., acrodermatitis enteropathica [102], and these cellular alterations are ameliorated by supplementation with micromolar levels of zinc [9]
Summary
Introduction published maps and institutional affilVimentin is a type III intermediate filament protein that forms robust filaments in mesenchymal cells. The intermediate filament network maintains an intimate crosstalk with the other cytoskeletal systems (microfilaments and microtubules) and modulates essential cell properties, from cell size to migration, division, and elasticity [1,2,3]. Vimentin is involved in disease by various mechanisms, including acting as a cellular receptor for pathogens, such as bacteria and viruses [4], regulating the immune response [5], or playing a key role in the epithelial–mesenchymal transition during tumorigenesis [6], for which it is considered a critical drug target [4,7,8]. The vimentin network finely responds to electrophilic and oxidative stress, a function in which its single cysteine residue (Cys328) plays an important role [9,10]. 4-hydroxynonenal, cyclopentenone prostaglandins, and nitrated phospholipids induce marked vimentin reorganization [10,11]. iations.
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