Molecular Insight into the Regulation of Vimentin by Cysteine Modifications and Zinc Binding

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.

Unique Role of Vimentin Networks in Compression Stiffening of Cells and Protection of Nuclei from Compressive Stress

“…Figure J,K was taken from the GFP-vimentin expressing fibroblast just before (Figure J) and after (Figure K) application of 50 nN compressive force, showing that no visible vimentin cage change. This result also suggests that the rate of deformation is faster than any possible modifications caused by cell signaling, which has been shown to post-translationally alter VIF network mechanics, − and the observed difference in compressibility between WT mEFs and vim null mEFs cannot be attributed to such alterations.…”

Intracellular pH modulates vimentin remodeling in response to oxidants

“…Briefly, vimentin at 4 µM diluted with buffers at different pHs, and incubated at 24 o C in the presence of 1 mM H2O2 or 1 mM diamide for 1 h, or with 20 µM Mal-B for 15 min. The buffers at different pHs used were near the pKa values previously calculated by us [23]. Given the fact that DTT can react with electrophilic compounds and chelate metals [47], reactions with Mal-B were carried out with vimentin subjected to DTT elimination, while for modifications with diamide and H2O2, the final concentration of DTT was kept below 0.1 mM.…”

“Block and attack” strategy for tumor therapy through ZnO2/siRNA/NIR‐mediating Zn2+‐overload and amplified oxidative stress

“…Cystine residues play important roles in proteins, including binding to various metal ions. [54][55][56] In particular, cysteine residues have a high affinity for Zn 2+ , and the Zn 2+ -cysteine complexes play a key role in mediation of protein structure, catalysis and regulation (Figure 4F). [57,58] Mutations in cysteine may distort the conformational structure of Oria1, leading to down-regulation of Oria1 expression (Figure 4G).…”