Monico, A. | bioRxiv Science

bioRxiv2021

Elucidating vimentin interaction with zinc ions and its interplay with oxidative modifications through crosslinking assays and molecular dynamics simulations

The intermediate filament protein vimentin is involved in essential cellular processes, including cell division and stress responses. Vimentin oxidative modifications impact network reorganization and its single cysteine residue, Cys328, acts as a redox sensor. Vimentin binds zinc, which influences its assembly by undefined mechanisms. Here, results from combined biochemical and molecular dynamics studies 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 a close proximity of cysteine residues in certain vimentin associations, validated by our computational models. Notably, micromolar zinc concentrations selectively prevent Cys328 alkylation and crosslinking. These effects are not mimicked by magnesium, consistent with the fewer magnesium ions hosted at the cysteine region. Altogether, our results pinpoint the region surrounding Cys328, highly conserved in type III intermediate filaments, as a hot spot for zinc binding, which modulates Cys328 reactivity and vimentin assembly.

biochemistry↗

bioRxiv2021

Dynamic posttranslational modifications of cytoskeletal proteins unveil hot spots under nitroxidative stress

The cytoskeleton is a supramolecular structure consisting of interacting protein networks that support cell dynamics in essential processes such as migration and division, as well as in responses to stress. Fast cytoskeletal remodeling is achieved with the participation of regulatory proteins and posttranslational modifications (PTMs). Redox-related PTMs are emerging as critical players in cytoskeletal regulation. Here we used a cellular model of mild nitroxidative stress in which a peroxynitrite donor induced transient changes in the organization of three key cytoskeletal proteins, i.e., vimentin, actin and tubulin. Nitroxidative stress-induced reconfiguration of intermediate filaments, microtubules and actin structures were further correlated with their PTM profiles and dynamics of the PTM landscape. Using high-resolution mass spectrometry, 62 different PTMs were identified and relatively quantified in vimentin, actin and tubulin proteins, including 12 enzymatic, 13 oxidative and 2 nitric oxide-derived modifications as well as 35 modifications by carbonylated lipid peroxidation products, thus evidencing the occurrence of a chain reaction with formation of reactive species and the activation of multiple signaling pathways. Our results unveil the presence of certain modifications under basal conditions and their modulation in response to stress in a target-, residue- and reactive species-dependent manner. Moreover, we identified protein PTM "hot spots", such as the single cysteine residue of vimentin, supporting its role in PTM cross-talk and redox sensing. Finally, identification of novel PTMs in these proteins may pave the way for unveiling novel cytoskeleton regulatory mechanisms.

cell biology↗