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.

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent commonly used in protein purification, both to eliminate contaminating divalent cations and to inhibit protease activity. For a number of subsequent applications EDTA needs to be exhaustively removed. Most purification methods rely in extensive dialysis and/or gel filtration in order to exchange or remove protein buffer components, including metal chelators. We report here that dialysis protocols, even as extensive as those typically employed for protein refolding, may not effectively remove EDTA, which is reduced only by approximately two-fold and it also persists after spin-column gel filtration, as determined by NMR and by colorimetric methods. Remarkably, the most efficient removal was achieved by ultrafiltration, after which EDTA became virtually undetectable. These results highlight a potentially widespread source of experimental variability affecting free divalent cation concentrations in protein applications.

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, 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 numerous reactive species and 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. Thus, some modifications accumulated during the experiment whereas others varied transiently. Moreover, we identified protein PTM “hot spots”, such as the single cysteine residue of vimentin, which was detected in seven modified forms, thus, supporting its role in PTM crosstalk and redox sensing. Finally, identification of novel PTMs in these proteins paves the way for unveiling new cytoskeleton regulatory mechanisms.

Four new diterpenes were isolated from the methanolic extract of Euphorbia piscatoria, two ent-abietanes (1, 2) and two lathyrane-type macrocyclic diterpenes (3, 4), along with three known diterpenes (5-7). Their structures were characterized by spectroscopic methods, mainly 1D and 2D NMR ((1)H, (13)C, DEPT, COSY, HMBC, HMQC, and NOESY) experiments. Compound 2, with an unusual structure, might be considered intermediate in the biosynthesis of ent-abietane α,β-unsaturated lactones, commonly found in Euphorbia species. Therefore, a possible biogenetic pathway is proposed. The MDR reversal potential of macrocyclic diterpenes 3-5 was evaluated through a drug combination assay, using the L5178Y mouse T lymphoma cell line transfected with the human MDR1 gene. Compounds 3-5 were able to enhance, synergistically, the antiproliferative activity of doxorubicin (combination indexes < 0.5). Moreover, compounds 1-6 were also assessed for their antiproliferative activity on human MDR cancer cell models, namely gastric, pancreatic, and colon. Weak antiproliferative activity was observed for compounds 1 (IC50 = 66.02 ± 7.10 µM) and 4 (IC50 = 39.51 ± 3.82 µM) on the MDR gastric cell line.

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.