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

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.

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, 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.

Zinc Differentially Modulates the Assembly of Soluble and Polymerized Vimentin.

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress. We previously showed that vimentin interacts with zinc, which affects its assembly and redox sensing. Here, we used vimentin wt and C328S, an oxidation-resistant mutant showing improved NaCl-induced polymerization, to assess the impact of zinc on soluble and polymerized vimentin by light scattering and electron microscopy. Zinc acts as a switch, reversibly inducing the formation of vimentin oligomeric species. High zinc concentrations elicit optically-detectable vimentin structures with a characteristic morphology depending on the support. These effects also occur in vimentin C328S, but are not mimicked by magnesium. Treatment of vimentin with micromolar ZnCl2 induces fibril-like particles that do not assemble into filaments, but form aggregates upon subsequent addition of NaCl. In contrast, when added to NaCl-polymerized vimentin, zinc increases the diameter or induces lateral association of vimentin wt filaments. Remarkably, these effects are absent or attenuated in vimentin C328S filaments. Therefore, the zinc-vimentin interaction depends on the chemical environment and on the assembly state of the protein, leading to atypical polymerization of soluble vimentin, likely through electrostatic interactions, or to broadening and lateral association of preformed filaments through mechanisms requiring the cysteine residue. Thus, the impact of zinc on vimentin assembly and redox regulation is envisaged.

Cucurbalsaminones A-C, Rearranged Triterpenoids with a 5/6/3/6/5-Fused Pentacyclic Carbon Skeleton from Momordica balsamina, as Multidrug Resistance Reversers.

Three new triterpenoids, cucurbalsaminones A-C (1-3), featuring a unique 5/6/3/6/5-fused pentacyclic carbon skeleton, named cucurbalsaminane, were isolated from a methanol extract of Momordica balsamina. Their structures were elucidated by spectroscopic methods and corroborated, for 1, by structure solution using single-crystal X-ray diffraction analysis. A hypothetical biogenetic pathway for these compounds is proposed. Compounds 1-3 were evaluated for their P-glycoprotein (P-gp/ABCB1) modulation ability, using a mouse T-lymphoma MDR1-transfected cell model by the rhodamine-123 accumulation assay, and displayed potent multidrug resistance (MDR)-reversing activity.

Integrated approaches to unravel the impact of protein lipoxidation on macromolecular interactions.

Protein modification by lipid derived reactive species, or lipoxidation, is increased during oxidative stress, a common feature observed in many pathological conditions. Biochemical and functional consequences of lipoxidation include changes in the conformation and assembly of the target proteins, altered recognition of ligands and/or cofactors, changes in the interactions with DNA or in protein-protein interactions, modifications in membrane partitioning and binding and/or subcellular localization. These changes may impact, directly or indirectly, signaling pathways involved in the activation of cell defense mechanisms, but when these are overwhelmed they may lead to pathological outcomes. Mass spectrometry provides state of the art approaches for the identification and characterization of lipoxidized proteins/residues and the modifying species. Nevertheless, understanding the complexity of the functional effects of protein lipoxidation requires the use of additional methodologies. Herein, biochemical and biophysical methods used to detect and measure functional effects of protein lipoxidation at different levels of complexity, from in vitro and reconstituted cell-like systems to cells, are reviewed, focusing especially on macromolecular interactions. Knowledge generated through innovative and complementary technologies will contribute to comprehend the role of lipoxidation in pathophysiology and, ultimately, its potential as target for therapeutic intervention.

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

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.

Lathyrol and epoxylathyrol derivatives: Modulation of Cdr1p and Mdr1p drug-efflux transporters of Candida albicans in Saccharomyces cerevisiae model.

Macrocyclic diterpenes were previously found to be able to modulate the efflux pump activity of Candida albicans multidrug transporters. Most of these compounds were jatrophanes, but only a few number of lathyrane-type diterpenes was evaluated. Therefore, the aim of this study was to evaluate the ability of nineteen structurally-related lathyrane diterpenes (1-19) to overcome the drug-efflux activity of Cdr1p and Mdr1p transporters of C. albicans, and get some insights on their structure-activity relationships. The transport assay was performed by monitoring Nile Red (NR) efflux in a Saccharomyces cerevisiae strain overexpressing the referred efflux pumps from C. albicans. Moreover, a chemosensitization assay was performed in order to evaluate the type of interaction between the inhibitory compounds and the antifungal drug fluconazole. Compounds 1-13 were previously isolated from Euphorbia boetica or obtained by derivatization, and compounds 14-19 were prepared by chemical transformations of compound 4. In the transport assays, compounds 14-19 revealed the strongest inhibitory activity of the Cdr1p efflux pump, ranging from 65 to 85%. Concerning Mdr1p efflux pump, the most active compounds were 1, 3, 6, 8, and 12 (75-85%). When used in combination with fluconazole, epoxyboetirane K (2) and euphoboetirane N (18) revealed synergistic effects in the AD-CDR1 yeast strain, overexpressing the Cdr1p transporter, through their ability to reduce the effective concentration of the antifungal drug by 23- and 52-fold, respectively.

Drawbacks of Dialysis Procedures for Removal of EDTA.

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.

Overcoming Multidrug Resistance in Candida albicans: Macrocyclic Diterpenes from Euphorbia Species as Potent Inhibitors of Drug Efflux Pumps.

Thirteen macrocyclic diterpenes (1-13) of the jatrophane and lathyrane types, either isolated from Euphorbia species or obtained by chemical derivatization, were evaluated for their ability to inhibit the drug efflux activity of Candida albicans CaCdr1p and CaMdr1p multidrug transporters overexpressed in a Saccharomyces cerevisiae strain. Their inhibitory potential was assessed through a functional assay of Nile Red accumulation monitored by flow cytometry. A chemosensitization assay, using the checkerboard method, was also performed with the active compounds in order to evaluate their type of interaction with fluconazole.In the transport assay, most compounds were found to inhibit both transporters, most likely as non-substrates, as shown by relative resistance indices close to unity. In contrast, the jatrophanes euphopubescenol (10) and euphomelliferene A (11) were selective for CaMdr1p and CaCdr1p, respectively. Moreover, when used in combination with fluconazole, compounds 12 and 13 displayed strong synergistic interactions (FICI = 0.071) against the yeast strain overexpressing CaMdr1p, decreasing the MIC80 of the antifungal agent 13-fold. Both compounds were also able to reduce the effective concentration of this antifungal agent by 4- to 8-fold against an azole-resistant clinical isolate of C. albicans (F5).

Diterpenes from Euphorbia piscatoria: synergistic interaction of Lathyranes with doxorubicin on resistant cancer cells.

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,ß-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.