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Intermediate filaments are critical for cell and tissue homeostasis and for stress responses. Cytoplasmic intermediate filaments form versatile and dynamic assemblies that interconnect cellular organelles, participate in signaling and protect cells and tissues against stress. Here we have focused on their involvement in redox signaling and oxidative stress, which arises in numerous pathophysiological situations. We pay special attention to type III intermediate filaments, mainly vimentin, because it provides a physical interface for redox signaling, stress responses and mechanosensing. Vimentin possesses a single cysteine residue that is a target for multiple oxidants and electrophiles. This conserved residue fine tunes vimentin assembly, response to oxidative stress and crosstalk with other cellular structures. Here we integrate evidence from the intermediate filament and redox biology fields to propose intermediate filaments as redox sentinel networks of the cell. To support this, we appraise how vimentin detects and orchestrates cellular responses to oxidative and electrophilic stress.

Intermediate filaments (IFs) are cytoskeletal elements involved in mechanotransduction and in the integration of cellular responses. They are versatile structures and their assembly and organization are finely tuned by posttranslational modifications. Among them, type III IFs, mainly vimentin, have been identified as targets of multiple oxidative and electrophilic modifications. A characteristic of most type III IF proteins is the presence in their sequence of a single, conserved cysteine residue (C328 in vimentin), that is a hot spot for these modifications and appears to play a key role in the ability of the filament network to respond to oxidative stress. Current structural models and experimental evidence indicate that this cysteine residue may occupy a strategic position in the filaments in such a way that perturbations at this site, due to chemical modification or mutation, impact filament assembly or organization in a structure-dependent manner. Cysteine-dependent regulation of vimentin can be modulated by interaction with divalent cations, such as zinc, and by pH. Importantly, vimentin remodeling induced by C328 modification may affect its interaction with cellular organelles, as well as the cross-talk between cytoskeletal networks, as seems to be the case for the reorganization of actin filaments in response to oxidants and electrophiles. In summary, the evidence herein reviewed delineates a complex interplay in which type III IFs emerge both as targets and modulators of redox signaling.

Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent i

Frontotemporal dementia is the second most prevalent type of early-onset dementia and up to 40% of cases are familial forms. One of the genes mutated in patients is CHMP2B, which encodes a protein found in a complex important for maturation of late endosomes, an essential process for recycling membrane proteins through the endolysosomal system. Here, we have generated a CHMP2B-mutated human embryonic stem cell line using genome editing with the purpose to create a human in vitro Frontotemporal dementia disease model. To date, most studies have focused on neuronal alterations; however, we present a new co-culture system in which neurons and astrocytes are independently generated from human embryonic stem cells and combined in co-cultures. With this approach, we have identified alterations in the endolysosomal system of Frontotemporal dementia astrocytes, a higher capacity of astrocytes to uptake and respond to glutamate, and a neuronal network hyperactivity as well as excessive synchronization. Overall, our data indicates that astrocyte alterations precede neuronal impairments and could potentially trigger neuronal network changes, indicating the important and specific role of astrocytes in disease development.

Alexander disease is a rare neurodegenerative disorder caused by mutations in the glial fibrillary acidic protein, a type III intermediate filament protein expressed in astrocytes. Both early (infantile or juvenile) and adult onsets of the disease are known and, in both cases, astrocytes present characteristic aggregates, named Rosenthal fibers. Mutations are spread along the glial fibrillary acidic protein sequence disrupting the typical filament network in a dominant manner. Although the presence of aggregates suggests a proteostasis problem of the mutant forms, this behavior is also observed when the expression of wild-type glial fibrillary acidic protein is increased. Additionally, several isoforms of glial fibrillary acidic protein have been described to date, while the impact of the mutations on their expression and proportion has not been exhaustively studied. Moreover, the posttranslational modification patterns and/or the protein-protein interaction networks of the glial fibrillary acidic protein mutants may be altered, leading to functional changes that may modify the morphology, positioning, and/or the function of several organelles, in turn, impairing astrocyte normal function and subsequently affecting neurons. In particular, mitochondrial function, redox balance and susceptibility to oxidative stress may contribute to the derangement of glial fibrillary acidic protein mutant-expressing astrocytes. To study the disease and to develop putative therapeutic strategies, several experimental models have been developed, a collection that is in constant growth. The fact that most cases of Alexander disease can be related to glial fibrillary acidic protein mutations, together with the availability of new and more relevant experimental models, holds promise for the design and assay of novel therapeutic strategies.

The intermediate filament protein vimentin plays a key role in cell signaling and stress sensing, as well as an integrator of cytoskeletal dynamics. The vimentin monomer consists of a central rod-like domain and intrinsically disordered head and tail domains. Although the organization of vimentin oligomers in filaments is beginning to be understood, the precise disposition of the tail region remains to be elucidated. Here we observed that electrophilic stress-induced condensation shielded vimentin from recognition by antibodies against specific segments of the tail domain. A detailed characterization revealed that vimentin tail segments are differentially exposed at distinct subcellular locations, both in basal and stress conditions. The 411–423 segment appeared accessible in all cell areas, correlating with vimentin abundance. In contrast, the 419–438 segment was more scantily recognized in perinuclear vimentin and lipoxidative stress-induced bundles, and better detected in peripheral filaments, where it appeared to protrude further from the filament core. These differences persisted in mitotic cells. Interestingly, both tail segments showed closer accessibility in calyculin A-treated cells and phosphomimetic mutants of the C-terminal region. Our results lead us to hypothesize the presence of at least two distinct arrangements of vimentin tail in cells: an “extended” conformation (accessible 419–438 segment), preferentially detected in peripheral areas with looser filaments, and a “packed” conformation (shielded 419–438 segment), preferentially detected at the cell center in robust filaments and lipoxidative stress-induced bundles. These different arrangements could be putatively interconverted by posttranslational modifications, contributing to the versatility of vimentin functions and/or interactions.

Complex lipids, phospholipids (PLs) and triacylglycerides (TAGs), are prone to modifications induced by reactive nitrated species and reactive oxygen species, generating a range of nitrated, nitrosated or nitroxidized derivatives, as nitro PLs and nitro TAGs. These modified lipids (epilipids) have been reported in vitro and in vivo using lipidomics approaches. However, their detection in living systems remains a challenge hampered by its complexity, high structural diversity, and low abundance. The advances in high-resolution mass spectrometry combined with the higher sensitivity of the instruments like Orbitrap-based mass spectrometers opened new opportunities for the detection of these modified complex lipids. This review summarizes the challenges and findings behind the identification of nitrated, nitrosated and nitroxidized PLs and TAGs fragmentation fingerprints based on collision-induced dissociation (CID) and higher energy CID (HCD) MS/MS approaches. Following what has already been reported for nitrated fatty acids, these complex lipids are found to act as endogenous mediators with potential electrophilic properties and can express bioactivities such as anti-inflammatory and antioxidant actions. This information can be used to design untargeted and targeted lipidomics strategies for these modified complex lipids in biological samples as well as in pathological, food and industrial settings, further unveiling their biological and signalling roles.

The SARS-CoV-2 Spike protein mediates docking of the virus onto cells prior to viral invasion. Several cellular receptors facilitate SARS-CoV-2 Spike docking at the cell surface, of which ACE2 plays a key role in many cell types. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Nevertheless, the potential colocalization of vimentin with Spike and its receptors on the cell surface has not been explored. Here we have assessed the binding of Spike protein constructs to several cell types. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which consistently colocalized with endogenous ACE2, but sparsely with a lipid raft marker. Vimentin immunoreactivity mostly appeared as spots or patches unevenly distributed at the surface of diverse cell types. Of note, vimentin could also be detected in extracellular particles and in the cytoplasm underlying areas of compromised plasma membrane. Interestingly, although overall colocalization of vimentin-positive spots with ACE2 or Spike was moderate, a selective enrichment of the three proteins was detected at elongated structures, positive for acetylated tubulin and ARL13B. These structures, consistent with primary cilia, concentrated Spike binding at the top of the cells. Our results suggest that a vimentin-Spike interaction could occur at selective locations of the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking.

The type III intermediate filament proteins vimentin and GFAP are modulated by oxidants and electrophiles, mainly through perturbation of their single cysteine residues. Desmin, the type III intermediate filament protein specific to muscle cells, is critical for muscle homeostasis, playing a key role in sarcomere organization and mitochondrial function. Here, we have studied the impact of oxidants and cysteine-reactive agents on desmin behavior. Our results show that several reactive species and drugs induce covalent modifications of desmin in vitro, of which its single cysteine residue, C333, is an important target. Moreover, stimuli eliciting oxidative stress or lipoxidation, including H2O2, 15-deoxy-prostaglandin J2, and CoCl2-elicited chemical hypoxia, provoke desmin disorganization in H9c2 rat cardiomyoblasts transfected with wild-type desmin, which is partially attenuated in cells expressing a C333S mutant. Notably, in cells lacking other cytoplasmic intermediate filaments, network formation by desmin C333S appears less efficient than that of desmin wt, especially when these proteins are expressed as fluorescent fusion constructs. Nevertheless, in these cells, the desmin C333S organization is also protected from disruption by oxidants. Taken together, our results indicate that desmin is a target for oxidative and electrophilic stress, which elicit desmin remodeling conditioned by the presence of its single cysteine residue.

The metabolism and intercellular transfer of glutathione or its precursors may play an important role in cellular defense against oxidative stress, a common hallmark of neurodegeneration. In the 1990s, several studies in the Neurobiology field led to the widely accepted notion that astrocytes produce large amounts of glutathione that serve to feed neurons with precursors for glutathione synthesis. This assumption has important implications for health and disease since a reduction in this supply from astrocytes could compromise the capacity of neurons to cope with oxidative stress. However, at first glance, this shuttling would imply a large energy expenditure to get to the same point in a nearby cell. Thus, are there additional underlying reasons for this expensive mechanism? Are neurons unable to import and/or synthesize the three non-essential amino acids that are the glutathione building blocks? The rather oxidizing extracellular environment favors the presence of cysteine (Cys) as cystine (Cis), less favorable for neuronal import. Therefore, it has also been proposed that astrocytic GSH efflux could induce a change in the redox status of the extracellular space nearby the neurons, locally lowering the Cis/Cys ratio. This astrocytic glutathione release would also increase their demand for precursors, stimulating Cis uptake, which these cells can import, further impacting the local decline of the Cis/Cys ratio, in turn, contributing to a more reduced extracellular environment and subsequently favoring neuronal Cys import. Here, we revisit the experimental evidence that led to the accepted hypothesis of astrocytes acting as suppliers of neuronal glutathione precursors, considering recent data from the Human Protein Atlas. In addition, we highlight some potential drawbacks of this hypothesis, mainly supported by heterogeneous cellular models. Finally, we outline additional and more cost-efficient possibilities by which astrocytes could support neuronal glutathione l

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 oxidederived 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.

The Spike protein from SARS-CoV-2 mediates docking of the virus onto cells and contributes to viral invasion. Several cellular receptors are involved in SARS-CoV-2 Spike docking at the cell surface, including ACE2 and neuropilin. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Here we have explored the binding of Spike protein constructs to several cell types using low-temperature immunofluorescence approaches in live cells, to minimize internalization. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which showed scarce colocalization with a lipid raft marker, but consistent coincidence with ACE2. Under our conditions, vimentin immunoreactivity appeared as spots or patches unevenly distributed at the surface of diverse cell types. Remarkably, several observations including potential antibody internalization and adherence to cells of vimentin-positive structures present in the extracellular medium exposed the complexity of vimentin cell surface immunoreactivity, which requires careful assessment. Notably, overall colocalization of Spike and vimentin signals markedly varied with the cell type and the immunodetection sequence. In turn, vimentin-positive spots moderately colocalized with ACE2; however, a particular enrichment was detected at elongated structures positive for acetylated tubulin, consistent with primary cilia, which also showed Spike binding. Thus, these results suggest that vimentin-ACE2 interaction could occur at selective locations near the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking.

Protein lipoxidation is a non-enzymatic post-translational modification that consists of the covalent addition of reactive lipid species to proteins. This occurs under basal conditions but increases in situations associated with oxidative stress. Protein targets for lipoxidation include metabolic and signalling enzymes, cytoskeletal proteins, and transcription factors, among others. There is strong evidence for the involvement of protein lipoxidation in disease, including atherosclerosis, neurodegeneration, and cancer. Nevertheless, the involvement of lipoxidation in cellular regulatory mechanisms is less understood. Here we review basic aspects of protein lipoxidation and discuss several features that could support its role in cell signalling, including its selectivity, reversibility, and possibilities for regulation at the levels of the generation and/or detoxification of reactive lipids. Moreover, given the great structural variety of electrophilic lipid species, protein lipoxidation can contribute to the generation of multiple structurally and functionally diverse protein species. Finally, the nature of the lipoxidised proteins and residues provides a frameshift for a complex interplay with other post-translational modifications, including redox and redox-regulated modifications, such as oxidative modifications and phosphorylation, thus strengthening the importance of detailed knowledge of this process.

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.

Vimentin is an intermediate filament protein that plays key roles in integration of cytoskeletal functions, and therefore in basic cellular processes such as cell division and migration. Consequently, vimentin has complex implications in pathophysiology. Vimentin is required for a proper immune response, but it can also act as an autoantigen in autoimmune diseases or as a damage signal. Although vimentin is a predominantly cytoplasmic protein, it can also appear at extracellular locations, either in a secreted form or at the surface of numerous cell types, often in relation to cell activation, inflammation, injury or senescence. Cell surface targeting of vimentin appears to associate with the occurrence of certain posttranslational modifications, such as phosphorylation and/or oxidative damage. At the cell surface, vimentin can act as a receptor for bacterial and viral pathogens. Indeed, vimentin has been shown to play important roles in virus attachment and entry of severe acute respiratory syndrome-related coronavirus (SARS-CoV), dengue and encephalitis viruses, among others. Moreover, the presence of vimentin in specific virus-targeted cells and its induction by proinflammatory cytokines and tissue damage contribute to its implication in viral infection. Here, we recapitulate some of the pathophysiological implications of vimentin, including the involvement of cell surface vimentin in interaction with pathogens, with a special focus on its role as a cellular receptor or co-receptor for viruses. In addition, we provide a perspective on approaches to target vimentin, including antibodies or chemical agents that could modulate these interactions to potentially interfere with viral pathogenesis, which could be useful when multi-target antiviral strategies are needed.

Intermediate filaments (IFs) play key roles in cell mechanics, signaling and homeostasis. Their assembly and dynamics are finely regulated by posttranslational modifications. The type III IFs, vimentin, desmin, peripherin and glial fibrillary acidic protein (GFAP), are targets for diverse modifications by oxidants and electrophiles, for which their conserved cysteine residue emerges as a hot spot. Pathophysiological examples of these modifications include lipoxidation in cell senescence and rheumatoid arthritis, disulfide formation in cataracts and nitrosation in endothelial shear stress, although some oxidative modifications can also be detected under basal conditions. We previously proposed that cysteine residues of vimentin and GFAP act as sensors for oxidative and electrophilic stress, and as hinges influencing filament assembly. Accumulating evidence indicates that the structurally diverse cysteine modifications, either per se or in combination with other posttranslational modifications,elicit specific functional outcomes inducing distinct assemblies or network rearrangements, including filament stabilization, bundling or fragmentation. Cysteine-deficient mutants are protected from these alterations but show compromised cellular performance in network assembly and expansion, organelle positioning and aggresome formation, revealing the importance of this residue. Therefore, the high susceptibility to modification of the conserved cysteine of type III IFs and its cornerstone position in filament architecture sustains their role in redox sensing and integration of cellular responses. This has deep pathophysiological implications and supports the potential of this residue as a drug target.