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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.

The vimentin network displays remarkable plasticity to support basic cellular functions and reorganizes during cell division. Here, we show that in several cell types vimentin filaments redistribute to the cell cortex during mitosis, forming a robust framework interwoven with cortical actin and affecting its organization. Importantly, the intrinsically disordered tail domain of vimentin is essential for this redistribution, which allows normal mitotic progression. A tailless vimentin mutant forms curly bundles, which remain entangled with dividing chromosomes leading to mitotic catastrophes or asymmetric partitions. Serial deletions of vimentin tail domain gradually impair cortical association and mitosis progression. Disruption of f-actin, but not of microtubules, causes vimentin bundling near the chromosomes. Pathophysiological stimuli, including HIV-protease and lipoxidation, induce similar alterations. Interestingly, full filament formation is dispensable for cortical association, which also occurs in vimentin particles. These results unveil implications of vimentin dynamics in cell division through its interplay with the actin cortex.

Introduction: Beta-lactams (BL) are the main cause of allergic drug reactions mediated by specific immunological mechanisms. Reactions can be IgE or effector T-cell mediated. The new antigenic determinants are recognized by the immunological system in the context of the common betalactam structure or the specific differences in the side chains of the antibiotics of this family plus the protein carrier., Areas covered: We have reviewed the recent clinical literature concerning new clinical entities, the progress in diagnosis including the difficulties for in in vivo and or in vitro testing as well as the new algorithms proposed for delabelling subjects classified as allergic to beta-lactams, and recommendations for desensitization procedures., Expert opinion: The knowledge gained over the last years on beta-lactam hypersensitivity has enabled a better understanding and management of cases with allergic reactions to beta-lactams.

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 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.

BACKGROUND: Selective reactions to clavulanic acid (CLV) account for around 30% of immediate reactions after administration of amoxicillin-CLV. Currently, no immunoassay is available for detecting specific IgE to CLV, and its specific recognition in patients with immediate reactions has only been demonstrated by basophil activation testing, however with suboptimal sensitivity. The lack of knowledge regarding the structure of the drug that remains bound to proteins (antigenic determinant) is hampering the development of in vitro diagnostics. We aimed to identify the antigenic determinants of CLV as well as to evaluate their specific IgE recognition and potential role for diagnosis., METHODS: Based on complex CLV degradation mechanisms, we hypothesized the formation of two antigenic determinants for CLV, AD-I (N-protein, 3-oxopropanamide) and AD-II (N-protein, 3-aminopropanamide), and designed different synthetic analogs to each one. IgE recognition of these structures was evaluated in basophils from patients with selective reactions to CLV and tolerant subjects. In parallel, the CLV fragments bound to proteins were identified by proteomic approaches., RESULTS: Two synthetic analogs of AD-I were found to activate basophils from allergic patients. This determinant was also detected bound to lysines 195 and 475 of CLV-treated human serum albumin. One of these analogs was able to activate basophils in 59% of patients whereas CLV only in 41%. Combining both results led to an increase in basophil activation in 69% of patients, and only in 12% of controls., CONCLUSION: We have identified AD-I as one CLV antigenic determinant, which is the drug fragment that remains protein-bound.

Cells employ multiple defence mechanisms to sustain a wide range of stress conditions associated with accumulation of modified self-biomolecules leading to lipo- and proteotoxicity. One of such mechanisms involves activation of the autophagy-lysosomal pathway for removal and degradation of modified lipids, proteins and even organelles. Biomolecules carbonylation, an irreversible oxidative modification, occurs in a variety of pathological conditions and is generally viewed as a marker of oxidative stress. Here, we used a model of rat primary cardiac cells to elucidate the role of autophagy-lysosomal pathway in the turnover of carbonylated biomolecules. Cells treated with inhibitors of autophagy-lysosomal degradation and primed with a short pulse of mild nitroxidative stress were studied using fluorescent microscopy and accumulation of carbonylated biomolecules in droplets- or vesicle-like structures was observed. Furthermore, systems-wide analysis of proteome regulation using relative label free quantification approach revealed the most significant alterations in cells treated with protease inhibitors. Interestingly, down-regulation of insulin signalling was among the most enriched pathway, as revealed by functional annotation of regulated proteins.

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.

Despite the international guidelines on the containment of the coronavirus disease 2019 (COVID-19) pandemic, the European scientific community was not sufficiently prepared to coordinate scientific efforts. To improve preparedness for future pandemics, we have initiated a network of nine European-funded Cooperation in Science and Technology (COST) Actions that can help facilitate inter-, multi-, and trans-disciplinary communication and collaboration.

Homeostasis of the main cellular methyl donor, S-adenosylmethionine, depends on the methionine cycle, which is at the crossroads of metabolic pathways involved in the synthesis and regulation of a large variety of compounds and processes. Impairment of the methionine cycle is detected in a vast majority of liver diseases that concur with oxidative stress. The complex regulation of the pathway is exerted from transcriptional to protein oligomerization and activity levels, subcellular distribution being recently added to the play. Oxidative stress performs a role at all of these regulatory levels either directly (e.g., eliciting protein modifications) or indirectly (e.g., decreasing recycling of vitamin B12). Therefore, the interest in deciphering the mechanisms through which medical interventions could revert methionine cycle impairment, contributing to liver disease treatment. In this line, the control of subcellular distribution has emerged as a putative target for new and existent antioxidants, their evaluation requiring a large research effort.

Salmonella uses Type 3 secretion systems (T3SSs) to deliver virulence factors, called effectors, into host cells during infection. The T3SS effectors promote invasion into host cells and the generation of a replicative niche. SopB is a T3SS effector that plays an important role in Salmonella pathogenesis through its lipid phosphatase activity. Here, we show that SopB mediates the recruitment of Rho GTPases (RhoB, RhoD, RhoH, and RhoJ) to bacterial invasion sites. RhoJ contributes to Salmonella invasion, and RhoB and RhoH play an important role in Akt activation. R‐Ras1 also contributes to SopB‐dependent Akt activation by promoting the localised production of PI(3,4)P2/PI(3,4,5)P3. Our studies reveal new signalling factors involved in SopB‐dependent Salmonella infection.

Endothelial dysfunction has been widely associated with oxidative stress, glucotoxicity and lipotoxicity and underlies the development of cardiovascular diseases (CVDs), atherosclerosis and diabetes. In such pathological conditions, lipids are emerging as mediators of signalling pathways evoking key cellular responses as expression of proinflammatory genes, proliferation and apoptosis. Hence, the assessment of lipid profiles in endothelial cells (EC) can provide valuable information on the molecular alterations underlying CVDs, atherosclerosis and diabetes. We performed a lipidomic approach based on hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) for the analysis of the phospholipidome of bovine aortic EC (BAEC) exposed to oxidative (H2O2), glycative (glucose), or lipoxidative (4-hydroxynonenal, HNE) stress. The phospholipid (PL) profile was evaluated for the classes PC, PE, PS, PG, PI, SM, LPC and CL. H2O2 induced a more acute adaptation of the PL profile than glucose or HNE. Unsaturated PL molecular species were up-regulated after 24h incubation with H2O2, while an opposite trend was observed in glucose- and HNE-treated cells. This study compared, for the first time, the adaptation of the phospholipidome of BAEC upon different induced biochemical stresses. Although further biological studies will be necessary, our results unveil specific lipid signatures in response to characteristic types of stress.

Despite the international guidelines on the containment of the coronavirus disease 2019 (COVID-19) pandemic, the European scientific community was not sufficiently prepared to coordinate scientific efforts. To improve preparedness for future pandemics, we have initiated a network of nine European-funded Cooperation in Science and Technology (COST) Actions that can help facilitate inter-, multi-, and trans-disciplinary communication and collaboration.

Nitrated phospholipids have recently been detected in vitro and in vivo and associated with beneficial health effects. They were identified and quantified in biological samples by lipidomics methodologies using liquid chromatography-collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) acquired with the linear ion trap mass spectrometer. Only a few studies have used higher-energy collision dissociation (HCD)-MS/MS in high-resolution Orbitraps to characterize nitrated phosphatidylserines and nitrated cardiolipins, highlighting the marked differences in the fragmentation patterns when using CID or HCD fragmentation methods. In this study, we aimed to evaluate the fragmentation of nitrated phosphatidylcholine and nitrated phosphatidylethanolamine species under HCD-MS/MS. We studied the effect of normalized collision energy (NCE) in the fragmentation pattern to identify the best acquisition conditions and reporter ions to detect nitrated phospholipids. The results showed that the intensity of the typical neutral loss of nitrous acid (HNO2) diminishes with increasing NCE, becoming non-detectable for a higher NCE. Thus, the loss of HNO2 could not be the most suitable ion/fragment for the characterization of nitrated phospholipids under HCD. In HCD-MS/MS new fragment ions were identified, corresponding to the nitrated fatty acyl chains, NO2-RCOO−, (NO2-RCOOH-H2O + H)+, and (NO2-RCOOH + H)+, suggested as potential reporter ions to detect nitrated phospholipids when using the HCD-MS/MS lipidomics analysis.

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.