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Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAP R239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAP R239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

The intermediate filament protein vimentin performs an essential role in cytoskeletal interplay and dynamics, mechanosensing and cellular stress responses. In pathology, vimentin is a key player in tumorigenesis, fibrosis and infection. Vimentin filaments undergo distinct and versatile reorganizations, and behave as redox sensors. The vimentin monomer possesses a central α-helical rod domain flanked by N- and C-terminal low complexity domains. Interactions between this type of domains play an important function in the formation of phase-separated biomolecular condensates, which in turn are critical for the organization of cellular components. Here we show that several oxidants, including hydrogen peroxide and diamide, elicit the remodeling of vimentin filaments into small particles. Oxidative stress elicited by diamide induces a fast dissociation of filaments into circular, motile dots, which requires the presence of the single vimentin cysteine residue, C328. This effect is reversible, and filament reassembly can occur within minutes of oxidant removal. Diamide-elicited vimentin droplets recover fluorescence after photobleaching. Moreover, fusion of cells expressing differentially tagged vimentin allows the detection of dots positive for both tags, indicating that vimentin dots merge upon cell fusion. The aliphatic alcohol 1,6-hexanediol, known to alter interactions between low complexity domains, readily dissolves diamide-elicited vimentin dots at low concentrations, in a C328 dependent manner, and hampers reassembly. Taken together, these results indicate that vimentin oxidation promotes a fast and reversible filament remodeling into biomolecular condensate-like structures, and provide primary evidence of its regulated phase separation. Moreover, we hypothesize that filament to droplet transition could play a protective role against irreversible damage of the vimentin network by oxidative stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Agencia Estatal Consejo Superior de Investigaciones Científicas. Published by Elsevier B.V. All rights reserved.)

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., (© 2024 The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: None

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 H 2 O 2 , 15-deoxy-prostaglandin J 2 , and CoCl 2 -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.

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 it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a "sensor" transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Frontotemporal dementia (FTD) 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 FTD 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 FTD 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., Competing Interests: The authors report no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

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 levels, including its shuttling in extracellular vesicles.

Alexander disease is a fatal neurological disorder caused by mutations in the intermediate filament protein Glial Fibrillary Acidic Protein (GFAP), which is key for astrocyte homeostasis. These mutations cause GFAP aggregation, astrocyte dysfunction and neurodegeneration. Remarkably, most of the known GFAP mutations imply a change by more nucleophilic amino acids, mainly cysteine or histidine, which are more susceptible to oxidation and lipoxidation. Therefore, we hypothesized that a higher susceptibility of Alexander disease GFAP mutants to oxidative or electrophilic damage, which frequently occurs during neurodegeneration, could contribute to disease pathogenesis. To address this point, we have expressed GFP-GFAP wild type or the harmful Alexander disease GFP-GFAP R239C mutant in astrocytic cells. Interestingly, GFAP R239C appears more oxidized than the wild type under control conditions, as indicated both by its lower cysteine residue accessibility and increased presence of disulfide-bonded oligomers. Moreover, GFP-GFAP R239C undergoes lipoxidation to a higher extent than GFAP wild type upon treatment with the electrophilic mediator 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ). Importantly, GFAP R239C filament organization is altered in untreated cells and is earlier and more severely disrupted than GFAP wild type upon exposure to oxidants (diamide, H 2 O 2 ) or electrophiles (4-hydroxynonenal, 15d-PGJ 2 ), which exacerbate GFAP R239C aggregation. Furthermore, H 2 O 2 causes reversible alterations in GFAP wild type, but irreversible damage in GFAP R239C expressing cells. Finally, we show that GFAP R239C expression induces a more oxidized cellular status, with decreased free thiol content and increased mitochondrial superoxide generation. In addition, mitochondria show decreased mass, increased colocalization with GFAP and altered morphology. Notably, a GFP-GFAP R239H mutant recapitulates R239C-elicited alterations whereas an R239G mutant induces a milder phenotype. Together, our results outline a deleterious cycle involving altered GFAP R239C organization, mitochondrial dysfunction, oxidative stress, and further GFAP R239C protein damage and network disruption, which could contribute to astrocyte derangement in Alexander disease., (Copyright © 2022 Consejo Superior de Investigaciones Científicas. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lois-Bermejo, González-Jiménez, Duarte, Pajares and Pérez-Sala.)

Allergic reactions to antibiotics are a major concern in the clinic. ß -lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α -enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α -enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α -enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α -enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α -enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 González-Morena, Sánchez-Gómez, Vida, Pérez-Inestrosa, Salas, Montañez, Altomare, Aldini, Pajares and Pérez-Sala.)

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., (Copyright © 2021. Published by Elsevier B.V.)

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., (Copyright © 2020. Published by Elsevier B.V.)

Serum and cellular proteins are targets for the formation of adducts with the β-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other β-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions., (Copyright © 2020 Pajares, Zimmerman, Sánchez-Gómez, Ariza, Torres, Blanca, Cañada, Montañez and Pérez-Sala.)

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) 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-PGJ 2 , 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., (Copyright © 2019 Consejo Superior de Investigaciones Científicas (Spain). Published by Elsevier B.V. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a progressive disease, with a median survival of 3-5 years following diagnosis. Lung remodeling by invasive fibroblasts is a hallmark of IPF. In this study, we demonstrate that inhibition of vimentin intermediate filaments (VimIFs) decreases the invasiveness of IPF fibroblasts and confers protection against fibrosis in a murine model of experimental lung injury. Increased expression and organization of VimIFs contribute to the invasive property of IPF fibroblasts in connection with deficient cellular autophagy. Blocking VimIF assembly by pharmacologic and genetic means also increases autophagic clearance of collagen type I. Furthermore, inhibition of expression of collagen type I by siRNA decreased invasiveness of fibroblasts. In a bleomycin injury model, enhancing autophagy in fibroblasts by an inhibitor of VimIF assembly, withaferin A (WFA), protected from fibrotic lung injury. Additionally, in 3D lung organoids, or pulmospheres, from patients with IPF, WFA reduced the invasiveness of lung fibroblasts in the majority of subjects tested. These studies provide insights into the functional role of vimentin, which regulates autophagy and restricts the invasiveness of lung fibroblasts.

Significance: Transsulfuration allows conversion of methionine into cysteine using homocysteine (Hcy) as an intermediate. This pathway produces S-adenosylmethionine (AdoMet), a key metabolite for cell function, and provides 50% of the cysteine needed for hepatic glutathione synthesis. The route requires the intake of essential nutrients (e.g., methionine and vitamins) and is regulated by their availability. Transsulfuration presents multiple interconnections with epigenetics, adenosine triphosphate (ATP), and glutathione synthesis, polyol and pentose phosphate pathways, and detoxification that rely mostly in the exchange of substrates or products. Major hepatic diseases, rare diseases, and sensorineural disorders, among others that concur with oxidative stress, present impaired transsulfuration. Recent Advances: In contrast to the classical view, a nuclear branch of the pathway, potentiated under oxidative stress, is emerging. Several transsulfuration proteins regulate gene expression, suggesting moonlighting activities. In addition, abnormalities in Hcy metabolism link nutrition and hearing loss., Critical Issues: Knowledge about the crossregulation between pathways is mostly limited to the hepatic availability/removal of substrates and inhibitors. However, advances regarding protein-protein interactions involving oncogenes, identification of several post-translational modifications (PTMs), and putative moonlighting activities expand the potential impact of transsulfuration beyond methylations and Hcy., Future Directions: Increasing the knowledge on transsulfuration outside the liver, understanding the protein-protein interaction networks involving these enzymes, the functional role of their PTMs, or the mechanisms controlling their nucleocytoplasmic shuttling may provide further insights into the pathophysiological implications of this pathway, allowing design of new therapeutic interventions. Antioxid. Redox Signal. 29, 408-452.

Asthma and rhinitis are two of the main clinical manifestations of allergy, in which increased reactive oxygen or electrophilic species can play a pathogenic role. Aldose reductase (AKR1B1) is involved in aldehyde detoxification and redox balance. Recent evidence from animal models points to a role of AKR1B1 in asthma and rhinitis, but its involvement in human allergy has not been addressed. Here, the putative association of allergic rhinitis and asthma with AKR1B1 variants has been explored by analysis of single-strand variants on the AKR1B1 gene sequence in 526 healthy subjects and 515 patients with allergic rhinitis, 366 of whom also had asthma. We found that the rs2229542 variant, introducing the p.Lys90Glu mutation, was significantly more frequent in allergic patients than in healthy subjects. Additionally, in cells transfected with expression vectors carrying the wild-type or the p.Lys90Glu variant of AKR1B1, the mutant consistently attained lower protein levels than the wild-type and showed a compromised thermal stability. Taken together, our results show that the rs2229542 variant associates with asthma and rhinitis, and hampers AKR1B1 protein levels and stability. This unveils a connection between the genetic variability of aldose reductase and allergic processes., (© 2018 Wiley Periodicals, Inc.)

Triflusal is a platelet aggregation inhibitor chemically related to acetylsalicylic acid, which is used for the prevention and/or treatment of vascular thromboembolisms, which acts as a prodrug. Actually, after oral administration it is absorbed primarily in the small intestine, binds to plasma proteins (99%) and is rapidly biotransformed in the liver into its deacetylated active metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). In healthy humans, the half-life of triflusal is ca. 0.5 h, whereas for HTB it is ca. 35 h. From a pharmacological point of view, it is interesting to note that HTB is itself highly active as a platelet anti-aggregant agent. Indeed, studies on the clinical profile of both drug and metabolite have shown no significant differences between them. It has been evidenced that HTB displays ability to induce photoallergy in humans. This phenomenon involves a cell-mediated immune response, which is initiated by covalent binding of a light-activated photosensitizer (or a species derived therefrom) to a protein. In this context, small proteins like ubiquitin could be appropriate models for investigating covalent binding by means of MS/MS and peptide fingerprint analysis. In previous work, it was shown that HTB forms covalent photoadducts with isolated lysine. Interestingly, ubiquitin contains seven lysine residues that could be modified by a similar reaction. With this background, the aim of the present work is to explore adduct formation between the triflusal metabolite and ubiquitin as model protein upon sunlight irradiation, combining proteomic and photophysical (fluorescence and laser flash photolysis) techniques. Photophysical and proteomic analysis demonstrates monoadduct formation as the major outcome of the reaction. Interestingly, addition can take place at any of the ε-amino groups of the lysine residues of the protein and involves replacement of the trifluoromethyl moiety with a new amide function. This process can in principle occur with other trifluoroaromatic compounds and may be responsible for the appearance of undesired photoallergic side effects.

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Unlabelled: Electrophilic eicosanoids are endogenous mediators that play complex roles in signaling and adaptive responses. Basal generation of these species contributes to cell homeostasis, whereas their increased generation under situations of oxidative stress may contribute to cell adaptation or cytoprotection, and, if generated in amounts surpassing the cell detoxification mechanisms, may elicit cytotoxicity. Electrophilic prostaglandins with cyclopentenone structure (cyPG) have been shown to exert protective effects in numerous models of inflammation and tissue injury, as well as antitumoral effects. Protein covalent modification is a key mechanism of action of these species. We have used tagged electrophilic PG, mainly cyPG, as tools to characterize electrophilic lipid-protein interactomes. cyPG with different structure elicit the modification of subsets of proteins comprising both common and selective targets. Among the targets identified are proteins involved in inflammatory responses, the modification of which contributes to the anti-inflammatory effects of cyPG, signaling proteins, and proteins involved in chemoprevention, chemoresistance or tumorigenesis. Thus, some cyPG targets, including glutathione transferase and aldo-keto reductases, have broad implications both in redox signaling and as drug targets. We have observed that cyPG effects are highly dependent on cell type, context, and levels of both cyPG and endogenous antioxidants, in particular GSH. Moreover, the ability of cyPG to display biphasic or dual actions and the nature of the proteins modified depend on these factors. A detailed knowledge of cyPG targets and of the factors that modulate protein modification will contribute to the understanding of their mechanisms of action and define their potential as leads for drug discovery, but may also shed light on redox signaling mechanisms., Funding: MINECO SAF2012-36519, ISCIII RD12/0013/0008 (Spain), EU COST Action CM1001., (Copyright © 2014. Published by Elsevier Inc.)

Cyclopentenone prostaglandins (cyPG) are endogenous lipid mediators involved in the resolution of inflammation and the regulation of cell proliferation and cellular redox status. Upon exogenous administration they have shown beneficial effects in models of inflammation and tissue injury, as well as potential antitumoral actions, which have raised a considerable interest in their study for the development of therapeutic tools. Due to their electrophilic nature, the best-known mechanism of action of these mediators is the covalent modification of proteins at cysteine residues through Michael addition. Identification of cyPG targets through proteomic approaches, including MS/MS analysis to pinpoint the modified residues, is proving critical to characterize their mechanisms of action. Among the targets of cyPG are proinflammatory transcription factors, proteins involved in cell defense, such as the regulator of the antioxidant response Keap1 and detoxifying enzymes like GST, and key signaling proteins like Ras proteins. Moreover, cyPG may interact with redox-active small molecules, such as glutathione and hydrogen sulfide. Much has been learned about cyPG in the past few years and this knowledge has also contributed to clarify both pharmacological actions and signaling mechanisms of these and other electrophilic lipids. Given the fact that many cyPG targets are involved in or are targets for redox regulation, there is a complex interplay with redox-induced modifications. Here we address the modification of protein cysteine residues by cyPG elucidated by proteomic studies, paying special attention to the interplay with redox signaling., (Copyright © 2013 Wiley Periodicals, Inc.)

Glutathione S transferase P1-1 plays a key role in the metabolism of inflammatory mediators and drugs, thus modulating the inflammatory response. Active GSTP1-1 is a homodimer with cysteine residues close to the active site that can undergo oligomerization in response to stress, a process that affects enzyme activity and interactions with signaling and redox-active proteins. Cyclopentenone prostaglandins (cyPG) are endogenous reactive lipid mediators that participate in the regulation of inflammation and may covalently modify proteins through Michael addition. cyPG with dienone structure, which can bind to vicinal cysteines, induce an irreversible oligomerization of GSTP1-1. Here we have characterized the oligomeric state of GSTP1-1 in Jurkat cells treated with 15-deoxy-Δ12,14-PGJ 2 (15d-PGJ 2 ). 15d-PGJ 2 induces both reversible and irreversible GSTP1-1 oligomerization as shown by blue-native 2D electrophoresis. Interestingly, GSTP1-1 dimers were the main species detected by analytical gel filtration chromatography in control cells, whereas only oligomers, compatible with a tetrameric association state, were found in 15d-PGJ 2 -treated cells. cyPG-induced GSTP1-1 oligomerization also occurred in cell-free systems. Therefore, we employed this model to assess the effects of endogenous reactive species and drugs. Inflammatory mediators, such as 15d-PGJ 2 and Δ12-PGJ 2 , and drugs like chlorambucil, phenylarsine oxide or dibromobimane elicited whereas ethacrynic acid hampered GSTP1-1 oligomerization or intra-molecular cross-linking in cell-free systems, yielding GSTP1-1 species specific for each compound. These observations situate GSTP1-1 at the cross-roads of inflammation and drug action behaving as a target for both inflammatory mediators and reactive drugs, which induce or reciprocally modulate GSTP1-1 oligomerization or conformation.

Transcription factors of the peroxisome proliferator-activated receptor (PPAR) family are ligand-activated receptors that play key roles in lipid metabolism and inflammation. The γ isoform (PPARγ) is involved in adipocyte differentiation, insulin sensitization, and vascular pathophysiology, including inflammation and atherosclerosis, for which it is considered an important drug target. PPARγ ligands display varied structures and include fatty acids, electrophilic lipids, and certain drugs. These agonists promote conformational changes allowing interaction of PPARγ with coactivators and hence transcriptional regulation. Here we present a panoply of methods to study PPARγ interactions with ligands and activation in vitro and in cells. The first method is based on the competition of the fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) with PPARγ ligands for the ligand binding pocket, allowing detection and quantification of ligand binding to PPARγ. This method is specific for PPARγ while ANS displays negligible interaction with other nuclear receptors such as PPARα and retinoid X receptor α (RXRα). The ANS competition assay has been validated through comparison of the affinities determined for well-known PPARγ ligands by this method with those reported in the literature. We also describe here gel-based competition assays that show limited performance with non-covalently bound ligands. In addition, we present a fluorescence anisotropy assay to analyze PPARγ activation by ligands in vitro through their capacity of eliciting PPARγ interaction with a fluorescently labeled peptide derived from one of its coactivators (SRC-1). Finally, we show cell-based assays to investigate PPARγ activation by interaction with its ligands. We believe that combined approaches using ANS, fluorescent coactivator peptides, and in-cell assays to monitor PPARγ binding and interactions may provide valuable strategies for the identification and characterization of PPARγ ligands.

Cyclopentenone prostaglandins play a modulatory role in inflammation, in part through their ability to covalently modify key proinflammatory proteins. Using mesangial cells as a cellular model of inflammation we have observed that 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) exerts a biphasic effect on cell activation by cytokines, with nanomolar concentrations eliciting an amplification of nitric oxide (NO) production and iNOS and COX-2 levels, and concentrations of 5 μM and higher inhibiting proinflammatory gene expression. An analog of 15d-PGJ(2) lacking the cyclopentenone structure (9,10-dihydro-15d-PGJ(2)) showed reduced ability to elicit both types of effects, suggesting that the electrophilic nature of 15d-PGJ(2) is important for its biphasic action. Interestingly, the switch from stimulatory to inhibitory actions occurred within a narrow concentration range and correlated with the ability of 15d-PGJ(2) to induce heme oxygenase 1 and γ-GCSm expression. These events are highly dependent on the triggering of the antioxidant response, which is considered as a sensor of thiol group modification. Indeed, the levels of the master regulator of the antioxidant response Nrf2 increased upon treatment with concentrations of 15d-PGJ(2) above 5 μM, an effect that could not be mimicked by 9,10-dihydro-15d-PGJ(2). Thus, an interplay of redox and electrophilic signalling mechanisms can be envisaged by which 15d-PGJ(2), as several other redox mediators, could contribute both to the onset and to the resolution of inflammation in a context or concentration-dependent manner., (© 2012 Bentham Science Publishers)

Polymorphisms in drug-related enzymes and receptors are often strongly related to altered drug response and to the risk of developing drug intolerance. Aspirin, usually available as an over-the-counter drug, is one of the most used drugs worldwide and is a common cause of drug intolerance events. Aspirin undergoes polymorphic metabolism. Among the enzymes involved in aspirin biodisposition a major role is played by the enzymes UDP-glucuronosyltransferase UGT1A6, cytochrome P450 CYP2C9 and the xenobiotic/medium chain fatty acid:CoA ligase ACSM2, although other UGTs and ACSMs enzymes may significantly contribute to aspirin metabolism. UGT1A6, CYP2C9 and ACSM2 are polymorphic, as well as PTGS1 and PTGS2, the genes coding for the enzymes cyclo-oxygenases COX1 and COX2, respectively. The present review analyzes the importance of genetic variations in enzymes involved in the metabolism and in the effects of aspirin and common polymorphisms related to aspirin intolerance, and it raises hypotheses on genetic factors related to altered response to aspirin that require further investigation. Major polymorphisms related to aspirin biodisposition are rs2070959, rs1105879 and rs6759892 for the UGT1A6 gene, rs1133607 for the ACSM2 gene, and rs1799853, rs1057910, rs28371686, rs9332131 and rs28371685 for the CYP2C9 gene. Regarding aspirin effects, major PGTS1 targets are rs3842787 and rs5789 for European subjects, and rs3842789 and rs3842792 for African subjects. For the PTGS2 gene nonsynonymous SNPs are likely to be of low relevance because of the influence of transcriptional and posttranscriptional factors. Combined studies for the above mentioned polymorphisms and those corresponding to other genes related to aspirin intolerance will provide excellent tools to identify individuals with a high risk to develop intolerance to aspirin.

The effect of prostaglandins on the development of papillomas has been investigated in mice receiving prostaglandins E2 (PGE2) or the cyclopentenone 15-deoxy-delta(12,14)-PGJ2 (15dPGJ2) topically, using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced tetradecanoylphorbol acetate (TPA)-promoted model of skin carcinogenesis. The presence of 15dPGJ2 during DMBA and TPA treatment inhibited apoptosis and increased the rate, number, size and vascularization of the papillomas, some of them progressing into carcinomas. Moreover, skin sections from mice treated for one week with DMBA and 15dPGJ2 showed a much reduced rate of apoptotic cells, and an enhanced expression of vascular epithelial growth factor when compared with animals receiving DMBA, with or without PGE2. The analysis of molecular events in the MCA3D keratinocyte cell line showed that 15dPGJ2 activated Ras and improved cell viability by inhibiting DMBA-dependent apoptosis. In addition to this, cell adhesion was impaired in MCA3D keratinocytes co-treated with 15dPGJ2 and DMBA, at the same time when the expression of cyclooxygenase-2 (COX-2) was observed under these conditions. These effects mediated by 15dPGJ2 might contribute to understand the role of COX-2 metabolites in carcinogenesis, leading to an increase of cell viability after mutagenic injury and therefore in the progression of tumors.

The cyclopentenone prostaglandin 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) has been shown to display protective effects against renal injury or inflammation. In cultured mesangial cells (MC), 15d-PGJ2 inhibits the expression of proinflammatory genes and modulates cell proliferation. Therefore, cyclopentenone prostaglandins (cyPG) have been envisaged as a promise in the treatment of renal disease. The effects of 15d-PGJ2 may be dependent on or independent from its role as a peroxisome proliferator-activated receptor agonist. It was shown recently that an important determinant for the peroxisome proliferator-activated receptor-independent effects of 15d-PGJ2 is the capacity to modify proteins covalently and alter their function. However, a limited number of protein targets have been identified to date. Herein is shown that a biotinylated derivative of 15d-PGJ2 recapitulates the effects of 15d-PGJ2 on the stress response and inhibition of inducible nitric oxide synthase levels and forms stable adducts with proteins in intact MC. Biotinylated 15d-PGJ2 was then used to identify proteins that potentially are involved in cyPG biologic effects. Extracts from biotinylated 15d-PGJ2-treated MC were separated by two-dimensional electrophoresis, and the spots of interest were analyzed by mass spectrometry. Identified targets include proteins that are regulated by oxidative stress, such as heat-shock protein 90 and nucleoside diphosphate kinase, as well as proteins that are involved in cytoskeletal organization, such as actin, tubulin, vimentin, and tropomyosin. Biotinylated 15d-PGJ2 binding to several targets was confirmed by avidin pull-down. Consistent with these findings, 15d-PGJ2 induced early reorganization of vimentin and tubulin in MC. The cyclopentenone moiety and the presence of cysteine were important for vimentin rearrangement. These studies may contribute to the understanding of the mechanism of action and therapeutic potential of cyPG.

In acute inflammation, the transcription factor NF-kappaB is activated and increases the expression of multiple pro-inflammatory genes. Agonists of peroxisome proliferator activated receptors (PPAR) have been reported to exert antiinflammatory effects in various systems. In keeping with such an antiinflammatory role, it was found that several PPAR agonists, including Wy14,643, clofibrate, carbaprostacyclin, and ciglitazone inhibited NF-kappaB activity and increased IkappaBalpha levels in cytokine-stimulated mesangial cells (MC). Activation of NF-kappaB has been found to be crucial to the cytokine-elicited expression of inducible nitric oxide synthase (iNOS). Despite the inhibitory effect of PPAR agonists on NF-kappaB activity, this study provides experimental data demonstrating that these agonists amplify cytokine-elicited NO generation in MC, potentiating iNOS protein expression approximately threefold. The upregulation of iNOS expression occurred at the mRNA level and apparently did not result from iNOS mRNA stabilization. Clofibrate and ciglitazone amplified the cytokine-elicited stimulation of a 16-Kb human iNOS promoter construct in stably transfected MC, suggesting that PPAR agonists potentiate iNOS induction through transcriptional mechanisms. MC express all three PPAR proteins. However, iNOS potentiation did not correlate with increased PPAR activity. In addition, Wy14,643-induced amplification of cytokine-elicited iNOS levels also occurred in RAW264.7 macrophages and in human epithelial Caco-2 and HT-29 cells. The observation that these epithelial cell lines express an inactive, truncated PPARalpha variant suggests that a classical PPARalpha agonist, such as Wy14,643, may act through PPARalpha-independent mechanisms. In conclusion, these results show that, despite reducing NF-kappaB activity, PPAR agonists may amplify the expression of certain NF-kappaB-dependent genes that are relevant to the inflammatory process, like iNOS.