Type III intermediate filaments in redox interplay: key role of the conserved cysteine residue.

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.

The redox-responsive roles of intermediate filaments in cellular stress detection, integration and mitigation.

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.

Desmin Reorganization by Stimuli Inducing Oxidative Stress and Electrophiles: Role of Its Single Cysteine Residue.

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.

Vimentin single cysteine residue acts as a tunable sensor for network organization and as a key for actin remodeling in response to oxidants and electrophiles.

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.

Appraising the Role of Astrocytes as Suppliers of Neuronal Glutathione Precursors.

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 GFAP R239C mutant shows increased susceptibility to lipoxidation and elicits mitochondrial dysfunction and oxidative stress.

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-Δ12,14-prostaglandin J2 (15d-PGJ2). 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, H2O2) or electrophiles (4-hydroxynonenal, 15d-PGJ2), which exacerbate GFAP R239C aggregation. Furthermore, H2O2 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.

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

The intermediate filament protein vimentin is involved in essential cellular processes, including cell division and stress responses, as well as in the pathophysiology of cancer, pathogen infection, and autoimmunity. The vimentin network undergoes marked reorganizations in response to oxidative stress, in which modifications of vimentin single cysteine residue, Cys328, play an important role, and is modulated by zinc availability. However, the molecular basis for this regulation is not fully understood. Here, we show that Cys328 displays a low pKa, supporting its reactivity, and is readily alkylated and oxidized in vitro. Moreover, combined oxidation and crosslinking assays and molecular dynamics simulations support that zinc ions interact with Cys328 in its thiolate form, whereas Glu329 and Asp331 stabilize zinc coordination. Vimentin oxidation can induce disulfide crosslinking, implying the close proximity of Cys328 from neighboring dimers in certain vimentin conformations, supported by our computational models. Notably, micromolar zinc concentrations prevent Cys328 alkylation, lipoxidation, and disulfide formation. Moreover, zinc selectively protects vimentin from crosslinking using short-spacer cysteine-reactive but not amine-reactive agents. These effects are not mimicked by magnesium, consistent with a lower number of magnesium ions hosted at the cysteine region, according to molecular dynamics simulations. Importantly, the region surrounding Cys328 is involved in interaction with several drugs targeting vimentin and is conserved in type III intermediate filaments, which include glial fibrillary acidic protein and desmin. Altogether, our results identify this region as a hot spot for zinc binding, which modulates Cys328 reactivity. Moreover, they provide a molecular standpoint for vimentin regulation through the interplay between cysteine modifications and zinc availability.

Dynamic posttranslational modifications of cytoskeletal proteins unveil hot spots under nitroxidative stress.

The cytoskeleton is a supramolecular structure consisting of interacting protein networks that support cell dynamics in essential processes such as migration and division, as well as in responses to stress. Fast cytoskeletal remodeling is achieved with the participation of regulatory proteins and posttranslational modifications (PTMs). Redox-related PTMs are emerging as critical players in cytoskeletal regulation. Here we used a cellular model of mild nitroxidative stress in which a peroxynitrite donor induced transient changes in the organization of three key cytoskeletal proteins, i.e., vimentin, actin and tubulin. Nitroxidative stress-induced reconfiguration of intermediate filaments, microtubules and actin structures were further correlated with their PTM profiles and dynamics of the PTM landscape. Using high-resolution mass spectrometry, 62 different PTMs were identified and relatively quantified in vimentin, actin and tubulin, including 12 enzymatic, 13 oxidative and 2 nitric oxide-derived modifications as well as 35 modifications by carbonylated lipid peroxidation products, thus evidencing the occurrence of a chain reaction with formation of numerous reactive species and activation of multiple signaling pathways. Our results unveil the presence of certain modifications under basal conditions and their modulation in response to stress in a target-, residue- and reactive species-dependent manner. Thus, some modifications accumulated during the experiment whereas others varied transiently. Moreover, we identified protein PTM "hot spots", such as the single cysteine residue of vimentin, which was detected in seven modified forms, thus, supporting its role in PTM crosstalk and redox sensing. Finally, identification of novel PTMs in these proteins paves the way for unveiling new cytoskeleton regulatory mechanisms.

Protein Lipoxidation: Basic Concepts and Emerging Roles.

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.

Type III intermediate filaments as targets and effectors of electrophiles and oxidants.

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.

Zinc Differentially Modulates the Assembly of Soluble and Polymerized Vimentin.

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

Amoxicillin Inactivation by Thiol-Catalyzed Cyclization Reduces Protein Haptenation and Antibacterial Potency.

Serum and cellular proteins are targets for the formation of adducts with the ß-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 ß-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.

Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division.

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.

Insight into the cellular effects of nitrated phospholipids: Evidence for pleiotropic mechanisms of action.

Nitrated phospholipids have been recently identified in biological systems and showed to display anti-oxidant and anti-inflammatory potential in models of inflammation in vitro. Here, we have explored the effects of nitrated 1-palmitoyl-2-oleyl-phosphatidyl choline (NO2-POPC) in cellular models. We have observed that NO2-POPC, but not POPC, induces cellular changes consisting in cytoskeletal rearrangement and cell shrinking, and ultimately, loss of cell adhesion or impaired cell attachment. NO2-POPC releases NO in vitro and induces accumulation of NO in cells. Nevertheless, the effects of NO2-POPC are not superimposable with those of NO donors, which points to distinctive mechanisms of action. Notably, they show a stronger parallelism, although not complete overlap, with the effects of nitrated fatty acids. Interestingly, redistribution of vimentin by NO2-POPC is attenuated in a C328S mutant, thus indicating that this residue may be a target for direct or indirect modification in NO2-POPC-treated cells. Additionally, NO2-POPC interacts with several typical lipoxidation targets in vitro, including vimentin and PPARγ constructs, likely through cysteine residues. Therefore, nitrated phospholipids emerge as potential novel electrophilic lipid mediators with selective actions.

Vimentin intermediate filament assembly regulates fibroblast invasion in fibrogenic lung injury.

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.

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

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress, which induce extensive reorganization of the vimentin cytoskeletal network. Here, we have investigated the mechanisms underlying these effects. In vitro, electrophilic lipids, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and 4-hydroxynonenal (HNE), directly bind to vimentin, whereas the oxidant diamide induces disulfide bond formation. Mutation of the single vimentin cysteine residue (Cys328) blunts disulfide formation and reduces lipoxidation by 15d-PGJ2, but not HNE. Preincubation with these agents differentially hinders NaCl-induced filament formation by wild-type vimentin, with effects ranging from delayed elongation and increased filament diameter to severe impairment of assembly or aggregation. Conversely, the morphology of vimentin Cys328Ser filaments is mildly or not affected. Interestingly, preformed vimentin filaments are more resistant to electrophile-induced disruption, although chemical modification is not diminished, showing that vimentin (lip)oxidation prior to assembly is more deleterious. In cells, electrophiles, particularly diamide, induce a fast and drastic disruption of existing filaments, which requires the presence of Cys328. As the cellular vimentin network is under continuous remodeling, we hypothesized that vimentin exchange on filaments would be necessary for diamide-induced disruption. We confirmed that strategies reducing vimentin dynamics, as monitored by FRAP, including cysteine crosslinking and ATP synthesis inhibition, prevent diamide effect. In turn, phosphorylation may promote vimentin disassembly. Indeed, treatment with the phosphatase inhibitor calyculin A to prevent dephosphorylation intensifies electrophile-induced wild-type vimentin filament disruption. However, whereas a phosphorylation-deficient vimentin mutant is only partially protected from disorganization, Cys328Ser vimentin is virtually resistant, even in the presence of calyculin A. Together, these results indicate that modification of Cys328 and vimentin exchange are critical for electrophile-induced network disruption.

Salmonella exploits host Rho GTPase signalling pathways through the phosphatase activity of SopB.

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.

Asthma and allergic rhinitis associate with the rs2229542 variant that induces a p.Lys90Glu mutation and compromises AKR1B1 protein levels.

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.

The cysteine residue of glial fibrillary acidic protein is a critical target for lipoxidation and required for efficient network organization.

The type III intermediate filament protein glial fibrillary acidic protein (GFAP) contributes to the homeostasis of astrocytes, where it co-polymerizes with vimentin. Conversely, alterations in GFAP assembly or degradation cause intracellular aggregates linked to astrocyte dysfunction and neurological disease. Moreover, injury and inflammation elicit extensive GFAP organization and expression changes, which underline reactive gliosis. Here we have studied GFAP as a target for modification by electrophilic inflammatory mediators. We show that the GFAP cysteine, C294, is targeted by lipoxidation by cyclopentenone prostaglandins (cyPG) in vitro and in cells. Electrophilic modification of GFAP in cells leads to a striking filament rearrangement, with retraction from the cell periphery and juxtanuclear condensation in thick bundles. Importantly, the C294S mutant is resistant to cyPG addition and filament disruption, thus highlighting the critical role of this residue as a sensor of oxidative damage. However, GFAP C294S shows defective or delayed network formation in GFAP-deficient cells, including SW13/cl.2 cells and GFAP- and vimentin-deficient primary astrocytes. Moreover, GFAP C294S does not effectively integrate with and even disrupts vimentin filaments in the short-term. Interestingly, short-spacer bifunctional cysteine crosslinking produces GFAP-vimentin heterodimers, suggesting that a certain proportion of cysteine residues from both proteins are spatially close. Collectively, these results support that the conserved cysteine residue in type III intermediate filament proteins serves as an electrophilic stress sensor and structural element. Therefore, oxidative modifications of this cysteine could contribute to GFAP disruption or aggregation in pathological situations associated with oxidative or electrophilic stress.

Mammalian Sulfur Amino Acid Metabolism: A Nexus Between Redox Regulation, Nutrition, Epigenetics, and Detoxification.

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.