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.

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.

Vimentin Tail Segments Are Differentially Exposed at Distinct Cellular Locations and in Response to Stress.

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.

Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia.

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

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.

Assessing Amphiphilic ABAB Zn(II) Phthalocyanines with Enhanced Photosensitization Abilities in In Vitro Photodynamic Therapy Studies Against Cancer.

We have previously demonstrated that singlet oxygen photosensitization abilities of Zn(II) phthalocyanines (Zn(II)Pcs) are enhanced through α-functionalization with bulky fluorinated substituents (i.e., bis(trifluoromethyl)phenyl units) at facing positions of ABAB Zn(II)Pcs, where A and B refer to differently functionalized isoindoles. In this work, we have prepared the Zn(II)Pc ABAB 1 endowed with hydrophilic triethylene glycol monomethyl ether (i.e., at the A isoindoles) to provide solubility in aqueous media, together with its A3B and A4 counterparts, and compared their ability to behave as photosensitizers for photodynamic therapy. All photophysical data, aggregation studies and preliminary in vitro biological assays in cell cultures of SCC-13 (squamous cell carcinoma) and HeLa (cervical cancer cells), have proved ABAB 1 as the best photosensitizer of the series.

Fluorine-substituted tetracationic ABAB-phthalocyanines for efficient photodynamic inactivation of Gram-positive and Gram-negative bacteria.

Herein, we report the synthesis and characterization of new amphiphilic phthalocyanines (Pcs), the study of their singlet oxygen generation capabilities, and biological assays to determine their potential as photosensitizers for photodynamic inactivation of bacteria. In particular, Pcs with an ABAB geometry (where A and B refer to differently substituted isoindole constituents) have been synthesized. These molecules are endowed with bulky bis(trifluoromethylphenyl) groups in two facing isoindoles, which hinder aggregation and favour singlet oxygen generation, and pyridinium or alkylammonium moieties in the other two isoindoles. In particular, two water-soluble Pc derivatives (PS-1 and PS-2) have proved to be efficient in the photoinactivation of S. aureus and E. coli, selected as models of Gram-positive and Gram-negative bacteria.