Redox, cysteines, and kinases-A triad sustaining myeloid leukemia.

Reactive oxygen species (ROS) work as a second messenger, modulating cell response and establishing homeostasis. Abrupt changes in ROS are used to modulate transient cell response to different stimuli, from viral infection to inflammation. Chronic exposure to high ROS concentration can cause cellular damage and promote the development of diseases. Leukemogenesis is adapted to high concentrations of ROS, hijacking the ROS system, and uses kinase cascades to promote survival advantages. The oxidation-reduction (redox) machinery is composed of enzymes that orchestrate all classes of protein and use available Cys as transmitters and sensors, to disseminate stress signals through cells via kinase cascades. Myeloid leukemias (MLs) are known for being a heterogeneous disease, and clonal diversity is remarkably characterized by differences in the activation of kinase-regulated signaling cascades to provide survival advantage. Stress-activated kinase cascades and other cascades are regulated by the ROS system. Several studies present nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and the ER-resident NOX4 as key elements of ROS activity in healthy myeloid cells and myeloid leukemia. Targeting ROS presents an attractive therapeutic strategy for (MLs) patients, but the boundaries between pro-apoptotic and anti-apoptotic ROS concentrations are not well established. Detailed understanding of the signaling switches that determine cell fate needs to be well understood. This work explores several aspects of the redox system and thiol-mediated reactions with focus on kinase signaling in myeloid cancers and highlights some of the challenges.

Functional Divergence in the Affinity and Stability of Non-Canonical Cysteines and Non-Canonical Disulfide Bonds: Insights from a VHH and VNAR Study.

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

The hydrophobicity of the CARD8 N-terminus tunes inflammasome activation.

Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress.

Redox regulation of gene expression: proteomics reveals multiple previously undescribed redox-sensitive cysteines in transcription complexes and chromatin modifiers.

Redox signalling is crucial for regulating plant development and adaptation to environmental changes. Proteins with redox-sensitive cysteines can sense oxidative stress and modulate their functions. Recent proteomics efforts have comprehensively mapped the proteins targeted by oxidative modifications. The nucleus, the epicentre of transcriptional reprogramming, contains a large number of proteins that control gene expression. Specific redox-sensitive transcription factors have long been recognized as key players in decoding redox signals in the nucleus and thus in regulating transcriptional responses. Consequently, the redox regulation of the nuclear transcription machinery and its cofactors has received less attention. In this review, we screened proteomic datasets for redox-sensitive cysteines on proteins of the core transcription complexes and chromatin modifiers in Arabidopsis thaliana. Our analysis indicates that redox regulation affects every step of gene transcription, from initiation to elongation and termination. We report previously undescribed redox-sensitive subunits in transcription complexes and discuss the emerging challenges in unravelling the landscape of redox-regulated processes involved in nuclear gene transcription.

Assessing the mechanism of fast-cycling cancer-associated mutations of Rac1 small Rho GTPase.

Rho-GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer-associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all-atom molecular dynamics simulations on wild-type (wt) and oncogenic isoforms of this protein in GDP- and GTP-bound states. Our results unprecedentedly elucidate that P29Q/S-induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg2+ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer-associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.

Application of AlphaFold models in evaluating ligandable cysteines across E3 ligases.

Proteolysis Targeting Chimeras (PROTACs) are an emerging therapeutic modality and chemical biology tools for Targeted Protein Degradation (TPD). PROTACs contain a ligand targeting the protein of interest, a ligand recruiting an E3 ligase and a linker connecting these two ligands. There are over 600 E3 ligases known so far, but only a handful have been exploited for TPD applications. A key reason for this is the scarcity of ligands binding various E3 ligases and the paucity of structural data available, which complicates ligand design across the family. In this study, we aim to progress PROTAC discovery by proposing a shortlist of E3 ligases that can be prioritized for covalent targeting by performing systematic structural ligandability analysis on a chemoproteomic dataset of potentially reactive cysteines across hundreds of E3 ligases. One of the goals of this study is to apply AlphaFold (AF) models for ligandability evaluations, as for a vast majority of these ligases an experimental structure is not available in the protein data bank (PDB). Using a combination of pocket features, AF model quality and additional aspects, we propose a shortlist of E3 ligases and corresponding cysteines that can be prioritized to potentially discover covalent ligands and expand the PROTAC toolbox.

Arsenite S-Adenosylmethionine Methyltransferase Is Responsible for Antimony Biomethylation in Nostoc sp. PCC7120.

Antimony (Sb) biomethylation is an important but uninformed process in Sb biogeochemical cycling. Methylated Sb species have been widely detected in the environment, but the gene and enzyme for Sb methylation remain unknown. Here, we found that arsenite S-adenosylmethionine methyltransferase (ArsM) is able to catalyze Sb(III) methylation. The stepwise methylation by ArsM forms mono-, di-, and trimethylated Sb species. Sb(III) is readily coordinated with glutathione, forming the preferred ArsM substrate which is anchored on three conserved cysteines. Overexpressing arsM in Escherichia coli AW3110 conferred resistance to Sb(III) by converting intracellular Sb(III) into gaseous methylated species, serving as a detoxification process. Methylated Sb species were detected in paddy soil cultures, and phylogenetic analysis of ArsM showed its great diversity in ecosystems, suggesting a high metabolic potential for Sb(III) methylation in the environment. This study shows an undiscovered microbial process methylating aqueous Sb(III) into the gaseous phase, mobilizing Sb on a regional and even global scale as a re-emerging contaminant.

The Molecular Frequency, Conservation and Role of Reactive Cysteines in Plant Lipid Metabolism.

Cysteines (Cys) are chemically reactive amino acids containing sulfur that play diverse roles in plant biology. Recent proteomics investigations in Arabidopsis thaliana have revealed the presence of thiol post-translational modifications (PTMs) in several Cys residues. These PTMs are presumed to impact protein structure and function, yet mechanistic data regarding the specific Cys susceptible to modification and their biochemical relevance remain limited. To help address these limitations, we have conducted a wide-ranging analysis by integrating published datasets encompassing PTM proteomics (comparing S-sulfenylation, persulfidation, S-nitrosylation and S-acylation), genomics and protein structures, with a specific focus on proteins involved in plant lipid metabolism. The prevalence and distribution of modified Cys residues across all analyzed proteins is diverse and multifaceted. Nevertheless, by combining an evaluation of sequence conservation across 100+ plant genomes with AlphaFold-generated protein structures and physicochemical predictions, we have unveiled structural propensities associated with Cys modifications. Furthermore, we have identified discernible patterns in lipid biochemical pathways enriched with Cys PTMs, notably involving beta-oxidation, jasmonic acid biosynthesis, fatty acid biosynthesis and wax biosynthesis. These collective findings provide valuable insights for future investigations targeting the mechanistic foundations of Cys modifications and the regulation of modified proteins in lipid metabolism and other metabolic pathways.

Defining the Cell Surface Cysteinome using Two-step Enrichment Proteomics.

The plasma membrane proteome is a rich resource of functional and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surface Cysteinome. Our "Cys-Surf" platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of LDL particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome.

Cooperative oxygen binding in beta-semihemoglobins caused by a chemical modification in the alpha1beta1 interface.

A beta-semihemoglobin is an alpha-beta dimer of hemoglobin (Hb) in which the beta-subunit carries heme, while the alpha-subunit is heme-less, in apo form. It is characterised by displaying a high affinity for oxygen, and absence of cooperative binding of oxygen. We have modified chemically the residue beta112Cys (G14), located adjacent to the alpha1beta1 interface, and studied the impact of such a modification on the oligomeric state and oxygenation properties of the derivatives. We also studied the impact of modifying beta93Cys (F9) since its modification was unavoidable. For this, we used N-Ethyl maleimide and iodoacetamide. For the alkylation of beta112Cys (G14) in isolated subunits, we used N-Ethyl maleimide, iodoacetamide, or additionally, 4,4'-Dithiopyridine. Seven native and chemically modified beta-subunit derivatives were prepared and analysed. Only those derivatives treated with iodoacetamide showed oxygenation properties that were indistinguishable from those of native beta-subunits. These derivatives were then converted into their respective semihemoglobin forms, and four additional derivatives were prepared and analysed .in terms of ligation-linked oligomeric state, and oxygenation function, and contrasted against native Hb and unmodified beta-subunits. Strikingly, beta-semiHbs with modifications in beta112Cys showed indications of cooperative oxygen binding in various degrees, which suggested the possibility of assembly of two beta-semiHbs. The derivative modified with 4-Thiopyridine in beta112Cys showed a highly cooperative binding of oxygen (nmax = 1.67). A plausible allosteric scheme that could explain allostery in beta-semiHb system is suggested.

Inhibition of the Mitochondrial Carnitine/Acylcarnitine Carrier by Itaconate through Irreversible Binding to Cysteine 136: Possible Pathophysiological Implications.

BACKGROUND: The carnitine/acylcarnitine carrier (CAC) represents the route of delivering acyl moieties to the mitochondrial matrix for accomplishing the fatty acid ß-oxidation. The CAC has a couple of Cys residues (C136 and C155) most reactive toward ROS and redox signaling compounds such as GSH, NO, and H2S. Among physiological compounds reacting with Cys, itaconate is produced during inflammation and represents the connection between oxidative metabolism and immune responses. The possible interaction between the CAC and itaconate has been investigated. METHODS: the modulatory effects of itaconate on the transport activity of the native and recombinant CAC were tested using the proteoliposome experimental model together with site-directed mutagenesis and computational analysis. RESULTS: Itaconate reacts with the CAC causing irreversible inhibition. Dose-response experiment performed with the native and recombinant protein showed IC50 for itaconate of 11 ± 4.6 mM and 8.4 ± 2.9 mM, respectively. The IC50 decreased to 3.8 ± 1.0 mM by lowering the pH from pH 7.0 to pH 6.5. Inhibition kinetics revealed a non-competitive type of inhibition. C136 is the main target of itaconate, as demonstrated by the increased IC50 of mutants in which this Cys was substituted by Val. The central role of C136 was confirmed by covalent docking. Administration of dimethyl itaconate to HeLa cells inhibited the CAC transport activity, suggesting that itaconate could react with the CAC also in intact cells.

Structural changes in NOTCH3 induced by CADASIL mutations: Role of cysteine and non-cysteine alterations.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease that results from mutations in NOTCH3. How mutations in NOTCH3 ultimately result in disease is not clear, although there is a predilection for mutations to alter the number of cysteines of the gene product, supporting a model in which alterations of conserved disulfide bonds of NOTCH3 drives the disease process. We have found that recombinant proteins with CADASIL NOTCH3 EGF domains 1 to 3 fused to the C terminus of Fc are distinguished from wildtype proteins by slowed mobility in nonreducing gels. We use this gel mobility shift assay to define the effects of mutations in the first three EGF-like domains of NOTCH3 in 167 unique recombinant protein constructs. This assay permits a readout on NOTCH3 protein mobility that indicates that (1) any loss of cysteine mutation in the first three EGF motifs results in structural abnormalities; (2) for loss of cysteine mutants, the mutant amino acid residue plays a minimal role; (3) the majority of changes that result in a new cysteine are poorly tolerated; (4) at residue 75, only cysteine, proline, and glycine induce structural shifts; (5) specific second mutations in conserved cysteines suppress the impact of loss of cysteine CADASIL mutations. These studies support the importance of NOTCH3 cysteines and disulfide bonds in maintaining normal protein structure. Double mutant analysis suggests that suppression of protein abnormalities can be achieved through modification of cysteine reactivity, a potential therapeutic strategy.

Ionic Liquid-Mediated Approach for the Synthesis of Site-Specific Thioether Conjugates.

Site-specific conjugation approaches are of great importance in drug discovery, notably for the synthesis of biochemical probes or molecular conjugates for targeted delivery. Herein, we report a mild ionic liquid (IL)-mediated thiolation technique that relies on the use of 1,3-ethyl-methyl imidazolium acetate, [C2 mim][OAc] as a solvent and precursor to generate activated IL, as well as a solvent for the conjugation reaction. First, a focused library of active ILs was prepared for functionalizing/conjugating cysteine-containing small molecules and unprotected peptides. Interestingly, a bifunctional active IL could also be successfully employed as a linker for the conjugation of peptides lacking Cys. This study sets the ground for further investigation of the use of active ILs for modifying, labeling or conjugating larger and more complex therapeutic modalities such as proteins and antibodies.

The cysteine S-H stretching mode reports on long-range conformational changes in pyruvate oxidase from E. coli.

The pyruvate oxidases from Escherichia coli (EcPOX) and Lactobacillus plantarum (LpPOX) are both thiamin-dependent flavoenzymes. Their sequence and structure are closely related, and they catalyse similar reactions-but they differ in their activity pattern: LpPOX is always highly active, EcPOX only when activated by lipids or limited proteolysis, both involving the protein's C-terminal 23 residues (the 'α-peptide'). Here, we relate the redox-induced infrared (IR) difference spectrum of EcPOX to its unusual activation mechanism. The IR difference spectrum of EcPOX is marked by contributions from the protein backbone, reflecting major conformational changes. A rare sulfhydryl (-SH) difference signal indicates changes in the vicinity of cysteines. We could pin the Cys-SH difference signal to Cys88 and Cys494, both being remote from the moving α-peptide and the redox-active flavin cofactor. Yet, when the α-peptide is proteolytically removed, the Cys-SH difference signal disappears, together with several difference signals in the amide range. The remaining IR signature of the permanently activated EcPOXΔ23 is strikingly similar to the simpler signature of LpPOX. The loss of the α-peptide 'transforms' the catalytically complex EcPOX into the catalytically 'simpler' LpPOX.

The Mycotoxin Patulin Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier (SLC25A20) by Interaction with Cys136 Implications for Human Health.

The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [3H]-carnitineex/carnitinein antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC50 derived from the dose-response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.

Redox stress in COVID-19: Implications for hematologic disorders.

COVID-19 is the respiratory illness caused by the beta coronavirus SARS-CoV-2. COVID-19 is complicated by an increased risk for adverse thrombotic events that promote organ failure and death. While the mechanism of action for SARS-CoV-2 is still being understood, how SARS-CoV-2 infection impacts the redox environment in hematologic conditions is unclear. In this review, the redox mechanisms contributing to SARS-CoV-2 infection, coagulopathy and inflammation are briefly discussed. Specifically, sources of oxidant generation by hematopoietic and non-hematopoietic cells are identified with special emphasis on leukocytes, platelets, red cells, and endothelial cells. Furthermore, reactive cysteines in SARS-CoV-2 are also discussed with respect to oxidative cysteine modification and current therapeutic implications. Lastly, sickle cell disease will be discussed as a hematologic disorder with a pre-existing prothrombotic redox condition that complicates treatment strategies for COVID-19. An understanding of the redox mechanism may identify potential targets for COVID-19-mediated thrombosis in hematologic disorders.

Emerging chemistry and biology in protein glutathionylation.

Protein S-glutathionylation serves a regulatory role in proteins and modulates distinct biological processes implicated in health and diseases. Despite challenges in analyzing the dynamic and reversible nature of S-glutathionylation, recent chemical and biological methods have significantly advanced the field of S-glutathionylation, culminating in selective identification and detection, structural motif analysis, and functional studies of S-glutathionylation. This review will highlight emerging studies of protein glutathionylation, beginning by introducing biochemical tools that enable mass spectrometric identification and live-cell imaging of S-glutathionylation. Next, it will spotlight recent examples of S-glutathionylation regulating physiology and inflammation. Lastly, we will feature two emerging lines of glutathionylation research in cryptic cysteine glutathionylation and protein C-glutathionylation.

Destabilization of the SHP2 and SHP1 protein tyrosine phosphatase domains by a non-conserved "backdoor" cysteine.

Protein tyrosine phosphatases (PTPs) are critical regulators of cellular signal transduction that catalyze the hydrolytic dephosphorylation of phosphotyrosine in substrate proteins. Among several conserved features in classical PTP domains are an active-site cysteine residue that is necessary for catalysis and a "backdoor" cysteine residue that can serve to protect the active-site cysteine from irreversible oxidation. Curiously, two biologically important phosphatases, Src homology domain-containing PTPs 2 and 1 (SHP2 and SHP1), each contain an additional backdoor cysteine residue at a position of the PTP domain that is occupied by proline in almost all other classical PTPs (position 333 in human SHP2 numbering). Here we show that the presence of cysteine 333 significantly destabilizes the fold of the PTP domains in the SHPs. We find that replacement of cysteine 333 with proline confers increased thermal stability on the SHP2 and SHP1 PTP domains, as measured by temperature-dependent activity assays and differential scanning fluorimetry. Conversely, we show that substantial destabilization of the PTP-domain fold is conferred by introduction of a non-natural cysteine residue in a non-SHP PTP that contains proline at the 333 position. It has previously been suggested that the extra backdoor cysteine of the SHP PTPs may work in tandem with the conserved backdoor cysteine to provide protection from irreversible oxidative enzyme inactivation. If so, our current results suggest that, during the course of mammalian evolution, the SHP proteins have developed extra protection from oxidation at the cost of the thermal instability that is conferred by the presence of their PTP domains' second backdoor cysteine.

Intravenous caval leiomyomatosis in the setting of renal cell carcinoma - A case report.

Uterine leiomyomas are common, benign neoplasms of the uterine smooth muscle. Leiomyomatosis is uncommon and causes development of multiple leiomyomas that can manifest as intravascular leiomyomatosis (IVL). We present the case of a 46-year-old female with IVL extending from the right gonadal vein to the right atrium and pulmonary arteries with an independent renal cell carcinoma of the right kidney. She underwent successful open right radical nephrectomy, inferior vena caval tumor thrombectomy and pulmonary embolectomy. While there was initial concern for hereditary renal cell carcinoma, final histologic testing did not support the diagnosis.

Prominent role of cysteine residues C49 and C343 in regulating Plasmodium falciparum pyruvate kinase activity.

The protozoan parasite Plasmodium falciparum causes the most severe form of malaria and is highly dependent on glycolysis. Glycolytic enzymes were shown to be massively redox regulated, inter alia via oxidative post-translational modifications (oxPTMs) of their cysteine residues. In this study, we identified P. falciparum pyruvate kinase (PfPK) C49 and C343 as amino acid residues essentially involved in maintaining structural and functional integrity of the enzyme. The mutation of these cysteines resulted in an altered substrate affinity, lower enzymatic activities, and, as studied by X-ray crystallography, conformational changes within the A-domain where the substrate binding site is located. Although the loss of a cysteine evoked an impaired catalysis in both mutants, the effects observed for mutant C49A were more severe: multiple conformational changes, caused by the loss of two hydrogen bonds, impeded proper substrate binding and thus the transfer of phosphate upon catalysis.