Structural insights into redox-active cysteine residues of the Src family kinases.
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ABSTRACT:
SUBMITTER: Heppner DE
PROVIDER: S-EPMC8022254 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.
| S-EPMC3047142 | biostudies-literature
Project description:ABSTRACT We recently identified the novel persulfide sensor SqrR that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus. SqrR binds to the promoter regions of target genes to repress their expression in the absence of sulfide, and the repressor activity is negated by sulfide treatment. SqrR has 3 cysteine residues, 2 of which are conserved in SqrR homologs from other bacteria: Cys41 and Cys107. SqrR forms an intramolecular tetrasulfide bond between Cys41 and Cys107 when exposed to persulfide, which results in loss of the DNA-binding activity in vitro. Here, we address the mechanism through which these cysteine residues are modified by persulfides. We show that the predicted pKa value of Cys107, as revealed by a putative SqrR structural model, is lower than that of Cys41. Furthermore, C41S SqrR in which Cys41 was changed to serine forms an intermolecular disulfide-bond between Cys107 of 2 SqrRs, suggesting high nucleophilic reactivity of Cy107. These data suggest that Cys107 and Cys41 function as attacking Cys and resolving Cys, respectively; this occurs during tetrasulfide-bond formation of WT SqrR, when it is exposed to persulfide.
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Project description:Protein kinases, key regulators of intracellular signal transduction, have emerged as an important class of drug targets. Chemical proteomic tools that facilitate the functional interrogation of protein kinase active sites are powerful reagents for studying the regulation of this large enzyme family and performing inhibitor selectivity screens. Here we describe a new crosslinking strategy that enables rapid and quantitative profiling of protein kinase active sites in lysates and live cells. Applying this methodology to the SRC-family kinases (SFKs) SRC and HCK led to the identification of a series of conformation-specific, ATP-competitive inhibitors that have a distinct preference for the autoinhibited forms of these kinases. Furthermore, we show that ligands that have this selectivity are able to modulate the ability of the regulatory domains of SRC and HCK to engage in intermolecular binding interactions. These studies provide insight into the regulation of this important family of tyrosine kinases.
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Project description:The mitochondrion is the primary energy generator of a cell and is a central player in cellular redox regulation. Mitochondrial reactive oxygen species (mtROS) are the natural byproducts of cellular respiration that are critical for the redox signaling events that regulate a cell's metabolism. These redox signaling pathways primarily rely on the reversible oxidation of the cysteine residues on mitochondrial proteins. Several key sites of this cysteine oxidation on mitochondrial proteins have been identified and shown to modulate downstream signaling pathways. To further our understanding of mitochondrial cysteine oxidation and to identify uncharacterized redox-sensitive cysteines, we coupled mitochondrial enrichment with redox proteomics. Briefly, differential centrifugation methods were used to enrich for mitochondria. These purified mitochondria were subjected to both exogenous and endogenous ROS treatments and analyzed by two redox proteomics methods. A competitive cysteine-reactive profiling strategy, termed isoTOP-ABPP, enabled the ranking of the cysteines by their redox sensitivity, due to a loss of reactivity induced by cysteine oxidation. A modified OxICAT method enabled a quantification of the percentage of reversible cysteine oxidation. Initially, we assessed the cysteine oxidation upon treatment with a range of exogenous hydrogen peroxide concentrations, which allowed us to differentiate the mitochondrial cysteines by their susceptibility to oxidation. We then analyzed the cysteine oxidation upon inducing reactive oxygen species generation via the inhibition of the electron transport chain. Together, these methods identified the mitochondrial cysteines that were sensitive to endogenous and exogenous ROS, including several previously known redox-regulated cysteines and uncharacterized cysteines on diverse mitochondrial proteins.
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Project description:Here, we introduce the concept of the "seleno effect" in the study of oxidoreductases that catalyze thiol/disulfide exchange reactions. In these reactions, selenium can replace sulfur as a nucleophile, electrophile, or leaving group, and the resulting change in rate (the seleno effect) is defined as kS/ kSe. In solution, selenium accelerates the rate of thiol/disulfide exchange regardless of its chemical role (e.g., nucleophile or electrophile). Here we show that this is not the case for enzyme catalyzed reactions and that the magnitude of the seleno effect can differentiate the role of each sulfur atom of a disulfide bond between that of an electrophile or leaving group. We used selenium for sulfur substitution to study the thiol/disulfide exchange step that occurs between the N-terminal redox center and the C-terminal disulfide-containing ?-hairpin motif of Plasmodium falciparum thioredoxin reductase (PfTrxR), which has the sequence Gly-Cys535-Gly-Gly-Gly-Lys-Cys540-Gly. We assayed a truncated PfTrxR enzyme missing this C-terminal tail for disulfide-reductase activity using synthetic peptide substrates in which either Cys535 or Cys540 was replaced with selenocysteine (Sec). The results show that substitution of Cys535 with Sec resulted in a nearly 9-fold decrease in the rate of reduction, while substitution of Cys540 resulted in a 1.5-fold increase in the rate of reduction. We also produced full-length, semisynthetic enzymes in which Sec replaced either of these two Cys residues and observed similar results using E. coli thioredoxin as the substrate. In this assay, the observed seleno effect ( kS/ kSe) for the C535U mutant was 7.4, and that for the C540U mutant was 0.2.
| S-EPMC5866731 | biostudies-literature
Project description:Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases.
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Project description:CD20 is an integrate membrane protein expressed on the surface of normal and malignant B-cells. It is an excellent molecular target for monoclonal antibodies (mAbs) that are widely used in the treatment of non-Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL). Anti-CD20-directed therapies are the most effective, successful and widely used therapeutic monoclonal antibodies, routinely incorporated into all phases of conventional treatment, including first-line, maintenance and salvage treatment. Nonetheless, the overall response rates to treatment have been reported to be 46-67% for rituximab, and in patients relapsing after initial rituximab treatment the response rate is ~40%. A number of mechanisms have been proposed to account for this inefficiency, including modulation of surface CD20 levels, occurring due to both transcriptional and posttranscriptional regulations. Our observations strongly imply that the observed CD20 down-regulation relies on transcriptional mechanism and delineate new perspectives in the field of CD20 regulation. Since many of the new therapeutic agents selectively inhibit specific signaling pathways, combining the compounds that target different mechanisms of cell growth and survival is a particularly attractive approach. However, optimal combinations of novel treatment modalities with already existing ones should take into account potential ”off-target” activity yet to be identified that could unexpectedly result in high toxicity and/or impaired outcomes of treatment
2014-12-31 | GSE50929 | GEO
Project description:Thiol-dependent redox systems are involved in regulation of diverse biological processes, such as response to stress, signal transduction, and protein folding. The thiol-based redox control is provided by mechanistically similar, but structurally distinct families of enzymes known as thiol oxidoreductases. Many such enzymes have been characterized, but identities and functions of the entire sets of thiol oxidoreductases in organisms are not known. Extreme sequence and structural divergence makes identification of these proteins difficult. Thiol oxidoreductases contain a redox-active cysteine residue, or its functional analog selenocysteine, in their active sites. Here, we describe computational methods for in silico prediction of thiol oxidoreductases in nucleotide and protein sequence databases and identification of their redox-active cysteines. We discuss different functional categories of cysteine residues, describe methods for discrimination between catalytic and noncatalytic and between redox and non-redox cysteine residues and highlight unique properties of the redox-active cysteines based on evolutionary conservation, secondary and three-dimensional structures, and sporadic replacement of cysteines with catalytically superior selenocysteine residues.
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Project description:Cell adhesion molecules, including integrins, cadherins, and claudins (CLDNs), are known to activate Src-family kinases (SFKs) that organize a variety of physiological and pathological processes; however, the underlying molecular basis remains unclear. Here, we identify the SFK members that are coupled with the CLDN6-adhesion signaling. Among SFK subtypes, BLK, FGR, HCK, and SRC were highly expressed in F9 cells and concentrated with CLDN6 along cell borders during epithelial differentiation. Immunoprecipitation assay showed that BLK and SRC, but not FGR or HCK, form a complex with CLDN6 via the C-terminal cytoplasmic domain. We also demonstrated, by pull-down assay, that recombinant BLK and SRC proteins directly bind to the C-terminal cytoplasmic domain of CLDN6 (CLDN6C). Unexpectedly, both recombinant SFK proteins recognized the CLDN6C peptide in a phosphotyrosine-independent manner. Furthermore, by comparing phenotypes of F9:Cldn6:Blk-/- and F9:Cldn6:Src-/- cells with those of wild-type F9 and F9:Cldn6 cells, we revealed that BLK and SRC are essential for CLDN6-triggered cellular events, namely epithelial differentiation and the expression of retinoid acid receptor target genes. These results indicate that selective SFK members appear to participate in the CLDN-adhesion signaling.
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Project description:Abstract: Malignant mesothelioma (MM) is a deadly tumor mainly caused by exposure to asbestos. Unfortunately, no current treatment is able to change significantly the natural history of the disease, which has a poor prognosis in the majority of patients. The non-receptor tyrosine kinase SRC and other SRC family kinase (SFK) members are frequently hyperactivated in many cancer types, including MM. Several works have indeed suggested that SFKs underlie MM cell proliferation, survival, motility, and invasion, overall affecting multiple oncogenic pathways. Consistently, SFK inhibitors effectively counteracted MM cancerous features at the preclinical level. Dasatinib, a multi-kinase inhibitor targeting SFKs, was also assessed in clinical trials either as second-line treatment for patients with unresectable MM or, more recently, as a neoadjuvant agent in patients with resectable MM. Here, we provide an overview of the molecular mechanisms implicating SFKs in MM progression and discuss possible strategies for a more successful clinical application of SFK inhibitors. Our aim is to stimulate discussion and further consideration of these agents in better designed preclinical and clinical studies to make the most of another class of powerful antitumoral drugs, which too often are lost in translation when applied to MM.
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