Project description:Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to the systemic disease candidiasis. Its ability to adopt various morphological forms, such as unicellular yeasts, filamentous pseudohyphae and hyphae, contributes to its ability to survive within the host. It has been suggested that the antioxidant glutathione is involved in the filamentation process. We investigated S-glutathionylation, the reversible binding of glutathione to proteins, and the functional consequences on C. albicans metabolic remodeling during the yeast-to-hyphae transition. Our work provided evidence for the specific glutathionylation of mitochondrial proteins involved in bioenergetics pathways in filamentous forms and a regulation of the main enzyme of the glyoxylate cycle, isocitrate lyase, by glutathionylation. Our proteomic data demonstrate that the binding of one glutathione molecule to isocitrate lyase in the hyphal forms leads to enzyme inactivation which was reversed by glutaredoxin treatment. We also assessed the effect of alternative carbon sources on glutathione levels and isocitrate lyase activity. Changes in nutrient availability led to morphological flexibility and was related to perturbations in glutathione levels and isocitrate lyase activity, confirming the key role of the maintenance of intracellular redox status in the adaptive metabolic strategy of the pathogen.
Project description:We engineered a cell system in which the glutathione synthetase (GS) mutant was expressed that catalyzed the formation of a glutathione analogy from azido-alanine to profile changes of glutathionylome in CD38-overexpressing cells.
Project description:Protein-S-glutathionylation (PSSG) is a post-translational modification involving the conjugation of glutathione to reactive protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing the reversible deglutathionylation reaction. Mammalian GLRX only requires the N-terminal cysteine in the thioredoxin domain to carry out deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, and oxidation of GLRX itself has been reported in close association with aggregation and loss of activity. Further, the specific cysteines within GLRX that are vulnerable to oxidative modification and their relative reactivities with oxidants remain unknown. Herein, we utilized various molecular modeling approaches coupled with site-directed mutagenesis of each cysteine both individually and in combination to address how the five cysteines present in GLRX influence the enzyme’s biological activity and susceptibility to dimerization and inactivation. We report that mutation of cysteine 26 (C26) to serine of murine GLRX1 confers a higher rate of deglutathionylation. We further demonstrate that C8 and C83 are targets for PSSG in vitro while C79 is less vulnerable due to steric hindrance. Hydrogen peroxide also caused inactivation of GLRX that was only protected against with mutation of C8 or C83. Both experimental and modeling evidence indicates C8 contributes to dimer formation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased biological activity and resistance to aggregation and oxidative inactivation. Overall, these results from our corroborated experimental and computational studies have valuable implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.
Project description:Prader-Willi syndrome (PWS) is a multisystem disorder caused by loss of expression of a cluster of paternally-expressed, imprinted genes. Neonatal failure to thrive is followed by childhood-onset hyperphagia, obesity, neurobehavioral abnormalities, and hormonal deficits. Prior evidence from a mouse model with a deletion of the orthologous PWS-domain identified abnormal pancreatic islet development with deficient insulin secretion, hypoglucagonemia, and postnatal onset of progressive, lethal hypoglycemia. To investigate PWS-genes in β-cell secretory function, we used CRISPR/Cas9 genome-editing to generate isogenic, clonal INS-1 insulinoma lines with 3.16 Mb deletions of the silent, maternal (control) or active, paternal (PWS) alleles. A significant reduction in basal and glucose-stimulated insulin secretion signifies a cell autonomous insulin secretion deficit in PWS β-cells. Parallel proteome and transcriptome studies revealed reduced levels of secreted peptides and for eleven endoplasmic reticulum (ER) chaperones, including HSPA5 and HSP90B1. In contrast to dosage compensation previously seen for ER chaperones in Hspa5 or Hsp90b1 gene knockouts, compensation is precluded by the widespread deficiency of ER chaperones in PWS cells. Remarkably, but consistent with reduced ER chaperone levels, PWS β-cells are more sensitive to ER stress activation of all three regulatory pathways (XBP1, eIF2α-P, ATF6-N). Therefore, a coordinated, chronic deficit of ER chaperones in PWS β-cells is hypothesized to lead to a delay in ER transit and/or folding of insulin and other cargo along the secretory pathway. These findings provide insight into the pathophysiological basis of hormone deficits in PWS and indicate key roles for PWS-imprinted genes in β-cell secretory function.
Project description:We utilized the well-characterized murine T cell transfer model of colitis to find specific alterations in the intestinal luminal proteome associated with inflammation. Mass spectrometry proteomic analysis of colonic samples permitted the identification of ~10,000-12,000 unique peptides that corresponded to 5610 protein clusters identified across three groups, including the colitic Rag1 -/- T cell recipients, isogenic Rag1 -/- controls, as well as wild-type mice. Bioinformatic analyses on host and microbial proteins found specific proteins and GO term functionalities unique to each group, as well as GO terms shared across the three cohorts. We further demonstrated that the colitic mice exhibited a significant increase in Proteobacteria and Verrucomicrobia that was substantiated with 16S rDNA sequencing.
Project description:Mammalian TFEB and TFE3, as well as their ortholog in C. elegans HLH-30, play an important role in mediating cellular response to a variety of stress conditions, including nutrient deprivation, oxidative stress and pathogen infection. In this study we identify a novel mechanism of TFEB/HLH-30 regulation through a cysteine-mediated redox switch. Under stress conditions, TFEB-C212 undergoes oxidation, allowing the formation of intermolecular disulfide bonds that result in TFEB oligomerization. TFEB oligomers display increased resistance to mTORC1-mediated inactivation and are more stable under prolonged stress conditions. Mutation of the only cysteine residue present in HLH-30 (C284) significantly reduced its activity, resulting in developmental defects and increased pathogen susceptibility. Therefore, cysteine oxidation represents a new type of TFEB post-translational modification that functions as a molecular switch to link changes in redox balance with expression of TFEB/HLH-30 target genes.
Project description:Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite’s histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy. This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi.
Project description:Drak2¬-deficient (Drak2-/-) mice are resistant to multiple models of autoimmunity, yet effectively eliminate pathogens and tumors. Thus, DRAK2 is an ideal target to treat autoimmune diseases. However, the mechanisms by which DRAK2 contributes to autoimmunity, particularly type 1 diabetes (T1D), remain unresolved. Our data indicate that DRAK2 contributes to autoimmunity in multiple ways by regulating thymic Treg development and by impacting the sensitivity of conventional T cells to Treg-mediated suppression.
Project description:A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be non-enzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme Sirtuin 5 (SIRT5). Here, we identify glutarylation of the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH). We show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We then demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic data indicate a novel role for SIRT5 in regulation of amino acid metabolism. Together, these data suggest a model whereby a feedback loop exists within the lysine/tryptophan oxidation pathway, in which glutaryl-CoA is produced, in turn inhibiting GCDH function. This inhibition is relieved by SIRT5 deacylation activity.
Project description:Recently, we elucidated T. urticae’s repertoire of secreted salivary proteins, revealing several members of expanded protein families with unknown functions [PMID: 27703040]. In this study, mite salivary secretions were additionally examined using a peptidomics approach.