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A conserved cysteine-based redox mechanism sustains TFEB/HLH-30 activity under persistent stress

ABSTRACT: 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 in worms. 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.

SUBMITTER: Dr. Rosa Puertollano

PROVIDER: S-SCDT-EMBOJ-2020-105793 | biostudies-other |

REPOSITORIES: biostudies-other

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A conserved cysteine-based redox mechanism sustains TFEB/HLH-30 activity under persistent stress.

Project description:Mammalian TFEB and TFE3, as well as their ortholog in Caenorhabditis 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 in worms. 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.

| S-EPMC7849306 | biostudies-literature

A conserved cysteine-based redox mechanism sustains TFEB/HLH-30 activity under persistent stress

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.

2021-01-22 | PXD022258 | Pride

A Water-Bridged Cysteine-Cysteine Redox Regulation Mechanism in Bacterial Protein Tyrosine Phosphatases.

Project description:The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains highlights the need to develop more efficacious and potent drugs. However, this goal is dependent on a comprehensive understanding of Mtb virulence protein effectors at the molecular level. Here, we used a post-expression cysteine (Cys)-to-dehydrolanine (Dha) chemical editing strategy to identify a water-mediated motif that modulates accessibility of the protein tyrosine phosphatase A (PtpA) catalytic pocket. Importantly, this water-mediated Cys-Cys non-covalent motif is also present in the phosphatase SptpA from Staphylococcus aureus, which suggests a potentially preserved structural feature among bacterial tyrosine phosphatases. The identification of this structural water provides insight into the known resistance of Mtb PtpA to the oxidative conditions that prevail within an infected host macrophage. This strategy could be applied to extend the understanding of the dynamics and function(s) of proteins in their native state and ultimately aid in the design of small-molecule modulators.

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The conserved regulator of autophagy and innate immunity hlh-30/TFEB mediates tolerance of enterohemorrhagic Escherichia coli in Caenorhabditis elegans.

Project description:Infection with antibiotic-resistant bacteria is an emerging life-threatening issue worldwide. Enterohemorrhagic Escherichia coli O157: H7 (EHEC) causes hemorrhagic colitis and hemolytic uremic syndrome via contaminated food. Treatment of EHEC infection with antibiotics is contraindicated because of the risk of worsening the syndrome through the secreted toxins. Identifying the host factors involved in bacterial infection provides information about how to combat this pathogen. In our previous study, we showed that EHEC colonizes in the intestine of Caenorhabditis elegans. However, the host factors involved in EHEC colonization remain elusive. Thus, in this study, we aimed to identify the host factors involved in EHEC colonization. We conducted forward genetic screens to isolate mutants that enhanced EHEC colonization and named this phenotype enhanced intestinal colonization (Inc). Intriguingly, four mutants with the Inc phenotype showed significantly increased EHEC-resistant survival, which contrasts with our current knowledge. Genetic mapping and whole-genome sequencing (WGS) revealed that these mutants have loss-of-function mutations in unc-89. Furthermore, we showed that the tolerance of unc-89(wf132) to EHEC relied on HLH-30/TFEB activation. These findings suggest that hlh-30 plays a key role in pathogen tolerance in C. elegans.

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Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence.

Project description:Mammalian thioredoxin reductases (TrxR) are homodimers, homologous to glutathione reductase (GR), with an essential selenocysteine (SeCys) residue in an extension containing the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. In the oxidized enzyme, we demonstrated two nonflavin redox centers by chemical modification and peptide sequencing: one was a disulfide within the sequence -Cys(59)-Val-Asn-Val-Gly-Cys(64), identical to the active site of GR; the other was a selenenylsulfide formed from Cys(497)-SeCys(498) and confirmed by mass spectrometry. In the NADPH reduced enzyme, these centers were present as a dithiol and a selenolthiol, respectively. Based on the structure of GR, we propose that in TrxR, the C-terminal Cys(497)-SeCys(498) residues of one monomer are adjacent to the Cys(59) and Cys(64) residues of the second monomer. The reductive half-reaction of TrxR is similar to that of GR followed by exchange from the nascent Cys(59) and Cys(64) dithiol to the selenenylsulfide of the other subunit to generate the active-site selenolthiol. Characterization of recombinant mutant rat TrxR with SeCys(498) replaced by Cys having a 100-fold lower k(cat) for Trx reduction revealed the C-terminal redox center was present as a dithiol when the Cys(59)-Cys(64) was a disulfide, demonstrating that the selenium atom with its larger radius is critical for formation of the unique selenenylsulfide. Spectroscopic redox titrations with dithionite or NADPH were consistent with the structure model. Mechanisms of TrxR in reduction of Trx and hydroperoxides have been postulated and are compatible with known enzyme activities and the effects of inhibitors, like goldthioglucose and 1-chloro-2,4-dinitrobenzene.

| S-EPMC18523 | biostudies-literature

Protein S-mycothiolation functions as redox-switch and thiol protection mechanism in Corynebacterium glutamicum under hypochlorite stress.

Project description:Protein S-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in Firmicutes bacteria. Here we used transcriptomics to analyze the NaOCl stress response in the mycothiol (MSH)-producing Corynebacterium glutamicum. We further applied thiol-redox proteomics and mass spectrometry (MS) to identify protein S-mycothiolation.Transcriptomics revealed the strong upregulation of the disulfide stress ?(H) regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH.We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol- and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives.

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DAF-16/FOXO and HLH-30/TFEB function as combinatorial transcription factors to promote stress resistance and longevity.

Project description:The ability to perceive and respond to harmful conditions is crucial for the survival of any organism. The transcription factor DAF-16/FOXO is central to these responses, relaying distress signals into the expression of stress resistance and longevity promoting genes. However, its sufficiency in fulfilling this complex task has remained unclear. Using C. elegans, we show that DAF-16 does not function alone but as part of a transcriptional regulatory module, together with the transcription factor HLH-30/TFEB. Under harmful conditions, both transcription factors translocate into the nucleus, where they often form a complex, co-occupy target promoters, and co-regulate many target genes. Interestingly though, their synergy is stimulus-dependent: They rely on each other, functioning in the same pathway, to promote longevity or resistance to oxidative stress, but they elicit heat stress responses independently, and they even oppose each other during dauer formation. We propose that this module of DAF-16 and HLH-30 acts by combinatorial gene regulation to relay distress signals into the expression of specific target gene sets, ensuring optimal survival under each given threat.

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Age- and stress-associated C. elegans granulins impair lysosomal function and induce a compensatory HLH-30/TFEB transcriptional response.

Project description:The progressive failure of protein homeostasis is a hallmark of aging and a common feature in neurodegenerative disease. As the enzymes executing the final stages of autophagy, lysosomal proteases are key contributors to the maintenance of protein homeostasis with age. We previously reported that expression of granulin peptides, the cleavage products of the neurodegenerative disease protein progranulin, enhance the accumulation and toxicity of TAR DNA binding protein 43 (TDP-43) in Caenorhabditis elegans (C. elegans). In this study we show that C. elegans granulins are produced in an age- and stress-dependent manner. Granulins localize to the endolysosomal compartment where they impair lysosomal protease expression and activity. Consequently, protein homeostasis is disrupted, promoting the nuclear translocation of the lysosomal transcription factor HLH-30/TFEB, and prompting cells to activate a compensatory transcriptional program. The three C. elegans granulin peptides exhibited distinct but overlapping functional effects in our assays, which may be due to amino acid composition that results in distinct electrostatic and hydrophobicity profiles. Our results support a model in which granulin production modulates a critical transition between the normal, physiological regulation of protease activity and the impairment of lysosomal function that can occur with age and disease.

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