Detection of Protein S‐Sulfhydration by a Tag‐Switch Technique

Zou, Dehui; Macinkovic, Igor; Devarie‐Baez, Nelmi O.; Pan, Jia; Park, Chung‐Min; Carroll, Kate S.; Filipovic, Milos R.; Xian, Ming

doi:10.1002/anie.201305876

Cited by 249 publications

(296 citation statements)

References 35 publications

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“…This assay is based on detection of protein gel mobility shifts upon reduction of persulfurated cysteine labelled with synthetic maleimide-peptide and was validated by direct identification of persulfide through mass spectrometry analysis. Compared with the other techniques described for identification of persulfide [46][47][48] , the peptidemaleimide assay provides additional advantages, including the small number of steps required before analysis, its selectivity and owing to its ratiometric nature it provides an accurate quantification of the ratio of persulfurated versus non-persulfurated species. Moreover, it is the first assay enabling determination of the number of persulfurated cysteine residues.…”

Section: Discussionmentioning

confidence: 99%

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

et al. 2015

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Friedreich's ataxia is a severe neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein that stimulates iron-sulfur (Fe-S) cluster biogenesis. In mammals, the primary steps of Fe-S cluster assembly are performed by the NFS1-ISD11-ISCU complex via the formation of a persulfide intermediate on NFS1. Here we show that frataxin modulates the reactivity of NFS1 persulfide with thiols. We use maleimide-peptide compounds along with mass spectrometry to probe cysteine-persulfide in NFS1 and ISCU. Our data reveal that in the presence of ISCU, frataxin enhances the rate of two similar reactions on NFS1 persulfide: sulfur transfer to ISCU leading to the accumulation of a persulfide on the cysteine C104 of ISCU, and sulfur transfer to small thiols such as DTT, L-cysteine and GSH leading to persulfuration of these thiols and ultimately sulfide release. These data raise important questions on the physiological mechanism of Fe-S cluster assembly and point to a unique function of frataxin as an enhancer of sulfur transfer within the NFS1-ISD11-ISCU complex.

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Section: Discussionmentioning

confidence: 99%

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

et al. 2015

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“…glyceraldehyde-3-phosphate dehydrogenase) in cell lysates and cells exposed to Na 2 S (91). In this proof-of-concept study, the authors also identified heat shock protein 70 as a novel target of protein S-sulfhydration (91). The probe-labeled proteins also can be captured and subject to MS-based proteomic analysis.…”

Section: The Expanding Landscape Of the Thiol Redox Proteomementioning

confidence: 97%

“…To overcome this limitation, Xian and colleagues recently developed a selective two-step approach to detect protein Ssulfhydration (Fig. 4E) (91,92). In the first step, nucleophilic protein thiol species (-SH and -SSH) are tagged with the electrophile, methylsulfonyl benzathiazole.…”

Section: The Expanding Landscape Of the Thiol Redox Proteomementioning

confidence: 99%

The Expanding Landscape of the Thiol Redox Proteome

Yang

Carroll

Liebler

2016

Molecular & Cellular Proteomics

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Cysteine occupies a unique place in protein chemistry. The nucleophilic thiol group allows cysteine to undergo a broad range of redox modifications beyond classical thiol-disulfide redox equilibria, including S-sulfenylation (-SOH), S-sulfinylation (-SO 2 H), S-sulfonylation (-SO 3 H), S-nitrosylation (-SNO), S-sulfhydration (-SSH), S-glutathionylation (-SSG), and others. Emerging evidence suggests that these post-translational modifications (PTM) are important in cellular redox regulation and protection against oxidative damage. Identification of protein targets of thiol redox modifications is crucial to understanding their roles in biology and disease. However, analysis of these highly labile and dynamic modifications poses challenges. Recent advances in the design of probes for thiol redox forms, together with innovative mass spectrometry based chemoproteomics methods make it possible to perform global, site-specific, and quantitative analyses of thiol redox modifications in complex proteomes.

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“…To detect protein persulfidation, a tagswitch assay was used as previously described (35,36). Briefly, protein extracts were prepared from either whole flies (three flies per sample, 100 μL extraction buffer) for persulfidation analysis of CSE overexpression effect under control of actin driver in a nondisease background or fly heads (25 heads per sample, 65 μL of extraction buffer) for persulfidation analysis of CSE overexpression effect under control of a glass multiple reporter (GMR) driver in the SCA3 background.…”

Section: Protein Persulfidation Assaymentioning

confidence: 99%

Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3

Snijder

Baratashvili

Grzeschik

et al. 2015

Mol Med

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Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.

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Detection of Protein S‐Sulfhydration by a Tag‐Switch Technique

Cited by 249 publications

References 35 publications

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

The Expanding Landscape of the Thiol Redox Proteome

Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3

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