Dataset Information

Site-Specific Proteomic Mapping Identifies Selectively Modified Regulatory Cysteine Residues in Functionally Distinct Protein Networks.

ABSTRACT: S-Acylation, S-glutathionylation, S-nitrosylation, and S-sulfenylation are prominent, chemically distinct modifications that regulate protein function, redox sensing, and trafficking. Although the biological significance of these modifications is increasingly appreciated, their integration in the proteome remains unknown. Novel mass spectrometry-based technologies identified 2,596 predominately unique sites in 1,319 mouse liver proteins under physiological conditions. Structural analysis localized the modifications in unique, evolutionary conserved protein segments, outside commonly annotated functional regions. Contrary to expectations, propensity for modification did not correlate with biophysical properties that regulate cysteine reactivity. However, the in vivo chemical reactivity is fine-tuned for specificity, demonstrated by the nominal complementation between the four modifications and quantitative proteomics which showed that a reduction in S-nitrosylation is not correlated with increased S-glutathionylation. A comprehensive survey uncovered clustering of modifications within biologically related protein networks. The data provide the first evidence for the occurrence of distinct, endogenous protein networks that undergo redox signaling through specific cysteine modifications.

SUBMITTER: Gould NS

PROVIDER: S-EPMC4515171 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Site-Specific Proteomic Mapping Identifies Selectively Modified Regulatory Cysteine Residues in Functionally Distinct Protein Networks.

Gould Neal S NS Evans Perry P Martínez-Acedo Pablo P Marino Stefano M SM Gladyshev Vadim N VN Carroll Kate S KS Ischiropoulos Harry H

Chemistry & biology 20150709 7

S-Acylation, S-glutathionylation, S-nitrosylation, and S-sulfenylation are prominent, chemically distinct modifications that regulate protein function, redox sensing, and trafficking. Although the biological significance of these modifications is increasingly appreciated, their integration in the proteome remains unknown. Novel mass spectrometry-based technologies identified 2,596 predominately unique sites in 1,319 mouse liver proteins under physiological conditions. Structural analysis localiz ...[more]

PMID: 26165157

Similar Datasets

Project description:Persulfide dioxygenases (PDOs) are abundant in Bacteria and also crucial for H2S detoxification in mitochondria. One of the two pdo-genes of the acidophilic bacterium Acidithiobacillus caldus was expressed in Escherichia coli. The protein (AcPDO) had 0.77 ± 0.1 Fe/subunit and an average specific sulfite formation activity of 111.5 U/mg protein (Vmax) at 40°C and pH 7.5 with sulfur and GSH following Michaelis-Menten kinetics. KM for GSH and Kcat were 0.5 mM and 181 s-1, respectively. Glutathione persulfide (GSSH) as substrate gave a sigmoidal curve with a Vmax of 122.3 U/mg protein, a Kcat of 198 s-1 and a Hill coefficient of 2.3 ± 0.22 suggesting positive cooperativity. Gel permeation chromatography and non-denaturing gels showed mostly tetramers. The temperature optimum was 40-45°C, the melting point 63 ± 1.3°C in thermal unfolding experiments, whereas low activity was measurable up to 95°C. Site-directed mutagenesis showed that residues located in the predicted GSH/GSSH binding site and in the central hydrogen bond networks including the iron ligands are essential for activity. Among these, the R139A, D141A, and H171A variants were inactive concomitant to a decrease of their melting points by 3-8 K. Other variants were inactivated without significant melting point change. Two out of five cysteines are likewise essential, both of which lie presumably in close proximity at the surface of the protein (C87 and C224). MalPEG labeling experiments suggests that they form a disulfide bridge. The reducing agent Tris(2-carboxyethyl)phosphine was inhibitory besides N-ethylmaleimide and iodoacetamide suggesting an involvement of cysteines and the disulfide in catalysis and/or protein stabilization. Mass spectrometry revealed modification of C87, C137, and C224 by 305 mass units equivalent to GSH after incubation with GSSH and with GSH in case of the C87A and C224A variants. The results of this study suggest that disulfide formation between the two essential surface-exposed cysteines and Cys-S-glutathionylation serve as a protective mechanism against uncontrolled thiol oxidation and the associated loss of enzyme activity.

Project description:Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5'-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

Dataset Information

Site-Specific Proteomic Mapping Identifies Selectively Modified Regulatory Cysteine Residues in Functionally Distinct Protein Networks.

Publications

Site-Specific Proteomic Mapping Identifies Selectively Modified Regulatory Cysteine Residues in Functionally Distinct Protein Networks.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets