Quantification of Protein Sulfenic Acid Modifications Using Isotope‐Coded Dimedone and Iododimedone

Seo, Young Ho; Carroll, Kate S.

doi:10.1002/ange.201007175

Cited by 22 publications

(20 citation statements)

References 23 publications

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“…After reaction of sulfenic acids with dimedone, the total mass should increase by +138 Da due to irreversible and selective condensation, which can be observed via mass spectrometry (34). Addition of dimedone to the air-sensitive species under normal physiological conditions did not result in any modification of the protein, probably due to a lack of access by the dimedone to the sulfenic group in the folded protein.…”

Section: Resultsmentioning

confidence: 96%

Copper–sulfenate complex from oxidation of a cavity mutant ofPseudomonas aeruginosaazurin

Sieracki

Tian

Hadt

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Metal-sulfenate centers are known to play important roles in biology and yet only limited examples are known due to their instability and high reactivity. Herein we report a copper-sulfenate complex characterized in a protein environment, formed at the active site of a cavity mutant of an electron transfer protein, type 1 blue copper azurin. Reaction of hydrogen peroxide with Cu(I)-M121G azurin resulted in a species with strong visible absorptions at 350 and 452 nm and a relatively low electron paramagnetic resonance g z value of 2.169 in comparison with other normal type 2 copper centers. The presence of a side-on copper-sulfenate species is supported by resonance Raman spectroscopy, electrospray mass spectrometry using isotopically enriched hydrogen peroxide, and density functional theory calculations correlated to the experimental data. In contrast, the reaction with Cu(II)-M121G or Zn(II)-M121G azurin under the same conditions did not result in Cys oxidation or copper-sulfenate formation. Structural and computational studies strongly suggest that the secondary coordination sphere noncovalent interactions are critical in stabilizing this highly reactive species, which can further react with oxygen to form a sulfinate and then a sulfonate species, as demonstrated by mass spectrometry. Engineering the electron transfer protein azurin into an active copper enzyme that forms a copper-sulfenate center and demonstrating the importance of noncovalent secondary sphere interactions in stabilizing it constitute important contributions toward the understanding of metal-sulfenate species in biological systems.bioinorganic chemistry | metalloprotein design | protein engineering | blue copper proteins | posttranslational modification T he presence of cysteine sulfenic acid (Cys-SOH) as a product of posttranslational oxidation of the cysteine thiol side chain has been found to play important roles in biology (1-6) in the context of metal coordination (7), enzyme-protein regulation (8, 9), redox signaling (1, 5), and gene regulation (10-13). Unlike its higher oxidation state counterparts, i.e., sulfinic (Cys-SO 2 ) and sulfonic (Cys-SO 3 ) acids, the sulfenic acid is inherently unstable and highly reactive and thus requires stabilization to operate in a controlled context in biological systems (1-5). For example, a crystal structure of a sulfenic acid form of SarZ, a redox active global transcriptional regulator in Staphylococcus aureus, revealed that cysteine sulfenic acid is stabilized through two hydrogen bonds with surrounding residues, and its reversible oxidation-reduction allows redox-mediated virulence regulation in S. aureus (13).Because of the inherent instability and high reactivity, few examples of cysteine sulfenic acid have been observed in metalbinding sites in proteins (7,14). Nature has evolved proteins to allow metal ions to coordinate sulfenic acids, resulting in interesting functions. For example, nitrile hydratase, a metalloenzyme which has long found utility in the industrial synthesis of acrylamide (15...

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Section: Resultsmentioning

confidence: 96%

Copper–sulfenate complex from oxidation of a cavity mutant ofPseudomonas aeruginosaazurin

Sieracki

Tian

Hadt

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Frozen human kidney and liver tissues from five individuals were rapidly fractionated into microsomal fractions in deaerated buffer and labeled using an isotope-coded ICDID strategy for relative quantitation ( Fig. 1) (32). After excising the 40-60 kDa P450 region and digesting the proteins with trypsin, we identified 347 modified proteins in the kidney microsomes at a 5% peptide false discovery rate (FDR) ( Table 2, Supplemental Table 2, Fig.…”

Section: Identification Of Sulfenylated Drug Metabolizing Enzymes In mentioning

confidence: 99%

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration

Albertolle

Phan

Pozzi

et al. 2018

Molecular & Cellular Proteomics

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2The abbreviations used are: CID, collision-induced dissociation; DTT, dithiothreitol; ER, endoplasmic reticulum; FDR, (peptide) false discovery rate; FMO, microsomal flavin-containing monooxygenase; HCD, higher energy collisional dissociation; HEPPS, 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonate; ICDID, isotope-coded dimedone/iododimedone (labeling); MAO, monoamine oxidase; P450 or CYP, cytochrome P450; TCEP, tris(carbethoxymethyl)phosphine;UGT, UDP-glucuronyl transferase. SUMMARYThe lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. et al. (2017) J. Biol. Chem. 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.) (INTRODUCTION)The endoplasmic reticulum (ER) is a site of protein translation, posttranslational processing, and small molecule metabolism (1). Posttranslational processing includes glycosylation of proteins for sorting, disulfide bond formation, and specific proteolytic cleavages.The ER lumen is an oxidizing environment that aids in the production of disulfide bonds, which is maintained at a homeostatic level by both small molecules (ascorbate) and proteins (protein disulfide isomerases) (2, 3). The cytosolic side of the ER remains a reducing environment to preserve the normal function of integral ER proteins.Cytochromes P450 (CYP, P450) are found in the cytoplasmic side of the ER and are most well-known for the ability to metabolize xenobiotics and important endogenous substrates (e.g., steroids and vitamins) (4). These proteins have been of interest in the pharmaceutical industry since their initial discovery, and enzymes in P450 Subfamilies 1A, 2C, 2D, and 3A are involved in the metabolism and clearance of a large majority of small molecule drugs currently approved for clinical use in humans (4). P450 regulation involves genetic and epigenetic aspects, as well as transcriptional regulation by bot...

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“…This is a very impressive number given the expected low abundance of this transient modification; however, no specific sites of modification were reported, leaving the identities of the reactive cysteines undefined. To determine the relative levels of sulfenic acid between samples, a d 0 and d 6 isotope coded pair of dimedone analog was recently reported (87).…”

Section: Oxmrm: Quantitative Cysteine Oxidation Analysis By Mrm-multimentioning

confidence: 99%

Regulatory Control or Oxidative Damage? Proteomic Approaches to Interrogate the Role of Cysteine Oxidation Status in Biological Processes

Held

Gibson

2012

Molecular & Cellular Proteomics

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Oxidation is a double-edged sword for cellular processes and its role in normal physiology, cancer and aging remains only partially understood. Although oxidative stress may disrupt biological function, oxidation-reduction (redox) reactions in a cell are often tightly regulated and play essential physiological roles. Cysteines lie at the interface between these extremes since the chemical properties that make specific thiols exquisitely redox-sensitive also predispose them to oxidative damage by reactive oxygen or nitrogen species during stress. Thus, these modifications can be either under reversible redox regulatory control or, alternatively, a result of reversible or irreversible oxidative damage. In either case, it has become increasingly important to assess the redox status of protein thiols since these modifications often impact such processes as catalytic activity, conformational alterations, or metal binding. To better understand the redox changes that accompany protein cysteine residues in complex biological systems, new experimental approaches have been developed to identify and characterize specific thiol modifications and/or changes in their overall redox status. In this review, we describe the recent technologies in redox proteomics that have pushed the boundaries for detecting and quantifying redox cysteine modifications in a cellular context. While there is no one-size-fits-all analytical solution, we highlight the rationale, strengths, and limitations of each technology in order to effectively apply them to specific biological questions. Several technological limitations still remain unsolved, however these approaches and future developments play an important role toward understanding the interplay between oxidative stress and redox signaling in health and disease. Molecular & Cellular Proteomics 11: 10.1074/mcp.R111.013037, 1-14, 2012. CYSTEINE: AN UNCOMMONLY REACTIVE AMINO ACIDThe nucleophilic sulfur atom allows cysteines to undergo a broad range of chemical modifications. These modifications include redox reactions, lipid acylation, and metal binding motifs that are important for protein structure, localization, regulation, and catalysis. Metal binding and oxidation play a role in protein structure through iron-sulfur (Fe-S) clusters, zinc fingers (ZF) 1 , and disulfide bonding, among others. Catalytic cysteines are essential to the function of numerous enzymes such as the E1 and E2 ligases of ubiquitin and ubiquitin-like proteins; the HECT domain of ubiquitin E3 ligases; SENP family sumo proteases; the tyrosine phosphatases protein phosphatase 1b (PTP1b) and PTEN; and many others including antioxidants in the thioredoxin, glutaredoxin, and peroxiredoxin families.Multiple thiol chemistries can converge to regulate the function of individual cysteines in a biological context. An example of the interconnection between the catalytic and redox properties of a cysteine is found in the cysteine-dependent aspartate-directed protease family of caspases. Essential to apoptosis, caspases are cysteine pr...

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Quantification of Protein Sulfenic Acid Modifications Using Isotope‐Coded Dimedone and Iododimedone

Cited by 22 publications

References 23 publications

Copper–sulfenate complex from oxidation of a cavity mutant ofPseudomonas aeruginosaazurin

Copper–sulfenate complex from oxidation of a cavity mutant ofPseudomonas aeruginosaazurin

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration

Regulatory Control or Oxidative Damage? Proteomic Approaches to Interrogate the Role of Cysteine Oxidation Status in Biological Processes

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