In-silico structural characterization and phylogenetic analysis of Nucleoside diphosphate kinase: A novel antiapoptotic protein of Porphyromonas gingivalis.

The Nucleoside diphosphate kinase (NDK) protein of Porphyromonas gingivalis (P. gingivalis) plays a crucial role in immune evasion and inhibition of apoptosis in host cells and has the potential to cause cancer. However, its structure has not yet been characterized. We used an in-silico approach to determine the 3D structure of the P. gingivalis NDK. Furthermore, structural characterization and functional annotation were performed using computational approaches. The 3D structure of NDK was predicted through homology modeling. The structural domains predicted for the model protein belong to the NDK family. Structural alignment of prokaryotic and eukaryotic NDKs with the model protein revealed the conservation of the domain region. Structure-based phylogenetic analysis depicted a significant evolutionary relationship between the model protein and the prokaryotic NDK. Functional annotation of the model confirmed structural homology, exhibiting similar enzymatic functions as NDK, including ATP binding and nucleoside diphosphate kinase activity. Furthermore, molecular dynamic (MD) simulation technique stabilized the model structure and provides a thermo-stable protein structure that can be used as a therapeutic target for further studies.

The Mechanism of Action of Lactoferrin - Nucleoside Diphosphate Kinase Complex in Combating Biofilm Formation.

BACKGROUND: The ESKAPE group of pathogens which comprise of multidrug resistant bacteria, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species are the cause of deadly nosocomial infections all over the world. While these pathogens have developed robust strategies to resist most antibiotics, their ability to form biofilms is one of their most combative properties. Hence there is an urgent need to discover new antibacterial agents which could prevent or destroy the biofilms made by these bacteria. Though it has been established that lactoferrin (LF), a potent iron binding antibacterial, antifungal, and antiviral protein displays anti-biofilm properties, its mechanisms of action, in addition to its iron chelation property, still remains unclear. OBJECTIVE: The binding and inhibition studies of LF with the enzyme Nucleoside diphosphate Kinase (NDK) and its elastase cleaved truncated 12 kDa fragment (12-NDK). METHODS: The characterization studies of NDK and 12-NDK using florescence spectroscopy, dynamic light scattering, size exclusion chromatography and ADP-glo Kinase Assay. Inhibition studies of LF-NDK using ADP-glo kinase assay, Surface Plasmon Resonance and Biofilm inhibition studies. RESULTS: NDK and 12-NDK were cloned, expressed and purified from Acinetobacter baumannii and Pseudomonas aeruginosa. The characterization studies revealed NDK and 12-NDK from both species are stable and functional. The inhibition studies of LF-NDK revealed stable binding and inhibition of kinase activity by LF. CONCLUSION: The binding and inhibition studies have shown that while LF binds with both the NDK and their truncated forms, it tends to have a higher binding affinity with the truncated 12 kDa fragments, resulting in their decreased kinase activity. This study essentially gives a new direction to the field of inhibition of biofilm formation, as it proves that LF has a novel mechanism of action in other than iron sequestration.

Crystal structure and characterization of nucleoside diphosphate kinase from Vibrio cholerae.

Nucleoside diphosphate kinases (NDK) are ubiquitous enzymes that catalyse the transfer of the γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphate (NDPs), to maintain appropriate NTP levels in cells. NDKs are associated with signal transduction, cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility. The critical role of NDK in bacterial virulence renders it a potential drug target. The present manuscript reports crystal structure and functional characterization of Vibrio cholerae NDK (VNDK). The 16 kDa VNDK was crystallized in a solution containing 30% PEG 4000, 100 mM Tris-HCl pH 8.5 and 200 mM sodium acetate in orthorhombic space group P212121 with unit cell parameters a = 48.37, b = 71.21, c = 89.14 Å, α = ß = Î³ = 90° with 2 molecules in asymmetric unit. The crystal structure was solved by molecular replacement and refined to crystallographic Rfactor and Rfree values of 22.8% and 25.8% respectively. VNDK exists as both dimer and tetramer in solution as confirmed by size exclusion chromatography, glutaraldehyde crosslinking and small angle X-ray scattering while the crystal structure appears to be a dimer. The biophysical characterization states that VNDK has kinase and DNase activity with maximum stability at pH 8-9 and temperature up to 40 °C. VNDK shows elevated thermolability as compared to other NDK and shows preferential binding with GTP rationalized using computational studies.

Structure, Folding and Stability of Nucleoside Diphosphate Kinases.

Nucleoside diphosphate kinases (NDPK) are oligomeric proteins involved in the synthesis of nucleoside triphosphates. Their tridimensional structure has been solved by X-ray crystallography and shows that individual subunits present a conserved ferredoxin fold of about 140 residues in prokaryotes, archaea, eukaryotes and viruses. Monomers are functionally independent from each other inside NDPK complexes and the nucleoside kinase catalytic mechanism involves transient phosphorylation of the conserved catalytic histidine. To be active, monomers must assemble into conserved head to tail dimers, which further assemble into hexamers or tetramers. The interfaces between these oligomeric states are very different but, surprisingly, the assembly structure barely affects the catalytic efficiency of the enzyme. While it has been shown that assembly into hexamers induces full formation of the catalytic site and stabilizes the complex, it is unclear why assembly into tetramers is required for function. Several additional activities have been revealed for NDPK, especially in metastasis spreading, cytoskeleton dynamics, DNA binding and membrane remodeling. However, we still lack the high resolution structural data of NDPK in complex with different partners, which is necessary for deciphering the mechanism of these diverse functions. In this review we discuss advances in the structure, folding and stability of NDPKs.

Inosine 5'-diphosphate, a molecular decoy rescues Nucleoside diphosphate kinase from c-MYC G-Quadruplex unfolding.

BACKGROUND: The transcription-inhibitory G-Quadruplex(Pu27-GQ) at c-MYC promoter is challenging to target due to structural heterogeneity. Nucleoside diphosphate kinase (NM23-H2) specifically binds and unfolds Pu27-GQ to increase c-MYC transcription. Here, we used Inosine 5'-diphosphate (IDP) to disrupt NM23-H2-Pu27-GQ interactions and arrest c-MYC transcription without compromising NM23-H2-mediated kinase properties. METHODS: Site-directed mutagenesis,31P-NMR and STD-NMR studies delineate the epitope of NM23-H2-IDP complex and characterize specific amino acids in NM23-H2 involved in Pu27-GQ and IDP interactions. Immunoprecipitations and phosphohistidine-immunoblots reveal how IDP blocks NM23-H2-Pu27 association to downregulate c-MYC transcription in MDAMB-231 cells exempting NM23-H2-mediated kinase properties. RESULTS: NMR studies show that IDP binds to the Guanosine diphosphate-binding pocket of NM23-H2 (KD = 5.0 ± 0.276 µM). Arg88-driven hydrogen bonds to the terminal phosphate of IDP restricts P-O-P bond-rotation increasing its pKa (∆pKa = 0.85 ± 0.0025).9-inosinyl moiety of IDP is stacked over Phe60 phenyl ring driving trans-conformation of inosine and axial geometry of pyrophosphates. Chromatin immunoprecipitations revealed that these interactions rescue NM23-H2-driven Pu27-GQ unfolding, which triggers Nucleolin recruitment and lowers Sp1 occupancy at c-MYC promoter stabilizing Pu27-GQ. This silences c-MYC transcription that reduces c-MYC-Sp1 association amplifying Sp1 recruitment across P21 promoter stimulating P21 transcription and G2/M arrest. CONCLUSIONS: IDP synergizes the effects of Pu27-GQ-interacting compounds to abrogate c-MYC transcription and induce apoptosis in MDAMB-231 cells by disrupting NM23-H2-Pu27-GQ interactions without affecting NM23-H2-mediated kinase properties. GENERAL SIGNIFICANCE: Our study provides a pragmatic approach for developing NM23-H2-targeting regulators to rescue NM23-H2 binding at structurally ambiguous Pu27-GQ that synergizes the anti-tumorigenic effects of GQ-based therapeutics with minimized off-target effects.

Characterization of a Schistosoma mansoni NDPK expressed in sexual and digestive organs.

Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.

Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases by Flow Injection Analysis with High-Resolution Mass Spectrometry.

Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.

Structure and analysis of nucleoside diphosphate kinase from Borrelia burgdorferi prepared in a transition-state complex with ADP and vanadate moieties.

Nucleoside diphosphate kinases (NDKs) are implicated in a wide variety of cellular functions owing to their enzymatic conversion of NDP to NTP. NDK from Borrelia burgdorferi (BbNDK) was selected for functional and structural analysis to determine whether its activity is required for infection and to assess its potential for therapeutic inhibition. The Seattle Structural Genomics Center for Infectious Diseases (SSGCID) expressed recombinant BbNDK protein. The protein was crystallized and structures were solved of both the apoenzyme and a liganded form with ADP and vanadate ligands. This provided two structures and allowed the elucidation of changes between the apo and ligand-bound enzymes. Infectivity studies with ndk transposon mutants demonstrated that NDK function was important for establishing a robust infection in mice, and provided a rationale for therapeutic targeting of BbNDK. The protein structure was compared with other NDK structures found in the Protein Data Bank and was found to have similar primary, secondary, tertiary and quaternary structures, with conserved residues acting as the catalytic pocket, primarily using His132 as the phosphohistidine-transfer residue. Vanadate and ADP complexes model the transition state of this phosphoryl-transfer reaction, demonstrating that the pocket closes when bound to ADP, while allowing the addition or removal of a γ-phosphate. This analysis provides a framework for the design of potential therapeutics targeting BbNDK inhibition.

Two-Step Membrane Binding of NDPK-B Induces Membrane Fluidity Decrease and Changes in Lipid Lateral Organization and Protein Cluster Formation.

Nucleoside diphosphate kinases (NDPKs) are crucial elements in a wide array of cellular physiological or pathophysiological processes such as apoptosis, proliferation, or metastasis formation. Among the NDPK isoenzymes, NDPK-B, a cytoplasmic protein, was reported to be associated with several biological membranes such as plasma or endoplasmic reticulum membranes. Using several membrane models (liposomes, lipid monolayers, and supported lipid bilayers) associated with biophysical approaches, we show that lipid membrane binding occurs in a two-step process: first, initiation by a strong electrostatic adsorption process and followed by shallow penetration of the protein within the membrane. The NDPK-B binding leads to a decrease in membrane fluidity and formation of protein patches. The ability of NDPK-B to form microdomains at the membrane level may be related to protein-protein interactions triggered by its association with anionic phospholipids. Such accumulation of NDPK-B would amplify its effects in functional platform formation and protein recruitment at the membrane.

Biophysical Characterization of the Nucleoside Diphosphate Kinase of Leishmania major and Effect of the P95S Mutation.

Nucleoside diphosphate kinases (NDK; EC 2.7.4.6) are enzymes required for maintaining intracellular levels of nucleosides triphosphates (NTP) through transfer the γ-phosphoryl group from a NTP to a NDP. The enzyme is associated with several biological functions including prevention of host ATP-mediated cytolysis during pathogenic infections. Here we present the biophysical characterization of NDK from Leishmania major and the effect of a mutation on the protein structure in solution. The structural stability was analyzed since this secreted protein may act in different microenvironments at various stages of the parasite life cycle. LmNDK and P95S mutant were subjected to denaturation with pH and guanidine. Structural transitions were monitored by circular dichroism and intrinsic fluorescence tryptophan emission. Our results showed that the LmNDK is more structurally stable than other described NDKs and that the catalytically active P95S mutant in the Kpn loop presented a decrease in protein stability, indicating the importance of this proline for maintenance of the LmNDK structure.

In vivo protein cross-linking.

In the cell, homo- and hetero-associations of polypeptide chains evolve and take place within subcellular compartments that are crowded with many other cellular macromolecules. In vivo chemical cross-linking of proteins is a powerful method to examine changes in protein oligomerization and protein-protein interactions upon cellular events such as signal transduction. This chapter is intended to provide a guide for the selection of cell membrane permeable cross-linkers, the optimization of in vivo cross-linking conditions, and the identification of specific cross-links in a cellular context where the frequency of random collisions is high. By combining the chemoselectivity of the homo-bifunctional cross-linker and the length of its spacer arm with knowledge on the protein structure, we show that selective cross-links can be introduced specifically on either the dimer or the hexamer form of the same polypeptide in vitro as well as in vivo, using the human type B nucleoside diphosphate kinase as a protein model.

Structural comparison of highly similar nucleoside-diphosphate kinases: Molecular explanation of distinct membrane-binding behavior.

NDPK-A, NDPK-B and NDPK-D are three enzymes which belong to the NDPK group I isoforms and are not only involved in metabolism process but also in transcriptional regulation, DNA cleavage, histidine protein kinase activity and metastasis development. Those enzymes were reported to bind to membranes either in mitochondria where NDPK-D influences cardiolipin lateral organization and is thought to be involved in apoptotic pathway or in cytosol where NDPK-A and NDPK-B membrane association was shown to influence several cellular processes like endocytosis, cellular adhesion, ion transport, etc. However, despite numerous studies, the role of NDPK-membrane association and the molecular details of the binding process are still elusive. In the present work, a comparative study of the three NDPK isoforms allowed us to show that although membrane binding is a common feature of these enzymes, mechanisms differ at the molecular scale. NDPK-A was not able to bind to model membranes mimicking the inner leaflet of plasma membrane, suggesting that its in vivo membrane association is mediated by a non-lipidic partner or other partners than the studied phospholipids. On the contrary, NDPK-B and NDPK-D were shown to bind efficiently to liposomes mimicking plasma membrane and mitochondrial inner membrane respectively but details of the binding mechanism differ between the two enzymes as NDPK-B binding necessarily involved an anionic phospholipid partner while NDPK-D can bind either zwitterionic or anionic phospholipids. Although sharing similar secondary structure and homohexameric quaternary arrangement, tryptophan fluorescence revealed fine disparities in NDPK tertiary structures. Interfacial behavior as well as ANS fluorescence showed further dissimilarities between NDPK isoforms, notably the presence of distinct accessible hydrophobic areas as well as different capacity to form Gibbs monolayers related to their surface activity properties. Those distinct features may contribute to explain the differences in the protein behavior towards membrane binding.

Elimination and utilization of oxidized guanine nucleotides in the synthesis of RNA and its precursors.

Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and by the oxidation of GTP can be used for RNA synthesis. This misincorporation is prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well as 8-oxo-GDP to 8-oxo-GMP. When (14)C-labeled 8-oxo-GTP was applied to CaCl2-permeabilized cells of a mutT(-) mutant strain, it could be incorporated into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA.

Protein structure prediction using global optimization by basin-hopping with NMR shift restraints.

Computational methods that utilize chemical shifts to produce protein structures at atomic resolution have recently been introduced. In the current work, we exploit chemical shifts by combining the basin-hopping approach to global optimization with chemical shift restraints using a penalty function. For three peptides, we demonstrate that this approach allows us to find near-native structures from fully extended structures within 10,000 basin-hopping steps. The effect of adding chemical shift restraints is that the α and ß secondary structure elements form within 1000 basin-hopping steps, after which the orientation of the secondary structure elements, which produces the tertiary contacts, is driven by the underlying protein force field. We further show that our chemical shift-restraint BH approach also works for incomplete chemical shift assignments, where the information from only one chemical shift type is considered. For the proper implementation of chemical shift restraints in the basin-hopping approach, we determined the optimal weight of the chemical shift penalty energy with respect to the CHARMM force field in conjunction with the FACTS solvation model employed in this study. In order to speed up the local energy minimization procedure, we developed a function, which continuously decreases the width of the chemical shift penalty function as the minimization progresses. We conclude that the basin-hopping approach with chemical shift restraints is a promising method for protein structure prediction.

Structural and docking studies of a nucleoside diphosphate kinase 1 (CsNDPK1) from tea [Camellia sinensis (L.) O. Kuntze].

Nucleoside diphosphate kinase (NDPK, EC 2.7.4.6) is a housekeeping gene, which functions in the general homeostasis of cellular nucleoside triphosphate (NTP) pools. Among the various NDPK isoforms, cytosolic NDPK1 has been shown to be the main NDPK isoform in plants, accounting for more than 70 % of total NDPK activity in plants. For the first time, a full-length cDNA (697 bp), designated as CsNDPK1 was cloned from tea leaves and consisted of a 448-bp open reading frame (ORF) encoding a 147-amino-acid polypeptide with calculated molecular mass of 16.1 kDa and a pI of 6.3. Homology modeling of CsNDPK1 shows that the presented tea NDPK1 also contains several motifs, binding and catalytic sites which are highly conserved among other NDPKs. Docking studies of CsNDPK1 with its substrates (NTPs) are discussed in detail. In summary, we describe a reliable model of CsNDPK1 that can be used in structure-based protein-protein interaction studies for identifying its potential role in intracellular communication and its physiological significance in tea.

An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of Aquifex aeolicus.

The nucleoside diphosphate kinase (Ndk) catalyzes the reversible transfer of the γ-phosphate from nucleoside triphosphate to nucleoside diphosphate. Ndks form hexamers or two types of tetramers made of the same building block, namely, the common dimer. The secondary interfaces of the Type I tetramer found in Myxococcus xanthus Ndk and of the Type II found in Escherichia coli Ndk involve the opposite sides of subunits. Up to now, the few available structures of Ndk from thermophiles were hexameric. Here, we determined the X-ray structures of four crystal forms of the Ndk from the hyperthermophilic bacterium Aquifex aeolicus (Aa-Ndk). Aa-Ndk displays numerous features of thermostable proteins and is made of the common dimer but it is a tetramer of Type I. Indeed, the insertion of three residues in a surface-exposed spiral loop, named the Kpn-loop, leads to the formation of a two-turn α-helix that prevents both hexamer and Type II tetramer assembly. Moreover, the side chain of the cysteine at position 133, which is not present in other Ndk sequences, adopts two alternate conformations. Through the secondary interface, each one forms a disulfide bridge with the equivalent Cys133 from the neighboring subunit. This disulfide bridge was progressively broken during X-ray data collection by radiation damage. Such crosslinks counterbalance the weakness of the common-dimer interface. A 40% decrease of the kinase activity at 60°C after reduction and alkylation of the protein corroborates the structural relevance of the disulfide bridge on the tetramer assembly and enzymatic function.

Physiological and biochemical characterization of three nucleoside diphosphate kinase isozymes from rice (Oryza sativa L.).

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. In this study, we examined the subcellular localization, tissue-specific gene expression, and enzymatic characteristics of three rice NDPK isozymes (OsNDPK1-OsNDPK3). Sequence comparison of the three OsNDPKs suggested differential subcellular localization. Transient expression of green fluorescence protein-fused proteins in onion cells indicated that OsNDPK2 and OsNDPK3 are localized to plastid and mitochondria respectively, while OsNDPK1 is localized to the cytosol. Expression analysis indicated that all the OsNDPKs are expressed in the leaf, leaf sheath, and immature seeds, except for OsNDPK1, in the leaf sheath. Recombinant OsNDPK2 and OsNDPK3 showed lower optimum pH and higher stability under acidic pH than OsNDPK1. In ATP formation, all the OsNDPKs displayed lower K(m) values for the second substrate, ADP, than for the first substrate, NTP, and showed lowest and highest K(m) values for GTP and CTP respectively.

Synthesis of an NDPK phosphocarrier domain peptide containing a novel triazolylalanine analogue of phosphohistidine using click chemistry.

Click phosphorylation of a propargylated unprotected peptide and phosphoryl azide using chaotrope-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition enabled a high-yielding and rapid synthesis of a nucleoside diphosphate kinase (NDPK) phosphocarrier domain. The synthesis showcases a valuable synthetic platform for the synthesis of biologically relevant phosphopeptide analogues.

Novel oxidative modifications in redox-active cysteine residues.

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.