Cristina M. Furdui

Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species

Isoform-specific signaling of Akt, a major signaling hub and a prominent therapeutic target, remained poorly defined until recently. Subcellular distribution, tissue-specific expression, substrate specificity, and posttranslational modifications are believed to underlie isoform-specific signaling of Akt. The studies reported here show inhibition of Akt2 activity under physiologically relevant conditions of oxidation created by PDGF-induced reactive oxygen species. Combined MS and functional assays identified Cys124 located in the linker region between the N-terminal pleckstrin homology domain and the catalytic kinase domain as one of the unique regulatory redox sites in Akt2 with functional consequence on PDGF-stimulated glucose uptake. A model is proposed describing the consequence of increased endogenous oxidation induced by extracellular cues such as PDGF on Akt2 activity.

Use of dimedone-based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox-sensitive proteins.

Sulfenic acids, formed as transient intermediates during the reaction of cysteine residues with peroxides, play significant roles in enzyme catalysis and regulation, and are also involved in the redox regulation of transcription factors and other signaling proteins. Therefore, interest in the identification of protein sulfenic acids has grown substantially in the past few years. Dimedone, which specifically traps sulfenic acids, has provided the basis for the synthesis of a novel group of compounds that derivatize 1,3-cyclohexadione, a dimedone analogue, with reporter tags such as biotin for affinity capture and fluorescent labels for visual detection. These reagents allow identification of the cysteine sites and proteins that are sensitive to oxidation and permit identification of the cellular conditions under which such oxidations occur. We have shown that these compounds are reactive and specific toward sulfenic acids and that the labeled proteins can be detected at high sensitivity using gel analysis or mass spectrometry. Here, we further characterize these reagents, showing that the DCP-Bio1 incorporation rates into three sulfenic acid containing proteins, papaya papain, Escherichia coli fRMsr, and the Salmonella typhimurium peroxiredoxin AhpC, are significantly different and, in the case of fRMsr, are unaffected by changes in buffer pH from 5.5 and 8.0. We also provide protocols to label protein sulfenic acids in cellular proteins, either by in situ labeling of intact cells or by labeling at the time of lysis. We show that the addition of alkylating reagents and catalase to the lysis buffer is critical in preventing the formation of sulfenic acid subsequent to cell lysis. Data presented herein also indicate that the need to standardize, as much as possible, the protein and reagent concentrations during labeling. Finally, we introduce several new test or control proteins that can be used to evaluate labeling procedures and efficiencies.

The Precise Sequence of FGF Receptor Autophosphorylation Is Kinetically Driven and Is Disrupted by Oncogenic Mutations

The order of FGFR1 tyrosine autophosphorylation is kinetically controlled and determined by primary and tertiary structures. Is Timing Everything? Five tyrosine residues in the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1) undergo autophosphorylation, a process that both enhances its kinase activity and provides binding sites for downstream signaling molecules. Lew et al. investigated the mechanisms underlying this sequential and precisely ordered autophosphorylation and determined that it was under kinetic control, with the order of phosphorylation depending on the location of individual tyrosines within the primary and tertiary structures of the FGFR1 kinase domain. Intriguingly, the order in which these tyrosine residues underwent autophosphorylation was disrupted by a glioblastoma-derived, oncogenic FGFR1 point mutation. The authors postulate that such mutations may also alter the temporal recruitment of downstream signaling molecules and this may contribute to their oncogenic activity. Autophosphorylation of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1) is mediated by a sequential and precisely ordered three-stage autophosphorylation reaction. First-stage autophosphorylation of an activation loop tyrosine leads to 50- to 100-fold stimulation of kinase activity and is followed by second-stage phosphorylation of three additional tyrosine residues, which are binding sites for signaling molecules. Finally, third-stage phosphorylation of a second activation loop tyrosine leads to an additional 10-fold stimulation of FGFR1 catalytic activity. In this report, we show that sequential autophosphorylation of five tyrosines in the FGFR1 kinase domain is under kinetic control, mediated by both the amino acid sequence surrounding the tyrosines and their locations within the kinase structure, and, moreover, that phosphoryl transfer is the rate-limiting step. Furthermore, the strict order of autophosphorylation is disrupted by a glioblastoma-derived, oncogenic FGFR1 point mutation in the kinase domain. We propose that disrupted stepwise activation of tyrosine autophosphorylation caused by oncogenic and other activating FGFR mutations may lead to aberrant activation of and assembly of signaling molecules by the activated receptor.

Strained cycloalkynes as new protein sulfenic acid traps.

Protein sulfenic acids are formed by the reaction of biologically relevant reactive oxygen species with protein thiols. Sulfenic acid formation modulates the function of enzymes and transcription factors either directly or through the subsequent formation of protein disulfide bonds. Identifying the site, timing, and conditions of protein sulfenic acid formation remains crucial to understanding cellular redox regulation. Current methods for trapping and analyzing sulfenic acids involve the use of dimedone and other nucleophilic 1,3-dicarbonyl probes that form covalent adducts with cysteine-derived protein sulfenic acids. As a mechanistic alternative, the present study describes highly strained bicyclo[6.1.0]nonyne (BCN) derivatives as concerted traps of sulfenic acids. These strained cycloalkynes react efficiently with sulfenic acids in proteins and small molecules yielding stable alkenyl sulfoxide products at rates more than 100× greater than 1,3-dicarbonyl reagents enabling kinetic competition with physiological sulfur chemistry. Similar to the 1,3-dicarbonyl reagents, the BCN compounds distinguish the sulfenic acid oxoform from the thiol, disulfide, sulfinic acid, and S-nitrosated forms of cysteine while displaying an acceptable cell toxicity profile. The enhanced rates demonstrated by these strained alkynes identify them as new bioorthogonal probes that should facilitate the discovery of previously unknown sulfenic acid sites and their parent proteins.

Chemical approaches to detect and analyze protein sulfenic acids

Orchestration of many processes relying on intracellular signal transduction is recognized to require the generation of hydrogen peroxide as a second messenger, yet relatively few molecular details of how this oxidant acts to regulate protein function are currently understood. This review describes emerging chemical tools and approaches that can be applied to study protein oxidation in biological systems, with a particular emphasis on a key player in protein redox regulation, cysteine sulfenic acid. While sulfenic acids (within purified proteins or simple mixtures) are detectable by physical approaches like X-ray crystallography, nuclear magnetic resonance and mass spectrometry, the propensity of these moieties to undergo further modification in complex biological systems has necessitated the development of chemical probes, reporter groups and analytical approaches to allow for their selective detection and quantification. Provided is an overview of techniques that are currently available for the study of sulfenic acids, and some of the biologically meaningful data that have been collected using such approaches.

Water-soluble triarylphosphines as biomarkers for protein S-nitrosation.

S-Nitrosothiols (RSNOs) represent an important class of post-translational modifications that preserve and amplify the actions of nitric oxide and regulate enzyme activity. Several regulatory proteins are now verified targets of cellular S-nitrosation, and the direct detection of S-nitrosated residues in proteins has become essential to better understand RSNO-mediated signaling. Current RSNO detection depends on indirect assays that limit their overall specificity and reliability. Herein, we report the reaction of S-nitrosated cysteine, glutathione, and a mutated C165S alkyl hydroperoxide reductase with the water-soluble phosphine tris(4,6-dimethyl-3-sulfonatophenyl)phosphine trisodium salt hydrate (TXPTS). A combination of NMR and MS techniques reveals that these reactions produce covalent S-alkylphosphonium ion adducts (with S-P(+) connectivity), TXPTS oxide, and a TXPTS-derived aza-ylide. Mechanistically, this reaction may proceed through an S-substituted aza-ylide or the direct displacement of nitroxyl from the RSNO group. This work provides a new means for detecting and quantifying S-nitrosated species in solution and suggests that phosphines may be useful tools for understanding the complex physiological roles of S-nitrosation and its implications in cell signaling and homeostasis.

Identification of Intact Protein Thiosulfinate Intermediate in the Reduction of Cysteine Sulfinic Acid in Peroxiredoxin by Human Sulfiredoxin

The reversible oxidation of the active site cysteine in typical 2-Cys peroxiredoxins (Prx) to sulfinic acid during oxidative stress plays an important role in peroxide-mediated cell signaling. The catalytic retroreduction of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Prx}\mathrm{-}\mathrm{SO}_{2}^{-}\) \end{document} by sulfiredoxin (Srx) has been proposed to proceed through two novel reaction intermediates, a sulfinic phosphoryl ester and protein-based thiosulfinate. Two scenarios for the repair mechanism have been suggested that differ in the second step of the reaction. The attack of Srx or GSH on the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Prx}\mathrm{-}\mathrm{SO}_{2}\mathrm{PO}_{3}^{2-}\) \end{document} intermediate would result in either the formation of Prx-Cys-S(=O)–S-Cys-Srx or the formation of Prx-Cys-S(=O)–S-G thiosulfinates, respectively. To elucidate the mechanism of Prx repair, we monitored the reduction of human \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{PrxII}\mathrm{-}\mathrm{SO}_{2}^{-}\) \end{document} using rapid chemical quench methodology and electrospray ionization time-of-flight mass spectrometry. An 18O exchange study revealed that the Prx sulfinic acid phosphoryl ester is rapidly formed and hydrolyzed (k = 0.35 min–1). Furthermore, we observed the exclusive formation of a thiosulfinate linkage between Prx and Srx (k = 1.4 min–1) that collapses to the disulfide-bonded Srx-Prx species (k = 0.14 min–1). Thus, the kinetic and chemical competences of the first two steps in the Srx reaction have been demonstrated. It is clear, however, that GSH may influence thiosulfinate formation and that GSH and Srx may play additional roles in the resolution of the thiosulfinate intermediate.

A simple and effective strategy for labeling cysteine sulfenic acid in proteins by utilization of β-ketoesters as cleavable probes

β-ketoesters are robust probes for labeling sulfenic acid (-SOH) proteins allowing quantitative cleavage of the tag for improved analysis of the labeled peptides by MS.

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Background: Human 2-Cys peroxiredoxins (Prxs) are differentially susceptible to inactivation by H2O2. Results: Engineered Prx2 and Prx3 variants demonstrate that C-terminal residues modulate the extent of hyperoxidation. Conclusion: Rapid disulfide bond formation protects Prx3 from inactivation. Significance: The reactivity of Prx3 with H2O2 is important for understanding its protective role in the mitochondria. Peroxiredoxins (Prxs) detoxify peroxides and modulate H2O2-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1–4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H2O2 to form Cys sulfinic acid (Cys-SO2H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H2O2 and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.

Thiol‐blocking electrophiles interfere with labeling and detection of protein sulfenic acids

Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post‐translational process that is implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag‐switch techniques. Common to both approaches is chemical blocking of free thiols using reactive electrophiles to prevent post‐lysis oxidation or other thiol‐mediated cross‐reactions. These reagents are used in large excess, and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol‐blocking electrophiles, iodoacetamide, N‐ethylmaleimide and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine ‐SOR (product) species are partially or fully susceptible to reduction by dithiothreitol, tris(2‐carboxyethyl)phosphine and ascorbate, regenerating protein thiols, or, in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S‐nitrosothiols are discussed.