Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides

Reaction of radicals in the presence of O2, and singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, and that this can result in inactivation of thiol-dependent enzymes. The major route for the cellular removal of damaged proteins is via catabolism mediated by proteosomal and lysosomal pathways; cysteine proteases (cathepsins) play a key role in the latter system. We hypothesized that inactivation of cysteine proteases by hydroperoxide-containing oxidised proteins may contribute to the accumulation of modified proteins within cells. We show here that thiol-dependent cathepsins, either isolated or in cell lysates, are rapidly and efficiently inactivated by amino acid, peptide, and protein hydroperoxides in a time- and concentration-dependent manner; this occurs with similar efficacy to equimolar H2O2. Inactivation involves reaction of the hydroperoxide with Cys residues as evidenced by thiol loss and formation of sulfenic acid intermediates. Structurally related, non-thiol-dependent cathepsins are less readily inactivated by these hydroperoxides. This inhibition, by oxidized proteins, of the system designed to remove modified proteins, may contribute to the accumulation of damaged proteins in cells subject to oxidative stress.     

Amino acid, peptide, and protein hydroperoxides and their decomposition products modify the activity of the 26S proteasome

Proteins are major biological targets for oxidative damage within cells because of their high abundance and rapid rates of reaction with radicals and singlet oxygen. These reactions generate high yields of hydroperoxides. The turnover of both native and modified/damaged proteins is critical for maintaining cell homeostasis, with this occurring via the proteasomal and endosomal-lysosomal systems; the former is of particular importance for intracellular proteins. In this study we have examined whether oxidation products generated on amino acids, peptides, and proteins modulate 26S proteasome activity. We show that oxidation products, and particularly protein hydroperoxides, are efficient inhibitors of the 26S proteasome tryptic and chymotryptic activities, with this depending, at least in part, on the presence of hydroperoxide groups. Removal of these species by reduction significantly reduces proteasome inhibition. This loss of activity is accompanied by a loss of thiol residues, but an absence of radical formation, consistent with molecular, rather than radical, reactions being responsible for proteasome inhibition. Aldehydes also seem to play a role in the inhibition of chymotryptic activity, with this prevented by treatment with NaBH(4), which reduces these groups. Inhibition occurred at hydroperoxide concentrations of ≥1μM for oxidized amino acids and peptides and ≥10μM for oxidized proteins, compared with ca. 100μM for H(2)O(2), indicating that H(2)O(2) is a much less effective inhibitor. These data indicate that the formation of oxidized proteins within cells may modulate cell function by interfering with the turnover of native proteins and the clearance of modified materials.     

Modulating protein activity and cellular function by methionine residue oxidation

The sulfur-containing amino acid residue methionine (Met) in a peptide/protein is readily oxidized to methionine sulfoxide [Met(O)] by reactive oxygen species both in vitro and in vivo. Methionine residue oxidation by oxidants is found in an accumulating number of important proteins. Met sulfoxidation activates calcium/calmodulin-dependent protein kinase II and the large conductance calcium-activated potassium channels, delays inactivation of the Shaker potassium channel ShC/B and L-type voltage-dependent calcium channels. Sulfoxidation at critical Met residues inhibits fibrillation of atherosclerosis-related apolipoproteins and multiple neurodegenerative disease-related proteins, such as amyloid beta, α-synuclein, prion, and others. Methionine residue oxidation is also correlated with marked changes in cellular activities. Controlled key methionine residue oxidation may be used as an oxi-genetics tool to dissect specific protein function in situ.     

Redox regulation of the protein tyrosine phosphatase PTP1B in cancer cells

The oxidation and inactivation of protein tyrosine phosphatases is one mechanism by which reactive oxygen species influence tyrosine phosphorylation-dependent signaling events and exert their biological functions. In the present study, we determined the redox status of endogenous protein tyrosine phosphatases in HepG2 and A431 human cancer cells, in which reactive oxygen species are produced constitutively. We used mass spectrometry to assess the state of oxidation of the catalytic cysteine residue of endogenous PTP1B and show that this residue underwent both reversible and irreversible oxidation to high stoichiometry in response to intrinsic reactive oxygen species production. In addition, our data show that the oxidation of PTP1B is specific to the active site Cys, with the other Cys residues in the protein remaining in a reduced state. Treatment of these cells with diphenyleniodonium, an inhibitor of NADPH oxidases, decreased reactive oxygen species levels. This resulted in inhibition of protein tyrosine phosphatase oxidation, concomitant with decreased tyrosine phosphorylation of cellular proteins and inhibition of anchorage-independent cell growth. Therefore, our data also suggest that the high level of intrinsic reactive oxygen species may contribute to the transformed phenotype of HepG2 and A431 cells via constitutive inactivation of cellular protein tyrosine phosphatases.     

Development of a modified in-gel assay to identify protein tyrosine phosphatases that are oxidized and inactivated in vivo

Protein tyrosine phosphatases (PTPs) comprise a superfamily of enzymes that control a diverse array of signal transduction pathways. However, the function and regulation of many of these enzymes remain undefined. Previous studies have shown that the optimal tyrosine phosphorylation response to various exogenous stimuli requires the production of reactive oxygen species (ROS). It has been proposed that ROS might transiently inactivate inhibitory PTPs, thus facilitating tyrosine phosphorylation-dependent signaling. Interestingly, the unique chemistry of the invariant, active site Cys residue located in the signature motif renders it highly susceptible to oxidation, leading to the inactivation of PTPs. We have developed a novel strategy to identify those PTPs that are oxidized and therefore, inactivated in response to extracellular stimuli. Iodoacetic acid (IAA) was used to alkylate selectively the thiolate anion of the active site Cys in the reduced PTPs. In contrast, any PTPs in which the active site Cys had been oxidized in response to the stimulus were resistant to alkylation. Following this key step to differentiate between the two pools of PTPs, the oxidized phosphatases were reduced back to the active state during the process of a standard in-gel PTP activity assay. This novel technique revealed, for the first time, that multiple cellular PTPs were indeed oxidized and inactivated in response to exogenous hydrogen peroxide. We have used this technique extensively to show that the ligand-stimulated production of intracellular hydrogen peroxide reversibly regulates the activity of specific PTPs in vivo. By defining the precise PTP targets of intracellular oxidants, the mechanistic details of signal transduction can be delineated. Due to the potential use of this method in finding the molecular targets of intracellular oxidants in diverse signaling pathways, we describe here the theoretical background and the detailed protocols of the modified in-gel PTP assay.     

Mass spectrometry-based analyses for identifying and characterizing S-nitrosylation of protein tyrosine phosphatases

All members in the protein tyrosine phosphatase (PTP) family of enzymes contain an invariant Cys residue which is absolutely indispensable for catalysis. Due to the unique microenvironment surrounding the active center of PTPs, this Cys residue exhibits an unusually low pKa characteristic, thus being highly susceptible to oxidation or S-nitrosylation. While oxidation-dependent regulation of PTP activity has been extensively examined, the molecular details and biological consequences of PTP S-nitrosylation remain unexplored. We hypothesized that the catalytic Cys residue is targeted by proximal nitric oxide (NO) and its derivatives collectively termed reactive nitrogen species (RNS), leading to nitrosothiol formation concomitant with reversible inactivation of PTPs. To test this hypothesis, we have developed novel strategies to examine the redox status of Cys residues of purified PTP1B that was exposed to NO donor S-Nitroso-N-penicillamine (SNAP). A gel-based method in conjunction with mass spectrometry (MS) analysis revealed that the catalytic Cys215 of PTP1B was reversibly modified when PTP1B was briefly treated with SNAP. In order to further identify the exact mode of NO-induced modification, we employed an online LC-ESI-MS/MS analysis incorporating a mass difference-based, data-dependent acquisition function that effectively mapped the S-nitrosylated Cys residues. Our results demonstrated that treating PTP1B with SNAP led to S-nitrosothiol formation of the catalytic Cys215. Interestingly, SNAP-induced modifications were strictly reversible as highly oxidized Cys derivatives (Cys-SO(2)H or Cys-SO(3)H) were not identified by MS analyses. Thus, the methods introduced in this study provide direct evidence to prove the direct link between S-nitrosylation of the catalytic Cys residue and reversible inactivation of PTPs.     

Conjugation of glutathione to oxidized tyrosine residues in peptides and proteins

Tyrosine residues are sensitive to oxidation and can be converted to hydroperoxides either by superoxide reacting with the Tyr radical or by singlet oxygen. These hydroperoxides rearrange to bicyclic derivatives that are readily reduced to more stable hydroxides. The aromatic character of tyrosine is lost, but the product contains an α-β unsaturated carbonyl group and is, therefore, an electrophile. We have generated hydroxide derivatives of several Tyr-containing peptides and shown using liquid chromatography/mass spectrometry that they undergo Michael addition with GSH. For Tyr-Gly, rate constants of 9.2 and 11.8 m(-1)min(-1) were measured for the two chromatographically distinct isomers. Unusual for GSH addition to an electrophile, the reaction is reversible, with a half-life of many hours for the reverse reaction. These kinetics indicate that with a typical cellular concentration of 5 mm GSH, >95% Tyr-Gly hydroxide would become conjugated with a half-life of ～15 min. Sperm whale myoglobin forms a hydroperoxide on Tyr-151 in a hydrogen peroxide/superoxide-dependent reaction. We show that its hydroxide derivative reacts with GSH to form a conjugate. Detection of the conjugate required stabilization by reduction; otherwise, the reverse reaction occurred during tryptic digestion and analysis. Our findings represent a novel mechanism for peptide or protein glutathionylation involving a carbon-sulfur cross-link between oxidized Tyr and Cys. As with other electrophiles, the oxidized Tyr should undergo a similar reaction with Cys residues in proteins to give intramolecular or intermolecular protein cross-links. This mechanism could give rise to protein cross-linking in conditions of oxidative stress.     

Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide

The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-β peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation is due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP.     

Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo

We have investigated the regulation of protein tyrosine phosphatases (PTPs) by reactive oxygen species (ROS) in a cellular environment. We demonstrate that multiple PTPs were reversibly oxidized and inactivated following treatment of Rat-1 cells with H(2)O(2) and that inhibition of PTP function was important for ROS-induced mitogenesis. Furthermore, we show transient oxidation of the SH2 domain containing PTP, SHP-2, in response to PDGF that requires association with the PDGFR. Our results indicate that SHP-2 inhibits PDGFR signaling and suggest a mechanism by which autophosphorylation of the PDGFR occurs despite its association with SHP-2. The data suggest that several PTPs may be regulated by oxidation and that characterization of this process may define novel links between specific PTPs and particular signaling pathways in vivo.     

Redox regulation of a soybean tyrosine-specific protein phosphatase

Plant protein tyrosine phosphatases (PTPs) are important in regulating cellular responses to redox change through their reversible inactivation under oxidative conditions. Studies on the soybean (Glycine max) GmPTP have shown that, compared with its mammalian counterparts, the plant enzyme is relatively insensitive to inactivation by H2O2 but hypersensitive (k(inact) = 559 M(-1) s(-1)) to S-glutathionylation (thiolation) promoted by the presence of oxidized glutathione (GSSG). Through a combination of chemical and mutational modification studies, three of the seven cysteine residues of GmPTP have been identified by mass spectrometry as being able to inactivate the enzyme when thiolated by GSSG or alkylated with iodoacetamide. Conserved Cys 266 was shown to be essential for catalysis but surprisingly resistant to S-modification, whereas the regulatory Cys 78 and Cys 176 were readily thiolated and/or alkylated. Mutagenesis of these cysteines showed that all three residues were in proximity of each other, regulating each's reactivity to S-modifying agents. Through a combination of protein modification and kinetic experiments, we conclude that the inactivation of GmPTP by GSSG is regulated at two levels. Cys 176 appears to be required to promote the formation of the reduced form of Cys 266, which is otherwise unreactive. When thiolated, Cys 176 immediately inactivates the enzyme, and this is followed by the thiolation of Cys 78, which undergoes a slow disulfide exchange with Cys 266 giving rise to a Cys 78-Cys 266 disulfide. We speculate that this two-tiered protection is required for regulation of GmPTP under highly oxidizing conditions.     

A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase

A cDNA, PHCC-TPx, specifying a protein highly homologous to known phospholipid hydroperoxide glutathione peroxidases was isolated from a Chinese cabbage cDNA library. PHCC-TPx encodes a preprotein of 232 amino acids containing a putative N-terminal chloroplast targeting sequence and three conserved Cys residues (Cys(107), Cys(136), and Cys(155)). The mature form of enzyme without the signal peptide was expressed in Escherichia coli, and the recombinant protein was found to utilize thioredoxin (Trx) but not GSH as an electron donor. In the presence of a Trx system, the protein efficiently reduces H(2)O(2) and organic hydroperoxides. Complementation analysis shows that overexpression of the PHCC-TPx restores resistance to oxidative stress in yeast mutants lacking GSH but fails to complement mutant lacking Trx, suggesting that the reducing agent of PHCC-TPx in vivo is not GSH but is Trx. Mutational analysis of the three Cys residues individually replaced with Ser shows that Cys(107) is the primary attacking site by peroxide, and oxidized Cys(107) reacts with Cys(155)-SH to make an intramolecular disulfide bond, which is reduced eventually by Trx. Tryptic peptide analysis by matrix-assisted laser desorption and ionization time of flight mass spectrometry shows that Cys(155) can form a disulfide bond with either Cys(107) or Cys(136).     

Redox regulation of protein-tyrosine phosphatases

The protein-tyrosine phosphatases (PTPs) form a large family of signaling proteins with essential functions in embryonic development and adult physiology. The PTPs are characterized by an absolutely conserved catalytic site cysteine with a low pK_a due to its microenvironment, making it vulnerable to oxidation. PTPs are differentially oxidized and inactivated in vitro and in living cells. Many cellular stimuli induce a shift in the cellular redox state towards oxidation and evidence is accumulating that at least part of the cellular responses to these stimuli are due to specific, transient inactivation of PTPs, indicating that PTPs are important sensors of the cellular redox state.     

Oxidative inactivation of protein tyrosine phosphatase 1B by organic hydroperoxides

Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that play a central role in cell signaling. Organic hydroperoxides cause thiol-reversible, oxidative inactivation of PTP1B in a manner that mirrors the endogenous signaling agent hydrogen peroxide.     

Inactivation of Enzymes by Linoleic Acid Hydroperoxides and Linoleic Acid

The inactivation of the enzymes by linoleic acid hydroperoxides (LAHPO) was tested in connection with the toxicity of oxidized fat. At the same time, the inhibition of enzyme activities by linoleic acid was also tested. Ribonuclease (RNase), trypsin, chymotrypsin and pepsin which are considered to be simple proteins and not to be SH-enzymes were chosen as the enzymes. RNase was largely inhibited by LAHPO, but the other enzymes were inhibited by linoleic acid as well as LAHPO. The inhibition of each enzyme occurred at different pH. This fact may show that the inhibition occurs by binding of such hydrophobic compounds to the enzyme, and that the surface exposition of hydrophobic region may depend on the pH. Not only the reaction of some specific amino acid residue in the protein molecules with LAHPO, but also the binding of these hydrophobic compounds must be remembered in the mechanism of inhibition.     

Screening combinatorial libraries by mass spectrometry. 2. Identification of optimal substrates of protein tyrosine phosphatase SHP-1

Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. In this work, we have developed a novel combinatorial library method, termed "enzyme-catalyzed loss of isotope peak signal enhancement (ECLIPSE)", to determine the substrate specificity of PTPs. This method involves partial labeling of pY at a nonbridging phosphate oxygen atom with 50% (18)O ((16)O/(18)O = 1:1). A 361-member solution-phase peptide library with randomization at the -1 and -2 positions (relative to pY), RNNXXpYA-NH(2) (X = 19 alpha-amino acids except for Cys), was synthesized with the partially (18)O-labeled pY by the split-synthesis method. Each member of the resulting pY peptide library appeared as a doublet peak in the mass spectrum (m/z m and m + 2.0043). Limited treatment of the library with a PTP removed the mass-degenerate phosphoryl group from the most preferred substrates to generate products as singlet peaks, which were readily identified and sequenced by tandem mass spectrometry. Screening of the pY library against the catalytic domain of SHP-1 revealed that SHP-1 prefers an acidic residue at the -2 position, with aspartic acid being slightly better than glutamic acid. At the -1 position, SHP-1 also prefers an acidic residue, although a variety of other amino acids are also tolerated. On the other hand, positively charged residues at these positions render the corresponding peptides very poor substrates of SHP-1. Several selected peptides were individually synthesized and assayed against SHP-1, and the kinetic data confirmed the screening results. These results demonstrate that ECLIPSE is a viable method for studying the substrate specificity of PTPs.     

Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation

Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 A resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H(2)O(2)-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.     

Inactivation of glutathione peroxidase by superoxide radical

The selenium-containing glutathione peroxidase, when in its active reduced form, was inactivated during exposure to the xanthine oxidase reaction. Superoxide dismutase completely prevented this inactivation, whereas catalase, hydroxyl radical scavengers, or chelators did not, indicating that O2 was the responsible agent. Conversion of GSH peroxidase to its oxidized form, by exposure to hydroperoxides, rendered it insensitive toward O2. The oxidized enzyme regained susceptibility toward inactivation by O2 when reduced with GSH. The inactivation by O2 could be reversed by GSH; however, sequential exposure to O2 and then hydroperoxides caused irreversible inactivation. Reactivity toward CN- has been used as a measure of the oxidized form of GSH peroxidase, whereas reactivity toward iodoacetate has been taken as an indicator of the reduced form. By these criteria both O2 and hydroperoxides convert the reduced form to oxidized forms. A mechanism involving oxidation of the selenocysteine residue at the active site has been proposed to account for these observations.     

Selective degradation of oxidatively modified protein substrates by the proteasome

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. Oxidants produce modifications to proteins leading to loss of function (or gain of undesirable function) and very often to an enhanced degradation of the oxidized proteins. For several years it has been known that the proteasome is involved in the degradation of oxidized proteins. This review summarizes our knowledge about the recognition of oxidized protein substrates by the proteasome in in vitro systems and its applicability to living cells. The majority of studies in the field agree that the degradation of mildly oxidized proteins is an important function of the proteasomal system. The major recognition motif of the substrates seems to be hydrophobic surface patches that are recognized by the 20S 'core' proteasome. Such hydrophobic surface patches are formed by partial unfolding and exposure of hydrophobic amino acid residues during oxidation. Oxidized proteins appear to be relatively poor substrates for ubiquitination, and the ubiquitination system does not seem to be involved in the recognition or targeting of oxidized proteins. Heavily oxidized proteins appear to first aggregate (new hydrophobic and ionic bonds) and then to form covalent cross-links that make them highly resistant to proteolysis. The inability to degrade extensively oxidized proteins may contribute to the accumulation of protein aggregates during diseases and the aging process.     

Separation, detection, and quantification of hydroperoxides formed at side-chain and backbone sites on amino acids, peptides, and proteins

Hydroperoxides are major reaction products of radicals and singlet oxygen with amino acids, peptides, and proteins. However, there are few data on the distribution of hydroperoxides in biological samples and their sites of formation on peptides and proteins. In this study we show that normal-or reversed-phase gradient HPLC can be employed to separate hydroperoxides present in complex systems, with detection by postcolumn oxidation of ferrous xylenol orange to the ferric species and optical detection at 560 nm. The limit of detection (10-25 pmol) is comparable to chemiluminescence detection. This method has been used to separate and detect hydroperoxides, generated by hydroxyl radicals and singlet oxygen, on amino acids, peptides, proteins, plasma, and intact and lysed cells. In conjunction with EPR spin trapping and LC/MS/MS, we have obtained data on the sites of hydroperoxide formation. A unique fingerprint of hydroperoxides formed at alpha-carbon (backbone) positions has been identified; such backbone hydroperoxides are formed in significant yields only when the amino acid is part of a peptide or protein. Only side-chain hydroperoxides are detected with free amino acids. These data indicate that free amino acids are poor models of protein damage induced by radicals or other oxidants.     

Taurine chloramine-induced inactivation of cofilin protein through methionine oxidation

Cofilin regulates reorganization of actin filaments (F-actin) in eukaryotes. A recent finding has demonstrated that oxidation of cofilin by taurine chloramine (TnCl), a physiological oxidant derived from neutrophils, causes cofilin to translocate to the mitochondria inducing apoptosis (F. Klamt et al. Nat. Cell Biol. 11:1241–1246; 2009). Here we investigated the effect of TnCl on biological activities of cofilin in vitro. Our data show that TnCl-induced oxidation of recombinant human cofilin-1 inhibits its F-actin-binding and depolymerization activities. Native cofilin contains four free Cys and three Met residues. Incubation of oxidized cofilin with DTT does not lead to its reactivation. A double Cys to Ala mutation on the two C-terminal Cys shows similar biological activities as the wild type, but does not prevent the TnCl-induced inactivation. In contrast, incubation of oxidized cofilin with methionine sulfoxide reductases results in its reactivation. Phosphorylation is known to inhibit cofilin activities. We found that Met oxidation also prevents phosphorylation of cofilin, which is reversed by incubating oxidized cofilin with methionine sulfoxide reductases. Interestingly, intact protein mass spectrometry of the oxidized mutant indicated one major oxidation product with an additional mass of 16 Da, consistent with oxidation of one specific Met residue. This residue was identified as Met-115 by peptide mapping and tandem mass spectrometry. It is adjacent to Lys-114, a known residue on globular-actin-binding site, implying that oxidation of Met-115 disrupts the globular-actin-binding site of cofilin, which causes TnCl-induced inactivation. The findings identify Met-115 as a redox switch on cofilin that regulates its biological activity.