Structural insights into the homology and differences between mouse protein tyrosine phosphatase-sigma and human protein tyrosine phosphatase-sigma

Protein tyrosine phosphatases PTP-sigma (PTPσ) plays an important role in the development of the nervous system and nerve regeneration. Although cumulative studies about the function of PTPσ have been reported, yet limited data have been reported about the crystal structure and in vitro activity of mouse PTPσ. Here we report the crystal structure of mouse PTPσ tandem phosphatase domains at 2.4 ? resolution. Then we compared the crystal structure of mouse PTPσ with human PTPσ and found that they are very similar, superimposing with a root mean square deviation of 0.45 ? for 517 equivalent Cα atoms. But some residues in mouse PTPσ form loops while corresponding residues in human PTPσ form β-sheets or α-helices. Furthermore, we also compared in vitro activities of mouse PTPσ with human PTPσ and found that mouse PTPσ has 25-fold higher specific activity than human PTPσ does toward O-methyl fluorescein phosphate (OMFP) as the substrate. However, there is no significant activity difference between the mouse and the human enzyme detected with p-nitrophenylphosphate (pNPP) as the substrate. Mouse PTPσ and human PTPσ have different substrate specificities toward OMFP and pNPP as substrates. This work gives clues for further study of PTPσ.     

PTPσ promotes retinal neurite outgrowth non‐cell‐autonomously

The receptor‐like protein tyrosine phosphatase (RPTP) PTPσ controls the growth and targeting of retinal axons, both in culture and in ovo. Although the principal actions of PTPσ have been thought to be cell‐autonomous, the possibility that RPTPs related to PTPσ also have non‐cell‐autonomous signaling functions during axon development has also been supported genetically. Here we report that a cell culture substrate made from purified PTPσ ectodomains supports retinal neurite outgrowth in cell culture. We show that a receptor for PTPσ must exist on retinal axons and that binding of PTPσ to this receptor does not require the known, heparin binding properties of PTPσ. The neurite‐promoting potential of PTPσ ectodomains requires a basic amino acid domain, previously demonstrated in vitro as being necessary for ligand binding by PTPσ. Furthermore, we demonstrate that heparin and oligosaccharide derivatives as short as 8mers, can specifically block neurite outgrowth on the PTPσ substrate, by competing for binding to this same domain. This is the first direct evidence of a non‐cell‐autonomous, neurite‐promoting function of PTPσ and of a potential role for heparin‐related oligosaccharides in modulating neurite promotion by an RPTP. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Regulation of PTP1B via glutathionylation of the active site cysteine 215.

The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicating that the inactivated form of the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic forms and hinder the regulatory efficiency if it is not converted to a more stable and reversible end product such as a glutathionyl derivative. Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.     

Protein tyrosine phosphatase σ regulates the synapse number of zebrafish olfactory sensory neurons

The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks. To investigate the functional role of protein tyrosine phosphatase (PTP) σ, we employed an olfactory sensory neuron (OSN)-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos. Knockdown of PTPσ enhanced the accumulation of synaptic vesicles in the axon terminals of OSNs. The exaggerated accumulation of synaptic vesicles was restored to the normal level by the OSN-specific expression of PTPσ, indicating that presynaptic PTPσ is responsible for the regulation of synaptic vesicle accumulation. Consistently, transient expression of a dominant-negative form of PTPσ in OSNs enhanced the accumulation of synaptic vesicles. The exaggerated accumulation of synaptic vesicles was reproduced in transgenic zebrafish lines carrying an OSN-specific expression vector of the dominant-negative PTPσ. By electron microscopic analysis of the transgenic line, we found the significant increase of the number of OSN-mitral cell synapses in the central zone of the olfactory bulb. The density of docked vesicles at the active zone was also increased significantly. Our results suggest that presynaptic PTPσ controls the number of OSN-mitral cell synapses by suppressing their excessive increase.     

Redox regulation of MAP kinase phosphatase 3

MAP kinase phosphatase 3 (MKP3) is a protein tyrosine phosphatase (PTP) for which in vivo evidence suggests that regulation can occur by oxidation and/or reduction of the active site cysteine. Using kinetics and mass spectrometry, we have probed the biochemical details of oxidation of the active site cysteine in MKP3, with particular focus on the mechanism of protection from irreversible inactivation to the sulfinic or sulfonic acid species. Like other PTPs, MKP3 was found to be rapidly and reversibly inactivated by mild treatment with hydrogen peroxide. We demonstrate that unlike the case for some PTPs, the sulfenic acid of the active site cysteine in MKP3 is not stabilized in the active site but instead is rapidly trapped in a re-reducible form. Unlike the case for other PTPs, the sulfenic acid in MKP3 does not form a sulfenyl-amide species with its neighboring residue or a disulfide with a single proximate cysteine. Instead, multiple cysteines distributed in both the N-terminal substrate-binding domain (Cys147 in particular) and the C-terminal catalytic domain (Cys218) are capable of rapidly and efficiently trapping the sulfenic acid as a disulfide. Our results extend the diversity of mechanisms utilized by PTPs to prevent irreversible oxidation of their active sites and expand the role of the N-terminal substrate recognition domain in MKP3 to include redox regulation.     

Catalytic and chemical competence of regulation of cdc25 phosphatase by oxidation/reduction

Cdc25 phosphatases belong to the family of protein tyrosine phosphatases (PTPs) that contain an active-site cysteine and form a phosphocysteine intermediate. Recently, oxidation/reduction of active-site cysteines of PTPs, including Cdc25, has been proposed to serve as a form of reversible regulation for this class of enzymes. Here we provide in vitro evidence that supports the chemical and kinetic competence for oxidation/reduction of the active-site cysteines of Cdc25B and Cdc25C as a mechanism of regulation. Using kinetic measurements and mass spectrometry, we have found that the active-site cysteines of the Cdc25's are highly susceptible to oxidation. The rate of thiolate conversion to the sulfenic acid by hydrogen peroxide for Cdc25B is 15-fold and 400-fold faster than that for the protein tyrosine phosphatase PTP1B and the cellular reductant glutathione, respectively. If not for the presence of an adjacent (back-door) cysteine in proximity to the active-site cysteine in the Cdc25's, the sulfenic acid would rapidly oxidize further to the irreversibly inactivated sulfinic acid, as determined by using kinetic partitioning and mass spectrometry with mutants of these back-door cysteines. Thus, the active-site cysteine is protected by rapid intramolecular disulfide formation with the back-door cysteines in the wild-type enzymes. These intramolecular disulfides can then be rapidly and effectively rereduced by thioredoxin/thioredoxin reductase but not glutathione. Thus, the chemistry and kinetics of the active-site cysteines of the Cdc25's support a physiological role for reversible redox-mediated regulation of the Cdc25's, important regulators of the eukaryotic cell cycle.     

Protein Tyrosine Phosphatase σ in Proteoglycan-Mediated Neural Regeneration Regulation

The receptor-type protein tyrosine phosphatase PTPσ mediates neural development and regeneration. Early studies on the ligands of PTPσ identified heparan sulfate proteolycan (HSPG) as a ligand. Binding of HSPG to PTPσ plays a critical role in axon guidance and synapse formation. PTPσ is also a receptor for chondroitin sulfate proteoglycan (CSPG). CSPG is deposited in high concentration at sites of neural injury. The deposited CSPG inhibits neural regeneration and axonal growth via PTPσ. The crystal structure of N-terminal immunoglobulin-like domains of PTPσ shows that the glycan binding site forms an elliptical surface patch of ～35 by 24 Å, which interacts with sulfate groups of HSPG and CSPG. In this review, we focus on the structural and functional mechanisms for the neural regeneration regulation by different types of proteoglycans. We also discuss recent results on induction of neural regeneration in the stroke model and neural transplantation. The mechanistic understanding of relationships between proteoglycans and PTPσ provides new therapeutic opportunities against diseases with impaired neural regeneration.     

The requirement of reversible cysteine sulfenic acid formation for T cell activation and function

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naive CD8(+) T cell activation and proliferation. We observed that, within minutes of T cell activation, naive CD8(+) T cells increased ROI levels in a manner dependent upon Ag concentration. Increased ROI resulted in elevated levels of cysteine sulfenic acid in the total proteome. Analysis of specific proteins revealed that the protein tyrosine phosphatases SHP-1 and SHP-2, as well as actin, underwent increased sulfenic acid modification following stimulation. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation, increasing concentrations of 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures. Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation, calcium flux, cell growth, and proliferation of naive CD8(+) and CD4(+) T cells. We also found that TNF-alpha production by effector and memory CD8(+) T cells was more sensitive to the inhibition of reversible cysteine sulfenic acid formation than IFN-gamma. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8(+) T cells are able to modulate signaling, proliferation, and function.     

Synthesis of chemical probes to map sulfenic acid modifications on proteins

Cysteine sulfenic acids in proteins can be identified by their ability to form adducts with dimedone, but this reagent imparts no spectral or affinity tag for subsequent analyses of such tagged proteins. Given its similar reactivity toward cysteine sulfenic acids, 1,3-cyclohexadione was synthetically modified to an alcohol derivative and linked to fluorophores based on isatoic acid and 7-methoxycoumarin. The resulting compounds retain full reactivity and specificity toward cysteine sulfenic acids in proteins, allowing for incorporation of the fluorescent label into the protein and "tagging" it based on its sulfenic acid redox state. Control experiments using dimedone further show the specificity of the reaction of 1,3-diones with protein sulfenic acids in aqueous media. These new compounds provide the basis for an improved method for the detection of protein sulfenic acids.     

New inhibitors of iron-containing nitrile hydratases

There is growing evidence in the literature emphasizing the significance of the post-translational modification of cysteine thiols to sulfenic acids (SOH), which have been found in a number of proteins. Crystallographic and mass spectrometric evidence has shown the presence of this group in an inactive form of the industrially important enzyme nitrile hydratase (NHase). This oxidized cysteine is unique in that it forms part of the coordination sphere of the low-spin iron III at the active site of the enzyme. The presence of this unstable sulfenic group in the active form of NHase is the subject of some controversy. To try to detect this function in NHase, we have studied the inhibitory effect on nitrile hydration of reagents known to react with sulfenic acids. Two NHases were studied, namely, Rhodococcus rhodochrous R312 NHase and Comamonas testosteroni NI1 NHase, and the reagents used were meta-chlorocarbonyldicyano-phenylhydrazone (m-ClCP), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), and 2-nitro-5-thiocyanato-benzoic acid (NTBA). Following this approach we report three novel inhibitors of NHases. In addition, we report thiocyanate reagents that can be used to monitor NHase activity spectroscopically.     

Postsynaptic TrkC and presynaptic PTPσ function as a bidirectional excitatory synaptic organizing complex

Neurotrophin receptor tyrosine kinases (Trks) have well-defined trophic roles in nervous system development through kinase activation by neurotrophins. Yet Trks have typical cell-adhesion domains and express noncatalytic isoforms, suggesting additional functions. Here we discovered noncatalytic TrkC in an unbiased hippocampal neuron-fibroblast coculture screen for proteins that trigger differentiation of neurotransmitter release sites in axons. All TrkC isoforms, but not TrkA or TrkB, function directly in excitatory glutamatergic synaptic adhesion by neurotrophin-independent high-affinity trans binding to axonal protein tyrosine phosphatase receptor PTPσ. PTPσ triggers and TrkC mediates clustering of postsynaptic molecules in dendrites, indicating bidirectional synaptic organizing functions. Effects of a TrkC-neutralizing antibody that blocks TrkC-PTPσ interaction and TrkC knockdown in culture and in?vivo reveal essential roles of TrkC-PTPσ in glutamatergic synapse formation. Thus, postsynaptic TrkC trans interaction with presynaptic PTPσ generates bidirectional adhesion and recruitment essential for excitatory synapse development and positions these signaling molecules at the center of synaptic pathways.     

Catalytic inactivation of protein tyrosine phosphatase CD45 and protein tyrosine phosphatase 1B by polyaromatic quinones

Polyaromatic quinones, such as the environmental pollutants 9,10-phenanthrenediones, elicit a wide range of responses including growth inhibition, immune suppression, and glucose normalization in diabetic models. Yet the molecular mechanisms behind these effects remain controversial. Here we report that many of them are oxygen-dependent and catalytic inactivators of protein tyrosine phosphatases (PTP). Under aerobic conditions, the PTP inactivation by 2-nitro-9,10-phenanthrenedione followed a pseudo-first-order process, with the rate of inactivation increasing nearly linearly with increasing inhibitor concentration, yielding apparent inactivation rate constants of 4300, 387, and 5200 M(-1) s(-1) at pH 7.2 against CD45, PTP1B, and LAR, respectively. The rate of CD45 inactivation increased approximately 25-fold from pH 6.0 to 7.5, with complete inactivation achieved using a catalytic amount (0.05 molar equiv) of the inhibitor. The quinone-catalyzed CD45 inactivation was prevented by catalase or superoxide dismutase. Inactivated CD45 after (125)I-9,10-phenanthrenedione treatment carried no radioactivity, indicating the absence of a stable inhibitor/enzyme complex. The activity of inactivated CD45 was partially restored ( approximately 10%) by hydroxylamine or dithiothreitol, supporting the presence of a small population of sulfenic acid or sulfenyl-amide species. Treatment of PTP1B with 2-nitro-9,10-phenanthrenedione resulted in the specific and sequential oxidation of the catalytic cysteine to the sulfinic and sulfonic acid. These results suggest that reactive oxygen species and the semiquinone radical, continuously generated during quinone-catalyzed redox cycling, mediate the specific catalytic cysteine oxidation. Naturally occurring quinones may act as efficient regulators of protein tyrosine phosphorylation in biological systems. Aberrant phosphotyrosine homeostasis resulting from continued polyaromatic hydrocarbon quinone exposure may play a significant role in their disease etiology.     

Discovery of potent inhibitors of receptor protein tyrosine phosphatase sigma through the structure-based virtual screening

Receptor protein tyrosine phosphatase sigma (PTPσ) has proved to be a promising target for the development of therapeutics for the treatment of neurological diseases. Here, we report the first example for a successful application of the structure-based virtual screening to identify the novel small-molecule inhibitors of PTPσ. These inhibitors revealed high potencies with the associated IC₅₀ values ranging from 0.1 to 1.3 μM and were also screened for having desirable physicochemical properties as a drug candidate. Therefore, they deserve consideration for further development by structure–activity relationship studies to develop therapeutics for neurological diseases. Structural features relevant to the stabilization of the newly identified inhibitors in the active site of PTPσ are discussed in detail.     

Characterization of the yeast peroxiredoxin Ahp1 in its reduced active and overoxidized inactive forms using NMR

Peroxiredoxins (Prx's) are a superfamily of thiol-specific antioxidant proteins present in all organisms and involved in the hydroperoxide detoxification of the cell. The catalytic cysteine of Prx's reduces hydroperoxides and is transformed into a transient sulfenic acid (Cys-SOH). At high hydroperoxide concentration, the sulfenic acid can be overoxidized into a sulfinate, or even a sulfonate. We present here the first peroxiredoxin characterization by solution NMR of the Saccharomyces cerevisiae alkylhydroperoxide reductase (Ahp1) in its reduced and in vitro overoxidized forms. NMR (15)N relaxation data and ultracentrifugation experiments indicate that the protein behaves principally as a homodimer (2 x 19 kDa) in solution, regardless of the redox state. In vitro treatment of Ahp1 by a large excess of tBuOOH leads to an inactive form, with the catalytic cysteine overoxidized into sulfonate, as demonstrated by (13)C NMR. Depending on the amino acid sequence of their active site, Prx's are classified into five different families. In this classification, Ahp1 is a member of the scarcely studied D-type Prx's. Ahp1 is unique among the D-type Prx's in its ability to form an intermolecular disulfide. The peptidic sequence of Ahp1 was analyzed and compared to other D-type Prx sequences.     

Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite.

Peroxiredoxins are antioxidant enzymes whose peroxidase activity depends on a redox-sensitive cysteine residue at the active center. In this study we investigated properties of the active center cysteine of bovine 1-Cys peroxiredoxin using a recombinant protein (BRPrx). The only cysteine residue of the BRPrx molecule was oxidized rapidly by an equimolar peroxide or peroxynitrite to the cysteine sulfenic acid. Approximate rates of oxidation of BRPrx by different peroxides were estimated using selenium glutathione peroxidase as a competitor. Oxidation of the active center cysteine of BRPrx by H2O2 proceeded only several times slowly than that of the selenocysteine of glutathione peroxidase. The rate of oxidation varied depending on peroxides tested, with H2O2 being about 7 and 80 times faster than tert-butyl hydroperoxide and cumene hydroperoxide, respectively. Peroxynitrite oxidized BRPrx slower than H2O2 but faster than tert-butyl hydroperoxide. Further oxidation of the cysteine sulfenic acid of BRPrx to higher oxidation states proceeded slowly. Oxidized BRPrx was reduced by dithiothreitol, dihydrolipoic acid, and hydrogen sulfide, and demonstrated peroxidase activity (about 30 nmol/mg/min) with these reductants as electron donors. beta-Mercaptoethanol formed a mixed disulfide and did not support peroxidase activity. Oxidized BRPrx did not react with glutathione, cysteine, homocysteine, N-acetyl-cysteine, and mercaptosuccinic acid.     

Facile synthesis and biological evaluation of a cell-permeable probe to detect redox-regulated proteins

We have developed an improved synthesis for the cell-permeable, sulfenic acid probe DAz-1. Using DAz-1, we detect sulfenic acid modifications in the cell-cycle regulatory phosphatase Cdc25A. In addition, we show that DAz-1 has superior potency in cells compared to a biotinylated derivative. Collectively, these findings set the stage for the development of activity-based inhibitors of Cdc25 cell-cycle phosphatases, which are sensitive to the redox state of the active-site cysteine and demonstrate the advantage of bioorthogonal conjugation methods to detect protein sulfenic acids in cells.     

Activity of the tetrapyrrole regulator CrtJ is controlled by oxidation of a redox active cysteine located in the DNA binding domain

CrtJ from Rhodobacter capsulatus is a regulator of genes involved in the biosynthesis of haem, bacteriochlorophyll, carotenoids as well as structural proteins of the light harvesting‐II complex. Fluorescence anisotropy‐based DNA‐binding analysis demonstrates that oxidized CrtJ exhibits ～ 20‐fold increase in binding affinity over that of reduced CrtJ. Liquid chromatography electrospray tandem ionization mass spectrometric analysis using DAz‐2, a sulfenic acid (–SOH)‐specific probe, demonstrates that exposure of CrtJ to oxygen or to hydrogen peroxide leads to significant accumulation of a sulfenic acid derivative of Cys420 which is located in the helix–turn–helix (HTH) motif. In vivo labelling with 4‐(3‐azidopropyl)cyclohexane‐1,3‐dione (DAz‐2) shows that Cys420 also forms a sulfenic acid modification in vivo when cells are exposed to oxygen. Moreover, a Cys420 to Ala mutation leads to a ～ 60‐fold reduction of DNA binding activity while a Cys to Ser substitution at position 420 that mimics a cysteine sulfenic acid results in a ～ 4‐fold increase in DNA binding activity. These results provide the first example where sulfenic acid oxidation of a cysteine in a HTH‐motif leads to differential effects on gene expression.     

Methionine sulfoxide reductase: Chemistry,substrate binding,recycling process and oxidase activity

Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a sulfenic acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the sulfenic acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the sulfenic acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form.     

Protein sulfenic acid formation: from cellular damage to redox regulation

Protein sulfenic acid formation has long been regarded as unwanted damage caused by reactive oxygen species (ROS). However, over the past 10 years, accumulating evidence has shown that the reversible oxidation of cysteine thiol groups to sulfenic acid functions as a redox-based signal transduction mechanism. Here, we review the mechanisms of sulfenic acid formation by ROS. We present some of the most important roles played by sulfenic acids in living cells as well as the pathways that regulate sulfenic acid formation. We highlight the experimental tools that have been developed to study the cellular sulfenome and show how computational approaches might help to better understand the mechanisms of sulfenic acid formation.