Low Molecular Weight Protein Tyrosine Phosphatase Research Articles

Low Molecular Weight Protein Tyrosine Phosphatases Control Antibiotic Production in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) possesses a low molecular weight protein tyrosine phosphatase (LMW-PTP), PtpA, that affects the production of undecylprodigionsin (RED) and actinorhodin (ACT). In this study we identified another LMW-PTP called sco3700. Tyrosine phosphatase activity of the purified Sco3700 was established using para-nitrophenyl phosphate and the tyrosine-phosphorylated protein PtkA from Bacillus subtilis as substrates. The optimum pH for the Sco3700 phosphatase activity was 6.8, and KM for pNPP was 14.3 mM compared to pH 6.0 and KM0.75 mM for PtpA. The potential of Sco3700 to participate, alongside PtpA, in the regulation of S. coelicolor antibiotic production was investigated. Hence, S. coelicolor A3(2) strains with ptpA and sco3700 overexpression were constructed and characterized for growth, RED and ACT production. We did observe an increase in volumetric productivity of ACT in the ptpA over expression strain. Furthermore, a significantly earlier onset of ACT production was observed when ptpA was over expressed. Sco3700 overexpression had a pleiotropic effect on the cell, and the strain exhibited lower productivities and final concentrations of antibiotics. We conclude that Sco3700 is indeed a tyrosine phosphatase, and it contributes to regulation of antibiotic production in S. coelicolor affecting the timing of onset of the antibiotic production.

Loss of TAK1 increases cell traction force in a ROS-dependent manner to drive epithelial–mesenchymal transition of cancer cells

Epithelial–mesenchymal transition (EMT) is a crucial step in tumor progression, and the TGFβ–SMAD signaling pathway as an inductor of EMT in many tumor types is well recognized. However, the role of non-canonical TGFβ–TAK1 signaling in EMT remains unclear. Herein, we show that TAK1 deficiency drives metastatic skin squamous cell carcinoma earlier into EMT that is conditional on the elevated cellular ROS level. The expression of TAK1 is consistently reduced in invasive squamous cell carcinoma biopsies. Tumors derived from TAK1-deficient cells also exhibited pronounced invasive morphology. TAK1-deficient cancer cells adopt a more mesenchymal morphology characterized by higher number of focal adhesions, increase surface expression of integrin α5β1 and active Rac1. Notably, these mutant cells exert an increased cell traction force, an early cellular response during TGFβ1-induced EMT. The mRNA level of ZEB1 and SNAIL, transcription factors associated with mesenchymal phenotype is also upregulated in TAK1-deficient cancer cells compared with control cancer cells. We further show that TAK1 modulates Rac1 and RhoA GTPases activities via a redox-dependent downregulation of RhoA by Rac1, which involves the oxidative modification of low-molecular weight protein tyrosine phosphatase. Importantly, the treatment of TAK1-deficient cancer cells with Y27632, a selective inhibitor of Rho-associated protein kinase and antioxidant N-acetylcysteine augment and hinders EMT, respectively. Our findings suggest that a dysregulated balance in the activation of TGFβ–TAK1 and TGFβ–SMAD pathways is pivotal for TGFβ1-induced EMT. Thus, TAK1 deficiency in metastatic cancer cells increases integrin:Rac-induced ROS, which negatively regulated Rho by LMW-PTP to accelerate EMT.

Low Molecular Weight Protein Tyrosine Phosphatase Isoforms Regulate Breast Cancer Cells Migration through a RhoA Dependent Mechanism

Low molecular weight protein tyrosine phosphatase (LMW-PTP) has been associated with cell proliferation control through dephosphorylation and inactivation of growth factor receptors such as PDGF-R and EphA2, and with cellular adhesion and migration through p190RhoGap and RhoA. We aim to clarify the role of two main LMW-PTP isoforms in breast cancer tumorigenesis. We used a siRNA-mediated loss-of-function in MDA-MB-435 breast cancer cell line to study the role of the two main LMW-PTP isoforms, fast and slow, in breast cancer tumorigenesis and migration. Our results show that the siRNAs directed against total LMW-PTP and LMW-PTP slow isoform enhanced cell motility in an invasive breast cancer cell line, MDA-MB-435, with no changes in the proliferation and invasive potential of cells. The total LMW-PTP knockdown caused a more pronounced increase of cell migration. Suppression of total LMW-PTP decreased RhoA activation and suppression of the LMW-PTP slow isoform caused a small but significant increase in RhoA activation. We propose that the increase or decrease in RhoA activation induces changes in stress fibers formation and consequently alter the adhesive and migratory potential of cells. These findings suggest that the two main isoforms of LMW-PTP may act differentially, with the fast isoform having a more prominent role in tumor cell migration. In addition, our results highlight functional specificity among LMW-PTP isoforms, suggesting hitherto unknown roles for these proteins in breast cancer biology. Novel therapeutic approaches targeting LMW-PTP, considering the expression of these two isoforms and not LMW-PTP as a whole, should be investigated.

Identification of new inhibitors for low molecular weight protein tyrosine phosphatase isoform B

The National Cancer Institute Diversity Set II (1356 compounds) and Diversity Set III (1597 compounds) were screened via in silico methods as potential inhibitors of low molecular weight protein tyrosine phosphatase (LWM-PTP) isoform B (EC 3.1.3.48). Those candidates that demonstrated comparable or better docking scores than that of pyridoxal 5′-phosphate (PLP), one of the most potent known inhibitors of LMW-PTP with a competitive inhibitor dissociation constant (Kis) of 7.6μM (pH 5.0), were analyzed via in vitro kinetic assays against LMW-PTP isoform B. While none of the compounds tested in vitro was significantly better that PLP, five compounds showed comparable inhibition. These five compounds are very diverse in structure and represent new therapeutic leads for inhibition of this isozyme.

Thioredoxin-Interacting Protein Expression Is Required for VEGF-Mediated Angiogenic Signal in Endothelial Cells

Thioredoxin-interacting protein (TXNIP) contributes to cellular redox-state homeostasis via binding and inhibiting thioredoxin (TRX). Increasing evidence suggests that cellular redox homeostasis regulates vascular endothelial growth factor (VEGF)-mediated signaling. This study aims to examine the redox-dependant role of TXNIP in regulating VEGF-mediated S-glutathionylation and angiogenic signaling. TXNIP-knockout mice (TKO) or wild-type (WT) treated with the reduced glutathione (GSH)-precursor, N-acetyl cysteine (WT-NAC, 500 mg/kg) were compared to WT using hypoxia-induced neovascularization model. In response to hypoxia, retinas from TKO and WT-NAC mice showed significant decreases in reparative revascularization and pathological neovascularization with similar VEGF expression compared with WT. VEGF failed to stimulate vascular sprouting from aortic rings of TKO compared to WT mice. TKO mice or WT+NAC experienced reductive stress as indicated by twofold increase in TRX reductase activity and fourfold increase in reduced-GSH levels compared with WT. In human microvascular endothelial (HME) cells, VEGF stimulated co-precipitation between vascular endothelial growth factor receptor 2 (VEGFR2) with low molecular weight protein tyrosine phosphatase (LMW-PTP). Silencing TXNIP expression blunted VEGF-induced oxidation of GSH and S-glutathionylation of the LMW-PTP in HME cells. These effects were associated with impaired VEGFR2 phosphorylation that culminated in inhibiting cell migration and tube formation. Overexpression of TXNIP restored VEGFR2 phosphorylation and cell migration in TKO-endothelial cells. TXNIP expression is required for VEGF-mediated VEGFR2 activation and angiogenic response in vivo and in vitro. TXNIP expression regulates VEGFR-2 phosphorylation via S-glutathionylation of LMW-PTP in endothelial cells. Our results provide novel mechanistic insight into modulating TXNIP expression as a potential therapeutic target in diseases characterized by aberrant angiogenesis.

Targeting the Arginine Phosphatase YwlE with a Catalytic Redox-Based Inhibitor

Protein phosphatases are critical regulators of cellular signaling in both eukaryotes and prokaryotes. The majority of protein phosphatases dephosphorylate phosphoserine/phosphothreonine or phosphotyrosine residues. Recently, however, YwlE, a member of the low-molecular weight protein tyrosine phosphatase (LMW-PTP) family, was shown to efficiently target phosphoarginine. YwlE shares several sequence motifs with this family including the C(X)4 CR(S/T) motif that is crucial for catalysis and redox regulation of the enzyme. Herein we confirm that Cys9 and Cys14 play important roles in YwlE catalysis and regulation. On the basis of these observations, we designed and synthesized a YwlE inhibitor, denoted cyc-SeCN-amidine, that irreversibly inhibits YwlE (kinact/KI = 310 M(-1) min(-1)) by inducing disulfide bond formation between the two active site cysteine residues. Interestingly, inactivation appears to be catalytic, since the compound is neither destroyed nor altered after enzyme inhibition. Although the exact mechanism of disulfide induction remains elusive, we propose several potential mechanisms accounting for the cyc-SeCN-amidine mediated inhibition of YwlE. These findings could stimulate the design of similar selenium-based compounds targeting other redox-sensitive enzymes.

Alr5068, a Low-Molecular-Weight protein tyrosine phosphatase, is involved in formation of the heterocysts polysaccharide layer in the cyanobacterium Anabaena sp. PCC 7120

The filamentous cyanobacterium Anabaena sp. PCC 7120 forms nitrogen-fixing heterocysts after deprivation of combined nitrogen. Under such conditions, vegetative cells provide heterocysts with photosynthate and receive fixed nitrogen from the latter. Heterocyst envelope contains a glycolipid layer and a polysaccharide layer to restrict the diffusion of oxygen into heterocysts. Low-Molecular-Weight protein tyrosine phosphatases (LMW-PTPs) are involved in the biosynthesis of exopolysaccharides in bacteria. Alr5068, a protein from Anabaena sp. PCC 7120, shows significant sequence similarity with LMW-PTPs. In this study we characterized the enzymatic properties of Alr5068 and showed that it can dephosphorylate several autophosphorylated tyrosine kinases (Alr2856, Alr3059 and All4432) of Anabaena sp. PCC 7120 in vitro. Several conserved residues among LMW-PTPs are shown to be essential for the phosphatase activity of Alr5068. Overexpression of alr5068 results in a strain unable to survive under diazotrophic conditions, with the formation of morphologically mature heterocysts detached from the filaments. Overexpression of an alr5068 allele that lost phosphatase activity led to the formation of heterocyst with an impaired polysaccharide layer. The alr5068 gene was upregulated after nitrogen step-down and its mutation affected the expression of hepA and hepC, two genes necessary for the formation of the heterocyst envelope polysaccharide (HEP) layer. Our results suggest that Alr5068 is associated with the production of HEP in Anabaena sp. PCC 7120.

Sa1694 Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) Is Upregulated in Primary Colorectal Cancer and Affects Cancer Signaling Pathways

Background: c9orf140 is a novel gene that has been recently isolated and indentified by the mRNA differential display (mRNAD), which is associated with cell proliferation and tumorigenesis in Gastric caner. Moreover, it was reported that the expression of c9orf140 is significantly elevated in colorectal caner (CRC) tissues when compared with normal tissues. However, the role of c9orf140 in the invasion of CRC and the mechanism of c9orf140 overexpression are still unclear. Method: c9orf140, EZH2, beta-catanin, STAT5 and pSTAT5 expressions were measured by Western Blot and immunohistochemical staining. To establish stable cells, CRC cells were infected with c9orf140 shRNA, c9orf140 overexpression or control lentivirus. Stable cell lines with different protein or shRNA expressions were introduced into nude mice respectively via tail veins injection. The photos were taken by the NightOWL II LB 983 in vivo Imaging system. Chromatin Immunoprecipitation (ChIP) and Luciferase reporter gene assay were carried out according to the protocol of manufactures. Result: there is almost no expression of C9orf140 in a normal human colon epithelial cell line, CRL1790, but the expression of C9orf140 is significantly increased in all CRC cell line especially in those highly invasive CRC cells. Immunofluorence analysis showed that C9orf140 is mainly detected in the cytoplasm of CRC HCT116 and HT29 cells. Immunohistochemical analysis of 150 patients' tissue sections showed that the staining of C9orf140 was found at higher expression levels in CRC samples than normal samples. Furthermore, the expression of C9orf140 was gradually increased from well differentiated to poor differentiated in CRC tissues. Knockdown of C9orf140 dramatically increased E-cadherin expression and decreased N-cadherin expression. Knockdown of C9orf140 significantly reduced the CRC cell invasion. More lung metastasis and shorter overall survival were detected after overexpression of C9orf140 compared with control in vivo. Knockdown of C9orf140 dramatically increased overall survival and decreased metastasis of lung in vivo. Western Blot data showed that C9orf140 expressions may be regulated by EZH2, STAT5 and β-catanin. Moreover, Immunoprecipitation, ChIP and Luciferase reporter gene assay showed that EZH2, STAT5 and βcatanin may colocalized together and directly bind to the promoter of C9orf140 gene to upregulate this gene expression in CRC cells. Conclusion: we firstly revealed the role of C9orf140 in the metastatic progression of CRC in vitro and in vivo. C9orf140-induced CRC cell migration and invasion may depend on promote the progression of EMT. Furthermore, we firstly identified that c9orf140 expression may be directly regulated by EZH2, STAT5 and β-catanin. Our data may provide potential targets to prevent and/or treat aggressive CRC.

The role of low-molecular-weight protein tyrosine phosphatase (LMW-PTP ACP1) in oncogenesis

Protein tyrosine phosphorylation is a crucial cellular event that is involved in the most important processes of cellular metabolism. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a tyrosine phosphatase that presents two active distinct isoforms and is regulated through cysteine oxidation and tyrosine phosphorylation. This enzyme has been linked to tumorigenesis, but its role is considered controversial: it may be considered oncogenic or anti-oncogenic depending on its interaction with different substrates. Furthermore, recent studies have demonstrated that LMW-PTP is involved in epithelial cell migration, a characteristic of tumor cells. This fact strengthens the importance of this enzyme in the oncogenic process and opens new avenues for future research. The study of LMW-PTP and its pathways may enhance therapeutic strategies that target tyrosine phosphorylation and its substrates. In this review, we try to clarify the importance of this protein in carcinogenesis through the analysis of LMW-PTP interaction with different substrates.

SmLMWPTP, a teleost low molecular weight protein tyrosine phosphatase, inhibits the immune response of peripheral blood leukocytes in a manner that depends on the conserved P-loop

Protein tyrosine phosphatases (PTPs) are a family of enzymes that play a key role in cellular signal transduction. Low molecular weight PTPs (LMWPTPs) are a subfamily of PTPs that are characterized by the presence of a conserved phosphate-binding loop (P-loop) with the signature sequence of (V/I)CXGNXCRS. To date, very little study on teleost LMWPTPs has been documented, and, as a result, the function of LMWPTPs in fish is essentially unknown. In this study, we identified a LMWPTP from turbot (Scophthalmus maximus) and examined its biological activity and functionality. The turbot LMWPTP (SmLMWPTP) is composed of 158 residues and possesses a typical P-loop sequence in the form of 12VCLGNICRS20. Purified recombinant SmLMWPTP (rSmLMWPTP) exhibited apparent phosphatase activity, which was optimal at pH 5 and 50°C. The activity of SmLMWPTP was abolished when C13 and, in particular, R19 of the P-loop were mutated. SmLMWPTP expression was detected in a wide range of tissues and upregulated by bacterial and viral infection. Subcellular localization analysis showed that SmLMWPTP was secreted by peripheral blood leukocytes (PBL) into the extracellular milieu. When PBL were treated with rSmLMWPTP, the cells exhibited significant reductions in (i) proliferative and respiratory burst activity, (ii) expression levels of multiple immune relevant genes, and (iii) phagocytic activity. In contrast, the mutant SmLMWPTP bearing R19 mutation had no effect on PBL activity. Taken together, these results indicate that SmLMWPTP is a secreted PTP that exerts a negative regulatory effect on the innate immune response of PBL in a manner that depends on the structural integrity of the P-loop.

The Apo-structure of the Low Molecular Weight Protein-tyrosine Phosphatase A (MptpA) from Mycobacterium tuberculosis Allows for Better Target-specific Drug Development

Protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases co-regulate cellular processes. In pathogenic bacteria, they are frequently exploited to act as key virulence factors for human diseases. Mycobacterium tuberculosis, the causative organism of tuberculosis, secretes a low molecular weight PTP (LMW-PTP), MptpA, which is required for its survival upon infection of host macrophages. Although there is otherwise no sequence similarity of LMW-PTPs to other classes of PTPs, the phosphate binding loop (P-loop) CX(5)R and the loop containing a critical aspartic acid residue (D-loop), required for the catalytic activity, are well conserved. In most high molecular weight PTPs, ligand binding to the P-loop triggers a large conformational reorientation of the D-loop, in which it moves ∼10 Å, from an "open" to a "closed" conformation. Until now, there have been no ligand-free structures of LMW-PTPs described, and hence the dynamics of the D-loop have remained largely unknown for these PTPs. Here, we present a high resolution solution NMR structure of the free form of the MptpA LMW-PTP. In the absence of ligand and phosphate ions, the D-loop adopts an open conformation. Furthermore, we characterized the binding site of phosphate, a competitive inhibitor of LMW-PTPs, on MptpA and elucidated the involvement of both the P- and D-loop in phosphate binding. Notably, in LMW-PTPs, the phosphorylation status of two well conserved tyrosine residues, typically located in the D-loop, regulates the enzyme activity. PtkA, the kinase complementary to MptpA, phosphorylates these two tyrosine residues in MptpA. We characterized the MptpA-PtkA interaction by NMR spectroscopy to show that both the P- and D-loop form part of the binding interface.

Cloning, purification, crystallization and preliminary X-ray analysis of two low-molecular-weight protein tyrosine phosphatases from Vibrio cholerae.

Low-molecular-weight protein tyrosine phosphatases (LMWPTPs) are small cytoplasmic enzymes of molecular weight ∼18 kDa that belong to the large family of protein tyrosine phosphatases (PTPs). Despite their wide distribution in both prokaryotes and eukaryotes, their exact biological role in bacterial systems is not yet clear. Two low-molecular-weight protein tyrosine phosphatases (VcLMWPTP-1 and VcLMWPTP-2) from the Gram-negative bacterium Vibrio cholerae have been cloned, overexpressed, purified by Ni(2+)-NTA affinity chromatography followed by gel filtration and used for crystallization. Crystals of VcLMWPTP-1 were grown in the presence of ammonium sulfate and glycerol and diffracted to a resolution of 1.6 Å. VcLMWPTP-2 crystals were grown in PEG 4000 and diffracted to a resolution of 2.7 Å. Analysis of the diffraction data showed that the VcLMWPTP-1 crystals had symmetry consistent with space group P3(1) and that the VcLMWPTP-2 crystals had the symmetry of space group C2. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient for the VcLMWPTP-1 crystals was estimated to be 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.4%. The corresponding values for the VcLMWPTP-2 crystals, assuming the presence of two molecules in the asymmetric unit, were 2.77 Å(3) Da(-1) and 55.62%, respectively.

Knocking Down Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) Reverts Chemoresistance through Inactivation of Src and Bcr-Abl Proteins

The development of multidrug resistance (MDR) limits the efficacy of continuous chemotherapeutic treatment in chronic myelogenous leukemia (CML). Low molecular weight protein tyrosine phosphatase (LMW-PTP) is up-regulated in several cancers and has been associated to poor prognosis. This prompted us to investigate the involvement of LMW-PTP in MDR. In this study, we investigated the role of LMW-PTP in a chemoresistant CML cell line, Lucena-1. Our results showed that LMW-PTP is highly expressed and 7-fold more active in Lucena-1 cells compared to K562 cells, the non-resistant cell line. Knocking down LMW-PTP in Lucena-1 cells reverted chemoresistance to vincristine and imatinib mesylate, followed by a decrease of Src and Bcr-Abl phosphorylation at the activating sites, inactivating both kinases. On the other hand, overexpression of LMW-PTP in K562 cells led to chemoresistance to vincristine. Our findings describe, for the first time, that LMW-PTP cooperates with MDR phenotype, at least in part, through maintaining Src and Bcr-Abl kinases in more active statuses. These findings suggest that inhibition of LMW-PTP may be a useful strategy for the development of therapies for multidrug resistant CML.

Tyrosine phosphorylation and bacterial virulence

Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria. In this article, we review the structure and function of bacterial tyrosine kinases and phosphatases. Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes (bacterial tyrosine (BY) kinases) that are characterized by the presence of Walker motifs. The reverse reaction is catalyzed by three classes of enzymes: the eukaryotic-like phosphatases (PTPs) and dual-specific phosphatases; the low molecular weight protein-tyrosine phosphatases (LMW-PTPs); and the polymerase–histidinol phosphatases (PHP). Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates, thereby contributing to bacterial pathogenicity. Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development. The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii. Ltp1 is upregulated by contact with S. gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2.

S-Glutathionylation of LMW-PTP regulates VEGF-mediated FAK activation and endothelial cell migration

Although promising, the ability to regulate angiogenesis through delivery of VEGF remains an unrealized goal. We have shown previously that physiological levels of peroxynitrite (1 µM) are required for a VEGF-mediated angiogenic response, yet the redox-regulated mechanisms that govern the VEGF signal remain unexplored. We assessed the impact of VEGF and peroxynitrite on modifying redox-state, the level of reduced-glutathione (GSH) and S-glutathionylation on regulation of the low molecular weight protein tyrosine phosphatase (LMW-PTP) and focal adhesion kinase (FAK), which are key mediators of VEGF-mediated cell migration. Stimulation of human microvascular endothelial (HME) cells with VEGF (20 ng/ml) or peroxynitrite (1 µM) caused an immediate and reversible negative-shift in the cellular redox-state and thiol oxidation of LMW-PTP, which culminated in cell migration. VEGF causes reversible S-glutathionylation of LMW-PTP, which inhibits its phosphorylation and activity, and causes the transient activation of FAK. Modulating the redox-state using decomposing peroxynitrite (FeTPPS, 2.5 µM) or the GSH-precursor [N-acetylcysteine (NAC), 1 mM] caused a positive-shift of the redox-state and prevented VEGF-mediated S-glutathionylation and oxidative inhibition of LMW-PTP. NAC and FeTPPS prevented the activation of FAK, its association with LMW-PTP and cell migration. Inhibiting LMW-PTP expression markedly enhanced FAK activation and cell migration. Although mild oxidative stress achieved by combining VEGF with 0.1-0.2 mM peroxynitrite augmented cell migration, an acute shift to oxidative stress achieved by combining VEGF with 0.5 mM peroxynitrite induced and sustained FAK activation, and LMW-PTP S-glutathionylation, resulting in LMW-PTP inactivation and inhibited cell migration. In conclusion, our findings demonstrate that a balanced redox-state is required for VEGF to facilitate reversible S-glutathionylation of LMW-PTP, FAK activation and endothelial cell migration. Shifting the redox-state to reductive stress or oxidative stress inhibited the VEGF-mediated angiogenic response.

Abstract P315: S-Glutathiolation of Low-Molecular-Weight PTP Regulates VEGF-Angiogenic Signal

While reparative angiogenesis improves outcome of stroke and myocardial infarction; pathological angiogenesis aggravates tumors, psoriasis and diabetic retinopathy. We have previously shown that low levels of peroxynitrite (PN, 1µM) are required to mediate VEGF’s angiogenic signal. Prior studies showed that low molecular weight protein tyrosine phosphatase (LMW-PTP) can regulate activation of VEGFR2 and focal adhesion kinase (FAK), key proteins involved in VEGF angiogenic signal. Here, we test the hypothesis that VEGF-induced peroxynitrite regulates LMW-PTP activity via reversible S-glutathiolation. Our results using human microvascular endothelial (HME) cells showed that VEGF (20 ng/ml) caused immediate (1,5 min) and transient negative shift in redox-state assessed by reduced-glutathione (GSH) level that was restored back to baseline by 15–30 minutes. In parallel, total free thiols of LMW-PTP; assessed by 5-iodoacetamide; were immediately oxidized (1, 5 min) and recovered after 15–30 minutes. PN (1μM) mimicked VEGF’ actions by inducing oxidative inhibition of LMW-PTP and FAK phosphorylation that peaked at 15 min. Studies using anti-GSH antibody demonstrated strong s-glutathiolation of LMW-PTP thiols that peaked at (5–10min) under non-reducing conditions and were completely abrogated in DTT-treated samples. These effects were associated with impaired tyrosine phosphorylation of LMW-PTP. PN caused concentration-dependent significant inhibition of LMW-PTP phosphatase activity reaching maximum at 0.5mM. Under pathological conditions recapitulated by combining with peroxynitrite (0.5mM), VEGF caused immediate yet permanent negative shift in cellular redox-state, LMW-PTP inactivation and sustained FAK activation in HME cells. Modulating cellular redox-state to reductive stress using N-acetyl cysteine (NAC, 1mM) or FeTPPS (2.5μM) prevented VEGF-induced angiogenesis in vitro and in vivo using hypoxia-induced neovascularization mouse model. Taken together, shifting redox-state to oxidative stress or reductive stress can impair VEGF’s signal and achieving a balanced redox-state is critical to facilitate VEGF’ angiogenic signal.

A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the functional properties of this hypothetical protein, open reading frame slr0328 was expressed in Escherichia coli. The purified recombinant protein, SynPTP, displayed its catalytic phosphatase activity towards several tyrosine, but not serine, phosphorylated exogenous protein substrates. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Kinetic analysis indicated that the K(m) and V(max) values for SynPTP towards p-nitrophenyl phosphate are similar to those of other known bacterial low molecular weight PTPs. Mutagenic alteration of the predicted catalytic cysteine of PTP, Cys(7), to serine abolished enzyme activity. Using a combination of immunodetection, mass spectrometric analysis and mutagenically altered Cys(7)SerAsp(125)Ala-SynPTP, we identified PsaD (photosystem I subunit II), CpcD (phycocyanin rod linker protein) and phycocyanin-α and -β subunits as possible endogenous substrates of SynPTP in this cyanobacterium. These results indicate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803.

DHEA attenuates PDGF-induced phenotypic proliferation of vascular smooth muscle A7r5 cells through redox regulation

It is known that dehydroepiandrosterone (DHEA) inhibits a phenotypic switch in vascular smooth muscle cells (VSMC) induced by platelet-derived growth factor (PDGF)-BB. However, the mechanism behind the effect of DHEA on VSMC is not clear. Previously we reported that low molecular weight-protein tyrosine phosphatase (LMW-PTP) dephosphorylates PDGF receptor (PDGFR)-β via a redox-dependent mechanism involving glutathione (GSH)/glutaredoxin (GRX)1. Here we demonstrate that the redox regulation of PDGFR-β is involved in the effect of DHEA on VSMC. DHEA suppressed the PDGF-BB-dependent phosphorylation of PDGFR-β. As expected, DHEA increased the levels of GSH and GRX1, and the GSH/GRX1 system maintained the redox state of LMW-PTP. Down-regulation of the expression of LMW-PTP using siRNA restored the suppression of PDGFR-β-phosphorylation by DHEA. A promoter analysis of GRX1 and γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme of GSH synthesis, showed that DHEA up-regulated the transcriptional activity at the peroxisome proliferator-activated receptor (PPAR) response element, suggesting PPARα plays a role in the induction of GRX1 and γ-GCS expression by DHEA. In conclusion, the redox regulation of PDGFR-β is involved in the suppressive effect of DHEA on VSMC proliferation through the up-regulation of GSH/GRX system.

ACP1 genotype, glutathione reductase activity, and riboflavin uptake affect cardiovascular risk in the obese

Erythrocyte acid phosphatase (ACP locus 1), also known as low–molecular-weight protein tyrosine phosphatase, has previously been associated to glycemia, dyslipidemia, and obesity. In this study, ACP1 genotype and activity were tested in 318 women aged 19 to 83 (mean, 51.74 ± 13.44) years. ACP1 genotype was found to directly correlate to glutathione reductase activity ( P < .001) and levels of low-density lipoprotein cholesterol ( P = .038). Glutathione reductase activity was in turn found to correlate to a series of cardiovascular risk factors such as systolic arterial pressure ( P < .001), total cholesterol levels ( P = .018), and low-density lipoprotein cholesterol levels ( P = .039). A possible protective effect of ACP1 genotype AA against these cardiovascular risk factors was observed in this study. Furthermore, this work hypothesizes that nutritional riboflavin uptake becomes more crucial as body mass index increases, to counteract oxidative stress and minimize cardiovascular risk. This might be especially true in ACP1 genotypes AC, BC, and CC, which might possibly show the least endogenous protection against oxidative stress.

A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation

Objective Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels. Design hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays. Results LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day ( p < 0.001) of culturing, decreasing thereafter. Conclusions Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. In this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation.