Inactivation Of PTP1B Research Articles

Abstract 12127: Protein Disulfide Isomerase A1 as Novel Redox Sensor in VEGFR2 Signaling and Angiogenesis

Background: VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. However, role of PDIA1 in ROS-dependent VEGFR2 signaling and angiogenesis is entirely unknown. Results: Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or early endosome marker Rab5, but not late endosome marker Rab7, at the perinuclear region in human ECs in response to VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration (43.3%), proliferation (52.8%) and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Experiments using “redox-dead” mutant PDIA1 with replacement of the active four Cys residues with Ser revealed that VEGF-induced PDIA1 CysOH formation promoted angiogenic responses by increasing phosphorylation of VEGFR2 at Y1175 via oxidative inactivation of PTP1B. In vivo, Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic CD31 positive ECs of ischemic muscles, and that limb perfusion recovery and neovascularization (48%) in response to ischemic injury were significantly impaired in EC-specific Pdia1 conditional knockout mice. Conclusion: PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs via oxidative inactivation of PTP1B, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases

ABCB10 exports mitochondrial biliverdin, driving metabolic maladaptation in obesity.

Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.

Qualitative Differences in Protection of PTP1B Activity by the Reductive Trx1 or TRP14 Enzyme Systems upon Oxidative Challenges with Polysulfides or H2O2 Together with Bicarbonate.

Protein tyrosine phosphatases (PTPs) can be regulated by several redox-dependent mechanisms and control growth factor-activated receptor tyrosine kinase phosphorylation cascades. Reversible oxidation of PTPs is counteracted by reductive enzymes, including thioredoxin (Trx) and Trx-related protein of 14 kDa (TRP14), keeping PTPs in their reduced active states. Different modes of oxidative inactivation of PTPs concomitant with assessment of activating reduction have been little studied in direct comparative analyses. Determining PTP1B activities, we here compared the potency of inactivation by bicarbonate-assisted oxidation using H2O2 with that of polysulfide-mediated inactivation. Inactivation of pure PTP1B was about three times more efficient with polysulfides as compared to the combination of bicarbonate and H2O2. Bicarbonate alone had no effect on PTP1B, neither with nor without a combination with polysulfides, thus strengthening the notion that bicarbonate-assisted H2O2-mediated inactivation of PTP1B involves formation of peroxymonocarbonate. Furthermore, PTP1B was potently protected from polysulfide-mediated inactivation by either TRP14 or Trx1, in contrast to the inactivation by bicarbonate and H2O2. Comparing reductive activation of polysulfide-inactivated PTP1B with that of bicarbonate- and H2O2-treated enzyme, we found Trx1 to be more potent in reactivation than TRP14. Altogether we conclude that inactivation of PTP1B by polysulfides displays striking qualitative differences compared to that by H2O2 together with bicarbonate, also with regard to maintenance of PTP1B activity by either Trx1 or TRP14.

New insights into TNFα/PTP1B and PPARγ pathway through RNF213- a link between inflammation, obesity, insulin resistance, and Moyamoya disease.

Diabetic patients are always at a higher risk of ischemic diseases like coronary artery diseases. One such ischemic carotid artery disease is Moyamoya disease (MMD) associated with diabetes Type I and II, but the causality was unclear. Ring Finger Protein 213 (RNF213) is the major susceptible gene for MMD. To understand the association between diabetes mellitus and MMD we chose the major players from both of the anomalies: insulin and RNF213. But before establishing the role of RNF213 in the insulin-regulating pathway we had to understand the involvement of RNF213 within different biological systems. For this, we have adopted a preliminary computational approach to find the prominent interactions of RNF213. Our first objective was to construct an interactome for RNF213. We have analyzed several curated databases and adapted a list of RNF213 interacting partners to develop its interactome. Then to understand the involvement of this interactome in biological functions we have analyzed major biological pathways, biological processes, and prominent clusters related to this interactome through a computational approach. Then to develop a pathway that might give clues for RNF213 involvement in the insulin regulatory pathway we have validated the intercluster and intracluster predictions and identified a regulatory pathway for RNF213. RNF213 interactome was observed to be involved in adaptive immunity with 4 major clusters; one of the clusters involved TNFα. The immune system involves several pathways, and therefore at this point, we have chosen an event-based strategy to obtain an explicit target. Immunity is mediated by pro-inflammatory cytokines like TNFα. TNFα-mediated inflammation, obesity, and insulin resistance are associated. Therefore we chose to explore the role of RNF213 in TNFα-mediated inflammation in macrophages and inflammation-mediated insulin-resistance in adipocytes. We have observed an enhancement of RNF213 gene expression by LPS mediated pro-inflammatory stimuli and suppression by PPARγ-mediated anti-inflammatory, insulin-sensitizing stimuli in macrophages, and also in adipocytes. Administration of the pro-inflammatory cytokine TNFα was able to impede the reduction in RNF213 expression during adipogenesis and this effect was observed to be mediated by PTP1B. Inactivation of PTP1B abolished RNF213 expression which in turn enhanced the adipogenesis process through enhanced PPARγ. Constitutive expression of RNF213 suppressed the adipocyte differentiation by the inhibition of PPARγ. We could show the regulation of RNF213 by TNFα/PTP1B pathway and PPARγ. The constitutive expression of RNF213 during adipogenesis appears to be an adipostatic measure that obese patients acquire to inhibit further adipogenesis. This is verified in silico by analyzing the gene expression data obtained from the Gene Expression Omnibus database, which showed a higher expression of RNF213 in adipose tissue samples of obese people. Overall this study gives new insights into the TNFα-mediated pathway in adipogenesis and suggests the role of RNF213 in adipogenesis via this pathway.

Characterization of protein expression of thyroid-responsive genes in cardiomyocyte PTP1B knockout mice in cardiac hypertrophy

Excessive Reactive Oxygen Species (ROS) production is involved in cardiac pathologies such as hypertrophy. Protein tyrosine phosphatases are regulated by reversible oxidation via ROS production. We demonstrated that in murine hearts undergoing hypertrophy, ROS production was associated with inactivation of the protein tyrosine phosphatase PTP1B. Moreover, PTP1B cardiac knockout (cKO) mice showed an exacerbated cardiac hypertrophy when subjected to pressure overload by transverse aortic constriction (TAC). Preventing thyroid hormone synthesis reversed the PTP1B-inactivation-driven pathological remodeling. Our aim was to investigate the protein expression of thyroid hormone-responsive genes possibly involved in the hypertrophic phenotype of TAC PTP1B cKO mice and in the bradycardia observed in cKO mice. 4 weeks post-surgery, TAC and sham hearts were harvested from control or PTP1B cKO mice for immunobloting from protein lysates. The protein expression of β-Myosin Heavy Chain (MHC), thyroid receptor alpha1 and beta 1 (TRα1, TRβ1), hyperpolarization-activated cyclic nucleotide-gated channels2/4 (HCN2, HCN4) and sarcoplasmic reticulum calcium ATPase2 (SERCA2) were studied. TAC hearts from PTP1B cKO mice showed a decreased expression of β-MHC and an increased expression of TRα1. TRβ1 was upregulated in both control and hypertrophic PTP1B cKO mice but SERCA2 or HCN4 protein levels did not show any significant differences between the two groups, subjected to TAC or not. Thyroid hormone signaling is altered following PTP1B inactivation in the heart and appears to occur mainly through changes in TRs isoforms expression. The limited sample size from our study ( n = 2) did not allow us to conclude definitely about HCN4 and SERCA2 expressions. Additional experiments will need to be performed to gain further insight on molecular mechanisms whereby PTP1B inactivation affects cardiac function and thyroid hormone signaling.

OP-22 - Bicarbonate is required for hydrogen peroxide-dependent inactivation of PTP1B in presence of the Trx system and peroxiredoxin

Protein tyrosine phosphatases need to be inhibited by oxidation of their active site cysteine during growth factor signaling. It is not clear how this inhibition is mediated but bicarbonate (HCO3-) has been shown to potentiate PTP1B inactivation. Therefore we investigated the effects of HCO3- and H2O2 on PTP1B activity in the presence of a reducing system constituted of Prx2, Trx1 and TrxR1. Recombinant PTP1B, TrxR1, Trx1 and Prx2 together with NADPH were used to mimic cellular growth factor-dependent PTP1B regulation in vitro. Effects of HCO3- and H2O2 exposure were assessed by PTP1B activity analysis. We found that HCO3-- and H2O2 inactivated PTP1B also in the presence of the complete TrxR1 / Trx1 / Prx system. Interestingly, a dynamic PTP1B inactivation pattern mimicking an oxidative burst was observed. The capacity for PTP1B reactivation was proportional to either TrxR1 or Trx1 concentrations. In summary, HCO3- and H2O2 in combination potently negatively regulates PTP1B activity. TrxR1 was previously shown to prevent H2O2 dependent PTP1B inactivation. Here we found that HCO3- with H2O2 together potently overcomes TrxR1- and Trx1 -mediated protection of PTP1B and that its subsequent reactivation is dependent upon Prx2, where H2O2 clearance becomes a key regulatory event.

Thioredoxin reductase 1 and NADPH directly protect protein tyrosine phosphatase 1B from inactivation during H2O2 exposure

Regulation of growth factor signaling involves reversible inactivation of protein tyrosine phosphatases (PTPs) through the oxidation and reduction of their active site cysteine. However, there is limited mechanistic understanding of these redox events and their co-ordination in the presence of cellular antioxidant networks. Here we investigated interactions between PTP1B and the peroxiredoxin 2 (Prx2)/thioredoxin 1 (Trx1)/thioredoxin reductase 1 (TrxR1) network. We found that Prx2 becomes oxidized in PDGF-treated fibroblasts, but only when TrxR1 has first been inhibited. Using purified proteins, we also found that PTP1B is relatively insensitive to inactivation by H2O2 but found no evidence for a relay mechanism in which Prx2 or Trx1 facilitates PTP1B oxidation. Instead, these proteins prevented PTP1B inactivation by H2O2. Intriguingly, we discovered that TrxR1/NADPH directly protects PTP1B from inactivation when present during the H2O2 exposure. This protection was dependent on the concentration of TrxR1 and independent of Trx1 and Prx2. The protection was blocked by auranofin and required an intact selenocysteine residue in TrxR1. This activity likely involves reduction of the sulfenic acid intermediate form of PTP1B by TrxR1 and is therefore distinct from the previously described reactivation of end-point oxidized PTP1B, which requires both Trx1 and TrxR1. The ability of TrxR1 to directly reduce an oxidized phosphatase is a novel activity that can help explain previously observed increases in PTP1B oxidation and PDGF receptor phosphorylation in TrxR1 knockout cells. The activity of TrxR1 is therefore of potential relevance for understanding the mechanisms of redox regulation of growth factor signaling pathways.

Phospho-Regulation of Soma-to-Axon Transcytosis of Neurotrophin Receptors

Axonal targeting of signaling receptors is essential for neuronal responses to extracellular cues. Here, we report that retrograde signaling by target-derived nerve growth factor (NGF) is necessary for soma-to-axon transcytosis of TrkA receptors in sympathetic neurons, and we define the molecular underpinnings of this positive feedback regulation that enhances neuronal sensitivity to trophic factors. Activated TrkA receptors are retrogradely transported in signaling endosomes from distal axons to cell bodies, where they are inserted on soma surfaces and promote phosphorylation of resident naive receptors, resulting in their internalization. Endocytosed TrkA receptors are then dephosphorylated byPTP1B, an ER-resident protein tyrosine phosphatase, prior to axonal transport. PTP1B inactivation prevents TrkA exit from soma and causes receptor degradation, suggesting a "gatekeeper" mechanism that ensures targeting of inactive receptors to axonsto engage with ligand. In mice, PTP1B deletion reduces axonal TrkA levels and attenuates neuron survival and target innervation under limiting NGF (NGF+/-) conditions.

Abstract 88: Cardiac-specific Deletion of Protein Tyrosine Phosphatase 1B Exacerbates Pressure Overload-induced Hypertrophy and Heart Failure in Mice

Cardiac hypertrophy involves the re-expression of a foetal gene program that occurs when cardiomyocytes are continuously exposed to stresses. The enlargement initially improves cardiac function, however, this compensatory hypertrophy predisposes individuals to arrhythmias, pathological hypertrophy and heart failure. Given the importance of reactive oxygen species (ROS) in the transition from cardiac hypertrophy to heart failure and the documented inhibition of PTPs by ROS, we hypothesized and explored whether specific PTPs could act as checkpoints in this process. We have identified PTP1B as a target of ROS in hearts undergoing hypertrophy. To better understand the role of PTP1B inhibition in cardiac hypertrophy, we generated cardiomyocyte-specific PTP1B knockout (PTP1B cKO) mice. Subjecting PTP1B cKO mice to pressure overload (PO) caused a dramatic left ventricular dilation and several distinctive features of heart failure when compared to control mice subjected to PO for the same period. Characterization of the mRNAs expressed in the hypertrophy-associated foetal gene program revealed that although PO led to increased mRNA levels of ANF and BNP, the increased expression of β-MHC observed in control mice subjected to PO was compromised in PTP1B cKO-PO mice. Since PTP1B inactivation can lead to the inactivation of AGO2 and compromise miRNA-mediated mRNAs repression, we investigated whether PTP1B regulated AGO2 phosphorylation and association with mRNAs in this context. We observed that AGO2 phosphotyrosine-393 levels were elevated and that AGO2 was a substrate of PTP1B in myocytes and in hearts undergoing hypertrophy. We also found changes in AGO2-mRNA associations between control- and PTP1B cKO-PO hearts and identified MED13 as a regulator of β-MHC expression that was differentially regulated by AGO2 in PTP1B cKO-PO hearts. Since increased expression of β-MHC contributes to the compensatory response that initially improves cardiac function, we will propose a model in which PTP1B inhibition regulates AGO2 activity and contributes to heart failure.

Inactivation of protein tyrosine phosphatases by dietary isothiocyanates.

Isothiocyanates are bioactive dietary phytochemicals that react readily with protein thiol groups. We find that isothiocyanates are time-dependent inactivators of cysteine-dependent protein tyrosine phosphatases (PTPs). Rate constants for the inactivation of PTP1B and SHP-2 by allyl isothiocyanate and sulforaphane range from 2 to 16 M(-1)s(-1). Results in the context of PTP1B are consistent with a mechanism involving covalent, yet reversible, modification of the enzyme's active site cysteine residue.

0294 : PTP1B endothelial gene deletion limits cardiovascular dysfunction and mortality during experimental septic shock

Sepsis-associated myocardial and vascular dysfunction is one of the main causes of mortality in critically ill patients, for which no efficacious therapies exist. We recently showed that PTP1B gene deletion affords myocardial protection in experimental heart failure. However, whether PTP1B inactivation opposes sepsis-associated myocardial dysfunction is unknown. Thus, we assessed the effect of endothelial PTP1B gene specific deletion on left ventricular (LV) function and inflammation in mice with endotoxinic shock. We used endoPTP1B-/- mice obtained with classical Cre-loxP system. To obtain endoPTP1B-/- we crossed Tie2-Cre (endothelial-specific promoter) with PTP1B-floxed (flox). Septic shock is induced by intraperitoneal injection of lipopolysaccharide (LPS) followed by a subcutaneous fluid resuscitation at 1 and 5 hours after LPS injection. Differents studies were realized at 8 hours after LPS injection. LV function was evaluated by echocardiography. Vascular function was evaluated by arteriography. Plasma samples were collected to measure the production of inflammatory cytokines by Elisa multiplex assay. Plasmatic levels of TNF-α and IL-6 are increased 8 hours after LPS injection showing a systemic inflammatory response with no significant difference between the two groups. Flow-mediated dilatation (FMD) assessed in isolated-perfused mesenteric arteries was abolished by LPS in flox mice and was improved in endoPTP1B-/- mice. Left ventricular systolic function is altered by LPS injection. In flox mice, LPS induce severe vascular dysfunction shown by the decreased fractional shortening and cardiac index which was improved in endoPTP1B-/- mice. Endo PTP1B-/- mice showed a marked decrease in mortality. Finally, endothelial PTP1B deletion in mice causes an improvement in cardiovascular function and survival. Additional molecular biological studies will be conducted to understand the mechanisms involved in inflammation, oxidative stress and endoplasmic reticulum stress.

Proteinase-activated receptor 1- and 4-promoted migration of Hep3B hepatocellular carcinoma cells depends on ROS formation and RTK transactivation.

There is growing evidence for a role of proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, in cancer. We have previously shown that PAR1 and PAR4 are able to promote the migration of hepatocellular carcinoma (HCC) cells suggesting a function in HCC progression. In this study, we assessed the underlying signalling mechanisms. Using Hep3B liver carcinoma cells, RTK activation was assessed by Western blot employing phospho-RTK specific antibodies, ROS level were estimated by H2DCF-DA using confocal laser scanning microscopy, and measurement of PTP activity was performed in cell lysates using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a substrate. Thrombin, the PAR1 selective agonist peptide TFLLRN-NH2 (PAR1-AP), and the PAR4 selective agonist peptide, AYPGKF-NH2 (PAR4-AP), induced a significant increase in Hep3B cell migration that could be blocked by inhibitors targeting formation of reactive oxygen species (ROS), or activation of hepatocyte-growth factor receptor (Met), or platelet-derived growth factor receptor (PDGFR), respectively. The involvement of these intracellular effectors in PAR1/4-initiated migratory signalling was further supported by the findings that individual stimulation of Hep3B cells with the PAR1-AP and the PAR4-AP induced an increase in ROS production and the transactivation of Met and PDGFR. In addition, PAR1- and PAR4-mediated inhibition of total PTP activity and specifically PTP1B. ROS inhibition by N-acetyl-L-cysteine prevented the inhibition of PTP1B phosphatase activity induced by PAR1-AP and the PAR4-AP, but had no effect on PAR1/4-mediated activation of Met and PDGFR in Hep3B cells. Collectively, our data indicate that PAR1 and PAR4 activate common promigratory signalling pathways in Hep3B liver carcinoma cells including activation of the receptor tyrosine kinases Met and PDGFR, the formation of ROS and the inactivation of PTP1B. However, PAR1/4-triggered Met and PDGFR transactivation seem to be mediated independently from the ROS-PTP1B signalling module.

New pROSpects for PTP1B: micro-Managing Oncogene-Induced Senescence

Oncogene-induced senescence (OIS) provides an important, but incompletely understood, barrier to tumorigenesis. In this issue, Yang etal. (2014) surprisingly report that inactivation of PTP1B by reactive oxygen species is essential for OIS, via effects on AGO2 and microRNA maturation.

Chromium-containing traditional Chinese medicine, Tianmai Xiaoke Tablet improves blood glucose through activating insulin-signaling pathway and inhibiting PTP1B and PCK2 in diabetic rats

Chromium is an essential mineral that is thought to be necessary for normal glucose homeostasis. Numerous studies give evidence that chromium picolinate can modulate blood glucose and insulin resistance. The main ingredient of Tianmai Xiaoke (TMXK) Tablet is chromium picolinate. In China, TMXK Tablet is used to treat type 2 diabetes. This study investigated the effect of TMXK on glucose metabolism in diabetic rats to explore possible underlying molecular mechanisms for its action. Diabetes was induced in rats by feeding a high-fat diet and subcutaneously injection with a single dose of streptozotocin (50 mg/kg, tail vein). One week after streptozotocin-injection, model rats were divided into diabetic group, low dose of TMXK group and high dose of TMXK group. Eight normal rats were used as normal control. After 8 weeks of treatment, skeletal muscle was obtained and was analyzed using Roche NimbleGen mRNA array and quantitative polymerase chain reaction (qPCR). Fasting blood glucose, oral glucose tolerance test and homeostasis model assessment of insulin resistance (HOMA-IR) index were also measured. The authors found that the administration of TMXK Tablet can reduce the fasting blood glucose and fasting insulin level and HOMA-IR index. The authors also found that 2 223 genes from skeletal muscle of the high-dose TMXK group had significant changes in expression (1 752 increased, 471 decreased). Based on Kyoto encyclopedia of genes and genomes pathway analysis, the most three significant pathways were "insulin signaling pathway", "glycolysis/gluconeogenesis" and "citrate cycle (TCA)". qPCR showed that relative levels of forkhead box O3 (FoxO3), phosphoenolpyruvate carboxykinase 2 (Pck2), and protein tyrosine phosphatase 1B (Ptp1b) were significantly decreased in the high-dose TMXK group, while v-akt murine thymoma viral oncogene homolog 1 (Akt1) and insulin receptor substrate 2 (Irs2) were increased. Our data show that TMXK Tablet reduces fasting glucose level and improves insulin resistance in diabetic rats. The mechanism may be linked to the inactivation of PTP1B and PCK enzymes, or through intracellular pathways, such as the insulin signaling pathway.

Effect of oxidative stress on protein tyrosine phosphatase 1B in scleroderma dermal fibroblasts

Platelet-derived growth factor (PDGF) and its receptor, PDGFR, promote fibrosis in systemic sclerosis (SSc; scleroderma) dermal fibroblasts, and such cells in scleroderma skin lesions produce excessive reactive oxygen species (ROS). PDGFR is phosphorylated upon PDGF stimulation, and is dephosphorylated by protein tyrosine phosphatases (PTPs), including PTP1B. This study was undertaken to determine whether the thiol-sensitive PTP1B is affected by ROS in SSc dermal fibroblasts, thereby enhancing the phosphorylation of PDGFR and synthesis of type I collagen. This study also sought to investigate the effect of a thiol antioxidant, N-acetylcysteine (NAC), in SSc. Fibroblasts were isolated from the skin of patients with diffuse SSc and normal healthy donors for cell culture experiments and immunofluorescence analyses. A phosphate release assay was used to determine the activity of PTP1B. Levels of ROS and type I collagen were significantly higher and amounts of free thiol were significantly lower in SSc fibroblasts compared to normal fibroblasts. After stimulation with PDGF, not only were PDGFR and ERK-1/2 phosphorylated to a greater extent, but also the ability to produce PTP1B was hampered in SSc fibroblasts. The activity of PTP1B was significantly inactivated in SSc fibroblasts as a result of cysteine oxidation by the raised levels of ROS, which was confirmed by the oxidation of multiple PTPs, including PTP1B, in SSc fibroblasts. Decreased expression of PTP1B in normal fibroblasts led to increased expression of type I collagen. Treatment of the cells with NAC restored the activity of PTP1B, improved the profile of PDGFR phosphorylation, decreased the numbers of tyrosine-phosphorylated proteins and levels of type I collagen, and scavenged ROS in SSc fibroblasts. This study describes a new mechanism by which ROS may promote a profibrotic phenotype in SSc fibroblasts through the oxidative inactivation of PTP1B, leading to pronounced activation of PDGFR. The study also presents a novel molecular mechanism by which NAC may act on ROS and PTP1B to provide therapeutic benefit in SSc.

Inactivating PTP1B upon ER stress

The gaseous signalling molecule hydrogen sulphide (H2S) regulates a range of cellular processes by causing the sulphydration of Cys residues (that is, the formation of Cys–SSH groups) in target proteins. Protein Tyr phosphatases (PTPs) use an essential Cys (Cys215) in their catalytic site to dephosphorylat e Tyr residues in target proteins. Krishnan et al. now report that the production of H2S upon endo­ plasmic reticulum (ER) stress results in the sulphydration and consequent inhibition of PTP1B, and that this promotes signalling by one branch of the unfolded protein response (UPR) pathway. The authors first measured PTP1B phosphatase activity in vitro, in the presence or absence of H2S, observing that PTP1B can be inhibited by this molecule. PTP1B inactivation by H2S is reversible, as its catalytic activity was restored by the addition of reducing agents that are known to reverse sulphydration. Using mass spectrometry, the authors established that Cys215 is specifically sulphydrated in response to H2S, and further experiments confirmed that PTP1B is sulphydrated at Cys215 in vivo. Together, these data reveal that PTP1B is sulphydrated and that its catalytic activity is inhibited by this modification. But, in what physiological set­ ting might sulphydration regulate PTP1B activity? PTP1B is localized to the ER and has been implicated in UPR signalling, which is activated in response to ER stress. As the production of H2S by the enzyme cystathionine γ­lyase (CSE) has also P O S TT R A N S L AT I O N A L M O D I F I C AT I O N

Regulation of ROS signal transduction by NADPH oxidase 4 localization.

Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER). ER localization of Nox4 is critical for the regulation of protein tyrosine phosphatase (PTP) 1B, also an ER resident, through redox-mediated signaling. Nox4-mediated oxidation and inactivation of PTP1B in the ER serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking and specifically acts to terminate EGF signaling. Consistent with this notion, PTP1B oxidation could also be modulated by ER targeting of antioxidant enzymes but not their untargeted counterparts. These data indicate that the specificity of intracellular ROS-mediated signal transduction may be modulated by the localization of Nox isoforms within specific subcellular compartments.

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.

Chemical Knockdown of Protein-tyrosine Phosphatase 1B by 1,2-Naphthoquinone through Covalent Modification Causes Persistent Transactivation of Epidermal Growth Factor Receptor

1,2-Naphthoquinone (1,2-NQ), an atmospheric contaminant, causes the contraction of guinea pig trachea through the activation of epidermal growth factor receptor (EGFR) by inhibiting protein-tyrosine phosphatases (PTPs). Phosphorylation of EGFR is negatively regulated by PTPs, but details of the mechanism by which 1,2-NQ inhibits PTPs have not been elucidated. Results described in this report demonstrate that 1,2-NQ forms covalent bonds with PTP1B after exposure to human epithelial A431 cells. In this study, a concentration-dependent phosphorylation of EGFR was found to be coupled to the reduction of PTP activity in the cells. The reduction in PTP activity was due to the irreversible modification of PTP1B, and when PTP1B was overexpressed by the cells, the 1,2-NQ-mediated EGFR phosphorylation was suppressed. Studies with purified PTP1B and 1,2-NQ showed that the reduction in enzyme activity was due to a nucleophilic attack by the quinone on the enzyme, to form covalent bonds. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry analysis and mutation experiments revealed that PTP1B inactivation was primarily due to covalent attachment of the quinone to Cys-121 of the enzyme, with binding to His-25 and Cys-215 as well. Collectively, the results show that covalent attachment of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged transactivation of EGFR in the cells.

Kinetics and Mechanism of Protein Tyrosine Phosphatase 1B Inactivation by Acrolein

Human cells are exposed to the electrophilic alpha,beta-unsaturated aldehyde acrolein from a variety of sources. The reaction of acrolein with functionally critical protein thiol residues can yield important biological consequences. Protein tyrosine phosphatases (PTPs) are an important class of cysteine-dependent enzymes whose reactivity with acrolein previously has not been well-characterized. These enzymes catalyze the dephosphorylation of phosphotyrosine residues on proteins via a phosphocysteine intermediate. PTPs work in tandem with protein tyrosine kinases to regulate a number of critically important mammalian signal transduction pathways. We find that acrolein is a potent time-dependent inactivator of the enzyme PTP1B ( k inact = 0.02 +/- 0.005 s (-1) and K I = 2.3 +/- 0.6 x 10 (-4) M). The enzyme activity does not return upon gel filtration of the inactivated enzyme, and addition of the competitive phosphatase inhibitor vanadate slows inactivation of PTP1B by acrolein. Together, these observations suggest that acrolein covalently modifies the active site of PTP1B. Mass spectrometric analysis reveals that acrolein modifies the catalytic cysteine residue at the active site of the enzyme. Aliphatic aldehydes such as glyoxal, acetaldehyde, and propanal are relatively weak inactivators of PTP1B under the conditions employed here. Similarly, unsaturated aldehydes such as crotonaldehyde and 3-methyl-2-butenal bearing substitution at the alkene terminus are poor inactivators of the enzyme. Overall, the data suggest that enzyme inactivation occurs via conjugate addition of the catalytic cysteine residue to the carbon-carbon double bond of acrolein. The results indicate that inactivation of PTPs should be considered as a possible contributor to the diverse biological activities of acrolein and structurally related alpha,beta-unsaturated aldehydes.