T-cell Protein Tyrosine Phosphatase Research Articles

The Potent Inhibitors of Protein Tyrosine Phosphatase 1B from the Fruits of Melaleuca leucadendron.

Background:Melaleuca leucadendron (Myrtaceae) is a kind of fruit used as Indonesian medicinal component and recorded in Jamu (tonic made of medical herbs) prescription records for the diabetes treatment. Its methanol extract exhibited a strong inhibitory activity with the half maximal inhibitory concentration (IC50) value of 2.05 μg/mL, while it is the same value with positive control RK-682.Objective:To isolate the chemical constituents of M. leucadendron and to evaluate their activity against protein tyrosine phosphatase 1B (PTP1B). Further, determine their toxicity potential against T-cell protein tyrosine phosphatase (TCPTP).Materials and Methods:Methanol extract was fractionated using silica column chromatography, and the obtained fraction was purified using Sephadex 20-LH. The structure of isolated compounds was identified based on 1H and 13Nuclear Magnetic Resonance Spectrometry. Furthermore, the compounds were examined against PTP1B and TCPTP.Results:Methanol extract of M. leucadendron (Myrtaceae) afforded two triterpenes: Betulinic acid and ursolic acid in high quantities. Both compounds exhibited a strong inhibitory activity against PTP1B inhibition with IC50 value of 1.5 and 2.3 μg/mL, respectively (positive control RK-682, IC50 = 2.05 μg/mL). Their activity toward TCPTP, on the other hand, were at 2.4 and 3.1 μg/mL, respectively. Based on this purification work, betulinic acid and ursolic acid presented 7.6% and 2.4%, respectively, as markedly M. leucadendron most potential for betulinic acid source among Indonesian plants. The result should have demonstrated that the antidiabetes of M. dendron could be through the inhibition of PTP1B.SUMMARY Melaleuca leucadendron is a good source for ursolic acid.Confirming traditional use for type II diabetes via PTP1B inhibition.

Synthesis of Aromatic Amide Derivatives and Their Biological Evaluation against Protein Tyrosine Phosphatase 1B and Scr Homology-2 Domain Containing Protein Tyrosine Phosphatase-2

To explore the chemical space of protein tyrosine phosphatase 1B (PTP1B) inhibitors by changing bis-aromatic amide moiety into aromatic amide moiety, a series of aromatic amide derivatives were designed, synthesized and their biological activities were evaluated against PTP1B and Scr homology-2 domain containing protein tyrosine phosphatase-2 (SHP2). Among them, compound 3c displayed moderate inhibitory activity with IC50 of (5.13±0.21) μmol/L against PTP1B and showed two times selectivity for other related PTPs. Interestingly, compound 12 [IC50=(7.47±1.26) μmol/L] showed moderate inhibitory activity against SHP2 and 2-fold selectivity against PTP1B, T-cell protein tyrosine phosphatase (TCPTP) or Src homology-2 domain containing protein tyrosine phosphatase-1 (SHP1) respectively, and offered a novel scaffold to develop new SHP2 inhibitors.

Abstract 4567: Activation of T-cell protein tyrosine phosphatase suppresses keratinocyte survival and proliferation following UVB irradiation

Abstract Chronic exposure of UV radiation can contribute to the development of skin cancer by promoting PTK signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces PTP inactivation. In the present work, we report that TC-PTP activity is stimulated during the initial response to UVB irradiation which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. In corroboration with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer. Note: This abstract was not presented at the meeting. Citation Format: Hyunseung Lee, Mihwa Kim, Liza D. Morales, Thomas J. Slaga, Dae Joon Kim. Activation of T-cell protein tyrosine phosphatase suppresses keratinocyte survival and proliferation following UVB irradiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4567. doi:10.1158/1538-7445.AM2015-4567

Discovery of novel, potent, selective and cellular active ADC type PTP1B inhibitors via fragment-docking-oriented de novel design

Fragment-docking-oriented de novel design for both the catalytic site and the C phosphotyrosine binding site led to the discovery of novel scaffold and chemical easy N-(2,5-diethoxy-phenyl)-methanesulfonamide based phosphotyrosine mimetics that when incorporated into ureas are high potent and selective inhibitors of protein tyrosine phosphatase 1B. Among them, compound 15 was shown to be the most potent PTP1B inhibitor with great selectivity over the highly homologous T-cell protein tyrosine phosphatase.

VSL#3 Probiotic Stimulates T‐Cell Protein Tyrosine Phosphatase‐Mediated Protection of Intestinal Epithelial Barrier Function

VSL#3 is a probiotic cocktail that has shown efficacy in treating murine colitis and patients with ulcerative colitis. VSL#3 can also exert a beneficial effect on epithelial barrier function which is dysregulated in inflammatory bowel disease (IBD). We have previously shown that the enzyme T-cell protein tyrosine phosphatase (TCPTP), encoded by the IBD candidate gene PTPN2, protects intestinal epithelial barrier function. The aim of this study was to identify if VSL#3 activates TCPTP and protects intestinal epithelial cells (IEC) from the effects of the IBD-associated pro-inflammatory cytokine interferon-gamma (IFN-γ). Administration of VSL#3 to T84 or HT-29 IEC polarized monolayers increased expression and enzymatic activity of TCPTP in a dose-dependent and time-dependent manner. VSL#3 reduced IFN-γ signaling, as reflected by decreased phosphorylation of STAT-1, in a TCPTP-dependent manner. In addition, VSL#3 alone increased IEC barrier integrity and promoted recovery of barrier function in IFN-γ pre-treated IEC monolayers. VSL#3 conditioned medium also promoted partial recovery from IFN-γ-induced barrier defects. Using a stable TCPTP-deficient HT-29 cell line, we observed that the recovery of TER induced by VSL#3 in IFN-γ treated cells was TCPTP-dependent. In conclusion, the probiotic VSL#3 increased activity and expression of TCPTP to reduce inflammatory signaling and correct epithelial barrier defects.

The interaction of the Hepatitis C Virus with intra-cellular signaling converts EGF into a potent inducer of the production of CXCR2-ligands by the infected host cell

Introduction: To establish persistent infection the hepatitis C virus (HCV) must have evolved strategies to escape antiviral and innate immunity and to influence the inflammatory response. Migration of the different cell types of adaptive and innate immunity is largely controlled by chemokines. Hence chemokines are decisive for the local composition of the different types of immune- or inflammatory cells and therefore play a major role for the formation of the local inflammatory- or immune-milieu. It is therefore not surprising that HCV has evolved strategies to gain influence on the expression of chemokines. The molecular mechanisms, which are employed by HCV to modify the expression of these factors, are incompletely understood. Likewise the influence of HCV on the regulation of chemokine expression in response to different stimuli is not clear. The present study investigates the impact of HCV on the production of the major chemo-attractants of neutrophils, the CXCR2 ligands CXCL1, 2, 3 and 8, in its host cell upon stimulation with the growth factor EGF. Methods: Huh-7 cells either infected with the HCV cc strain JC1 or harbouring the HCV subgenomic replicon were used and the impact of HCV on cellular signalling and gene expression was analysed employing targeted gene knockdown by siRNA, inhibitor-studies, rtPCR and Immunoblot-analysis. Results: Expression of the CXCR2 ligands in the hepatoma cell lines Huh-7 or Huh-7.5 is not or only weakly influenced upon stimulation with EGF. Therefore, the observation was surprising, that in the presence of either the sub-genomic or the full-length HCV replicon or upon infection of Huh-7.5 cells with the HCVcc strain JC1 EGF is converted in to a potent inducer of the different CXCR2 ligands. The further analysis of the molecular mechanism involved in this phenomenon revealed that with respect to the regulation of CXCL8 the NS3/4A mediated down-regulation of the endogenous feedback inhibitor of the EGF receptor (EGFR), the ubiquitously expressed T cell protein-tyrosine phosphatase (TC-PTP) and subsequent sensitization of the EGFR is important. Furthermore inhibitor studies and studies using targeted gene knockdown by specific siRNA suggest that downstream of the EGFR activation of the mitogen activated protein kinase (MAPK) activating protein kinase (MEK)1 and of its down-stream effector kinases the extracellular signal stimulated kinase (Erk)1 and 2 play a role for EGF inducible expression of CXCL8 in the presence of HCV. Interestingly this mechanism is not responsible for the EGFR-dependent up-regulation of the other CXCR2 ligands CXCL-1, 2 and 3 in HCV infected cells upon stimulation with EGF. Conclusion: The study provide evidence that HCV utilizes EGFR and EGFR-dependent signalling not only as cofactors for viral entry and replication but also interferes with EGF-dependent signalling thereby modifying the inter-cellular communication of the infected host cell.

Activation of T-cell Protein-tyrosine Phosphatase Suppresses Keratinocyte Survival and Proliferation following UVB Irradiation

Chronic exposure to UV radiation can contribute to the development of skin cancer by promoting protein-tyrosine kinase (PTK) signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces protein-tyrosine phosphatase (PTP) inactivation. In the present work, we report that T-cell PTP (TC-PTP) activity is stimulated during the initial response to UVB irradiation, which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. Corroborating with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure, and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer.

Phosphonate derivatives of tetraazamacrocycles as new inhibitors of protein tyrosine phosphatases.

α,α-Difluoro-β-ketophosphonated derivatives of tetraazamacrocycles were synthesized and found to be potential inhibitors of protein tyrosine phosphatases. N-Substituted conjugates of cyclam and cyclen with bioisosteric phosphonate groups displayed good activities toward T-cell protein tyrosine phosphatase with IC50 values in the micromolar to nanomolar range and showed selectivity over PTP1B, CD45, SHP2, and PTPβ. Kinetic studies indicated that the inhibitors can occupy the region of the active site of TC-PTP. This study demonstrates a new approach which employs tetraazamacrocycles as a molecular platform for designing inhibitors of protein tyrosine phosphatases.

The adaptor TRAF3 restrains the lineage determination of thymic regulatory T cells by modulating signaling via the receptor for IL-2.

The number of Foxp3+ regulatory T (Treg) cells must be tightly controlled to efficiently suppress autoimmunity while not impairing normal immune responses. Here we show that the adapter molecule TRAF3 is intrinsically required for restraining lineage determination of thymic Treg cells. T cell-specific TRAF3 deficiency resulted in a 2-3 fold increase of Treg cell frequency, due to more efficient transition from T precursors to Foxp3+ Treg cells. TRAF3 dampened interleukin-2 (IL-2) signaling by facilitating recruitment of T cell protein tyrosine phosphatase (TCPTP) to the IL-2 receptor complex, resulting in dephosphorylation of the signaling molecules Jak1 and Jak3 and negative regulation of Jak-STAT5 signaling. Our results identify a role for TRAF3 as an important negative regulator of IL-2 receptor signaling that impacts Treg cell development.

Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling

Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-β mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-β receptor (TβRII), which in turn promotes a TβRI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within TβRII is considered the principal regulatory mechanism of TβRII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate TβRII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the TβRII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated TβRII tail tyrosine residues, resulting in inhibition of TβR-dependent fibrotic signaling. The collagen-binding receptor integrin α1β1 was required for recruitment of TCPTP to the TβRII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of TβRII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin α1β1 and TβRII that is essential for TβRII-mediated SMAD activation and fibrotic signaling pathways.

Stable PTPN2 knockdown in human intestinal epithelial cell lines increases claudin‐2 expression and activation of MEK/ERK and PI3K signaling (1111.7)

Stable PTPN2 Knockdown in Human Intestinal Epithelial Cell Lines increases Claudin‐2 expression and activation of MEK/ERK and PI3K signaling Moorthy Krishnan and Declan F McCole, Division of Biomedical Sciences, University of California, Riverside, Riverside, CA 92521.Genome‐wide association studies identified protein tyrosine phosphatase non‐receptor type‐2 (PTPN2) as a susceptibility gene for inflammatory bowel disease (IBD), Diabetes and Celiac disease. Increased permeability of the intestinal epithelium is believed to be an important factor contributing to IBD. The aim of this study was to generate a stable PTPN2‐deficient intestinal epithelial cell line to investigate the role of PTPN2 and its protein product, T‐cell protein tyrosine phosphatase (TCPTP), in protecting epithelial barrier function. We developed Caco2‐BBE and HT‐29 intestinal epithelial cell lines (IECs) stably expressing short hairpin RNA for PTPN2. Western blotting and densitometry indicated that TCPTP protein expression was reduced by 50 ± 0.3% in PTPN2‐depleted Caco2‐BBE and HT‐29 cells, while there was a 3.5 ± 1.3 and 4.2 ± 1.5 fold increased expression of claudin‐2 in Caco2‐BBE and HT‐29 cells respectively. Furthermore, knockdown of PTPN2 resulted in decreased transepithelial electrical resistance (TER) (76 ± 3% and 50 ± 4%) in Caco2‐BBE and HT‐29 cells respectively) compared to control, and this correlated with the observed increase in claudin‐2 expression. Interestingly, Western blot and immunofluorescence studies showed that basal phosphorylation of ERK1/2 and Akt was increased in PTPN2 KD cells. In conclusion, we report an inverse relationship between PTPN2 (TCPTP) and claudin‐2 expression associated with increased activation of MEK/ERK and PI3K pathways in stable PTPN2‐deficient human IECs.Grant Funding Source: Supported by NIH R01DK091281

Synthesis and evaluation of several oleanolic acid glycoconjugates as protein tyrosine phosphatase 1B inhibitors

Sixteen novel oleanolic acid triterpenoid saponins were synthesized in an efficient and practical strategy, and their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) and selectivity over T-cell protein tyrosine phosphatase (TCPTP) were evaluated in vitro. The preliminary structure–activity relationship studies demonstrated that sugar-substituted moiety attached to the C-3 and C-28 positions of OA scaffold greatly affected the inhibitory activity against PTP1B and the selectivity over TCPTP. All the compounds showed inhibitory potencies, and compounds 1h, 1i and 1j exhibited remarkably potent inhibitory activities against PTP1B with IC50 values of 1.03, 0.78 and 3.12 μM, respectively. More significantly, compound 1h showed greater than 4 folds selectivity over highly homologous TCPTP. In parallel, the lipophilicity evaluation of all synthesized compounds was tested as a prediction for pharmacological potency. According to the predicted log P values, the predicted Log P results showed that lipophilicity may correlate with the evaluated biological potency.

Pancreatic T cell protein-tyrosine phosphatase deficiency ameliorates cerulein-induced acute pancreatitis

BackgroundAcute pancreatitis (AP) is a common clinical problem whose incidence has been progressively increasing in recent years. Onset of the disease is trigged by intra-acinar cell activation of digestive enzyme zymogens that induce autodigestion, release of pro-inflammatory cytokines and acinar cell injury. T-cell protein tyrosine phosphatase (TCPTP) is implicated in inflammatory signaling but its significance in AP remains unclear.ResultsIn this study we assessed the role of pancreatic TCPTP in cerulein-induced AP. TCPTP expression was increased at the protein and messenger RNA levels in the early phase of AP in mice and rats. To directly determine whether TCPTP may have a causal role in AP we generated mice with pancreatic TCPTP deletion (panc-TCPTP KO) by crossing TCPTP floxed mice with Pdx1-Cre transgenic mice. Amylase and lipase levels were lower in cerulein-treated panc-TCPTP KO mice compared with controls. In addition, pancreatic mRNA and serum concentrations of the inflammatory cytokines TNFα and IL-6 were lower in panc-TCPTP KO mice. At the molecular level, panc-TCPTP KO mice exhibited enhanced cerulein-induced STAT3 Tyr705 phosphorylation accompanied by a decreased cerulein-induced NF-κB inflammatory response, and decreased ER stress and cell death.ConclusionThese findings revealed a novel role for pancreatic TCPTP in the progression of cerulein-induced AP.

Cleavage of the T Cell Protein Tyrosine Phosphatase by the Hepatitis C Virus Nonstructural 3/4A Protease Induces a Th1 to Th2 Shift Reversible by Ribavirin Therapy

Ribavirin has proven to be a key component of hepatitis C therapies both involving IFNs and new direct-acting antivirals. The hepatitis C virus-mediated interference with intrahepatic immunity by cleavage of mitochondrial antiviral signaling protein (MAVS) and T cell protein tyrosine phosphatase (TCPTP) suggests an avenue for compounds that may counteract these effects. We therefore studied the effects of ribavirin, with or without inhibition of the nonstructural (NS)3/4A protease, on intrahepatic immunity. The intrahepatic immunity of wild-type and NS3/4A-transgenic mice was determined by Western blot, ELISA, flow cytometry, and survival analysis. Various MAVS or TCPTP constructs were injected hydrodynamically to study their relevance. Ribavirin pretreatment was performed in mice expressing a functional or inhibited NS3/4A protease to analyze its effect on NS3/4A-mediated changes. Intrahepatic NS3/4A expression made mice resistant to TNF-α-induced liver damage and caused an alteration of the intrahepatic cytokine (IFN-γ and IL-10) and chemokine (CCL3, CCL17, CCL22, CXCL9, and CXCL11) profiles toward an anti-inflammatory state. Consistent with this, the number of intrahepatic Th1 cells and IFN-γ(+) T cells in NS3/4A-transgenic mice decreased, whereas the amount of Th2 cells increased. These effects could be reversed by injection of uncleavable TCPTP but not uncleavable MAVS and were absent in a mouse expressing a nonfunctional NS3/4A protease. Importantly, the NS3/4A-mediated effects were reversed by ribavirin treatment. Thus, cleavage of TCPTP by NS3/4A induces a shift of the intrahepatic immune response toward a nonantiviral Th2-dominated immunity. These effects are reversed by ribavirin, supporting that ribavirin complements the effects of direct-acting antivirals as an immunomodulatory compound.

T-cell protein tyrosine phosphatase (TCPTP) regulates phosphorylation of Txk, a tyrosine kinase of the Tec family

Phosphorylation and dephosphorylation of intracellular enzymes, such as the T-cell-specific Tec family protein kinase Txk, are critically involved in the regulation of a number of cellular functions. Although PTPs located in the nucleus are limited, TCPTP is known to be one such nuclear phosphatase, due to its nuclear localization signal sequences. Here, we investigate the role of TCPTP in the regulation of Txk phosphorylation/dephosphorylation in Txk-transfected Cos7 and Jurkat cells. Nuclear-type TCPTP (TC45) was present in both the nuclear and cytoplasmic compartments of Cos7 cells transfected with TC45. Cytoplasmic-type TCPTP (TC48) was localized in the cytoplasmic compartment of Cos7 cells transfected with TC48. We observed that expression of TC45 dephosphorylated Txk in the nuclei of Cos7 cells. TC48 expression did not dephosphorylate Txk; rather, it enhanced and sustained Txk phosphorylation in the nuclei of Cos7 cells. Phosphorylation of Txk increased 60 minutes after lectin stimulation in Jurkat cells transfected with TCPTP-specific small interfering RNA (siRNA), which efficiently knocked down endogenous TCPTP. TCPTP may play a crucial role in the regulation of Txk phosphorylation status in T-cells after stimulation. Rec./11/5/2014, Acc.11/19/2014, pp240-246

TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication.

Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperature-sensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCα and PKCδ, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health.

1H-2,3-Dihydroperimidine Derivatives: A New Class of Potent Protein Tyrosine Phosphatase 1B Inhibitors

A series of 1H-2,3-dihydroperimidine derivatives was designed, synthesized, and evaluated as a new class of inhibitors of protein tyrosine phosphatase 1B (PTP1B) with IC50 values in the micromolar range. Compounds 46 and 49 showed submicromolar inhibitory activity against PTP1B, and good selectivity (3.48-fold and 2.10-fold respectively) over T-cell protein tyrosine phosphatases (TCPTP). These results have provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

Inhibition of PTPN2 by PTP inhibitor V

Protein phosphorylation is closely linked to critical pathways in numerous human diseases, which is regulated by the coordinated activities of kinases and phosphatases. Protein phosphorylation occurs predominantly on serine, threonine, and tyrosine residues of proteins and plays important roles in the regulation of physiological processes including gene expression, proliferation, differentiation, cell cycle arrest, and apoptosis. About 30% of proteins are phosphorylated in the mammalian cell. In case of eukaryotic cells, tyrosine phosphorylation occupies only 0.01-0.05% of total protein phosphorylation, while most of protein phosphorylation occurs on serine or threonine residues. However, growth factor stimulation or tumorigenesis enhances the level of tyrosine phosphorylation up to 1-2% of total protein phosphorylation. Protein phosphorylation is a reversible process that is controlled by protein kinases and protein phosphatases. In particular, protein tyrosine phosphatases (PTPs) are involved in most signaling pathways and thus play important roles in eukaryotes. PTPs superfamily comprises 107 enzymes in human genome. Based on the amino acid sequences of their catalytic domains, the PTPs can be grouped into four main families. Since PTPs play critical roles for cell homeostasis and thus diseases, chemical inhibitors that regulate PTPs have been extensively investigated to be used as therapeutic reagents. PTP inhibitor V, also known as phenyl hydrazono pyrazolone sulfate 1 (PHPS1), is 4-{N'-[3-(4-nitrophenyl)-5-oxo-1phenyl-1,5-dihydro-pyrazol-(4Z)-ylidene]-hydrazino}-benzenesulfonic acid (Fig. 1(a)). PTP inhibitor V was originally identified as a potent cell-permeable inhibitor that acts as active-site targeting, reversible, competitive inhibitor of SHP2 with the IC50 value of 2.1 μM. 5 PTP inhibitor V inhibits ECPTP, PTP1B and SHP-1 (IC50 = 5.4, 19, 30 μM, respectively). To investigate the effect of PTP inhibitor V on other PTPs, we performed in vitro phosphatase assays with purified recombinant PTPs (Table 1). We found that protein tyrosine phosphatase non-receptor type 2 (PTPN2) was inhibited by PTP inhibitor V while other PTPs were not. PTPN2 that is also called T cell protein tyrosine phosphatase (TC-PTP) belongs to class I Cys-based PTPs as the intracellular non-receptor PTPs with a high degree of sequence and structural homology within the catalytic domain. The mRNA levels of PTPN2 increase at G1 phase and return to basal level after G1 phase during cell cycle progression. 8

Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease

The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication. We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease.

Spermidine Stimulates T Cell Protein-tyrosine Phosphatase-mediated Protection of Intestinal Epithelial Barrier Function

The gene locus encoding protein-tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with inflammatory bowel disease. Expression of the PTPN2 gene product, T cell protein-tyrosine phosphatase (TCPTP), in intestinal epithelial cells has been shown to play an important role in the protection of epithelial barrier function during periods of inflammation by acting as a negative regulator of the proinflammatory cytokine IFN-γ. Therefore, agents that increase the activity of TCPTP are of general interest as modifiers of inflammatory signaling events. A previous study demonstrated that the small molecule spermidine is a selective activator of TCPTP in vitro. The aim of this study was to investigate whether activation of TCPTP by spermidine was capable of alleviating IFN-γ-induced, proinflammatory signaling and barrier dysfunction in human intestinal epithelial cells. Studies revealed that treatment of T84 and HT29/cl.19A colonocytes with spermidine increased both TCPTP protein levels and enzymatic activity, correlating with a decrease in the phosphorylation of the signal transducers and activators of transcription 1 and 3, downstream mediators of IFN-γ signaling, upon coadministration of spermidine to IFN-γ-treated cells. On a functional level, spermidine protected barrier function in the setting of inflammation, restricting the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ in coincubation experiments. These data implicate spermidine as a potential therapeutic agent to treat conditions associated with elevated IFN-γ signaling and a faulty mucosal barrier.