ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.

Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3ß decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.

An Insight into MptpB Inhibitors as a Key Strategy to Treat MDR and XDRTuberculosis.

Tuberculosis (TB) is a chronic, air-borne infectious disease caused by Mycobacterium tuberculosis (Mtb), which prominently affects the lungs and usually manifests in other organs. TB is preventable and curable but what makes it challenging is the emergence of resistance to the available treatment options. MDR-continued TB's expansion is one of the world's most pressing and difficult problems. Mtb revives via the reciprocity between Mycobacterium and host signalling pathways. Mtb secretes a virulence component called Mycobacterium tuberculosis protein tyrosine phosphatase (MptpB), which helps to survive against host macrophages. It indicates that targeting secreted virulence factors offers more benefits to circumvent the emergence of resistance. Many effective inhibitors of MptpA and MptpB have been discovered, providing a solid foundation for future research and development. Aside from possessing a structurally unique binding site in the Mtb enzyme, MptpB's minimal resemblance to other human phosphatases provides a broad platform for improving selectivity over host PTPs. We believe that addressing several parts of infection processes in the host and bacteria with combination therapy is the greatest way to reduce treatment burden and medication resistance. We have discussed the recent potent, selective, and efficacious MptpB inhibitors, such as natural and marine-based, isoxazole- linked carboxylic acid-based, oxamic acid-based, and lactone-based inhibitors, as potential strategies for treating TB.

1H-1,2,3-Triazole-4H-chromene-D-glucose hybrid compounds: Synthesis and inhibitory activity against Mycobacterium tuberculosis protein tyrosine phosphatase B.

A series of 1H-1,2,3-triazole-4H-chromene-D-glucose hybrid compounds 7a-w were synthesized using click chemistry of 2-amino-7-propargyloxy-4H-chromene-3-carbonitriles 5a-w. CuNPs@montmorillonite was used as a catalyst in the presence of DIPEA as an additive for this chemistry. All synthesized 1H-1,2,3-triazoles were examined for in vitro inhibition against Mycobacterium tuberculosis protein tyrosine phosphatase B (MtbPtpB). Nine 1H-1,2,3-triazoles, including 7c-e, 7h, 7i, and 7r-t, displayed remarkable inhibitory activity against MtbPtpB with IC50 < 10 µM; compound 7t exhibited the most potent inhibition in vitro with an IC50 value of 0.61 µM. Kinetic studies of the three most active compounds, 7c,h,t, showed their competitive inhibition toward the MtbPtpB enzyme. Induced-fit docking and MM-GBSA studies on the enzyme (PDB: 2OZ5) revealed that the most active compound 7t was more effective against MtbPtpB. Residues Arg64, Arg136, Ash165, Arg166, and Arg63 in the binding pocket were identified as potential ligand-binding hot-spot residues for ligand 7t. The binding free energy calculation by the MM-GBSA approach for ligand 7t indicated that Coulomb, lipophilic, and van der Waals energy terms are major contributors to the inhibitor binding. Furthermore, the stability of the ligand-protein complex and the structural insights into the mode of binding were confirmed by 300-ns molecular dynamics simulation of 7t/2OZ5.

Disruption of Mitochondrial-associated ER membranes by HIV-1 tat protein contributes to premature brain aging.

INTRODUCTION: Mitochondrial-associated ER membranes (MAMs) control many cellular functions, including calcium and lipid exchange, intracellular trafficking, and mitochondrial biogenesis. The disruption of these functions contributes to neurocognitive disorders, such as spatial memory impairment and premature brain aging. Using neuronal cells, we demonstrated that HIV-1 Tat protein deregulates the mitochondria. METHODS& RESULTS: To determine the mechanisms, we used a neuronal cell line and showed that Tat-induced changes in expression and interactions of both MAM-associated proteins and MAM tethering proteins. The addition of HIV-1 Tat protein alters expression levels of PTPIP51 and VAPB proteins in the MAM fraction but not the whole cell. Phosphorylation of PTPIP51 protein regulates its subcellular localization and function. We demonstrated that the Tat protein promotes PTPIP51 phosphorylation on tyrosine residues and prevents its binding to VAPB. Treatment of the cells with a kinase inhibitor restores the PTPIP51-VAPB interaction and overcomes the effect of Tat. CONCLUSION: These results suggest that Tat disrupts the MAM, through the induction of PTPIP51 phosphorylation, leading to ROS accumulation, mitochondrial stress, and altered movement. Hence, we concluded that interfering in the MAM-associated cellular pathways contributes to spatial memory impairment and premature brain aging often observed in HIV-1-infected patients.

MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE 1 mediates root sensing of serotonin through jasmonic acid signaling and modulating reactive oxygen species.

Serotonin (5-hydroxytryptamine) acts as a neurotransmitter in mammals and is widely distributed in the plant kingdom, where it influences root growth and defense. Mitogen-Activated Protein Kinases (MAPKs) and MAPK phosphatases (MKPs) play critical functions in decoding hormonal signalling, but their possible roles in mediating serotonin responses await investigation. In this report, we unveiled positive roles for the MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE1 (MKP1) in the inhibition of the primary root growth, cell division, meristem structure, and differentiation events in Arabidopsis seedlings. mkp1 mutants were less sensitive to jasmonic acid applications that halted primary root growth in wild-type (WT) plants, and consistently, the neurotransmitter activated the expression of the JASMONATE ZIM-domain (JAZ) proteins JAZ1 and JAZ10, two critical proteins orchestrating jasmonic acid signalling. This effect correlated with exacerbated production of endogenous reactive oxygen species (ROS) in the WT, a process constitutively manifested in mkp1 mutants. These data help to clarify the relationship between serotonin and growth/defense trade-offs, and reveal the importance of the MAPK pathway in root development through ROS production.

Oxidation of the Mycobacterium tuberculosis key virulence factor protein tyrosine phosphatase A (MptpA) reduces its phosphatase activity.

The Mycobacterium tuberculosis tyrosine-specific phosphatase MptpA and its cognate kinase PtkA are prospective targets for anti-tuberculosis drugs as they interact with the host defense response within the macrophages. Although both are structurally well-characterized, the functional mechanism regulating their activity remains poorly understood. Here, we investigate the effect of post-translational oxidation in regulating the function of MptpA. Treatment of MptpA with H2 O2 /NaHCO3 , mimicking cellular oxidative stress conditions, leads to oxidation of the catalytic cysteine (C11) and to a conformational rearrangement of the phosphorylation loop (D-loop) by repositioning the conserved tyrosine 128 (Y128) and generating a temporarily inactive preclosed state of the phosphatase. Thus, the catalytic cysteine in the P-loop acts as a redox switch and regulates the phosphatase activity of MptpA.

Downregulation of secreted frizzled-related protein 4 inhibits hypoxia/reoxygenation injury in diabetic cardiomyocytes by protein tyrosine phosphatase nonreceptor type 12.

Myocardial ischemia-reperfusion injury in diabetic patients leads to an increased incidence of complications and mortality. Secreted frizzled-related protein 4 (SFRP4) plays a critical role in diabetic myocardial ischemia-reperfusion. This paper aims to uncover the underlying mechanisms of SFRP4 in hypoxia/reoxygenation (H/R) injury of diabetic myocardial cells. An in vitro ischemia/reperfusion (I/R) injury model was established using high glucose-induced H9c2 cardiomyocytes. Expression of SFRP4 was detected by real-time reverse transcriptase-polymerase chain reaction and Western blotting. After transfection of SFRP4, the binding of SFRP4 to protein tyrosine phosphatase nonreceptor type 12 (PTPN12) was predicted by database and verified by co-immunoprecipitation assay. P13 K/AKT protein levels were examined by Western blotting. PTPN12 levels were tested by RT-qPCR and Western blotting, cell viability by Cell Counting Kit-8, lactose dehydrogenase kit, terminal dUTP nick-end labeling assay, and cell inflammation and oxidative stress by Western blotting and enzyme linked immunosorbent assay. After overexpression of PTPN12, the experiments for cell viability, inflammation and oxidative stress were repeated once more. SFRP4 expression was upregulated in a high-glucose-stimulated H/R cardiomyocyte model. The interference of SFRP4 promoted cell viability, inhibited the inflammatory and oxidative stress response of H/R cardiomyocytes induced by high glucose. SFRP4 interacted with PTPN12 and inhibited the PI3K/AKT signaling pathway. PTPN12 overexpression reversed the inhibitory effect of sh-SFRP4 on H/R cardiomyocyte damage induced by high glucose. Downregulation of SFRP4 inhibited H/R cell damage in diabetic cardiomyocytes by binding to PTPN12.

Role of Protein Tyrosine Phosphatase in Regulation of Cell Signaling Cascades Affecting Tumor Cell Growth: A Future Perspective as Anti-Cancer Drug Target.

Protein Tyrosine Phosphatase (PTP) superfamily is a key enzyme involved in the regulation of growth-related cell signaling cascades, such as the RAS/MAPK pathway, that directly affect cancer cell growth and metastasis. Several studies have indicated that the drug resistance observed in several late-stage tumors might also be affected by the levels of PTP in the cell. Hence, these phosphatases have been in the limelight for the past few decades as potential drug targets and several promising drug candidates have been developed, even though none of these drugs have reached the market yet. In this review, we explore the potential of PTP as a viable anti-cancer drug target by studying PTPs, their regulation of several key cancer cell signaling pathways, and how their levels affect various types of cancer. Furthermore, we present the current scenario of PTP as a molecular target and the various challenges faced in the development of PTP-targeting anti-cancer drugs.

PRL3 induces polypoid giant cancer cells eliminated by PRL3-zumab to reduce tumor relapse.

PRL3, a unique oncotarget, is specifically overexpressed in 80.6% of cancers. In 2003, we reported that PRL3 promotes cell migration, invasion, and metastasis. Herein, firstly, we show that PRL3 induces Polyploid Giant Cancer Cells (PGCCs) formation. PGCCs constitute stem cell-like pools to facilitate cell survival, chemo-resistance, and tumor relapse. The correlations between PRL3 overexpression and PGCCs attributes raised possibilities that PRL3 could be involved in PGCCs formation. Secondly, we show that PRL3+ PGCCs co-express the embryonic stem cell markers SOX2 and OCT4 and arise mainly due to incomplete cytokinesis despite extensive DNA damage. Thirdly, we reveal that PRL3+ PGCCs tolerate prolonged chemotherapy-induced genotoxic stress via suppression of the pro-apoptotic ATM DNA damage-signaling pathway. Fourthly, we demonstrated PRL3-zumab, a First-in-Class humanized antibody drug against PRL3 oncotarget, could reduce tumor relapse in 'tumor removal' animal model. Finally, we confirmed that PGCCs were enriched in relapse tumors versus primary tumors. PRL3-zumab has been approved for Phase 2 clinical trials in Singapore, US, and China to block all solid tumors. This study further showed PRL3-zumab could potentially serve an 'Adjuvant Immunotherapy' after tumor removal surgery to eliminate PRL3+ PGCC stem-like cells, preventing metastasis and relapse.

Low molecular weight protein tyrosine phosphatase (LMW-PTP2) protein can potentially modulate virulence of the parasite Entamoeba histolytica.

The Entamoeba histolytica parasite is the causative agent of amebiasis, infecting approximately 1% of the world population and causing 100,000 deaths per year. It binds to Fibronectin (FN), activating signaling pathways regulated by kinases and phosphatases. EhLMW-PTPs genes from E. histolytica encode for Low Molecular Weight Tyrosine Phosphatases expressed in trophozoites and amoebic cysts. The role of these phosphatases in the virulence of the parasite has not yet been well characterized. Our results showed a differential expression of the EhLMW-PTPs, at the mRNA and protein levels, in an asynchronous trophozoites culture. Furthermore, we observed that trophozoites transfected that overexpressed EhLMW-PTP2 phagocytized fewer erythrocytes, possibly due to decreased phagocytic cups, and showed deficiencies in adherence to FN and less cytopathic effect. These analyzes suggest that the parasite's EhLMW-PTPs have an essential role in the mechanisms of proliferation, adhesion, and phagocytosis, regulating its pathogenicity.

Neuroprotective effects of a protein tyrosine phosphatase inhibitor against hippocampal excitotoxic injury.

Neuronal excitotoxicity is the neuronal cell death arising from prolonged exposure to glutamate and the associated excessive influx of ions into the cell. Sodium orthovanadate (Na3VO4,) competitively inhibits the protein tyrosine phosphatases that affect intracellular protein phosphorylation. No study has examined the role of protein tyrosine phosphatases in kainic acid (KA)-induced excitotoxic injury using sodium orthovanadate. Thus, the present study was conducted to determine the neuroprotective effects of sodium orthovanadate on KA-induced neuronal death in organotypic hippocampal slice culture. We also performed an in vivo electrophysiology study in Sprague-Dawley rats to observe the function of surviving cells after sodium orthovanadate treatment in KA-induced excitotoxicity. Rats were anaesthetized with sodium pentobarbital and KA was injected unilaterally in CA3 of the hippocampus by microinjection-cannula. Neuronal cell death, as assessed by propidium iodide uptake, was reduced by 10 and 25 µM sodium orthovanadate treatment (24 and 48 h) compared with the KA-only group. Sodium orthovanadate enhanced survival signals by increasing levels of phospho-Akt and superoxide dismutase. In addition, sodium orthovanadate treatment reduced calcineurin level for neuronal protection, which regulates activation of cellular calcium caused by KA-induced injury. In vivo results showed that sodium orthovanadate treatment elicited resistance to KA-induced behavior seizures and significantly reduced the duration of epileptiform discharges. In addition, sodium orthovanadate treatment (25 mM) significantly prevented the increase in power spectra induced by KA injection. These results suggest that sodium orthovanadate decreases the acute effects of KA, thereby inducing neuroprotective effects with reduced reactive oxygen species and cellular Ca2+. Thus, sodium orthovanadate may protect hippocampal neurons against excitotoxicity, and surviving neurons may function to reduce seizures.

MptpB Promotes Mycobacteria Survival by Inhibiting the Expression of Inflammatory Mediators and Cell Apoptosis in Macrophages.

Tuberculosis is a severe contagious disease caused by Mycobacterium tuberculosis (Mtb). To develop new vaccines and medicine against TB, there is an urgent need to provide insights into the mechanisms by which Mtb induces tuberculosis. In this study, we found that secreted Mtb virulence factor MptpB significantly enhanced the survival of H37Rv in macrophages. MptpB suppressed the production of iNOS, the expression of inflammatory factors IL-1ß and IL-6, as well as the apoptosis of the macrophage in Mtb infected RAW264.7 cells. Mechanism investigation showed that MptpB simultaneously hampered the NF-κB and MAPK signal pathways, evidenced by its blocking of p65, IKKα, Erk1/2, and p38 phosphorylation induced by Mtb infection. MptpB also inhibited host cell p53 expression. The results demonstrated that MptpB contributed to the survival of H37Rv by inhibiting host inflammatory responses and apoptosis through impeding the NF-κB and MAPK signal pathways and p53 expression in the macrophage.

Effects of hPTPß inhibitor on microcirculation of rat cremaster muscle flap following ischemia-reperfusion injury.

INTRODUCTION: Inhibition of protein tyrosine phosphatases (PTP) enhances endothelial receptor tyrosine kinases activation and may have beneficial effects on vessel growth and improve blood flow to ischemic tissue. The purpose of this study is to determine influence of hPTPß inhibitors on ischemia-reperfusion injury in muscle flap. MATERIALS AND METHODS: Following cremaster muscle dissection, 60 rats divided into 10 experimental groups (placebo and treatment groups following 0, 1, 2, 3, and 4 h of ischemia). Following group-specific treatment (placebo/hPTPß inhibitor, 15 mg/kg), 2 h of reperfusion is initiated. Observations are performed at 4 h after completion of reperfusion and microcirculatory hemodynamics and leukocyte-endothelial activation were recorded. RESULTS: Administration of hPTPß inhibitor showed preservation of capillary perfusion in group subjected to 2 h of ischemia when compared with placebo (P < .05). The effect of hPTPß inhibitor on mean venule diameter was found to be altered by duration of ischemia and this effect was statistically significant (P < .05). Treated ischemic groups (1 h, 2 h, and 3 h) showed decreased activation of rolling, sticking, and transmigrating leukocytes compared to respective placebo groups at all time points. The differences were significant for transmigrating leukocytes after 2 h and 3 h of ischemia (P < .05). Endothelial edema index was also significantly reduced in 2 h ischemia group (P < .05). CONCLUSION: Administration of hPTP inhibitors after submission of tissue to subcritical ischemia (1-2 h) improved functional capillary perfusion and decreased leukocyte-endothelial activation after 4 h after reperfusion. These results indicate that hPTP inhibitor has a potential postischemic therapeutic effect applied after tissue ischemia just before the reperfusion injury.

Druggability analysis and classification of protein tyrosine phosphatase active sites.

Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability.

Human recombinant cementum attachment protein (hrPTPLa/CAP) promotes hydroxyapatite crystal formation in vitro and bone healing in vivo.

Cementum extracellular matrix is similar to other mineralized tissues; however, this unique tissue contains molecules only present in cementum. A cDNA of these molecules, cementum attachment protein (hrPTPLa/CAP) was cloned and expressed in a prokaryotic system. This molecule is an alternative splicing of protein tyrosine phosphatase-like A (PTPLa). In this study, we wanted to determine the structural and functional characteristics of this protein. Our results indicate that hrPTPLa/CAP contains a 43.2% α-helix, 8.9% ß-sheet, 2% ß-turn and 45.9% random coil secondary structure. Dynamic light scattering shows that this molecule has a size distribution of 4.8 nm and aggregates as an estimated mass of 137 kDa species. AFM characterization and FE-SEM studies indicate that this protein self-assembles into nanospheres with sizes ranging from 7.0 to 27 nm in diameter. Functional studies demonstrate that hrPTPLa/CAP promotes hydroxyapatite crystal nucleation: EDS analysis revealed that hrPTPLa/CAP-induced crystals had a 1.59 ± 0.06 Ca/P ratio. Further confirmation with MicroRaman spectrometry and TEM confirm the presence of hydroxyapatite. In vivo studies using critical-size defects in rat cranium showed that hrPTPLa/CAP promoted 73% ± 2.19% and 87% ± 1.97% new bone formation at 4 and 8 weeks respectively. Although originally identified in cementum, PTPLa/CAP is very effective at inducing bone repair and healing and therefore this novel molecule has a great potential to be used for mineralized tissue bioengineering and tissue regeneration.

Pharmacological effects of JTT-551, a novel protein tyrosine phosphatase 1B inhibitor, in diet-induced obesity mice.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of leptin signaling as well as insulin signaling. JTT-551 is a new PTP1B inhibitor, which is reported to improve glucose metabolism by enhancement of insulin signaling. We have evaluated an antiobesity effect of JTT-551 using diet-induced obesity (DIO) mice. A single administration of JTT-551 was provided to DIO mice with or without leptin, and DIO mice were given food containing JTT-551 for six weeks. A single administration of JTT-551 with leptin treatment enhanced the food inhibition and the signal transducer and activator of transcription 3 (STAT3) phosphorylation in hypothalamus. Moreover, chronic administration of JTT-551 showed an antiobesity effect and an improvement of glucose and lipid metabolism in DIO mice. JTT-551 shows an antiobesity effect possibly by enhancement of leptin signaling and could be useful in the treatment of type 2 diabetes and obesity.

A systems toxicology approach identifies Lyn as a key signaling phosphoprotein modulated by mercury in a B lymphocyte cell model.

Network and protein-protein interaction analyses of proteins undergoing Hg²âº-induced phosphorylation and dephosphorylation in Hg²âº-intoxicated mouse WEHI-231 B cells identified Lyn as the most interconnected node. Lyn is a Src family protein tyrosine kinase known to be intimately involved in the B cell receptor (BCR) signaling pathway. Under normal signaling conditions the tyrosine kinase activity of Lyn is controlled by phosphorylation, primarily of two well known canonical regulatory tyrosine sites, Y-397 and Y-508. However, Lyn has several tyrosine residues that have not yet been determined to play a major role under normal signaling conditions, but are potentially important sites for phosphorylation following mercury exposure. In order to determine how Hg²âº exposure modulates the phosphorylation of additional residues in Lyn, a targeted MS assay was developed. Initial mass spectrometric surveys of purified Lyn identified 7 phosphorylated tyrosine residues. A quantitative assay was developed from these results using the multiple reaction monitoring (MRM) strategy. WEHI-231 cells were treated with Hg²âº, pervanadate (a phosphatase inhibitor), or anti-Ig antibody (to stimulate the BCR). Results from these studies showed that the phosphoproteomic profile of Lyn after exposure of the WEHI-231 cells to a low concentration of Hg²âº closely resembled that of anti-Ig antibody stimulation, whereas exposure to higher concentrations of Hg²âº led to increases in the phosphorylation of Y-193/Y-194, Y-501 and Y-508 residues. These data indicate that mercury can disrupt a key regulatory signal transduction pathway in B cells and point to phospho-Lyn as a potential biomarker for mercury exposure.

Avaliação enzimática e funcional de fosfatases associadas à virulência em trofozoítos de entamoeba histolytica / Evaluation of functional enzyme and phosphatases associated with virulence in trophozoites of entamoeba histolytica

Entamoeba histolytica, o agente etiológico da amebíase, constitui a segunda maior causa de morte por protozooses, sendo considerado um grave problema de saúde pública, particularmente em países em desenvolvimento. A morte celular induzida por este parasito entérico é dependente de contato e mediada por proteínas específicas, as quais modulam diversos eventos fisiológicos no hospedeiro. Dentre os mecanismos reguladores de tais respostas está a desfosforilação de grupos fosfotirosil de proteínas, através da atuação de proteína tirosina fosfatases (PTPases), tanto expressas na superfície celular como secretados pelo trofozoíto. Alguns estudos demonstram uma rápida diminuição nos níveis de fosforilação de resíduos tirosina em proteínas de células-alvo após o contato com E. histolytica. Estas PTPases têm sido descritas como tendo importante papel na patogênese da amebíase. O objetivo deste estudo foi analisar o perfil de atividade fosfatásica em diferentes cepas de E. histolytica, assim como caracterizar a sua sensibilidade a conhecidos inibidores de proteína tirosina fosfatases, e os efeitos destes moduladores em mecanismos envolvidos na patogênese, a exemplo de fagocitose e efeito citopático. Inicialmente, a sensibilidade aos moduladores da via de sinalização celular em trofozoítos de E. histolytica (cepas ICB – 452, ICB – CSP e HM1:IMSS) foi caracterizada através de avaliação da proliferação celular, onde inóculos de parasitos foram incubados em presença ou ausência de inibidores de PTPases. Dentre os moduladores testados, o derivado de vanádio bisperoxo (1, 10 - fenantrolina) oxovanadato de potássio (bpVphen) e o óxido de fenilarsina (PAO) demonstraram efetiva capacidade antiproliferativa. Estas células apresentaram uma maior sensibilidade ao PAO em comparação ao bpV(phen), com valores de IC50 para a cepa HM1:IMSS de 0,9 μM e 38,4 μM, respectivamente. A análise bioquímica revelou que há uma maior atividade secretória e ectofosfatásica na linhagem HM1:IMSS (24,48 nM p-NP/40 min./3 x 106 células e 297 nM p-NP/40 min./2 x 105 células, respectivamente), e uma redução desta atividade foi detectada na presença dos derivados de vanádio na superfície do parasito (31,3 nM p-NP/40 min./2 x 105 células). O mesmo não foi observado após a adição de PAO. A análise da eritrofagocitose e da destruição da monocamada de células epiteliais da linhagem MDCK, importantes marcadores de virulência neste patógeno, demonstrou significativa redução destes processos em trofozoítos tratados com os inibidores ortovanadato de sódio (OVS) e o bpV(phen), indicando a participação de PTPases nos mesmos. Estas mesmas alterações foram observadas após o tratamento do parasito com PAO, porém dados ultraestruturais de citoquímica enzimática não indicam a redução da atividade fosfatásica por este composto. Estes dados foram confirmados após a avaliação da atividade das frações purificadas de homogenatos solubilizados de trofozoítos de E. histolytica, onde detectou-se a redução dos níveis de atividade enzimática após a adição de vanadato e seus derivados à reação, o que não pôde ser observado após o acréscimo de PAO. Os resultados obtidos indicam que PTPases estão diretamente envolvidas em importantes funções celulares exercidas por trofozoítos de Entamoeba histolytica. Ademais, novos estudos que visem à elucidação dos possíveis modos de ação do composto PAO neste patógeno poderiam contribuir, consideravelmente, com o entendimento da biologia do parasito e, consequentemente, dos mecanismos patogênicos da amebíase.

HIV-1 enhancing effect of prostatic acid phosphatase peptides is reduced in human seminal plasma.

We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.

Prostatic acid phosphatase reduces thermal sensitivity and chronic pain sensitization by depleting phosphatidylinositol 4,5-bisphosphate.

Prostatic acid phosphatase (PAP) is expressed in nociceptive dorsal root ganglion (DRG) neurons, functions as an ectonucleotidase, and generates adenosine extracellularly. Here, we found that PAP inhibits noxious thermal sensitivity and sensitization that is associated with chronic pain through sustained activation of the adenosine A(1) receptor (A(1)R) and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)). In mice, intrathecal injection of PAP reduced PIP(2) levels in DRGs, inhibited thermosensation through TRPV1, and enduringly reduced thermal hyperalgesia and mechanical allodynia caused by inflammation, nerve injury, and pronociceptive receptor activation. This included inhibitory effects on lysophosphatidic acid, purinergic (ATP), bradykinin, and protease-activated (thrombin) receptors. Conversely, PIP(2) levels were significantly elevated in DRGs from Pap(-/-) mice, and this correlated with enhanced thermal hyperalgesia and mechanical allodynia in Pap(-/-) mice. To directly test the importance of PIP(2) in nociception, we intrathecally injected PIP(2) into mice. This transiently (2 h) elevated PIP(2) levels in lumbar DRGs and transiently (2 h) enhanced thermosensation. Additionally, thermal hyperalgesia and mechanical allodynia were enduringly enhanced when PIP(2) levels were elevated coincident with injury/pronociceptive receptor stimulation. Nociceptive sensitization was not affected if PIP(2) levels were elevated in the absence of ongoing pronociceptive receptor stimulation. Together, our data suggest that PIP(2) levels in DRGs directly influence thermosensation and the magnitude of nociceptive sensitization. Moreover, our data suggest there is an underlying "phosphoinositide tone" that can be manipulated by an adenosine-generating ectonucleotidase. This tone regulates how effectively acute nociceptive insults promote the transition to chronic pain.