Protein tyrosine phosphatase 1B is a regulator of alpha-actinin4 in the glomerular podocyte.

Glomerular podocytes are instrumental for the barrier function of the kidney, and podocyte injury contributes to proteinuria and the deterioration of renal function. Protein tyrosine phosphatase 1B (PTP1B) is an established metabolic regulator, and the inactivation of this phosphatase mitigates podocyte injury. However, there is a paucity of data regarding the substrates that mediate PTP1B actions in podocytes. This study aims to uncover novel substrates of PTP1B in podocytes and validate a leading candidate. To this end, using substrate-trapping and mass spectroscopy, we identified putative substrates of this phosphatase and investigated the actin cross-linking cytoskeletal protein alpha-actinin4. PTP1B and alpha-actinin4 co-localized in murine and human glomeruli and transiently transfected E11 podocyte cells. Additionally, podocyte PTP1B deficiency in vivo and culture was associated with elevated tyrosine phosphorylation of alpha-actinin4. Conversely, reconstitution of the knockdown cells with PTP1B attenuated alpha-actinin4 tyrosine phosphorylation. We demonstrated co-association between alpha-actinin4 and the PTP1B substrate-trapping mutant, which was enhanced upon insulin stimulation and disrupted by vanadate, consistent with an enzyme-substrate interaction. Moreover, we identified alpha-actinin4 tandem tyrosine residues 486/487 as mediators of its interaction with PTP1B. Furthermore, knockdown studies in E11 cells suggest that PTP1B and alpha-actinin4 are modulators of podocyte motility. These observations indicate that PTP1B and alpha-actinin4 are likely interacting partners in a signaling node that modulates podocyte function. Targeting PTP1B and plausibly this one of its substrates may represent a new therapeutic approach for podocyte injury that warrants additional investigation.

Application of hybrid biophysical-biochemical methods to unravel the molecular basis for auto-inhibition and activation of protein tyrosine phosphatase TCPTP/PTPN2.

Since the discovery of protein tyrosine phosphorylation as one of the critical post-translational modifications, it has been well known that the activity of protein tyrosine kinases (PTKs) is tightly regulated. On the other hand, protein tyrosine phosphatases (PTPs) are often regarded to act constitutively active, but recently we and others have shown that many PTPs are expressed in an inactive form due to allosteric inhibition by their unique structural features. Furthermore, their cellular activity is highly regulated in a spatiotemporal manner. In general, PTPs share a conserved catalytic domain comprising about 280 residues that is flanked by either an N-terminal or a C-terminal non-catalytic segment, which differs significantly in size and structure from each other and is known to regulate specific PTP's catalytic activity. The well-characterized non-catalytic segments can be globular or intrinsically disordered. In this work, we have focused on the T-Cell Protein Tyrosine Phosphatase (TCPTP/PTPN2) and demonstrated how the hybrid biophysical-biochemical methods can be applied to unravel the underlying mechanism through which TCPTP's catalytic activity is regulated by the non-catalytic C-terminal segment. Our analysis showed that TCPTP is auto-inhibited by its intrinsically disordered tail and trans-activated by Integrin alpha-1's cytosolic region.

Active-site cysteine 215 sulfonation targets protein tyrosine phosphatase PTP1B for Cullin1 E3 ligase-mediated degradation.

Reactive oxygen species (ROS), released as byproducts of mitochondrial metabolism or as products of NADPH oxidases and other processes, can directly oxidize the active-site cysteine (Cys) residue of protein tyrosine phosphatases (PTPs) in a mammalian cell. Robust degradation of irreversibly oxidized PTPs is essential for preventing accumulation of these permanently inactive enzymes. However, the mechanism underlying the degradation of these proteins was unknown. In this study, we found that the active-site Cys215 of endogenous PTP1B is sulfonated in H9c2 cardiomyocytes under physiological conditions. The sulfonation of Cys215 led PTP1B to exhibit a conformational change, and drive the subsequent ubiquitination and degradation of this protein. We then discovered that Cullin1, an E3 ligase, interacts with the Cys215-sulfonated PTP1B. The functional impairment of Cullin1 prevented PTP1B from oxidation-dependent ubiquitination and degradation in H9c2 cells. Moreover, delivery of the terminally oxidized PTP1B resulted in proteotoxicity-caused injury in the affected cells. In conclusion, we elucidate how sulfonation of the active-site Cys215 can direct turnover of endogenous PTP1B through the engagement of ubiquitin-proteasome system. These data highlight a novel mechanism that maintains PTP homeostasis in cardiomyocytes with constitutive ROS production.

The catalytic activity of TCPTP is auto-regulated by its intrinsically disordered tail and activated by Integrin alpha-1.

T-Cell Protein Tyrosine Phosphatase (TCPTP, PTPN2) is a non-receptor type protein tyrosine phosphatase that is ubiquitously expressed in human cells. TCPTP is a critical component of a variety of key signaling pathways that are directly associated with the formation of cancer and inflammation. Thus, understanding the molecular mechanism of TCPTP activation and regulation is essential for the development of TCPTP therapeutics. Under basal conditions, TCPTP is largely inactive, although how this is achieved is poorly understood. By combining biomolecular nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and chemical cross-linking coupled with mass spectrometry, we show that the C-terminal intrinsically disordered tail of TCPTP functions as an intramolecular autoinhibitory element that controls the TCPTP catalytic activity. Activation of TCPTP is achieved by cellular competition, i.e., the intrinsically disordered cytosolic tail of Integrin-α1 displaces the TCPTP autoinhibitory tail, allowing for the full activation of TCPTP. This work not only defines the mechanism by which TCPTP is regulated but also reveals that the intrinsically disordered tails of two of the most closely related PTPs (PTP1B and TCPTP) autoregulate the activity of their cognate PTPs via completely different mechanisms.

Crystal Structure of TCPTP Unravels an Allosteric Regulatory Role of Helix α7 in Phosphatase Activity.

The T-cell protein tyrosine phosphatase (TCPTP/PTPN2) targets a broad variety of substrates across different subcellular compartments. In spite of that, the structural basis for the regulation of TCPTP's activity remains elusive. Here, we investigated whether the activity of TCPTP is regulated by a potential allosteric site in a comparable manner to its most similar PTP family member (PTP1B/PTPN1). We determined two crystal structures of TCPTP at 1.7 and 1.9 Å resolutions that include helix α7 at the TCPTP C-terminus. Helix α7 has been functionally characterized in PTP1B and was identified as its allosteric switch. However, its function is unknown in TCPTP. Here, we demonstrate that truncation or deletion of helix α7 reduced the catalytic efficiency of TCPTP by ∼4-fold. Collectively, our data supports an allosteric role of helix α7 in regulation of TCPTP's activity, similar to its function in PTP1B, and highlights that the coordination of helix α7 with the core catalytic domain is essential for the efficient catalytic function of TCPTP.

Putative role of natural products as Protein Kinase C modulator in different disease conditions.

INTRODUCTION: Protein kinase C (PKC) is a promising drug target for various therapeutic areas. Natural products derived from plants, animals, microorganisms, and marine organisms have been used by humans as medicine from prehistoric times. Recently, several compounds derived from plants have been found to modulate PKC activities through competitive binding with ATP binding site, and other allosteric regions of PKC. As a result fresh race has been started in academia and pharmaceutical companies to develop an effective naturally derived small-molecule inhibitor to target PKC activities. Herein, in this review, we have discussed several natural products and their derivatives, which are reported to have an impact on PKC signaling cascade. METHODS: All information presented in this review article regarding the regulation of PKC by natural products has been acquired by a systematic search of various electronic databases, including ScienceDirect, Scopus, Google Scholar, Web of science, ResearchGate, and PubMed. The keywords PKC, natural products, curcumin, rottlerin, quercetin, ellagic acid, epigallocatechin-3 gallate, ingenol 3 angelate, resveratrol, protocatechuic acid, tannic acid, PKC modulators from marine organism, bryostatin, staurosporine, midostaurin, sangivamycin, and other relevant key words were explored. RESULTS: The natural products and their derivatives including curcumin, rottlerin, quercetin, ellagic acid, epigallocatechin-3 gallate, ingenol 3 angelate, resveratrol, bryostatin, staurosporine, and midostaurin play a major role in the management of PKC activity during various disease progression. CONCLUSION: Based on the comprehensive literature survey, it could be concluded that various natural products can regulate PKC activity during disease progression. However, extensive research is needed to circumvent the challenge of isoform specific regulation of PKC by natural products.

Targeting protein tyrosine phosphatase PTP-PEST (PTPN12) for therapeutic intervention in acute myocardial infarction.

AIMS: The myocardial ischaemia/reperfusion (I/R) injury is almost inevitable since reperfusion is the only established treatment for acute myocardial infarction (AMI). To date there is no effective strategy available for reducing the I/R injury. Our aim was to elucidate the mechanisms underlying myocardial I/R injury and to develop a new strategy for attenuating the damage it causes. METHODS AND RESULTS: Using a mouse model established by ligation of left anterior descending artery, we found an increase in activity of protein tyrosine phosphatases (PTPs) in myocardium during I/R. Treating the I/R-mice with a pan-PTP inhibitor phenyl vinyl sulfone attenuated I/R damage, suggesting PTP activation to be harmful in I/R. Through analysing RNAseq data, we showed PTPs being abundantly expressed in mouse myocardium. By exposing primary cardiomyocytes ablated with specific endogenous PTPs by RNAi to hypoxia/reoxygenation (H/R), we found a role that PTP-PEST (PTPN12) plays to promote cell death under H/R stress. Auranofin, a drug being used in clinical practice for treating rheumatoid arthritis, may target PTP-PEST thus suppressing its activity. We elucidated the molecular basis for Auranofin-induced inactivation of PTP-PEST by structural studies, and then examined its effect on myocardial I/R injury. In the mice receiving Auranofin before reperfusion, myocardial PTP activity was suppressed, leading to restored phosphorylation of PTP-PEST substrates, including ErbB-2 that maintains the survival signalling of the heart. In line with the inhibition of PTP-PEST activity, the Auranofin-treated I/R-mice had smaller infarct size and better cardiac function. CONCLUSIONS: PTP-PEST contributes to part of the damages resulting from myocardial I/R. The drug Auranofin, potentially acting through the PTP-PEST-ErbB-2 signalling axis, reduces myocardial I/R injury. Based on this finding, Auranofin could be used in the development of new treatments that manage I/R injury in patients with AMI.

A clinical study on Virechana Karma (therapeutic purgation) over the gut flora with special reference to obesity.

BACKGROUND: Altered gut flora is associated with the pathogenesis of both intestinal and extra­intestinal disorders. Aetiology of obesity is associated with mechanisms such as short chain fatty acid production, stimulation of hormones, chronic low­grade inflammation, lipoprotein and bile acid metabolism and increased endocannabinoid. Receptor system tone have been suggested to explain the role of gut microbiota of obesity. The Panchakarma (Ayurvedic purification methods) claims the management of metabolic disorders hence this work provides the target specific evidence for the clinical studies. The proposed project is aimed to explore the particular molecular mechanism and, to make this therapy more evidence based. Hence, it was hypothesized that Panchakarma­based intervention such as Virechana Karma (therapeutic purgation) may influence microbiota and help in the management of the obesity. MATERIALS AND METHODS: The study was conducted to explore the effect of Virechana Karma over the gut flora; therefore, total of 19 patients with Madhyama Koshtha diagnosed with obesity were included and received the intervention. Before and after Virechana, a stool sample was collected and processed for the enterobacterial repetitive intergenic consensus ­polymerase chain reaction to find the changes over the facultative aerobic bacteria. RESULTS: It was found that Virechana is effective in the management of the obesity as it helps to reduce colonization of aerobic bacteria. After Virechana and after follow­up also, it showed the correction of the gut flora dysbiosis, thus initiated the weight loss mechanism in the body, resulting in diminution in the signs and symptoms of obesity. CONCLUSION: Virechana is effective in the management of the obesity due to reduction in the Escherichia coli colonization and is effective over the gut flora dysbiosis.

Cross-sectional Study on Visual Inspection with Acetic Acid and Pap Smear Positivity Rates According to Sociodemographic Factors Among Rural Married Women of Bareilly (Uttar Pradesh).

BACKGROUND: It is possible to prevent deaths due to cervical cancer through screening and treatment. Cervical cytology which is a standard screening tool in developed countries fails as a screening method in low-resource countries due to financial and technical constraints. OBJECTIVE: To determine the prevalence of pre-malignant lesions of the cervix by VIA and Pap smear test among rural married women and to find out association of socio demographic factors with positive screening test results. METHOD: A community based cross-sectional study was carried out among rural married women in the field practice area of a tertiary health care center. A pre-designed questionnaire was administered to collect information on socio-demographic characteristics from 550 women. They were tested for the presence of pre-malignant lesions of the cervix using VIA and Pap smear as screening tools. RESULTS: Out of 550 study participants, total 37 patients were found positive, out of which 7, 17 & 13 patients were found positive by Pap smear alone, VIA test alone, and by both these tests respectively. Moderate agreement (k=0.498) was found between these two tests by applying Kappa statistics at 95% confidence interval. The VIA and Pap smear tests were positive among 5.5% and 3.6% study subjects respectively. The positivity rate was found to be more in the age group of >50 years, Hindu, SC/ST caste, joint family, professional and, upper class. CONCLUSION: The prevalence of pre-malignant lesions of the cervix by VIA test was 5.5% while 3.6% pre-malignant lesion was detected by Pap smear method. VIA and Pap smear positivity rates among rural married women.

Clinical evaluation of Lekhaniya Kashaya Vasti in the management of Sthaulya (obesity).

BACKGROUND: Obesity is considered the world's oldest metabolic disorder. It is not a single disease entity, but a syndrome with many causes including combination of genetic, nutritional and sociological factors. The World Health Organization (WHO) considers obesity as "Insidious, creeping pandemic which is now engulfing the entire world". Diet and life-style play a significant role both in the development and control of obesity Sthaulya (obesity). In Ayurveda, Acharyas have mentioned about the use of Lekhaniya Vasti to manage the Sthaulya. AIM: To evaluate the efficacy of Lekhaniya Kashaya Vasti in patients of Sthaulya. MATERIALS AND METHODS: A total of 70 patients of Sthaulya were registered. Further they were divided into 2 groups each having 35 patients. In Group I (Lekhaniya Kashaya Vasti) group out of 35 patients 32 and in Group II (Pathya) group out of 35 patients 33 completed the follow-up. RESULTS: In Group I, mean change was observed in body mass index (P < 0.001), waist hip ratio (P < 0.001). Overweight (P < 0.001), Kshudraswas (breathlessness) (P < 0.001) and Nidraadhikyata (excessive sleep) (P < 0.001) which is statistically significant in comparison with Group II. CONCLUSION: Trial drug is very good combination for Medoghna activity.

A quality control program within a clinical trial Consortium for PCR protocols to detect Plasmodium species.

Malaria parasite infections that are only detectable by molecular methods are highly prevalent and represent a potential transmission reservoir. The methods used to detect these infections are not standardized, and their operating characteristics are often unknown. We designed a proficiency panel of Plasmodium spp. in order to compare the accuracy of parasite detection of molecular protocols used by labs in a clinical trial consortium. Ten dried blood spots (DBSs) were assembled that contained P. falciparum, P. vivax, P. malariae, and P. ovale; DBSs contained either a single species or a species mixed with P. falciparum. DBS panels were tested in 9 participating laboratories in a masked fashion. Of 90 tests, 68 (75.6%) were correct; there were 20 false-negative results and 2 false positives. The detection rate was 77.8% (49/63) for P. falciparum, 91.7% (11/12) for P. vivax, 83.3% (10/12) for P. malariae, and 70% (7/10) for P. ovale. Most false-negative P. falciparum results were from samples with an estimated ≤ 5 parasites per µl of blood. Between labs, accuracy ranged from 100% to 50%. In one lab, the inability to detect species in mixed-species infections prompted a redesign and improvement of the assay. Most PCR-based protocols were able to detect P. falciparum and P. vivax at higher densities, but these assays may not reliably detect parasites in samples with low P. falciparum densities. Accordingly, formal quality assurance for PCR should be employed whenever this method is used for diagnosis or surveillance. Such efforts will be important if PCR is to be widely employed to assist malaria elimination efforts.

Safety, efficacy and population pharmacokinetics of fixed-dose combination of artesunate-mefloquine in the treatment of acute uncomplicated Plasmodium falciparum malaria in India.

BACKGROUND & OBJECTIVES: India has switched over to artemisinin-based combination therapy (ACT) for the treatment of acute uncomplicated Plasmodium falciparum malaria and the ACT used in the national programme is artesunate + sulphadoxine-pyrimethamine. Since the efficacy of ACT is dependent also on the partner drug, there is a need to evaluate and deploy multiple ACTs. METHODS: This multicentre, single-arm, open-label clinical trial was carried out to assess the efficacy, safety and population pharmacokinetics of a fixed dose combination (FDC) artesunate mefloquine (ASMQ) in P. falciparum infected, Indian adults at Panjim, Goa, and Mangalore, Karnataka between December 2007 and November 2008. RESULTS: A total of 77 patients (males 74) were screened and enrolled: 42 at Goa and 35 at Mangalore with a median age of 25 yr (range 18-55 yr). One patient failed in treatment on D53, a PCR proven new infection, seven developed recurrent vivax parasitaemia and 11 did not have a parasitological endpoint. By per protocol analysis, the D63 cure rate was 58/59 (98.3; 95% C.I. 90.9-99.9%), and 58/58, with PCR correction. ASMQ was well-tolerated and no serious adverse events were reported. INTERPRETATION & CONCLUSION: The study showed that the ASMQ FDC was efficacious and well-tolerated for the treatment of acute, uncomplicated P. falciparum malaria in highly endemic, chloroquine resistant areas of Goa and Mangalore. It is a viable option for India.

Arterolane maleate plus piperaquine phosphate for treatment of uncomplicated Plasmodium falciparum malaria: a comparative, multicenter, randomized clinical trial.

BACKGROUND: Artemisinin-based combination therapy is the first-line treatment for uncomplicated falciparum malaria. This study assessed the antimalarial efficacy and safety of a combination of 150 mg of arterolane maleate and 750 mg of piperaquine phosphate (AM-PQP) in comparison to Coartem (artemether and lumefantrine) in patients with acute uncomplicated P. falciparum malaria. METHODS: In this open-label, randomized, multicentric, parallel group clinical trial, 240 patients were randomized to receive AM-PQP (160 patients) or Coartem (80 patients). Patients with P. falciparum monoinfection and initial parasite densities ranging from 1000 to 100 000 asexual parasites/µL of blood were followed for 28 days. Polymerase chain reaction-corrected adequate clinical and parasitologic response on day 28, parasite clearance time, and fever clearance time were evaluated. RESULTS: A total of 151 (94.4%) of 160 patients in the AM-PQP group completed the trial, while 77 (96.3%) of 80 patients in the Coartem group completed the trial. No treatment failure was noted in the AM-PQP group, while one patient receiving Coartem failed treatment on day 28. There was no difference in the median parasite clearance time (30 hours in both groups) or median fever clearance time (24 hours in both groups) after administration of the 2 study treatments. CONCLUSIONS: The available data support the evaluation of a drug combination in a larger population as a fixed-dose combination. Clinical Trials Registration. CTRI/2007/091/000031.

Monitoring antimalarial drug resistance in India via sentinel sites: outcomes and risk factors for treatment failure, 2009-2010.

OBJECTIVE: To describe India's National Antimalarial Drug Resistance Monitoring System, measure the efficacy of first-line malaria treatments, and determine risk factors for treatment failure. METHODS: In 2009-2010, prospective studies with 28 days of follow-up were conducted at 25 sentinel sites. Patients infected with Plasmodium falciparum were given artesunate plus sulfadoxine-pyrimethamine (AS+SP); those infected with P. vivax were given chloroquine. Polymerase chain reaction was used to distinguish post-treatment reinfection from treatment failure. Isolates of P. falciparum were checked for dhfr and dhps mutations. FINDINGS: Overall, 1664 patients were enrolled. Kaplan-Meier survival analysis showed an efficacy of 98.8% for AS+SP. Most patients with P. falciparum parasitaemia cleared their parasitaemias within 24 hours of treatment initiation, but six, including four with treatment failure, remained parasitaemic after 72 hours. Double mutants in dhfr were found in 68.4% of the genotyped isolates. Triple or quadruple mutants in dhfr and mutations in dhps were rare. A daily dose of artesunate of < 3 mg per kg of body weight, age of less than 5 years, and fever at enrolment were associated with an increased risk of treatment failure. Chloroquine remained 100% efficacious and generally cleared P. vivax parasitaemias within 48 hours. Vomiting (seen in 47 patients) was the most common adverse event. CONCLUSION: India's National Antimalarial Drug Resistance Monitoring System provides wide coverage. The first-line antimalarials used in the country remain safe and efficacious. The treatment of malaria in young children and the relative benefits of age- and weight-based dosing need further exploration.

Clinical efficacy of Rasona Pinda in the management of Amavata (rheumatoid arthritis).

In the present clinical study, 63 patients of Amavata were registered from the Kayachikitsa out patient department/indoor patient department (OPD/IPD) of Sir Sunder Lal Hospital (Indian Medicine Wing), IMS, BHU, Varanasi-5. In group I (Rasona Pinda), 27 patients completed the study of a total of 33patients registered in the group (six patients dropped out mid-therapy). In group II (control group), 23 patients completed all three follow-ups out of 30 patients (there were seven dropouts in mid-therapy). In group I, complete remission in 29.6%, major improvement in 59.3% and minor improvement in change font so as to appear 11.1% were observed. In group II, complete remission in 13%, major improvement in 21.7%, minor improvement in 39.1% and unchanged in 26.9% of the patients were observed.