Phosphotyrosine Protein Phosphatase Research Articles

Dual PTP1B/TC-PTP Inhibitors: Biological Evaluation of 3-(Hydroxymethyl)cinnoline-4(1H)-Ones

Dual inhibitors of protein phosphotyrosine phosphatase 1B (PTP1B)/T-cell protein phosphotyrosine phosphatase (TC-PTP) based on the 3-(hydroxymethyl)-4-oxo-1,4-dihydrocinnoline scaffold have been identified. Their dual affinity to both enzymes has been thoroughly corroborated by in silico modeling experiments. The compounds have been profiled in vivo for their effects on body weight and food intake in obese rats. Likewise, the effects of the compounds on glucose tolerance, insulin resistance, as well as insulin and leptin levels, have been evaluated. In addition, the effects on PTP1B, TC-PTP, and Src homology region 2 domain-containing phosphatase-1 (SHP1), as well as the insulin and leptin receptors gene expressions, have been assessed. In obese male Wistar rats, a five-day administration of all studied compounds led to a decrease in body weight and food intake, improved glucose tolerance, attenuated hyperinsulinemia, hyperleptinemia and insulin resistance, and also compensatory increased expression of the PTP1B and TC-PTP genes in the liver. The highest activity was demonstrated by 6-Chloro-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 3) and 6-Bromo-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 4) with mixed PTP1B/TC-PTP inhibitory activity. Taken together, these data shed light on the pharmacological implications of PTP1B/TC-PTP dual inhibition, and on the promise of using mixed PTP1B/TC-PTP inhibitors to correct metabolic disorders.

Bombyxin-stimulated ecdysteroidogenesis in relation to sugar transporter/trehalase expressions in Bombyx prothoracic glands

Our previous studies showed that bombyxin stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs) during a long-term incubation period in a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. In the present study, we further investigated the downstream signaling cascade in bombyxin-stimulated PGs. Our results showed that upon treatment with bombyxin, expression levels of the sugar transport 1 (St1) and St4 genes and trehalase 1 (Treh1) gene, but not ecdysteroid biosynthesis genes were greatly enhanced compared to the controls. Treatment with LY294002 (an inhibitor of PI3K) reduced the enhanced St1 and Treh1 expression levels, clearly indicating the involvement of PI3K. Treatment with 1 mM of mpV(pic) (a potent inhibitor of protein phosphotyrosine phosphatase and activator of insulin receptor (InR) kinase) also stimulated expression levels of the St1 and Treh1 genes, thus further confirming the involvement of the InR. Determining Treh enzyme activity showed that bombyxin treatment stimulated Treh enzyme activity in time- and PI3K-dependent manners. Validamycin A (a Treh inhibitor) blocked bombyxin-stimulated Treh enzyme activity and partly decreased bombyxin-stimulated ecdysteroidogenesis. A specific sugar transport inhibitor (cytochalasin B) and a glycolysis inhibitor (2-deoxy-D-glucose (2-DG)) also reduced bombyxin-stimulated ecdysteroidogenesis. Taken together, these results indicated that increased expressions of Sts and Treh1 and enhanced Treh enzyme activity downstream of InR/PI3K are involved in bombyxin-stimulated ecdysteroidogenesis in B. mori PGs.

Characterization and biological activity of new 4-oxo-1,4-dihydrocinnoline-based inhibitors of the tyrosine phosphatase PTP1B and TCPTP

Functional disorders in obesity are largely due to a decrease in tissue sensitivity to insulin and leptin. One of the ways to restore it is inhibition of protein phosphotyrosine phosphatase 1B (PTP1B) and T-cell protein phosphotyrosine phosphatase (TCPTP), negative regulators of the insulin and leptin signaling. Despite progress in the development of inhibitors of these phosphatases, commercial preparations based on them have not been developed yet, and the mechanisms of action are poorly understood. The aim of the work was to study the effect of new derivatives of 4-oxo-1,4-dihydrocinnoline (PI04, PI06, PI07) on the activity of PTP1B and TCPTP, as well as to study the effect of their five-day administration (i.p., 10 mg/kg/day) to Wistar rats with diet-induced obesity on body weight and fat, metabolic and hormonal parameters, and gene expression of phosphatase and insulin and leptin receptors in the liver. It has been shown that PI04 is a mild, low selective inhibitor of both phosphatases (PTP1B, IC50=3.42(2.60-4.51) μM; TCPTP, IC50=4.16(3.49-4.95) μM), while PI06 and PI07 preferentially inhibit PTP1B (IC50=3.55 (2.63-4.78) μM) and TCPTP (IC50=1.45(1.18-1.78) μM), respectively. PI04 significantly reduced food intake, body weight and fat, attenuated hyperglycemia, normalized glucose tolerance, basal and glucose-stimulated levels of insulin and leptin, and insulin resistance index. Despite the anorexigenic effect, PI06 and PI07 were less effective, having little effect on glucose homeostasis and insulin sensitivity. PI04 significantly increased the expression of the PTP1B and TCPTP genes and decreased the expression of the insulin and leptin receptor genes. PI06 and PI07 had little effect on these indicators. Thus, PI04, the inhibitor of PTP1B and TCPTP phosphatases, restored metabolic and hormonal parameters in obese rats with greater efficiency than inhibitors of PTP1B (PI06) and TCPTP (PI07). This indicates the prospect of creating mixed PTP1B/TCPTP inhibitors for correction of metabolic disorders.

LMW-PTP modulates glucose metabolism in cancer cells

BackgroundLow Molecular Weight Phosphotyrosine Protein Phosphatase (LMW-PTP) is an enzyme involved not only in tumor onset and progression but also in type 2 diabetes. A recent review shows that LMW-PTP acts on several RTK (receptor tyrosine kinase) such as PDGFR, EGFR, EphA2, Insulin receptor. It is well described also its interaction with cSrc. It is noteworthy that most of these conclusions are based on the use of cell lines expressing low levels of LMW-PTP. The aim of the present study was to discover new LMW-PTP substrates in aggressive human tumors where the over-expression of this phosphatase is a common feature. MethodsWe investigated, by proteomic analysis, the protein phosphorylation pattern of A375 human melanoma cells silenced for LMW-PTP. Two-dimensional electrophoresis (2-DE) analysis, followed by western blot was performed using anti-phosphotyrosine antibodies, in order to identify differentially phosphorylated proteins. ResultsProteomic analysis pointed out that most of the identified proteins belong to the glycolytic metabolism, such as α-enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, suggesting an involvement of LMW-PTP in glucose metabolism. Assessment of lactate production and oxygen consumption demonstrated that LMW-PTP silencing enhances glycolytic flux and slow down the oxidative metabolism. In particular, LMW-PTP expression affects PKM2 tyrosine-phosphorylation and nuclear localization, modulating its activity. ConclusionAll these findings propose that tumor cells are subjected to metabolic reprogramming after LMW-PTP silencing, enhancing glycolytic flux, probably to compensate the inhibition of mitochondrial metabolism. General significanceOur results highlight the involvement of LMW-PTP in regulating glucose metabolism in A375 melanoma cells.

4-oxo-1,4-dihydrocinnoline Derivative with Phosphatase 1B Inhibitor Activity Enhances Leptin Signal Transduction in Hypothalamic Neurons

Most of regulatory effects of leptin on feeding behavior and energy metabolism are implemented via the hypothalamic leptin system. Attenuation of its activity leads to hyperphagia, obesity and other metabolic disorders. To prevent these pathological conditions, it is necessary to develop approaches aimed at restoring the leptin system. Most promising of them is creating efficient inhibitors of protein phosphotyrosine phosphatase PTP1B, a negative regulator of leptin signaling. The aim of this work was to synthesize a new compound, ethyl-3-(hydroxymethyl)-4-oxo-1,4-dihydrocinnoline-6-carboxylate (PI-04), a 4-oxo-1,4-dihydrocinnoline derivative with a PTP1B inhibitor activity, and to study its effect on IRS2- and STAT3-dependent leptin pathways in culture of hypothalamic neurons isolated from 18-day-old rat embryos. It was shown that PI-04 enhances the stimulatory effect of leptin on phosphorylation of the IRS2 protein at the Ser731 residue and of the STAT3 transcriptional factor at the Tyr705 residue, suggesting a potentiation of functional responses of hypothalamic neurons to leptin in the presence of this compound. The potentiating effect of PI-04 on leptin signaling was implemented at micromolar concentrations when this substance had virtually no effect on neuronal survival. The data obtained are promising in terms of creating phosphatase PTP1B inhibitors based on 4-oxo-1,4-dihydrocinnoline, as well as the possibility of their further use to prevent and correct metabolic disorders caused by attenuated activity of the hypothalamic leptin system.

Protein phosphotyrosine phosphatase 1B: Structure, function, role in the development of metabolic disorders and their correction by the enzyme inhibitors

Protein phosphotyrosine phosphatase 1B (PPTP1B) dephosphorylates receptor and nonreceptor forms of tyrosine kinases, causing the inhibition of their activity and thus regulating appropriate signaling cascades. Increased PPTP1B activity leads to insulin and leptin resistance, being among the causes of type 2 diabetes mellitus and many other metabolic and functional disorders. Selective PPTP1B inhibitors normalize functions of insulin, leptin and some other systems comprising different forms of tyrosine kinases as signaling components, and their development is a promising approach to treat and prevent metabolic disorders. Currently, an active search is in progress for “binary” PPTP1B inhibitors able to interact simultaneously with the catalytic and allosteric sites of the enzyme, providing thereby high efficiency and selectivity of their action. This review focuses on the status quo of the problem of studying the structure, functions and regulatory properties of PPTP1B, its role in the development of metabolic disorders, as well as on recent advances in designing selective PPTP1B inhibitors.

Focus on O-phosphohydroxylysine, O-phosphohydroxyproline, N 1-phosphotryptophan and S-phosphocysteine.

The synthesis and chemistry of the lesser-known phosphoamino acids, O-phosphohydroxylysine, O-phosphohydroxyproline, N 1-phosphotryptophan and S-phosphocysteine are described in detail. In addition, where anything at all is known, the biological synthesis, occurrence and functions of these phosphoamino acids are described. Of these phosphoamino acids, only N 1-phosphotryptophan has not been reported to occur in proteins; however, apart from the roles of S-phosphocysteine in the sugar transporter component (EII) and in catalysis by protein phosphotyrosine phosphatase, little is currently known about the biological roles of the phosphoamino acids when they occur as post-translational modifications.

Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells: IMPLICATIONS IN VEGF-DEPENDENT ANGIOGENESIS AND DIABETIC WOUND HEALING

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Overexpression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B overexpression. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGF-induced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA-transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes.

Tyrosine 601 of Bacillus subtilis DnaK Undergoes Phosphorylation and Is Crucial for Chaperone Activity and Heat Shock Survival.

In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple Stable Isotope Labeling by Amino acids in Cell culture-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type (WT), were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the WT dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

PTP1B in Calpain Dependent Feedback Regulation of VEGFR2 in Endothelial Cells: Implication in VEGF‐Dependent Angiogenesis and Diabetic Wound Healing

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF‐induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF‐induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via cleavage and activation of its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF‐induced angiogenesis, especially in diabetic wound healing. Over‐expression of PTP1B inhibited VEGF‐induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells (BAECs), while PTP1B siRNA increased both VEGFR2 and Akt phosphorylation in response to VEGF stimulation. Calpain inhibitor induced VEGFR2 activation can be completely blocked by PTP1B over‐expression. These data implicate calpain/PTP1B feedback regulation of VEGFR2. We next examined a potential role of PTP1B in VEGF‐induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA transfected mice also had augmented endothelial outgrowth. To further define the role of PTP1B in physiological angiogenesis in vivo, wound healing assay in diabetic mice was carried out. Furthermore, we found that PTP1B over‐expression or calpain inhibition significantly deteriorated wound healing in STZ‐induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop. A critical role of PTP1B as a negative regulator in VEGF‐induced angiogenesis is revealed. Modulation of local calpain and PTP1B activities may prove beneficial in the treatment of defective wound healing in diabetes.Support or Funding InformationThis study has been supported by National Heart, Lung and Blood Institute (HL088975).

A Hybrid Mechanism for the Synechocystis Arsenate Reductase Revealed by Structural Snapshots during Arsenate Reduction

Evolution of enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the "hybrid" mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the enzyme reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family.

Identification of thiamine monophosphate hydrolyzing enzymes in chicken liver

In animals, thiamine monophosphate (TMP) is an intermediate on the path of thiamine diphosphate, the coenzyme form of vitamin B1, degradation. The enzymes involved in TMP metabolism in animal tissues are not identified hitherto. The aim of this work was to study TMP hydrolysis in chicken liver. Two phosphatases have been found to contribute to TMP hydrolysis in liver homogenate. The first one, possessing a maximal activity at pH 6.0, is soluble, whereas the second one represents a membrane-bound enzyme with a pH optimum of 9.0. Membrane-bound TMPase activity was enhanced 1.7-fold by 5 mM Mg2+ ions and strongly inhibited by levamisole in uncompetitive manner with K1 of 53 μM, indicating the involvement of alkaline phosphatase. An apparent Km of alkaline phosphatase for TMP was calculated from the Hanes plot to be 0.6 mM. The soluble TMPase has an apparent Km of 0.7 mM; this enzyme is Mg2+ independent and insensitive to levamisole. As estimated by gel filtration on a Toyopearl HW-55 column, the soluble enzyme has a molecular mass of 17.8 kDa, TMPase activity being eluted simultaneously with peaks of flavinmononucleotide and p-nitrophenyl phosphatase activity. Thus, TMP appears to be a physiological substrate for a low-molecular weight acid phosphatase, also known as low-molecular-weight protein phosphotyrosine phosphatase.

Antitumor activity of irradiated riboflavin on human renal carcinoma cell line 786-O.

Riboflavin (vitamin B2) is a precursor for coenzymes involved in energy production, biosynthesis, detoxification, and electron scavenging. Previously, we demonstrated that irradiated riboflavin (IR) has potential antitumoral effects against human leukemia cells (HL60), human prostate cancer cells (PC3), and mouse melanoma cells (B16F10) through a common mechanism that leads to apoptosis. Hence, we here investigated the effect of IR on 786-O cells, a known model cell line for clear cell renal cell carcinoma (CCRCC), which is characterized by high-risk metastasis and chemotherapy resistance. IR also induced cell death in 786-O cells by apoptosis, which was not prevented by antioxidant agents. IR treatment was characterized by downregulation of Fas ligand (TNF superfamily, member 6)/Fas (TNF receptor superfamily member 6) (FasL/Fas) and tumor necrosis factor receptor superfamily, member 1a (TNFR1)/TNFRSF1A-associated via death domain (TRADD)/TNF receptor-associated factor 2 (TRAF) signaling pathways (the extrinsic apoptosis pathway), while the intrinsic apoptotic pathway was upregulated, as observed by an elevated Bcl-2 associated x protein/B-cell CLL/lymphoma 2 (Bax/Bcl-2) ratio, reduced cellular inhibitor of apoptosis 1 (c-IAP1) expression, and increased expression of apoptosis-inducing factor (AIF). The observed cell death was caspase-dependent as proven by caspase 3 activation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. IR-induced cell death was also associated with downregulation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian)/protein serine/threonine kinase B/extracellular signal-regulated protein kinase 1/2 (Src/AKT/ERK1/2) pathway and activation of p38 MAP kinase (p38) and Jun-amino-terminal kinase (JNK). Interestingly, IR treatment leads to inhibition of matrix metalloproteinase-2 (MMP-2) activity and reduced expression of renal cancer aggressiveness markers caveolin-1, low molecular weight phosphotyrosine protein phosphatase (LMWPTP), and kinase insert domain receptor(a type III receptor tyrosine kinase) (VEGFR-2). Together, these results show the potential of IR for treating cancer.

Effects of extremely low frequency magnetic fields on NGF induced neuronal differentiation of PC12 cells.

Extremely low-frequency magnetic fields (ELF-MFs) affect various cellular processes and systems, such as cell proliferation, differentiation and metabolic pathways. The present study investigated ELF-MFs effect on nerve growth factor (NGF) induced neuronal differentiation of PC12 cells using proteomic applications to understand its role in the enhancement of neuronal differentiation. After 50 Hz, 1 mT ELF-MFs 5-day exposure on NGF induced PC12 cells, it was observed to increase neurite length as well as an increase in the number of neurite bearing cells. It was also discovered that there was a decrease in proliferation activity, which is associated with an increase in differentiated cells. Neuronal differentiation related mRNA levels and protein levels were increased in NGF induced PC12 cells. Compared with NGF induced group, ELF-MFs stimulated PC12 cells had different protein expression as measured with two-dimensional electrophoresis (2-DE) gels. Consequently six differentially expressed spots were detected between the 2-DE maps, which were identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF LC/MS/MS) as: peripherin, neurosecretory protein nerve growth factor inducible (VGF8a) precursor, dnaK-type molecular chaperone sp72-ps1 (HSP72-psI), low molecular weight (Mr) phosphotyrosine protein phosphatase isoenzyme AcP1 (LMW-PTP/ACP1), Tubulin alpha-1A (TUBA1A) chain, outcome predictor in acute leukemia 1 homolog (OPA1L). The identification of these proteins provides clues to the mechanism of ELF-MFs stimulation on NGF induced PC12 cells that occur during neuronal differentiation and may contribute to the development novel treatments for neurodegenerative diseases.

Proteomic analysis indicates that overexpression and nuclear translocation of lactoylglutathione lyase (GLO1) is associated with tumor progression in murine fibrosarcoma

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma.

Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs

BackgroundThe failure of Mycobacterium bovis Bacille Calmette-Guérin to impart satisfactory protection against adult pulmonary tuberculosis has necessitated the development of more effective TB vaccines. The assumption that the vaccine strain should be antigenically as similar as possible to the disease causing pathogen has led to the evaluation of M.tuberculosis mutants as candidate tuberculosis vaccines.Methods/Principal FindingsIn this study, we have generated a mutant of M.tuberculosis (Mtb∆mms) by disrupting 3 virulence genes encoding a mycobacterial secretory acid phosphatase (sapM) and two phosphotyrosine protein phosphatases (mptpA and mptpB) and have evaluated its protective efficacy in guinea pigs. We observed that Mtb∆mms was highly attenuated in THP-1 macrophages. Moreover, no bacilli were recovered from the lungs and spleens of guinea pigs after 10 weeks of Mtb∆mms inoculation, although, initially, the mutant exhibited some growth in the spleens. Subsequently, when Mtb∆mms was evaluated for its protective efficacy, we observed that similar to BCG vaccination, Mtb∆mms exhibited a significantly reduced CFU in the lungs of guinea pigs when compared with the unvaccinated animals at 4 weeks after challenge. In addition, our observations at 12 weeks post challenge demonstrated that Mtb∆mms exhibited a more sustainable and superior protection in lungs as compared to BCG. However, the mutant failed to control the hematogenous spread as the splenic bacillary load between Mtb∆mms vaccinated and sham immunized animals was not significantly different. The gross pathological observations and histopathological observations corroborated the bacterial findings. Inspite of disruption of phosphatase genes in MtbΔmms, the lipid profiles of M.tuberculosis and MtbΔmms were identical indicating thereby that the phenotype of the mutant was ascribed to the loss of phosphatase genes and the influence was not related to any alteration in the lipid composition.Conclusions/SignificanceThis study highlights the importance of M.tuberculosis mutants in imparting protection against pulmonary TB.

Phospho-tyrosine phosphatase inhibitor Bpv(Hopic) enhances C2C12 myoblast migration in vitro. Requirement of PI3K/AKT and MAPK/ERK pathways

Muscle progenitor cell migration is an important step in skeletal muscle myogenesis and regeneration. Migration is required for muscle precursors to reach the site of damage and for the alignment of myoblasts prior to their fusion, which ultimately contributes to muscle regeneration. Limited spreading and migration of donor myoblasts are reported problems of myoblast transfer therapy, a proposed therapeutic strategy for Duchenne Muscular Dystrophy, warranting further investigation into different approaches for improving the motility and homing of these cells. In this article, the effect of protein phospho-tyrosine phosphatase and PTEN inhibitor BpV(Hopic) on C2C12 myoblast migration and differentiation was investigated. Applying a wound healing migration model, it is reported that 1μM BpV(Hopic) is capable of enhancing the migration of C2C12 myoblasts by approximately 40% in the presence of myotube conditioned media, without significantly affecting their capacity to differentiate and fuse into multinucleated myotubes. Improved migration of myoblasts treated with 1μM BpV(Hopic) was associated with activation of PI3K/AKT and MAPK/ERK pathways, while their inhibition with either LY294002 or UO126, respectively, resulted in a reduction of C2C12 migration back to control levels. These results propose that bisperoxovanadium compounds may be considered as potential tools for enhancing the migration of myoblasts, while not reducing their differentiation capacity and underpin the importance of PI3K/AKT and MAPK/ERK signalling for the process of myogenic progenitor migration.

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Comparative Proteomic Profiling of Human Breast Cell Lines

Although epidemiological studies have suggested that the isoflavone genistein found in soy may be protective against breast cancer, its effects on molecular processes involved in cell proliferation have not been clearly elucidated. Using comparative shotgun proteomics, we examined the proteome of non‐cancerous breast MCF10A cells, estrogen receptor‐positive breast cancer T47D cells, and Hras transfected MCF10A cells after treatment with 3, 10, and 30 μM genistein for 18 h. While genistein induced G1‐phase arrest in MCF10A‐Hras and G2/M‐phase arrest in T47D cells, it did not alter the cell cycle distribution in MCF10A cells. The arrest in G1‐phase of MCF10A‐Hras cells coincided with downregulation of the proliferation marker Ki67, epidermal growth factor receptor, various components of the mitogen‐activated protein kinase family of enzymes and low molecular weight phosphotyrosine protein phosphatase (ACP1), The arrest in G2/M‐phase of T47D cells was accompanied by upregulation of caspase‐8 and mSH2, suggesting that genistein may induce a G2/M checkpoint/proapoptotic response involving the mismatch repair system. Taken together, these findings suggest a differential response to genistein in cancerous cells and non‐malignant epithelial cells, and demonstrate the utility of comparative shotgun proteomics for investigating the cancer preventative effects of bioactive components.

Low Molecular Weight Phosphotyrosine Protein Phosphatases as Emerging Targets for the Design of Novel Therapeutic Agents

ADVERTISEMENT RETURN TO ISSUEPREVPerspectiveNEXTLow Molecular Weight Phosphotyrosine Protein Phosphatases as Emerging Targets for the Design of Novel Therapeutic AgentsRosanna Maccari* and Rosaria OttanàView Author Information Dipartimento Farmaco-Chimico, Faculty of Pharmacy, University of Messina, Polo Universitario dell’Annunziata, 98168 Messina, Italy* Telephone: +39906766406. Fax: +39906766402. E-mail: [email protected]Cite this: J. Med. Chem. 2012, 55, 1, 2–22Publication Date (Web):October 11, 2011Publication History Received13 May 2011Published online26 October 2011Published inissue 12 January 2012https://doi.org/10.1021/jm200607gCopyright © 2011 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views3185Altmetric-Citations26LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (4 MB) Get e-AlertsSUBJECTS:Cell and molecular biology,Inhibitors,Monomers,Peptides and proteins,Post-translational modification Get e-Alerts