Serine-threonine Protein Phosphatase Research Articles

PF396 HISTONE DEACETYLASE INHIBITOR PANOBINOSTAT TARGETS CALCINEURIN/NFATC1 SIGNALING AND ABROGATES THE RESISTANCE OF PH+ LEUKEMIA CELLS TO DASATINIB

Background:BCR‐ABL‐targeted tyrosine kinase inhibitors (TKI) revolutionized the outcome of patients inflicted with CML and Ph+ B‐ALL. However, TKI resistance continues to pose challenges for both CML and ALL.Aims:We hypothesized that overcoming the BM microenvironment‐mediated protection of Ph+ leukemic cells from TKI‐mediated apoptosis may further enhance the responsiveness to TKI therapy.Methods:In vitro experiments using BCR‐ABL‐positive ALL (BV‐173, NALM1 and NALM20) and CML (K562 and LAMA84) cell lines.Results:Dasatinib treatment significantly suppressed the growth of BCR‐ABL‐positive ALL and CML cell lines (p < 0.01). However, co‐culture with bone marrow stromal cells (BMSCs) protected both ALL and CML cells from dasatinib‐mediated toxicity. Furthermore, dasatinib treatment promoted significant upregulation of the chemokine receptor CXCR4, on both mRNA and cell surface levels. Elevated CXCR4 expression was accompanied by increased responsiveness of ALL and CML cells to CXCL12 stimulation, resulting in strong and sustained phosphorylation of Erk1/2 and Akt and increased adhesion capacity to BMSCs. Consequently, dasatinib‐induced upregulation of CXCR4 promotes the stroma‐mediated survival advantage of Ph+ leukemia cells upon TKI therapy.Next, in order to overcome the CXCR4‐mediated stromal protection, we choose to combine dasatinib with the histone deacetylase inhibitor panobinostat, for its known ability to deplete CXCR4 in AML cells. Single‐agent treatment with panobinostat demonstrated significant inhibition of Ph+ ALL and CML cell growth at low nanomolar concentrations (p < 0.01). Importantly, combination of panobinostat with dasatinib synergized (CI<0.5), effectively overcoming the protection provided by BMSCs and inducing apoptosis of Ph+ leukemia cells, as demonstrated by phosphatidylserine externalization and mitochondrial depolarization. Moreover, panobinostat/dasatinib treatment suppressed cell cycle progression, inducing subG0/G1 DNA fragmentation. Furthermore, combining panobinostat with dasatinib significantly reduced CXCR4 surface levels and CXCL12‐mediated responses, including Erk1/2 and Akt activation in Ph+ leukemic cells. Notably, panobinostat/dasatininb combination significantly inhibited soft agar colony formation of K562 cells.To elucidate the molecular mechanisms, we performed gene and protein expression analysis. Panobinostat, alone or in combination with dasatinib, significantly down‐regulated the protein levels of calcineurin, a serine–threonine protein phosphatase previously implicated in ALL and CML pathogenesis, as well as of NFATc1, a critical effector of the calcineurin signaling cascade, and NFATc1‐regulated target genes. Additionally, inhibiting calcineurin activity with cyclosporin A (CsA) decreased both basal and dasatinib‐induced CXCR4 surface levels and sensitized leukemic cells to dasatinib, overcoming the protection of the BMSCs. In addition, combining CsA with panobinostat demonstrated synergistic (CI<0.5) anti‐leukemic effect that results in deeper suppression of NFATc1‐regulated target genes. We thus link the effect of panobinostat with calcineurin‐dependent downregulation of CXCR4 and NFTAc1 signaling, blocking the ability of the leukemic cells to respond to CXCL12‐mediated stromal support and enhancing their response to BCR‐ABL inhibition.Summary/Conclusion:Taken together, our results identify calcineurin/NFATc1 signaling pathway as a novel target of panobinostat in Ph+ leukemia cells. Based on these findings, HDAC inhibition with panobinostat may be effective strategy for facilitating the anti‐leukemic activity of TKI therapy.

Transcriptome analysis of finger millet (Eleusine coracana (L.) Gaertn.) reveals unique drought responsive genes

Finger millet (Eleusine coracana (L.) Gaertn.), an important C4 species is known for its stress hardiness and nutritional significance. To identify novel drought responsive mechanisms, we generated transcriptome data from leaf tissue of finger millet, variety GPU-28, exposed to gravimetrically imposed drought stress so as to simulate field stress conditions. De novo assembly based approach yielded 80,777 and 90,830 transcripts from well-irrigated (control) and drought-stressed samples, respectively. A total of 1790 transcripts were differentially expressed between the control and drought-stress treatments. Functional annotation and pathway analysis indicated activation of diverse drought-stress signalling cascade genes such as serine threonine protein phosphatase 2A (PP2A), calcineurin B-like interacting protein kinase31 (CIPK31), farnesyl pyrophosphate synthase (FPS), signal recognition particle receptor α (SRPR α) etc. The basal regulatory genes such as TATA-binding protein (TBP)-associated factors (TAFs) werefound to be drought responsive, indicating that genes associated with housekeeping or basal regulatory processes are activated underdrought in finger millet. A significant portion of the expressed genes was uncharacterized, belonging to the category of proteins of unknown functions (PUFs). Among the differentially expressed PUFs, we attempted to assign putative function for a few, using anovel annotation tool, Proteins of Unknown Function Annotation Server. Analysis of PUFs led to the discovery of novel drought responsive genes such as pentatricopeptide repeat proteins and tetratricopeptide repeat proteins that serve as interaction modules in multiprotein interactions. The transcriptome data generated can be utilized for comparative analysis, and functional validation of the genes identified would be useful to understand the drought adaptive mechanisms operating under field conditions in finger millet, as has been already attempted for a few candidates such as CIPK31 and TAF6. Such an attempt is needed to enhance the productivity of finger millet under water-limited conditions, and/or to adopt the implicated mechanisms in other related crops.

MiR-650 promotes motility of anaplastic thyroid cancer cells by targeting PPP2CA.

Aberrant expression of miRNAs is crucial in several tissues tumorigenesis including thyroid. Recent studies demonstrated that miR-650 plays different role depending on the cancer type. Herein, we investigated the role of miR-650 in thyroid carcinoma. The expression of miR-650 was analyzed in human thyroid tissues by q-RT-PCR. Anaplastic (8505C, CAL62, SW1736) and papillary (TPC-1) thyroid cancer cell lines were used to dissect the role of miR-650 on malignant hallmarks of transformation. Label-free proteomic analysis was exploited to unravel the targets of miR-650, while luciferase reporter assay and functional experiments were performed to confirm a selected target. Spearman's rank correlation test was used to assess the association between miR-650 and its target in human thyroid cancer tissues. miR-650 is over-expressed in anaplastic (ATC) thyroid carcinoma where it enhances cell migration and invasion. Proteomic label-free and bioinformatics analysis revealed that the serine-threonine protein phosphatase 2 catalytic subunit alpha (PPP2CA) is a target of miR-650; these finding were confirmed by luciferase assay. Restoration of PPP2CA mRNA, deprived of its 3'UTR, is able to revert the malignant phenotype induced by miR-650 in HEK-293 cells. Importantly, PPP2CA is down-regulated in ATC tissues and is inversely correlated with miR-650. miR-650 displayed oncogenic activity in ATC cells through targeting PPP2CA phosphatase. These results suggest that miR-650/PPP2CA axis could be modulated to interfere with motile ability of thyroid carcinoma cells.

Synergism of myocardial β-adrenoceptor blockade and I1-imidazoline receptor-driven signaling: Kinase-phosphatase switching

Recently identified imidazoline receptors of the first type (I1Rs) on the cardiomyocyte's sarcolemma open a new field in calcium signaling research. In particular, it is interesting to investigate their functional interaction with other well-known systems, such as β-adrenergic receptors. Here we investigated the effects of I1Rs activation on L-type voltage-gated Ca2+-currents under catecholaminergic stress induced by the application of β-agonist, isoproterenol. Pharmacological agonist of I1Rs (I1-agonist), rilmenidine, and the putative endogenous I1-ligand, agmatine, have been shown to effectively reduce Ca2+-currents potentiated by isoproterenol. Inhibitory analysis shows that the ability to suppress voltage-gated Ca2+-currents by rilmenidine and agmatine is fully preserved in the presence of the protein kinase A blocker (PKA), which indicates a PKA-independent mechanism of their action. The blockade of NO synthase isoforms with 7NI does not affect the intrinsic effects of agmatine and rilmenidine, which suggests NO-independent signaling pathways triggered by I1Rs. A nonspecific serine/threonine protein phosphatase (STPP) inhibitor, calyculin A, abrogates effects of rilmenidine or agmatine on the isoproterenol-induced Ca2+-currents. Direct measurements of phosphatase activity in the myocardial tissues showed that activation of the I1Rs leads to stimulation of STPP, which could be responsible for the I1-agonist influences. Obtained data clarify peripheral effects that occur during activation of the I1Rs under endogenous catecholaminergic stress, and can be used in clinical practice for more precise control of heart contractility in some cardiovascular pathologies.

The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells.

Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future.

Protein Serine/Threonine Phosphatases: Keys to Unlocking Regulators and Substrates.

Protein serine/threonine phosphatases (PPPs) are ancient enzymes, with distinct types conserved across eukaryotic evolution. PPPs are segregated into types primarily on the basis of the unique interactions of PPP catalytic subunits with regulatory proteins. The resulting holoenzymes dock substrates distal to the active site to enhance specificity. This review focuses on the subunit and substrate interactions for PPP that depend on short linear motifs. Insights about these motifs from structures of holoenzymes open new opportunities for computational biology approaches to elucidate PPP networks. There is an expanding knowledge base of posttranslational modifications of PPP catalytic and regulatory subunits, as well as of their substrates, including phosphorylation, acetylation, and ubiquitination. Cross talk between these posttranslational modifications creates PPP-based signaling. Knowledge of PPP complexes, signaling clusters, as well as how PPPs communicate with each other in response to cellular signals should unlock the doors to PPP networks and signaling "clouds" that orchestrate and coordinate different aspects of cell physiology.

Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase

The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC.

A serine/threonine phosphatase 1 of Streptococcus suis type 2 is an important virulence factor.

Streptococcus suis is regarded as one of the major pathogens of pigs, and Streptococcus suis type 2 (SS2) is considered a zoonotic bacterium based on its ability to cause meningitis and streptococcal toxic shock-like syndrome in humans. Many bacterial species contain genes encoding serine/threonine protein phosphatases (STPs) responsible for dephosphorylation of their substrates in a single reaction step. This study investigated the role of stp1 in the pathogenesis of SS2. An isogenic stp1 mutant (Δstp1) was constructed from SS2 strain ZJ081101. The Δstp1 mutant exhibited a significant increase in adhesion to HEp-2 and bEnd.3 cells as well as increased survival in RAW264.7 cells, as compared to the parent strain. Increased survival in macrophage cells might be related to resistance to reactive oxygen species since the Δstp1 mutant was more resistant than its parent strain to paraquat-induced oxidative stress. However, compared to parent strain virulence, deletion of stp1 significantly attenuated virulence of SS2 in mice, as shown by the nearly double lethal dose 50 value and the lower bacterial load in organs and blood in the murine model. We conclude that Stp1 has an essential role in SS2 virulence.

Salinity induced differential methylation patterns in contrasting cultivars of foxtail millet (Setaria italica L.).

Genome-wide methylation analysis of foxtail millet cultivars contrastingly differing in salinity tolerance revealed DNA demethylation events occurring in tolerant cultivar under salinity stress, eventually modulating the expression of stress-responsive genes. Reduced productivity and significant yield loss are the adverse effects of environmental conditions on physiological and biochemical pathways in crop plants. In this context, understanding the epigenetic machinery underlying the tolerance traits in a naturally stress tolerant crop is imperative. Foxtail millet (Setaria italica) is known for its better tolerance to abiotic stresses compared to other cereal crops. In the present study, methylation-sensitive amplified polymorphism (MSAP) technique was used to quantify the salt-induced methylation changes in two foxtail millet cultivars contrastingly differing in their tolerance levels to salt stress. The study highlighted that the DNA methylation level was significantly reduced in tolerant cultivar compared to sensitive cultivar. A total of 86 polymorphic MSAP fragments were identified, sequenced and functionally annotated. These fragments showed sequence similarity to several genes including ABC transporter, WRKY transcription factor, serine threonine-proteinphosphatase, disease resistance, oxidoreductases, cell wall-related enzymes and retrotransposon and transposase like proteins, suggesting salt stress-induced methylation in these genes. Among these, four genes were chosen for expression profiling which showed differential expression pattern between both cultivars of foxtail millet. Altogether, the study infers that salinity stress induces genome-wide DNA demethylation, which in turn, modulates expression of corresponding genes.

Pharmacological Activation of Protein Phosphatase 2 A (PP2A): A Novel Strategy to Fight Against Human Malignancies?

The serine-threonine protein phosphatase 2A (PP2A) regulates multiple cell signaling cascades and its inactivation by viral oncoproteins, mutation of specific structural subunits or upregulation of the cellular endogenous inhibitors may contribute to malignant transformation by regulating specific phosphorylation events. Pharmacological modulation of PP2A activity is becoming an attractive strategy for cancer treatment. Some compounds targeting PP2A are able to induce PP2A reactivation and subsequent cell death in several types of cancer. We undertook a search of bibliographic databases for peer-reviewed articles focusing on the main item of the review. We selected articles published in indexed journals. The quality of retrieved papers was appraised using the standard bibliometric indicators. One hundred and fourteen papers were included in the review. Twenty-seven papers gave an overview of structure and physiological role of PP2A. Twenty-five papers outlined the role of PP2A in tumor suppression. Forty papers analyzed the mechanism involved in PP2A reactivation by synthetic compounds, and twenty-two papers outlined the capability of natural compounds of restoring PP2A activity and how this could be beneficial. Findings analyzed in this review underline the central role of PP2A as a regulator of cell growth and survival, hence its function as tumor suppressor. The discovery that some compounds, either synthetic or natural, are capable of reactivating PP2A opens up new perspectives for future strategies to fully exploit therapeutic potential in human cancer. Thus, this review could also be of particular interest to pharmaceutical or biotechnology companies for drug design and targeted delivery.

The Effects of Microcystins (Cyanobacterial Heptapeptides) on the Eukaryotic Cytoskeletal System.

Microcystins (MCYs) are cyanobacterial heptapeptides known for their high toxicity in eukaryotic cells and for their potential human health hazards. They are potent and specific inhibitors of type 1 and 2A, serine-threonine protein phosphatases (PP1 and PP2A) and as such, interfere with key cellular and metabolic events. Moreover, they induce oxidative stress involving reactive oxygen species (ROS) generation. Their cytoskeletal effects involve both mitotic and differentiated eukaryotic cells. The main objective of the present review is to summarize the most important cytoskeletal effects of MCY on human, animal and plant cells known to date and to give an insight into the cellular and molecular background of these alterations. Disruptions of microtubule (MTs), microfilament (MF) and intermediate filament (IF) organization have all been described, having consequences on cell shape, tissue integrity and functionality and mitotic division. Most of these subcellular changes are closely related to PP1 and PP2A inhibition and involve misfunctioning of cytoskeleton associated proteins. However, several cytoskeletal alterations are likely to be related to the induction of oxidative stress. MCY induced changes in MT, MF and IF assembly may have severe human health consequences. The main target of cyanotoxin in human/ animal cells is liver and cytoskeletal disruption alters structure and functioning of hepatocytes. However, many other cell types undergo alterations similar to those observed in hepatocytes. Both PP1/PP2A inhibition and ROS generation are involved and the activation of mitogen activated protein kinases (MAPKs) seems to play a crucial role in the molecular events leading to cytoskeletal disruption.

PP2A(Cdc55)'s role in reductional chromosome segregation during achiasmate meiosis in budding yeast is independent of its FEAR function.

PP2ACdc55 is a highly conserved serine-threonine protein phosphatase that is involved in diverse cellular processes. In budding yeast, meiotic cells lacking PP2ACdc55 activity undergo a premature exit from meiosis I which results in a failure to form bipolar spindles and divide nuclei. This defect is largely due to its role in negatively regulating the Cdc Fourteen Early Anaphase Release (FEAR) pathway. PP2ACdc55 prevents nucleolar release of the Cdk (Cyclin-dependent kinase)-antagonising phosphatase Cdc14 by counteracting phosphorylation of the nucleolar protein Net1 by Cdk. CDC55 was identified in a genetic screen for monopolins performed by isolating suppressors of spo11Δ spo12Δ lethality suggesting that Cdc55 might have a role in meiotic chromosome segregation. We investigated this possibility by isolating cdc55 alleles that suppress spo11Δ spo12Δ lethality and show that this suppression is independent of PP2ACdc55’s FEAR function. Although the suppressor mutations in cdc55 affect reductional chromosome segregation in the absence of recombination, they have no effect on chromosome segregation during wild type meiosis. We suggest that Cdc55 is required for reductional chromosome segregation during achiasmate meiosis and this is independent of its FEAR function.

Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling.

Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine-threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species-generating NADPH oxidase-4 (Nox4) is induced downstream of ATF4, binds to a PP1-targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4-regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia-reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4-GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.

Addressing the Potential Role of Fingolimod in Cancer Therapy

Fingolimod is a well known immunomodulator used by oral route in relapsing multiple sclerosis patients. Upon phosphorylation by sphingosine kinase 2 (SPHK2), fingolimod binds to one or more of at least five G protein coupled receptors known as S1PR1-5, thus it causes S1PRs internalization and consequent sequestration of lymphocytes in lymphoid organs. Fingolimod also affects other signaling pathways. In particular, this drug is able to activate the serine-threonine protein phosphatase 2A (PP2A) which regulates multiple cell signaling cascades by virtue of its phosphatase activity. PP2A loss-of-function may represent one of the major events contributing to cancer development and progression. Hence, there is a need for therapeutic PP2A reactivation. In this regard, fingolimod is revealing a promising candidate for cancer treatment due to its ability to reactivate PP2A, reduce cell viability and promote apoptosis. However, the appropriate dosage and safety remain a challenge.

Protein phosphatase 2A plays an important role in migration of bone marrow stroma cells.

Administration of bone marrow stroma cells (BMSCs) has the potential to ameliorate degenerative disorders and to repair injured sites. The homing of transplanted BMSCs to damaged tissues is a critical property of engraftment. Therefore, it is important to understand signal molecules controlling migration of BMSCs. Here, we demonstrate that serine-threonine protein phosphatase 2A (PP2A) is responsive to migration of BMSCs. Pharmacological Inhibition of PP2A, using okadaic acid (OA), leads to attenuated cell migration in rat primary BMSCs both in the absence or presence of stromal cell-derived factor-1 (SDF-1). Consistent with the above findings, knockdown of the main catalytic subunit PP2Acα using small interfering RNA also attenuates chemotaxis of BMSCs. On the other hand, cell viability of BMSCs remains unchanged with OA treatment or knockdown of PP2Acα subunit. Moreover, we observed an upregulation of PP2A-B55β in transcription level after SDF-1 treatment, indicating their potential role as the functioning regulatory subunit of PP2A phosphatase in BMSCs migration model. Collectively, these data provide first insight into the modulation of BMSCs migration by PP2A phosphatase activity and lay a foundation for exploring PP2A signaling as a modulating target for BMSCs transplantation.

Crystal Structure of Okadaic Acid Binding Protein 2.1: A Sponge Protein Implicated in Cytotoxin Accumulation.

Okadaic acid (OA) is a marine polyether cytotoxin that was first isolated from the marine sponge Halichondria okadai. OA is a potent inhibitor of protein serine/threonine phosphatases (PP) 1 and 2A, and the structural basis of phosphatase inhibition has been well investigated. However, the role and mechanism of OA retention in the marine sponge have remained elusive. We have solved the crystal structure of okadaic acid binding protein 2.1 (OABP2.1) isolated from H. okadai; it has strong affinity for OA and limited sequence homology to other proteins. The structure revealed that OABP2.1 consists of two α-helical domains, with the OA molecule deeply buried inside the protein. In addition, the global fold of OABP2.1 was unexpectedly similar to that of aequorin, a jellyfish photoprotein. The presence of structural homologues suggested that, by using similar protein scaffolds, marine invertebrates have developed diverse survival systems adapted to their living environments.

Focused library with a core structure extracted from natural products and modified: application to phosphatase inhibitors and several biochemical findings.

Synthesis of a focused library is an important strategy to create novel modulators of specific classes of proteins. Compounds in a focused library are composed of a common core structure and different diversity structures. In this Account, we describe our design and synthesis of libraries focused on selective inhibitors of protein phosphatases (PPases). We considered that core structures having structural and electronic features similar to those of PPase substrates, phosphate esters, would be a reasonable choice. Therefore, we extracted core structures from natural products already identified as PPase inhibitors. Since many PPases share similar active-site structures, such phosphate-mimicking core structures should interact with many enzymes in the same family, and therefore the choice of diversity structures is pivotal both to increase the binding affinity and to achieve specificity for individual enzymes. Here we present case studies of application of focused libraries to obtain PPase inhibitors, covering the overall process from selection of core structures to identification and evaluation of candidates in the focused libraries. To synthesize a library focused on protein serine-threonine phosphatases (PPs), we chose norcantharidin as a core structure, because norcantharidin dicarboxylate shows a broad inhibition profile toward several PPs. From the resulting focused library, we identified a highly selective PP2B inhibitor, NCA-01. On the other hand, to find inhibitors of dual-specificity protein phosphatases (DSPs), we chose 3-acyltetronic acid extracted from natural product RK-682 as a core structure, because its structure resembles the transition state in the dephosphorylation reaction of DSPs. However, a highly selective inhibitor was not found in the resulting focused library. Furthermore, an inherent drawback of compounds having the highly acidic 3-acyltetronic acid as a core structure is very weak potency in cellulo, probably due to poor cell membrane permeability. Therefore, we next modified the core structure from acidic to neutral by transformation to the enamine derivative and constructed a second-generation focused library (RE derivatives). The resulting compounds showed dramatically improved cell membrane permeability and inhibitory selectivity and included VHR (vaccinia VH1-related)-selective RE12 and CDC25A/B (cell division cycle 25A/B)-selective RE44. These inhibitors act on target enzymes in cellulo and do not generate reactive oxygen species, which is a potential problem with quinoid-type inhibitors of CDC25s. The cellular activity of RE12 was further improved by replacement of the side chain to afford RE176, which showed more potent antiproliferative activity than RE12 against HeLa cells. The dramatic change of inhibitory selectivity obtained by core structure modification from 3-acyltetronic acid to its enamine derivative was associated with a change in the mode of action. Namely, RE derivatives were found to be noncompetitive inhibitors with respect to a small-molecular substrate of CDC25A/B, whereas RK-682 was a competitive inhibitor of VHR. We identified the binding site of RE derivatives on the CDC25A as a pocket adjacent to the active site; this appears to be a promising target site for development of further novel inhibitors of CDC25s.

Biochemical and molecular characterization of the calcineurin in Echinococcus granulosus larval stages

Calcineurin (CaN) is a Ca2+-calmodulin activated serine–threonine protein phosphatase that couples the local or global calcium signals, thus controlling important cellular functions in physiological and developmental processes. The aim of this study was to characterize CaN in Echinococcus granulosus (Eg-CaN), a human cestode parasite of clinical importance, both functionally and molecularly. We found that the catalytic subunit isoforms have predicted sequences of 613 and 557 amino acids and are substantially similar to those of the human counterpart, except for the C-terminal end. We also found that the regulatory subunit consists of 169 amino acids which are 87% identical to the human ortholog. We cloned a cDNA encoding for one of the two catalytic subunit isoforms of CaN (Eg-can-A1) as well as the only copy of the Eg-can-B gene, both constitutively transcribed in all Echinococcus larval stages and responsible for generating a functionally active heterodimer. Eg-CaN native enzyme has phosphatase activity, which is enhanced by Ca2+/Ni2+ and reduced by cyclosporine A and Ca2+ chelators. Participation of Eg-CaN in exocytosis was demonstrated using the FM4-64 probe and Eg-CaN-A was immunolocalized in the cytoplasm of tegumental cells, suckers and excretory bladder of protoscoleces. We also showed that the Eg-can-B transcripts were down-regulated in response to low Ca2+ intracellular level, in agreement with decreased enzyme activity. Confocal microscopy revealed a striking pattern of Eg-CaN-A in discrete fluorescent spots in the protoscolex posterior bladder and vesicularized protoscoleces beginning the vesicular differentiation. In contrast, Eg-CaN-A was undetectable during the pre-microcyst closing stage while a high DDX-like RNA helicase expression was evidenced. Finally, we identified and analyzed the expression of CaN-related endogenous regulators.

Physiological performance, secondary metabolite and expression profiling of genes associated with drought tolerance in Withania somnifera.

Physiological, biochemical, and gene expression responses under drought stress were studied in Withania somnifera. Photosynthesis rate, stomatal conductance, transpiration rate, relative water content, chlorophyll content, and quantum yield of photosystems I and II (PSI and PSII) decreased in response to drought stress. Comparative expression of genes involved in osmoregulation, detoxification, signal transduction, metabolism, and transcription factor was analyzed through quantitative RT-PCR. The genes encoding 1-pyrroline-5-carboxylate synthetase (P5CS), glutathione S-transferase (GST), superoxide dismutase (SOD), serine threonine-protein kinase (STK), serine threonine protein phosphatase (PSP), aldehyde dehydrogenase (AD), leucoanthocyanidin dioxygenase/anthocyanin synthase (LD/AS), HSP, MYB, and WRKY have shown upregulation in response to drought stress condition in leaf tissues. Enhanced detoxification and osmoregulation along with increased withanolides production were also observed under drought stress. The results of this study will be helpful in developing stress-tolerant and high secondary metabolite yielding genotypes.

CONTROL OF ATRIAL GAP JUNCTION CONDUCTANCE BY INTRACELLULAR PHOSPHATASES

Myocardial action potential conduction velocity is a function of gap junction (GJ) electrical conductance (Gj). The phosphorylation state of GJ proteins, in particular Cx43 determines Gj and the role of protein phosphatases (PPs) remains unclear. This study aims to investigate the role of serine-threonine PPs in regulating Gj, in particular the Ca2+-dependent PP, calcineurin, and the Ca2+-independent PPs, PP1 and PP2A. Left atrial strips were dissected from male, Dunkin-Hartley guinea pigs to estimate Gj by measuring the frequency-dependent intracellular conductance1 in control solution (Na=149.4 mM), or low-Na solution (Na=29.4 mM) to raise [Ca2+]i. Inhibitors to calcinuerin, (cyclosporine-A; 5 µM; calcineurin auto-inhibitory peptide; CAIP, 50 µM); PPI (tautomycin, 5 nM) or PP2A (okadaic acid, 2 nM; fostreicin 100 nM) were used. Western blots were conducted on inhibitor perfused tissue. Values are mean±SEM, normalised to control. Differences were tested by ANOVA; null hypotheses rejected at p Low-Na significantly and reversibly decreased Gj, and in control conditions individual inhibitors had no effect. Cyclosporine-A and CAIP diminished the effects of low-Na (Gj: low-Na, 55±5.8% low-Na+ cyclosporine-A (n=6): 85±9.7%; low-Na+CAIP (n=3): 104±10.2% control). Fostreicin also reduced the low-Na effect (low-Na: 54±2.5%; low-Na+fostreicin (n=6): 77±6.1% control) but not tautomycin (low-Na: 65±4.9%; low-Na+TTM (n=7): 66±3.9%). Phosphorylation of Cx43 during low-Na was diminished by CysA, CAIP and FST. The phosphatases calcineurin and PP2A mediate the decrease of Gj in the left atrium during elevated [Ca2+]i. Connexin43 is one of the targets for calcineurin and PP2A.