Dysfunction of programmed embryo senescence is linked to genetic developmental defects.

Developmental senescence is a form of programmed senescence that contributes to morphogenesis during embryonic development. We showed recently that the SIX1 homeoprotein, an essential regulator of organogenesis, is also a repressor of adult cellular senescence. Alterations in the SIX/EYA pathway are linked to the human branchio-oto-renal (BOR) syndrome, a rare congenital disorder associated with defects in the ears, kidneys and branchial arches. Here, we have used Six1-deficient mice, an animal model of the BOR syndrome, to investigate whether dysfunction of senescence underpins the developmental defects associated with SIX1 deficiency. We have focused on the developing inner ear, an organ with physiological developmental senescence that is severely affected in Six1-deficient mice and BOR patients. We show aberrant levels and distribution of senescence markers in Six1-deficient inner ears concomitant with defective morphogenesis of senescent structures. Transcriptomic analysis and ex vivo assays support a link between aberrant senescence and altered morphogenesis in this model, associated with deregulation of the TGFß/BMP pathway. Our results show that misregulation of embryo senescence may lead to genetic developmental disorders, significantly expanding the connection between senescence and disease.

Conservation and divergence of meiotic DNA double strand break forming mechanisms in Arabidopsis thaliana.

In the current meiotic recombination initiation model, the SPO11 catalytic subunits associate with MTOPVIB to form a Topoisomerase VI-like complex that generates DNA double strand breaks (DSBs). Four additional proteins, PRD1/AtMEI1, PRD2/AtMEI4, PRD3/AtMER2 and the plant specific DFO are required for meiotic DSB formation. Here we show that (i) MTOPVIB and PRD1 provide the link between the catalytic sub-complex and the other DSB proteins, (ii) PRD3/AtMER2, while localized to the axis, does not assemble a canonical pre-DSB complex but establishes a direct link between the DSB-forming and resection machineries, (iii) DFO controls MTOPVIB foci formation and is part of a divergent RMM-like complex including PHS1/AtREC114 and PRD2/AtMEI4 but not PRD3/AtMER2, (iv) PHS1/AtREC114 is absolutely unnecessary for DSB formation despite having a conserved position within the DSB protein network and (v) MTOPVIB and PRD2/AtMEI4 interact directly with chromosome axis proteins to anchor the meiotic DSB machinery to the axis.

Credentialing and Pharmacologically Targeting PTP4A3 Phosphatase as a Molecular Target for Ovarian Cancer.

High grade serous ovarian cancer (OvCa) frequently becomes drug resistant and often recurs. Consequently, new drug targets and therapies are needed. Bioinformatics-based studies uncovered a relationship between high Protein Tyrosine Phosphatase of Regenerating Liver-3 (PRL3 also known as PTP4A3) expression and poor patient survival in both early and late stage OvCa. PTP4A3 mRNA levels were 5-20 fold higher in drug resistant or high grade serous OvCa cell lines compared to nonmalignant cells. JMS-053 is a potent allosteric small molecule PTP4A3 inhibitor and to explore further the role of PTP4A3 in OvCa, we synthesized and interrogated a series of JMS-053-based analogs in OvCa cell line-based phenotypic assays. While the JMS-053 analogs inhibit in vitro PTP4A3 enzyme activity, none were superior to JMS-053 in reducing high grade serous OvCa cell survival. Because PTP4A3 controls cell migration, we interrogated the effect of JMS-053 on this cancer-relevant process. Both JMS-053 and CRISPR/Cas9 PTP4A3 depletion blocked cell migration. The inhibition caused by JMS-053 required the presence of PTP4A3. JMS-053 caused additive or synergistic in vitro cytotoxicity when combined with paclitaxel and reduced in vivo OvCa dissemination. These results indicate the importance of PTP4A3 in OvCa and support further investigations of the lead inhibitor, JMS-053.

Phosphatase of regenerating liver-3 regulates cancer cell metabolism in multiple myeloma.

Cancer cells often depend on microenvironment signals from molecules such as cytokines for proliferation and metabolic adaptations. PRL-3, a cytokine-induced oncogenic phosphatase, is highly expressed in multiple myeloma cells and associated with poor outcome in this cancer. We studied whether PRL-3 influences metabolism. Cells transduced to express PRL-3 had higher aerobic glycolytic rate, oxidative phosphorylation, and ATP production than the control cells. PRL-3 promoted glucose uptake and lactate excretion, enhanced the levels of proteins regulating glycolysis and enzymes in the serine/glycine synthesis pathway, a side branch of glycolysis. Moreover, mRNAs for these proteins correlated with PRL-3 expression in primary patient myeloma cells. Glycine decarboxylase (GLDC) was the most significantly induced metabolism gene. Forced GLDC downregulation partly counteracted PRL-3-induced aerobic glycolysis, indicating GLDC involvement in a PRL-3-driven Warburg effect. AMPK, HIF-1α, and c-Myc, important metabolic regulators in cancer cells, were not mediators of PRL-3's metabolic effects. A phosphatase-dead PRL-3 mutant, C104S, promoted many of the metabolic changes induced by wild-type PRL-3, arguing that important metabolic effects of PRL-3 are independent of its phosphatase activity. Through this study, PRL-3 emerges as one of the key mediators of metabolic adaptations in multiple myeloma.

Biallelic loss-of-function HACD1 variants are a bona fide cause of congenital myopathy.

Congenital myopathies include a wide range of genetically determined disorders characterized by muscle weakness that usually manifest shortly after birth. To date, two different homozygous loss-of-function variants in the HACD1 gene have been reported to cause congenital myopathy. We identified three patients manifesting with neonatal-onset generalized muscle weakness and motor delay that carried three novel homozygous likely pathogenic HACD1 variants. The two of these changes (c.373_375+2delGAGGT and c.785-1G>T) were predicted to introduce splice site alterations, while one is a nonsense change (c.458G>A). The clinical presentation of our and the previously reported patients was comparable, including the temporally progressive improvement that seems to be characteristic of HACD1-related myopathy. Our findings conclusively confirm the implication of HACD1 in the pathogenesis of congenital myopathies, corroborate the main phenotypic features, and further define the genotypic spectrum of this genetic form of myopathy. Importantly, the genetic diagnosis of HACD1-related myopathy bears impactful prognostic value.

Inhibition of p70 isoforms of S6K1 induces anoikis to prevent transformed human hepatocyte growth.

AIMS: The mTOR/S6K1 signaling axis, known for cell growth regulation, is hyper-activated in multiple cancers. In this study, we have examined the mechanisms for ribosomal protein p70-S6 kinase 1 (S6K1) associated transformed human hepatocyte (THH) growth regulation. MAIN METHODS: THH were treated with p70-S6K1 inhibitor and analyzed for cell viability, cell cycle distribution, specific marker protein expression by western blot, and tumor inhibition in a xenograft mouse model. We validated our results by knockdown of p70-S6K1 using specific siRNA. KEY FINDINGS: p70-S6K1 inhibitor treatment caused impairment of in vitro hepatocyte growth, and arrested cell cycle progression at the G1 phase. Further, p70-S6K1 inhibitor treatment exhibited a decrease in FAK and Erk activation, followed by altered integrin-ß1 expression, caspase 8, and PARP cleavage appeared to be anoikis like growth inhibition. p70-S6K1 inhibitor also depolymerized actin microfilaments and diminished active Rac1/Cdc42 complex formation for loss of cellular attachment. Similar results were obtained with other transformed human hepatocyte cell lines. p70-S6K1 inhibition also resulted in a reduced phospho-EGFR, Slug and Twist; implicating an inhibition of epithelial-mesenchymal transition (EMT) state. A xenograft tumor model, generated from implanted THH in nude mice, following intraperitoneal injection of S6K1 inhibitor prevented further tumor growth. SIGNIFICANCE: Our results suggested that p70-S6K1 inhibition alters orchestration of cell cycle progression, induces cell detachment, and sensitizes hepatocyte growth impairment. Targeting p70 isoform of S6K1 by inhibitor may prove to be a promising approach together with other therapies for hepatocellular carcinoma (HCC) treatment.

Unifying the mechanism of mitotic exit control in a spatiotemporal logical model.

The transition from mitosis into the first gap phase of the cell cycle in budding yeast is controlled by the Mitotic Exit Network (MEN). The network interprets spatiotemporal cues about the progression of mitosis and ensures that release of Cdc14 phosphatase occurs only after completion of key mitotic events. The MEN has been studied intensively; however, a unified understanding of how localisation and protein activity function together as a system is lacking. In this paper, we present a compartmental, logical model of the MEN that is capable of representing spatial aspects of regulation in parallel to control of enzymatic activity. We show that our model is capable of correctly predicting the phenotype of the majority of mutants we tested, including mutants that cause proteins to mislocalise. We use a continuous time implementation of the model to demonstrate that Cdc14 Early Anaphase Release (FEAR) ensures robust timing of anaphase, and we verify our findings in living cells. Furthermore, we show that our model can represent measured cell-cell variation in Spindle Position Checkpoint (SPoC) mutants. This work suggests a general approach to incorporate spatial effects into logical models. We anticipate that the model itself will be an important resource to experimental researchers, providing a rigorous platform to test hypotheses about regulation of mitotic exit.

An allosteric interaction controls the activation mechanism of SHP2 tyrosine phosphatase.

SHP2 is a protein tyrosine phosphatase (PTP) involved in multiple signaling pathways. Mutations of SHP2 can result in Noonan syndrome or pediatric malignancies. Inhibition of wild-type SHP2 represents a novel strategy against several cancers. SHP2 is activated by binding of a phosphopeptide to the N-SH2 domain of SHP2, thereby favoring dissociation of the N-SH2 domain and exposing the active site on the PTP domain. The conformational transitions controlling ligand affinity and PTP dissociation remain poorly understood. Using molecular simulations, we revealed an allosteric interaction restraining the N-SH2 domain into a SHP2-activating and a stabilizing state. Only ligands selecting for the activating N-SH2 conformation, depending on ligand sequence and binding mode, are effective activators. We validate the model of SHP2 activation by rationalizing modified basal activity and responsiveness to ligand stimulation of several N-SH2 variants. This study provides mechanistic insight into SHP2 activation and may open routes for SHP2 regulation.

PRL-2 phosphatase is required for vascular morphogenesis and angiogenic signaling.

Protein tyrosine phosphatases are essential modulators of angiogenesis and have been identified as novel therapeutic targets in cancer and anti-angiogenesis. The roles of atypical Phosphatase of Regenerative Liver (PRL) phosphatases in this context remain poorly understood. Here, we investigate the biological function of PRL phosphatases in developmental angiogenesis in the postnatal mouse retina and in cell culture. We show that endothelial cells in the retina express PRL-2 encoded by the Ptp4a2 gene, and that inducible endothelial and global Ptp4a2 mutant mice exhibit defective retinal vascular outgrowth, arteriovenous differentiation, and sprouting angiogenesis. Mechanistically, PTP4A2 deletion limits angiogenesis by inhibiting endothelial cell migration and the VEGF-A, DLL-4/NOTCH-1 signaling pathway. This study reveals the importance of PRL-2 as a modulator of vascular development.

Screening of Bombyx mori brain proteins interacting with protein tyrosine phosphatase of BmNPV.

In this study, glutathione-S-transferase pull-down combined with mass spectrometry techniques were used to identify the candidate proteins interacting with protein tyrosine phosphatase of the Bombyx Mori nucleopolyhedrovirus in the B. mori (BmNPV-PTP) brain. A total of 36 proteins were identified from BmNPV-PTP coprecipitate samples by searching the NCBI_Bombyx Mori database with the original mass spectrum data. Among those proteins, the interaction between BmNPV-PTP and B. mori cyclophilin A may accelerate the apoptosis of certain nerve cells involved in regulating behavior, and thus may be an inducer of enhanced locomotor activity (ELA). After the BmNPV invasion, BmNPV-PTP binding to peripheral-type benzodiazepine receptors may initiate a series of abnormal cascades of the nervous system, which results in abnormal hyperactive behavior in B. mori. Besides this, vacuolar ATP synthase catalytic subunit A, annexin, and several enzymes for energy conversion were identified, which may play a role in enhancing viral entry and infectivity and provide energy for enhancing the locomotor activity of B. mori. In general, the results of this study will facilitate the understanding of the molecular mechanisms underlying the ELA of B. mori larva induced by BmNPV.

The Role of Protein Tyrosine Phosphatases in Inflammasome Activation.

Inflammasomes are multi-protein complexes that mediate the activation and secretion of the inflammatory cytokines IL-1ß and IL-18. More than half a decade ago, it has been shown that the inflammasome adaptor molecule, ASC requires tyrosine phosphorylation to allow effective inflammasome assembly and sustained IL-1ß/IL-18 release. This finding provided evidence that the tyrosine phosphorylation status of inflammasome components affects inflammasome assembly and that inflammasomes are subjected to regulation via kinases and phosphatases. In the subsequent years, it was reported that activation of the inflammasome receptor molecule, NLRP3, is modulated via tyrosine phosphorylation as well, and that NLRP3 de-phosphorylation at specific tyrosine residues was required for inflammasome assembly and sustained IL-1ß/IL-18 release. These findings demonstrated the importance of tyrosine phosphorylation as a key modulator of inflammasome activity. Following these initial reports, additional work elucidated that the activity of several inflammasome components is dictated via their phosphorylation status. Particularly, the action of specific tyrosine kinases and phosphatases are of critical importance for the regulation of inflammasome assembly and activity. By summarizing the currently available literature on the interaction of tyrosine phosphatases with inflammasome components we here provide an overview how tyrosine phosphatases affect the activation status of inflammasomes.

Mechanism of PRL2 phosphatase-mediated PTEN degradation and tumorigenesis.

Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) levels are frequently found reduced in human cancers, but how PTEN is down-regulated is not fully understood. In addition, although a compelling connection exists between PRL (phosphatase of regenerating liver) 2 and cancer, how this phosphatase induces oncogenesis has been an enigma. Here, we discovered that PRL2 ablation inhibits PTEN heterozygosity-induced tumorigenesis. PRL2 deficiency elevates PTEN and attenuates AKT signaling, leading to decreased proliferation and increased apoptosis in tumors. We also found that high PRL2 expression is correlated with low PTEN level with reduced overall patient survival. Mechanistically, we identified PTEN as a putative PRL2 substrate and demonstrated that PRL2 down-regulates PTEN by dephosphorylating PTEN at Y336, thereby augmenting NEDD4-mediated PTEN ubiquitination and proteasomal degradation. Given the strong cancer susceptibility to subtle reductions in PTEN, the ability of PRL2 to down-regulate PTEN provides a biochemical basis for its oncogenic propensity. The results also suggest that pharmacological targeting of PRL2 could provide a novel therapeutic strategy to restore PTEN, thereby obliterating PTEN deficiency-induced malignancies.

Inhibition of striatal-enriched protein tyrosine phosphatase (STEP) activity reverses behavioral deficits in a rodent model of autism.

Autism spectrum disorders (ASDs) are highly prevalent childhood illnesses characterized by impairments in communication, social behavior, and repetitive behaviors. Studies have found aberrant synaptic plasticity and neuronal connectivity during the early stages of brain development and have suggested that these contribute to an increased risk for ASD. STEP is a protein tyrosine phosphatase that regulates synaptic plasticity and is implicated in several cognitive disorders. Here we test the hypothesis that STEP may contribute to some of the aberrant behaviors present in the VPA-induced mouse model of ASD. In utero VPA exposure of pregnant dams results in autistic-like behavior in the pups, which is associated with a significant increase in the STEP expression in the prefrontal cortex. The elevated STEP protein levels are correlated with increased dephosphorylation of STEP substrates GluN2B, Pyk2 and ERK, suggesting upregulated STEP activity. Moreover, pharmacological inhibition of STEP rescues the sociability, repetitive and abnormal anxiety phenotypes commonly associated with ASD. These data suggest that STEP may play a role in the VPA model of ASD and STEP inhibition may have a potential therapeutic benefit in this model.

Endogenous siRNAs promote proteostasis and longevity in germline-less Caenorhabditis elegans.

How lifespan and the rate of aging are set is a key problem in biology. Small RNAs are conserved molecules that impact diverse biological processes through the control of gene expression. However, in contrast to miRNAs, the role of endo-siRNAs in aging remains unexplored. Here, by combining deep sequencing and genomic and genetic approaches in Caenorhabditis elegans, we reveal an unprecedented role for endo-siRNA molecules in the maintenance of proteostasis and lifespan extension in germline-less animals. Furthermore, we identify an endo-siRNA-regulated tyrosine phosphatase, which limits the longevity of germline-less animals by restricting the activity of the heat shock transcription factor HSF-1. Altogether, our findings point to endo-siRNAs as a link between germline removal and the HSF-1 proteostasis and longevity-promoting somatic pathway. This establishes a role for endo siRNAs in the aging process and identifies downstream genes and physiological processes that are regulated by the endo siRNAs to affect longevity.

ATP synthase, an essential enzyme in growth and multiplication is modulated by protein tyrosine phosphatase in Mycobacterium tuberculosis H37Ra.

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase (PtpA) has so far been known to control intracellular survival of mycobacteria; whereas the ATP synthase which is essential for mycobacterial growth has recently been contemplated in developing a breakthrough anti-TB drug, diarylquinoline. Since both of these enzymes have been established as validated drug targets; we report a robust and functional relationship between these two enzymes through a series of experiments using Mtb H37Ra. In the present study we report that the mycobacterial ATP synthase alpha subunit is regulated by PtpA. We generated gene knock-out for the enzyme PtpA and subjected to determine the mycobacterial replication and the proteome profile of wild type, mutant (ΔptpA) and complemented (ΔptpA:ptpA) strains of Mtb H37Ra. A substantial amount of decrease in the protein level of ATP synthase alpha subunit (AtpA) in case of mutant H37Ra was observed, while the levels of the enzyme were either increased or remained unchanged, in wild type and in the complemented strains.

Regulation of the ERK signalling pathway in the developing mouse blastocyst.

Activation of the ERK signalling pathway is essential for the differentiation of the inner cell mass (ICM) during mouse preimplantation development. We show here that ERK phosphorylation occurs in ICM precursor cells, in differentiated primitive endoderm (PrE) cells as well as in the mature, formative state epiblast (Epi). We further show that DUSP4 and ETV5, factors often involved in negative-feedback loops of the FGF pathway, are differently regulated. Whereas DUSP4 presence clearly depends on ERK phosphorylation in PrE cells, ETV5 localises mainly to Epi cells. Unexpectedly, ETV5 accumulation does not depend on direct activation by ERK but requires NANOG activity. Indeed ETV5, like Fgf4 expression, is not present in Nanog mutant embryos. Our results lead us to propose that in pluripotent early Epi cells, NANOG induces the expression of both Fgf4 and Etv5 to enable the differentiation of neighbouring cells into the PrE while protecting the Epi identity from autocrine signalling.

Branch-restricted localization of phosphatase Prl-1 specifies axonal synaptogenesis domains.

Central nervous system (CNS) circuit development requires subcellular control of synapse formation and patterning of synapse abundance. We identified the Drosophila membrane-anchored phosphatase of regenerating liver (Prl-1) as an axon-intrinsic factor that promotes synapse formation in a spatially restricted fashion. The loss of Prl-1 in mechanosensory neurons reduced the number of CNS presynapses localized on a single axon collateral and organized as a terminal arbor. Flies lacking all Prl-1 protein had locomotor defects. The overexpression of Prl-1 induced ectopic synapses. In mechanosensory neurons, Prl-1 modulates the insulin receptor (InR) signaling pathway within a single contralateral axon compartment, thereby affecting the number of synapses. The axon branch-specific localization and function of Prl-1 depend on untranslated regions of the prl-1 messenger RNA (mRNA). Therefore, compartmentalized restriction of Prl-1 serves as a specificity factor for the subcellular control of axonal synaptogenesis.

Intracellular Zn2+ Signaling Facilitates Mossy Fiber Input-Induced Heterosynaptic Potentiation of Direct Cortical Inputs in Hippocampal CA3 Pyramidal Cells.

Repetitive action potentials (APs) in hippocampal CA3 pyramidal cells (CA3-PCs) backpropagate to distal apical dendrites, and induce calcium and protein tyrosine kinase (PTK)-dependent downregulation of Kv1.2, resulting in long-term potentiation of direct cortical inputs and intrinsic excitability (LTP-IE). When APs were elicited by direct somatic stimulation of CA3-PCs from rodents of either sex, only a narrow window of distal dendritic [Ca2+] allowed LTP-IE because of Ca2+-dependent coactivation of PTK and protein tyrosine phosphatase (PTP), which renders non-mossy fiber (MF) inputs incompetent in LTP-IE induction. High-frequency MF inputs, however, could induce LTP-IE at high dendritic [Ca2+] of the window. We show that MF input-induced Zn2+ signaling inhibits postsynaptic PTP, and thus enables MF inputs to induce LTP-IE at a wide range of [Ca2+]i values. Extracellular chelation of Zn2+ or genetic deletion of vesicular zinc transporter abrogated the privilege of MF inputs for LTP-IE induction. Moreover, the incompetence of somatic stimulation was rescued by the inhibition of PTP or a supplement of extracellular zinc, indicating that MF input-induced increase in dendritic [Zn2+] facilitates the induction of LTP-IE by inhibiting PTP. Consistently, high-frequency MF stimulation induced immediate and delayed elevations of [Zn2+] at proximal and distal dendrites, respectively. These results indicate that MF inputs are uniquely linked to the regulation of direct cortical inputs owing to synaptic Zn2+ signaling.SIGNIFICANCE STATEMENT Zn2+ has been mostly implicated in pathological processes, and the physiological roles of synaptically released Zn2+ in intracellular signaling are little known. We show here that Zn2+ released from hippocampal mossy fiber (MF) terminals enters postsynaptic CA3 pyramidal cells, and plays a facilitating role in MF input-induced heterosynaptic potentiation of perforant path (PP) synaptic inputs through long-term potentiation of intrinsic excitability (LTP-IE). We show that the window of cytosolic [Ca2+] that induces LTP-IE is normally very narrow because of the Ca2+-dependent coactivation of antagonistic signaling pairs, whereby non-MF inputs become ineffective in inducing excitability change. The MF-induced Zn2+ signaling, however, biases toward facilitating the induction of LTP-IE. The present study elucidates why MF inputs are more privileged for the regulation of PP synapses.

The role of pseudophosphatases as signaling regulators.

Pseudophosphatases are atypical members of the protein tyrosine phosphatase superfamily. Mutations within their catalytic signature motif render them catalytically inactive. Despite this lack of catalytic function, pseudophosphatases have been implicated in various diseases such as Charcot Marie-Tooth disorder, cancer, metabolic disorder, and obesity. Moreover, they have roles in various signaling networks such as spermatogenesis, apoptosis, stress response, tumorigenesis, and neurite differentiation. This review highlights the roles of pseudophosphatases as essential regulators in signaling cascades, providing insight into the function of these catalytically inactive enzymes.

The roles of protein tyrosine phosphatases in bone-resorbing osteoclasts.

Maintaining the proper balance between osteoblast-mediated production of bone and its degradation by osteoclasts is essential for health. Osteoclasts are giant phagocytic cells that are formed by fusion of monocyte-macrophage precursor cells; mature osteoclasts adhere to bone tightly and secrete protons and proteases that degrade its matrix. Phosphorylation of tyrosine residues in proteins, which is regulated by the biochemically-antagonistic activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is central in regulating the production of osteoclasts and their bone-resorbing activity. Here we review the roles of individual PTPs of the classical and dual-specificity sub-families that are known to support these processes (SHP2, cyt-PTPe, PTPRO, PTP-PEST, CD45) or to inhibit them (SHP1, PTEN, MKP1). Characterizing the functions of PTPs in osteoclasts is essential for complete molecular level understanding of bone resorption and for designing novel therapeutic approaches for treating bone disease.