PME-1 Research Articles

Synergistic suppression of BDNF via epigenetic mechanism deteriorating learning and memory impairment caused by Mn and Pb co-exposure

Microglia, the resident immune cells of the central nervous system (CNS), play a dual role in neurotoxicity by releasing the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome and brain-derived neurotrophic factor (BDNF) in response to environmental stress. Suppression of BDNF is implicated in learning and memory impairment induced by exposure to manganese (Mn) or lead (Pb) individually. Methyl CpG Binding Protein 2 (MeCp2) and its phosphorylation status are related to BDNF suppression. Protein phosphatase2A (PP2A), a member of the serine/threonine phosphatases family, dephosphorylates substrates based on the methylation state of its catalytic C subunit (PP2Ac). However, the specific impairment patterns and molecular mechanisms resulting from co-exposure to Mn and Pb remain unclear. Therefore, the purpose of this study was to explore the effects of Mn and Pb exposure, alone and in combination, on inducing neurotoxicity in the hippocampus of mice and BV2 cells, and to determine whether simultaneous exposure to both metals exacerbate their toxicity. Our findings reveal that co-exposure to Mn and Pb leads to severe learning and memory impairment in mice, which correlates with the accumulation of metals in the hippocampus and synergistic suppression of BDNF. This suppression is accompanied by up-regulation of the epigenetic repressor MeCp2 and its phosphorylation status, as well as demethylation of PP2Ac. Furthermore, inhibition of PP2Ac demethylation using ABL127, an inhibitor for its protein phosphatase methylesterase1 (PME1), or knockdown of MeCp2 via siRNA transfection in vitro effectively increases BDNF expression and mitigates BV2 cell damage induced by Mn and Pb co-exposure. We also observe abnormal activation of microglia characterized by enhanced release of the NLRP3 inflammasome, Casepase-1 and pro-inflammatory cytokines IL-1β, in the hippocampus of mice and BV2 cells. In summary, our experiments demonstrate that simultaneous exposure to Mn and Pb results in more severe hippocampus-dependent learning and memory impairment, which is attributed to epigenetic suppression of BDNF mediated by PP2A regulation.

The luciferase-based in vivo protein–protein interaction assay revealed that CHK1 promotes PP2A and PME-1 interaction

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein-protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, in vitro phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.

Transcriptome analysis revealed that PME-1 suppresses inflammatory signaling, activates PI3K/Akt signaling, and promotes epithelial-mesenchymal transition

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase that belongs to the type2A protein phosphatase family with PP4 and PP6. PP2A functions as a trimeric holoenzyme, and the composition of the trimer is regulated by the methyl-esterification (methylation) of PP2A. Demethylation of PP2A is catalyzed by protein phosphatase methyl-esterase-1 (PME-1). Despite the physiological and pathophysiological importance of PME-1, the impact of changes in PME-1 expression on the transcriptome has not been reported. This study provides transcriptome data to gain a comprehensive understanding of the effects of PME-1 knockout on intracellular signaling of mouse embryonic fibroblasts. Our data showed that PME-1 suppresses inflammatory signaling, activates PI3K/Akt signaling, and promotes epithelial-mesenchymal transition.

PME-1 sensitizes glioblastoma cells to oxidative stress-induced cell death by attenuating PP2A-B55α-mediated inactivation of MAPKAPK2-RIPK1 signaling

Glioblastoma (GBM) is the most common primary brain tumor in adults. Current standard therapy is surgery followed by radiotherapy, with concurrent and adjuvant temozolomide chemotherapy. GBM is characterized by almost uniformly fatal outcomes, highlighting the unmet clinical need for more efficient, biomarker-guided treatments. Protein phosphatase methylesterase-1 (PME-1), a regulator of the tumor suppressive phosphatase PP2A, promotes PP2A demethylation and inactivation, and is overexpressed in 44% of GBM, associated with increased tumor grade and cellular proliferation. Here, we aimed to investigate how reactive oxygen species (ROS), a frequent by-product of radiotherapy and temozolomide chemotherapy, regulate PP2A function via its methylesterase PME-1, and how PME-1 overexpression impacts the response of GBM cells to oxidative stress. We found that in two glioblastoma cell lines, U87MG and U251MG, expression of PME-1 is positively correlated with the sensitivity of the cells to H2O2 or t-BHP-induced oxidative stress. Experiments using the irreversible pharmacologic PME-1 inhibitor, AMZ30, and different PME-1 mutants, revealed that the methylesterase function, the PP2A binding capacity, and the nuclear localization of PME-1 are all important for the sensitizing effect of PME-1 expression. Furthermore, we identified increased nuclear localization of the PP2A-B55α subunit, increased binding of PP2A-B55α to PME-1, and increased B55α-bound PP2A-C demethylation upon oxidative stress. Lastly, we uncovered increased stress-induced phosphorylation and activity of MAPKAPK2 and RIPK1 in PME-1 overexpressing U87MG cells, which caused the observed sensitization to t-BHP treatment. Our data reveal a novel role for PME-1 in oxidative stress-induced GBM cell death, regulating nuclear PP2A-B55α activity and MAPKAPK2-RIPK1 signaling. Patients with GBM tumors overexpressing PME-1, although having a worse prognosis due to increased cellular proliferation of the tumor, could actually be more responsive to oxidative stress-inducing therapies.

A Novel Role of PP2A Methylation in the Regulation of Tight Junction Assembly and Integrity.

Tight junctions (TJs) are multiprotein complexes essential for cell polarity and the barrier function of epithelia. The major signaling molecule, protein serine/threonine phosphatase 2A (PP2A), interacts with the TJ and modulates the phosphorylation state of TJ proteins. An important PP2A regulatory mechanism involves leucine carboxyl methyltransferase-1 (LCMT1)-dependent methylation and protein phosphatase methylesterase-1 (PME1)-mediated demethylation of its catalytic subunit on Leu309. Here, using MDCK cells, we show that overexpression of LCMT1, which enhances cellular PP2A methylation, inhibits TJ formation, induces TJ ruffling, and decreases TJ barrier function. Conversely, overexpression of PME1 accelerates TJ assembly and enhances TJ barrier function. PME1-dependent PP2A demethylation increases during early Ca2+-dependent junctional assembly. Inhibition of endogenous PME1 delays the initial Ca2+-mediated redistribution of TJ proteins to cell-cell contacts and affects TJ morphology and barrier function. Manipulating one-carbon metabolism modulates TJ assembly, at least in part by affecting PP2A methylation state. The integrity of PP2A methylation is critical for proper targeting of PP2A to the TJ. It is necessary for PP2A complex formation with the TJ proteins, occludin and ZO-1, and proteins of the PAR complex, Par3 and atypical protein kinase C ζ (aPKCζ), which play a key role in development of cell polarity. Expression of a methylation incompetent PP2A mutant induces defects in TJ assembly and barrier function. aPKCζ-mediated Par3 phosphorylation is also required for targeting of the PP2A ABαC holoenzyme to the TJ. Our findings provide the first evidence for a role of LCMT1, PME1 and PP2A methylation/demethylation processes in modulating TJ assembly and functional integrity. They also position PP2A at the interface of one-carbon metabolism and the regulation of key TJ and polarity proteins that become deregulated in many human diseases.

Hsa_circ_0050102 regulates the pancreatic cancer development via miR-218-5p/PPME1 axis.

BackgroundPancreatic cancer (PC) is a malignancy worldwide. Circular RNAs (circRNAs) affects the growth of PC, nonetheless the mechanism is blurry. Here, we reconnoitered the parts of hsa_circ_0050102 in PC.MethodsHsa_circ_0050102, microRNA‐218‐5p (miR‐218‐5p) and protein phosphatase methylesterase 1 (PPME1) abundances were indicated by quantitative RT‐PCR or Western blot. Moreover, the cell functions were uncovered. Additionally, the relation of miR‐218‐5p and hsa_circ_0050102 or PPME1 was identified by dual‐luciferase reporter assay. Ultimately, the mice teats were utilized to quantity the part of hsa_circ_0050102.ResultsHsa_circ_0050102 and PPME1 contents were increased, and the miR‐218‐5p was dwindled in PC. Hsa_circ_0050102 lack subdued cell vitality, colony formation, cell migration and invasion, and angiogenesis, but endorsed cell apoptosis in PC cells. Furthermore, miR‐218‐5p was established to block the development of PC cells via PPME1. Hsa_circ_0050102 bound to miR‐218‐5p to adjust the content of PPME1.ConclusionHsa_circ_0050102 expedited the expansion of PC through growing PPME1 abundance by adjusting miR‐218‐5p.

Hyperphosphorylation of Tau Due to the Interference of Protein Phosphatase Methylesterase-1 Overexpression by MiR-125b-5p in Melatonin Receptor Knockout Mice.

Melatonin has been indicated to ameliorate tau hyperphosphorylation in the pathogenesis of tau diseases, but the role of melatonin-receptor signal transduction has not been clearly discovered. In this study, we found intensive tau hyperphosphorylation in melatonin receptor knockout mice. Bielschowsky silver staining showed ghostlike neurofibrillary tangles in melatonin receptor-2 knockout (MT2KO) as well as melatonin receptors-1 and -2 knockout (DKO) mice, and an argyrophilic substance was deposited in melatonin receptor-1 knockout (MT1KO) mice. Furthermore, we found significantly decreased activity of protein phosphatase 2A (PP2A) by Western blot and enzyme-linked immunosorbent assay (ELISA), which was partly due to the overexpression of protein phosphatase methylesterase-1 (PME-1), but not glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5 (CDK5) or protein kinase B (Akt). Finally, we observed a significant increase in cyclic adenosine monophosphate (cAMP) and a decrease in miR-125b-5p levels in MT1KO, MT2KO and DKO mice. Using a luciferase reporter assay, we discovered that miR-125b-5p largely decreased the expression of firefly luciferase by interfering with the 3′UTR of PME-1. Furthermore, miR-125b-5p mimics significantly decreased the expression of PME-1, while miR-125b-5p inhibitor induced tau hyperphosphorylation. These results show that melatonin-receptor signal transduction plays an important role in tau hyperphosphorylation and tangle formation.

Long non-coding RNA HOTAIR/microRNA-761 sponge regulates PPME1 and further influences cell biological functions in thyroid carcinoma.

BackgroundMost well‐differentiated thyroid carcinomas display good therapeutic outcomes, but there are still some patients who are not sensitive to the general treatments lose their treatment opportunities. Thus, it is important to understand the molecular mechanisms that cause thyroid carcinoma, so as to find effective diagnostic and therapeutic targets.Aim of the studyTo explore the role of homeobox transcript antisense RNA (HOTAIR) in thyroid carcinoma through protein phosphatase methylesterase 1 (PPME1) by sponging microRNA 761 (miR‐761).MethodsThe regulation network amongst HOTAIR, miR‐761 and PPME1 was predicted by online sources. RT‐PCR was conducted to evaluate the expression of HOTAIR and miR‐761 in tumor tissues. Clinical data was collected and analyzed by Chi‐square test. Cell apoptosis and proliferation was evaluated using three types of cancer cells (HTh‐7, CAL‐62, BCPAP) after treated with si‐HOTAIR and miR‐761inhibitor. The binding site among HOTAIR, miR‐761 and PPME1 was verified by dual luciferase reporter assay. PPME1 expression was measured after HOTAIR and miR‐761 were suppressed by western blot. Survival time was measured in nude mice using log‐rank test.ResultsHOTAIR was expressed to a significantly greater extent than miR‐761 in thyroid tumor tissues (P < .001). miR‐761 and PPME1 were negatively correlated (coef = −1.91, P < .001). HOTAIR competitively binds to miR‐761 and miR‐761 directly targets PPME1. HOTAIR was highly correlated with TNM (χ 2 = 5.797, P = .016), tumor size (χ 2 = 7.955, P = .005) and lymphatic metastasis (χ 2 = 6.0, P = .014). HOTAIR promoted cell proliferation and inhibited cell apoptosis, whereas miR‐761 did not. HOTAIR elevated and miR‐761 suppressed PPME1 expression. HOTAIR expression appears to affect the survival time in vivo.ConclusionHOTAIR regulated thyroid cancer cells by binding to miR‐761 through PPME1.

Pseudoginsenoside-F11 ameliorates okadiac acid-induced learning and memory impairment in rats via modulating protein phosphatase 2A

We have reported that pseudoginsenoside-F11 (PF11) can significantly improve the cognitive impairments in several Alzheimer’s disease (AD) models, but the mechanism has not been fully elucidated. In the present study, the effects of PF11 on AD, in particular the underlying mechanisms related with protein phosphatase 2A (PP2A), were investigated in a rat model induced by okadaic acid (OA), a selective inhibitor of PP2A. The results showed that PF11 treatment dose-dependently improved the learning and memory impairments in OA-induced AD rats. PF11 could significantly inhibit OA-induced tau hyperphosphorylation, suppress the activation of glial cells, alleviate neuroinflammation, thus rescue the neuronal and synaptic damage. Further investigation revealed that PF11 could regulate the protein expression of methyl modifying enzymes (leucine carboxyl methyltransferase-1 and protein phosphatase methylesterase-1) in the brain, thus increase methyl-PP2A protein expression and indirectly increase the activity of PP2A. Molecular docking analysis, structural alignment and in vitro results showed that PF11 was similar in the shape and electrostatic field feature to a known activator of PP2A, and could directly bind and activate PP2A. In conclusion, the present data indicate that PF11 can ameliorate OA-induced learning and memory impairment in rats via modulating PP2A.

Effects of carboxyl-terminal methylation on holoenzyme function of the PP2A subfamily.

Phosphoprotein Phosphatases (PPPs) are enzymes highly conserved from yeast and human and catalyze the majority of the serine and threonine dephosphorylation in cells. To achieve substrate specificity and selectivity, PPPs form multimeric holoenzymes consisting of catalytic, structural/scaffolding, and regulatory subunits. For the Protein Phosphatase 2A (PP2A)-subfamily of PPPs, holoenzyme assembly is at least in part regulated by an unusual carboxyl-terminal methyl-esterification, commonly referred to as 'methylation'. Carboxyl-terminal methylation is catalyzed by Leucine carboxyl methyltransferase-1 (LCMT1) that utilizes S-adenosyl-methionine (SAM) as the methyl donor and removed by protein phosphatase methylesterase 1 (PME1). For PP2A, methylation dictates regulatory subunit selection and thereby downstream phosphorylation signaling. Intriguingly, there are four families of PP2A regulatory subunits, each exhibiting different levels of methylation sensitivity. Thus, changes in PP2A methylation stoichiometry alters the complement of PP2A holoenzymes in cells and creates distinct modes of kinase opposition. Importantly, selective inactivation of PP2A signaling through the deregulation of methylation is observed in several diseases, most prominently Alzheimer's disease (AD). In this review, we focus on how carboxyl-terminal methylation of the PP2A subfamily (PP2A, PP4, and PP6) regulates holoenzyme function and thereby phosphorylation signaling, with an emphasis on AD.

Expression Pattern and Prognostic Utility of PME-1 in Patients with Hepatocellular Carcinoma.

PurposeHepatocellular carcinoma (HCC) remains one of the most common malignancies. While there is lack of markers capable of predicting which patients are at risk of aggressive course of the disease. Although a few protein phosphatase methyl-esterase-1 (PME-1) tumor-promoting mechanisms have been reported, the role of PME-1 in cancer including HCC occurrence and progression remains to be elucidated. The aim of this study was to explore the expression pattern and relationship between PME-1 with the pathological parameters in patients with HCC.MethodsPME-1 expression was assessed from HCC tissue chips via immunohistochemistry. Chi-square test was used to identify the association between PME-1 staining and clinicopathological variables of HCC patients. Kaplan–Meier analysis and Cox regression analysis were performed to draw survival curves and verify the independent prognostic factors of HCC patients, respectively.ResultsWe found that PME-1 expression was significantly higher in HCC tumor tissues compared with non-tumor tissues (P < 0.001). Furthermore, high expression level of PME-1 was significantly associated with differentiation (P = 0.047), tumor number (P = 0.001), intrahepatic or extrahepatic metastasis (P = 0.018), and recurrence (P = 0.001). Kaplan–Meier analysis revealed that high expression level of PME-1 was associated with shorter survival (P < 0.001). Univariate analysis with Log-rank test revealed that PME-1 expression status was significantly correlated with overall survival (P < 0.001). Furthermore, multivariate models with Cox proportional hazards analysis showed that high expression of PME-1 was a statistically independent predictive factor of poor prognosis in HCC patients (hazard ratio, 3.429; 95% confidence interval, 1.369–8.589; P = 0.009).ConclusionIn conclusion, these findings indicated that PME-1 expression was associated with aggressive pathological features and worse oncological outcomes, suggesting its potential therapeutic value for patients with HCC.

Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1

Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies.

Inhibition of protein phosphatase methylesterase 1 dysregulates MAP kinase signaling and attenuates muscle cell differentiation.

Protein phosphatase methylesterase 1 has been identified as a novel gene in skeletal muscle that is upregulated in response to neurogenic atrophy in mice. Western blot analysis confirms that Ppme1 is expressed during both muscle cell proliferation and differentiation. Additionally, the Ppme1 promoter is active in muscle cells, while mutation of a conserved E-box element prevents full induction of the Ppme1 reporter gene, suggesting that Ppme1 is transcriptionally regulated by myogenic regulatory factors. Interestingly, immunofluorescence analysis indicates that Ppme1 is localized to both the cytoplasm and the nucleus, while cell fractionation shows that Ppme1 is found only in the cytoplasm. Functional studies reveal that inhibition of Ppme1 using ABL127 or AMZ30 attenuates muscle cell differentiation. Interestingly, inhibition of Ppme1 by ABL127 led to a significant increase in AP-1 reporter activity, as well as, increases in ERK1/2, c-Jun, Ppme1, and PP2A protein levels in differentiating muscle cells. In contrast, AMZ30 treated cells showed a significant decrease in AP-1 reporter activity and a decrease in ERK1/2 and p38 phosphorylation levels. Finally, co-immunoprecipitation studies show that ABL127, but not AMZ30, causes disruption of the endogenous interaction between Ppme1 and PP2A. The data in this study show for the first time that Ppme1 is expressed in skeletal muscle and is upregulated in response to neurogenic atrophy. Furthermore, these findings suggest that Ppme1 may act as a sentinel of the MAP kinase signaling pathway and may indirectly regulate the ERK1/2 and p38 branches via a non-canonical mechanism leading to inhibition of muscle cell differentiation.

Prolyl oligopeptidase inhibition activates autophagy via protein phosphatase 2A

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurodegenerative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclin1-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson’s disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of death-associated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.

Glycogen synthase kinase-3β suppresses the expression of protein phosphatase methylesterase-1 through β-catenin.

Protein phosphatase 2A (PP2A) is the major tau phosphatase. Its activity toward tau is regulated by the methylation of PP2A catalytic subunit (PP2Ac) at Leu309. Protein phosphatase methylesterase-1 (PME-1) demethylates PP2Ac and suppresses its activity. We previously found that glycogen synthase kinase-3β (GSK-3β) suppresses PME-1 expression. However, the underlying molecular mechanism is unknown. In the present study, we analyzed the promoter of PME-1 gene and found that human PME-1 promoter contains two lymphoid enhancer binding factor-1/T-cell factor (LEF1/TCF) cis-elements in which β-catenin serves as a co-activator. β-catenin acted on these two cis-elements and promoted PME-1 expression. GSK-3β phosphorylated β-catenin and suppressed its function in promoting PME-1 expression. Inhibition and activation of GSK-3β by PI3K-AKT pathway promoted and suppressed, respectively, PME-1 expression in primary cultured neurons, SH-SY5Y cells and in the mouse brain. These findings suggest that GSK-3β phosphorylates β-catenin and suppresses its function on PME-1 expression, resulting in an increase of PP2Ac methylation.

Identification and Characterization of Protein Phosphatase Methylesterase 1 (Ppme1) in Skeletal Muscle

Skeletal muscle atrophy results from a range of physiological conditions, including immobilization, denervation, glucocorticoid treatment, cancer, and aging. To provide a better understanding of the pathway of skeletal muscle atrophy, a previous study conducted a microarray analysis of muscle from mice undergoing neurogenic atrophy in response to denervation as well as dexamethasone exposure. The data generated from this microarray identified a suite of several hundred predicted genes that displayed differential expression under conditions of muscle wasting, including protein phosphatase methylesterase‐1 (Ppme1). The Ppme1 gene was observed to be transcriptionally upregulated in response to neurogenic skeletal muscle atrophy (i.e. denervation). To confirm that Ppme1 is expressed in muscle, qPCR was performed using RNA isolated from cultured muscle cells at different stages of proliferation and differentiation and expression was found to be relatively constant in both proliferating and differentiated cells. In addition, Ppme1 protein levels were analyzed by Western blot analysis and found to be expressed at a constant level at different time points throughout muscle cell proliferation and differentiation. Additionally, the cDNA of Ppme1 was fused in frame with GFP and subcellular localization was analyzed by confocal fluorescent microscopy in proliferating myoblasts. To assess the transcriptional activity of the Ppme1 gene, fragments of the promoter were cloned, fused to a reporter gene, transfected into C2C12 cells, and found to be highly transcriptionally active. Interestingly, the Ppme1 reporter gene constructs were not significantly affected by ectopic expression of myogenic regulatory facts such as MyoD1 and myogenin. Finally, mouse myoblast cells treated with an inhibitor of Ppme1 displayed reduced AP‐1 reporter activity as well as delayed muscle cell differentiation. The results presented here help contribute new insights into the molecular mechanisms of muscle atrophy and may provide a better understanding of the genome‐wide transcriptional changes and cellular events that occur during skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrogen peroxide redistributes the localization of protein phosphatase methylesterase 1

AimsProtein phosphatase methylesterase-1 (PME-1) is a serine hydrolase that catalyzes protein phosphatase 2A (PP2A) demethylation and negatively regulates its activity. PME-1 is compartmentalized within cells to precisely control the demethylation of PP2A. This study investigated the localization of PME-1 in human fibroblast cells (HDF) under oxidative stress. Main methodsAlkaline demethylation and peptide competition assays were applied to detect the methylation sensitivity of anti-PP2Ac. The localization of PME-1, leucine carboxyl methyltransferase 1 (LCMT1), demethylated-phosphorylated-PP2Ac (dem-p-PP2Ac) and total PP2Ac was determined by immunofluorescence analysis, and protein expression was measured by Western blot. A HEK293 cell line stably expressing constructed PME-1-EGFP was used to dynamically monitor the nuclear export of PME-1 under oxidative stress. Key resultsAfter hydrogen peroxide (H2O2) treatment, the protein expression of PME-1 remained unchanged, while PME-1 facilitated redistribution from the nucleus to the cytoplasm in HDF according to immunofluorescence analysis. In constructed HEK293 cells, the EGFP-tagged PME-1 was exported from the nucleus to the cytoplasm after H2O2 treatment, and nuclear export was eliminated after leptomycin B additions. Our observation of dem-p-PP2Ac species relocation from the nucleus to the cytoplasm under oxidative stress is consistent with the redistribution patterns of PME-1. Antioxidant N-acetyl cysteine can reverse the nuclear to cytoplasmic ratio of PME-1 proteins and dem-p-PP2Ac after H2O2 exposure. SignificanceWe found that PME-1 is exported from the nucleus to the cytoplasm upon H2O2 treatment and redistributes dem-p-PP2Ac in subcellular compartments. These findings offer new insight into the regulation of PME-1 localization and PP2A demethylation under oxidative stress.

Cornel Iridoid Glycoside Inhibits Tau Hyperphosphorylation via Regulating Cross-Talk Between GSK-3β and PP2A Signaling.

Neurofibrillary pathology contributes to neuronal dysfunction and correlates with the clinical progression of Alzheimer’s disease (AD). Tau phosphorylation is mainly regulated by a balance of glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) activities. Cornel iridoid glycoside (CIG) is a main component extracted from Cornus officinalis. The purpose of this study was to investigate the effects of CIG on GSK-3β and PP2A, thus to explore the mechanisms of CIG to inhibit tau hyperphosphorylation. The rat model of tau hyperphosphorylation was established by intraventricular injection of wortmannin and GF-109203X (GFX) to activate GSK-3β. The results showed that intragastrical administration of CIG inhibited tau hyperphosphorylation in the brain of rats induced by wortmannin/GFX. The results in vivo and in vitro exhibited that CIG inhibited tau hyperphosphorylation and GSK-3β over-activation. In the mechanism of action, CIG’s attenuating GSK-3β activity was found to be dependent on PI3K/AKT signaling pathway. PP2A catalytic C subunit (PP2Ac) siRNA abrogated the effect of CIG on PI3K/AKT/GSK-3β. Additionally and crucially, we also found that CIG inhibited the demethylation of PP2Ac at Leu309 in vivo and in vitro. It enhanced PP2A activity, decreased tau hyperphosphorylation, and protected cell morphology in okadaic acid (OA)-induced cell model in vitro. PP2Ac siRNA abated the inhibitory effect of CIG on tau hyperphosphorylation. Moreover, CIG inhibited protein phosphatase methylesterase-1 (PME-1) and demethylation of PP2Ac, enhanced PP2A activity, and decreased tau hyperphosphorylation in PME-1-transfectd cells. Taken together, CIG inhibited GSK-3β activity via promoting P13K/AKT and PP2A signaling pathways. In addition, CIG also elevated PP2A activity via inhibiting PME-1-induced PP2Ac demethylation to inhibit GSK-3β activity, thus regulated the cross-talk between GSK-3β and PP2A signaling and consequently inhibited tau hyperphosphorylation. These results suggest that CIG may be a promising agent for AD therapy.

H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling in HEK293 cells.

Mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that functions as an ATP and amino acid sensor to govern cell growth and proliferation by mediating mitogen- and nutrient-dependent signal transduction. Protein phosphatase 2A (PP2A), a ubiquitously expressed serine/threonine phosphatase, negatively regulates mTOR signaling. Methylation of PP2A is catalyzed by leucine carboxyl methyltransferase-1 (LCMT1) and reversed by protein phosphatase methylesterase 1 (PME-1), which regulates PP2A activity and substrate specificity. However, whether PP2A methylation is related to mTOR signaling is still unknown. In this study, we examined the effect of PP2A methylation on mTOR signaling in HEK293 cells under oxidative stress. Our results show that oxidative stress induces PP2A demethylation and inhibits the mTORC1 signaling pathway. Next, we examined two strategies to block PP2A demethylation under oxidative stress. One strategy was to prevent PP2A demethylation using a PME-1 inhibitor; the other strategy was to activate PP2A methylation via overexpression of LCMT1. The results show that both the PME-1 inhibitor and LCMT1 overexpression prevent the mTORC1 signaling suppression induced by oxidative stress. Additionally, LCMT1 overexpression rescued cell viability and the mitochondrial membrane potential decrease in response to oxidative stress. These results demonstrate that H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling. These findings provide a novel mechanism for the regulation of PP2A demethylation and mTORC1 signaling under oxidative stress.

Leucine Carboxyl Methyltransferase Downregulation and Protein Phosphatase Methylesterase Upregulation Contribute Toward the Inhibition of Protein Phosphatase 2A by α-Synuclein.

The pathology of Parkinson’s disease (PD) is characterized by intracellular neurofibrillary tangles of phosphorylated α-synuclein (α-syn). Protein phosphatase 2A (PP2A) is responsible for α-syn dephosphorylation. Previous work has demonstrated that α-syn can regulate PP2A activity. However, the mechanisms underlying α-syn regulation of PP2A activity are not well understood. In this study, we found that α-syn overexpression induced increased α-syn phosphorylation at serine 129 (Ser129), and PP2A inhibition, in vitro and in vivo. α-syn overexpression resulted in PP2A demethylation. This demethylation was mediated via downregulated leucine carboxyl methyltransferase (LCMT-1) expression, and upregulated protein phosphatase methylesterase (PME-1) expression. Furthermore, LCMT-1 overexpression, or PME-1 inhibition, reversed α-syn-induced increases in α-syn phosphorylation and apoptosis. In addition to post-translational modifications of the catalytic subunit, regulatory subunits are involved in the regulation of PP2A activity. We found that the levels of regulatory subunits which belong to the PPP2R2 subfamily, not the PPP2R5 subfamily, were downregulated in the examined brain regions of transgenic mice. Our work identifies a novel mechanism to explain how α-syn regulates PP2A activity, and provides the optimization of PP2A methylation as a new target for PD treatment.