Phosphatase Research Articles

Design and synthesis of novel benzoic acid derivatives as striatal-enriched protein tyrosine phosphatase (STEP) inhibitors with neuroprotective properties.

As a central nervous system-specific member of the protein tyrosine phosphatase (PTP) family, the striatal-enriched protein tyrosine phosphatase (STEP) is an attractive drug target for neurodegenerative diseases. Here, we reported the discovery of a series of benzoic acid derivatives as new STEP inhibitors. Among them, compound 14b exhibited good STEP inhibitory activity and displayed selectivity against other PTPs. The neuroprotective activity of compound 14b was evaluated against glutamate-induced oxidative cell death in HT22cells. Results indicated that compound 14b co-treatment prevented cell death and reduced cellular ROS accumulation. Compound 14b inhibited cell apoptosis by upregulating BCL-2 expression and downregulating BAX and C-caspase3 expression. Moreover, compound 14b was also found to provide neuroprotection to primary cortical neurons after oxygen-glucose deprivation/reoxygenation (OGD/R). Further structural elaboration of compound 14b may provide new drug candidates for neurodegenerative diseases.

Multi-scale computational modeling to identify novel chemical scaffolds as trehalose-6-phosphate phosphatase inhibitors to combat Burkholderia pseudomallei

Multi-scale computational modeling to identify novel chemical scaffolds as trehalose-6-phosphate phosphatase inhibitors to combat Burkholderia pseudomallei

TC2N maintains stem cell-like characteristics to accelerate lung carcinogenesis by blockade of dual specificity protein phosphatase 3

BackgroundTandem C2 domains, nuclear (TC2N) is a protein that has been characterized to contain C2A domain, C2B domain, and a short C-terminus with a WHXL motif. In previous studies, we have uncovered the oncogenic role and mechanisms of TC2N in lung cancer: TC2N achieves this by inhibiting the p53 signaling pathway and activating the NF-kappaB signaling pathway. Beyond that, its precise function in tumorigenesis is not fully understood.MethodsTC2N-engineered mice model was used to assess the effect of TC2N knockout on normal lung and urethane-induced carcinogenesis. Tumor tissues of 395 lung cancer patients were subjected to tissue microarray and further assessed the associations of TC2N expression with tumor differentiation degree. The protein levels of TC2N and stem cell markers in cell lines and tissue specimens were monitored by WB and immunohistochemistry. In vitro cell assays were performed to assess the effect of TC2N ectopic expression on the stem cell-like characteristics of lung cancer cells. The downstream signaling pathway or target molecule of TC2N was mined using a combination of transcriptomics and proteomics, and the underlying mechanism was explored by WB and co-IP assays.ResultsHerein, TC2N appeared to have a strong effect in promoting lung tumorigenesis caused by urethane, whereas it seemed to lose its function in the normal lung. Meanwhile, we found that the functional differences of TC2N between lung tumor and normal lung were linked to its potential role in cancer cell stemness. Function-wise, TC2N overexpression maintained stem-like properties of lung cancer cell. Mechanism-wise, TC2N upregulated the phosphorylation of EGFR, ERK, STAT3 and FAK1 to activate these signaling pathways by the inhibition of DUSP3 phosphatase via a dual mechanism. Firstly, TC2N competes with EGFR, ERK, STAT3 and FAK1 for binding to DUSP3. This competition prevents these signaling molecules from being dephosphorylated by DUSP3, resulting in their sustained activation. Secondly, TC2N bind to DUSP3 and restrict the enzyme’s ability to dephosphorylate the signaling molecules.ConclusionsOverall, this study revealed a previously unknown role and mechanism of TC2N in the regulation of tumorigenesis and stemness in lung cancer cells.

Combined Graphene Oxide with 2-Methoxyestradiol for Effective Anticancer Therapy in-vitro Model.

This article describes the invention of graphene oxide (GO) or reduced graphene oxide (rGO) functionalised with 2-methoxy estradiol. The presence of polar hydroxyl groups enables the binding of 2-ME to GO/rGO through hydrogen bonds with epoxy and hydroxyl groups located on the surface and carbonyl and carboxyl groups located at the edges of graphene flake sheets. The patented method of producing the subject of the invention and the research results regarding its anticancer effectiveness via cytotoxicity in an in vivo model (against A375 melanoma and 143B osteosarcoma cells) are described. It was shown that the inhibition of PTP1B phosphotyrosine phosphatase is one of the mechanisms of action of GO functionalised with 2-ME (GO-2-ME). This is a very important result, considering the fact that 2-ME itself has no inhibitory properties against this phosphatase. Graphene oxide flakes embroidered with 2-methoxyestradiol molecules may be a promising solution, bringing a new and important effect in the form of improving the bioavailability of the therapeutic substance, ie 2-ME. An appropriate dosage of GO-2-ME/rGO-2-ME, in which GO/rGO is a carrier of 2-methoxyestradiol (2-ME), can ensure effective penetration of the active substance through biological boundaries/membranes and controlled modification of cell signalling, ultimately leading to the selective elimination of malignant cells.

The Sub-Acute Potential Risk of Oxamyl in Male Albino Rats.

The current study aimed to investigate the sub-acute effects of oxamyl on male Albino rats following oral administration of either 0.031 or 0.31 mg/kg/day for 14 consecutive days. The findings demonstrated that oxamyl produced a significant impact on most of the examined blood profile and biomarkers, along with a significant progressive and discernible alterations in the histology of organs. According to the results obtained, the potential mechanisms by which oxamyl causes its toxic effects on rats are identified as the inflammation indices, the inhibition of transaminases, alkaline phosphatase, and antioxidant enzymes, as well as the production of thiobarbituric acid reactive substances (TBARs) in organs following oxamyl treatment based on histopathological examinations. Due to the substantial genetic similarities between rats and humans, it is therefore anticipated that oxamyl will have comparable detrimental effects on humans.

Transendothelial migration of the Lyme disease spirochete involves spirochete internalization as an intermediate step through a transcellular pathway that involves Cdc42 and Rac1.

Despite its importance in pathogenesis, the hematogenous dissemination pathway of Borrelia burgdorferi is still largely uncharacterized. To probe the molecular details of transendothelial migration more easily, we studied this process using cultured primary or telomerase-immortalized human microvascular endothelial cells in a medium that maintains both the human cells and the spirochetes. In B. burgdorferi-infected monolayers, we observed ~55% of wild-type spirochetes crossing the monolayer. Microscopic characterization revealed entrance points across the cellular surface rather than at cellular junctions, supporting a transcellular route. In support of this pathway, locking the endothelial junctions using a vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibitor did not reduce transendothelial migration. We also used inhibitors to block the most common endocytic pathways to elucidate effectors that might be involved in B. burgdorferi uptake and/or transmigration. Directly inhibiting Cdc42 reduced spirochete transmigration by impeding internalization. However, blocking Rac1 alone dramatically reduced transmigration by ~84% and resulted in a concomitant doubling in spirochete accumulation in the cell. Our combined results support that B. burgdorferi internalization is an intermediate step in the transendothelial migration process, which requires both Cdc42 and Rac1; Cdc42 is needed for spirochete internalization, while Rac1 is required for cellular egress. These are the first two host proteins implicated in B. burgdorferi transmigration across endothelial cells.IMPORTANCELyme borreliosis is caused by Borrelia burgdorferi and related bacteria. It is the most common tick-transmitted illness in the Northern Hemisphere. The ability of this pathogen to spread to a wide variety of locations results in a diverse set of clinical manifestations, yet little is known regarding vascular escape of the spirochete, an important pathway for dissemination. Our current work has studied the traversal of B. burgdorferi across a monolayer of microvascular endothelial cells grown using a new culture system. We show that this occurs by passage of the spirochetes directly through cells rather than at cellular junctions and that internalization of B. burgdorferi is an intermediate step in transmigration. We also identify the first two host proteins, Cdc42 and Rac1, that are used by the spirochetes to promote traversal of the cellular monolayer. Our new experimental system also provides a new avenue for further studies of this important process.

Stress contingent changes in Hog1 pathway architecture and regulation in Candida albicans.

The Hog1 stress-activated protein kinase (SAPK) is a key mediator of stress resistance and virulence in Candida albicans. Hog1 activation via phosphorylation of the canonical TGY motif is mediated by the Pbs2 MAPKK, which itself is activated by the Ssk2 MAPKKK. Although this three-tiered SAPK signalling module is well characterised, it is unclear how Hog1 activation is regulated in response to different stresses. Functioning upstream of the Ssk2 MAPKKK is a two-component related signal transduction system comprising three sensor histidine kinases, a phosphotransfer protein Ypd1, and a response regulator Ssk1. Here, we report that Ssk1 is a master regulator of the Hog1 SAPK that promotes stress resistance and Hog1 phosphorylation in response to diverse stresses, except high osmotic stress. Notably, we find Ssk1 regulates Hog1 in a two-component independent manner by functioning to promote interactions between the Ssk2 and Pbs2 kinases. We propose this function of Ssk1 is important to maintain a basal level of Hog1 phosphorylation which is necessary for oxidative stress, but not osmotic stress, mediated Hog1 activation. We find that osmotic stress triggers robust Pbs2 phosphorylation which drives its dissociation from Ssk2. In contrast, Pbs2 is not robustly phosphorylated following oxidative stress and the Ssk1-mediated Ssk2-Pbs2 interaction remains intact. Instead, oxidative stress-stimulated increases in phosphorylated Hog1 is dependent on the inhibition of protein tyrosine phosphatases that negatively regulate Hog1 coupled with the Ssk1-mediated promotion of basal Hog1 activity. Furthermore, we find that inhibition of protein tyrosine phosphatases is linked to the hydrogen peroxide induced oxidation of these negative regulators in a mechanism that is partly dependent on thioredoxin. Taken together these data reveal stress contingent changes in Hog1 pathway architecture and regulation and uncover a novel mode of action of the Ssk1 response regulator in SAPK regulation.

A Study of the Effects of Wetland Degradation on Soil-Microbial-Extracellular Enzyme Carbon, Nitrogen, and Phosphorus and Their Ecological Stoichiometry

Due to the unique geographic location of A’er Xiang, there is a natural landscape where sandy land and lake-marsh wetlands coexist. However, the wetland degradation caused by the disturbance of anthropogenic activities has led to the change in land use. In this study, the spatial-temporal substitution method was used to select five sample plots: the original wetland converted to forest land for reuse area of five years and ten years; the original wetland converted to cropland for reuse area of five years and ten years; and the native wetland. It aims to investigate the variations in carbon, nitrogen, and phosphorus and their stoichiometric characteristics of soil-microorganisms-extracellular enzymes before and after reuse, and to analyze potential interactions among these elements. The results indicated that following wetlands degradation, changes in land use for five years did not significantly affect the content of soil organic carbon (TOC), total nitrogen (TN), or total phosphorus (TP). However, after ten years, both TOC and TN, except for TP, decreased significantly. Microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) contents in cropland were consistently higher than those in WL, showing a trend of first increasing and then decreasing with longer conversion periods. In contrast, forest land values were lower than in WL and increased as the conversion period lengthened. The microbial biomass phosphorus (MBP) content was ranked across the five sample sites as follows: 10 CL > 5 CL > 5 FL > 10 FL > WL. β-1,4-glucosidase (BG) activity was significantly increased after conversion to forest land and significantly decreased after conversion to cropland. β-1,4-N-glucosidase (NAG) and L-leucine aminopeptidase (LAP) activities were ranked as follows among the five sites: 5 FL > WL > 5 CL > 10 FL > 10 CL. Phosphatase (PHOS) activity showed no significant changes post-conversion, though it was consistently lower compared to WL.

Pharmacological inhibition of receptor protein tyrosine phosphatase β/ζ decreases Aβ plaques and neuroinflammation in the hippocampus of APP/PS1 mice.

Alzheimer's disease (AD) is a major neurodegenerative disorder that courses with chronic neuroinflammation. Pleiotrophin (PTN) is an endogenous inhibitor of Receptor Protein Tyrosine Phosphatase (RPTP) β/ζ which is upregulated in different neuroinflammatory disorders of diverse origin, including AD. To investigate the role of RPTPβ/ζ in neuroinflammation and neurodegeneration, we used eight-to ten-month-old APP/PS1 AD mouse model. They were administered intragastrically with MY10, an inhibitor of RPTPβ/ζ, at different doses (60 and 90mg/kg) every day for 14days. Treatment with 90mg/kg MY10 significantly reduced the number and size of amyloid beta (Aβ) plaques in the dorsal subiculum of the hippocampus of APP/PS1 mice. In addition, we observed a significant decrease in the number and size of astrocytes in both sexes and in the number of microglial cells in a sex-dependent manner. This suggests that RPTPβ/ζ plays an important role in modulating Aβ plaque formation and influences glial responses, which may contribute to improved Aβ clearance. In addition, MY10 treatment decreased the interaction of glial cells with Aβ plaques in the hippocampus of APP/PS1 mice. Furthermore, the analysis of proinflammatory markers in the hippocampus revealed that MY10 treatment decreased the mRNA levels of Tnfa and Hmgb1. Notably, treatment with MY10 increased Bace1 mRNA expression, which could be involved in enhancing Aβ degradation, and it decreased Mmp9 levels, which might reflect changes in the neuroinflammatory environment and impact Aβ plaque dynamics. These results support the therapeutic potential of inhibition of RPTPβ/ζ in modulating Aβ pathology and neuroinflammation in AD.

KAHA ligation as a platform for the rapid discovery of Protein Tyrosine phosphatase 1B (PTP1B) inhibitors.

We have successfully designed and assembled a 66-member library of protein tyrosine phosphatases (PTP) inhibitor candidates using α-ketoacid-hydroxylamine (KAHA) ligation. Subsequent in situ enzymatic screening revealed a potent hit (IC50=1.67μM) against PTP1B, which displayed 6.8- to 50-fold selectivity over other phosphatases.

In Obesity, Esophagogastric Junction Fat Impairs Esophageal Barrier Function and Dilates Intercellular Spaces via Hypoxia-Inducible Factor 2α.

Dilated intercellular space in esophageal epithelium, a sign of impaired barrier function, is a characteristic finding of gastroesophageal reflux disease that is also found in obese patients without gastroesophageal reflux disease. We explored molecular mechanisms whereby adipose tissue products might impair esophageal barrier integrity. Cultures of visceral fat obtained during foregut surgery from obese and nonobese patients were established. Monolayer and air-liquid interface cultures of human esophageal cells were grown with conditioned medium (CM) from fat cultures. RNA sequencing, enzyme-linked immunosorbent assay, western blot, immunostaining, histology, and analyses of barrier function were performed; inhibitors of hypoxia-inducible factor 2α (HIF-2α [PT2385]), caspase-1 (AC-YVAD-CHO), myosin light chain kinase (PIK), and myosin light chain phosphatase (permeant inhibitor of phosphatase 250) were applied; blebbistatin was used to disrupt actin-myosin interactions; N-acetylcysteine was used to scavenge reactive oxygen species. CM from esophagogastric junction fat of obese patients caused dilated intercellular space with impaired barrier function in esophageal air-liquid interface cultures; these effects were blocked by PT2385. CM from esophagogastric junction fat of obese patients induced reactive oxygen species production that activated HIF-2α in esophageal cells. RNA-sequencing analyses linked CM from esophagogastric junction fat of obese patient-induced HIF-2α increases with innate immune response pathways. Cross-talk between HIF-2α and caspase-1 in esophageal cells led to interleukin 1β secretion, and interleukin 1β/interleukin 1 receptor 1 signaling caused dilated intercellular space with impaired esophageal barrier function via actin-myosin interactions induced by myosin light chain phosphorylation. We have elucidated molecular mechanisms whereby visceral fat of obese patients can impair esophageal barrier integrity by secreting substances that generate reactive oxygen species, which activate HIF-2α in esophageal epithelial cells. These mechanisms could render the esophagus of obese individuals vulnerable to damage from acid and other noxious agents.

The landscape of intrinsically disordered proteins in Leishmania parasite: Implications for drug discovery

Proteins that lack three-dimensional structures are known as Intrinsically disordered proteins (IDPs). In this study, we aimed to identify intrinsically disordered proteins in the Leishmania donovani proteome using various predictors that can identify IDPs based on amino acid residues and charge hydropathy. Top identified IDPs are analyzed using STRING, PSP-Hunter, Deep Loc-2.0, and Alpha fold to understand the protein-protein interaction, phase separation, localization, and structural assessment of those proteins. From this study, we found that >50 % of Leishmania donovani proteome has proteins or regions of proteins that are intrinsically disordered with VSL2 score >0.5; most proteins interact with many other proteins with PPI enrichment p-value <1.0e-16. Few proteins, such as Protein phosphatase inhibitor, UMSBP, and Zinc knuckle, have redox-sensitive regions. Functional disorder profiles of identified IDPs showed MoRFs and predicted protein domains. HASPB, UTP11, Nucleolar protein 12, and UMSBP have a high probability of undergoing phase separation. Localization studies showed that most of these proteins are in the cytoplasm and nucleus. Our present study of identifying IDPs in Leishmania proteome yields significant information on druggable targets and can serve as a basis for further studies to identify unexplored pathways.

Guanylate Kinase 1 Deficiency: A Novel and Potentially Treatable Mitochondrial DNA Depletion/Deletions Disease.

Mitochondrial DNA (mtDNA) depletion/deletions syndrome (MDDS) comprises a group of diseases caused by primary autosomal defects of mtDNA maintenance. Our objective was to study the etiology of MDDS in 4 patients who lack pathogenic variants in known genetic causes. Whole exome sequencing of the probands was performed to identify pathogenic variants. We validated the mitochondrial defect by analyzing mtDNA, mitochondrial dNTP pools, respiratory chain activities, and GUK1 activity. To confirm pathogenicity of GUK1 deficiency, we expressed 2 GUK1 isoforms in patient cells. We identified biallelic GUK1 pathogenic variants in all 4 probands who presented with ptosis, ophthalmoparesis, and myopathic proximal limb weakness, as well as variable hepatopathy and altered T-lymphocyte profiles. Muscle biopsies from all probands showed mtDNA depletion, deletions, or both, as well as reduced activities of mitochondrial respiratory chain enzymes. GUK1 encodes guanylate kinase, originally identified as a cytosolic enzyme. Long and short isoforms of GUK1 exist. We observed that the long isoform is intramitochondrial and the short is cytosolic. In probands' fibroblasts, we noted decreased GUK1 activity causing unbalanced mitochondrial dNTP pools and mtDNA depletion in both replicating and quiescent fibroblasts indicating that GUK1 deficiency impairs de novo and salvage nucleotide pathways. Proband fibroblasts treated with deoxyguanosine and/or forodesine, a purine phosphatase inhibitor, ameliorated mtDNA depletion, indicating potential pharmacological therapies. Primary GUK1 deficiency is a new and potentially treatable cause of MDDS. The cytosolic isoform of GUK1 may contribute to the T-lymphocyte abnormality, which has not been observed in other MDDS disorders. ANN NEUROL 2024;96:1209-1224.

A versatile and automatic on-line screening method: Transverse diffusion of laminar flow profiles-based capillary electrophoresis for exploring PTP1B inhibitors in natural products.

Natural products (NPs) play an important role in drug discovery and drug development due to their diverse chemical properties and biological activities. In the present work, an on-line capillary electrophoresis (CE) method was developed and applied to screen protein tyrosine phosphatase 1B (PTP1B) inhibitors in NPs. As a generic technique, transverse diffusion of laminar flow profiles (TDLFP) was utilized to mix all reactants in the capillary for on-line enzymatic reaction. The procedures of TDLFP were optimized in terms of the concentration of PTP1B, injection order of the plugs, PTP1B injection time, overall plugs' length, and temperature set point. After validation, this developed on-line method was used for kinetic constant determination and inhibitor screening. As a result, the proposed method was validated with good repeatability and a short analysis time of 3 min. The obtained Km value was as 3.66 ± 0.97 mM. The IC50 value of the phosphatase inhibitor Na3VO4 was determined as 98.50 ± 24.82 µM. An enhanced inhibition effect was found by the combined extract of Morus alba L. and quercetin/caffeic acid which could be a synergistic effect on PTP1B. Afterwards, molecular docking was performed for mechanism clarification. According to the docking results, it is speculated that the compounds in the extract of M. alba L. contribute to the enhanced effect through occupation of the catalytic region and substrate recognition site. In conclusion, a universal and automatic on-line method using CE was developed and applied for inhibitor screening. Owing to the merits of automation and low consumption of samples, this method can be an alternative for inhibitor screening of other dephosphorylating enzymes, especially some valuable enzymes.

Colorimetric and photothermal dual-mode sensing platform for point-of-care testing of acid phosphatase activity and inhibitor screening based on oxidase-mimetic activity of Pt-Ni alloy nanoparticles

It is vital to exploit sensitive and portable means for the reliable assay of acid phosphatase (ACP) activity and its inhibitors screening in clinical diagnosis and drug therapy. Herein, the Pt-Ni alloy nanoparticles (Pt-Ni ANPs) with enhanced oxidase-mimetic activity were successfully prepared and applied to oxidize the achromatous 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxTMB. The oxTMB, as an effective photothermal agent, can transform photon energy to heat under an 808 nm laser illumination. The absorbance and temperature of the reaction solution increased significantly when TMB was oxidized to oxTMB by the oxidase-like Pt-Ni ANPs. However, the ACP can hydrolyze 2-Phospho-L-ascorbic acid trisodium salt (AAP) to produce ascorbic acid (AA), and then the production of AA, as a powerful reductant, can efficiently reduce oxTMB and react with singlet oxygen (1O2) to suppress the oxidase-mimicking activity of Pt-Ni ANPs, causing an obvious decrease in absorption spectrum and temperature of the assay system. A colorimetric and photothermal dual-mode detection platform was established for ACP activity assay by integrating colorimetric and temperature signals. Moreover, the potential application of this strategy was also verified by sensing ACP activity in human serum samples and its inhibitor screening. This dual-mode sensing platform not only improves sensitivity and precision but also meets the needs of diversity detection, which offers new insights for efficient monitoring of ACP activity and inhibitor screening in resource-poor settings.

Antiproliferative and Apoptotic Potential of Chicoric Acid, a Major Phytoconstituent of Cichorium intybus, against Colon Cancer Cells: A Possible Inhibition of CDC25 Phosphatases

Background: Over the years, plants have been utilized worldwide for the treatment of cancer in the traditional system of medicine, notably in the majority of developing nations. The use of chemotherapeutic drugs is frequently associated with lethal side effects when treating cancer. Therefore, the panacea to the vicious side effects linked to synthetic pharmaceuticals is the alternative usage of widely accessible and affordable therapeutic approaches. Chicoric acid, a major constituent of Cichorium intybus, has recently been shown to exhibit antioxidant, anti-inflammatory, antidiabetic, antiviral, neuroprotective, hepatoprotective, and anticancer properties. Purpose: Except for a few preliminary studies, no detailed study on the chemotherapeutic efficacy of chicoric acid against cancer cells has been done till now. Therefore, the aim of this study is to assess the potential of chicoric acid against colon cancer cells. Methods: Different in vitro assays like MTT, DAPI staining, rhodamine staining, H2DCFDA staining, caspase activity, and cell analysis were performed on colon cancer HCT116 cells to assess their growth inhibitory properties. Results: According to the results, chicoric acid dose-dependently suppressed the growth of colon cancer HCT116 cells by inducing cell cycle arrest at G0/G1 phase and apoptosis via both intrinsic and extrinsic pathways. In silico findings unveiled the cell cycle regulatory capacity of chicoric acid through binding CDC25 phosphatases, implying its potential as an antiproliferative agent capable of suppressing cell growth. Conclusion: Collectively, the results suggested strong chemopreventive potential of chicoric acid against colon cancer cells. Thus, chicoric acid may turn out to be an excellent anticancer agent, which may be explored in the future for the therapeutic drug development against cancer.

Abstract 4113617: Single-cell Analysis Reveals Endothelial Cell CD45 Deficiency Prevents Endothelial-to-Mesenchymal Transition (EndMT) in Atherosclerosis

Introduction: Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells (ECs). However, a recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells induced expression of EndMT marker genes. Also, the CD45 phosphatase inhibitor blocked EndMT activities in TGFβ1-treated ovine mitral valve endothelial cells. EndMT contributes to atherosclerotic pathobiology and is associated with more complex plaques. We identified CD45-positive ECs undergoing EndMT in atherosclerotic ApoE-deficient (ApoE -/- ) mice. Here, we aim to investigate whether endothelial CD45-specific deletion can prevent EndMT in a mouse model of atherosclerosis. Methods and Results: We generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE -/- background (C57BL/6) and fed these mice a Western diet for 16 weeks. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA-seq. Cells populated 15 major clusters after integrating the single-cell transcriptomic profiles. In the EC-iCD45KO group, the population of endothelial, resident macrophage, and mesenchymal-endothelial transition cells increased while vascular smooth muscle, EndMT, and fibroblast cells decreased. In EndMT cells, EC markers (Cdh5, Cldn5, Pecam1) increased, but SMC markers (Acta2) and EndMT markers (Col1a2 and Col3a1）decreased. In addition, we characterized EndMT cells into two sub-clusters: 1 and 2. EC-specific CD45 deletions reduced the cell numbers in the EndMT2 sub-cluster in atherosclerotic mice but had less impact on the EndMT1 sub-cluster. FGFR pathways were potentiated while TGFβ1 and Tgfbr2 decreased in EndMT1 sub-cluster， and the genes controlling endothelial cell development were upregulated but the genes controlling actin cytoskeleton organization were downregulated in both EndMT1 and 2 sub-clusters. Conclusion: Our single-cell analysis indicates that the loss of endothelial CD45 protects against EndMT-driven atherosclerosis, inhibiting TGFβ and EndoMT marker expression while stimulating FGFR pathways. Consistently, EC-iCD45KO/ApoE -/- mice showed reduced plaque macrophages, expression of cell adhesion molecules, foam cell formation, and lesion development when compared to ApoE -/- controls. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndMT and atherosclerosis.

A mechanism for hypoxia-induced inflammatory cell death in cancer.

Hypoxic cancer cells resist many antineoplastic therapies and can seed recurrence1,2. We previously found that either deficiency or inhibition of protein-tyrosine phosphatase (PTP1B) promotes human epidermal growth factor receptor 2-positive breast cancer cell death in hypoxia by activation of RNF213 (ref. 3), a large protein with multiple AAA-ATPase domains and two ubiquitin ligase domains (RING and RZ) implicated in Moyamoya disease, lipotoxicity and innate immunity4. Here we report that PTP1B and ABL1/2 reciprocally control RNF213 tyrosine phosphorylation and, consequently, its oligomerization and RZ domain activation. The RZ domain ubiquitylates and induces the degradation of the major NF-κB regulator CYLD/SPATA2. Decreased CYLD/SPATA2 levels lead to NF-κB activation and induction of the NLRP3 inflammasome which, together with hypoxia-induced endoplasmic reticulum stress, triggers pyroptotic cell death. Consistent with this model, CYLD deletion phenocopies, whereas NLRP3 deletion blocks, the effects of PTP1B deficiency on human epidermal growth factor receptor 2-positive breast cancer xenograft growth. Reconstitution studies with RNF213 mutants confirm that the RZ domain mediates tumour cell death. In concert, our results identify a unique, potentially targetable PTP1B-RNF213-CYLD-SPATA2 pathway critical for the control of inflammatory cell death in hypoxic tumours, provide new insights into RNF213 regulation and have potential implications for the pathogenesis of Moyamoya disease, inflammatory disorders and autoimmune disease.

A coordinated kinase and phosphatase network regulates Stu2 recruitment to yeast kinetochores.

Cells coordinate diverse events at anaphase onset, including separase activation, cohesin cleavage, chromosome separation, and spindle reorganization. Regulation of the XMAP215 family member and microtubule polymerase, Stu2, at the metaphase-anaphase transition determines a specific redistribution from kinetochores to spindle microtubules. We show that cells modulate Stu2 kinetochore-microtubule localization by Polo-like kinase1/Cdc5-mediated phosphorylation of T866, near the Stu2 C-terminus, thereby promoting dissociation from the kinetochore Ndc80 complex. Cdk/Cdc28 likely primes Cdc5:Stu2 interaction. Cdc28 activity is also required for Stu2 nuclear import. PP2ACdc55 actively opposes Cdc5 activity on Stu2T866 during metaphase. This counter-regulation allows for switchlike redistribution of Stu2pT866 at anaphase onset when separase inhibits PP2ACdc55. Blocking Stu2T866 phosphorylation disrupts anaphase spindle progression, and we infer that PP2ACdc55 regulates the mitotic spindle by dephosphorylating Stu2 and other MAPs. These data support a model in which increased phosphorylation at anaphase onset results from phosphatase inhibition and point to a larger regulatory network that facilitates rapid cytoskeletal modulation required for anaphase spindle maintenance.

PPM1G Inhibits Epithelial-Mesenchymal Transition in Cholangiocarcinoma by Catalyzing TET1 Dephosphorylation for Destabilization to Impair Its Targeted Demethylation of the CLDN3 Promoter.

Ten-eleven translocation protein 1 (TET1) functions as an epigenetic regulatory molecule, mediating the majority of DNA demethylation, and plays a role in the development of different types of cancers by regulating the expression of proto-oncogenes and oncogenes. Here it is found that TET1 is highly expressed in cholangiocarcinoma (CCA) and is associated with a poor prognosis. In addition, TET1 promotes claudin-3 (CLDN3) transcription by targeting the CLDN3 promoter region between -16 and 512 for demethylation. PPM1G functions as a protein dephosphorylase, catalyzing the dephosphorylation of TET1. This results in the destabilization of the TET1 protein, thereby impairing the targeting of the CLDN3 promoter for demethylation. Two phosphatase inhibitors, staurosporine and AZD0156, inhibit epithelial-to-mesenchymal transition (EMT) in cholangiocarcinoma cells by suppressing TET1 expression. In conclusion, it is also demonstrated that PPM1G can be employed as a therapeutic target to impede the progression of CCA by catalyzing the dephosphorylation of TET1, which diminishes the capacity of TET1 to target the CLDN3 promoter to activate transcription and inhibit EMT in CCA.