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Journal of Virology Nov 2023As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and...
As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and mortality rates. Initially identified in Hong Kong in 2012, the virus has rapidly spread worldwide. During PDCoV infection, the virus employs evasion mechanisms to evade host surveillance, while the host mounts corresponding responses to impede viral replication. Our research has revealed that PDCoV infection down-regulates the expression of PGAM5 to promote virus replication. In contrast, PGAM5 degrades PDCoV N through autophagy by interacting with the cargo receptor P62 and the E3 ubiquitination ligase STUB1. Additionally, PGAM5 interacts with MyD88 and TRAF3 to activate the IFN signal pathway, resulting in the inhibition of viral replication.
Topics: Animals; Coronavirus Infections; Interferon Type I; Signal Transduction; Swine; Swine Diseases; Ubiquitin-Protein Ligases; Virus Replication; Coronavirus Nucleocapsid Proteins; Deltacoronavirus; Phosphoprotein Phosphatases; Mitochondrial Proteins; Down-Regulation; Immune Evasion; RNA-Binding Proteins; Proteolysis
PubMed: 37882521
DOI: 10.1128/jvi.01470-23 -
Cold Spring Harbor Molecular Case... Jun 2023PPP2 syndrome type R5D, or Jordan's syndrome, is a neurodevelopmental disorder caused by pathogenic missense variants in , a β-subunit of the Protein Phosphatase 2A...
PPP2 syndrome type R5D, or Jordan's syndrome, is a neurodevelopmental disorder caused by pathogenic missense variants in , a β-subunit of the Protein Phosphatase 2A (PP2A). The condition is characterized by global developmental delays, seizures, macrocephaly, ophthalmological abnormalities, hypotonia, attention disorder, social and sensory challenges often associated with autism, disordered sleep, and feeding difficulties. Among affected individuals, there is a broad spectrum of severity, and each person only has a subset of all associated symptoms. Some, but not all, of the clinical variability is due to differences in the genotype. These suggested clinical care guidelines for the evaluation and treatment of individuals with PPP2 syndrome type R5D are based on data from 100 individuals reported in the literature and from an ongoing natural history study. As more data are available, particularly for adults and regarding treatment response, we anticipate that revisions to these guidelines will be made.
Topics: Adult; Humans; Intellectual Disability; Jordan; Neurodevelopmental Disorders; Autistic Disorder; Syndrome; Reference Standards; Protein Phosphatase 2
PubMed: 37339871
DOI: 10.1101/mcs.a006285 -
American Journal of Respiratory Cell... Nov 2023
Topics: Humans; Protein Phosphatase 2; Emphysema
PubMed: 37552790
DOI: 10.1165/rcmb.2023-0263ED -
Acta Neuropathologica Feb 2024Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a common cause of familial ALS, accounting for around 4% of the cases. Understanding the mechanisms by which mutant FUS becomes toxic to neurons can provide insight into the pathogenesis of both familial and sporadic ALS. We have previously observed that overexpression of wild-type or ALS-mutant FUS in Drosophila motor neurons is toxic, which allowed us to screen for novel genetic modifiers of the disease. Using a genome-wide screening approach, we identified Protein Phosphatase 2A (PP2A) and Glycogen Synthase Kinase 3 (GSK3) as novel modifiers of FUS-ALS. Loss of function or pharmacological inhibition of either protein rescued FUS-associated lethality in Drosophila. Consistent with a conserved role in disease pathogenesis, pharmacological inhibition of both proteins rescued disease-relevant phenotypes, including mitochondrial trafficking defects and neuromuscular junction failure, in patient iPSC-derived spinal motor neurons (iPSC-sMNs). In FUS-ALS flies, mice, and human iPSC-sMNs, we observed reduced GSK3 inhibitory phosphorylation, suggesting that FUS dysfunction results in GSK3 hyperactivity. Furthermore, we found that PP2A acts upstream of GSK3, affecting its inhibitory phosphorylation. GSK3 has previously been linked to kinesin-1 hyperphosphorylation. We observed this in both flies and iPSC-sMNs, and we rescued this hyperphosphorylation by inhibiting GSK3 or PP2A. Moreover, increasing the level of kinesin-1 expression in our Drosophila model strongly rescued toxicity, confirming the relevance of kinesin-1 hyperphosphorylation. Our data provide in vivo evidence that PP2A and GSK3 are disease modifiers, and reveal an unexplored mechanistic link between PP2A, GSK3, and kinesin-1, that may be central to the pathogenesis of FUS-ALS and sporadic forms of the disease.
Topics: Animals; Humans; Mice; Amyotrophic Lateral Sclerosis; Glycogen Synthase Kinase 3; Protein Phosphatase 2; RNA-Binding Protein FUS; Neurodegenerative Diseases; Kinesins; Motor Neurons; Drosophila; Mutation
PubMed: 38363426
DOI: 10.1007/s00401-024-02689-y -
Acta Pharmacologica Sinica Jan 2024Acute kidney injury (AKI) is a worldwide public health problem characterized by the massive loss of tubular cells. However, the precise mechanism for initiating tubular...
Acute kidney injury (AKI) is a worldwide public health problem characterized by the massive loss of tubular cells. However, the precise mechanism for initiating tubular cell death has not been fully elucidated. Here, we reported that phosphoglycerate mutase 5 (PGAM5) was upregulated in renal tubular epithelial cells during ischaemia/reperfusion or cisplatin-induced AKI in mice. PGAM5 knockout significantly alleviated the activation of the mitochondria-dependent apoptosis pathway and tubular apoptosis. Apoptosis inhibitors alleviated the activation of the mitochondria-dependent apoptosis pathway. Mechanistically, as a protein phosphatase, PGAM5 could dephosphorylate Bax and facilitate Bax translocation to the mitochondrial membrane. The translocation of Bax to mitochondria increased membrane permeability, decreased mitochondrial membrane potential and facilitated the release of mitochondrial cytochrome c (Cyt c) into the cytoplasm. Knockdown of Bax attenuated PGAM5 overexpression-induced Cyt c release and tubular cell apoptosis. Our results demonstrated that the increase in PGAM5-mediated Bax dephosphorylation and mitochondrial translocation was implicated in the development of AKI by initiating mitochondrial Cyt c release and activating the mitochondria-dependent apoptosis pathway. Targeting this axis might be beneficial for alleviating AKI.
Topics: Mice; Animals; Cytochromes c; Phosphoglycerate Mutase; bcl-2-Associated X Protein; Apoptosis; Mitochondria; Acute Kidney Injury; Carrier Proteins; Phosphoprotein Phosphatases
PubMed: 37684381
DOI: 10.1038/s41401-023-01151-1 -
Cell Chemical Biology Dec 2023An emerging strategy for the therapeutic targeting of protein phosphatases involves the use of compounds that interfere with the binding of regulatory subunits or...
An emerging strategy for the therapeutic targeting of protein phosphatases involves the use of compounds that interfere with the binding of regulatory subunits or substrates. However, high-throughput screening strategies for such interfering molecules are scarce. Here, we report on the conversion of the NanoBiT split-luciferase system into a robust assay for the quantification of phosphatase subunit and substrate interactions in cell lysates. The assay is suitable to screen small-molecule libraries for interfering compounds. We designed and validated split-luciferase sensors for a broad range of PP1 and PP2A holoenzymes, including sensors that selectively report on weak interaction sites. To facilitate efficient hit triaging in large-scale screening campaigns, deselection procedures were developed to eliminate assay-interfering molecules with high fidelity. As a proof-of-principle, we successfully applied the split-luciferase screening tool to identify small-molecule disruptors of the interaction between the C-terminus of PP1β and the ankyrin-repeat domain of the myosin-phosphatase targeting subunit MYPT1.
Topics: Protein Phosphatase 1; Protein Binding; Myosin-Light-Chain Phosphatase; Phosphorylation
PubMed: 37625414
DOI: 10.1016/j.chembiol.2023.07.018 -
Circulation Research Jan 2024Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include...
BACKGROUND
Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized.
METHODS
Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes.
RESULTS
We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions.
CONCLUSIONS
Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling.
Topics: Animals; Mice; Calcineurin; Cardiomegaly; Chromatin; Epigenesis, Genetic; Histones; Myocytes, Cardiac; Transferases; Ventricular Remodeling
PubMed: 38084599
DOI: 10.1161/CIRCRESAHA.123.323458 -
Cell Death & Disease Feb 2024Copper ions play a crucial role as cofactors for essential enzymes in cellular processes. However, when the intracellular concentration of copper ions exceeds the...
Copper ions play a crucial role as cofactors for essential enzymes in cellular processes. However, when the intracellular concentration of copper ions exceeds the homeostatic threshold, they become toxic to cells. In our study, we demonstrated that elesclomol, as a carrier of copper ions, caused an upregulation of protein phosphatase 1 regulatory subunit 15 A (PPP1R15A), which plays a role in regulating substrate selectivity of protein phosphatase 1 during cuproptosis. Mechanistically, we investigated that PPP1R15A activated translation initiation by dephosphorylating eukaryotic translation initiation factor 2 subunit alpha at the S51 residue through protein phosphatase 1 and phosphorylating eukaryotic translation initiation factor 4E binding protein 1 at the T70 residue. In addition, PPP1R15A reduced H3K4 methylation by altering the phosphorylation of histone methyltransferases, which led to the silencing of MYC and G2M phase arrest.
Topics: Humans; Copper; Ions; Neoplasms; Phosphoproteins; Phosphorylation; Protein Biosynthesis; Protein Phosphatase 1; Cell Cycle Checkpoints; Apoptosis; Peptide Chain Initiation, Translational
PubMed: 38365764
DOI: 10.1038/s41419-024-06489-w -
The Journal of Clinical Investigation Nov 2023Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine...
Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine-rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium "DASH-like" diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.
Topics: Animals; Mice; Blood Pressure; Solute Carrier Family 12, Member 3; Protein Serine-Threonine Kinases; Sodium Chloride; Potassium, Dietary; Kidney; Hypertension; Potassium; Phosphorylation
PubMed: 37676724
DOI: 10.1172/JCI158498 -
The Journal of Biological Chemistry Sep 2023Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is...
Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.
Topics: Humans; Autism Spectrum Disorder; HEK293 Cells; Mechanistic Target of Rapamycin Complex 1; Phosphorylation; Protein Phosphatase 2; Proteomics; Ribosomal Protein S6; Abnormalities, Multiple
PubMed: 37572851
DOI: 10.1016/j.jbc.2023.105154