-
Autophagy Aug 2023Acetaminophen (APAP) overdose is the predominant cause of drug-induced liver injury worldwide. The macroautophagy/autophagy-lysosomal pathway (ALP) is involved in the...
Acetaminophen (APAP) overdose is the predominant cause of drug-induced liver injury worldwide. The macroautophagy/autophagy-lysosomal pathway (ALP) is involved in the APAP hepatotoxicity. TFEB (transcription factor EB) promotes the expression of genes related to autophagy and lysosomal biogenesis, thus, pharmacological activation of TFEB-mediated ALP may be an effective therapeutic approach for treating APAP-induced liver injury. We aimed to reveal the effects of narirutin (NR), the main bioactive constituents isolated from citrus peels, on APAP hepatotoxicity and to explore its underlying mechanism. Administration of NR enhanced activities of antioxidant enzymes, improved mitochondrial dysfunction and alleviated liver injury in APAP-treated mice, whereas NR did not affect APAP metabolism and MAPK/JNK activation. NR enhanced TFEB transcriptional activity and activated ALP in an MTOR complex 1 (MTORC1)-independent but PPP3/calcineurin-dependent manner. Moreover, knockout of or knockdown of PPP3CB/CNA2 (protein phosphatase 3, catalytic subunit, beta isoform) in the liver abolished the beneficial effects of NR on APAP overdose. Mechanistically, NR bound to PPP3CB via PRO31, LYS61 and PRO347 residues and enhanced PPP3/calcineurin activity, thereby eliciting dephosphorylation of TFEB and promoting ALP, which alleviated APAP-induced oxidative stress and liver injury. Together, NR protects against APAP-induced liver injury by activating a PPP3/calcineurin-TFEB-ALP axis, indicating NR may be a potential agent for treating APAP overdose. ALP: autophagy-lysosomal pathway; APAP: acetaminophen; APAP-AD: APAP-protein adducts; APAP-Cys: acetaminophen-cysteine adducts; CAT: catalase; CETSA: cellular thermal shift assay; CQ: chloroquine; CYP2E1: cytochrome P450, family 2, subfamily e, polypeptide 1; CYCS/Cyt c: cytochrome c, somatic; DARTS: drug affinity responsive target stability assay; ENGASE/NAG: endo-beta-N-acetylglucosaminidase; GOT1/AST: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALT: glutamic pyruvic transaminase, soluble; GSH: glutathione; GPX/GSH-Px: glutathione peroxidase; K: dissociation constant; Leu: leupeptin; MCOLN1: mucolipin 1; MTORC1: MTOR complex 1; NAC: -acetylcysteine; NAPQI: N-acetyl--benzoquinoneimine; NFAT: nuclear factor of activated T cells; NR: narirutin; OA: okadaic acid; RRAG: Ras related GTP binding; ROS: reactive oxygen species; PPP3CB/CNA2: protein phosphatase 3, catalytic subunit, beta isoform; PPP3R1/CNB1: protein phosphatase 3, regulatory subunit B, alpha isoform (calcineurin B, type I); SOD: superoxide dismutase; SPR: surface plasmon resonance analysis; TFEB: transcription factor EB.
Topics: Mice; Animals; Calcineurin; Acetaminophen; Autophagy; Chemical and Drug Induced Liver Injury, Chronic; Liver; Glutathione; Mechanistic Target of Rapamycin Complex 1; TOR Serine-Threonine Kinases
PubMed: 36779633
DOI: 10.1080/15548627.2023.2179781 -
Cell Jul 2023Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly...
Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruits the serine/threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.
Topics: Phosphoric Monoester Hydrolases; Proteins; Phytophthora; Plants; Protein Processing, Post-Translational; Protein Phosphatase 2; Plant Diseases
PubMed: 37369204
DOI: 10.1016/j.cell.2023.05.049 -
Cell Reports Sep 2023Alternative splicing (AS) has been implicated in cell cycle regulation and cancer, but the underlying mechanisms are poorly understood. The poly(U)-binding splicing...
Alternative splicing (AS) has been implicated in cell cycle regulation and cancer, but the underlying mechanisms are poorly understood. The poly(U)-binding splicing factor 60 (PUF60) is essential for embryonic development and is overexpressed in multiple types of cancer. Here, we report that PUF60 promotes mitotic cell cycle and lung cancer progression by controlling AS of the cell division cycle 25C (CDC25C). Systematic analysis of splicing factors deregulated in lung adenocarcinoma (LUAD) identifies that elevated copy number and expression of PUF60 correlate with poor prognosis. PUF60 depletion inhibits LUAD cell-cycle G2/M transition, cell proliferation, and tumor development. Mechanistically, PUF60 knockdown leads to exon skipping enriched in mitotic cell cycle genes, including CDC25C. Exon 3 skipping in the full-length CDC25C results in nonsense-mediated mRNA decay and a decrease of CDC25C protein, thereby inhibiting cell proliferation. This study establishes PUF60 as a cell cycle regulator and an oncogenic splicing factor in lung cancer.
Topics: Humans; Adenocarcinoma of Lung; Alternative Splicing; cdc25 Phosphatases; Cell Cycle; Cell Division; Cell Line, Tumor; Lung Neoplasms; RNA Splicing Factors
PubMed: 37682709
DOI: 10.1016/j.celrep.2023.113041 -
Molecular Therapy : the Journal of the... Jun 2023The available targeted therapies for gastric cancer (GC) are still limited, so it is important to discover novel molecules as potential treatment options. Proteins or...
The available targeted therapies for gastric cancer (GC) are still limited, so it is important to discover novel molecules as potential treatment options. Proteins or peptides encoded by circular RNAs (circRNAs) are increasingly reported to play essential roles in malignancies. The aim of the present study was to identify an undiscovered protein encoded by circRNA and explore its key role and molecular mechanism in GC progression. CircMTHFD2L (hsa_circ_0069982) was screened and validated as a downregulated circRNA with coding potential. The protein encoded by circMTHFD2L, named CM-248aa, was identified for the first time by immunoprecipitation and mass spectrometry. CM-248aa was significantly downregulated in GC, while its low expression was associated with advanced tumor-node-metastasis (TNM) stage and histopathological grade. Low expression of CM-248aa could be an independent risk factor for poor prognosis. Functionally, CM-248aa, instead of circMTHFD2L suppressed the proliferation and metastasis of GC in vitro and in vivo. Mechanistically, CM-248aa competitively targeted the acidic domain of SET nuclear oncogene (SET) and acted as an endogenous inhibitor of the SET-protein phosphatase 2A interaction to promote dephosphorylation of AKT, extracellular signal-regulated kinase, and P65. Our discovery revealed that CM-248aa could be a potential prognostic biomarker and endogenous therapeutic option for GC.
Topics: Humans; RNA, Circular; Stomach Neoplasms; RNA; Protein Phosphatase 2; Gene Expression Regulation, Neoplastic; Cell Line, Tumor; Cell Proliferation; MicroRNAs
PubMed: 37101395
DOI: 10.1016/j.ymthe.2023.04.013 -
Cell Death & Disease Nov 2023The mechanism underlying acute kidney injury (AKI) and AKI-to-Chronic kidney disease (CKD) transition remains unclear, but mitochondrial dysfunction may be a key driving...
The mechanism underlying acute kidney injury (AKI) and AKI-to-Chronic kidney disease (CKD) transition remains unclear, but mitochondrial dysfunction may be a key driving factor. Literature reports suggest that dual-specificity phosphatase 1 (DUSP1) plays a critical role in maintaining mitochondrial function and structural integrity. In this study, ischemic Acute Kidney Injury (AKI) and post-ischemic fibrosis models were established by clamping the renal pedicle with different reperfusion times. To investigate the role of DUSP1, constitutional Dusp1 knockout mice and tubular-specific Sting knockout mice were used. Mitochondrial damage was assessed through electron microscopy observation, measurements of mitochondrial membrane potential, mtDNA release, and BAX translocation. We found that Dusp1 expression was significantly upregulated in human transplant kidney tissue and mouse AKI tissue. Dusp1 gene deletion exacerbated acute ischemic injury, post-ischemic renal fibrosis, and tubular mitochondrial dysfunction in mice. Mechanistically, DUSP1 could directly bind to JNK, and DUSP1 deficiency could lead to aberrant phosphorylation of JNK and BAX mitochondria translocation. BAX translocation promoted mitochondrial DNA (mtDNA) leakage and activated the cGAS-STING pathway. Inhibition of JNK or BAX could inhibit mtDNA leakage. Furthermore, STING knockout or JNK inhibition could significantly mitigate the adverse effects of DUSP1 deficiency in ischemic AKI model. Collectively, our findings suggest that DUSP1 is a regulator for the protective response during AKI. DUSP1 protects against AKI by preventing BAX-induced mtDNA leakage and blocking excessive activation of the cGAS-STING signaling axis through JNK dephosphorylation.
Topics: Animals; Humans; Mice; Acute Kidney Injury; bcl-2-Associated X Protein; DNA, Mitochondrial; Dual Specificity Phosphatase 1; Kidney; Mice, Knockout; Mitochondria; Nucleotidyltransferases; Reperfusion Injury
PubMed: 37935658
DOI: 10.1038/s41419-023-06247-4 -
Blood Dec 2023PPM1D encodes a phosphatase that is recurrently activated across cancer, most notably in therapy-related myeloid neoplasms. However, the function of PPM1D in...
PPM1D encodes a phosphatase that is recurrently activated across cancer, most notably in therapy-related myeloid neoplasms. However, the function of PPM1D in hematopoiesis and its contribution to tumor cell growth remain incompletely understood. Using conditional mouse models, we uncover a central role for Ppm1d in hematopoiesis and validate its potential as a therapeutic target. We find that Ppm1d regulates the competitive fitness and self-renewal of hematopoietic stem cells (HSCs) with and without exogenous genotoxic stresses. We also show that although Ppm1d activation confers cellular resistance to cytotoxic therapy, it does so to a lesser degree than p53 loss, informing the clonal competition phenotypes often observed in human studies. Notably, loss of Ppm1d sensitizes leukemias to cytotoxic therapies in vitro and in vivo, even in the absence of a Ppm1d mutation. Vulnerability to PPM1D inhibition is observed across many cancer types and dependent on p53 activity. Importantly, organism-wide loss of Ppm1d in adult mice is well tolerated, supporting the tolerability of pharmacologically targeting PPM1D. Our data link PPM1D gain-of-function mutations to the clonal expansion of HSCs, inform human genetic observations, and support the therapeutic targeting of PPM1D in cancer.
Topics: Adult; Humans; Animals; Mice; Tumor Suppressor Protein p53; Protein Phosphatase 2C; DNA Damage; Mutation; Phosphoric Monoester Hydrolases; Cell Cycle
PubMed: 37595362
DOI: 10.1182/blood.2023020331 -
Cell Death and Differentiation Sep 2023Impaired transcription factor EB (TFEB) function and deficient autophagy activity have been shown to aggravate intervertebral disc (IVD) degeneration (IDD), yet the...
Impaired transcription factor EB (TFEB) function and deficient autophagy activity have been shown to aggravate intervertebral disc (IVD) degeneration (IDD), yet the underlying mechanisms remain less clear. Protein posttranslational modifications (PTMs) are critical for determining TFEB trafficking and transcriptional activity. Here, we demonstrate that TFEB activity is controlled by protein methylation in degenerated nucleus pulposus cells (NPCs), even though TFEB itself is incapable of undergoing methylation. Specifically, protein phosphatase 1 catalytic subunit alpha (PPP1CA), newly identified to dephosphorylate TFEB, contains a K141 mono-methylated site. In degenerated NPCs, increased K141-methylation of PPP1CA disrupts its interaction with TEFB and subsequently blocks TEFB dephosphorylation and nuclear translocation, which eventually leads to autophagy deficiency and NPC senescence. In addition, we found that the PPP1CA-mediated targeting of TFEB is facilitated by the protein phosphatase 1 regulatory subunit 9B (PPP1R9B), which binds with PPP1CA and is also manipulated by K141 methylation. Further proteomic analysis revealed that the protein lysine methyltransferase suppressor of variegation 3-9 homologue 2 (SUV39H2) is responsible for the K141 mono-methylation of PPP1CA. Targeting SUV39H2 effectively mitigates NPC senescence and IDD progression, providing a potential therapeutic strategy for IDD intervention.
Topics: Humans; Methylation; Lysine; Intervertebral Disc Degeneration; Protein Phosphatase 1; Proteomics; Autophagy; Histone-Lysine N-Methyltransferase; Protein Processing, Post-Translational; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
PubMed: 37605006
DOI: 10.1038/s41418-023-01210-4 -
Clinical and Experimental Hypertension... Dec 2023As a common and frequently occurring disease, heart failure has been paid more and more attention, but the mechanism of its occurrence and development is still unclear....
OBJECTIVES
As a common and frequently occurring disease, heart failure has been paid more and more attention, but the mechanism of its occurrence and development is still unclear. This study investigated that PGAM5 expression levels in heart failure and its underlying mechanisms in vivo and in vitro.
METHODS
The inhibition of PGAM5 mRNA expression levels in patients with heart failure was compared with the normal group.
RESULTS
The serum of PGAM5 mRNA expression was negative correlation with collagen I and collagen III in patients with heart failure. PGAM5 mRNA and protein expression in the heart tissue of mice with heart failure were down-regulated at a time-dependent rate. The inhibition of PGAM5 presented heart failure in the model. PGAM5 reduced inflammation and inhibited ROS-induced oxidative stress in models of heart failure. PGAM5 reduced Ferroptosis in models of heart failure. PGAM5 regulated Keap1/Nrf2 signaling pathway. IP also showed that PGAM5 protein combined with the Keap1 protein. PGAM5 could increase Keap1 protein ubiquitination. Keap1 inhibition affected the effects of PGAM5 in model of heart failure.
CONCLUSIONS
We conclude that the protection of PGAM5 reduced ROS-induced oxidative stress and ferroptosis by the Keap1/Nrf2 signaling pathway in heart failure, suggesting that targeting this mechanism of PGAM5 may be a feasible strategy to treat heart failure.
Topics: Mice; Animals; Kelch-Like ECH-Associated Protein 1; Reactive Oxygen Species; NF-E2-Related Factor 2; Ferroptosis; Oxidative Stress; Heart Failure; Phosphoprotein Phosphatases
PubMed: 36780919
DOI: 10.1080/10641963.2022.2162537 -
Biochimica Et Biophysica Acta. Reviews... Sep 2023Protein phosphatase 2A (PP2A) inactivation is common in cancer, leading to sustained activation of pro-survival and growth-promoting pathways. PP2A consists of a... (Review)
Review
Protein phosphatase 2A (PP2A) inactivation is common in cancer, leading to sustained activation of pro-survival and growth-promoting pathways. PP2A consists of a scaffolding A-subunit, a catalytic C-subunit, and a regulatory B-subunit. The functional complexity of PP2A holoenzymes arises mainly through the vast repertoire of regulatory B-subunits, which determine both their substrate specificity and their subcellular localization. Therefore, a major challenge for developing more effective therapeutic strategies for cancer is to identify the specific PP2A complexes to be targeted. Of note, the development of small molecules specifically directed at PP2A-B56α has opened new therapeutic avenues in both solid and hematological tumors. Here, we focus on the B56/PR61 family of PP2A regulatory subunits, which have a central role in directing PP2A tumor suppressor activity. We provide an overview of the mechanisms controlling the formation and regulation of these complexes, the pathways they control, and the mechanisms underlying their deregulation in cancer.
Topics: Humans; Protein Phosphatase 2; Protein Processing, Post-Translational; Catalytic Domain; Neoplasms; Holoenzymes
PubMed: 37437699
DOI: 10.1016/j.bbcan.2023.188953 -
MBio Oct 2023Pyrin, a unique cytosolic receptor, initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors like YopE and YopT. Understanding pyrin...
Pyrin, a unique cytosolic receptor, initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors like YopE and YopT. Understanding pyrin regulation is crucial due to its association with dysregulated inflammatory responses, including Familial Mediterranean Fever (FMF), linked to pyrin gene mutations. FMF mutations historically acted as a defense mechanism against plague. Negative regulation of pyrin through PKN phosphorylation is well established, with using the YopM effector to promote pyrin phosphorylation and counteract its activity. This study highlights the importance of phosphoprotein phosphatase activity in positively regulating pyrin inflammasome assembly in phagocytic cells of humans and mice. Oligomeric murine pyrin has S205 phosphorylated before inflammasome assembly, and this study implicates the dephosphorylation of murine pyrin S205 by two catalytic subunits of PP2A in macrophages. These findings offer insights for investigating the regulation of oligomeric pyrin and the balance of kinase and phosphatase activity in pyrin-associated infectious and autoinflammatory diseases.
Topics: Humans; Animals; Mice; Inflammasomes; Pyrin; Protein Processing, Post-Translational; Macrophages; Phosphoprotein Phosphatases; Mutation
PubMed: 37787552
DOI: 10.1128/mbio.02066-23