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Nature Aug 2022Multiple studies have established associations between human gut bacteria and host physiology, but determining the molecular mechanisms underlying these associations has...
Multiple studies have established associations between human gut bacteria and host physiology, but determining the molecular mechanisms underlying these associations has been challenging. Akkermansia muciniphila has been robustly associated with positive systemic effects on host metabolism, favourable outcomes to checkpoint blockade in cancer immunotherapy and homeostatic immunity. Here we report the identification of a lipid from A. muciniphila's cell membrane that recapitulates the immunomodulatory activity of A. muciniphila in cell-based assays. The isolated immunogen, a diacyl phosphatidylethanolamine with two branched chains (a15:0-i15:0 PE), was characterized through both spectroscopic analysis and chemical synthesis. The immunogenic activity of a15:0-i15:0 PE has a highly restricted structure-activity relationship, and its immune signalling requires an unexpected toll-like receptor TLR2-TLR1 heterodimer. Certain features of the phospholipid's activity are worth noting: it is significantly less potent than known natural and synthetic TLR2 agonists; it preferentially induces some inflammatory cytokines but not others; and, at low doses (1% of EC) it resets activation thresholds and responses for immune signalling. Identifying both the molecule and an equipotent synthetic analogue, its non-canonical TLR2-TLR1 signalling pathway, its immunomodulatory selectivity and its low-dose immunoregulatory effects provide a molecular mechanism for a model of A. muciniphila's ability to set immunological tone and its varied roles in health and disease.
Topics: Akkermansia; Cell Membrane; Cytokines; Homeostasis; Humans; Immunity; Inflammation Mediators; Phosphatidylethanolamines; Structure-Activity Relationship; Toll-Like Receptor 1; Toll-Like Receptor 2
PubMed: 35896748
DOI: 10.1038/s41586-022-04985-7 -
Molecular Cell Oct 2022The covalent conjugation of ubiquitin family proteins is a widespread post-translational protein modification. In the ubiquitin family, the ATG8 subfamily is exceptional...
The covalent conjugation of ubiquitin family proteins is a widespread post-translational protein modification. In the ubiquitin family, the ATG8 subfamily is exceptional because it is conjugated mainly to phospholipids. However, it remains unknown whether other ubiquitin family proteins are also conjugated to phospholipids. Here, we report that ubiquitin is conjugated to phospholipids, mainly phosphatidylethanolamine (PE), in yeast and mammalian cells. Ubiquitinated PE (Ub-PE) accumulates at endosomes and the vacuole (or lysosomes), and its level increases during starvation. Ub-PE is also found in baculoviruses. In yeast, PE ubiquitination is catalyzed by the canonical ubiquitin system enzymes Uba1 (E1), Ubc4/5 (E2), and Tul1 (E3) and is reversed by Doa4. Liposomes containing Ub-PE recruit the ESCRT components Vps27-Hse1 and Vps23 in vitro. Ubiquitin-like NEDD8 and ISG15 are also conjugated to phospholipids. These findings suggest that the conjugation to membrane phospholipids is not specific to ATG8 but is a general feature of the ubiquitin family.
Topics: Animals; Endosomal Sorting Complexes Required for Transport; Liposomes; Mammals; Phosphatidylethanolamines; Phospholipids; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination
PubMed: 36044902
DOI: 10.1016/j.molcel.2022.08.008 -
Nature Reviews. Molecular Cell Biology Aug 2023Cellular membranes function as permeability barriers that separate cells from the external environment or partition cells into distinct compartments. These membranes are... (Review)
Review
Cellular membranes function as permeability barriers that separate cells from the external environment or partition cells into distinct compartments. These membranes are lipid bilayers composed of glycerophospholipids, sphingolipids and cholesterol, in which proteins are embedded. Glycerophospholipids and sphingolipids freely move laterally, whereas transverse movement between lipid bilayers is limited. Phospholipids are asymmetrically distributed between membrane leaflets but change their location in biological processes, serving as signalling molecules or enzyme activators. Designated proteins - flippases and scramblases - mediate this lipid movement between the bilayers. Flippases mediate the confined localization of specific phospholipids (phosphatidylserine (PtdSer) and phosphatidylethanolamine) to the cytoplasmic leaflet. Scramblases randomly scramble phospholipids between leaflets and facilitate the exposure of PtdSer on the cell surface, which serves as an important signalling molecule and as an 'eat me' signal for phagocytes. Defects in flippases and scramblases cause various human diseases. We herein review the recent research on the structure of flippases and scramblases and their physiological roles. Although still poorly understood, we address the mechanisms by which they translocate phospholipids between lipid bilayers and how defects cause human diseases.
Topics: Humans; Lipid Bilayers; Phospholipids; Cell Membrane; Glycerophospholipids; Phosphatidylserines
PubMed: 37106071
DOI: 10.1038/s41580-023-00604-z -
Nature Jul 2021T follicular helper (T) cells are crucial for B cell-mediated humoral immunity. Although transcription factors such as BCL6 drive the differentiation of T cells, it is...
T follicular helper (T) cells are crucial for B cell-mediated humoral immunity. Although transcription factors such as BCL6 drive the differentiation of T cells, it is unclear whether and how post-transcriptional and metabolic programs enforce T cell programming. Here we show that the cytidine diphosphate (CDP)-ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of T cells and humoral immunity. Using in vivo CRISPR-Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI-enzymes in the CDP-ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)-as selective post-transcriptional regulators of T cell differentiation that act by promoting the surface expression and functional effects of CXCR5. T cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP-ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a (which mediates the CDP-choline pathway), in activated T cells impairs the differentiation of T cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on B cells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for T cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs.
Topics: Animals; B-Lymphocytes; CRISPR-Cas Systems; Cell Differentiation; Cytidine Diphosphate; Female; Gene Expression Regulation; Humans; Immunity, Humoral; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphatidylethanolamines; Phosphotransferases (Alcohol Group Acceptor); RNA Nucleotidyltransferases; Receptors, CXCR5; Signal Transduction; T-Lymphocytes, Helper-Inducer
PubMed: 34234346
DOI: 10.1038/s41586-021-03692-z -
Molecular Cell May 2021Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8...
Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.
Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagosomes; Autophagy; Autophagy-Related Protein 8 Family; Autophagy-Related Proteins; Cysteine Endopeptidases; Female; HCT116 Cells; HEK293 Cells; HeLa Cells; Humans; Influenza A virus; Macrolides; Male; Mice; Microtubule-Associated Proteins; Monensin; Phagocytosis; Phosphatidylethanolamines; Phosphatidylserines; Protein Processing, Post-Translational; RAW 264.7 Cells; Signal Transduction
PubMed: 33909989
DOI: 10.1016/j.molcel.2021.03.020 -
Acta Pharmaceutica Sinica. B Aug 2023Nonalcoholic steatohepatitis (NASH) is a spectrum of chronic liver disease characterized by hepatic lipid metabolism disorder. Recent reports emphasized the contribution...
Nonalcoholic steatohepatitis (NASH) is a spectrum of chronic liver disease characterized by hepatic lipid metabolism disorder. Recent reports emphasized the contribution of triglyceride and diglyceride accumulation to NASH, while the other lipids associated with the NASH pathogenesis remained unexplored. The specific purpose of our study was to explore a novel pathogenesis and treatment strategy of NASH profiling the metabolic characteristics of lipids. Herein, multi-omics techniques based on LC-Q-TOF/MS, LC-MS/MS and MS imaging were developed and used to screen the action targets related to NASH progress and treatment. A methionine and choline deficient (MCD) diet-induced mouse model of NASH was then constructed, and lignans extract (SLE) was applied to alleviate hepatic damage by regulating the lipid metabolism-related enzymes CES2A and CYP4A14. Hepatic lipidomics indicated that MCD-diet led to aberrant accumulation of phosphatidylethanolamines (PEs), and SLE could significantly reduce the accumulation of intrahepatic PEs. Notably, exogenous PE (18:0/18:1) was proved to significantly aggravate the mitochondrial damage and hepatocyte apoptosis. Supplementing PE (18:0/18:1) also deteriorated the NASH progress by up regulating intrahepatic proinflammatory and fibrotic factors, while PE synthase inhibitor exerted a prominent hepatoprotective role. The current work provides new insights into the relationship between PE metabolism and the pathogenesis of NASH.
PubMed: 37655337
DOI: 10.1016/j.apsb.2023.04.009 -
Drug Design, Development and Therapy 2022Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia...
BACKGROUND
Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury.
METHODS
We generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting.
RESULTS
DHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR).
CONCLUSION
This research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment.
Topics: Animals; Coenzyme A; Ferroptosis; Flavonols; Glucose; Infarction, Middle Cerebral Artery; Iron; Ligases; Lipids; Mammals; Oxygen; Phosphatidylethanolamine Binding Protein; Phospholipid Hydroperoxide Glutathione Peroxidase; Phosphotransferases (Alcohol Group Acceptor); Rats; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; TOR Serine-Threonine Kinases; Water
PubMed: 36118165
DOI: 10.2147/DDDT.S378786 -
Cell Metabolism Mar 2024Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs, and DGAT2 inhibitors are under...
Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs, and DGAT2 inhibitors are under investigation for the treatment of fatty liver disease. Here, we show that DGAT2 inhibition also suppressed SREBP-1 cleavage, reduced fatty acid synthesis, and lowered TG accumulation and secretion from liver. DGAT2 inhibition increased phosphatidylethanolamine (PE) levels in the endoplasmic reticulum (ER) and inhibited SREBP-1 cleavage, while DGAT2 overexpression lowered ER PE concentrations and increased SREBP-1 cleavage in vivo. ER enrichment with PE blocked SREBP-1 cleavage independent of Insigs, which are ER proteins that normally retain SREBPs in the ER. Thus, inhibition of DGAT2 shunted diacylglycerol into phospholipid synthesis, increasing the PE content of the ER, resulting in reduced SREBP-1 cleavage and less hepatic steatosis. This study reveals a new mechanism that regulates SREBP-1 activation and lipogenesis that is independent of sterols and SREBP-2 in liver.
Topics: Animals; Mice; Diacylglycerol O-Acyltransferase; Endoplasmic Reticulum; Liver; Non-alcoholic Fatty Liver Disease; Phosphatidylethanolamines; Sterol Regulatory Element Binding Protein 1; Triglycerides
PubMed: 38340721
DOI: 10.1016/j.cmet.2024.01.011 -
Molecular Cancer Jul 2022Increasing evidence has demonstrated that circular RNAs (circRNAs) are implicated in cancer progression. However, the aberrant expression and biological functions of...
Circular RNA circPOLR2A promotes clear cell renal cell carcinoma progression by facilitating the UBE3C-induced ubiquitination of PEBP1 and, thereby, activating the ERK signaling pathway.
BACKGROUND
Increasing evidence has demonstrated that circular RNAs (circRNAs) are implicated in cancer progression. However, the aberrant expression and biological functions of circRNAs in clear cell renal cell carcinoma (cRCC) remain largely elusive.
METHOD
Differentially expressed circRNAs in cRCC were filtered via bioinformatics analysis. Aberrant circPOLR2A expression was validated in cRCC tissues and cell lines via qRT-PCR. Sanger sequencing was used to identify the backsplicing site of circPOLR2A. In vitro and in vivo functional experiments were performed to evaluate the role of circPOLR2A in cRCC malignancy. RNA pull-down, mass spectrometry, RIP, FISH and immunofluorescence assays were used to identify and validate the circPOLR2A-interacting proteins. Ubiquitination modification and interaction between proteins were detected via Co-IP and western blotting. The m6A modification in circPOLR2A was validated by the meRIP assay.
RESULTS
Bioinformatics analysis revealed that circPOLR2A was highly expressed in cRCC tissues and metastatic cRCC tissues. CircPOLR2A expression was associated with tumor size and TNM stage in cRCC patients. In vitro and in vivo functional assays revealed that circPOLR2A accelerated cRCC cell proliferation, migration, invasion and angiogenesis, while inhibiting apoptosis. Further mechanistic research suggested that circPOLR2A could interact with UBE3C and PEBP1 proteins, and that UBE3C could act as a specific ubiquitin E3 ligase for the PEBP1 protein. The UBE3C/circPOLR2A/PEBP1 protein-RNA ternary complex enhanced the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein which could inactivate the ERK signaling pathway. Rescue experiments revealed that the PEBP1 protein was the functional downstream target of circPOLR2A. Furthermore, m6A modification in circPOLR2A was confirmed, and the m6A reader YTHDF2 could regulate circPOLR2A expression.
CONCLUSION
Our study demonstrated that circPOLR2A modulated the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein, and further activated the ERK pathway during cRCC progression and metastasis. The m6A reader, YTHDF2, regulated circPOLR2A expression in cRCC. Hence, circPOLR2A could be a potential target for the diagnosis and treatment of cRCC.
Topics: Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Humans; Kidney Neoplasms; MAP Kinase Signaling System; Phosphatidylethanolamine Binding Protein; RNA, Circular; Transcription Factors; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 35840930
DOI: 10.1186/s12943-022-01607-8 -
Aging and Disease Jun 2023Locating on endoplasmic reticulum and mitochondria associated membrane, Phosphatidylethanolamine N-methyltransferase (PEMT), catalyzes phosphatidylethanolamine... (Review)
Review
Locating on endoplasmic reticulum and mitochondria associated membrane, Phosphatidylethanolamine N-methyltransferase (PEMT), catalyzes phosphatidylethanolamine methylation to phosphatidylcholine. As the only endogenous pathway for choline biosynthesis in mammals, the dysregulation of PEMT can lead to imbalance of phospholipid metabolism. Dysregulation of phospholipid metabolism in the liver or heart can lead to deposition of toxic lipid species that adversely result in dysfunction of hepatocyte/cardiomyocyte. Studies have shown that PEMT mice increased susceptibility of diet-induced fatty liver and steatohepatitis. However, knockout of PEMT protects against diet-induced atherosclerosis, diet-induced obesity, and insulin resistance. Thus, novel insights to the function of PEMT in various organs should be summarized. Here, we reviewed the structural and functional properties of PEMT, highlighting its role in the pathogenesis of obesity, liver diseases, cardiovascular diseases, and other conditions.
PubMed: 37191416
DOI: 10.14336/AD.2022.1025