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The Journal of Cell Biology Oct 2023In mammalian cells, misfolded glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are cleared out of the ER to the Golgi via a constitutive and a...
In mammalian cells, misfolded glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are cleared out of the ER to the Golgi via a constitutive and a stress-inducible pathway called RESET. From the Golgi, misfolded GPI-APs transiently access the cell surface prior to rapid internalization for lysosomal degradation. What regulates the release of misfolded GPI-APs for RESET during steady-state conditions and how this release is accelerated during ER stress is unknown. Using mutants of prion protein or CD59 as model misfolded GPI-APs, we demonstrate that inducing calnexin degradation or upregulating calnexin-binding glycoprotein expression triggers the release of misfolded GPI-APs for RESET. Conversely, blocking protein synthesis dramatically inhibits the dissociation of misfolded GPI-APs from calnexin and subsequent turnover. We demonstrate an inverse correlation between newly synthesized calnexin substrates and RESET substrates that coimmunoprecipitate with calnexin. These findings implicate competition by newly synthesized substrates for association with calnexin as a key factor in regulating the release of misfolded GPI-APs from calnexin for turnover via the RESET pathway.
Topics: Animals; Calnexin; Cell Membrane; Glycosylphosphatidylinositols; Mammals; Molecular Chaperones; Prions; Endoplasmic Reticulum; Golgi Apparatus; Protein Folding; GPI-Linked Proteins
PubMed: 37702712
DOI: 10.1083/jcb.202108160 -
Cell Communication and Signaling : CCS Oct 2023Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including...
BACKGROUND
Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC).
METHODS
Here, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport.
RESULTS
Following twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus.
CONCLUSION
Overall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs. Video Abstract.
Topics: Mice; Animals; Humans; Sphingomyelins; Sphingomyelin Phosphodiesterase; Annexin A5; Reactive Oxygen Species; Phosphatidylserines; Ceramides; Retinal Ganglion Cells
PubMed: 37904133
DOI: 10.1186/s12964-023-01291-1 -
The EMBO Journal Dec 2023Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the...
Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.
Topics: Calcium; alpha-Synuclein; Plasma Membrane Calcium-Transporting ATPases; Cell Membrane; Adenosine Triphosphatases; Binding Sites
PubMed: 37916890
DOI: 10.15252/embj.2022111122 -
Scientific Reports Nov 2023Nonalcoholic fatty liver disease (NAFLD) is characterized by an increase in hepatic lipid accumulation due to impaired lipid metabolism. Although a correlation was found...
Nonalcoholic fatty liver disease (NAFLD) is characterized by an increase in hepatic lipid accumulation due to impaired lipid metabolism. Although a correlation was found between NAFLD and sphingosine-1-phosphate (S1P), the role of the sphingolipid remains controversial. The aim of this study was to investigate any involvement of S1P in steatosis using its analog FTY720P and HepG2 cells. Lipid accumulation was induced by incubating the cells in a mixture of oleic and palmitic acid, and was quantified using Oil Red O. The involvement of signaling mediators was studied using pharmacological inhibitors and western blot analysis. FTY720P increased lipid accumulation, but this increase wasn't maintained in the presence of inhibitors of S1PR3, Gq, SREBP, mTOR, PI3K, and PPARγ indicating their involvement in the process. The results revealed that FTY720P binds to S1PR3 which activates sequentially Gq, PI3K, and mTOR leading to an increase in SREBP expression and PPARγ activation. It was concluded that in presence of a high level of fatty acids, lipid accumulation is increased in hepatocytes by the exogenously added FTY720P.
Topics: Humans; Hep G2 Cells; Non-alcoholic Fatty Liver Disease; Sterol Regulatory Element Binding Protein 1; PPAR gamma; Liver; TOR Serine-Threonine Kinases; Lipid Metabolism; Lysophospholipids; Phosphatidylinositol 3-Kinases
PubMed: 37953311
DOI: 10.1038/s41598-023-46011-4 -
Proceedings of the National Academy of... Nov 2023() enzymes cleave phosphatidylinositol 4,5-bisphosphate ( producing and (diacylglycerol). modulates the function of many ion channels, while and regulate...
() enzymes cleave phosphatidylinositol 4,5-bisphosphate ( producing and (diacylglycerol). modulates the function of many ion channels, while and regulate intracellular Ca levels and protein phosphorylation by protein kinase C, respectively. enzymes are under the control of G protein coupled receptor signaling through direct interactions with G proteins and and have been shown to be coincidence detectors for dual stimulation of and -coupled receptors. are aqueous-soluble cytoplasmic enzymes but partition onto the membrane surface to access their lipid substrate, complicating their functional and structural characterization. Using newly developed methods, we recently showed that activates by recruiting it to the membrane. Using these same methods, here we show that increases the catalytic rate constant, , of . Since stimulation of by depends on an autoinhibitory element (the X-Y linker), we propose that produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the and complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate activity. The structures rationalize a finding in the enzyme assay, that costimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity of is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation.
Topics: Hydrolysis; Phospholipase C beta; GTP-Binding Proteins; Phosphatidylinositols
PubMed: 37991948
DOI: 10.1073/pnas.2315011120 -
The Journal of Cell Biology Aug 2023As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces...
As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces cerevisiae, closed mitosis allows visualization of NE biogenesis during mitosis. During this period, the SUMO E3 ligase Siz2 binds the inner nuclear membrane (INM) and initiates a wave of INM protein SUMOylation. Here, we show these events increase INM levels of phosphatidic acid (PA), an intermediate of phospholipid biogenesis, and are necessary for normal mitotic NE membrane expansion. The increase in INM PA is driven by the Siz2-mediated inhibition of the PA phosphatase Pah1. During mitosis, this results from the binding of Siz2 to the INM and dissociation of Spo7 and Nem1, a complex required for the activation of Pah1. As cells enter interphase, the process is then reversed by the deSUMOylase Ulp1. This work further establishes a central role for temporally controlled INM SUMOylation in coordinating processes, including membrane expansion, that regulate NE biogenesis during mitosis.
Topics: Cell Nucleus; Mitosis; Nuclear Envelope; Nuclear Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sumoylation; Organelle Biogenesis
PubMed: 37398994
DOI: 10.1083/jcb.202208137 -
Cell Discovery Nov 2023Lipid droplets (LDs) are dynamic lipid storage organelles that can sense and respond to changes in systemic energy balance. The size and number of LDs are controlled by...
Lipid droplets (LDs) are dynamic lipid storage organelles that can sense and respond to changes in systemic energy balance. The size and number of LDs are controlled by complex and delicate mechanisms, among which, whether and which SNARE proteins mediate LD fusion, and the mechanisms governing this process remain poorly understood. Here we identified a SNARE complex, syntaxin 18 (STX18)-SNAP23-SEC22B, that is recruited to LDs to mediate LD fusion. STX18 targets LDs with its transmembrane domain spanning the phospholipid monolayer twice. STX18-SNAP23-SEC22B complex drives LD fusion in adiposome lipid mixing and content mixing in vitro assays. CIDEC/FSP27 directly binds STX18, SEC22B, and SNAP23, and promotes the lipid mixing of SNAREs-reconstituted adiposomes by promoting LD clustering. Knockdown of STX18 in mouse liver via AAV resulted in smaller liver and reduced LD size under high-fat diet conditions. All these results demonstrate a critical role of the SNARE complex STX18-SNAP23-SEC22B in LD fusion.
PubMed: 37989733
DOI: 10.1038/s41421-023-00613-4 -
Frontiers in Bioscience (Landmark... Sep 2023Serine hydroxymethyltransferase () is a serine-glycine-one-carbon metabolic enzyme in which and encode the cytoplasmic and mitochondrial isoenzymes, respectively. and...
BACKGROUND
Serine hydroxymethyltransferase () is a serine-glycine-one-carbon metabolic enzyme in which and encode the cytoplasmic and mitochondrial isoenzymes, respectively. and are key players in cancer metabolic reprogramming, and thus are attractive targets for cancer therapy. However, the role of in patients with renal cell carcinoma (RCC) has not been fully elucidated. We aimed to systematically analyze the expression, gene regulatory network, prognostic value, and target prediction of and in patients with kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), and kidney renal papillary cell carcinoma (KIRP); elucidate the association between expression and RCC; and identify potential new targets for clinical RCC treatment.
METHODS
Several online databases were used for the analysis, including cBioPortal, TRRUST, GeneMANIA, GEPIA, Metascape, UALCAN, LinkedOmics, and TIMER.
RESULTS
and transcript levels were significantly down- and upregulated, respectively, in patients with KICH, KIRC, and KIRP, based on sample type, individual cancer stage, sex, and patient age. Compared to men, women with KIRC and KIRP showed significantly up- and downregulated transcript levels, respectively. However, transcript levels were significantly upregulated in the patients mentioned above. KIRC and KIRP patients with high expression had longer survival periods than those with low expression. In patients with KIRC, the findings were similar to those mentioned above. However, in KICH patients, the findings were the opposite regarding expression. versus were altered by 9% versus 3% (n = 66 KICH patients), 4% versus 4% (n = 446 KIRC patients), and 6% versus 7% (n = 280 KIRP patients). versus promoter methylation levels were significantly up- and downregulated in patients with KIRP versus KIRC and KIRP, respectively. , , and their neighboring genes (NG) formed a complex network of interactions. The molecular functions of and its NG in patients with KICH, KIRC, and KIRP, included clathrin adaptor, metalloendopeptidase, and GTPase regulator activities; lipid binding, active transmembrane transporter activity, and lipid transporter activity; and type I interferon receptor binding, integrin binding, and protein heterodimerization, respectively. Their respective Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were involved in lysosome activity, human immunodeficiency virus 1 infection, and endocytosis; coronavirus disease 2019 and neurodegeneration pathways (multiple diseases); and RIG-I-like receptor signaling pathway, cell cycle, and actin cytoskeleton regulation. The molecular functions of and its NG in patients with KICH, KIRC, and KIRP included cell adhesion molecule binding and phospholipid binding; protein domain-specific binding, enzyme inhibitor activity, and endopeptidase activity; and hormone activity, integrin binding, and protein kinase regulator activity, respectively. For patients with KIRC versus KIRP, the KEGG pathways were involved in cAMP and calcium signaling pathways versus microRNAs (MiRNAs) in cancer cells and neuroactive ligand-receptor interactions, respectively. We identified the key transcription factors of and its NG.
CONCLUSIONS
and expression levels were different in patients with RCC. and may be potential therapeutic and prognostic biomarkers in these patients. Transcription factor (MYC, STAT1, PPARG, AR, SREBF2, and SP3) and miRNA (miR-17-5P, miR-422, miR-492, miR-137, miR-30A-3P, and miR-493) regulations may be important strategies for RCC treatment.
Topics: Male; Humans; Female; Carcinoma, Renal Cell; Glycine Hydroxymethyltransferase; COVID-19; MicroRNAs; Kidney Neoplasms; Integrins; Lipids
PubMed: 37796681
DOI: 10.31083/j.fbl2809196 -
An iPSC-derived astrocyte model of fragile X syndrome exhibits dysregulated cholesterol homeostasis.Communications Biology Jul 2023Cholesterol is an essential membrane structural component and steroid hormone precursor, and is involved in numerous signaling processes. Astrocytes regulate brain...
Cholesterol is an essential membrane structural component and steroid hormone precursor, and is involved in numerous signaling processes. Astrocytes regulate brain cholesterol homeostasis and they supply cholesterol to the needs of neurons. ATP-binding cassette transporter A1 (ABCA1) is the main cholesterol efflux transporter in astrocytes. Here we show dysregulated cholesterol homeostasis in astrocytes generated from human induced pluripotent stem cells (iPSCs) derived from males with fragile X syndrome (FXS), which is the most common cause of inherited intellectual disability. ABCA1 levels are reduced in FXS human and mouse astrocytes when compared with controls. Accumulation of cholesterol associates with increased desmosterol and polyunsaturated phospholipids in the lipidome of FXS mouse astrocytes. Abnormal astrocytic responses to cytokine exposure together with altered anti-inflammatory and cytokine profiles of human FXS astrocyte secretome suggest contribution of inflammatory factors to altered cholesterol homeostasis. Our results demonstrate changes of astrocytic lipid metabolism, which can critically regulate membrane properties and affect cholesterol transport in FXS astrocytes, providing target for therapy in FXS.
Topics: Male; Animals; Mice; Humans; Fragile X Syndrome; Astrocytes; Induced Pluripotent Stem Cells; Lipid Metabolism; Cytokines; Homeostasis
PubMed: 37516746
DOI: 10.1038/s42003-023-05147-9 -
Proceedings of the National Academy of... Aug 2023Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for...
Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation.
Topics: Arabidopsis Proteins; Arabidopsis; NLR Proteins; Plant Immunity; Plants; Receptors, Immunologic; Cell Membrane; Plant Diseases; Carboxylic Ester Hydrolases
PubMed: 37523563
DOI: 10.1073/pnas.2222036120