UBXD8 lipidomics from whole cells (Part 2)
Ontology highlight
ABSTRACT:
ORGANISM(S): Human Homo Sapiens
TISSUE(S): Cultured Cells
SUBMITTER: 
John Purdy
PROVIDER: ST002422 | MetabolomicsWorkbench | Sun Jan 01 00:00:00 GMT 2023
REPOSITORIES: MetabolomicsWorkbench
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Project description:The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we investigate if the ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulates the lipidome of cells. LC-MS/MS lipidomics found significant changes in distinct lipid species in UBXD8 knockout cells, in particular in saturated or mono-unsaturated lipid species. Perturbation of contacts and inherent lipid synthesis is emerging as a hallmark in a variety of human disorders such as neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a manner that is dependent on UBXD8.
2022-12-30 | ST002421 | MetabolomicsWorkbench
Project description:The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites (ERMCS) serves as a platform for several critical cellular processes, particularly lipid synthesis. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we show that the p97 AAA-ATPase and its ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulate the prevalence of ERMCS. The p97-UBXD8 complex localizes to contacts and its loss increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics and lipidomics of ERMCS demonstrates alterations in proteins regulating lipid metabolism and a significant change in saturated or monounsaturated lipid species in UBXD8 knockout cells. We show that loss of p97-UBXD8 results in perturbed contacts due to an increase in membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. These aberrant contacts can be rescued by supplementation with unsaturated fatty acids or overexpression of SCD1. Notably, we find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a p97-UBXD8 dependent manner.
2023-01-25 | PXD039061 | Pride
Project description:The distinct activities of cellular organelles are dependent on the proper function of their membranes. Coordinated membrane biogenesis of the different organelles necessitates interorganelle transport of lipids from their site of synthesis to their destination membranes. Several proteins and trafficking pathways have been proposed to participate in lipid distribution, but despite the basic importance of this process, in vivo evidence linking the absence of putative transport pathways to specific transport defects remains scarce. An obvious reason for this scarcity is the near absence of in vivo lipid trafficking assays. Here we introduce a versatile method named METALIC (Mass tagging-Enabled TrAcking of Lipids In Cells) to track interorganelle lipid flux inside living cells. In this strategy, two enzymes, one directed to a “donor” and the other to an “acceptor” organelle, add two distinct mass tags to lipids. Mass spectrometry-based detection of lipids bearing the two mass tags is then used as a proxy for lipid exchange between the two organelles. By applying this approach to ER and mitochondria, we show that the ERMES and Vps13-Mcp1 complexes have lipid transport activity in vivo, and unravel their relative contributions to ER-mitochondria lipid exchange.
2022-03-22 | MTBLS3415 | MetaboLights
Project description:Organelles such as endoplasmic reticulum (ER) and mitochondria interact with each other at specialized domains on the ER known as mitochondria-associated membranes (MAMs). Here, using three-dimensional high-resolution imaging techniques, we show that the Sel1LHrd1 protein complex, the most conserved branch of ER-associated protein degradation (ERAD), exerts a profound impact on ER-mitochondria contacts and mitochondrial dynamics, at least in part, by regulating the turnover and hence the abundance of the MAM protein sigma receptor 1 (SigmaR1). Sel1L or Hrd1 deficiency in brown adipocytes impairs dynamic interaction between ER and mitochondria, leading to the formation of pleomorphic “megamitochondria” and, in some cases with penetrating ER tubule(s), in response to acute cold challenge. Mice with ERAD deficiency are cold sensitive and exhibit mitochondrial dysfunction in brown adipocytes. Mechanistically, endogenous SigmaR1 is targeted for proteasomal degradation by Sel1L-Hrd1 ERAD, whose accumulation in ERAD-deficient cells leads to mitofusin 2 (Mfn2) oligomerization, thereby linking ERAD to mitochondrial dynamics. Our study identifies Sel1L-Hrd1 ERAD as a critical determinant of ER-mitochondria contacts, thereby regulating mitochondrial dynamics and thermogenesis.
2020-04-24 | GSE145895 | GEO
Project description:Mitochondria-Rough-ER Contacts in The Liver Regulate Systemic Lipid Homeostasis
| PRJNA556401 | ENA
Project description:Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remained insufficiently characterized. Here we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers a dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.
2024-03-05 | PXD048553 | Pride
Project description:SEPN1 is a type II protein of the endoplasmic reticulum (ER) whose loss of function gives rise to a collection of debilitating autosomal recessive myopathies gathered under the umbrella term of SEPN1-related myopathy (RM). At the moment, SEPN1-RM lacks an effective pharmacological treatment; thus, the medical management of the disease is only supportive. The potentially fatal diaphragmatic weakness leading to respiratory insufficiency in patients still ambulant is the main reason for medical concerns. Thus, studies on SEPN1-RM pathogenesis are necessary to implement a targeted pharmacological therapy aimed at relieving the general muscle weakness and the more worrisome diaphragmatic dysfunction. Here, we show a physical and functional interaction between SEPN1 and the ER stress mediator ERO1 alpha (henceforth, ERO1). Both SEPN1 and ERO1 are involved in the redox regulation of proteins into the ER, although in an opposite way SEPN1 imposes a less oxidant ER poise while ERO1 a more oxidant one, conceivable with a reductase function of SEPN1 and an oxidase one of ERO1. Furthermore, both are mainly localized in a region of the ER in close contact with mitochondria termed mitochondria-associated membranes (MAMs) and their loss impacts oppositely on the short-range MAMs, thereby impinging ER-mitochondria Ca2+ dynamics, OXPHOS, and bioenergetics. We find that ERO1 depletion restores the impaired short-range MAMs due to SEPN1 loss together with mitochondrial ATP. ERO1 knockout in a mouse background of SEPN1 loss blunts ER stress and the consequent Unfolded Protein Response (UPR) while it rescues the diaphragmatic weakness by improving ER/mitochondria contacts, calcium dynamics, and OXPHOS. Importantly, treatments of SEPN1 knock out mice with the chemical chaperone tauroursodeoxycholic acid (TUDCA) mimic the results of ERO1 loss improving calcium dynamics, OXPHOS, ER/mitochondria contacts, thereby rescuing diaphragmatic weakness as well. In addition, TUDCA-treated SEPN1-RM patients-derived myoblasts show a dynamic dose-dependent increase in ATP levels under ER stress conditions suggesting improved mitochondrial function. Thus, our findings point to the ERO1 axis in the pathogenesis of SEPN1-RM thereby impinging on MAMs and mitochondria bioenergetics and recall for the efficacy of a pharmacology therapy with ad hoc ER stress/ERO1 inhibitors for SEPN1-RM.
2024-02-14 | E-MTAB-12460 | biostudies-arrayexpress
Project description:Digested dietary fats are taken up, processed and transported by enterocytes to supply the body with lipids. Most absorbed lipids are assembled into pre-chylomicrons in the endoplasmic reticulum (ER) of enterocytes, which are then transported to the Golgi for maturation and subsequent secretion to the circulation. The role of mitochondria in regulating intestinal lipid transport remains unknown. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with intestinal epithelial cell (IEC)-specific ablation of the mitochondrial-specific aspartyl - tRNA synthetase DARS2, as well as of the respiratory chain subunit SDHA or the assembly factor COX10 failed to thrive and showed massive accumulation of lipids within large lipid droplets (LDs) in enterocytes of the proximal small intestine (SI). Feeding a fat-free diet inhibited the formation of LDs in DARS2-deficient enterocytes, showing that accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in IECs. Moreover, DARS2-deficient enterocytes showed a distinct lack of mature chylomicrons concomitant with a disorganisation of the Golgi apparatus, suggesting that impaired ER to Golgi trafficking underlies impaired chylomicron production and secretion. Taken together, these results revealed a vital role of mitochondria in regulating dietary lipid transport in enterocytes, which is relevant for understanding the intestinal and nutritional defects observed in patients with mitochondrial defects.
2023-12-21 | PXD026934 | Pride
Project description:Contacts between organelles create microdomains that play major roles in regulating key intracellular activities and signaling pathways, but whether they also regulate systemic functions remains unknown. Here, we report the ultrastructural organization and dynamics of the inter-organellar contact established by rough-Endoplasmic Reticulum closely wrapped around the mitochondria (wrappER). To elucidate the in vivo function of this contact, mouse liver fractions enriched in wrappER-associated-mitochondria are analyzed by transcriptomics, proteomics and lipidomics. The biochemical signature of the wrappER points to a role in the biogenesis of very-low-density lipoproteins (VLDL). Altering wrappER-mitochondria contacts curtails VLDL secretion and increases hepatic fatty acids, lipid droplets and neutral lipid content. Conversely, acute liver-specific ablation of Mttp, the most upstream regulator of VLDL biogenesis, recapitulates this hepatic dyslipidemia phenotype and promotes remodelling of the wrappER-mitochondria contact. The discovery that liver wrappER-mitochondria contacts participate in VLDL biology suggests an involvement of inter-organelle contacts in systemic lipid homeostasis.
2021-03-15 | GSE134777 | GEO
Project description:Lipids comprise 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty-acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as novel therapeutics for MS. Fig. 2A. Mini-Array I: IgG antibody reactivity to various glycero-3-phosphocholine lipids in CSF samples from patients with relapsing remitting MS and from control patients with other neurological disease. Lipid hits with the lowest FDR (q=0.029) were clustered according to their reactivity profiles. 47 different lipids were custom-spotted in duplicate using the CAMAG Automatic TLC Sampler (ATS4) robot to spray 200 nl of 10 to 100 pmol of lipids onto PVDF membranes affixed to the surface of microscope slides. The slides were probed with cerebrospinal fluid (CSF) from 24 human patient samples. 25 slides total: 13 relapsing-remitting MS, 11 other neurological disease, and 1 secondary Ab alone (not included in this submission). CSF diluted 1/20. HRP-conjugated secondary Ab (goat anti-human IgM/IgG) diluted 1/175. ECL for 3 minutes.
2012-05-08 | E-GEOD-37670 | biostudies-arrayexpress