Lipid droplet membrane proteins ubiquitylation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Rattus Norvegicus (rat)
SUBMITTER: 
Paul Thomes
LAB HEAD: Paul Thomes
PROVIDER: PXD038662 | Pride | 2023-02-27
REPOSITORIES: Pride
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Project description:Lipid droplet (LD) function is regulated by a complement of integral and peripheral proteins that associate with the LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to U2OS and Huh7 cells identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins.
2017-12-22 | PXD007695 | Pride
Project description:Membrane contact sites (MCS) are interorganellar contacts that allow for the direct exchange of molecules such as lipids or Ca2+ between organelles, but can also be a mean for tethering of organelles. In mammals and yeast, LDs have been shown to engage in MCS with nearly all organelles in the cell, while in plants only LD-ER and LD-peroxisome contacts have been characterised. We here analyse three proteins, LD-localised SEED LIPID DROPLET PROTEIN (SLDP) 1 and 2 and PM-localised LIPID DROPLET PLASMA MEMBRANE ADAPTOR. Knockout of SLDP1 and 2, as well as knockout of LIPA lead to aberrant clustering of LDs in seedlings, while ectopic co-expression of SLDP and LIPA in Nicotiana tabacum pollen tubes is sufficient to reconstitute LD-PM tethering in this tissue, where LDs normally dynamically float in the cytosol stream. We propose a model, in which SLDP and LIPA interact and thereby form a tether to anchor a subset of LDs to the PM during post-germinative growth in Arabidopsis thaliana.
2022-06-09 | PXD022769 | Pride
Project description:Multilocular adipocytes are a hallmark of thermogenic adipose tissue, but the factors that enforce this cellular phenotype are unknown. Here, we show that an adipocyte-specific product of the Clstn3 locus (CLSTN3b) present only in placental mammals facilitates the rapid utilization of stored triglyceride by limiting lipid droplet (LD) size. CLSTN3b is an integral ER protein that localizes to ER-LD contact sites via a conserved hairpin domain. Mice lacking CLSTN3b have altered LD morphology and increased lipid accumulation in BAT, as well as heightened sensitivity to cold challenge, despite having no defect in adrenergic signaling. Conversely, forced expression of CLSTN3b promotes a multilocular LD phenotype in cultured cells and BAT and facilitates triglyceride utilization. Mechanistically, CLSTN3b associates with CIDE proteins and impairs their ability to transfer lipid between droplets, thereby limiting LD expansion. These findings define a molecular mechanism that maximizes LD surface area to facilitate lipid utilization in thermogenic adipocytes.
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Project description:Alzheimer’s disease is associated with disrupted circadian rhythms and clock gene expression. REV-ERBα (Nr1d1) is a circadian transcriptional repressor involved in the regulation of lipid metabolism and macrophage function. While global REV-ERBα deletion increases microglial activation and mitigates amyloid plaque formation, the cell-autonomous effects of microglial REV-ERBα on tau pathology are unexplored. Here, we show that microglial REV-ERBα deletion enhances inflammatory signaling, disrupts lipid metabolic processes, and causes lipid droplet (LD) accumulation specifically in male microglia. Inflammation and LD accumulation combine to inhibit microglial tau phagocytosis, which can be partially rescued by blockage of lipid droplet formation. Microglial REV-ERBα deletion exacerbates tau aggregation and neuroinflammation in P301S and AAV-P301L tauopathy models in male, but not female mice. These data demonstrate the importance of microglial lipid droplets in tau accumulation and reveal REV-ERBα as a therapeutically accessible, sex-dependent regulator of microglial inflammatory signaling, lipid metabolism, and tauopathy.
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Project description:Alzheimer’s disease is associated with disrupted circadian rhythms and clock gene expression. REV-ERBα (Nr1d1) is a circadian transcriptional repressor involved in the regulation of lipid metabolism and macrophage function. While global REV-ERBα deletion increases microglial activation and mitigates amyloid plaque formation, the cell-autonomous effects of microglial REV-ERBα deletion in healthy brain and in tauopathy are unexplored. Here, we show that microglial REV-ERBα deficient enhances inflammatory signaling, disrupts lipid metabolism, and causes lipid droplet (LD) accumulation specifically in male microglia. Inflammation and LD accumulation combine to inhibit microglial tau phagocytosis, which can be partially rescued by blockage of lipid droplet formation. Microglial REV-ERBα deletion exacerbates tau aggregation and neuroinflammation in P301S and AAV-P301L tauopathy models in male, but not female mice. These data demonstrate the importance of microglial lipid droplets in tau accumulation and reveal REV-ERBα as a therapeutically accessible, sex-dependent regulator of microglial inflammatory signaling, lipid metabolism, and tauopathy.
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Project description:Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Notably, LD accumulation is prevented, and tumor burden reduced in vivo upon concurrent inhibition of MYC signaling and glutamine metabolism. Furthermore, we identified the hypoxia inducible lipid droplet associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples, postulated HILPDA as a specific ccRCC biomarker. Together, these discoveries provide an attractive approach for development of new therapeutic interventions for the treatment of this type of renal cancer.
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Project description:Accumulating studies have shown that mitochondria-lipid droplet (LD) interaction is crucial for maintaining the homeostasis of lipid metabolism in the liver. However, the proteins involved in mitochondria-LD interaction and the functioning mechanism remain largely elusive. Here, we applied a systematic approach to characterize the proteome of mitochondria-LD contacts under normal or starvation condition by combining the bimolecular fluorescence complementary assay and a proximity labeling strategy. We first established a reporter of mitochondria-LD contacts in HepG2 cells and demonstrated that mitochondria-LD contacts are pretty sensitive to nutritional stresses. Furthermore, we uncovered 60 proteins are up-or down-regulated at contact sites upon starvation. Finally, we validated that SQLE translocates from endoplasmic reticulum to LDs and accumulates at mitochondria-LD contact sites upon starvation so as to utilize local ATP for cholesterol synthesis. This work also provides a versatile tool to profile the proteomes of any other inter-organelle membrane contacts in living cells.
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Project description:Autophagy is a lysosomal degradation pathway that mediates protein and organelle turnover and maintains cellular homeostasis. Autophagosomes transport cargo to lysosomes and their formation is dependent on an appropriate lipid supply. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet (LD) metabolism, resulting in an impaired fatty acid mobilization. The reduced LD-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network, which include increased ATG2B expression and ATG12-ATG5 conjugate formation as well as enhanced ATG2B and ATG9A phosphorylation. Phosphorylation of ATG9A directs this transmembrane protein to the site of autophagosome formation and this particular modification is sufficient to rescue autophagic activity under basal conditions in the absence of RAB18. However, it is incapable of enabling an increased autophagy under inductive conditions. Thus, we illustrate the role of RAB18 in connecting LDs and autophagy, further emphasize the importance of LD-derived lipids for the degradative pathway, and characterize an ATG9A phosphorylation-dependent autophagy rescue mechanism as an adaptive response that maintains autophagy under conditions of reduced LD-derived lipid availability.
2020-01-29 | PXD013719 | Pride
Project description:We describe a method that permits the identification of proteins that are modified by both SUMOylation and ubiquitylation to better understand the role of this crosstalk. The procedure requires 3 days when starting from cell pellets and can yield more than 8000 SUMO sites and 3500 ubiquitin sites from 16 mg of cell extract.
2018-03-27 | PXD006545 | Pride
Project description:The number of known proteins associated with plant lipid droplets (LDs) is small compared to other organelles. Many questions of LD biosynthesis and degradation remain open, also due to lack of candidate LD proteins whose characterization could help to elucidate their function in those processes. We performed a proteomic screen on LDs isolated from Nicotiana tabacum pollen tubes. Proteins that were highly enriched in the LD fraction compared to the total or cytosolic fraction where verified for LD localization via transient expression in tobacco pollen tubes. We also compared the isoforms of typical LD proteins found in the pollen tubes on a qualitative level to the isoforms found in tobacco seeds.
2018-08-13 | PXD009186 | Pride