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: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.
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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.
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2024-07-12 | PXD043670 | Pride
Project description:Lipid droplets (LDs) are dynamic organelles mediating lipid metabolism and diverse cellular processes. However, the interplay between hepatocyte LDs and hepatitis E remains poorly understood. Using targeted lipidomics and lipid profiling, we reveal in cellular and rodent models that hepatitis E virus (HEV) infection substantially increases hepatocyte LD biogenesis. Mechanistically, HEV pORF3 is a key LD biogenesis inducer and an essential factor for viral infectivity in vivo. pORF3 formed a unique LD organelle through its liquid-liquid phase-separation (LLPS) property, associating with enhancing cholesterol anabolic pathways, thereby facilitating the synthesis of triglycerides and cholesterol esters. Accordingly, deleting ORF3 or inhibiting LD biogenesis with LD-lowering agent atorvastatin substantially suppressed HEV infection in vivo. These findings position LDs as critical hubs for HEV infection, reveal lipid biogenesis as a crucial function of HEV infectivity, and suggest alternative strategies for HEV intervention.
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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.
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Project description:Lipid droplets (LDs) are subcellular organelles found in all kingdoms of life. While LDs are well known as intracellular depots for neutral lipid storage, there is a growing appreciation that they are much more dynamic in terms of their functions. For instance, recent work in yeast and mammalian systems have revealed that LDs are involved in stress response, protein sequestration, and development, and that these roles are mediated by distinct proteins located on the LD surface. However, few yeast and mammalian LD proteins have obvious homologs in plants. Indeed, relatively few plant LD proteins have been characterized, limiting our overall understanding of the molecular mechanisms underlying LD biogenesis, maintenance, and turnover in plant cells. To address this, we have performed proteomic surveys of LDs isolated from various Arabidopsis tissues, and here we discuss the characterization of one such newly identified LD protein, LIPID DROPLET PROTEIN OF SEEDS (LDPS). LDPS in Arabidopsis is annotated to be of unknown function and is expressed exclusively in developing and mature seeds, as well as in young seedlings. Notably, mature ldps mutant seeds have smaller LDs and less storage oil relative to wild-type seeds, while ectopic overexpression of LDPS in leaves leads to an increase in LD number. Furthermore, following germination, LDs in ldps mutant seedlings do not appear to fuse during their turnover, as they do in wild-type seedlings. Protein-protein interaction assays combined with protein co-expression experiments suggest that LDPS interacts with other known LD proteins, in particular, oleosins. Taken together, these findings and those from proteomics and lipidomics analyses of ldps mutant seeds indicate that LDPS plays a key role in LD biogenesis and regulating LD size and number in plant seeds.
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Project description:Emerging evidence indicates that lipid droplets (LDs) play important roles in lipid metabolism, energy homeostasis, and cell stress management. Notably, dysregulation of LDs is tightly linked to numerous diseases, including lipodystrophies, cancer, obesity, atherosclerosis and others. The pivotal physiological roles of LDs have led to an exploration of research in recent years. The functions of LDs are inherently connected to the composition of their proteome. Current methods for profiling LD proteins mostly utilize LD fractionation including those based on proximity-based labeling techniques. Glob-al profiling of the LD proteome in live cells without isolation of LDs is still challenging. Herein, we disclose two small-molecule chemical probes, termed LDF and LDPL. Both LDF/LDPL are small in size, could freely and specifically migrate within the lipid context of LDs. Consequently, they were successfully used for live-cell fluorescence imaging of LDs and from animal tissues. We further showed that LDPL was capable of large-scale profiling of LD proteome without the need of LD isolation. By using LDPL, 1549 high-confidence proteins, most of which could be annotated to prominent LD func-tions, were next identified. Importantly, further validation studies by using representative “hit” proteins revealed that CHMP6 and PRDX4 could act as the lipophagy receptor and lipolysis suppressor, respectively. Our results thus confirmed for the first time that LDPL is a powerful chemical tool for in situ profiling of LD proteomes. With the ability to provide a deeper understanding of LD proteomics from the native cellular environments, our newly developed strategy may be used in future to decipher the dynamics and molecular mechanism of LDs in various diseases.
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