Browsing by Subject "Lipid Droplets"
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Item Dissecting the Role of the Lipodystrophy Protein Seipin in the Biogenesis of the Lipid Droplet Organelle(2014-07-14) Cartwright, Bethany Rose; Sternweis, Paul C.; Chook, Yuh Min; Bickel, PerryLong thought to be little more than inert storage depots, lipid droplets have recently become recognized as unique, dynamic, regulated organelles that play an essential role in fat storage. Despite this increased interest, much remains unknown. Lipid droplets have been observed to emerge from the endoplasmic reticulum, but the available models for lipid droplet biogenesis are largely conceptual, with little to no evidence for specific mechanisms of droplet formation. Debate even continues within the field as to whether lipid droplet formation is a spontaneous process, driven by physicochemical and hydrophobic forces, or a regulated process driven by protein factors. The Goodman laboratory previously found evidence to suggest that seipin, mutated in the most severe cases of congenital generalized lipodystrophy, may be a key factor in the early stages of lipid droplet formation. Seipin resides at the junction between lipid droplets and the endoplasmic reticulum, and deletion of seipin results in both a drastic impediment to de novo droplet formation and a striking disorganization of droplet morphology. For my thesis work, I have explored several aspects of seipin’s role at the lipid droplet. I have studied the effects of seipin deletion on protein targeting to abnormal lipid droplets, through which I identified a unique effect of seipin on the regulation of lipase targeting. I have also analyzed the topology of the seipin complex itself through a series of deletion mutants, identifying regions that contribute to the localization, membrane association, and stability of the seipin complex. Furthermore, these studies have led to novel insights on the function of seipin, through the characterization of a remarkable N-terminal seipin mutation that presents with defects in droplet initiation but homogenous droplet morphology. I have therefore concluded that seipin plays two dissectible roles in lipid droplet formation: 1) promoting lipid droplet initiation and 2) regulating subsequent droplet morphology. Finally, I suggest hypotheses on the mechanisms by which seipin exerts these effects, proposing that the N-terminus of seipin may regulate lipin, a mouse lipodystrophy protein, to effect droplet initiation, while the bulk of the protein may serve to regulate the access of phospholipids to the lipid droplet surface.Item Molecular Dissection of Bsc2: A Novel Negative Regulator of Triglyceride Lipolysis for a Lipid Droplet Subpopulation(December 2023) Speer, Natalie Ortiz; Goodman, Joel M.; Henne, W. Mike; Friedman, Jonathan R.; Nicastro, DanielaEukaryotic cells store lipids in the form of triglyceride (TG) and sterol-ester (SE) in cytoplasmic organelles called lipid droplets (LDs). Distinct pools of LDs with unique surface proteomes exist in cells, but a pervasive question is how proteins localize to and convey functions to specific LD subsets. Here, we show the yeast protein Bsc2 localizes to a specific subset of TG-containing LDs, and reveal it negatively regulates TG lipolysis. Mechanistically, Bsc2 LD targeting requires TG, and LD targeting is mediated by specific N-terminal hydrophobic regions (HRs) sufficient for Bsc2 function. Molecular dynamics simulations reveal these Bsc2 HRs interact extensively with TG on modeled LDs, and adopt a specific conformation on TG-rich LDs versus SE-rich LDs or a modeled ER bilayer. Bsc2-deficient yeast display no defect in LD biogenesis, but exhibit enhanced TG lipolysis dependent on the major TG lipase Tgl3. Remarkably, over-expression of Bsc2, but not LD protein Pln1, causes TG accumulation without altering SE levels. Finally, we find that Bsc2-deficient cells display altered LD accumulation during stationary phase growth. We propose that Bsc2 is a novel regulator of TG lipolysis that localizes to a subset of TG-enriched LDs and locally regulates TG lipolysis.Item A Study in SCAR20 Neurologic Disorder Reveals Defective Cellular Lipid Homeostasis(2020-11-23) Datta, Sanchari; Goodman, Joel M.; Henne, W. Mike; Schmid, Sandra; DeBose-Boyd, Russell A.Fatty acids (FAs) are important cellular metabolites that are utilized by the cells to perform important functions such as the generation of ATP, membrane biosynthesis, and cell signaling. Dysregulation in FA processing and storage causes toxic FA accumulation which alters membrane compositions and contributes to metabolic and neurological disorders. Excess lipids are stored as lipid droplets (LDs) which sequester toxic FAs and serve as metabolic buffers to maintain lipid and energy homeostasis. LDs emerge from the endoplasmic reticulum (ER) but how their formation is regulated is not completely understood. Recently, we identified sorting nexin family protein Snx14, implicated in cerebellar ataxia disease SCAR20, as a novel factor that enriches at ER-LD contacts following exogenous FA treatment independently of Seipin and promotes FA-induced LD growth. Loss of Snx14 perturbs LD morphology whereas Snx14 overexpression extends ER-LD contacts and promotes LD biogenesis. Proximity-based APEX2 labeling revealed the enrichment of Snx14 at ER-LD contacts during LD biogenesis. Capitalizing on this APEX technology, we also utilize Snx14-APEX2 localization to dissect the protein composition of ER-LD contact sites. We identify proteins involved in fatty acid activation, desaturation, and triacylglycerol synthesis as being enriched at ER-LD contacts, indicating these contact sites serve as lipogenic sub-domains of the ER network. Furthermore, we identify the major delta-9 FA desaturase SCD1 as a key interacting partner of Snx14. Consistent with this, Snx14-deficient cells are hypersensitive to saturated fatty acid (SFA)-mediated lipotoxic cell death that compromises ER integrity. We show that SCD1 is upregulated in SNX14-KO cells, and Snx14-associated SFA hypersensitivity can be rescued by ectopic SCD1 overexpression. The lipid-associated PXA domain of Snx14 and its interaction with SCD1 are required for Snx14-mediated SFA protection function. Snx14 loss mimics SCD1 inhibition and causes accumulation of free FAs and increased membrane lipid saturation. Altogether these mechanistic insights reveal a role for ER-LD contacts as lipogenic ER sub-domains, and Snx14 as an ER-LD tether with a key role in maintaining cellular FA homeostasis through a functional interaction with SCD1, defects of which may underlie the neuropathology of SCAR20.