Showing papers by "Robert V. Farese published in 2018"
TL;DR: Surprisingly, amphipathic helices with large hydrophobic residues from many different proteins are capable of binding toLDs, which suggests that LD protein composition is additionally determined by mechanisms that selectively prevent proteins from binding LDs, such as macromolecular crowding at the LD surface.
155 citations
TL;DR: A structure and functional testing suggest a model in which seipin oligomers initially detect forming LDs in the ER via HHs and subsequently act as membrane anchors to enable lipid transfer and LD growth.
Abstract: Metabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases The formation of monolayer-bound LDs from the endoplasmic reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process In this study, we report a cryo-electron microscopy structure and functional characterization of Drosophila melanogaster seipin The structure reveals a ring-shaped dodecamer with the luminal domain of each monomer resolved at ∼40 A Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix (HH) positioned toward the ER bilayer and a β-sandwich domain with structural similarity to lipid-binding proteins This structure and our functional testing in cells suggest a model in which seipin oligomers initially detect forming LDs in the ER via HHs and subsequently act as membrane anchors to enable lipid transfer and LD growth
138 citations
01 Dec 2018
TL;DR: In this paper, the authors investigated how alterations in α-synuclein or lipid/fatty acid homeostasis affect each other, and found that excess oleic acid (OA, 18:1) caused αS inclusion formation, which was reversed by SCD inhibition.
Abstract: Summary In Parkinson’s disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.
60 citations
TL;DR: A knockin mouse model with a Grn mutation corresponding to the most prevalent human GRN mutation, a premature stop codon at arginine 493, is generated, establishing a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation.
Abstract: Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation.
46 citations
TL;DR: It is concluded that Rab18 is not a general, necessary component of the protein machinery involved in LD biogenesis or turnover, or the targeting of several proteins to LDs.
Abstract: Rab GTPases recruit peripheral membrane proteins and can define organelle identity. Rab18 localizes to the endoplasmic reticulum (ER) but also to lipid droplets (LDs), where it has been implicated in effector protein recruitment and in defining LD identity. Here, we studied Rab18 localization and function in a human mammary carcinoma cell line. Rab18 localized to the ER and to LD membranes on LD induction, with the latter depending on the Rab18 activation state. In cells lacking Rab18, LDs were modestly reduced in size and numbers, but we found little evidence for Rab18 function in LD formation, LD turnover on cell starvation, or the targeting of several proteins to LDs. We conclude that Rab18 is not a general, necessary component of the protein machinery involved in LD biogenesis or turnover.
33 citations
TL;DR: The genetic inactivation of DDHD2, as caused by HSP-associated mutations, substantially perturbs lipid homeostasis and the formation and content of LDs, underscoring the importance of triglyceride metabolism for normal CNS function and the key role thatDDHD2 plays in this process.
Abstract: Deleterious mutations in the serine lipase DDHD2 are a causative basis of complex hereditary spastic paraplegia (HSP, subtype SPG54) in humans. We recently found that DDHD2 is a principal triglyceride hydrolase in the central nervous system (CNS) and that genetic deletion of this enzyme in mice leads to ectopic lipid droplet (LD) accumulation in neurons throughout the brain. Nonetheless, how HSP-related mutations in DDHD2 relate to triglyceride metabolism and LD formation remains poorly understood. Here, we have characterized a set of HSP-related mutations in DDHD2 and found that they disrupt triglyceride hydrolase activity in vitro and impair the capacity of DDHD2 to protect cells from LD accumulation following exposure to free fatty acid, an outcome that was also observed with a DDHD2-selective inhibitor. We furthermore isolated and characterized LDs from brain tissue of DDHD2–/– mice, revealing that they contain both established LD-associated proteins identified previously in other organs and CNS-enric...
32 citations
TL;DR: In insulin-resistant mice, inhibition of hepatic PKC-λ/ι sufficient to correct hepatic abnormalities and hyperinsulinemia simultaneously reversed increases in Akt, atypical protein kinase C (aPKC), β-secretase, and Aβ1-40/42, and restored acute Akt activation.
18 citations
TL;DR: In high-fat-fed mice, inhibition of hepatic aPKC, not only restored Akt association with WD40/ProF and FoxO1/PGC-1α phosphorylation, but also diminished expression of SREBP-1c, PGC-1 α, PKC-ι and gluconeogenic and lipogenic enzymes, and corrected glucose intolerance and hyperlipidemia.
18 citations
TL;DR: The phosphoproteome of insulin-treated human hepatoma cells was analyzed and it was shown that palmitate interferes with insulin signaling to FoxO1, a key transcription factor regulating gluconeogenesis, and alteredFoxO1 cellular compartmentalization as a contributing mechanism for selective insulin resistance.
15 citations
TL;DR: It is concluded that hematopoietic progranulin deficiency promotes diet-induced atherosclerosis in Ldlr-/- mice, possibly due to increased exophagy-mediated cholesterol uptake.
14 citations
TL;DR: It is shown that FIT2 is a lipid phosphate phosphatase (LPP) enzyme that is required for maintaining the normal structure of the ER and uncovered an enzymatic role for FIT1 in ER lipid metabolism that is crucial for ER membrane homeostasis.
Abstract: The endoplasmic reticulum (ER) protein Fat-Induced Transcript 2 (FIT2) has emerged as a key factor in lipid droplet (LD) formation, although its molecular function is unknown. Highlighting its importance, FIT2 orthologs are essential in worms and mice, and FIT2 deficiency causes a deafness/dystonia syndrome in humans. Here we show that FIT2 is a lipid phosphate phosphatase (LPP) enzyme that is required for maintaining the normal structure of the ER. Recombinant FIT2 exhibits LPP activity in vitro and loss of this activity in cells leads to ER membrane morphological changes and ER stress. Defects in LD formation in FIT2 depletion appear to be secondary to membrane lipid abnormalities, possibly due to alterations in phospholipids required for coating forming LDs. Our findings uncover an enzymatic role for FIT2 in ER lipid metabolism that is crucial for ER membrane homeostasis.
20 Dec 2018
TL;DR: A model in which seipin oligomers initially detect forming LDs in the ER via hydrophobic helices and subsequently act as membrane anchors to enable lipid transfer and LD growth is suggested.
Abstract: Metabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases. The formation of monolayer bound LDs from the endoplasmic reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process. Here, we report a cryo-electron microscopy structure and functional characterization of D. melanogaster seipin. The structure reveals a ringshaped dodecamer, with the luminal domain of each monomer resolved at ~4.0 angstrom. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix positioned towards the ER bilayer, and a b-sandwich domain that has structural similarity with lipidbinding proteins. This structure, and our functional testing in cells, suggest a model in which seipin oligomers initially detect forming LDs in the ER via hydrophobic helices and subsequently act as membrane anchors to enable lipid transfer and LD growth.