Cellular energy homeostasis of oxidative tissue relies on a critical balance between fatty acid (FA) uptake from the environment and consumption by mitochondria oxidation. These processes are controlled by complex regulatory mechanisms that ensure energy needs are met while preventing buildup of toxic lipid intermediates and/or oxidized lipids. Transient formation of lipid droplets (LD) may protect mitochondria from lipotoxicity in physiological conditions such as fasting. However, with chronic excess FA levels as observed in obesity, LDs not only cease to be protective but may also contribute to pathology ( 1 ). Distinct features of cardiomyopathy appear in obese and diabetic type 2 patients, including accumulation of lipid droplets and long chain fatty acyl compounds (ceramides, acyl carnitines, and CoAs) in cardiomyocytes, which are Abstract Maintaining cellular lipid homeostasis is crucial to oxidative tissues, and it becomes compromised in obesity. Lipid droplets (LD) play a central role in lipid homeostasis by mediating fatty acid (FA) storage in the form of triglyceride, thereby lowering intracellular levels of lipids that mediate cellular lipotoxicity. LDs and mitochondria have interconnected functions, and anecdotal evidence suggests they physically interact. However, the mechanisms of interaction have not been identifi ed. Perilipins are LD-scaffolding proteins and potential candidates to play a role in their interaction with mitochondria. We examined the contribution of LD perilipin composition to the physical and metabolic interactions between LD and mitochondria using multiple techniques: confocal imaging, electron microscopy (EM), and lipid storage and utilization measurements. Using neonatal cardiomyocytes, reconstituted cell culture models, and rodent heart tissues, we found that perilipin 5 (Plin5) recruits mitochondria to the LD surface through a C-terminal region. Compared with control cells, Plin5-expressing cells show decreased LD hydrolysis, decreased palmitate ␤ -oxidation, and increased palmitate incorporation into triglycerides in basal conditions, whereas in stimulated conditions, LD hydrolysis inhibition is lifted and FA released for ␤ -oxidation. These results suggest that Plin5 regulates oxidative LD hydrolysis and controls local FA fl ux to protect mitochondria against excessive exposure to FA Abbreviations: AA, amino acid; ADFP, adipose differentiationrelated protein; ASM, acid-soluble metabolite; CHO-K1, Chinese hamster ovary; EM, electron microscopy; ER, endoplasmic reticulum; LD, lipid droplet; OCR, oxygen consumption rate; Plin1, perilipin 1, perilipin A; Plin2, perilipin 2, ADFP; Plin5, perilipin 5, Lipid Storage Droplet Protein 5; PPAR ␣ , peroxisome proliferator-activated receptor ␣ ; RNAi, RNA interference; TIP47, tail interacting protein. This work was supported by American Diabetes Association Career Development Award 1-05-CD-17 (C.S.); by National Institutes of Health Grant 1RO1-DK-075017 (C.S.); by American Heart Association Grant-in-Aid 11GRNT7600027 (C.S.); by the Ger...

Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, coimmunoprecipitation, [32 P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [ 32 P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL. Hydrolysis of triacylglycerol (TAG)3 stored in the lipid droplet (LD) compartment provides a convenient source of cellular fuel for energy production during conditions such as fasting. However, lipolysis can contribute to the build up of toxic lipid intermediates and/or oxidized lipids in pathological conditions when cellular lipid homeostasis is altered, e.g. with obesity (1). Therefore, TAG hydrolysis must be carefully controlled to meet tissue-specific requirements for energy or lipid substrates in both adipose and non-adipose tissues. A better understanding of the mechanisms by which cells control lipid mobilization is needed to design novel approaches for intervention and prevention of the pathophysiological consequences of obesity.During the past 10 years, key players in the lipolytic pathway of adipocytes were identified through the study of transgenic mouse models (2-8). Phenotypic analysis of hormone-sensitive lipase null mice suggested the existence of another lipase (7), leading to the identification of adipose triglyceride lipase (ATGL) (6, 9, 10). Characterization of ATGL null mice helped to establish the respective roles of these two lipases in the lipolytic cascade (7). Complete lipolysis requires three enzyme reactions t...

Background:The highly flexible C-terminal region of TDP-43 is implicated in disease pathology. Results: An amyloidogenic core was identified to be critical for TDP-43 aggregation. Conclusion: Helix-to-sheet structural transformation of the amyloidogenic core initiates TDP-43 aggregation and cytoplasmic inclusion formation. Significance: This is a potential therapeutic target for mitigating the TDP-43 proteinopathies.

Many bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how these changes power rotation of the flagellum, remain unknown. Here we present ~3 Å-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational direction.

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The mixed lineage kinase domain-like (MLKL) protein is a key factor in tumor necrosis factor-induced necroptosis. Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane disruption. However, our knowledge of how MLKL functions on membrane remains very limited. Here we demonstrate that MLKL forms cation channels that are permeable preferentially to Mg2+ rather than Ca2+ in the presence of Na+ and K+. Moreover, the N-terminal domain containing six helices (H1-H6) is sufficient to form channels. Using the substituted cysteine accessibility method, we further determine that helix H1, H2, H3, H5 and H6 are transmembrane segments, while H4 is located in the cytoplasm. Finally, MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity. The Mg2+-preferred permeability and five transmembrane segment topology distinguish MLKL from previously identified Mg2+-permeable channels and thus establish MLKL as a novel class of cation channels.

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

DNA polymerase ␤ (␤-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of ␤-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of ␤-pol that seem to interact with a known natural product ␤-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and ␤-pol null mouse fibroblasts, was next ascertained. The most active and ␤-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified ␤-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting.