Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27 −/− mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27 −/− mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27 −/− mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27 −/− and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27 −/− mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1α were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27 −/− mice. Remarkably, Fsp27 −/− MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.
The endoplasmic reticulum (ER)4 protein Scap is unique in nature because it serves as a cholesterol sensor that ensures the proper amount of cholesterol in membranes of animal cells (1, 2). The function of Scap derives from its ability to mediate the regulated transport of sterol regulatory element-binding proteins (SREBPs) from ER to Golgi. SREBPs are a family of three transcription factors that activate all of the genes necessary to produce cholesterol, fatty acids, and triglycerides (3). The SREBPs are synthesized as intrinsic transmembrane proteins of the ER. Immediately after their synthesis, the SREBPs bind to Scap, which serves as the nidus for incorporation into COPIIcoated vesicles, which bud from the ER and travel to the Golgi. There the SREBPs are processed sequentially by two proteases, thereby releasing the active transcriptional fragments that travel to the nucleus. When cholesterol accumulates in ER membranes, Scap binds the cholesterol and undergoes a conformational change that causes it to bind to Insig, an ER-resident protein (4). As a result of the conformational change and its stabilization by Insig (5), the Scap-SREBP complex is no longer incorporated into budding vesicles, and the active fragment cannot reach the nucleus. As a result, synthesis of cholesterol and fatty acids declines.The 1276 amino acids of Scap can be divided into two functional regions (see Fig. 1). The COOH-terminal domain of ϳ540 amino acids extends into the cytosol. It contains at least four WD repeat sequences that mediate its binding to SREBPs. The NH 2 -terminal region of ϳ735 amino acids is the membrane attachment domain. It contains eight ␣-helices separated by hydrophilic loops (6). Three of the loops (Loops 1, 6, and 7) are long enough to have significant structure. Helices 2-6 contain the Insig binding site (7,8). Loop 6, which faces the cytosol, contains the hexapeptide sequence MELADL, which serves as the binding site for the COPII proteins that cluster the Scap-SREBP complex into COPII-coated vesicles that bud from ER membranes (2, 9). When the cholesterol content of ER membranes exceeds a sharp threshold of 4 -5% of total lipids, the cholesterol binds to the membrane region of Scap (10), and this elicits a conformational change in Loop 6 that can be monitored by a protease protection assay (11). The change is reflected by the exposure of a novel arginine (Arg 505 ) to cleavage by trypsin (Fig. 1).The cholesterol-induced conformational change in Loop 6 causes the MELADL sequence to become inaccessible to COPII proteins, thereby precluding transport to the Golgi (2). Although the conformational change does not require Insig, binding to Insig stabilizes the inactive conformation, thereby lowering the threshold for cholesterol (10).Our previous cholesterol-binding studies were performed with a recombinant form of Scap that contained the entire membrane attachment domain (TM1-8) (1,12). Within this domain, the precise site of cholesterol binding was not established. In the current study, we localize the choleste...
Cholesterol homeostasis is mediated by Scap, a polytopic endoplasmic reticulum (ER) protein that transports sterol regulatory element-binding proteins from the ER to Golgi, where they are processed to forms that activate cholesterol synthesis. Scap has eight transmembrane helices and two large luminal loops, designated Loop1 and Loop7. We earlier provided indirect evidence that Loop1 binds to Loop7, allowing Scap to bind COPII proteins for transport in coated vesicles. When ER cholesterol rises, it binds to Loop1. We hypothesized that this causes dissociation from Loop7, abrogating COPII binding. Here we demonstrate direct binding of the two loops when expressed as isolated fragments or as a fusion protein. Expressed alone, Loop1 remained intracellular and membrane-bound. When Loop7 was co-expressed, it bound to Loop1, and the soluble complex was secreted. A Loop1-Loop7 fusion protein was also secreted, and the two loops remained bound when the linker between them was cleaved by a protease. Point mutations that disrupt the Loop1-Loop7 interaction prevented secretion of the Loop1-Loop7 fusion protein. These data provide direct documentation of intramolecular Loop1-Loop7 binding, a central event in cholesterol homeostasis.
Background: Scap controls cholesterol by transporting SREBPs from ER to Golgi in a sterol-sensitive fashion. Two luminal loops of Scap (Loops 1 and 7) may play a regulatory role. Results: A point mutation in Scap Loop 7 prevents interaction with Loop 1 and abolishes ER exit. Conclusion: Scap movement and activation of SREBPs require interaction between Loops 1 and 7. Significance: This work provides insight into how cells control cholesterol.
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