Browsing by Subject "Sterol Regulatory Element Binding Proteins"
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Item Alternative Processing of SREBP in Site 2 Protease and Scap Mutants During Larval Development in Drosophila Melanogaster(2009-01-14) Matthews, Krista Ann; Rawson, Robert B.Lipid metabolism is regulated by the membrane-bound transcription factor, sterol regulatory element binding protein (SREBP). SREBP requires release of the amino terminus from the membrane to activate transcription of genes involved in cholesterol and fatty acid synthesis. In response to low sterol levels, Scap escorts SREBP from the ER to the Golgi where it is cleaved by Site-1 and Site-2 proteases. The SREBP pathway is conserved in Drosophila despite these organisms being cholesterol auxotrophs. dSREBP is essential for activating genes involved in the uptake and synthesis of fatty acids which are required for rapid growth during larval development. I have demonstrated that processing of SREBP in Drosophila does not require the S2P or Scap, in contrast to the mammalian system. Flies lacking dS2P are viable and still process dSREBP. dS2P homozygotes were subviable, only emerging at 40% of the expected ratio. This phenotype can be rescued completely by supplementation with fatty acids. dSREBP activity was detected in the fat body of dS2P mutant larvae and to a lesser extent in the oenoctyes. Additionally, SREBP target genes were expressed at higher levels in dS2P homozygotes compared to dSREBP mutants, though less than wild type. dS2P mutants were viable due to alternative cleavage of dSREBP within the juxtamembrane region by the effector caspase, Drice. Flies lacking both dS2P and Drice, or the initiator caspase Dronc, exhibited an early larval lethality that could be rescued by lipid supplementation. Caspase cleavage was dependant upon the aspartic acid at residue 386 in dSREBP. dScap was not essential for larval growth or dSREBP processing in Drosophila. dScap mutants were relatively healthy, emerging at 70% of the expected numbers. dSREBP was actively cleaved in midgut and oenocytes, but significantly reduced in fat body. Levels of dSREBP mRNA and precursor were reduced in larvae lacking dScap, thus demonstrating that Drosophila SREBP is subject to feed-forward activation of its own transcription. Addition of soy lipids suppress dSREBP processing in dScap mutants, but whether this regulation is translational or post-translational is unknown. Furthermore, flies lacking both dScap and dS2P are viable, but survive less well than either single mutant alone. Membrane-bound intermediate dSREBP accumulates in double mutants, suggesting that dSREBP is processed normally by dS1P and dS2P in dScap single mutants. Thus, dScap mutants escape the larval lethality seen in dSREBP mutants due to alternative processing of dSREBP, but through different mechanism than that seen in dS2P mutants.Item Characterization of Drosophila Scap: Analysis of Mutants and Evidence for a Retention Factor(2011-08-10) Özdemir, Cafer; Rawson, Robert B.The SREBP pathway is one of the major regulators of lipid homeostasis and it is highly conserved among metazoans. SREBP is a transcription factor whose precursor is an endoplasmic reticulum (ER) transmembrane protein. In order to be activated it must travel to the Golgi apparatus via interaction with an escort protein, Scap. Scap, in turn can interact with components of the coatamer protein complex II (COPII) when lipid levels fall. In the Golgi, SREBP is cleaved sequentially by two proteases, S1P and S2P. By contrast to mammalian cells, which cannot survive without S2P or Scap, flies lacking Scap or S2P can activate SREBP. These mutants survive owing to non-canonical mechanisms of SREBP activation. Scap has a intrinsic tendency to travel to Golgi. In vertebrates, the ER retention factor, Insig, anchors the Scap:SREBP complex to the ER membrane when de novo lipid synthesis is not required. In Drosophila dSREBP pathway there is no Insig orthologues. However, our data suggest that there should be an analogous component that retains dScap in the ER. In order to discover the putative retention factor and other modifiers of the dSREBP, I set up a high through-put genome-wide screen. Employing luciferase as reporter, knocking-down each gene in genome through RNA interference will reveal the genes that modulate the activity of dSREBP.Item Fatty Acid Auxotrophy in Drosophila Larvae Lacking SREBP(2006-08-11) Kunte, Amit Sudhakar; Brown, Michael S.; Goldstein, Joseph L.A rapid increase in size is a major characteristic of larval development in Drosophila melanogaster. Such growth presumably requires the concomitant production of membrane lipids and is also accompanied by a significant accumulation of neutral lipid stores. Growing larvae must accumulate fatty acids to permit the synthesis of these lipids. Interestingly, wild type Drosophila can grow in the complete absence of exogenous fatty acids. This dissertation reports the finding that a lipogenic transcription factor, dSREBP (Drosophila Sterol Regulatory Element Binding Protein), is essential for the maintenance of this prototrophy. Drosophila larvae lacking dSREBP demonstrate a profound growth deficit in the second instar and die before reaching third instar. This is accompanied by transcriptional deficits in fatty acid synthetic genes. The growth deficit and lethality can be reversed by supplementing the culture medium with fatty acids. The most effective fatty acid, oleate, rescues 80 percent of dSREBP mutants to adulthood. Thus, a lack of dSREBP renders larvae auxotrophic for fatty acids. A reporter system demonstrates that dSREBP is active in tissues known to be involved in lipid metabolism- the fat body, oenocytes and anterior midgut. Finally, as expected of an end-product inhibited metabolic pathway, dSREBP activity can be suppressed by dietary supplementation with lipids. Thus, the dSREBP pathway coordinates endogenous synthesis with the dietary provision of exogenous lipids. These results establish Drosophila as a viable model for the genetic study of the SREBP pathway and provide the first evidence that, at an organismal level, the essential role of the pathway is the accumulation of lipids. The auxotrophic mutants and other reagents described here should be useful tools for further study of the SREBP pathway in particular and fatty acid metabolism in general.Item Insig-Mediated Regulation of Mammalian HMG COA Reductase Ubiquitnation and Degradation(2004-12-15) Sever, Navdar; Brown, Michael S.3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (HMGR) catalyzes the conversion of HMG CoA to mevalonate, which is the rate limiting step in the production of cholesterol and numerous nonsterol isoprenoid products. Mammalian HMGR is regulated by transcriptional and post-transcriptional feedback mechanisms. The transcriptional regulation is mediated by sterol regulatory element binding proteins (SREBPs), which are synthesized as inactive precursors in the endoplasmic reticulum (ER) membrane. In the absence of sterols, SREBP cleavage activating protein (SCAP) escorts SREBPs from ER to the Golgi apparatus, where SREBPs are cleaved by site 1 and site 2 proteases so as to release their amino terminal transcription factor domains to the nucleus. Sterols inhibit the exit of SCAP-SREBP complex from the ER by promoting the binding of two related polytopic ER membrane proteins, Insig-1 and Insig-2, to the membrane domain of SCAP. Insig-1, but not Insig-2, is an SREBP target gene, causing Insig-1 levels to drop in the presence of sterols, when it is expected to exert its action. The degradation of HMGR requires both sterols and a nonsterol product of the mevalonate pathway and the eight membrane spanning segments in its amino terminus. The membrane domains of HMGR and SCAP bear sequence similarity prompting the investigation of whether Insig proteins can also bind to HMGR. Indeed, Insig-1 and Insig-2 were found to interact with HMGR in a regulated manner and mediate its proteasomal degradation. This effect can be specifically inhibited by overexpressing the membrane domain of SCAP. Insigs were shown to promote the ubiquitination of HMGR on lysine 248 in the cytoplasmic loop between transmembrane segments 6 and 7. In an attempt to achieve a better understanding of the mechanism by which HMGR is degraded, a genetic approach was developed to select mutant somatic cells that cannot degrade HMGR in the presence of sterols. The isolation and characterization of Chinese hamster ovary cells deficient in Insig-1 confirmed the endogenous requirement of Insig-1 for HMGR degradation and revealed the role of differential regulation of Insig-1 and Insig-2 in terms of SREBP processing. These studies revealed a complex feedback regulatory system governing cholesterol homeostasis.Item Modalities of Cholesterol Binding and Modulation of the NPC Proteins and Scap(2011-12-14) Motamed, Massoud; Brown, Michael S.Low density lipoproteins (LDL) and related plasma lipoproteins deliver cholesterol to cells by receptor-mediated endocytosis. The lipoprotein is degraded in late endosomes and lysosomes, allowing cholesterol to be released. Export of cholesterol from late endosomes and lysosomes (hereafter referred to as lysosomes) requires two lysosomal proteins: Niemann-Pick C2 (NPC2), a soluble protein of 132 amino acids; and NPC1, a membrane protein with 13 putative membrane-spanning helices. Recessive loss-of-function mutations in either NPC2 or NPC1 produce NPC disease, which causes death owing to lipid accumulation in lysosomes of liver, brain, and lung. Consistent with their cholesterol export role, NPC2 and NPC1 both bind to cholesterol. The cholesterol binding site on NPC1 is located in the NH2-terminal domain (NTD), which projects into the lysosomal lumen. This domain, designated NPC1 (NTD), can be expressed in vitro as a soluble protein of 240 amino acids that maintains cholesterol binding activity. This thesis studies NPC2 in detail as summarized below. Despite a shared role as cholesterol binding proteins, NPC2 and NPC1 (NTD) bind to cholesterol in opposite orientations. The crystal structures of NPC2 and NPC1 (NTD) have been solved, and NPC2 binds cholesterol with the iso-octyl chain facing the interior of the protein, whereas, NPC1(NTD) binds cholesterol with the 3ß-hydroxyl facing the interior of the protein. Another striking difference is the kinetics of this cholesterol binding. NPC2 binds and releases cholesterol rapidly (half-time < 2 min at 4oC), while NPC1 (NTD) binds cholesterol very slowly (half-time > 2 hr at 4oC). However, NPC2 can stimulate the rate of cholesterol binding to NPC1 (NTD) (>15-fold in vitro). This stimulation of cholesterol binding to NPC1 (NTD) by NPC2 is believed to occur through a direct transfer of cholesterol from NPC2 to NPC1(NTD). Amino acid residues important for binding or transfer of cholesterol on NPC2 were identified through alanine scan mutagenesis. Residues that decreased binding thermodynamics and/or kinetics mapped to areas surrounding the binding pockets on the crystal structures; residues that decreased transfer, but not binding, mapped to discrete surface patches near the exposed residues of the binding pockets. These surface patches may be sites where the two proteins interact to transfer cholesterol. The most deleterious binding mutant was P120S, a residue in the cholesterol binding pocket; the most deleterious transfer mutant was V81D, a residue on the hydrophobic patch extending outward from the cholesterol binding pocket. The above mutants of NPC2 were unable to rescue LDL-stimulated cholesteryl ester synthesis in NPC2-deficient cells, in contrast to wild-type NPC2. Once LDL-derived cholesterol leaves the lysosomes, it is transported to the endoplasmic reticulum (ER), where it serves a regulatory role in cholesterol homeostasis. In the ER, these regulatory functions include activation of acetyl-coenzyme A acetyltransferase (ACAT), allowing for esterification of cholesterol for storage, and regulation of sterol regulatory element–binding protein (SREBP) localization, a transcription factor that regulates key enzymes for cholesterol synthesis. SREBP cleavage-activating protein (Scap) is the switch that controls SREBP, and therefore cholesterol synthesis. Scap senses cholesterol abundance in the ER and acts as an escort protein. In sterol depleted cells, Scap escorts SREBP to the Golgi complex, where two proteases cleave SREBP, thereby releasing its transcriptionally active domain so that it can go to the nucleus and activate transcription of genes involved in cholesterol synthesis and uptake. When cholesterol in abundant, the sterol binds to Scap and triggers a conformational change in the protein that prevents it from escorting SREBPs to the Golgi for proteolytic cleavage. Scap is a 1276 amino acid protein that consists of two domains: an N-terminal domain with 8 transmembrane spanning regions and a C-terminal domain that projects into the cytosol and associates with SREBPs. Previous studies have localized the cholesterol-binding activity of Scap to its membrane domain. Studies described in this thesis identify the cholesterol binding pocket in Scap and identify key residues that play an important role in the protein’s responsiveness to cholesterol binding. The first loop region of Scap (hereafter referred to as Scap(Loop1)) was purified as a recombinant protein and found to have cholesterol binding activity. The specificity of this sterol binding was determined through competition studies and shown to be physiologically relevant. Additionally, this binding affinity and specificity was similar to that of the membrane domain of Scap. Subsequently, alanine scan mutagenesis was performed on Scap(Loop1). Through this approach, several mutations of Scap were identified that constitutively adopt the cholesterol-bound state. This data demonstrates that Scap(Loop1) binds to cholesterol and that the binding then helps induce the conformational change required for Scap to anchor SREBP in ER membranes.Item Sterol Sensing by Two Luminal Loops in Scap(2014-04-14) Zhang, Yinxin; Thomas, Philip J.; Seemann, Joachim; Roth, Michael G.SREBP cleavage-activating protein (Scap) is an endoplasmic reticulum (ER) membrane protein that controls cholesterol homeostasis by transporting SREBPs from the ER to the Golgi complex. Transport is initiated when COPII proteins bind to Scap and cause the Scap/SREBP complex to enter COPII coated vesicles for transport to the Golgi. In the Golgi complex, two proteases cleave SREBP, thereby releasing its transcriptionally active domain so that it can move to the nucleus and activate transcription of genes involved in cholesterol synthesis and uptake. Scap is not only an escort protein, but also a cholesterol sensor. When cholesterol is abundant in ER membranes, the sterol binds to Scap and triggers a conformational change in the protein that prevents COPII proteins from binding to Scap. The Scap/SREBP complex cannot move to the Golgi and proteolytic cleavage is terminated. This cholesterol feedback inhibition is essential to control cholesterol metabolism in animals. Scap can be divided into two functional regions. The C-terminal cytosolic WD domain interacts with the regulatory domain of SREBPs. The N-terminal membrane attachment domain includes eight transmembrane helices (TM) joined by four small hydrophilic loops and three large loops. One large cytosolic loop (Loop 6) in Scap binds COPII proteins. The other two large loops (Loops 1 and 7) face the ER lumen. Previous studies localized the cholesterol-binding activity to the N-terminal membrane domain of Scap. Studies described in this thesis narrow down the cholesterol binding pocket to the first large luminal loop (Loop 1). Mutational analysis further suggests a direct interaction between luminal Loop 1 and Loop 7 to control Scap transport activity. Scap Loop 1 was purified as a recombinant protein and found to bind [3H]-cholesterol through an in vitro binding assay. The specificity of this binding was determined through competition studies with different unlabeled sterols. Importantly, the binding affinity and specificity of Loop 1 was similar to that of the entire Scap membrane domain. Subsequently, alanine scan mutagenesis was performed on luminal Loop1 and Loop7. Through this approach, two point mutations of Scap (Y234A in Loop 1 and Y640S in Loop 7) were identified that prevent its movement to the Golgi, thus abrogating the processing of SREBPs. Trypsin cleavage assays on the full-length Scap show that Loop 6 of Scap(Y234A) or Scap(Y640S) is always in the configuration that precludes COPII binding, even in sterol-depleted cells. When the Scap TM1-6 segment (containing Loop 1) and the TM7-end segment (containing Loop 7) are expressed in the same cells, the two proteins bind to each other as determined by co-immunoprecipitation. This binding does not occur when Loop 1 contains the Y234A mutation, or Loop 7 contains the Y640S mutation. These data support the model that luminal Loop 1 and luminal Loop 7 must interact in order for Scap movement to occur.