Switch-like Responses of Two Cholesterol Sensors Do Not Require Protein Oligomerization in Membranes

Many cellular processes are sensitive to levels of cholesterol in specific membranes and show a strongly sigmoidal dependence on membrane composition. The sigmoidal responses of the cholesterol sensors involved in these processes could arise from several mechanisms, including positive cooperativity (protein effects) and limited cholesterol accessibility (membrane effects). Here, we describe a sigmoidal response that arises primarily from membrane effects due to sharp changes in the chemical activity of cholesterol. Our models for eukaryotic membrane-bound cholesterol sensors are soluble bacterial toxins that show an identical switch-like specificity for endoplasmic reticulum membrane cholesterol. We show that truncated versions of these toxins fail to form oligomers but still show sigmoidal binding to cholesterol-containing membranes. The nonlinear response emerges because interactions between bilayer lipids control cholesterol accessibility to toxins in a threshold-like fashion. Around these thresholds, the affinity of toxins for membrane cholesterol varies by >100-fold, generating highly cooperative lipid-dependent responses independently of protein-protein interactions. Such lipid-driven cooperativity may control the sensitivity of many cholesterol-dependent processes.     

Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.     

Insig required for sterol-mediated inhibition of Scap/SREBP binding to COPII proteins in vitro

When added to living cells, sterols such as cholesterol and 25-hydroxycholesterol block the lateral movement of sterol regulatory element-binding proteins (SREBPs) into COPII-coated vesicles on endoplasmic reticulum (ER) membranes and thereby prevent the SREBPs from reaching the Golgi complex for processing to the mature forms that activate cholesterol synthesis. Sorting of SREBPs into COPII vesicles is mediated by Sar1 and the coat proteins Sec23 and Sec24. Here, we explore the mechanism of sterol inhibition in vitro through use of protein pull-down assays. We show that addition of cholesterol or 25-hydroxycholesterol to microsomal membranes in vitro blocks Sar1-dependent binding of the Sec23/24 complex to Scap, the SREBP escort protein. This in vitro inhibition is dependent on the presence of Insig-1, an ER resident protein that is necessary for sterol-mediated inhibition of Scap/SREBP transport in intact cells. Sec23/24 binding to Scap requires the hexapeptide sequence MELADL located in a cytoplasmic loop of Scap. This hexapeptide acts as a sterol-regulated ER sorting signal. These studies define the biochemical parameters responsible for regulated sorting of an ER membrane protein into COPII-coated vesicles.     

The Structural Basis of Cholesterol Accessibility in Membranes

Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.     

The Structural Basis of Cholesterol Accessibility in Membranes

Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.     

Identification of luminal Loop 1 of Scap protein as the sterol sensor that maintains cholesterol homeostasis

Cellular cholesterol homeostasis is maintained by Scap, an endoplasmic reticulum (ER) protein with eight transmembrane helices. In cholesterol-depleted cells, Scap transports sterol regulatory element-binding proteins (SREBPs) to the Golgi, where the active fragment of SREBP is liberated by proteases so that it can activate genes for cholesterol synthesis. When ER cholesterol increases, Scap binds cholesterol, and this changes the conformation of cytosolic Loop 6, which contains the binding site for COPII proteins. The altered conformation precludes COPII binding, abrogating movement to the Golgi. Consequently, cholesterol synthesis declines. Here, we identify the cholesterol-binding site on Scap as Loop 1, a 245-amino acid sequence that projects into the ER lumen. Recombinant Loop 1 binds sterols with a specificity identical to that of the entire Scap membrane domain. When tyrosine 234 in Loop 1 is mutated to alanine, Loop 6 assumes the cholesterol-bound conformation, even in sterol-depleted cells. As a result, full-length Scap(Y234A) cannot mediate SREBP processing in transfected cells. These results indicate that luminal Loop 1 of Scap controls the conformation of cytosolic Loop 6, thereby determining whether cells produce cholesterol.     

Peroxisome deficiency-induced ER stress and SREBP-2 pathway activation in the liver of newborn PEX2 knock-out mice

Disruption of the Pex2 gene leads to peroxisome deficiency and widespread metabolic dysfunction. We previously demonstrated that peroxisomes are critical for maintaining cholesterol homeostasis, using peroxisome-deficient Pex2(-/-) mice on a hybrid Swiss Webster×129S6/SvEv (SW/129) genetic background. Peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, leading to dysregulation of the endogenous sterol response mechanism. Herein, we demonstrate a more profound dysregulation of cholesterol homeostasis in newborn Pex2(-/-) mice congenic on a 129S6/SvEv (129) genetic background, and substantial differences between newborn versus postnatal Pex2(-/-) mice in factors that activate ER stress. These differences extend to relationships between activation of genes regulated by SREBP-2 versus PPARα. The SREBP-2 pathway is induced in neonatal Pex2(-/-) livers from 129 and SW/129 strains, despite normal hepatic cholesterol levels. ER stress markers are increased in newborn 129 Pex2(-/-) livers, which occurs in the absence of hepatic steatosis or accumulation of peroxins in the ER. Moreover, the induction of SREBP-2 and ER stress pathways is independent of PPARα activation in livers of newborn 129 and SW/129 Pex2(-/-) mice. Two-week-old wild-type mice treated with the peroxisome proliferator WY-14,643 show strong induction of PPARα-regulated genes and decreased expression of SREBP-2 and its target genes, further demonstrating that SREBP-2 pathway induction is not dependent on PPARα activation. Lastly, there is no activation of either SREBP-2 or ER stress pathways in kidney and lung of newborn Pex2(-/-) mice, suggesting a parallel induction of these pathways in peroxisome-deficient mice. These findings establish novel associations between SREBP-2, ER stress and PPARα pathway inductions.     

Cholesterol effects on BAX pore activation

The importance of BCL-2 family proteins in the control of cell death has been clearly established. One of the key members of this family, BAX, has soluble, membrane-bound, and membrane-integrated forms that are central to the regulation of apoptosis. Using purified monomeric human BAX, defined liposomes, and isolated human mitochondria, we have characterized the soluble to membrane transition and pore formation by this protein. For the purified protein, activation, but not oligomerization, is required for membrane binding. The transition to the membrane environment includes a binding step that is reversible and distinct from the membrane integration step. Oligomerization and pore activation occur after the membrane integration. In cells, BAX targets several intracellular membranes but notably does not target the plasma membrane while initiating apoptosis. When cholesterol was added to either the liposome bilayer or mitochondrial membranes, we observed increased binding but markedly reduced integration of BAX into both membranes. This cholesterol inhibition of membrane integration accounts for the reduction of BAX pore activation in liposomes and mitochondrial membranes. Our results indicate that the presence of cholesterol in membranes inhibits the pore-forming activity of BAX by reducing the ability of BAX to transition from a membrane-associated protein to a membrane-integral protein.     

Cholesterol accumulation inhibits ER to Golgi transport and protein secretion: studies of apolipoprotein E and VSVGt

Cholesterol excess is typical of various diseases including atherosclerosis. We have investigated whether cholesterol accumulation in the ER (endoplasmic reticulum) can inhibit exit of vesicular cargo and secretion of proteins by studying apoE (apolipoprotein E), a significant glycoprotein in human health and disease. CHO (Chinese hamster ovary) cells expressing human apoE under a cholesterol-independent promoter incubated with cholesterol-cyclodextrin complexes showed increased levels of cellular free and esterified cholesterol, inhibition of SREBP-2 (sterol-regulatory-element-binding protein 2) processing, and a mild induction of ER stress, indicating significant accumulation of cholesterol in the ER. Secretion of apoE was markedly inhibited by cholesterol accumulation, and similar effects were observed in cells enriched with lipoprotein-derived cholesterol and in primary human macrophages. Removal of excess cholesterol by a cyclodextrin vehicle restored apoE secretion, indicating that the transport defect was reversible. That cholesterol impaired protein trafficking was supported by the cellular accumulation of less sialylated apoE glycoforms, and by direct visualization of altered ER to Golgi transport of thermo-reversible VSVG (vesicular stomatitis virus glycoprotein) linked to GFP (green fluorescent protein). We conclude that intracellular accumulation of cholesterol in the ER reversibly inhibits protein transport and secretion. Strategies to correct ER cholesterol may restore homoeostatic processes and intracellular protein transport in conditions characterized by cholesterol excess.