Arun Radhakrishnan

Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Oxysterols block transport by binding to Insig

Cholesterol synthesis in animals is controlled by the regulated transport of sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum to the Golgi, where the transcription factors are processed proteolytically to release active fragments. Transport is inhibited by either cholesterol or oxysterols, blocking cholesterol synthesis. Cholesterol acts by binding to the SREBP-escort protein Scap, thereby causing Scap to bind to anchor proteins called Insigs. Here, we show that oxysterols act by binding to Insigs, causing Insigs to bind to Scap. Mutational analysis of the six transmembrane helices of Insigs reveals that the third and fourth are important for Insigs binding to oxysterols and to Scap. These studies define Insigs as oxysterol-binding proteins, explaining the long-known ability of oxysterols to inhibit cholesterol synthesis in animal cells.

Condensed complexes of cholesterol and phospholipids

Mixtures of dihydrocholesterol and phospholipids form immiscible liquids in monolayer membranes at the air-water interface under specified conditions of temperature and 2-dimensional pressure. In recent work it has been discovered that a number of these mixtures exhibit two upper miscibility critical points. Pairs of upper critical points can be accounted for by a theoretical model that implies the cooperative formation of molecular complexes of dihydrocholesterol and phospholipid molecules. These complexes are calculated to be present in the membranes both above and below the critical points. Below the critical points the complexes form a separate phase, whereas above the critical points the complexes are completely miscible with the other lipid components. The cooperativity of complex formation prompts the use of the terminology condensed complex.

Switch-like Control of SREBP-2 Transport Triggered by Small Changes in ER Cholesterol: A Delicate Balance

Animal cells control their membrane lipid composition within narrow limits, but the sensing mechanisms underlying this control are largely unknown. Recent studies disclosed a protein network that controls the level of one lipid-cholesterol. This network resides in the endoplasmic reticulum (ER). A key component is Scap, a tetrameric ER membrane protein that binds cholesterol. Cholesterol binding prevents Scap from transporting SREBPs to the Golgi for activation. Using a new method to purify ER membranes from cultured cells, we show that Scap responds cooperatively to ER cholesterol levels. When ER cholesterol exceeds 5% of total ER lipids (molar basis), SREBP-2 transport is abruptly blocked. Transport resumes when ER cholesterol falls below the 5% threshold. The 5% threshold is lowered to 3% when cells overexpress Insig-1, a Scap-binding protein. Cooperative interactions between cholesterol, Scap, and Insig create a sensitive switch that controls the cholesterol composition of cell membranes with remarkable precision.

NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes

Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann–Pick C1 (NPC1) and soluble Niemann–Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25–264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [3H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype.

Purified NPC1 protein: II. Localization of sterol binding to a 240-amino acid soluble luminal loop

Defects in Niemann-Pick, Type C-1 protein (NPC1) cause cholesterol, sphingolipids, phospholipids, and glycolipids to accumulate in lysosomes of liver, spleen, and brain. In cultured fibroblasts, NPC1 deficiency causes lysosomal retention of lipoprotein-derived cholesterol after uptake by receptor-mediated endocytosis. NPC1 contains 1278 amino acids that form 13 membrane-spanning helices and three large loops that project into the lumen of lysosomes. We showed earlier that NPC1 binds cholesterol and oxysterols. Here we localize the binding site to luminal loop-1, a 240-amino acid domain with 18 cysteines. When produced in cultured cells, luminal loop-1 was secreted as a soluble dimer. This loop bound [3H]cholesterol (Kd, 130 nm) and [3H]25-hydroxycholesterol (25-HC, Kd, 10 nm) with one sterol binding site per dimer. Binding of both sterols was competed by oxysterols (24-, 25-, and 27-HC). Unlabeled cholesterol competed strongly for binding of [3H]cholesterol, but weakly for [3H]25-HC binding. Binding of [3H]cholesterol but not [3H]25-HC was inhibited by detergents. We also studied NPC2, a soluble protein whose deficiency causes a similar disease phenotype. NPC2 bound cholesterol, but not oxysterols. Epicholesterol and cholesteryl sulfate competed for [3H]cholesterol binding to NPC2, but not NPC1. Glutamine 79 in luminal loop-1 of NPC-1 is important for sterol binding; a Q79A mutation abolished binding of [3H]cholesterol and [3H]25-HC to full-length NPC1. Nevertheless, the Q79A mutant restored cholesterol transport to NPC1-deficient Chinese hamster ovary cells. Thus, the sterol binding site on luminal loop-1 is not essential for NPC1 function in fibroblasts, but it may function in other cells where NPC1 deficiency produces more complicated lipid abnormalities.

Purified NPC1 Protein I. BINDING OF CHOLESTEROL AND OXYSTEROLS TO A 1278-AMINO ACID MEMBRANE PROTEIN

The Niemann-Pick, Type C1 protein (NPC1) is required for the transport of lipoprotein-derived cholesterol from lysosomes to endoplasmic reticulum. The 1278-amino acid, polytopic membrane protein has not been purified, and its mechanism of action is unknown. Unexpectedly, we encountered NPC1 in a search for a membrane protein that binds 25-hydroxycholesterol (25-HC) and other oxysterols. A 25-HC-binding protein was purified more than 14,000-fold from rabbit liver membranes and identified as NPC1 by mass spectroscopy. We prepared recombinant human NPC1 and confirmed its ability to bind oxysterols, including those with a hydroxyl group on the 24, 25, or 27 positions. Hydroxyl groups on the 7, 19, or 20 positions failed to confer binding. Recombinant human NPC1 also bound [3H]cholesterol in a reaction inhibited by Nonidet P-40 above its critical micellar concentration. Low concentrations of unlabeled 25-HC abolished binding of [3H]cholesterol, but the converse was not true, i.e. unlabeled cholesterol, even at high concentrations, did not abolish binding of [3H]25-HC. NPC1 is not required for the known regulatory actions of oxysterols. Thus, in NPC1-deficient fibroblasts 25-HC blocked the processing of sterol regulatory element-binding proteins and activated acyl-CoA:cholesterol acyltransferase in a normal fashion. The availability of assays to measure NPC1 binding in vitro may further the understanding of ways in which oxysterols regulate intracellular lipid transport.

Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells.

A handoff model has been proposed to explain the egress from lysosomes of cholesterol derived from receptor-mediated endocytosis of LDL. Cholesterol is first bound by soluble Niemann-Pick C2 (NPC2) protein, which hands off the cholesterol to the N-terminal domain of membrane-bound NPC1. Cells lacking NPC1 or NPC2 accumulate LDL-derived cholesterol in lysosomes and fail to deliver LDL cholesterol to the endoplasmic reticulum (ER) for esterification by acyl-CoA acyltransferase (ACAT) and for inhibition of sterol regulatory element-binding protein cleavage. Here, we support this model by showing that the cholesterol transport defect in NPC1 mutant cells is restricted to lysosomal export. Other cholesterol transport pathways appear normal, including the movement of cholesterol from the plasma membrane to the ER after treatment of cells with 25-hydroxycholesterol or sphingomyelinase. The NPC1 or NPC2 block in cholesterol delivery to the ER can be overcome by 2-hydroxypropyl-β-cyclodextrin, which leads to a marked increase in ACAT-mediated cholesterol esterification. The buildup of cholesteryl esters in the cytosol is expected to be much less toxic than the buildup of free cholesterol in the lysosomes of patients with mutations in NPC1 or NPC2.

Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis

When human fibroblasts take up plasma low density lipoprotein (LDL), its cholesterol is liberated in lysosomes and eventually reaches the endoplasmic reticulum (ER) where it inhibits cholesterol synthesis by blocking activation of SREBPs. This feedback protects against cholesterol overaccumulation in the plasma membrane (PM). But how does ER know whether PM is saturated with cholesterol? In this study, we define three pools of PM cholesterol: (1) a pool accessible to bind 125I-PFO*, a mutant form of bacterial Perfringolysin O, which binds cholesterol in membranes; (2) a sphingomyelin(SM)-sequestered pool that binds 125I-PFO* only after SM is destroyed by sphingomyelinase; and (3) a residual pool that does not bind 125I-PFO* even after sphingomyelinase treatment. When LDL-derived cholesterol leaves lysosomes, it expands PMs PFO-accessible pool and, after a short lag, it also increases the ERs PFO-accessible regulatory pool. This regulatory mechanism allows cells to ensure optimal cholesterol levels in PM while avoiding cholesterol overaccumulation. DOI: http://dx.doi.org/10.7554/eLife.02882.001

Ultra-high throughput rotary capillary array electrophoresis scanner for fluorescent DNA sequencing and analysis

We have constructed a rotary confocal fluorescence scanner and capillary array electrophoresis system that is designed to analyze over 1000 DNA sequencing or fragment sizing separations in parallel. Capillaries are arranged around the surface of a cylinder and a rotating objective in the middle of the cylinder excites and collects fluorescence from labeled DNA fragments as they pass the capillary detection window. The capillaries are pressure‐filled with a replaceable matrix and the samples are electrokinetically injected in parallel from a stainless steel microtiter plate at the cathode end. We demonstrate that the instrument is capable of producing four‐color data from all capillaries at a scan rate of 4 Hz (corresponding to a linear scan velocity of 121 cm/s). M13 sequencing data were obtained using a 128 capillary array mounted in half of the first quadrant of the scanner. In this initial run, read lengths greater than 500 bases were obtained in over 60% of the capillaries.

Accessibility of Cholesterol in Endoplasmic Reticulum Membranes and Activation of SREBP-2 Switch Abruptly at a Common Cholesterol Threshold

Recent studies have shown that cooperative interactions in endoplasmic reticulum (ER) membranes between Scap, cholesterol, and Insig result in switch-like control over activation of SREBP-2 transcription factors. This allows cells to rapidly adjust rates of cholesterol synthesis and uptake in response to even slight deviations from physiological set-point levels, thereby ensuring cholesterol homeostasis. In the present study we directly probe for the accessibility of cholesterol in purified ER membranes. Using a soluble cholesterol-binding bacterial toxin, perfringolysin O, we show that cholesterol accessibility increases abruptly at ∼5 mol % ER cholesterol, the same concentration at which SREBP-2 activation is halted. This switch-like change in cholesterol accessibility is observed not only in purified ER membranes but also in liposomes made from ER lipid extracts. The accessibility of cholesterol in membranes is related to its chemical activity. Complex formation between cholesterol and some ER phospholipids can result in sharp changes in cholesterol chemical activity and its accessibility to perfringolysin O or membrane sensors like Scap. The control of the availability of the cholesterol ligand to participate in cooperative Scap/cholesterol/Insig interactions further sharpens the sensitive switch that exerts precise control over cholesterol levels in cell membranes.