Spatial Distribution of the Pathways of Cholesterol Homeostasis in Human Retina

The retina is a light-sensitive tissue lining the inner surface of the eye and one of the few human organs whose cholesterol maintenance is still poorly understood. Challenges in studies of the retina include its complex multicellular and multilayered structure; unique cell types and functions; and specific physico-chemical environment.We isolated specimens of the neural retina (NR) and underlying retinal pigment epithelium (RPE)/choroid from six deceased human donors and evaluated them for expression of genes and proteins representing the major pathways of cholesterol input, output and regulation. Eighty-four genes were studied by PCR array, 16 genes were assessed by quantitative real time PCR, and 13 proteins were characterized by immunohistochemistry. Cholesterol distribution among different retinal layers was analyzed as well by histochemical staining with filipin. Our major findings pertain to two adjacent retinal layers: the photoreceptor outer segments of NR and the RPE. We demonstrate that in the photoreceptor outer segments, cholesterol biosynthesis, catabolism and regulation via LXR and SREBP are weak or absent and cholesterol content is the lowest of all retinal layers. Cholesterol maintenance in the RPE is different, yet the gene expression also does not appear to be regulated by the SREBPs and varies significantly among different individuals.This comprehensive investigation provides important insights into the relationship and spatial distribution of different pathways of cholesterol input, output and regulation in the NR-RPE region. The data obtained are important for deciphering the putative link between cholesterol and age-related macular degeneration, a major cause of irreversible vision loss in the elderly

REV-ERBα Participates in Circadian SREBP Signaling and Bile Acid Homeostasis

The nuclear receptor REV-ERBα shapes the daily activity profile of Sterol Response Element Binding Protein (SREBP) and thereby participates in the circadian control of cholesterol and bile acid synthesis in the liver

Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the synthesis of cholesterol

SummaryFeedback control of cholesterol synthesis is mediated in part by sterol-induced binding of HMG CoA reductase to Insig proteins in the endoplasmic reticulum (ER). Binding leads to ubiquitination and proteasomal degradation of reductase, a rate-controlling enzyme in cholesterol synthesis. Using in vitro and in vivo assays, we show that lanosterol, the first sterol intermediate in cholesterol synthesis, potently stimulates ubiquitination of reductase, whereas cholesterol has no effect at 10-fold higher concentrations. Lanosterol is not effective in mediating the other action of Insigs, namely to promote ER retention of SCAP-SREBP complexes, a reaction that is mediated directly by cholesterol. A pair of methyl groups located in the C4 position of lanosterol confers this differential response. These data indicate that buildup of cholesterol synthesis intermediates represses the pathway selectively at reductase and reveal a previously unappreciated link between feedback inhibition of reductase and carbon flow through the cholesterol synthetic pathway

Intramembrane glycine mediates multimerization of Insig-2, a requirement for sterol regulation in Chinese hamster ovary cells

Sterol-induced binding of endoplasmic reticulum (ER) membrane proteins Insig-1 and Insig-2 to SREBP cleavage-activating protein (Scap) and HMG-CoA reductase triggers regulatory events that limit cholesterol synthesis in animal cells. Binding of Insigs to Scap prevents proteolytic activation of sterol-regulatory element binding proteins (SREBPs), membrane-bound transcription factors that enhance cholesterol synthesis, by trapping Scap-SREBP complexes in the ER. Insig binding to reductase causes ubiquitination and subsequent proteasome-mediated degradation of the enzyme from ER membranes, slowing a rate-limiting step in cholesterol synthesis. Here, we report the characterization of mutant Chinese hamster ovary cells, designated SRD-20, that are resistant to 25-hydroxycholesterol, which potently inhibits SREBP activation and stimulates degradation of reductase. SRD-20 cells were produced by mutagenesis of Insig-1-deficient SRD-14 cells, followed by selection in 25-hydroxycholesterol. DNA sequencing reveals that SRD-20 cells harbor a point mutation in one Insig-2 allele that results in production of a truncated, nonfunctional protein, whereas the other allele contains a point mutation that results in substitution of glutamic acid for glycine-39. This glycine residue localizes to the first membrane-spanning segment of Insig-2 and is also present in the corresponding region of Insig-1. Mutant forms of Insig-1 and Insig-2 containing the Glu-to-Gly substitution fail to confer sterol regulation upon overexpressed Scap and reductase. These studies identify the intramembrane glycine as a key residue for normal sterol regulation in animal cells

Insig-mediated, Sterol-accelerated Degradation of the Membrane Domain of Hamster 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase in Insect Cells*

Sterol-accelerated degradation of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase is one of several mechanisms through which cholesterol synthesis is controlled in mammalian cells. This degradation results from sterol-induced binding of the membrane domain of reductase to endoplasmic reticulum membrane proteins called Insig-1 and Insig-2, which are carriers of a ubiquitin ligase called gp78. The ensuing gp78-mediated ubiquitination of reductase is a prerequisite for its rapid, 26 S proteasome-mediated degradation from endoplasmic reticulum membranes, a reaction that slows a rate-limiting step in cholesterol synthesis. Here, we report that the membrane domain of hamster reductase is subject to sterol-accelerated degradation in Drosophila S2 cells, but only when mammalian Insig-1 or Insig-2 are co-expressed. This degradation mimics the reaction that occurs in mammalian cells with regard to its absolute requirement for the action of Insigs, sensitivity to proteasome inhibition, augmentation by nonsterol isoprenoids, and sterol specificity. RNA interference studies reveal that this degradation requires the Drosophila Hrd1 ubiquitin ligase and several other proteins, including a putative substrate selector, which associate with the enzyme in yeast and mammalian systems. These studies define Insigs as the minimal requirement for sterol-accelerated degradation of the membrane domain of reductase in Drosophila S2 cells