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The Cholesterol-Regulated Stard4 Gene Encodes a Star-Related Lipid Transfer Protein with Two Closely Related Homologues, Stard5 and Stard6

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The Cholesterol-Regulated Stard4 Gene Encodes a Star-Related Lipid Transfer Protein with Two Closely Related Homologues, Stard5 and Stard6 The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer protein with two closely related homologues, StarD5 and StarD6 Raymond E. Soccio*, Rachel M. Adams*, Michael J. Romanowski†, Ephraim Sehayek*, Stephen K. Burley†‡§, and Jan L. Breslow*¶ *Laboratory of Biochemical Genetics and Metabolism, †Laboratories of Molecular Biophysics, and ‡Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021 Contributed by Jan L. Breslow, March 12, 2002 Using cDNA microarrays, we identified StarD4 as a gene whose modate a cholesterol molecule (6). The only other START expression decreased more than 2-fold in the livers of mice fed a domain with a known lipid ligand is the phosphatidylcholine high-cholesterol diet. StarD4 expression in cultured 3T3 cells was transfer protein (PCTP͞StarD2) (8). also sterol-regulated, and known sterol regulatory element bind- In this study, cDNA microarrays were used to identify cho- ing protein (SREBP)-target genes showed coordinate regulation. lesterol-regulated genes. As an in vivo physiological model, The closest homologues to StarD4 were two other StAR-related C57BL͞6 mice were fed a high-cholesterol diet to raise liver lipid transfer (START) proteins named StarD5 and StarD6. StarD4, cholesterol. StarD4 (START-domain-containing 4) was identi- StarD5, and StarD6 are 205- to 233-aa proteins consisting almost fied as a gene whose hepatic expression decreased more than entirely of START domains. These three constitute a subfamily 2-fold upon cholesterol feeding. StarD4 expression was coordi- among START proteins, sharing Ϸ30% amino acid identity with Ϸ nately regulated with known SREBP-target genes, suggesting one another, 20% identity with the cholesterol-binding START that StarD4 is also SREBP regulated. StarD5 and StarD6 were domains of StAR and MLN64, and less than 15% identity with identified on the basis of homology to StarD4, and the three phosphatidylcholine transfer protein (PCTP) and other START do- form a subfamily among START domain-containing proteins. mains. StarD4 and StarD5 were expressed in most tissues, with StarD4 and StarD5 were ubiquitously expressed, with highest highest levels in liver and kidney, whereas StarD6 was expressed exclusively in the testis. In contrast to StarD4, expression of StarD5 levels in liver and kidney, whereas StarD6 expression was limited and MLN64 was not sterol-regulated. StarD4, StarD5, and StarD6 to the testis. These three proteins may function in the intracel- may be involved in the intracellular transport of sterols or other lular shuttling of sterols or other lipids. lipids. Materials and Methods Animals and Diets. Six-week old C57͞BL6 mice (The Jackson holesterol is an essential component of mammalian cell Laboratory) were housed in a specific pathogen free, humidity- membranes and is the biosynthetic precursor for steroid C and temperature-controlled room with a 12-h light-dark cycle. hormones, bile acids, and vitamin D. Precursors and metabolites Mice were fed pelleted PicoLab Rodent Diet 20 (product code of cholesterol are involved in cellular signaling events (1). 5053), which contains 0.02% cholesterol (wt͞wt), or the same Animals obtain cholesterol from their diets and synthesize it de ͞ novo from acetate, but excess cellular free cholesterol is toxic, diet supplemented to 0.5% cholesterol (wt wt) (Harlan Teklad, and high plasma low density lipoprotein (LDL) cholesterol is Madison, WI). After 3 weeks, mice were fasted for 6 h, anes- associated with atherosclerotic vascular disease. Therefore, cho- thetized with ketamine, and killed during the last3hofthelight cycle. Livers were harvested, frozen in liquid nitrogen, and lesterol homeostasis is finely regulated to ensure an adequate Ϫ MEDICAL SCIENCES supply, yet avoid excess. Much of this regulation is transcrip- stored at 80°C. Liver cholesterol was assayed by gas chroma- tional and mediated by sterol regulatory element binding pro- tography (9). teins (SREBPs) and liver X receptors (LXRs) (1). When cellular sterols are abundant, SREBPs are inactive in the endoplasmic cDNA Microarrays. Fluorescent cDNA probes were synthesized by ␮ reticulum membrane, whereas LXR nuclear receptors bind their reverse transcribing (Invitrogen Superscript II) 100 g of liver total oxysterol ligands and activate genes involved in reverse choles- RNA (prepared by using RNeasy from Qiagen, Valencia, CA) in terol transport. Upon sterol depletion, LXRs are inactive but the presence of Cy3- or Cy5-labeled dUTP (Amersham Pharmacia SREBPs are cleaved by regulated proteolysis to release the Biotech). cDNA microarrays with Ϸ9,000 mouse expressed se- mature transcription factor domain, which translocates to the quence tags (ESTs) were a generous gift of Raju Kucherlapati at nucleus. SREBPs then bind promoter sterol-regulatory elements Albert Einstein College of Medicine (10). Array protocols are (SREs) to activate genes involved in the biosynthesis and uptake online at http:͞͞sequence.aecom.yu.edu͞bioinf͞microarray͞ of cholesterol and fatty acids (2). protocol4.html. Scanned arrays were analyzed with SCANALYZE Since cholesterol and other sterols are hydrophobic lipids, intracellular sterol transport is mediated either by vesicles or by soluble protein carriers (3). An example of the latter is the Abbreviations: StAR, steroidogenic acute regulatory protein; MLN64, protein of unknown ͞ function; PCTP, phosphatidylcholine transfer protein; START, StAR-related lipid transfer; steroidogenic acute regulatory protein (StAR StarD1), which StarD, START domain-containing; SREBP, sterol regulatory element binding protein; EST, delivers cholesterol to mitochondrial P450 side-chain-cleavage expressed sequence tag; RT-PCR, reverse transcription–PCR; UTR, untranslated region. enzymes in steroidogenic cells (4). There is a family of proteins Data deposition: The StarD4, StarD5, and StarD6 sequences reported in this paper have with homology to StAR, each containing a 200- 210-aa StAR- been deposited in the GenBank database (accession nos. AF480297–AF480305). related lipid transfer (START) domain (5). The START domain §Present address: Structural GenomiX, Inc., 10505 Roselle Street, San Diego, CA 92121. ͞ of MLN64 StarD3, which is 36% identical to StAR, has also ¶To whom reprint requests should be addressed. E-mail: [email protected]. been shown in vitro to bind cholesterol (6) and stimulate The publication costs of this article were defrayed in part by page charge payment. This steroidogenesis (7). The MLN64 START domain crystal struc- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. ture shows an internal hydrophobic tunnel that could accom- §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.052143799 PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 ͉ 6943–6948 Downloaded by guest on September 27, 2021 Table 1. Microarray genes down-regulated by dietary cholesterol Fold decrease on six arrays Mean fold GenBank decrease accession no. Gene product 1a 1b 2a 2b 3a 3b 4.6 AA237469 Isopentenyl diphosphate isomerase 3.3 7.6 2.4 3.6 4.9 5.9 2.8 AA239481 Uncharacterized EST (StarD4) 3.6 2.3 4.8 1.6 2.8 1.9 2.6 AA268608 Squalene epoxidase 3.2 2.7 2.8 1.3 2.9 (44) 2.5 AA061468 Hydroxymethylglutaryl-CoA synthase 2.6 0.9 5.2 2.0 2.4 2.1 2.0 AA116513 Fatty acid synthase 1.4 1.7 2.0 2.8 2.3 2.0 2.0 AA500330 Farnesyl pyrophosphate synthetase 1.2 1.7 2.0 2.8 2.3 2.0 Experiments 1–3 each compared liver gene expression in a pair of individual mice fed different diets (0.02% versus 0.5% cholesterol). Each experiment was performed on duplicate arrays (a, b) by reversing the Cy3 and Cy5 probe labeling. Since there was marked variability within and between experiments, the following criteria were used for regulated genes: expression differed 2-fold or greater on at least four of the six arrays with the higher expression level at least 25% over background. For each of the six genes down-regulated by the high-cholesterol diet, the fold regulation on each array is shown, as well the average from all six (the outlying value in parenthesis was eliminated). (by Michael Eisen, http:͞͞rana.lbl.gov͞EisenSoftware.htm), and splice junctions. The template was 10 ␮l of a 1:100 dilution of results were compiled by using Microsoft EXCEL and ACCESS. cDNA, and the standards were a serial dilution of cDNA. A 7700 Sequence Detection System (Applied Biosystems) was Cloning and PCR. Molecular cloning followed standard techniques used with the default thermal cycling profile (95°C for 10 min; using enzymes from New England Biolabs. Except where noted, 40 cycles of 95°C for 15 s, 60°C for 1 min; 4°C soak). The PCR reagents were Advantage cDNA polymerase (CLON- quencher dye (TAMRA, N,N,NЈ,NЈ-tetramethyl-6-carboxyrho- TECH), primers were from Gene Link (Hawthorne, NY; se- damine) was the passive reference. The threshold was set at 0.05 quences in Table 3, which is published as supporting information unit of normalized fluorescence, and a threshold cycle (Ct) was on the PNAS web site, www.pnas.org), and thermal cycling was measured in each well. Relative standard curves were plotted for on a Perkin–Elmer 9700. TA cloning of PCR products was each gene, and the mean Ct for each cDNA sample was expressed carried out with pCR-2.1-TOPO (Invitrogen). All DNA con- as an arbitrary value relative to standard. For each cDNA, values structs were sequence-verified. for genes of interest were normalized to the corresponding value for cyclophilin and expressed as a ratio. Groups were compared Multiple-Tissue Northern Blots. Radiolabeled DNA probes were by a two-tailed type 2 Student’s t test. synthesized from 20 ng of template by random priming using the Results DECA prime II kit (Ambion) and [32P]dATP (Perkin–Elmer). Blots were purchased and hybridized by using Express Hyb rapid Cholesterol Feeding cDNA Microarray. The initial experiment sought to identify hepatic genes regulated by dietary cholesterol.
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