The cholesterol-regulated StarD4 encodes a StAR-related lipid transfer 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 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) 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 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. hybridization buffer according to the manufacturer’s instruc- ͞ tions (CLONTECH). C57BL 6 male mice were fed a chow diet low (0.02%) or high (0.5%) in cholesterol for 3 weeks. Liver cholesterol was mea- sured for five mice on each diet, and the 0.5% cholesterol diet Cell Culture. Mouse 3T3-L1 cells (American Type Culture Col- Ϯ Ϯ lection) were maintained in Dulbecco’s modified Eagle’s me- raised the total liver cholesterol (1.87 0.23 to 4.47 0.85 ͞ ␮g͞mg of liver, P Ͻ 0.001), with an increase in both esterified dium (DME) with 10% FBS at 37°Cina5%CO2 95% air Ϯ Ϯ ␮ ͞ Ͻ Ϯ atmosphere. Cells were grown as fibroblast-like cells and were (0.48 0.08 to 2.64 0.81 g mg, P 0.001) and free (1.40 0.23 to 1.83 Ϯ 0.25 ␮g͞mg, P Ͻ 0.05) cholesterol. cDNA not differentiated to adipocytes. Lipoprotein-depleted serum microarrays were used to screen for genes whose hepatic ex- (LPDS) was prepared from FBS by ultracentrifugation. Stock pression changed upon cholesterol feeding. Among the six genes solutions were made for three reagents (Sigma): cholesterol (10 on the array down-regulated more than 2-fold by dietary cho- mg͞ml in ethanol), 25-hydroxycholesterol (0.5 mg͞ml in etha- lesterol (Table 1), five were known SREBP-target genes involved nol), and mevinolin (lovastatin, 0.8 mg͞ml). Subconfluent cells in cholesterol or fatty acid biosynthesis (2, 11, 12). The sixth was in 6-well plates were cultured for 20 h in one of three media: an EST (AA239481) not previously characterized. control (DME with 10% LPDS), sterols (DME with 10% LPDS, 10 ␮g͞ml cholesterol, and 1 ␮g͞ml 25-hydroxycholesterol), or ␮ ͞ Identification of the StarD4 Gene. To identify the novel gene, the lovastatin (DME with 10% LPDS and 1 g ml mevinolin). 1,114-bp insert of EST AA239481 was sequenced. There were no long protein-coding regions in any reading frame, suggesting the Real-Time Quantitative Reverse Transcription–PCR (RT-PCR). Total sequence was 3Ј untranslated region (3Ј UTR). BLAST searches RNA was isolated from cultured cells or frozen liver by using placed the EST sequence on a mouse bacterial artificial chro- RNeasy kits (Qiagen) and treated with DNase I (DNA-free from mosome (BAC) clone (AC020796), about 3 kb downstream of a Ambion, Austin, TX). Five micrograms of RNA was reverse 230-bp coding sequence homologous to START genes. This ␮ transcribed in 20 l by using random hexamers and Superscript 230-bp sequence (the coding part of exon 6, see below) was in II (Invitrogen). To perform relative quantification of gene multiple mouse ESTs, allowing assembly of a 675-bp ORF by in expression with a standard curve, the genes of interest and the silico EST walking. The mouse gene encodes a 224-aa protein endogenous control gene (cyclophilin) were amplified in sepa- consisting almost entirely of a START domain, so it was named rate tubes for each cDNA sample. Duplicate 50-␮l PCRs were stard4. The human STARD4 orthologue encodes 205 amino acids carried out with 1ϫ Jumpstart PCR buffer, 1.00 unit of Jump- 87% identical to the mouse protein. ␮ start Taq DNA polymerase (Sigma), 3.5 mM MgCl2, 200 M Mouse and human StarD4 ORFs were RT-PCR amplified each dNTP, 300 nM forward primer, 300 nM reverse primer, and from liver, cloned, and sequence-verified. The sequence of 100 nM 6-carboxyfluorescein (6FAM)-labeled TaqMan probe. mouse StarD4 from C57BL͞6 disagreed with some ESTs at ORF TaqMan primer pairs and probes (sequences in Table 3) were positions 121 and 152. Since other ESTs agreed with the designed with Primer Express (Applied Biosystems) to span C57BL͞6 sequence, StarD4 was cloned from a second inbred

6944 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.052143799 Soccio et al. Downloaded by guest on September 27, 2021 Fig. 2. Sterol regulation of StarD4 expression. (A) Regulation by dietary cholesterol in mouse liver. C57BL͞6 female mice were fed a semisynthetic diet with no cholesterol (0.00%, n ϭ 5) or the same diet supplemented to 0.50% cholesterol (n ϭ 5). StarD4 expression was measured in each liver by quanti- tative RT-PCR. (B and C) Regulation by sterols in cultured 3T3 cells. 3T3-L1 cells were cultured in one of three media: control, sterols, or statin (n ϭ 4 wells each, see Materials and Methods). Expression of StarD4, hydroxymethylglu- taryl-CoA reductase (HMGR) and synthase (HMGS), low density lipoprotein receptor (LDLR), and ATP:citrate lyase (ACLY) was measured in each well. All data (mean Ϯ SD) are normalized to cyclophilin and control condition set equal to 100% for each gene. *, P Ͻ 0.001 vs. control, **, P Ͻ 0.05 vs. control. Fig. 1. Multiple-tissue Northern blots of mouse StarD4. (a) Schematic of the mouse StarD4 mRNA, at least 5 kb long with 3.2 kb of 3Ј UTR between the ORF and EST AA239481 sequence. A mouse multiple-tissue Northern blot was A Subfamily of START Genes Including StarD4, StarD5, and StarD6. hybridized with three probes: StarD4 ORF (B), EST AA239481 insert (C), or BLAST searches against the complete and EST ␤-actin control for RNA loading (D). Longer exposures revealed the Ϸ5.5-kb StarD4 band in skeletal muscle. databases identified 15 START domain-containing genes. Brown fat inducible thioesterase (BFIT) gene is alternatively spliced to encode two C termini (14), so the amino acid mouse strain, FVB. Sequencing confirmed that these nucleotide sequences of 16 human START domains were aligned (Fig. 3A). and amino acid positions are indeed polymorphic between Two other uncharacterized START proteins, which were named strains: C57BL͞6 has guanine-121, cytosine-152 (Glu-41, Ala- StarD5 and StarD6, shared 26–32% identity with StarD4 and 51), whereas FVB has adenine-121, thymine-152 (Lys-41, Val- with one another. These three proteins formed a subfamily 51). Based on the StarD4 crystal structure (13), both side chains among START proteins, sharing only 16–21% identity to MLN64 and StAR and 14% or less with PCTP and other START

are surface exposed and do not contribute to the putative MEDICAL SCIENCES domains (Fig. 3B). A phylogenetic tree divided these human lipid-binding tunnel, so it is uncertain whether these polymor- START domains into six distinct subfamilies (Fig. 3C). The three phisms have functional consequences. novel proteins formed the StarD4 subfamily, and the nearest Northern blots were performed to verify that the array EST branch included StAR and MLN64. The other four subfamilies and ORF sequences were present on the same mRNA (Fig. 1A). were proteins with PCTP-like START domains, proteins with On a mouse multiple-tissue Northern blot, a StarD4 ORF probe Ϸ N-terminal Rho GTPase activator protein (RhoGAP) domains, hybridized to a predominant mRNA at 5.5 kb in all eight proteins with N-terminal acyl-CoA hydrolase (ACH) domains, tissues, with the highest levels in liver and kidney (Fig. 1B). The Ϸ and the hypothetical protein KIAA1300. Additional subfamilies array EST insert probe also hybridized to this 5.5-kb mRNA of START proteins are present only in plants (13). with the same relative tissue levels (Fig. 1C). The human and mouse sequences for StarD4, StarD5, and StarD6 were analyzed in silico by using resources available Sterol Regulation of StarD4 Expression in Vivo and in Vitro. To publicly through the National Center for Biotechnology Infor- confirm cholesterol-regulated expression of StarD4, quantitative mation (http:͞͞www.ncbi.nlm.nih.gov͞) or commercially RT-PCR primers and probes were designed. Upon feeding 0.5% through Celera Genomics (http:͞͞www.celera.com͞). Full- cholesterol, mouse liver StarD4 mRNA decreased 2- to 3-fold length coding sequences, UniGene clusters, and chromosomal (Fig. 2A), as expected from the microarray data. In addition, 3T3 positions were determined (Table 2). The three genes do not cells were cultured in sterols to repress SREBP activation, and reside together in a cluster, but the mouse and human ortho- StarD4 expression decreased 4-fold compared with the control logues for each gene reside in syntenic chromosomal regions. media. Conversely, when 3T3 cells were cultured in lovastatin (to While there is only one copy per genome of StarD4 and StarD5, inhibit cholesterol biosynthesis, deplete cellular sterols, and Celera mouse 10 includes an intronless StarD6 activate SREBPs), StarD4 expression increased 3.4-fold com- sequence, perhaps a processed pseudogene. Mouse StarD6 ORF pared with control media and 14-fold compared with sterol- PCR primers in different exons amplified a strong Ϸ700-bp containing media (Fig. 2B). Four known SREBP-target genes product from genomic DNA (data not shown), confirming this (2) showed the same pattern of regulation (Fig. 2C). intronless gene.

Soccio et al. PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 ͉ 6945 Downloaded by guest on September 27, 2021 Fig. 3. StarD4, StarD5, and StarD6 are a subfamily among START domains. (A) The START domains of 16 human proteins were aligned by using CLUSTALW. The colored bars above the alignment indicate positions with a strong consensus. Amino acid agreements with StarD4 are boxed, while agreements with consensus are colored yellow. (B) The percent amino acid identity for each pairwise comparison in the alignment. Yellow indicates the similarity between the StarD4 subfamily (violet) and the StAR͞MLN64 subfamily (blue). The remaining 9 proteins fall into four groups: PCTP-like (orange), Rho GTPase activator protein (RhoGAP) domain-containing (green), acyl-CoA hydrolase (ACH) domain-containing (pink), and other (gray). (C) A phylogenetic tree based on the alignment shows the same subfamilies. DNASTAR software (Lasergene, Madison, WI) was used for this analysis.

To determine the exonic organization of each gene, cDNA StarD5 or StarD6. Upstream in-frame stop codons allow contigs and genomic sequences were aligned to reveal introns identification of initiator codons in StarD4 and StarD6, that followed the GT-AG rule. The StarD4 subfamily genes whereas StarD5 has no upstream exons in over 180 mouse and were remarkably similar, with most splice junctions conserved human Unigene ESTs. The mouse StarD6 mRNA has a long, (Fig. 4, splice junction sequences in Table 4, which is published multiexon 5Ј UTR with multiple initiation codons, and such as supporting information on the PNAS web site). The exonic upstream AUGs may function as regulators of translation (15). organization is different for the MLN64, StAR, and PCTP Whereas most other START proteins have additional genes (not shown), supporting a distinct StarD4 subfamily. N-terminal domains (6), StarD4, StarD5, and StarD6 are Mouse StarD4 has 12 aa encoded by exon 1, but this exon is 205–233 aa proteins consisting almost entirely of START noncoding in human StarD4 and there are no counterparts in domains.

6946 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.052143799 Soccio et al. Downloaded by guest on September 27, 2021 Table 2. Gene information for human (h) and mouse (m) StarD4, StarD5, and StarD6 GenBank Genetic map Northern mRNA ORF Protein Gene accession no. UniGene cluster(s) Celera gene Chromosome position, cM length, kb length, bp length, aa

mStarD4 AF480297–8 Mm. 31508, 23344 mCG21633 18 12 Ϸ5.5 675 224 hStarD4 AF480299 Hs.162205 hCG37443 5q22 116–121 Ϸ5.5, Ϸ4.5 618 205 mStarD5 AF480302 Mm. 25702 mCG8260 7 41 Ϸ3.0, Ϸ1.5 642 213 hStarD5 AF480304 Hs. 172803 hCG27342 15q26 72–77 Ϸ3.0, Ϸ1.5 642 213 mStarD6 AF480303 Mm. 83623, 195613 mCG9256 18 44 Ϸ1.5 702 233 hStarD6 AF480305 Hs. 304542, 143962 hCG1643548 18q21 71–83 Ϸ1.5 663 220

cM, centimorgan.

Expression of StarD5 and StarD6. StarD5 and StarD6 ORFs were Three lines of evidence support the hypothesis that StarD4, RT-PCR amplified and cloned from liver and testis, respectively, StarD5, and StarD6 bind cholesterol or other sterols. First, like and used to probe mouse multiple-tissue Northern blots. Like many other genes in cholesterol metabolism, StarD4 is regulated StarD4, StarD5 messages showed ubiquitous expression, highest by sterols, and a functional sterol-regulatory element has been in liver and kidney (Fig. 5A). In contrast, StarD6 expression was identified in the StarD4 promoter (R.E.S., unpublished results). restricted to testis (Fig. 5 B and C). The mRNA lengths revealed Because SREBPs also regulate genes involved in the metabolism by Northern blots (Table 2) agree well with predictions based on of other lipids (2), nonsterols cannot be ruled out as potential the AAUAAA polyadenylation signals in the 3Ј UTRs (Fig. 4), ligands. Second, these three proteins are most homologous to and multiple messages appear to represent alternative sites of StAR and MLN64, which bind cholesterol, and more distantly RNA polyadenylation and cleavage. related to PCTP and other START domains, which bind phos- Finally, quantitative RT-PCR was performed to determine pholipids and perhaps other lipids. Third, the crystal structure of whether the widely expressed START genes StarD5 and MLN64 StarD4 shows a hydrophobic tunnel with dimensions similar to are sterol-regulated like StarD4. Neither StarD5 nor MLN64 those of MLN64 (13). StarD4, StarD5, and StarD6 may be cytosolic lipid carriers like PCTP (16), since all four lack expression was regulated in mouse liver upon cholesterol feeding additional N-terminal domains or other localization signals or in 3T3 cells upon culture in sterols (data not shown). [based on PROSITE searches (17)]. Discussion Given their ubiquitous expression, we speculate that StarD4 and StarD5 may be involved in fundamental processes such as This study describes StarD4, a sterol-regulated gene encoding intracellular sterol transport or cholesterol biosynthesis. While a StAR-related lipid transfer protein. StarD4 was initially some cholesterol transport is vesicular, cytosolic protein carriers identified by using cDNA microarrays, as liver StarD4 expres- have been proposed for other transport events, such as move- sion decreased more than 2-fold in mice fed a high-cholesterol ment of newly synthesized cholesterol to the plasma membrane diet. In cultured 3T3 cells, StarD4 expression was likewise (3) and plasma membrane cholesterol to the endocytic recycling coordinately regulated by sterols with known SREBP-target compartment (18). A START protein could also mediate sterol genes. Two other START proteins, StarD5 and StarD6, were transport to mitochondrial sterol 27-hydroxylase (Cyp27) (19). identified on the basis of homology to StarD4, and these three In cholesterol biosynthesis, squalene, lanosterol, and every sub- constituted a distinct subfamily of START proteins. StarD4 sequent intermediate are hydrophobic molecules that may re- and StarD5 were ubiquitously expressed, with highest levels in quire protein carriers. The squalene carrier supernatant protein liver and kidney, whereas StarD6 was expressed exclusively in factor (SPF) stimulates the enzymatic activity of squalene ep- the testis. oxidase, transfers squalene between membranes in vitro, and MEDICAL SCIENCES

Fig. 4. Exonic organization of StarD4, StarD5, and StarD6. Exons for the mouse (m) and human (h) genes, represented as boxes with lengths in nucleotides, were determined by alignment of cDNA with genomic DNA. Vertical bars show splice junctions that are conserved among the genes. Coding sequences are white and UTRs are gray. The 5Ј UTR lengths were based on the longest 5Ј sequences in each UniGene cluster, whereas the 3Ј UTR lengths were estimated on the basis of Northern blot mRNA sizes (Table 2). #, First upstream in-frame stop codon in 5Ј UTR; @, upstream AUG codon in 5Ј UTR; *, poly(A) signal (AAUAAA) in the 3Ј UTR. The distances from the stop codon for each poly(A) signal are indicated at the far right. The 5Ј end of human StarD6 is uncertain because very little sequence data are available for this gene.

Soccio et al. PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 ͉ 6947 Downloaded by guest on September 27, 2021 Testis-specific expression suggests a specialized role for StarD6, although a StAR-like role in steroidogenesis is unlikely because it is not expressed in the ovary. While it may also be expressed in Leydig or Sertoli cells, StarD6 appears to be present in the germ cells because several mouse ESTs have been cloned from spermatocyte or spermatid libraries. Cholesterol precursor sterols may have important roles in reproduction. Meiosis- activating sterols (MAS) stimulate resumption of meiosis (21), and testis-MAS (T-MAS, the cholesterol precursor 4,4-dimethyl- 5␣-cholesta-8,24-dien-3␤-ol) accumulates in postpubertal testis (22). Other postlanosterol sterols are surprisingly abundant in the testis and epididymis (23), where they may play a role in sperm maturation. If StarD6 binds a sterol involved in meiosis or germ cell development, this gene may be important for fertility. In summary, the lipid-binding protein StarD4 was identified by microarray analysis of gene expression in cholesterol-fed mouse liver. StarD5 and StarD6 were then identified on the basis of homology to StarD4, and these three genes form a subfamily within the START family. We hypothesize that the StarD4 subfamily is involved in the intracellular metabolism of choles- terol or other sterols, although it is possible that they bind other lipids instead. Since StarD4, StarD5, and StarD6 are only Ϸ30% identical to one another, each may bind specifically to a distinct lipid. Further studies including the generation of knockout mice should help to elucidate the physiological functions of these Fig. 5. Multiple-tissue Northern blots of StarD5 and StarD6. (A) Mouse blot hybridized with a mouse StarD5 ORF probe. (B) Mouse blot hybridized with a three proteins. mouse StarD6 ORF probe. (C) Human blot hybridized with a mouse StarD6 ORF probe (sm. int., small intestine; PBL, peripheral blood leukocyte). We thank Raju Kucherlapati, Joerg Heyer, and Sandra Merscher for assistance with cDNA microarray technology, Zhihua Han for help with Northern blots, Michael Sinensky and Doug Thewke for useful discus- stimulates cholesterol biosynthesis when overexpressed (20). sions, and Jonathan Smith, Kara Maxwell, and Leslie Castelo-Soccio for StarD4 could serve an analogous function for a postsqualene critical reading. This work was supported by National Institutes of Health sterol intermediate, as StarD4 shows coordinate regulation with Medical Scientist Training Program Grant GM07739 (R.E.S.) and by cholesterol biosynthetic enzymes. National Institutes of Health Grant HL32435 (J.L.B.).

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