Interaction of Hormone-sensitive Lipase with Steroidogeneic Acute Regulatory Protein
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m303934200
ISSN1083-351X
AutoresWen‐Jun Shen, Shailja Patel, Vanita Natu, Richard Hong, Jenny Wang, Salman Azhar, Fredric B. Kraemer,
Tópico(s)Pancreatic function and diabetes
ResumoHormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis. Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis. Neutral cholesteryl ester hydrolase activity can be demonstrated in most cells, including adipose tissue, adrenal, testes, placenta, macrophages, heart, skeletal and smooth muscles; steroidogenic tissues are especially enriched in this activity (1Kraemer F.B. Shen W.-J. J. Lipid Res. 2002; 43: 1585-1594Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Several lines of evidence suggest that hormone-sensitive lipase (HSL) 1The abbreviations used are: HSL, hormone-sensitive lipase; ALBP, adipocyte lipid-binding protein; Bt2cAMP, dibutyryl cAMP; CHO, Chinese hamster ovary; CMV, cytomegalovirus; GST, glutathione S-transferase; HDL3, human high density lipoprotein3; LDL, low density lipoprotein; StAR, steroidogenic acute regulatory protein; START domain, StAR-related lipid transfer domain; TGH, triacylglycerol hydrolase. is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. The most direct and convincing evidence comes from HSL knockout mice, where no detectable HSL protein and no neutral cholesteryl ester hydrolase activity are observed in the adrenal (2Kraemer F.B. Shen W.-J. Natu V. Patel S. Osuga J.-i. Ishibashi S. Azhar S. Endocrinology. 2002; 143: 801-806Crossref PubMed Scopus (51) Google Scholar) or testis (3Osuga J.-i. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoira F. Yahagi N. Kraemer F.B. Tsutsumi O. Yamada N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 787-792Crossref PubMed Scopus (503) Google Scholar). It is believed that through its action as a neutral cholesteryl ester hydrolase, HSL is involved in regulating intracellular cholesterol metabolism and, thus, contributing to a variety of pathways in which cells utilize cholesterol. The primary amino acid sequence of HSL is unrelated to any of the other known mammalian lipases; however, it shares some sequence similarity with liver arylacetamide deacetylase within its catalytic domain (4Probst M.R. Beer M. Beer D. Jenö D. Meter U.A. Randolfo G. J. Biol. Chem. 1994; 269: 21650-21656Abstract Full Text PDF PubMed Google Scholar). The C-terminal portion of HSL displays secondary structural homology with that of acetylcholinesterase and several fungal lipases (5Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and bacterial brefeldin A esterase (6Wei Y. Contreras J.A. Sheffield P. Osterlund T. Derewenda U. Kneusel R.E. Matern U. Holm C. Derewenda Z.S. Nat. Struct. Biol. 1999; 6: 340-345Crossref PubMed Scopus (147) Google Scholar), consisting of parallel β-sheets flanked by α-helical connections, which has allowed these proteins to be classified as α/β-hydrolases (7Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1845) Google Scholar). Using limited proteolysis, it has been suggested that HSL is composed of two major structural domains (8Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (138) Google Scholar, 9Østerlund T. Beussman D.J. Julenius K. Poon P.H. Linse S. Shabanowitz J. Hunt D.F. Schotz M.C. Derewenda Z.S. Holm C. J. Biol. Chem. 1999; 274: 15382-15388Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Based on sequence alignment, structural homology with fungal lipases, and mutational analyses, the C-terminal domain has been shown to contain the catalytic triad and other residues important in hydrolytic activity, as well as a 150-amino acid insert that has been termed the regulatory module because several serines located within this region have been shown to be phosphorylated (1Kraemer F.B. Shen W.-J. J. Lipid Res. 2002; 43: 1585-1594Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 10Østerlund T. Eur. J. Biochem. 2001; 268: 1899-1907Crossref PubMed Scopus (58) Google Scholar). The N-terminal domain in rat HSL constitutes the first 323 amino acids, which are encoded by exons 1–4, and displays no sequence or structural similarity with any other known proteins (8Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (138) Google Scholar, 9Østerlund T. Beussman D.J. Julenius K. Poon P.H. Linse S. Shabanowitz J. Hunt D.F. Schotz M.C. Derewenda Z.S. Holm C. J. Biol. Chem. 1999; 274: 15382-15388Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We (11Shen W.-J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5528-5532Crossref PubMed Scopus (182) Google Scholar) and others (12Syu L.-J. Saltiel A.R. Mol. Cell. 1999; 4: 109-115Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) have shown that HSL interacts specifically with intracellular proteins in adipose tissue. The interaction of HSL with adipocyte lipid-binding protein (ALBP) occurs through amino acid residues within the N-terminal domain; the physical interaction of ALBP with HSL increases the hydrolytic activity of HSL and protects HSL from product inhibition by fatty acids (13Shen W.-J. Liang Y. Hong R. Patel S. Natu V. Sridhar K. Jenkins A. Bernlohr D.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 49443-49448Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In the same way that the interaction of HSL with ALBP might help to facilitate the trafficking of fatty acids in adipose cells, HSL might interact with specific cholesterol carrier proteins in the adrenal and, thus, facilitate intracellular cholesterol trafficking to mitochondria for steroidogenesis. The steroidogenic acute regulatory protein (StAR) is synthesized as a 37-kDa protein that is targeted to and processed in mitochondria to a 30-kDa mature protein that facilitates the movement of cholesterol from the outer mitochondrial membrane to the cholesterol side chain cleavage enzyme (CYP11A1) on the inner mitochondrial membrane (14Miller W.L. Strauss III, J.F. J. Steroid Biochem. Mol. Biol. 1999; 69: 131-141Crossref PubMed Scopus (145) Google Scholar, 15Stocco D.M. Annu. Rev. Physiol. 2001; 63: 193-213Crossref PubMed Scopus (695) Google Scholar). Identification of mutations in humans (16Bose H.S. Sugawara T. Strauss III, J.F. Miller W.L. N. Engl. J. Med. 1996; 335: 1870-1878Crossref PubMed Scopus (525) Google Scholar) and mouse knockout experiments (17Caron K.M. Soo S.C. Wetsel W.C. Stocco D.M. Clark B.J. Parker K.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11540-11545Crossref PubMed Scopus (382) Google Scholar) have implicated StAR as a critical regulatory step in steroidogenesis. StAR has been shown to possess sterol transfer activity in vitro (18Kallen C.B. Billheimer J.T. Summers S.A. Stayrook S.E. Lewis M. Strauss III, J.F. J. Biol. Chem. 1998; 273: 26285-26288Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 19Petrescu A.D. Gallegos A.M. Okamura Y. Strauss III, J.F. Schroeder F. J. Biol. Chem. 2001; 276: 36970-36982Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The crystal structure of StAR shows that it has a classic lipid transporter-like structure (20Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (446) Google Scholar, 21Romanowski M.J. Soccio R.E. Breslow J.L. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6949-6954Crossref PubMed Scopus (141) Google Scholar), which is characterized by internal hydrophobic cavities, clefs, or tunnels. In this paper, we demonstrate a direct interaction of HSL with StAR in both in vitro and in vivo physiological conditions. HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in CHO cells results in higher hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from stored lipid droplets to mitochondria for steroidogenesis. Chemicals and Reagents—Reagents were obtained from the following sources. Bovine serum albumin (fraction V) was from Intergen Co., Purchase, NY. Fetal bovine serum was from Gemini Bio-Products, Inc., Calabasas, CA. Coon's F-12/Dulbecco's modified Eagle's media and Lipofectin reagent were from Invitrogen. ECL Western blotting detection reagents, horseradish peroxidase-linked whole antibody anti-rabbit IgG, GST-glutathione agarose beads, [35S]methionine, and cholesteryl [1-14C]oleate were from Amersham Biosciences. Protein assay reagent was from Bio-Rad. Nitrocellulose paper was from Schleicher & Schuell. The TNT®Transcription/Translation System was from Promega, Madison, WI. Sf9 cells, Sf21 cells, TNM-FH insect medium, baculovirus transfer vector pAcGHLT-A, and BaculoGold-linearized baculovirus DNA were from Pharmingen. Nickel-nitrilotriacetic acid-agarose was from Qiagen, Valencia, CA. The QuikChange mutagenesis kit was from Stratagene, La Jolla, CA. Organic solvents were from J. T. Baker, Phillipsburg, NJ. Thin layer chromatography (TLC) plates were from Whatman. Apolipoprotein E-free human high density lipoprotein3 (HDL3) was isolated and characterized as described previously (22Reaven E. Shi X.Y. Azhar S. J. Biol. Chem. 1990; 265: 19100-19111Abstract Full Text PDF PubMed Google Scholar). Deletional and Mutational Constructs of StAR—PCR was used to produce deletional constructs from the C terminus as well as N terminus of StAR (a kind gift from Dr. J. Strauss, III, University of Pennsylvania). For the StAR C-terminal deletional constructs, the 5′-primer was the Sp6 primer, and the 3′-primers were: StAR 1–225, 5′-GAT GAT GCT CTT GGG CAG CC; StAR 1–205, 5′-CCC GAA GTC TGT GTC CAT GCC; StAR 1–155, 5′-GAC CTT GAT CTC CTT GAC ATT GGG; StAR 1–100, 5′-ACT CTC CTT CTT CCA GCC CTC TTG; StAR 1–62, 5′-CCG AGA ACC GAG TAG AGA GCT CCG. For the N-terminal deletional constructs, the 3′-primer contained the 3′-sequence of StAR and a stop codon, 5′-TCCCGGGAATTCCTCAACACCTGGCTTCAGA. The 5′-primers were: StAR -62, 5′-GTA CCT AGG ATC CAG CTG GAA GAG ACT CTC TAC; StAR -100, 5′-GTA CCT AGG ATC CAG CAG CAG GAC AAT GGG GAC; StAR -155, 5′-GTA CCT AGG ATC CAG GTC CTG CAG AAG ATC GGA; StAR -205, 5′-GTA CCT AGG ATC CAG TTC GGG AAC ATG CCT GAG; StAR -225, 5′-GTA CCT AGG ATC CAG GTG CTT CAC CCG TTG GCT. The PCR products of the N-terminal deletional constructs were cloned into the BamHI and EcoRI sites of pcDNA3 vector. StAR truncations containing the internal fragment of StAR were made by amplifying the internal sequences and cloning into pcDNA3-HisB vector. The 5′-primer used for the StAR constructs containing the internal fragments was 5′-CAG CAG GAC AAT GGG GAC AAA GTG ATG. The 3′-primers were: StAR 100–182, 5′-TGA ACG GGG CCC CAC CAG GTT TCC TGC; StAR 100–221, 5′-TGA ACC GTG CAC CGC CCT GAT GAC; StAR 100–241, 5′-TGA CCA CGT AAG TTT GGT CTT AGA. The mutant construct in which amino acids 181–221 were deleted from StAR was generated with the QuikChange mutagenesis kit using the following primers: 5′-CCC CAC CAG GTT TCC TGC TGC CTC and 3′-CCC ACT TGC ATG GTG CTT CAC CCG. The identities of the mutant constructs were confirmed by DNA sequencing using an ABI prism DNA sequencer. In Vitro Protein-Protein Interaction—After sequence confirmation of the StAR mutants, the StAR constructs were in vitro translated with [35S]methionine using the TNT®Transcription/Translation System. GST-HSL and GST protein were produced as described previously (11Shen W.-J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5528-5532Crossref PubMed Scopus (182) Google Scholar). GST-HSL or GST alone was incubated with glutathione-agarose beads in buffer B (20 mm Tris, pH 8.0, 0.15 m NaCl, 1 mm EDTA, 0.5% Nonidet P-40). After a 1-h incubation at room temperature, the beads were washed three times in buffer B and then incubated with [35S]methionine-labeled StAR proteins. After a 1-h incubation at room temperature, the beads were washed five times in buffer B, and proteins that bound to the beads were eluted in SDS-PAGE sample buffer, separated on SDS in 10% PAGE, and visualized using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Immunoprecipitation and Immunoblotting—Male Sprague-Dawley rats (8 weeks old) were injected with saline or ACTH (25 μg) and the adrenals harvested 1 and 2 h after the injection. Adrenals were homogenized in TES buffer (20 mm Tris-HCL, 1 mm EDTA and 8% sucrose, with 2 μg/ml leupeptin, and okadaic acid) and centrifuged at 10,000 × g for 15 min. The supernatant was used for immunoprecipitation and protein determination. Immunoprecipitation of HSL was performed as described (11Shen W.-J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5528-5532Crossref PubMed Scopus (182) Google Scholar). A 250-μg aliquot was precleared with protein A beads and then incubated with an immunomatrix consisting of rabbit polyclonal anti-HSL/fusion protein IgG and protein A. After overnight incubation at 4 °C, the immune complex was centrifuged at 10,000 × g for 15 min and washed twice in phosphate-buffered saline with 0.05% bovine serum albumin and then twice in phosphate-buffered saline. The pellet was resuspended in SDS-PAGE loading buffer (0.063 m Tris-HCL, pH 6.8, with 1% 2-mercaptoethanol, 1% SDS, and 13% (v/v) glycerol), boiled for 5 min, electrophoresed on 15% SDS-PAGE, transferred to nitrocellulose paper, and immunoblotted with rabbit anti-StAR IgG (a kind gift from Dr. J. Strauss, III). Cell Culture and Transfection—CHO cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C under 5% CO2. For transient transfection experiments, cells were subcultured at a density of 2 × 105 cells/well in six well plates the day prior to incubation with 0.75 μg of pCMV-StAR or StAR mutants, 0.75 μg of pcDNA3-HSL, and 0.25 μg of pCMV-β-galactosidase in 10 μl of Lipofectin reagent following the procedure from the manufacturer. Cells were harvested 40 h after transfection for measurement of HSL activity. Y1-BS1 adrenocortical cells were grown in F-10 medium supplemented with 12.5% horse serum, 2.5% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C under 5% CO2. For transient transfection experiments, cells were subcultured at a density 1 × 106 cells/plate in 10-cm tissue culture plates the day prior to transfection. Each plate was transfected with either 5 μg of vector alone, pcDNA3-HSL, or pBK-CMV-TGH (a kind gift from Dr. Dennis Vance, University of Alberta, Canada) using Lipofectin reagent. 24 h after transfection, cells were incubated in medium containing lipoprotein-deficient serum supplemented with 500 μg of protein/ml HDL3 and 2.5 mm Bt2cAMP for 24 h. Cells were then changed to serum-free medium with 0.2% bovine serum albumin overnight and stimulated with 2.5 mm Bt2cAMP in the presence of 100 μm aminoglutethimide for 2 h before harvesting in TES buffer. Mitochondria Preparation—Mitochondria of Y1-BS1 cells were prepared as described (23DiBartolomeis M.J. Williams C. Jefcoate C.R. J. Biol. Chem. 1986; 261: 4432-4437Abstract Full Text PDF PubMed Google Scholar) with some modifications. Harvested Y1-BS1 cells were centrifuged at 200 × g for 10 min and then washed once with phosphate-buffered saline. The cell pellet was resuspended in a hypotonic buffer (10 mm Tris-HCL, pH 7.5, with 10 mm KCl, 0.5 mm EDTA) and cells were allowed to swell for 10 min at 4 °C. After the incubation, cells were gently broken with several passes in a loose fitting glass-Teflon homogenizer. Phosphate buffer and sucrose were added to final concentrations of 50 and 100 mm, respectively, to maintain an isotonic suspension. Aliquots of the homogenate were removed for total cholesterol and protein determinations. To prepare the mitochondrial fraction, the homogenates were centrifuged at 600 × g for 10 min at 4 °C, and the supernatants were again centrifuged at 10,000 × g for 15 min at 4 °C. The pellet containing the mitochondrial fraction was resuspended in mitochondrial stabilizing buffer (10 mm potassium phosphate, pH 7.2, 20 mm KCl, 15 mm triethanolamine hydrochloride, 0.1 mm EDTA, and 250 mm sucrose). Measurement of Cholesterol Content—Cholesteryl ester and free cholesterol content were measured using the Infinity" cholesterol measurement kit from Sigma after their separation by TLC. For TLC, lipids from isolated mitochondrial fractions were extracted by the method of Bligh and Dyer (24Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42828) Google Scholar). After centrifugation, the lower organic solvent phase was transferred, air dried, and dissolved in 50 μl of toluene. An aliquot was then applied to 20 × 20-cm TLC plates and developed sequentially with chloroform:methanol:water 60:40:10 (v/v/v) to 1 cm and then hexane:ether:acetic acid 85:15:2 (v/v/v) to 13 cm. Cholesterol and cholesteryl ester standards were applied on a separate lane on the TLC plates. The cholesterol and cholesteryl ester spots were visualized by brief exposure to iodine vapor, eluted, and measured enzymatically as described previously (2Kraemer F.B. Shen W.-J. Natu V. Patel S. Osuga J.-i. Ishibashi S. Azhar S. Endocrinology. 2002; 143: 801-806Crossref PubMed Scopus (51) Google Scholar). Other Measurements—Measurement of HSL activity was performed using a cholesteryl [14C]oleate emulsion as described previously (25Prokocimer P.G. Maze M. Vickery R.G. Kraemer F.B. Gandjei R. Hoffman B.B. Mol. Pharmacol. 1988; 33: 338-343PubMed Google Scholar). Protein concentration was assayed using Bio-Rad protein assay reagent. Recombinant GST-HSL was produced in baculovirus as described previously (11Shen W.-J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5528-5532Crossref PubMed Scopus (182) Google Scholar). Statistical Analysis—Data are expressed as the mean ± S.E. Statistical analyses were performed by unpaired two-tailed Student's t test using InStat (GraphPad Software, San Diego) software for Macintosh. HSL Interacts with StAR in Vitro—To determine whether HSL might interact with StAR, full-length StAR that was [35S]methionine-labeled by in vitro translation was incubated with either rat GST-HSL or with GST alone. The mixture was incubated with glutathione-agarose beads, and the proteins that bound to the beads were washed, eluted, and separated on SDS-PAGE, and then visualized. Results are shown in Fig. 1. Whereas GST protein alone does not appear to interact with StAR, GST-HSL was able to pull down StAR. Thus, these results suggest that HSL is capable of specifically interacting with StAR in vitro. HSL Interacts with StAR in Vivo—To document that the interaction of HSL with StAR occurs in vivo, male rats were injected with ACTH to induce StAR expression or with saline. The adrenals were harvested 1 and 2 h after the injection, and HSL was immunoprecipitated from extracts of the adrenals with anti-HSL antibodies. The immunoprecipitated complexes were separated on SDS-PAGE and immunoblotted with anti-StAR antibodies. As shown in Fig. 2, anti-StAR antibodies recognize StAR in extracts of control adrenals as a 30-kDa protein, reflecting the mature protein after its processing in mitochondria. Interestingly, the 30-kDa StAR is also detected in the immune complexes immunoprecipitated with anti-HSL antibodies from adrenal tissue prior to ACTH treatment (lane 2), supporting an interaction of HSL with StAR in vivo. 1 h after injection of ACTH, the 37-kDa StAR, which is the product initially synthesized in the endoplasmic reticulum prior to transport and processing in mitochondria to the 30-kDa form, is detected in the immune complexes of adrenal tissue immunoprecipitated with anti-HSL antibodies, confirming the interaction of HSL with StAR in vivo. By 2 h after ACTH injection, the 37-kDa product is no longer detected, reflecting the short half-life of the 37-kDa StAR protein. In contrast, adrenal tissues immunoprecipitated with nonimmune antibodies do not have any detectable StAR, establishing that StAR was not found in the anti-HSL immune complexes because of nonspecific interactions during the immunoprecipitation. The intense signal in the lane immunoprecipitated with nonimmune IgG represents IgG heavy chains that are detected by the chemiluminescence assay. The IgG heavy chains do not appear in the lanes immunoprecipitated with anti-HSL IgG because an immunomatrix consisting of anti-HSL IgG cross-linked to protein A was used which prevents free heavy chains from appearing in the supernatant after the pelleted immune complex is solubilized. Region of StAR Participating in the Interaction with HSL—To explore the structural determinants in StAR that are mediating its interaction with HSL, a series of C-terminal deletions, StAR 1–225, StAR 1–205, StAR 1–155, StAR 1–100, and StAR 1–62, was generated, and the interactions of these in vitro translated products with HSL were examined (Fig. 3). Interestingly, although none of the constructs interacted with GST alone, all constructs were specifically pulled down by GST-HSL, suggesting that the N-terminal 62 amino acids of StAR, which contains the signal that targets StAR to the mitochondria and which is cleaved in the mitochondria, can participate in the interaction with HSL. In view of the ability of the 30-kDa StAR to be coimmunoprecipitated with HSL, it is apparent that regions in addition to the N terminus of the protein must also be involved in the interaction with HSL. Therefore, a series of N-terminal deletions of StAR was generated and their ability to interact with HSL tested (Fig. 4). Deleting the first 62 amino acids of StAR did not affect its ability to be pulled down by GST-HSL, suggesting that the N terminus is not the only region participating in the interaction with HSL. Deleting the first 155 amino acids of StAR also did not affect its ability to be pulled down by GST-HSL; however, N-terminal deletions of the first 205 and 225 amino acids did eliminate the interaction. When C- and N-terminal deletions were combined (Fig. 5), StAR 100–241 and StAR 100–221 still retained their ability to bind to HSL; however, StAR 100–182 did not. Thus, it appears that sites within the first 62-amino acid N terminus and within the region between amino acids 182 and 205/221 are able to mediate the interaction of StAR with HSL.Fig. 4Interaction of HSL with N-terminal StAR deletions. N-terminal truncations of StAR were generated as described under "Experimental Procedures." A, pcDNA3-StAR 62–286 was in vitro translated and incubated with GST (lane 2) or GST-HSL (lane 3). B, pcDNA3-StAR 155–286 (lanes 1, 4, and 5), pcDNA3-StAR 205–286 (lanes 2, 6, and 7), and pcDNA3-StAR 225–286 (lanes 3, 8, and 9) were in vitro translated and incubated with GST (lanes 4, 6, and 8) or GST-HSL (lanes 5, 7, and 9) as described in Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Interaction of HSL with combined N- and C-terminal StAR deletions. StAR 100–241 (lanes 1, 4, and 5), StAR 100–221 (lanes 2, 6, and 7), and StAR 100–182 (lanes 3, 8, and 9) were in vitro translated and incubated with either GST (lanes 4, 6, and 8) or GST-HSL (lanes 5, 7, and 9) as described in Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) StAR Increases HSL Hydrolytic Activity—The interaction of ALBP with HSL is reported to increase the hydrolytic activity of the enzyme (13Shen W.-J. Liang Y. Hong R. Patel S. Natu V. Sridhar K. Jenkins A. Bernlohr D.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 49443-49448Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The ability of ALBP to increase substrate hydrolysis by HSL is not due entirely to the binding and sequestration of fatty acids by ALBP but is dependent on the physical interaction of ALBP with HSL, suggesting that the protein-protein interaction causes a conformational change or steric effect on the enzyme. Although StAR is not known to possess fatty acid binding properties, the fact that StAR does interact with HSL raised the possibility that the interaction of StAR with HSL might modulate HSL hydrolytic activity. To explore this possibility, HSL was expressed in CHO cells, and hydrolytic activity against cholesteryl ester was determined after coexpression of StAR or vector alone (Fig. 6). The amount of HSL expressed under these experimental conditions was similar, as determined by immunoblot (data not shown). Coexpression of HSL and StAR resulted in ∼75% increased hydrolysis (p < 0.001) of cholesteryl ester substrate. Thus, as observed with ALBP, the interaction of StAR with HSL can modulate the hydrolytic activity of the enzyme. To investigate whether there is a correlation between HSL-StAR protein-protein interaction in vitro and HSL function, various deletional mutants of StAR were coexpressed with HSL in CHO cells, and hydrolytic activity against cholesteryl ester was determined (Fig. 7). Deletions of the first 100 or 155 amino acids of StAR, constructs that bind HSL, stimulated hydrolytic activity against cholesteryl ester (p < 0.05) to an extent similar to that of full-length StAR (1–286). Deletion of the first 205 amino acids of StAR appeared to stimulate HSL activity, but this did not reach statistical significance; deletion of the first 225 amino acids, a construct that does not bind HSL, had no effect on HSL activity. Likewise, expression of StAR 100–182, which does not bind HSL, also did not stimulate HSL activity. However, small constructs that do bind HSL (StAR 100–221 and 100–241) also had no effect on HSL activity. Interestingly, a construct in which amino acids 181–221 were deleted from StAR also failed to stimulate HSL hydrolytic activity. Thus, it appears that the simple binding of StAR to HSL is insufficient to explain the effects on hydrolytic activity. HSL Augments Mitochondrial Cholesterol Content in the Presence of StAR—To explore the functional significance of the interaction of HSL with StAR and to test the hypothesis that the interaction of HSL with StAR might facilitate intracellular cholesterol trafficking to mitochondria for steroidogenesis in adrenal cells, we transfected Y1-BS1 adrenocortical cells with HSL or, as a control, triacylglycerol hydrolase. Triacylglycerol hydrolase is a neutral cytosolic lipase that has been reported to possess triacylglycerol (26Lehner R. Vance D.E. Biochem. J. 1999; 343: 1-10Crossref PubMed Scopus (116) Google Scholar) as well as cholesteryl ester (27Ghosh S. Mallonee D.H. Hylemon P.B. Grogan W.M. Biochim. Biophys. 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