The acyl chains of phosphoinositide PIP3 alter the structure and function of nuclear receptor steroidogenic factor-1

Abstract

Nuclear receptors are transcription factors that bind lipids, an event that induces a structural conformation of the receptor that favors interaction with transcriptional coactivators. The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) binds the signaling phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), and our previous crystal structures showed how the phosphoinositide headgroups regulate SF-1 function. However, what role the acyl chains play in regulating SF-1 structure remains unaddressed. Here, we used X-ray crystallography with in vitro binding and functional assays to examine how the acyl chains of PIP3 regulate human SF-1 ligand-binding domain structure and function. Altering acyl chain length and unsaturation regulates apparent binding of all tested phosphoinositides to SF-1. Mass spectrometry–based lipidomics data suggest C16 and C18 phospholipids preferentially associate with SF-1 expressed ectopically in bacteria. We then solved the 2.5 Å crystal structure of SF-1 bound to dioleoyl PIP3(18:1/18:1) to compare it with a matched structure of SF-1 bound to dipalmitoyl PIP3(16:0/16:0). The dioleoyl-bound structure was severely disordered in a specific SF-1 region associated with pathogenic human polymorphisms and within the coactivator-binding region critical for SF-1 function while inducing increased sensitivity to protease digestion in solution. Validating these structural observations, in vitro functional studies showed dioleoyl PIP3 induced 6-fold poorer affinity of a peroxisome proliferator-activated receptor gamma coactivator 1-alpha coactivator peptide for SF-1 compared with dipalmitoyl PIP3. Together, these data suggest the chemical nature of the phosphoinositide acyl chains controls the ordered state of specific, clinically important structural regions in SF-1, regulating SF-1 function in vitro.

Supplementary key wordsAbbreviations: AF2 (activation function 2), DBD (DNA-binding domain), LBD (ligand-binding domain), MRM (multiple reaction monitoring), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), SF-1 (Steroidogenic Factor-1)Nuclear receptors are a superfamily of DNA-binding transcription factors regulated by lipid ligands such as cholesterol-based steroids (Steroid binding and stabilization of glucocorticoid-receptor proteins from rat thymus., Blind R.D. Pineda-Torra I. Xu Y. Xu H.E. Garabedian M.J. Ligand structural motifs can decouple glucocorticoid receptor transcriptional activation from target promoter occupancy.), fatty acids (Bocos C. Göttlicher M. Gearing K. Banner C. Enmark E. Teboul M. Crickmore A. Gustaffson J.Å. Fatty acid activation of peroxisome proliferator-activated receptor (PPAR)., Schoonjans K. Staels B. Auwerx J. The peroxisome proliferator activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation.), and phospholipids (Chakravarthy M.V. Lodhi I.J. Yin L. V Malapaka R.R. Xu H.E. Turk J. Semenkovich C.F. Identification of a physiologically relevant endogenous ligand for PPARa in liver.) among other hydrophobic molecules such as xenobiotics (Blumberg B. Sabbagh W. Juguilon H. Bolado J. Van Meter C.M. Ong E.S. Evans R.M. SXR, a novel steroid and xenobiotic-sensing nuclear receptor.) and heme (Raghuram S. Stayrook K.R. Huang P. Rogers P.M. Nosie A.K. McClure D.B. Burris L.L. Khorasanizadeh S. Burris T.P. Rastinejad F. Identification of heme as the ligand for the orphan nuclear receptors REV-ERBα and REV-ERBβ.). Generally, nuclear receptors have important functions in metabolism, development, homeostasis, signaling, and reproduction in metazoans (Historical overview of nuclear receptors., Sonoda J. Pei L. Evans R.M. Nuclear receptors: Decoding metabolic disease.). One of the 48-member nuclear receptor superfamily in humans is steroidogenic factor-1 (SF-1, NR5A1), which plays an essential role in development and adult function of steroidogenic tissue (Minireview: Steroidogenic Factor 1: Its roles in differentiation, development, and disease.). SF-1 is required for proper regulation of gene expression (Ju Y. Mizutani T. Imamichi Y. Yazawa T. Matsumura T. Kawabe S. Kanno M. Umezawa A. Kangawa K. Miyamoto K. Nuclear receptor 5A (NR5A) family regulates 5-aminolevulinic acid synthase 1 (ALAS1) gene expression in steroidogenic cells., Luo X. Ikeda Y. Parker K.L. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation., Lala D.S. Rice D.A. Parker K.L. Steroidogenic factor i, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor i.) and is essential for sexual determination and development of the adrenals, gonads, and hypothalamus in mammals (Parker K.L. Schimmer B.P. Steroidogenic factor 1: A key determinant of endocrine development and function.). SF-1 is also a well-validated target for drug design efforts seeking to develop therapeutics for the treatment of adrenocortical carcinoma and endometriosis. Thus, understanding how this nuclear receptor is regulated can provide insight into several pathologies and physiological processes in humans.SF-1 binds many different classes of phospholipids with varying chemical composition of the headgroup and acyl chains. Ectopically expressed SF-1 in bacteria co-purifies and co-crystallizes with phosphatidylethanolamines and phosphatidylglycerols (Krylova I.N. Sablin E.P. Moore J. Xu R.X. Waitt G.M. MacKay J.A. Juzumiene D. Bynum J.M. Madauss K. Montana V. Lebedeva L. Suzawa M. Williams J.D. Williams S.P. Guy R.K. et al.Structural analyses reveal phosphatidyl inositols as ligands for the NR5A orphan receptors SF-1 and LRH-1.). SF-1 has also been co-crystallized with phosphatidylcholine (Sablin E.P. Blind R.D. Krylova I.N. Ingraham J.G. Cai F. Williams J.D. Fletterick R.J. Ingraham H.A. Structure of NR5A1 (SF-1) bound by different phospholipids: evidence for regulatory ligands.), whereas sphingosine was identified by mass spectrometry as specifically associated with SF-1 in mammalian cells (Urs A.N. Dammer E. Kelly S. Wang E. Merrill A.H. Sewer M.B. Steroidogenic factor-1 is a sphingolipid binding protein.), and sphingosine metabolic enzymes can regulate SF-1 activity (Lucki N.C. Li D. Bandyopadhyay S. Wang E. Merrill A.H. Sewer M.B. Acid ceramidase (ASAH1) represses steroidogenic factor 1-dependent gene transcription in H295R human adrenocortical cells by binding to the receptor.). There is also considerable evidence suggesting phosphoinositides as regulatory ligands for SF-1 (Crowder M.K. Seacrist C.D. Blind R.D. Phospholipid regulation of the nuclear receptor superfamily.). Despite the wide variety of phospholipid species that interact with SF-1, these lipids all share a common 1:1 stoichiometry, interacting within the same ligand-binding pocket at the C-terminal ligand binding domain (LBD) of SF-1, as shown by several X-ray crystal structures of the human and mouse orthologs of SF-1 (Weikum E.R. Liu X. Ortlund E.A. The nuclear receptor superfamily: A structural perspective.). These structures collectively show the acyl chains to be buried deep within the hydrophobic core of the SF-1 LBD, whereas the headgroups are largely solvent exposed, particularly true of the phosphoinositides PIP2 and PIP3 (Blind R.D. Sablin E.P. Kuchenbecker K.M. Chiu H.J. Deacon A.M. Das D. Fletterick R.J. Ingraham H.A. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor NR5A1 (SF-1).). Thus, a wide variety of phospholipids bind SF-1 at the same binding site and have potential to regulate SF-1 activity.Of all phospholipids that bind SF-1, the phosphoinositides bearing PI(4,5,)P2 (PIP2) and PI(3,4,5)P3 (PIP3) headgroups are among the most well characterized. Although sphingolipids have been identified as specifically associated with SF-1 biochemically purified from cell lines (Urs A.N. Dammer E. Kelly S. Wang E. Merrill A.H. Sewer M.B. Steroidogenic factor-1 is a sphingolipid binding protein.), there are no structures of SF-1 bound to any sphingolipid, and an endogenous ligand for SF-1 from mammalian tissue has yet to be identified. Phosphoinositides were suggested as endogenous ligands over 15 years ago (Krylova I.N. Sablin E.P. Moore J. Xu R.X. Waitt G.M. MacKay J.A. Juzumiene D. Bynum J.M. Madauss K. Montana V. Lebedeva L. Suzawa M. Williams J.D. Williams S.P. Guy R.K. et al.Structural analyses reveal phosphatidyl inositols as ligands for the NR5A orphan receptors SF-1 and LRH-1.), were among the first phospholipids to be tested for interaction with SF-1 (Krylova I.N. Sablin E.P. Moore J. Xu R.X. Waitt G.M. MacKay J.A. Juzumiene D. Bynum J.M. Madauss K. Montana V. Lebedeva L. Suzawa M. Williams J.D. Williams S.P. Guy R.K. et al.Structural analyses reveal phosphatidyl inositols as ligands for the NR5A orphan receptors SF-1 and LRH-1.), and bind with high affinity (Blind R.D. Sablin E.P. Kuchenbecker K.M. Chiu H.J. Deacon A.M. Das D. Fletterick R.J. Ingraham H.A. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor NR5A1 (SF-1).). The technical difficulties in detecting phosphoinositides by mass spectrometry remain a barrier to untargeted lipidomic approaches for identifying phosphoinositides unequivocally from mammalian cells and tissues using mass spectrometry (Kielkowska A. Niewczas I. Anderson K.E. Durrant T.N. Clark J. Stephens L.R. Hawkins P.T. A new approach to measuring phosphoinositides in cells by mass spectrometry.). Though not to the exclusion of other lipids (Urs A.N. Dammer E. Kelly S. Wang E. Merrill A.H. Sewer M.B. Steroidogenic factor-1 is a sphingolipid binding protein., Lucki N.C. Li D. Bandyopadhyay S. Wang E. Merrill A.H. Sewer M.B. Acid ceramidase (ASAH1) represses steroidogenic factor 1-dependent gene transcription in H295R human adrenocortical cells by binding to the receptor., Li D. Urs A.N. Allegood J. Leon A. Merrill A.H. Sewer M.B. Cyclic AMP-stimulated interaction between steroidogenic factor 1 and diacylglycerol kinase theta facilitates induction of CYP17.), there are several lines of evidence suggesting that the phosphoinositide PIP3 is a regulatory ligand for SF-1. Crystallographic studies showed atomic resolution details of how the PIP3 headgroup forms part of a novel regulatory surface on SF-1, which genetic studies demonstrated is required for full SF-1 function (Blind R.D. Sablin E.P. Kuchenbecker K.M. Chiu H.J. Deacon A.M. Das D. Fletterick R.J. Ingraham H.A. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor NR5A1 (SF-1).). Increased cellular levels of PIP3 correlate with increased SF-1 function in mammalian cells (Lin B.C. Suzawa M. Blind R.D. Tobias S.C. Bulun S.E. Scanlan T.S. Ingraham H.A. Stimulating the GPR30 estrogen receptor with a novel tamoxifen analogue activates NR5A and promotes endometrial cell proliferation.). The worm ortholog of SF-1 binds PIP3 in vitro and regulates phospholipid metabolism in vivo (Mullaney B.C. Blind R.D. Lemieux G.A. Perez C.L. Elle I.C. Faergeman N.J. Van Gilst M.R. Ingraham H.A. Ashrafi K. Regulation of C. elegans fat uptake and storage by acyl-CoA synthase-3 is dependent on NR5A family nuclear hormone receptor nhr-25.). Genetically or chemically inhibiting the nuclear inositol polyphosphate multikinase (Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity., Malabanan M.M. Blind R.D. Inositol polyphosphate multikinase (IPMK) in transcriptional regulation and nuclear inositide metabolism., Structural analyses of inositol phosphate second messengers bound to signaling effector proteins.) which generates PIP3 regulates SF-1 function in human cells, whereas simply decreasing total cellular PIP3 by inhibiting the p110-class of PI3 kinases does not have similar effects (Blind R.D. Suzawa M. Ingraham H.A. Direct modification and activation of a nuclear receptor - PIP2 complex by the inositol lipid kinase IPMK., Signaling through non-membrane nuclear phosphoinositide binding proteins in human health and disease.). Dipalmitoyl PIP3 has nanomolar affinity for SF-1, among the highest affinity of all phospholipids tested in previous work (Blind R.D. Sablin E.P. Kuchenbecker K.M. Chiu H.J. Deacon A.M. Das D. Fletterick R.J. Ingraham H.A. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor NR5A1 (SF-1).). Dipalmitoyl PIP3 also slightly enhances interaction of SF-1 with a peptide representing the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) transcriptional coactivator in vitro (Blind R.D. Sablin E.P. Kuchenbecker K.M. Chiu H.J. Deacon A.M. Das D. Fletterick R.J. Ingraham H.A. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor NR5A1 (SF-1).), also suggesting PIP3 can enhance SF-1 transcriptional activity (Signaling through non-membrane nuclear phosphoinositide binding proteins in human health and disease., Nuclear phosphoinositide regulation of chromatin.). Perhaps, the best evidence that SF-1 binds phosphoinositides in living cells comes from immunoprecipitated SF-1 from human cells, which contains PIP2 as detected by 32P radiolabeling by in vitro kinase reactions, and PIP2 associated with SF-1 was estimated to occupy about half the SF-1 sites in these studies (Blind R.D. Suzawa M. Ingraham H.A. Direct modification and activation of a nuclear receptor - PIP2 complex by the inositol lipid kinase IPMK.). Thus, a large body of growing evidence supports a model wherein PIP3 binds and activates SF-1, and the PIP3 headgroup participates in that activation.While PIP3 headgroup-mediated effects on SF-1 activation are well supported by several direct tests in previous work, what effect the acyl chains have on SF-1 structure has not been directly addressed. The closest comparison that might indicate how the acyl chains alter SF-1 structure comes from comparing the crystal structures of SF-1 bound to PE(16:0/16:0) and a co-repressor peptide (PDB:1YP0) (Li Y. Choi M. Cavey G. Daugherty J. Suino K. Kovach A. Bingham N.C. Kliewer S.A. Xu H.E. Crystallographic identification and functional characterization of phospholipids as ligands for the orphan nuclear receptor steroidogenic factor-1.) versus SF-1 bound to PE(16:1-18:1) and a different co-activator peptide (PDB:1YOW) (Krylova I.N. Sablin E.P. Moore J. Xu R.X. Waitt G.M. MacKay J.A. Juzumiene D. Bynum J.M. Madauss K. Montana V. Lebedeva L. Suzawa M. Williams J.D. Williams S.P. Guy R.K. et al.Structural analyses reveal phosphatidyl inositols as ligands for the NR5A orphan receptors SF-1 and LRH-1.). Although these models show differences in SF-1 structure, the different peptides make assigning any affect to acyl chain composition difficult. Still, these and other structures (Sablin E.P. Blind R.D. Krylova I.N. Ingraham J.G. Cai F. Williams J.D. Fletterick R.J. Ingraham H.A. Structure of NR5A1 (SF-1) bound by different phospholipids: evidence for regulatory ligands.) remain consistent with models in which the acyl chains might regulate SF-1 structure and thus the ability of SF-1 to interact with transcriptional coactivators.Here, we directly address the question of how the acyl chains of phosphoinositides regulate the structure and function of SF-1 for the first time, using a new protein-sequence and headgroup-matched crystal structure, combined with in vitro binding assays and additional functional assays. The data show the acyl chains of PIP3 regulate SF-1 structure and function in vitro. Our data suggest that longer and more complex acyl chains induce structural disorder in SF-1 that is detectable by X-ray crystallography, occurring in regions of SF-1 previously shown to be critical for function in human patients (Knarston I.M. Robevska G. van den Bergen J.A. Eggers S. Croft B. Yates J. Hersmus R. Looijenga L.H.J. Cameron F.J. Monhike K. Ayers K.L. Sinclair A.H. NR5A1 gene variants repress the ovarian-specific WNT signaling pathway in 46,XX disorders of sex development patients., Suntharalingham J.P. Buonocore F. Duncan A.J. Achermann J.C. DAX-1 (NR0B1) and steroidogenic factor-1 (SF-1, NR5A1) in human disease., Biason-Lauber A. Schoenle E.J. Apparently normal ovarian differentiation in a prepubertal girl with transcriptionally inactive steroidogenic factor 1 (NR5A1/SF-1) and adrenocortical insufficiency., Philibert P. Paris F. Lakhal B. Audran F. Gaspari L. Saâd A. Christin-Maître S. Bouchard P. Sultan C. NR5A1 (SF-1) gene variants in a group of 26 young women with XX primary ovarian insufficiency.). Although changes to any crystal structure could be because of crystallographic artifacts, the changes we observe directly correlated with predicted changes to SF-1 using in vitro functional assays. Together, these data suggest the PIP3 acyl changes can regulate SF-1, directly addressing the role of the phosphoinositide acyl chains in regulating SF-1 structure and function.Materials and Methods Phosphoinositide reagents

Chemically synthesized PIP3(18:1(9Z)/18:1(9Z)) (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-[phosphoinositol-3,4,5-trisphosphate]) was from Avanti Polar Lipids (Product number 850156, Alabaster, AL), and chemically synthesized PIP3(16:0/16:0) (1,2-dipalmitoyl-sn-glycero-3-[phosphoinositol-3,4,5-triphophosate]) was from Cayman Chemical Company (Ann Arbor, MI). All other phosphoinositides were purchased from Avanti. All lipids were stored lyophilized, in the dark at −20°C under vacuum.

 Protein expression and purificationThe human SF-1 (NR5A1, Accession NP_004950.2) DNA plasmid construct, all protein and peptide sequences, and purification procedure used here were identical to our previous crystallographic analysis of human SF-1 LBD bound to dipalmitoyl PIP3(16:0/16:0) (Blind R.D. Sablin E.P. Kuchenbecker K.M. Chiu H.J. Deacon A.M. Das D. Fletterick R.J. Ingraham H.A. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor NR5A1 (SF-1).), except that dioleoyl PIP3(18:1(9Z)/18:1(9Z)) was complexed with SF-1. Briefly, a 2CS Cys-lite mutant (C247S and C412S) of N-terminally 6X-HIS tagged human SF-1 LBD comprising amino acids 218–461 of human SF-1 (Blind R.D. Sablin E.P. Kuchenbecker K.M. Chiu H.J. Deacon A.M. Das D. Fletterick R.J. Ingraham H.A. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor NR5A1 (SF-1).) which included a TEV site to remove the HIS tag was used for all studies. This 2CS mutant had been previously shown to improve crystal diffraction quality when compared with the WT SF-1 LBD (Wang W. Zhang C. Marimuthu A. Krupka H.I. Tabrizizad M. Shelloe R. Mehra U. Eng K. Nguyen H. Settachatgul C. Powell B. Milburn M.V. West B.L. The crystal structures of human steroidogenic factor-1 and liver receptor homologue-1.). The SF-1 construct was expressed in BL21 E. coli DE3 bacteria and purified by immobilized metal affinity chromatography, and the HIS tag removed by overnight dialysis at 4°C under reducing conditions using 1 mg TEV per 10 mg SF-1 protein, desalted into buffer A (20 mM Hepes [7.5] with 2 mM CHAPS), and loaded onto a Capto-Q Hi-Trap ion exchange column, eluted isocratically with buffer A plus 300 mM NaCl, then desalted into 20 mM Hepes, all steps as previously described (Blind R.D. Sablin E.P. Kuchenbecker K.M. Chiu H.J. Deacon A.M. Das D. Fletterick R.J. Ingraham H.A. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor NR5A1 (SF-1).). To generate SF-1 LBD/PIP3 complexes, desalted SF-1 LBD in 20 mM Hepes (7.5) was incubated overnight at room temperature with 5:1 M excess of the phosphoinositide of interest (dioleoyl-PIP3 or dipalmitoyl-PIP3), nutating under nitrogen in borosilicate glass, the SF-1 LBD/PIP3 complexes were then separated from unexchanged SF-1 LBD using HiTrap-Q anion exchange chromatography (GE Biosciences) over a gradient of NaCl in 20 mM Hepes (7.5). The PIP3-bound SF-1 elutes at higher NaCl concentrations than SF-1 bound to ectopic bacterial phospholipid most likely because of the negative charges provided by the phosphates on PIP3 (Blind R.D. Sablin E.P. Kuchenbecker K.M. Chiu H.J. Deacon A.M. Das D. Fletterick R.J. Ingraham H.A. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor NR5A1 (SF-1)., Blind R.D. Suzawa M. Ingraham H.A. Direct modification and activation of a nuclear receptor - PIP2 complex by the inositol lipid kinase IPMK.

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