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1.
J Clin Invest ; 2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30507613

RESUMO

Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained ß cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.

2.
Sci Rep ; 8(1): 16672, 2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-30420721

RESUMO

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P2 (PIP2). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (ΔIPMK) are presented. Although the KM for PIP2 is identical between ΔIPMK and wild type, ΔIPMK has a 1.8-fold increase in kcat for PIP2, indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 Å crystal structure of ΔIPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative "ATP-clamp" in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the KM for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.

3.
J Lipid Res ; 2018 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-30201631

RESUMO

Phosphoinositide membrane signaling is critical for normal physiology, playing well-known roles in diverse human pathologies. The basic mechanisms governing phosphoinositide signaling within the nucleus, however, have remained deeply enigmatic owing to their presence outside the nuclear membranes. Over 40% of nuclear phosphoinositides can exist in this non-membrane state, held soluble in the nucleoplasm by nuclear proteins that remain largely unidentified. Recently, two nuclear proteins responsible for solubilizing phosphoinositides were identified - steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homolog-1 (LRH-1, NR5A2) - along with two enzymes that directly remodel these phosphoinositide/protein complexes - phosphatase and tensin homolog (PTEN, MMAC) and inositol polyphosphate multikinase (IPMK, ipk2). These new footholds now permit the assignment of physiological functions for nuclear phosphoinositides in human diseases, such as endometriosis, non-alcoholic fatty liver disease / steatohepatitis, glioblastoma and hepatocellular carcinoma. The unique nature of nuclear phosphoinositide signaling affords extraordinary clinical opportunities for new biomarkers, diagnostics and therapeutics. Thus, phosphoinositide biology within the nucleus may represent the next generation of low-hanging fruit for new drugs, not unlike what has occurred for membrane PI3-kinase drug development. This review connects recent basic science discoveries in nuclear phosphoinositide signaling to clinical pathologies, with the hope of inspiring development of new therapies.

4.
J Cell Physiol ; 233(1): 107-123, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28256711

RESUMO

Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear-driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology.


Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Cromatina/genética , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transcrição Genética
5.
Adv Biol Regul ; 63: 6-14, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27838257

RESUMO

Nuclear receptors are ligand-activated transcription factors whose diverse biological functions are classically regulated by cholesterol-based small molecules. Over the past few decades, a growing body of evidence has demonstrated that phospholipids and other similar amphipathic molecules can also specifically bind and functionally regulate the activity of certain nuclear receptors, suggesting a critical role for these non-cholesterol-based molecules in transcriptional regulation. Phosphatidylcholines, phosphoinositides and sphingolipids are a few of the many phospholipid like molecules shown to quite specifically regulate nuclear receptors in mouse models, cell lines and in vitro. More recent evidence has also shown that certain nuclear receptors can "present" a bound phospholipid headgroup to key lipid signaling enzymes, which can then modify the phospholipid headgroup with very unique kinetic properties. Here, we review the broad array of phospholipid/nuclear receptor interactions, from the perspective of the chemical nature of the phospholipid, and the cellular abundance of the phospholipid. We also view the data in the light of well established paradigms for phospholipid mediated transcriptional regulation, as well as newer models of how phospholipids might effect transcription in the acute regulation of complex nuclear signaling pathways. Thus, this review provides novel insight into the new, non-membrane associated roles nuclear phospholipids play in regulating complex nuclear events, centered on the nuclear receptor superfamily of transcription factors.


Assuntos
Regulação da Expressão Gênica , Fosfolipídeos/química , Receptores Citoplasmáticos e Nucleares/química , Fatores de Transcrição/química , Animais , Humanos , Ligantes , Camundongos , Modelos Moleculares , Fosfolipídeos/classificação , Fosfolipídeos/metabolismo , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/classificação , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
6.
Biochem Soc Trans ; 44(1): 279-85, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26862216

RESUMO

Inositol polyphosphate multikinase (IPMK, ipk2, Arg(82), ArgRIII) is an inositide kinase with unusually flexible substrate specificity and the capacity to partake in many functional protein-protein interactions (PPIs). By merging these two activities, IPMK is able to execute gene regulatory functions that are very unique and only now beginning to be recognized. In this short review, we present a brief history of IPMK, describe the structural biology of the enzyme and highlight a few recent discoveries that have shed more light on the role IPMK plays in inositide metabolism, nuclear signalling and transcriptional regulation.


Assuntos
Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Inositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transcrição Genética , Animais , Biocatálise , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/química
7.
J Struct Biol ; 192(3): 342-348, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26416531

RESUMO

The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.


Assuntos
Fosfatidilinositóis/química , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/ultraestrutura , Fator Esteroidogênico 1/ultraestrutura , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Receptor Nuclear Órfão DAX-1/química , Humanos , Modelos Moleculares , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Fator Esteroidogênico 1/química
8.
Proc Natl Acad Sci U S A ; 111(42): 15054-9, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25288771

RESUMO

The signaling phosphatidylinositol lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind nuclear receptor 5A family (NR5As), but their regulatory mechanisms remain unknown. Here, the crystal structures of human NR5A1 (steroidogenic factor-1, SF-1) ligand binding domain (LBD) bound to PIP2 and PIP3 show the lipid hydrophobic tails sequestered in the hormone pocket, as predicted. However, unlike classic nuclear receptor hormones, the phosphoinositide head groups are fully solvent-exposed and complete the LBD fold by organizing the receptor architecture at the hormone pocket entrance. The highest affinity phosphoinositide ligand PIP3 stabilizes the coactivator binding groove and increases coactivator peptide recruitment. This receptor-ligand topology defines a previously unidentified regulatory protein-lipid surface on SF-1 with the phosphoinositide head group at its nexus and poised to interact with other proteins. This surface on SF-1 coincides with the predicted binding site of the corepressor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region on chromosome X), and importantly harbors missense mutations associated with human endocrine disorders. Our data provide the structural basis for this poorly understood cluster of human SF-1 mutations and demonstrates how signaling phosphoinositides function as regulatory ligands for NR5As.


Assuntos
Fosfatidilinositóis/química , Fator Esteroidogênico 1/química , Aminoácidos/química , Animais , Transporte Biológico , Núcleo Celular/metabolismo , Cromatografia , Simulação por Computador , Cristalografia por Raios X , Elétrons , Humanos , Ligantes , Lipídeos/química , Camundongos , Modelos Moleculares , Conformação Molecular , Mutação , Mutação de Sentido Incorreto , Peptídeos/química , Transdução de Sinais , Solventes/química , Ressonância de Plasmônio de Superfície , Propriedades de Superfície , Temperatura Ambiente , Água/química
9.
Adv Biol Regul ; 54: 25-38, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24176936

RESUMO

An unresolved problem in biological signal transduction is how particular branches of highly interconnected signaling networks can be decoupled, allowing activation of specific circuits within complex signaling architectures. Although signaling dynamics and spatiotemporal mechanisms serve critical roles, it remains unclear if these are the only ways cells achieve specificity within networks. The transcription factor Steroidogenic Factor-1 (SF-1) is an excellent model to address this question, as it forms dynamic complexes with several chemically distinct lipid species (phosphatidylinositols, phosphatidylcholines and sphingolipids). This property is important since lipids bound to SF-1 are modified by lipid signaling enzymes (IPMK & PTEN), regulating SF-1 biological activity in gene expression. Thus, a particular SF-1/lipid complex can interface with a lipid signaling enzyme only if SF-1 has been loaded with a chemically compatible lipid substrate. This mechanism permits dynamic downstream responsiveness to constant upstream input, disentangling specific pathways from the full network. The potential of this paradigm to apply generally to nuclear lipid signaling is discussed, with particular attention given to the nuclear receptor superfamily of transcription factors and their phospholipid ligands.


Assuntos
Metabolismo dos Lipídeos , Animais , Humanos , Transdução de Sinais , Fator Esteroidogênico 1/metabolismo
10.
Sci Signal ; 5(229): ra44, 2012 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-22715467

RESUMO

Phosphatidylinositol 4,5-bisphosphate (PIP2) is best known as a plasma membrane-bound regulatory lipid. Although PIP2 and phosphoinositide-modifying enzymes coexist in the nucleus, their nuclear roles remain unclear. We showed that inositol polyphosphate multikinase (IPMK), which functions both as an inositol kinase and as a phosphoinositide 3-kinase (PI3K), interacts with the nuclear receptor steroidogenic factor 1 (SF-1) and phosphorylates its bound ligand, PIP2. In vitro studies showed that PIP2 was not phosphorylated by IPMK if PIP2 was displaced or blocked from binding to the large hydrophobic pocket of SF-1 and that the ability to phosphorylate PIP2 bound to SF-1 was specific to IPMK and did not occur with type 1 p110 PI3Ks. IPMK-generated SF-1-PIP3 (phosphatidylinositol 3,4,5-trisphosphate) was dephosphorylated by the lipid phosphatase PTEN. Consistent with the in vitro activities of IPMK and PTEN on SF-1-PIP(n), SF-1 transcriptional activity was reduced by silencing IPMK or overexpressing PTEN. This ability of lipid kinases and phosphatases to directly remodel and alter the activity of a non-membrane protein-lipid complex establishes a previously unappreciated pathway for promoting lipid-mediated signaling in the nucleus.


Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fator Esteroidogênico 1/metabolismo , Sítios de Ligação , Western Blotting , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Estrutura Molecular , Mutação , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Transdução de Sinais , Fator Esteroidogênico 1/química , Fator Esteroidogênico 1/genética , Especificidade por Substrato
11.
Biochem Biophys Res Commun ; 420(4): 839-44, 2012 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-22465009

RESUMO

Glucocorticoid (GC) induction of the tyrosine aminotransferase (TAT) gene by the glucocorticoid receptor (GR) is a classic model used to investigate steroid-regulated gene expression. Classic studies analyzing GC-induction of the TAT gene demonstrated that despite having very high affinity for GR, some steroids cannot induce maximal TAT enzyme activity, but the molecular basis for this phenomenon is unknown. Here, we used RT-PCR and chromatin immunoprecipitation to determine TAT mRNA accumulation and GR recruitment to the TAT promoter (TAT-GRE) in rat hepatoma cells induced by seven GR ligands: dexamethasone (DEX), cortisol (CRT), corticosterone (CCS), 11-deoxycorticosterone (DOC), aldosterone (ALD), progesterone (PRG) and 17-hydroxyprogesterone (17P). As expected, DEX, CRT, CCS and ALD all induced both TAT mRNA and GR recruitment to the TAT-GRE, while PRG and 17P did not. However, while DOC could not induce significant TAT mRNA, it did induce robust GR occupancy of the TAT-GRE. DOC also induced recruitment of the histone acetyltransferase p300 to the TAT-GRE as efficiently as DEX. These DOC-induced effects recapitulated at another GR target gene (sulfonyltransferase 1A1), and DOC also failed to promote the multiple changes in gene expression required for glucocorticoid-dependent 3T3-L1 adipocyte differentiation. Structural simulations and protease sensitivity assays suggest that DOC and DEX induce different conformations in GR. Thus, although steroids that bind GR with high affinity can induce GR and p300 occupancy of target promoters, they may not induce a conformation of GR capable of activating transcription.


Assuntos
Corticosteroides/química , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional/efeitos dos fármacos , 17-alfa-Hidroxiprogesterona/química , 17-alfa-Hidroxiprogesterona/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Corticosteroides/farmacologia , Aldosterona/química , Aldosterona/farmacologia , Animais , Linhagem Celular Tumoral , Corticosterona/química , Corticosterona/farmacologia , Desoxicorticosterona/química , Desoxicorticosterona/farmacologia , Dexametasona/química , Dexametasona/farmacologia , Hidrocortisona/química , Hidrocortisona/farmacologia , Ligantes , Camundongos , Estrutura Molecular , Progesterona/química , Progesterona/farmacologia , Conformação Proteica/efeitos dos fármacos , Ratos , Receptores de Glucocorticoides/química
12.
J Med Chem ; 54(7): 2266-81, 2011 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-21391689

RESUMO

The crystal structure of LRH-1 ligand binding domain bound to our previously reported agonist 3-(E-oct-4-en-4-yl)-1-phenylamino-2-phenyl-cis-bicyclo[3.3.0]oct-2-ene 5 is described. Two new classes of agonists in which the bridgehead anilino group from our first series was replaced with an alkoxy or 1-ethenyl group were designed, synthesized, and tested for activity in a peptide recruitment assay. Both new classes gave very active compounds, particularly against SF-1. Structure-activity studies led to excellent dual-LRH-1/SF-1 agonists (e.g., RJW100) as well as compounds selective for LRH-1 (RJW101) and SF-1 (RJW102 and RJW103). The series based on 1-ethenyl substitution was acid stable, overcoming a significant drawback of our original bridgehead anilino-substituted series. Initial studies on the regulation of gene expression in human cell lines showed excellent, reproducible activity at endogenous target genes.


Assuntos
Receptores Citoplasmáticos e Nucleares/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Fator Esteroidogênico 1/agonistas , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Homologia de Sequência de Aminoácidos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Fator Esteroidogênico 1/química , Fator Esteroidogênico 1/metabolismo , Ativação Transcricional/efeitos dos fármacos
13.
Cell Metab ; 12(4): 398-410, 2010 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-20889131

RESUMO

Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid-rich intestinal depots. Here, we show that ACS-3 functions in seam cells, epidermal cells anatomically distinct from sites of fat uptake and storage, and that acs-3 mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans.


Assuntos
Caenorhabditis elegans/metabolismo , Coenzima A Ligases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Gorduras/metabolismo , Fatores de Transcrição/fisiologia , Animais , Coenzima A Ligases/genética , Mucosa Intestinal/metabolismo , Lipídeos/biossíntese , Mutagênese Sítio-Dirigida , Receptores Citoplasmáticos e Nucleares
14.
Cancer Res ; 69(13): 5415-23, 2009 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-19549922

RESUMO

Estrogens and selective estrogen receptor (ER) modulators such as tamoxifen are known to increase uterine cell proliferation. Mounting evidence suggests that estrogen signaling is mediated not only by ERalpha and ERbeta nuclear receptors, but also by GPR30 (GPER), a seven transmembrane (7TM) receptor. Here, we report that primary human endometriotic H-38 cells express high levels of GPR30 with no detectable ERalpha or ERbeta. Using a novel tamoxifen analogue, STX, which activates GPR30 but not ERs, significant stimulation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways was observed in H-38 cells and in Ishikawa endometrial cancer cells expressing GPR30; a similar effect was observed in JEG3 choriocarcinoma cells. STX treatment also increased cellular pools of phosphatidylinositol (3,4,5) triphosphate, a proposed ligand for the nuclear hormone receptor SF-1 (NR5A1). Consistent with these findings, STX, tamoxifen, and the phytoestrogen genistein were able to increase SF-1 transcription, promote Ishikawa cell proliferation, and induce the SF-1 target gene aromatase in a GPR30-dependent manner. Our findings suggest a novel signaling paradigm that is initiated by estrogen activation of the 7TM receptor GPR30, with signal transduction cascades (PI3K and MAPK) converging on nuclear hormone receptors (SF-1/LRH-1) to modulate their transcriptional output. We propose that this novel GPR30/SF-1 pathway increases local concentrations of estrogen, and together with classic ER signaling, mediate the proliferative effects of synthetic estrogens such as tamoxifen, in promoting endometriosis and endometrial cancers.


Assuntos
Receptores Acoplados a Proteínas-G/genética , Fator Esteroidogênico 1/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Coriocarcinoma/genética , Coriocarcinoma/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Endometriose/genética , Estrogênios/farmacologia , Feminino , Genes Reporter , Humanos , Luciferases/genética , Camundongos , Camundongos Knockout , Gravidez , Receptores Estrogênicos/fisiologia , Receptores Acoplados a Proteínas-G/deficiência , Receptores Acoplados a Proteínas-G/fisiologia , Fator Esteroidogênico 1/genética , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia
15.
Mol Endocrinol ; 23(1): 25-34, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18988706

RESUMO

Despite the fact that many nuclear receptors are ligand dependent, the existence of obligate regulatory ligands is debated for some receptors, including steroidogenic factor 1 (SF-1). Although fortuitously bound bacterial phospholipids were discovered in the structures of the SF-1 ligand-binding domain (LBD), these lipids might serve merely as structural ligands. Thus, we examined whether exogenously added phospholipids would exchange for these bacterial lipids and bind to SF-1. Here, we report the first crystal structure of the SF-1 LBD bound by the exchanged phosphatidylcholine. Although the bound phosphatidylcholine phospholipid mimics the conformation of bound bacterial phosphoplipids, two surface loops, L2-3 and L11-12, surrounding the entrance to the pocket vary significantly between different SF-1 LBD structures. Based on this observation, we hypothesized that a bound ligand might control the conformations of loops L2-3 and L11-12, and that conserved residues in these dynamic loops could influence ligand binding and the receptor function. Consistent with this hypothesis, impaired phospholipid exchange and diminished transcriptional activity were observed for loop L11-12 SF-1 mutants and for the loop L2-3 human mutant R255L. The endocrine disease associated with this L2-3 mutation coupled with our cellular and biochemical data suggest that critical residues at the mouth of the ligand-binding pocket have evolved for efficient binding of phospholipid ligands and for achieving optimal SF-1 activity.


Assuntos
Fosfolipídeos/metabolismo , Fator Esteroidogênico 1/química , Fator Esteroidogênico 1/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Primers do DNA/genética , Evolução Molecular , Humanos , Técnicas In Vitro , Lecitinas/química , Lecitinas/metabolismo , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfolipídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fator Esteroidogênico 1/genética
16.
Mol Endocrinol ; 22(8): 1754-66, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18483179

RESUMO

The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.


Assuntos
Regulação da Expressão Gênica , Receptores de Glucocorticoides/metabolismo , Sequência de Aminoácidos , Anticorpos Fosfo-Específicos/farmacologia , Linhagem Celular Tumoral , Curcumina/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Ligantes , Complexo Mediador , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Peptídeos/química , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Estrutura Secundária de Proteína , Receptores de Glucocorticoides/química , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos
17.
J Steroid Biochem Mol Biol ; 109(1-2): 150-7, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18304804

RESUMO

The human glucocorticoid receptor (GR) is phosphorylated on its N-terminus at three major sites (S203, S211 and S226) within activation function 1 (AF1). Although GR has been shown to assemble at glucocorticoid responsive elements (GREs) in the presence of hormone, the timing and specificity of GR phospho-isoform recruitment to receptor target genes has not been established. Using chromatin immunoprecipitation (ChIP) and GR phosphorylation site-specific antibodies, we examined GR phospho-isoform recruitment to several glucocorticoid-induced genes including tyrosine aminotransferase (tat) and sulfonyltransferase-1A1 (sult) in rat hepatoma cells, and the glucocorticoid-induced leucine zipper (gilz) gene in human U2OS cells. GR P-S211 and GR P-S226 isoforms were efficiently recruited to the tat, sult and gilz GREs in a hormone-dependent manner. In contrast, the GR P-S203 isoform displayed no significant recruitment to any GREs of the genes analyzed, consistent with its lack of nuclear accumulation. Interestingly, the kinetics of GR P-S211 and GR P-S226 recruitment differed among genes. Our findings indicate that GR phospho-isoforms selectively occupy GR target genes, and suggests gene specific requirements for GR phosphorylation in receptor-dependent transcriptional activation.


Assuntos
Dexametasona/farmacologia , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Arilsulfotransferase/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Humanos , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Glucocorticoides/química , Fatores de Transcrição/genética , Tirosina Transaminase/genética
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