孤儿核受体类固醇生成因子1

孤儿核受体是核受体超家族中较独特的成员,它参与了糖类、脂类及胆固醇和类固醇激素的代谢,可能是体内细胞基本功能的重要调节因子。孤儿核受体SF－1最初作为肾上腺和性腺中的P450羟化酶必需的调节子而被鉴定,在类固醇组织、垂体和下丘脑腹内侧核均有表达,SF01在基础结构上具有不同于其他核受体的特征结构域,并广泛参与许多基因如类固醇合成酶类、Muellerian抑制性物质、黄体生成素β亚基启动子等的表达调控,基因剔除实验证实SF－1是肾上腺类固醇合成和性别分化中的一个关键调节因子。     

核孤儿受体在胆固醇分解代谢过程中的调控作用

由胆固醇合成胆汁酸有两条途径。一条是经典途径或中性途径[1] ,也是合成胆汁酸最主要的途径 ,其限速反应为胆固醇 7α 羟化酶 (由Ｃｙｐ7ａ编码 )催化胆固醇羟化为 7α 羟胆固醇的反应。近几年发现另一条胆汁酸合成的酸性途径[2 ] ,这一途径开始于胆固醇转变为氧固醇 (ｏｘｙｓｔｅｒｏｌ) ,然后经氧固醇 7α 羟化酶 (由Ｃｙｐ7ｂ编码 )催化产生 7α 羟氧固醇化合物 ,再融入胆汁酸合成经典途径的下游步骤。由于胆固醇在体内有重要作用 ,有效调节胆固醇分解代谢以维持胆固醇水平的动态平衡十分重要。研究发现 ,胆汁酸以及合成过程中的中间产…     

核受体辅活化子PNRC与孤儿核受体SF1相互作用位点的鉴定

为了阐明核受体辅活化子 (proline richnuclearreceptorcoactivatorprotein ,PNRC)在孤儿核受体类固醇生成因子 1(steroidogenicfactor1,SF1)基因表达调控中的作用 ,采用酵母双杂合分析、缺失突变技术和瞬时转染等研究方法鉴定了PNRC与SF1的相互作用位点 .结果显示 ,PNRC中氨基酸 2 78～ 30 0区域是与SF1相互作用的位点 .该区域富含脯氨酸 ,其中有 1个SH3结合模体 (motif) ,单独的SH3模体不足以与SF1产生有效的相互作用 .瞬时转染分析表明 ,PNRC 2 70 32 7对野生型PNRC的辅激活功能具有负显性抑制效应 .研究结果表明 ,含SH3结合模体的PNRC 2 78 30 0区域是与SF1相互作用的位点     

Defining a Canonical Ligand-Binding Pocket in the Orphan Nuclear Receptor Nurr1

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In Silico Adoption of an Orphan Nuclear Receptor NR4A1

A 4.1μs molecular dynamics simulation of the NR4A1 (hNur77) apo-protein has been undertaken and a previously undetected druggable pocket has become apparent that is located remotely from the ‘traditional’ nuclear receptor ligand-binding site. A NR4A1/bis-indole ligand complex at this novel site has been found to be stable over 1 μs of simulation and to result in an interesting conformational transmission to a remote loop that has the capacity to communicate with a NBRE within a RXR-α/NR4A1 heterodimer. Several features of the simulations undertaken indicate how NR4A1 can be affected by alternate-site modulators.     

核受体辅活化子PNRC与孤儿核受体SF1的相互作用有赖于SF1的AF-2功能结构域

核受体辅活化子PNRC(proline richnuclearreceptorcoregulatoryprotein ,富含脯氨酸的核受体辅调节蛋白 )可通过含SH3结合模体的PNRC2 78 30 0区域与孤儿核受体类固醇生成因子 1(steroido genicfactor 1,SF1)相互作用 .激活功能 2 (activationfunction 2 ,AF 2 )结构域在核受体配体依赖性转录激活中发挥了重要作用 ,为探讨AF 2结构域在SF1转录激活中的作用机制 ,采用酵母双杂合分析、缺失突变技术和瞬时转染等研究方法考察了AF 2结构域对SF1反式激活功能及SF1与PNRC相互作用的影响 .SF1的反式激活功能有赖于AF 2结构域 ,其机制是SF1AF 2结构域的突变严重影响了SF1与PNRC的有效相互作用 ,并消除了PNRC对SF1反式激活功能的辅激活作用 .结果表明 ,SF1与PNRC的相互作用有赖于AF 2的功能结构域     

Germ Cell Nuclear Factor: An Orphan Receptor in Search of a Function

Germ Cell Nuclear Factor (GCNF) is an orphan member of the nuclearreceptor gene superfamily. Much has been understood about thefunctioning of GCNF which represents a candidate receptor fora novel hormonal signalling pathway. GCNF is not closely relatedto other members of the nuclear receptor superfamily and formsits own branch within the superfamily tree. It has a uniqueexpression pattern that spans both embryonic and adult stagesof development. In the adult, it is expressed in the germ cells:oocytes and spermatogenic cells as well as specific neuronalcells within the brain. In the embryo, GCNF expression is turnedon after gastrulation in all germ layers the ectoderm, mesodermand endoderm. An antero-posterior gradient of GCNF is establishedin the neuroectoderm of the embryo, suggesting a role in regulationof neuronal and germ cell development. Regulation of physiologicalprocesses by a nuclear receptor is achieved through regulationof gene expression. GCNF is the only nuclear receptor to specifcallybind to DR0 hormone response elements to regulate gene expression.In the absense of a ligand, GCNF represses gene expression.GCNF is capable of regulating the expression of the protaminegenes in a response element-dependent manner. At present theligand for GCNF is unknown, but it is hypothesized that GCNFis a receptor for a novel hormonal signalling pathway that effectsits biological response by regulating the expression of a subsetof genes containing DR0 response elements.     

The Nuclear Orphan Receptor CAR-Retinoid X Receptor Heterodimer Activates the Phenobarbital-Responsive Enhancer Module of the CYP2B Gene

PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.     

Insulin Directly Regulates Steroidogenesis via Induction of the Orphan Nuclear Receptor DAX-1 in Testicular Leydig Cells

Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-β (phospho-IR-β), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.     

辅抑制子SMRT特异地抑制孤儿核受体hB1F／hLRH-1的转录活性

孤儿核受体hB1F (NR5A2 ,也称之为LRH 1、FTF或CPF)在胆汁酸合成代谢相关基因、乙型肝炎病毒基因和一些肝特异性基因的表达调控中起着非常重要的作用。为了认识hB1F转录激活的分子机制 ,对核受体辅抑制子SMRT在其中的作用进行了研究。利用与GAL4 DBD融合的hB1F所进行的报告基因分析发现 ,SMRT能够以剂量依赖的方式显著地抑制hB1F的反式激活能力 ,但对融合蛋白质的表达没有影响。而且 ,SMRT也能够明显地抑制hB1F响应的乙肝病毒增强子II 核心启动子的活性 ,对增强子II的点突变分析表明这种抑制作用主要是由hB1F介导的。共转染实验显示 ,SMRT在多种细胞中都表现出抑制作用。有意思的是 ,哺乳动物细胞的双杂交与体外的GST下拉 (pull down)分析都表明hB1F与SMRT之间不存在直接的相互作用。实验数据首次证实 ,辅抑制子SMRT可能通过间接的方式特异地抑制孤儿核受体hB1F的转录活性。     

Induction of a Nerve Growth Factor-Sensitive Kinase that Phosphorylates the DNA-Binding Domain of the Orphan Nuclear Receptor NGFI-B

Abstract: Nerve growth factor (NGF) induces the synthesis and the phosphorylation of the orphan nuclear receptor NGFI-B in PC12 cells. Previous work has shown that phosphorylation, by protein kinase A, of a specific serine in the DNA-binding domain inhibits its binding to the NGFI-B response element. Also, cytoplasmic extracts from PC12 cells phosphorylate this serine, and phosphorylation is greater in extracts from cells treated with NGF. The present work describes the induction, identification, and partial purification of a kinase (termed NGFI-B kinase I) from PC12 cell extracts that catalyzes this phosphorylation. Phosphorylation of the DNA-binding domain with this purified preparation inhibits its binding to the NGFI-B response element. The kinase is rapidly activated by treatment of the cells with NGF, and the activation lasts for at least several hours. It also is activated by fibroblast growth factor and epidermal growth factor (EGF), but the activation by EGF is quite transient. The kinase requires Mg²⁺ but will use Mn²⁺. The molecular mass of the kinase is 95–100 kDa, and it is different from protein kinase A, Fos kinase, or pp90 ^rsk . Comparison with a partially purified preparation of cyclic AMP response element-binding protein kinase, however, indicates that the two are either very similar or identical.