ENDOCRINOLOGY OF 17BETA-HYDROXYSTEROID DEHYDROGENASES

17β-羟基类固醇脱氢酶的内分泌学

• 批准号: 2905959
• 负责人: ALF STEFAN ANDERSSON
• 金额: $ 21.61万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2905959
• 关键词: enzyme activity; enzyme deficiency; enzyme mechanism; enzyme structure; gene mutation; hormone regulation /control mechanism; human fetus tissue; human genetic material tag; hydroxysteroid dehydrogenases; immunocytochemistry; in situ hybridization; isozymes; laboratory mouse; northern blottings; nucleic acid sequence; polymerase chain reaction; protein purification; protein structure function; pseudohermaphroditism; site directed mutagenesis; steroid hormone biosynthesis; transfection; western blottings

The objectives of the research proposed are to define the biochemistry, function, and physiological roles of the 17beta-hydroxysteroid dehydrogenases (17beta-HSDs), a group of isozymes crucial to the biosynthesis and activation/inactivation of sex steroid hormones. Over the last four years, we isolated and characterized cDNAs and genes that encode two distinct isozymes of 17beta-HSD, designated type 2 and type 3, the biochemical properties and tissue distributions of which are different and distinct. These findings pose a number of questions relating to the physiological roles of the two isozymes, and how their different biochemical properties influence these roles. The initial goal of the studies proposed is to conduct detailed analyses of the tissue distribution, cell type-specific expression and ontogeny of the two isozymes. These analyses will be accomplished by immunoblotting, immunohistochemistry, RNA blotting, in situ mRNA hybridization, and 17beta-HSD isozyme activity assays. The second objective is to purify the type 2 isozyme to homogeneity. The kinetic, pharmacological, and structural properties of the isozyme will be determined and related to its intracellular localization and function. The third goal is to identify and characterize key mutations in the 17beta-HSD type 3 gene among male pseudohermaphrodites caused by 17beta-HSD deficiency. some of these mutations give a gent that encodes an enzyme with modified Michaelis-Menten constants for substrate or cofactor; the study of these modifications will provide important information concerning the structural domains of the protein. The identification of mutations will be accomplished using the polymerase chain reaction followed b single strand DNA conformation polymorphism and sequence analysis of amplified DNA. The final objective of this research is to expand the structure-function information derived from naturally occurring mutations by creating, and expressing a limited number of site-directed mutations in the 17beta-HSD type 3 cDNA using the baculovirus system. These studies will be focused on identifying amino acid sequences that form the cofactor and substrate binding domains of the protein. Detailed kinetic, pharmacological and structure properties of the purified mutant enzymes will be compared with those of the normal enzyme.

这项研究的目标是确定生物化学， 17 β-羟基类固醇的功能和生理作用 酶（17 β-HSDs），一组对细胞增殖至关重要的同工酶， 性类固醇激素的生物合成和活化/失活。 超过 在过去的四年里，我们分离并鉴定了cDNA和基因， 编码两种不同的17 β-HSD同工酶，命名为2型 第三类，其生化特性和组织分布， 是不同的和独特的。 这些发现提出了一些问题 关于这两种同工酶的生理作用，以及它们如何 不同的生物化学性质影响这些作用。 最初的目标 其中一项研究是对组织进行详细的分析， 分布、细胞类型特异性表达和个体发育 同工酶 这些分析将通过免疫印迹完成， 免疫组织化学、RNA印迹、原位mRNA杂交， 和17 β-HSD同工酶活性测定。 第二个目标是 将2型同工酶纯化至均一。 动力学， 药理学，和同工酶的结构特性将是 确定并与其细胞内定位和功能相关。 第三个目标是识别和表征基因组中的关键突变。 17 β-HSD 3型基因在男性假两性畸形中的作用 17 β-HSD缺乏症。 其中一些突变会产生一种 编码一种具有修改的米氏常数的酶， 底物或辅因子;这些修饰的研究将提供 关于蛋白质结构域的重要信息。 突变的鉴定将使用 聚合酶链反应跟踪B单链DNA构象 扩增DNA的多态性和序列分析。 最终 本研究的目的是扩展结构-功能 通过创建从自然发生的突变中获得的信息， 并在细胞中表达有限数量的定点突变， 17 β-HSD 3型cDNA。 这些 研究将集中在确定氨基酸序列， 蛋白质的辅因子和底物结合域。 详细 纯化突变体的动力学、药理学和结构特性 将这些酶与正常酶进行比较。