Intragenic Suppressors of McCune-Albright Syndrome Mutations

McCune-Albright 综合征突变的基因内抑制因子

• 批准号: 7777980
• 负责人: Robin Pals Rylaarsdam
• 金额: $ 17.87万
• 依托单位: BENEDICTINE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-17 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777980
• 关键词: Adopted; Alleles; Amino Acid Substitution; Amino Acids; Basic Science; Binding; Biochemical; Biological Assay; Cell Culture Techniques; Cells; Codon Nucleotides; Cyclic AMP; Defect; Digestion; Disease; Drug Delivery Systems; Drug Design; Embryonic Development; Endocrine system; Engineering; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Exhibits; Foundations; GNAS gene; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Gene Expression; Genes; Genetic; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric G Protein Subunit; Hormones; Human; Hydrolysis; In Vitro; Libraries; Mammalian Cell; Maps; McCune-Albright Syndrome; Measures; Modification; Molecular Conformation; Mutate; Mutation; Neurotransmitters; Osteitis Fibrosa Disseminata; Patients; Pattern; Peptide Hydrolases; Pharmaceutical Preparations; Phenotype; Pilot Projects; Polyostotic fibrous dysplasia; Precocious Puberty; Protein Analysis; Protein Isoforms; Proteins; Puberty; Regulation; Relative (related person); Reporter Genes; Saccharomyces cerevisiae; Screening procedure; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Skeleton; Skin; Skin Pigmentation; Structure; Suppressor Mutations; Symptoms; Syndrome; System; Techniques; Triad Acrylic Resin; Trypsin; Tryptophan; Weight-Bearing state; Work; Yeast Model System; Yeasts; bone; design; expression vector; face bone structure; inorganic phosphate; malformation; migration; public health relevance; receptor; research study; response

DESCRIPTION (provided by applicant): McCune-Albright Syndrome (MAS) is a genetic syndrome classically defined by the triad of fibrous dysplasia of the bone, precocious puberty, and cafi-au-lait skin hyperpigentation, often accompanied by other disorders of a hyperfunctioning endocrine system. A single amino acid substitution in the heterotrimeric G-protein subunit Gs causes constitutive activation of its signaling pathways, as the G-protein cannot hydrolyze GTP to inactivate itself. The long-term goal of this project is to contribute to the rational design of drugs to treat MAS patients by identifying sites on the mutated protein which can inactivate the MAS isoform of Gs. These suppressing sites can then be developed as targets for drugs that achieve a similar modification of the G-protein's activity. We have recently used a yeast system to identify an intragenic suppressor of a mutation in the yeast G-protein homologous to the MAS defect. The goal of the proposed experiments is to extend these preliminary results in the analysis of the suppressor and the identification of more suppressor alleles. The specific aims are: 1) Analyze intragenic suppressors using in vitro biochemical analysis of proteins expressed in and purified from E. coli. Assays will measure the GTPase activity of the Gs isoforms using a colorimetric assay for free phosphate, and measure their ability to adopt active vs. inactive conformations by observing differential trypsin digestion patterns and/or tryptophan autofluorescence. 2) Express suppressor alleles in mammalian cells, and analyze the cells for changes in basal and receptor-stimulated cAMP levels, along with changes in expression of cAMP-regulated genes using a reporter gene. 3) Identify more suppressor alleles by screening a library of random mutations in the constitutively active G-protein, and by performing further site-directed mutagenesis of the site identified in the first intragenic suppressor. Mapping these suppressor mutations to the structure of Gs will provide important foundational information for the design of more effective drugs for MAS patients. PUBLIC HEALTH RELEVANCE: McCune-Albright Syndrome patients exhibit bone weakness in the weight-bearing skeleton, malformations of the facial bones, abnormally early beginning of puberty, and patches of hyperpigmented skin. This syndrome is one of many diseases caused by the loss of normal regulation of G-proteins, cellular components that are essential for the responses to many hormones and neurotransmitters. This project aims to provide important basic science information that can be used in the rational design of drugs for McCune-Albright Syndrome and other disorders cause by G-protein defects.

描述（由申请人提供）：麦库尼-奥尔布赖特综合征 (MAS) 是一种遗传综合征，经典定义为骨纤维发育不良、性早熟和咖啡牛奶皮肤色素沉着三联征，通常伴有内分泌系统功能亢进的其他疾病。异源三聚体 G 蛋白亚基 Gs 中的单个氨基酸取代会导致其信号通路的组成型激活，因为 G 蛋白不能水解 GTP 使其自身失活。该项目的长期目标是通过识别突变蛋白上可灭活 Gs 的 MAS 亚型的位点，为治疗 MAS 患者的药物的合理设计做出贡献。然后可以将这些抑制位点开发为药物的靶标，从而实现 G 蛋白活性的类似修饰。我们最近使用酵母系统来鉴定与 MAS 缺陷同源的酵母 G 蛋白突变的基因内抑制因子。所提出的实验的目标是将这些初步结果扩展到抑制子的分析和更多抑制子等位基因的鉴定中。具体目标是： 1) 对大肠杆菌中表达和纯化的蛋白质进行体外生化分析，分析基因内抑制因子。测定将使用游离磷酸盐的比色测定来测量 Gs 亚型的 GTP 酶活性，并通过观察不同的胰蛋白酶消化模式和/或色氨酸自发荧光来测量它们采用活性与非活性构象的能力。 2) 在哺乳动物细胞中表达抑制等位基因，并使用报告基因分析细胞的基础 cAMP 水平和受体刺激的 cAMP 水平的变化，以及 cAMP 调节基因表达的变化。 3) 通过筛选组成型活性 G 蛋白中的随机突变文库，并对第一个基因内抑制因子中鉴定的位点进行进一步的定点诱变，鉴定更多抑制等位基因。将这些抑制突变映射到 Gs 结构将为设计针对 MAS 患者更有效的药物提供重要的基础信息。 公共健康相关性：麦库尼-奥尔布赖特综合症患者表现出承重骨骼骨质无力、面部骨骼畸形、青春期异常提前开始以及皮肤色素沉着过度斑块。这种综合征是由 G 蛋白正常调节丧失引起的众多疾病之一，G 蛋白是对许多激素和神经递质做出反应所必需的细胞成分。该项目旨在提供重要的基础科学信息，可用于合理设计治疗 McCune-Albright 综合征和其他由 G 蛋白缺陷引起的疾病的药物。