Intragenic Suppressors of McCune-Albright Syndrome Mutations

描述（由申请人提供）：McCune-Albright综合征（MAS）是一种遗传综合征，经典定义为骨纤维异常增殖症、性早熟和咖啡牛奶皮肤增生三联征，通常伴有功能亢进的内分泌系统的其他疾病。异源三聚体G蛋白亚基Gs中的单个氨基酸取代导致其信号传导途径的组成性激活，因为G蛋白不能水解GTP以使其自身水解。该项目的长期目标是通过鉴定突变蛋白上可以取代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。这些抑制位点可以作为药物的靶点，实现G蛋白活性的类似修饰。我们最近使用了酵母系统，以确定一个基因内抑制基因的突变，在酵母G蛋白同源的MAS缺陷。本实验的目的是将这些初步结果推广到抑制基因的分析和更多抑制基因的鉴定。具体的目的是：1）利用体外生物化学方法分析大肠杆菌表达和纯化的蛋白质，分析基因内抑制因子。杆菌试验将使用游离磷酸盐的比色测定法测量Gs同种型的GTdR活性，并通过观察差异胰蛋白酶消化模式和/或色氨酸自发荧光来测量其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和受体刺激的cAMP水平的变化，沿着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，并通过对第一个基因内抑制基因中鉴定的位点进行进一步的定点诱变，鉴定更多的抑制基因等位基因。将这些抑制突变定位到Gs的结构上，将为设计更有效的治疗MAS患者的药物提供重要的基础信息。 公共卫生相关性：McCune-Albright综合征患者表现出承重骨骼的骨无力、面骨畸形、青春期异常早开始和皮肤色素沉着斑。这种综合征是由G蛋白的正常调节丧失引起的许多疾病之一，G蛋白是对许多激素和神经递质的反应必不可少的细胞成分。该项目旨在提供重要的基础科学信息，可用于合理设计McCune-Albright综合征和其他由G蛋白缺陷引起的疾病的药物。

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.