Intragenic Suppressors of McCune-Albright Syndrome Mutations

描述（由申请人提供）：麦库恩-奥尔布赖特综合征（MAS）是一种典型的遗传综合征，由骨质纤维发育不良、性早熟和皮肤色素沉着三合症定义，通常伴有内分泌系统功能亢进的其他疾病。异三聚体g蛋白亚基Gs中的单个氨基酸取代导致其信号通路的组成性激活，因为g蛋白不能水解GTP来使自身失活。该项目的长期目标是通过识别突变蛋白上可以灭活Gs的MAS亚型的位点，为合理设计治疗MAS患者的药物做出贡献。然后，这些抑制位点可以被开发为药物的靶标，从而实现对g蛋白活性的类似修饰。我们最近使用酵母系统鉴定了与MAS缺陷同源的酵母g蛋白突变的基因内抑制因子。该实验的目标是将这些初步结果扩展到抑制基因的分析和更多抑制基因等位基因的鉴定中。具体目的是：1)利用体外生化分析大肠杆菌中表达和纯化的蛋白质来分析基因内抑制因子。试验将使用游离磷酸盐比色法测量Gs亚型的GTPase活性，并通过观察胰蛋白酶消化模式和/或色氨酸自身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，利用报告基因分析细胞中cAMP基础水平和受体刺激水平的变化，以及cAMP调控基因的表达变化。3)通过筛选组成活性g蛋白的随机突变文库，并对在第一个基因内抑制基因中发现的位点进行进一步的定点突变，鉴定出更多的抑制等位基因。将这些抑制突变映射到Gs的结构将为设计更有效的MAS患者药物提供重要的基础信息。

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.