Intragenic Suppressors of McCune-Albright Syndrome Mutations

描述(申请人提供)：麦考恩-奥尔布赖特综合征(MAS)是一种遗传性综合征，经典的定义是骨纤维发育不良、性早熟和咖啡豆皮肤色素沉着三者，通常伴有其他功能亢进的内分泌系统障碍。异源三聚体G蛋白亚基Gs中的单个氨基酸替换导致其信号通路的结构性激活，因为G蛋白不能水解GTP来灭活自己。该项目的长期目标是通过确定突变蛋白上可以使Gs的MAS亚型失活的位点，为治疗MAS患者的药物的合理设计做出贡献。然后，这些抑制位点可以被开发成药物的靶点，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酵母G蛋白中与MAS缺陷同源的突变的基因内抑制物。拟议的实验的目标是将这些初步结果扩展到抑制基因的分析和识别更多的抑制基因。其具体目的是：1)利用在大肠杆菌中表达和纯化的蛋白质的体外生化分析来分析基因内抑制物。检测将使用游离磷的比色法来测量Gs异构体的GTPase活性，并通过观察不同的胰酶消化模式和/或色氨酸自发荧光来测量它们采用活性构象和非活性构象的能力。2)在哺乳动物细胞中表达抑制基因，并使用报告基因分析细胞基础和受体刺激的cAMP水平的变化，以及cAMP调节基因表达的变化。3)通过在成分活性G蛋白中筛选随机突变文库，以及通过对第一个基因内抑制子中确定的位点进行进一步的定点突变，来鉴定更多的抑制子等位基因。将这些抑制子突变映射到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。 公共卫生相关性：麦库恩-奥尔布赖特综合征患者表现为承重骨骼骨质薄弱，面部骨骼畸形，青春期异常提前，以及皮肤色素沉着斑块。这种综合征是由于G蛋白失去正常调节而导致的许多疾病之一，G蛋白是对许多激素和神经递质的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。

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.