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: 8116252
• 负责人: Robin Pals Rylaarsdam
• 金额: $ 4.48万
• 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: 2010-09-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8116252
• 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）：McCune-Albright综合征（MAS）是一种遗传综合征，其经典由骨骼的纤维发育异常三合会，早熟的青春期和CAFI-AU-au-Lait皮肤过度构图，通常由其他伴随性内分泌系统过度疾病伴随。异三聚体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蛋白无法将GTP水解为失活。该项目的长期目标是通过识别可能使GS的MAS同工型失活的突变蛋白上的位点来为治疗MAS患者的合理设计做出贡献。然后可以开发这些抑制位点作为对G蛋白活性进行类似修饰的药物的靶标。我们最近使用酵母系统来鉴定酵母G蛋白与MAS缺损同源的突变的基因内抑制剂。提出的实验的目的是在分析抑制器和识别更多抑制剂等位基因的情况下扩展这些初步结果。具体目的是：1）使用对大肠杆菌表达并纯化的蛋白质的体外生化分析分析基因内抑制子。测定法将使用比色测定法对GS同工型的GTPase活性进行自由磷酸盐的影响，并通过观察差异胰蛋白酶消化模式和/或色氨酸自动荧光来衡量其采用活性与非活动构象的能力。 2）在哺乳动物细胞中表达抑制等位基因，并分析细胞的基础和受体刺激的cAMP水平的变化，以及使用报告基因基因的cAMP调节基因表达的变化。 3）通过筛选组成型活性G蛋白中的随机突变库，并通过对第一个基因内抑制器中鉴定的位点进行进一步的位置定向诱变来识别更多的抑制等位基因。将这些抑制突变映射到GS结构中，将为MAS患者设计更有效的药物提供重要的基础信息。 公共卫生相关性：McCune-Albright综合征患者的体重骨骼，面部骨骼的畸形，青春期的早期开始和皮肤色素沉重的皮肤表现出骨骼虚弱。该综合征是由G蛋白正常调节的损失引起的众多疾病之一，这对于对许多激素和神经递质的反应至关重要。该项目旨在提供重要的基本科学信息，这些信息可用于用于麦克纳albright综合征的药物的合理设计，而其他疾病因G蛋白缺陷而导致的其他疾病。

项目成果

Three intragenic suppressors of a GTPase-deficient allele of GNAS associated with McCune-Albright syndrome.

与 McCune-Albright 综合征相关的 GNAS GTP 酶缺陷等位基因的三个基因内抑制因子。

• DOI: 10.1530/jme-13-0297
• 发表时间: 2014
• 期刊: Journal of molecular endocrinology
• 影响因子: 3.5
• 作者: Turcic,Kyle;Tobar-Rubin,Raquel;Janevska,Daniela;Carroll,Julie;Din,Eraj;Alvarez,Rebecca;Haick,Jennifer;Pals-Rylaarsdam,Robin
• 通讯作者: Pals-Rylaarsdam,Robin