Genome-edited uveal melanoma cell lines for investigating constitutively active GNAQ and GNA11

用于研究组成型活性 GNAQ 和 GNA11 的基因组编辑葡萄膜黑色素瘤细胞系

• 批准号: 9205498
• 负责人: PHILIP B WEDEGAERTNER
• 金额: $ 7.8万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-13 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9205498
• 关键词: Address; Adult; Amino Acids; Basic Science; Biological; Biological Assay; CRISPR/Cas technology; Cell Line; Cell Proliferation; Cell membrane; Cells; Cellular biology; Code; Cytoplasm; Development; Dissociation; Event; Exhibits; Fluorescence Microscopy; Fluorescence-Activated Cell Sorting; G-Protein-Coupled Receptors; G-substrate; GNAQ gene; GTP Binding; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genes; Goals; Guanosine Triphosphate; Heterotrimeric G Protein Subunit; Heterotrimeric GTP-Binding Proteins; Hydrolysis; Intracellular Membranes; Knowledge; Link; Lipids; Liver; Location; Malignant Neoplasms; Melanoma Cell; Mitogen-Activated Protein Kinases; Modeling; Modification; Molecular; Mutation; Neoplasm Metastasis; Oncogenic; Pathway interactions; Patients; Physiological Processes; Play; Predisposition; Property; Proteins; Regulation; Research; Role; Signal Pathway; Signal Transduction; Signaling Protein; Technology; Testing; Therapeutic Effect; Uveal Melanoma; Work; actionable mutation; base; cancer cell; effective therapy; experience; extracellular; genome editing; inhibitor/antagonist; live cell imaging; malignant neoplasm of eye; mutant; new therapeutic target; novel; novel therapeutic intervention; protein expression; protein function; public health relevance; research study; response; therapeutic target; tool; trafficking

 DESCRIPTION (provided by applicant): Heterotrimeric G proteins (αβγ) are well known for their function in linking G protein- coupled receptors (GPCRs) to intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological process. G proteins function as molecular switches whereby a GPCR promotes GDP release from the α subunit (Gα), followed by subsequent binding of GTP by Gα and dissociation of Gα and Gβγ. Gα-GTP and Gβγ can then regulate a variety of signaling proteins until the GTP hydrolysis activity of Gα turns off signaling by generating the inactive Gα-GDP, which then re- associates with Gβγ to complete the cycle of activation and inactivation. However, in uveal melanoma this tightly controlled G protein cycle is corrupted by a mutation in the closely related Gα, αq or α1. Such mutations, occurring in over 90% of uveal melanoma, generate a constitutively active αq or α11 in which GTP hydrolysis activity is abrogated, thereby locking the Gα in an active GTP bound state and turning αq or α11 into an oncogenic driver of this cancer. Uveal melanoma, the most common cancer of the eye in adults, metastasizes in up to 50% of uveal melanoma patients, and, once metastasis occurs, it is invariably fatal with an average survival of less than six months. There are currently no effective therapies for metastatic uveal melanoma, and thus there exists an urgent need to better understand the molecular mechanisms that promote the development of uveal melanoma. Based on numerous basic research studies on G protein function, it is clear that mutationally activated Gα exhibit key cell biology properties, such as changes in subcellular localization, trafficking, lipid modification, degradation and activation of unique signaling pathways, that set them apart from the wild type Gα counterpart. In uveal melanoma, it has been difficult to compare mutationally activated αq or α11 with wild type αq or α11 due to a lack of tools to differentiate between and effectively study the endogenous mutant versus wild type protein. Therefore, the objective of this project is to develop such a tool by using gene-editing approaches with uveal melanoma cell lines to generate cells in which the endogenous αq or α11, either the wild type or mutationally activated form, is fused in-frame to GFP. These genome-edited uveal melanoma cell lines will be a novel and much-needed tool to better understand how mutationally activated αq or α11 functions in uveal melanoma and to identify key aspects of regulation of mutationally activated αq or α11 that differ from that of wld type αq or α11.

 描述（由申请人提供）： 异三聚合物G蛋白（αβγ）在将G蛋白偶联受体（GPCR）与细胞内反应联系起来，从而在传播各种细胞外信号与无数物理过程的调节中发挥重要作用而闻名。 G蛋白充当分子开关，从而促进GPCR从α亚基（Gα）促进GDP释放，然后通过Gα与GTP结合并随后结合Gα和Gα和Gβγ。然后，Gα-GTP和Gβγ可以调节多种信号蛋白，直到GTP的GTP水解活性通过产生不活跃的Gα-GDP关闭信号传导，然后将其与Gβγ重新缔合以完成激活和灭活周期。然而，在卵巢黑色素瘤中，这种紧密控制的G蛋白周期因密切相关的Gα，αQ或α1的突变而损坏。这种突变发生在超过90％的紫veal黑色素瘤中，产生一个组成型活性的αQ或α11，其中GTP水解活性被废除，从而将Gα锁定在活跃的GTP结合状态下，并将αQ或α11转动到该癌症的癌症驱动器中。紫美黑色素瘤是成人眼睛最常见的眼癌，在多达50％的紫veal黑色素瘤患者中转移，一旦发生转移，它总是致命的，平均生存率小于小于 六个月。目前尚无有效的转移性卵子黑色素瘤的疗法，因此迫切需要更好地了解促进紫veal黑色素瘤发展的分子机制。基于许多关于G蛋白功能的基础研究，很明显，突变激活的Gα暴露的关键细胞生物学特性，例如亚细胞定位，运输，脂质修饰，降解和激活的变化 唯一的信号通路，这使它们与野生型Gα对应物区分开。在卵巢黑色素瘤中，很难将突变活化的αQ或α11与野生型αq或 α11由于缺乏区分和有效研究内源性突变体与野生型蛋白质的工具。因此，该项目的目的是通过使用紫veal黑色素瘤细胞系的基因编辑方法来开发这种工具，以生成细胞，其中内源性αQ或α11（野生型或突变激活形式）与GFP融合在一起。这些基因组编辑的紫veal黑色素瘤细胞系将是一种新颖且急需的工具，可以更好地了解突变活化的αQ或α11如何在卵巢黑色素瘤中起作用，并确定与WLDαQ或α11型的突变激活的αQ或α11调节的关键方面。