Structural and functional studies of the human RIO kinases

人类 RIO 激酶的结构和功能研究

• 批准号: 7264828
• 负责人: Nicole A LaRonde
• 金额: $ 16.35万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-21 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7264828
• 关键词: Address; Amino Acid Sequence; Base Sequence; Binding; Binding Sites; Biochemical; Biochemistry; Biogenesis; Cell Cycle Arrest; Cell Nucleolus; Cell Proliferation; Cells; Colon Carcinoma; Complex; Coupled; Crystallography; Development; Elements; Enzymes; Escherichia coli; Eukaryota; Eukaryotic Cell; Evaluation; Goals; Human; Insecta; Knock-out; Knowledge; Malignant Neoplasms; Mammalian Cell; Maps; Measures; Molecular; Molecular Conformation; Mus; Mutation; Nature; Oncogenes; Peptide Sequence Determination; Peptides; Phosphopeptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Plasmids; Process; Production; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; RNA; RNA Processing; RNA, Ribosomal, 18S; Rate; Reaction; Reporting; Research Personnel; Ribosomal Protein S6; Ribosomal RNA; Ribosomes; Riots; Roentgen Rays; Role; Sequence Alignment; Sequence Homology; Site; Site-Directed Mutagenesis; Source; Structure; Substrate Interaction; Substrate Specificity; Testing; Therapeutic; Up-Regulation; Work; X-Ray Crystallography; base; cancer cell; cancer therapy; cell growth; design; design and construction; inhibitor/antagonist; inorganic phosphate; melanoma; programs; size; small molecule; tumor progression

DESCRIPTION (provided by applicant): Cancer cell proliferation cannot occur in the absence of ribosome biogenesis. This is evidenced by an increase in the size of nucleoli in cancer cells and the upregulation of ribosome biogenesis by several oncogenes. Therefore, modulating the rate of ribosome synthesis could provide a means of blocking cancer progression. Ribosomal RNA (rRNA), which comprises most of the ribosome, are matured in a complex, stepwise process in the nucleoli. This involves several processing factors which are still poorly understood at the molecular level. My long term goal is to use X-ray crystallography and biochemistry to study the molecular mechanisms of rRNA processing factors. I will begin this work with the study of the RIO kinases, a group of ancient, highly conserved, atypical serine kinases reported to be necessary for the final maturation step of the 18S rRNA. The 18S rRNA is the RNA component of the small ribosomal subunit. Based on our previous structural studies of archaeal RIO kinases, I hypothesize the RIO kinases must interact with peptide substrates and ATP in a unique manner distinct from that seen for canonical protein kinases. To investigate this hypothesis, I propose to 1. Determine the substrate specificity of human Rio1 and Rio3, and 2. Determine the substrate binding site of human Rio1 and Rio3. This will demonstrate how RIO kinases interact with peptide substrate and identify residues on the substrate which direct phosphorylation by RIO kinases. I will address these aims by purifying human Rio1 and Rio3, determining the autophosphorylation sites on each protein, measuring the effect of site-directed mutations on the phosphoryl transfer reaction to evaluate the importance of specific residues, identifying putative substrates in mammalian cells based on phosphorylation site determination and solving the structure or human Rio1 and Rio3 with bound substrates. This work will provide the structural and functional information required for the successful inhibition of the RIO kinases, which will allow inhibition of rRNA processing. Since rRNA processing is a major step of ribosome biogenesis, and ribosome biogenesis is necessary for cancer progression, this may provide an opportunity for the development of cancer therapeutics. Therefore, this study will provide information for the evaluation of the RIO kinases as targets for the development of cancer treatment.

描述（由申请人提供）：在没有核糖体生物发生的情况下，癌细胞增殖不可能发生。癌细胞中核仁的大小增加以及几种肿瘤基因的核糖体生物发生的上调可以证明这一点。因此，调节核糖体合成速率可以提供阻断癌症进展的一种手段。核糖体RNA（RRNA）包括大多数核糖体，在核仁中的复杂的逐步过程中成熟。这涉及几个处理因子，这些因素在分子水平上仍未理解。我的长期目标是使用X射线晶体学和生物化学研究rRNA加工因子的分子机制。我将从对里约激酶的研究开始这项工作，里约激酶是一群古老的，高度保守的非典型丝氨酸激酶，据报道是18S rRNA的最终成熟步骤所必需的。 18S rRNA是小核糖体亚基的RNA成分。根据我们先前对古细菌激酶的结构研究，我假设里约激酶必须与肽底物和ATP相互作用，并以与规范蛋白激酶有关的独特方式与ATP相互作用。为了研究这一假设，我建议1。确定人Rio1和Rio3的底物特异性，以及2。确定人Rio1和Rio3的底物结合位点。这将证明里约激酶如何与肽底物相互作用并鉴定底物上的残基，这些残基通过rio激酶引导磷酸化。我将通过净化人Rio1和Rio3来解决这些目标，确定每种蛋白质上的自磷酸化位点，测量位置定向突变对磷酸化转移反应的影响，以评估特定残基的重要性，从而确定基于磷酸化位点的确定和解决结构和人类Rio 1和Rio 3和Rio 3和Rio 3和Rio 3和Rio 3和Rio 3和Rio3和Ry3和Ry3和Rio3和Rio3和Rio3和Rio3和Rio3和Rio3和Rio3和Rio3和Rio3和Rio3和Rio3 suid contection nece necented ut。 这项工作将提供成功抑制RIO激酶所需的结构和功能信息，这将允许抑制RRNA处理。由于rRNA加工是核糖体生物发生的主要步骤，核糖体生物发生对于癌症进展是必要的，因此这可能为癌症治疗剂的发展提供了机会。因此，这项研究将为Rio激酶作为癌症治疗的靶标提供信息。