UNIQUE AMINO DOMAINS AND AMP DEAMINASE ISOFORM DIVERSITY

独特的氨基结构域和 AMP 脱氨酶异构体多样性

• 批准号: 6177504
• 负责人: RICHARD L SABINA
• 金额: $ 19.63万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177504
• 关键词: AMP deaminase; X ray crystallography; actins; active sites; adenylate kinase; affinity labeling; enzyme deficiency; enzyme mechanism; enzyme structure; gene expression; hybrid enzyme; isozymes; muscle metabolism; myosins; phosphatidylinositols; phosphorylation; protein binding; protein isoforms; protein localization; protein protein interaction; protein sequence; protein structure function; recombinant proteins

AMP deaminase (AMPD; EC 3.5.4.6) is a diverse and highly regulated enzyme located at a branchpoint in the adenylate catabolic pathway. AMPD plays a key role during metabolic imbalances of energy supply and demand homeostasis by competing with 5' nucleotidase for available AMP. This role is underscored in striated muscle where inheritance of a prolonged survival in congestive hart failure. These opposing clinical consequences reflect a functional difference in adenylate catabolism in various striated muscles due, in part, to distinct regulatory features of AMPD isoforms. Emerging data show that divergent N-terminal domains in AMPD polypeptides alter physical and functional properties of the enzyme. The long-term objective of this project is to gain a comprehensive understanding of AMPD regulation in striated muscle with an emphasis on the functional significance for divergent N-terminal domains. The continued pursuit of this goal will be accomplished by expressing, isolating an characterizing wild type and genetically modified human AMPD recombinant enzymes that will be used to 1) define the structural bases and functional effects of phosphorylation and phosphoinositides on the catalytic and actomyosin binding properties of human AMPD recombinant isoforms. These analyses will also sere to assess the relationship between these two opposing regulators of AMPD catalytic activity, 2) determine the combined effects of a cassette-type alternative splicing event and a P43L substitution on the human AMPD1 recombinant enzyme. This latter information is central to hypotheses designed to explain clinical outcomes resulting from inheritance of the prevalent AMPD1 mutant allele, and 3) attempt to solve the crystal structure of the human AMPD1 recombinant enzyme. Detailed structural knowledge of AMPD1 will facilitate our understanding of the complex regulation of this enzyme. These combined efforts should provide critical information that may help explain clinical outcomes associated with a prevalent AMPD1 mutant allele.

AMP脱氨酶（AMP deaminase, EC 3.5.4.6）是一种多样且高度调控的酶，位于腺苷酸分解代谢途径的一个分支点。AMPD通过与5′核苷酸酶竞争可用的AMP，在能量供需平衡的代谢失衡中发挥关键作用。这一作用在横纹肌中得到强调，其中遗传在充血性心力衰竭中延长了生存期。这些相反的临床结果反映了不同横纹肌中腺苷酸分解代谢的功能差异，部分原因是AMPD亚型的不同调节特征。新出现的数据表明，AMPD多肽中不同的n端结构域改变了酶的物理和功能特性。该项目的长期目标是全面了解横纹肌中AMPD的调控，重点研究不同n端结构域的功能意义。为了实现这一目标，我们将通过表达和分离一种具有特征的野生型和转基因人AMPD重组酶来实现这一目标，这些酶将用于1)确定磷酸化和磷酸肌苷对人AMPD重组亚型的催化和肌动球蛋白结合特性的结构基础和功能影响。这些分析还将用于评估这两个相反的AMPD催化活性调节因子之间的关系，2)确定盒式选择性剪接事件和P43L取代对人AMPD1重组酶的综合影响。后一种信息对于解释普遍的AMPD1突变等位基因遗传导致的临床结果的假设至关重要，并且3)试图解决人类AMPD1重组酶的晶体结构。详细的AMPD1结构知识将有助于我们理解这种酶的复杂调控。这些共同的努力应该提供关键的信息，可能有助于解释与流行的AMPD1突变等位基因相关的临床结果。