Identifying and characterizing pathways of amino acid metabolism and signaling

氨基酸代谢和信号传导途径的识别和表征

• 批准号: RGPIN-2014-03687
• 负责人: Pulinilkunnil, Thomas
• 金额: $ 2.55万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566030
• 关键词: Identifying; characterizing; pathways; amino; acid

In muscle cells a balanced usage of nutrients such as glucose, fatty acids, ketones, amino acids and lactate is a prerequisite for optimal muscle growth. The longstanding view is that generation of energy within the power house of the cells (mitochondria) primarily from glucose and fatty acids stimulates muscle growth and survival. Amino acids particularly branched-chain amino acids (BCAA) such as leucine, isoleucine, and valine are mostly used for manufacturing proteins for growth within muscle but are also used for generating energy. However our understanding of how BCAA are utilized for generating energy within muscle cells is still lacking. Goals of our research are to determine the enzymes mediating this process, how these enzymes are regulated and the impact on the cell when BCAA are used as an energy source. We want to know whether BCAA compete with other nutrients for being consumed by the cell for energy and which pathways regulate the competition process. What is known so far is that, once inside the cell, BCAA are chemically converted to branch chain alpha ketoacids (BCKA) by virtue of the enzymes branch chain amino transferase (BCAT), branch chain ketodehydrogenase (BCKDH) and ketodehydrogenase kinase (BCKDK) to allow BCKA to enter mitochondria for use as an energy source. Despite the widespread presence of amino acid metabolizing enzymes in different tissues their impact on mitochondrial energy generation and function remains unexplored. Using cell biology, genetic and whole body approaches in zebrafish and mouse my research program will focus specifically on addressing two conceptual puzzles. Puzzle 1) to uncover how the nutrient triad glucose, fatty acid and BCAA biochemically communicate with each other resulting in nutrient transport and utilization within the mitochondria; Puzzle 2) to decipher whether within the cell different biomolecules such as ions, hormones, lipids, sugars and neurotransmitters employ the services of amino acids to govern cellular growth and survival. For puzzle 1, studies will be undertaken in zebrafish and mouse evaluating the effect of alterations in concentration and types of BCAA and BCKA on mitochondrial function in skeletal and cardiac muscle tissues. A method for analysing breakdown products of BCAA will be developed. In addition, conventional cell and molecular biology methodologies will be employed to evaluate the effects of altered BCAA and BCKA on murine and zebrafish amino acid metabolic enzymes (BCAT, BCKDH and BCKDK). To establish the contribution of these enzymes to cellular homeostasis, genetically mutated versions of these enzymes tagged with light emitting probes (fluorescence) will be engineered and expressed in murine cells and zebrafish. For resolving puzzle 2, cellular organelles from murine and zebrafish cells and/or tissues subjected to BCAA and BCKA treatment will be collected and subjected to discovery of genes and proteins influencing cellular size, proliferation and survival. To address how amino acids exert physiological effects of hormones, ions, sugars, and neuronal factors, mice and zebrafish will be administered biomolecules to mirror biological effects and cells and tissues harvested for molecular and physiological analysis. This research program will identify and characterize previously unrecognized proteins and novel molecular networks of cellular amino acid signaling, metabolism and physiology. The research will provide opportunities for graduate and undergraduate trainees to directly contribute to expanding our knowledge of amino acid biology, knowledge that will have direct impacts in the fields of mitochondrial biology, food science and human nutrition offering tremendous benefit to Canadian discovery science, education and innovation.

在肌肉细胞中，平衡使用营养素，如葡萄糖，脂肪酸，酮，氨基酸和乳酸盐是最佳肌肉生长的先决条件。长期以来的观点是，主要从葡萄糖和脂肪酸产生的能量在细胞的发电厂（线粒体）内刺激肌肉生长和存活。氨基酸，特别是支链氨基酸（BCAA），如亮氨酸，异亮氨酸和缬氨酸，主要用于制造肌肉内生长的蛋白质，但也用于产生能量。然而，我们对BCAA如何在肌肉细胞内产生能量的理解仍然缺乏。我们的研究目标是确定介导这一过程的酶，这些酶是如何被调节的，以及当BCAA被用作能量来源时对细胞的影响。我们想知道BCAA是否与其他营养素竞争被细胞消耗能量，以及哪些途径调节竞争过程。目前已知的是，一旦进入细胞，BCAA通过酶分支链氨基转移酶（BCAT）、分支链酮脱氢酶（BCKDH）和酮脱氢酶激酶（BCKDK）化学转化为分支链α酮酸（BCKA），以允许BCKA进入线粒体用作能量来源。尽管氨基酸代谢酶广泛存在于不同的组织中，但它们对线粒体能量产生和功能的影响仍未被探索。使用细胞生物学，遗传和全身的方法在斑马鱼和小鼠我的研究计划将特别侧重于解决两个概念上的难题。谜题1）揭示营养三联体葡萄糖、脂肪酸和支链氨基酸如何在线粒体内进行生物化学交流，从而导致营养物质的运输和利用;谜题2）破译细胞内不同的生物分子（如离子、激素、脂质、糖和神经递质）是否使用氨基酸来控制细胞的生长和存活。对于难题1，将在斑马鱼和小鼠中进行研究，评估BCAA和BCKA的浓度和类型的改变对骨骼肌和心肌组织中线粒体功能的影响。将开发一种分析BCAA分解产物的方法。此外，将采用常规细胞和分子生物学方法来评价改变的BCAA和BCKA对小鼠和斑马鱼氨基酸代谢酶（BCAT、BCKDH和BCKDK）的影响。为了确定这些酶对细胞内稳态的贡献，将在鼠细胞和斑马鱼中工程化并表达标记有发光探针（荧光）的这些酶的遗传突变形式。为了解决难题2，将收集来自经过BCAA和BCKA处理的鼠和斑马鱼细胞和/或组织的细胞器，并发现影响细胞大小、增殖和存活的基因和蛋白质。为了解决氨基酸如何发挥激素，离子，糖和神经元因子的生理作用，将向小鼠和斑马鱼施用生物分子以反映生物效应，并收获细胞和组织用于分子和生理分析。该研究计划将识别和表征以前未识别的蛋白质以及细胞氨基酸信号传导、代谢和生理学的新型分子网络。该研究将为研究生和本科生学员提供机会，直接有助于扩大我们对氨基酸生物学的知识，这些知识将对线粒体生物学，食品科学和人类营养领域产生直接影响，为加拿大发现科学，教育和创新带来巨大利益。