Functional characterization of a mitochondrial orphan enzyme

 描述(由申请人提供)：该提案的长期目标是破译线粒体孤儿酶的功能及其对未探索的代谢途径的调节，可能对人类疾病产生影响。线粒体是细胞内的细胞器，不仅包含合成ATP和燃料氧化的机制，而且还包含合成细胞生长所需构件的代谢酶。例如，一种关键的线粒体酶甲基丙二酰辅酶A变位酶(MUT)将氨基酸和脂肪中的支链碳链循环到三羧酸循环中。这一过程严重依赖于一种重要的辅酶维生素B12(B12)。本课题主要研究了一种广泛表达的未知功能的线粒体酶CLYBL在调节线粒体B12功能中所起的作用。最近的两项人类全基因组关联研究表明，CLYBL的一种功能丧失变异与亚临床B12缺乏症有关。这一建议建立在我们实验室进行的计算基因组学分析的基础上，该分析表明CLYBL位于一条新的线粒体代谢途径的核心，该途径与B12依赖的MUT功能有关。这项拟议的研究试图理解：(1)CLYBL的酶活性；(2)依赖CLYBL对线粒体B12功能的调节；以及(3)这种调节的分子机制。这些研究将利用实验室在先进的基于质谱学的代谢组学、计算基因组学和线粒体生理学方面的专业知识，结合我在生物化学和细胞生物学方面的技能，破译CLYBL的生理功能。对CLYBL的研究代表了“反向人类遗传学”的一个很好的例子。这项工作有望为线粒体生物化学提供新的见解，并对人类疾病产生潜在的影响。

 DESCRIPTION (provided by applicant): The long-term goal of the proposal is to decipher the function of mitochondrial orphan enzymes and their regulations on unexplored metabolic pathways with potential implications for human diseases. Mitochondria are intracellular organelles that house not only the machinery for ATP synthesis and fuel oxidation, but also metabolic enzymes for synthesis of building blocks for cell growth. For instance, a critical mitochondrial enzyme methylmalonyl-CoA mutase (MUT) recycles branched carbon chains from amino acids and lipids into tricarboxylic acid cycle. This process is critically dependent on an essential coenzyme vitamin B12 (B12). This project focuses on the role played by CLYBL, a ubiquitously expressed mitochondrial enzyme of unknown function, in regulating mitochondrial B12 function. A loss-of-function variant of CLYBL has been linked to subclinical B12 deficiency by two recent human genome-wide association studies. This proposal builds on the computational genomics analysis performed in our laboratory, which suggested CLYBL lies at the heart of a novel mitochondrial metabolic pathway connecting to B12-dependent MUT function. The proposed research seeks to understand: (1) enzymatic activities of CLYBL; (2) the CLYBL-dependent regulation of mitochondrial B12 function; and (3) molecular mechanism underlying this regulation. These studies will take advantage of the laboratory's expertise in advanced mass spectrometry-based metabolomics, computational genomics and mitochondrial physiology, combined with my skills in biochemistry and cell biology, to decipher the physiological function of CLYBL. This study of CLYBL represents a great example of "reverse human genetics". This work promises to shed new insights on mitochondrial biochemistry with potential implications for human diseases.