CARNITINE PALMITOYLTRANSFERASE I ISOFORM FUNCTION

描述（申请人摘要）：肉毒碱棕榈酰转移酶I（CPT-1） 催化线粒体FA氧化的限速步骤。催化 先前鉴定的CPT-I酶（B 1“肌肉”）和CPT-IA的活性 [肝]）可以被丙二酰辅酶A完全抑制， 这是由葡萄糖的可用性，细胞能量状态，和胰腺 内分泌激素。该机制影响葡萄糖与FA的相互作用 利用率CPT-1 N-和C-末端在胞质溶胶中的共居 所需的调节丙二酰辅酶A是通过酶的多位性， 线粒体外膜（OMM）：CPT-1A和B 1具有N-末端杂合线粒体 用2个跨膜结构域（TMD）靶向/终止转移信号。我们 假设存在并发现可能解释 持续活跃的心脏FA氧化。高达30%的心脏CPT-I mRNA 是新型B2变体，是CPT-IB选择性剪接的产物。经编码 B2同工酶具有完整的线粒体前导区和催化区，但只有一个偶联体 TMD，过表达的同工酶对mal-CoA不敏感。心脏表达 B2在围产期被诱导。该项目的目标是 确定CPT-I同工酶在细胞燃料代谢中的作用。动力学 将在B2之前和之后评估大鼠心脏线粒体CPT-I的特征 表情观察结果将与基于同工酶的预测进行比较 使用同种型特异性抗体通过免疫印迹判断的丰度，和 当使用重组质粒过量表达时，每种CPT-I同工酶的活性 腺病毒。CPT-IB同工酶表达对细胞代谢的影响将 使用B2表达前和表达后的心肌细胞测定，和 同工酶互补的CPT-I缺陷的成纤维细胞。[14 C]-FA氧化速率将 作为细胞mal-CoA含量的函数进行评估，通过 提供在生理范围内的中等葡萄糖、FA和胰岛素， 浓度的CPT-I同工酶对mal-CoA敏感性差异的基础 将使用放射性配体结合测定法与来自细胞的线粒体进行评估 表达每种同种型。这将与同工酶submito基因座相关， 拓扑并行策略：1.琼脂糖偶联的底物的功效和 mal-CoA（其为胞质限制性的）对同工酶活性的影响; 2.蛋白酶 [35 S]-CPT-I同工酶和衍生融合蛋白在转染后敏感性 体外Mito导入;和3. N-和C-末端表位：抗体相互作用。 特异性检测B2剪接的CPT-1B小基因报告基因将用于 作为分析的第一步， 替代CPT-IB拼接。我们假设之前未被发现的B2 尽管[mal-CoA]，同工酶仍有助于心脏FA氧化的持续活跃 这大大超过了Ki的已知酶，并部分解偶联 脂肪酸的氧化，从葡萄糖的可用性在这个组织。

DESCRIPTION (Applicant's abstract): Carnitine palmitoyltransferase I (CPT-1) catalyzes the rate-limiting step in mitochondrial FA oxidation. Catalytic activity of previously identified CPT-I enzymes (B 1 ['muscle'] & CPT-IA ['liver']) can be completely suppressed by malonyl-CoA, the concentration of which is governed by glucose availability, cell energy state, and pancreatic endocrine hormones. This mechanism effects reciprocal glucose vs FA utilization. The co-residence of CPT-I N- and C-termini in the cytosol necessary for regulation by malonyl-CoA is achieved by enzyme polytopy in the outer mito membrane (OMM): CPT-IA and B 1 have N-terminal hybrid mito targeting/stop transfer signals with 2 transmembrane domains (TMD). We hypothesized the existence of and found additional CPT-Is that may account for perpetually active cardiac FA oxidation. Up to 30 percent of cardiac CPT-I mRNA is the novel B2 variant, a product of alternative CPT-IB splicing. The encoded B2 isozyme has intact mito leader and catalytic domains, but only onecandidate TMD, and overexpressed isozyme is insensitive to mal-CoA. Cardiac expression of B2 is induced during the perinatal period. The objective of this project is to ascertain the role of CPT-I isozymes in cellular fuel metabolism. Kinetic features of rat heart mito CPT-I will be assessed before and after B2 expression. Observations will be compared with predictions based on isozyme abundance as judged by immunoblots using isoform-specific antibodies, and activities of each CPT-I isozyme when overexpressed using recombinant adenoviruses. The impact of CPT-IB isozyme expression on cell metabolism will be determined using cardiocytes pre- and post-B2 expression, and isozyme-complemented CPT-I-deficient fibroblasts. [14C]-FA oxidation rates will be assessed as a function of cellular mal-CoA content, to be modulated by providing medium glucose, FA, and insulin over a range of physiological concentrations. The basis of differential CPT-I isozyme sensitivity to mal-CoA will be assessed using radioligand binding assays with mitos from cells expressing each isoform. This will be correlated with isozyme submito loci and topology in parallel strategies: 1. Efficacy of Sepharose-coupled substrate and mal-CoA (which are cytosol-restricted) on isozyme activity; 2. Protease sensitivity of [35S]-CPT-I isozymes and derivative fusion proteins after in vitro mito import; and 3. N- and C-terminal epitope:antibody interactions. CPT-IB minigene reporters that specifically detect B2 splicing will be used to map intronic and exonic splicing enhancers as a first step in the analysis of alternative CPT-IB splcing. We hypothesize that the previously unrecognized B2 isozyme contributes to ceaseless brisk cardiac FA oxidation despite [mal-CoA] that vastly exceeds the Ki of the known enzymes, and to the partial uncoupling of FA oxidation from glucose availability in this tissue.