CARNITINE PALMITOYLTRANSFERASE I ISOFORM FUNCTION

说明(申请人摘要)：肉碱棕榈酰转移酶I(CPT-1) 催化线粒体FA氧化的限速步骤。催化作用 先前确定的CPT-I酶(B1[‘肌肉’]和CPT-IA)的活性 [‘肝’])可被丙二酰辅酶A完全抑制，其浓度 它受葡萄糖可获得性、细胞能量状态和胰腺的控制 内分泌荷尔蒙。这一机制影响葡萄糖与脂肪酸的相互作用 利用率。CPT-I N-末端和C-末端在胞浆中的共存 丙二酰辅酶A调节所必需的是通过酶的多态实现的 外膜(OMM)：CPT-IA和B1具有N端杂交MITO 靶向/停止带有2个跨膜结构域(TMD)的转移信号。我们 假设存在并发现了其他CPT-IS，这可能解释了 永久活跃的心脏FA氧化。高达30%的心脏CPT-I mRNA 是新型的B2变异体，是CPT-IB选择性剪接的产物。已编码的 B2同工酶具有完整的有丝分裂前导和催化区，但只有一个条件 TMD，过表达的同工酶对麦芽糖不敏感。心脏的表达 B2是在围产期诱导产生的。这个项目的目标是 确定CPT-I同工酶在细胞燃料代谢中的作用。动能 B2治疗前后大鼠心脏MITO CPT-I的变化 表情。观察结果将与基于同工酶的预测进行比较。 由免疫印迹使用异构体特异性抗体判断的丰度，以及 重组蛋白过表达时各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 用心肌细胞在B2前后的表达来确定，以及 同工酶互补的CPT-I缺陷成纤维细胞。[14C]-FA氧化率将 作为细胞MAL-CoA含量的函数进行评估，通过 在生理范围内提供中等葡萄糖、脂肪酸和胰岛素 浓度。差异CPT-I同工酶对丙二醛敏感性的基础 将使用放射性配基结合分析与细胞中的Mitos进行评估 表达每一种异构体。这将与同工酶Submito基因座和 并行策略中的拓扑学：1.琼脂糖偶联底物和 MAL-CoA(胞质受限)对同工酶活性的影响；2.蛋白水解酶 [35S]-CPT-I同工酶及其衍生融合蛋白在体内的敏感性 体外丝裂原导入；3.N-末端和C-末端表位：抗体相互作用。 专门检测B2剪接的CPT-IB微型基因记者将被用于 映射内含子和外显子剪接增强子作为分析的第一步 另一种CPT-IB拼接。我们假设之前未被识别的B2 同工酶有助于心脏FA的持续快速氧化，尽管[MAL-CoA] 这大大超过了已知的酶的KI，以及部分解偶联 从组织中葡萄糖的可获得性来判断FA氧化。

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.