Regulation of Cardiac Carnitine Palmitoyltransferase I

心脏肉毒碱棕榈酰转移酶 I 的调节

• 批准号: 6573333
• 负责人: Gebre Woldegiorgis
• 金额: $ 24.6万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6573333
• 关键词: active sites; aminoacid metabolism; carnitine; chimeric proteins; enzyme activity; enzyme inhibitors; fatty acid metabolism; gene targeting; genetically modified animals; heart function; heart metabolism; laboratory mouse; malonyl coA; myocardium; palmitates; protein binding; site directed mutagenesis; transferase

Plasma long-chain fatty acids are the primary fuel source for energy production in the normal heart. In the ischemic and reperfused myocardium, there is decreased utilization of fatty acids, and perfusion with fatty acids results in in situ membrane damage and cardiac dysfunction. Fatty acid-induced injury to the ischemic myocardium can be reduced by inhibition of carnitine palmitoyltransferase I (CPTI), a rate-limiting enzyme in beta-oxidation. For effective pharmacotherapy of defects in cardiac fatty acid oxidation, it is imperative that we understand the biochemical and molecular mechanisms regulating M-CPTI, a key enzyme in myocardial bioenergetics. Our working hypothesis, supported by recent mutagenesis studies, is that amino acids essential for malonyl- CoA inhibition and binding and for substrate binding and catalysis in M-CPTI reside in the N- and C-terminal regions, respectively. Furthermore, because of the essential role of M- CPTI in heart function, loss of the enzyme may result in death. Our specific aims are: (1) To identify specific amino acid residues important for malonyl-CoA inhibition and binding by deletion and substitution mutation analysis of chimeric constructs of M-CPTI and L-CPTI, and by cysteine scanning mutagenesis. (2) To map the substrate binding and catalytic site pocket of M-CPTI by site-directed mutagenesis, ligand binding, and intrinsic tryptophan fluorescence quenching studies. (3) To purify milligram quantities of P. pastoris- and E. coli-expressed M-CPTI and engineered fragments for structural characterization. (4) To generate a heart-specific conditional knockout mouse model for M-CPTI using the Cre-loxP system, and to determine its effect on embryonic lethality. The M-CPTI null mice will allow us to construct models that mimic human CPT deficiency diseases and to look for potential gene therapy through retro-transfection of the normal CPT gene.

血浆长链脂肪酸是正常心脏能量产生的主要燃料来源。 在缺血性和再生心肌中，脂肪酸的利用率降低，脂肪酸灌注会导致原位膜损伤和心脏功能障碍。脂肪酸诱导的缺血性心肌损伤可以通过抑制肉碱棕榈酰转移酶I（CPTI）来减少，这是β-氧化中的限速酶。 为了有效地对心脏脂肪酸氧化中的缺陷进行药物疗法，我们必须了解调节M-CPTI的生化和分子机制，M-CPTI是心肌生物能源的关键酶。 我们最近的诱变研究支持的我们的工作假设是，氨基酸对于MANONYL-COA抑制和结合以及M-CPTI中的底物结合和催化的氨基酸分别存在于N-和C末端区域。 此外，由于m cpti在心脏功能中的重要作用，酶的丧失可能导致死亡。我们的具体目的是：（1）确定对丙二酰-COA抑制和结合M-CPTI和L-CPTI的嵌合构建体以及半胱氨酸扫描诱变的特定氨基酸残基。 （2）通过定点诱变，配体结合和内在的色氨酸荧光淬灭研究来绘制M-CPTI的底物结合和催化位点袋。 （3）净化毫克少量的P. p. p. p. coli表达的M-CPTI和工程片段，以进行结构表征。 （4）使用CRE-LoxP系统为M-CPTI生成心脏特异性的条件基因敲除模型，并确定其对胚胎致死性的影响。 M-CPTI无效小鼠将使我们能够构建模拟人类CPT缺乏疾病的模型，并通过正常CPT基因的恢复转染来寻找潜在的基因治疗。