Violante Sara, Ijlst Lodewijk, van Lenthe Henk, de Almeida Isabel Tavares, Wanders Ronald J, Ventura Fátima V
Metabolism and Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences, iMed.UL, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
Biochim Biophys Acta. 2010 Sep;1802(9):728-32. doi: 10.1016/j.bbadis.2010.06.002. Epub 2010 Jun 9.
Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid beta-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2-C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8-C12) and long-chain (C14-C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (K(i) of 18.8 microM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.
在过去几年中，酰基肉碱已成为诊断线粒体脂肪酸β氧化（mFAO）和支链氨基酸氧化障碍的重要生物标志物，前提是它们反映了可能有毒的酰基辅酶A种类，这些物质在线粒体内酶阻断的上游积累。然而，这些中间体的来源仍知之甚少。肉碱穿梭系统的成员肉碱棕榈酰转移酶2（CPT2）有可能参与从积累的酰基辅酶A代谢产物在线粒体内合成酰基肉碱。为了解决这个问题，本文研究了CPT2的底物特异性谱。将表达人CPT2的酿酒酵母匀浆与饱和和不饱和的C2 - C26酰基辅酶A以及支链氨基酸氧化中间体一起孵育。通过电喷雾串联质谱（ESI - MS/MS）对产生的酰基肉碱进行定量。我们发现CPT2对中链（C8 - C12）和长链（C14 - C18）酰基辅酶A酯有活性，而对短链和极长链酰基辅酶A或支链氨基酸氧化中间体几乎没有活性。反式 - 2 - 烯酰基辅酶A中间体也被发现是CPT2的不良底物。进行的抑制研究表明，反式 - 2 - C16:1 - CoA可能作为CPT2的竞争性抑制剂（抑制常数K(i)为18.8微摩尔）。获得的结果清楚地表明，CPT2能够逆转其对中链和长链酰基辅酶A的生理机制，这导致了大多数mFAO障碍特有的异常酰基肉碱谱。CPT2对反式 - 2 - 烯酰基辅酶A处理不佳这一发现，可能解释了线粒体三功能蛋白缺陷患者的谱中不存在反式 - 2 - 烯酰肉碱（这是唯一它们会积累的缺陷），以及该疾病与其他长链mFAO障碍（如极长链酰基辅酶A脱氢酶缺乏症）临床特征之间的差异。
