Controlled overexpression of BCKD kinase expression: metabolic engineering applied to BCAA metabolism in a mammalian system.
Controlled overexpression of BCKD kinase expression: metabolic engineering applied to BCAA metabolism in a mammalian system.
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BCKD 激酶表达的受控过度表达：代谢工程应用于哺乳动物系统中的 BCAA 代谢。
DOI：
10.1006/mben.2000.0164
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发表时间：
2000
影响因子：
8.4
通讯作者：
Danner,DJ
中科院分区：
文献类型：
--
作者：
Doering,CB;Williams,IR;Danner,DJ
人类疾病的常见代谢并发症是不受控制的肌肉蛋白质分解或恶病质，其发生在患有慢性疾病如癌症、AIDS、肾衰竭和糖尿病的患者中。增加的支链氨基酸催化剂被认为是因果关系，并刺激了对调节这些氨基酸代谢方法的研究。在这里，我们证明了在哺乳动物细胞培养中强力霉素控制的支链α-酮酸脱氢酶（BCKD）激酶转基因的过表达。该激酶通过磷酸化作用使BCKD复合物活化，从而防止这些必需的调节代谢物的分解。在这项研究中，强力霉素治疗导致BCKD激酶蛋白增加10倍。转基因产生的激酶被迅速整合到线粒体内，并正确地发挥功能，使BCKD复合物稳定。达到的基础BCKD活性的最大降低为94%。出乎意料的是，尽管BCKD催化蛋白的表达没有观察到变化，但激酶过表达也降低了总BCKD活性。这些结果表明，人工调控支链氨基酸代谢是可能的，通过控制过表达的一个单一的内源性酶，并建议临床应用的可行性。
A common metabolic complication of human disease is uncontrolled muscle protein breakdown or cachexia, which occurs in patients with chronic diseases such as cancer, AIDS, renal failure, and diabetes. Increased branched-chain amino acid catabolism is implicated as causal and has stimulated the investigation of methods to regulate the metabolism of these amino acids. Here we demonstrate doxycycline-controlled overexpression of a branched-chain α-ketoacid dehydrogenase (BCKD) kinase transgene in mammalian cell culture. This kinase functions to inactivate the BCKD complex by phosphorylation, thus preventing the catabolism of these essential, regulatory metabolites. In this study, doxycycline treatment leads to a 10-fold increase in BCKD kinase protein. The transgene-generated kinase is rapidly incorporated within mitochondria and functions correctly to inactivate the BCKD complex. The maximum reduction in basal BCKD activity achieved was 94%. Unexpectedly, total BCKD activity was also decreased by kinase overexpression despite no observable change in expression of the BCKD catalytic proteins. These results demonstrate that artificial regulation of branched-chain amino acid metabolism is possible through the controlled overexpression of a single endogenous enzyme and suggest the feasibility of clinical applications.