Elizabeth A. Jennings, Megan M. Macdonald, Irina Romenskaia, Hao Yang, Grant A. Mitchell, Robert O. Ryan
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引用次数: 0
Abstract
The leucine catabolism pathway intermediate, trans-3-methylglutaconyl (3MGC) CoA, is considered to be the precursor of 3MGC acid, a urinary organic acid associated with specific inborn errors of metabolism (IEM). trans-3MGC CoA is an unstable molecule that can undergo a sequence of non-enzymatic chemical reactions that lead to either 3MGC acid or protein 3MGCylation. Herein, the susceptibility of trans-3MGC CoA to protein 3MGCylation was investigated. trans-3MGC CoA was generated through the activity of recombinant 3-methylcrotonyl CoA carboxylase (3MCCCase). Following enzyme incubations, reaction mixtures were spin-filtered to remove 3MCCCase. The recovered filtrates, containing trans-3MGC CoA, were then incubated in the presence of bovine serum albumin (BSA). Following this, sample aliquots were subjected to α-3MGC IgG immunoblot analysis to probe for 3MGCylated BSA. Experiments revealed a positive correlation between trans-3MGC CoA incubation temperature and 3MGCylated BSA immunoblot signal intensity. A similar correlation was observed between incubation time and 3MGCylated BSA immunoblot signal intensity. When trans-3MGC CoA hydratase (AUH) was included in incubations containing trans-3MGC CoA and BSA, 3MGCylated BSA immunoblot signal intensity decreased. Evidence that protein 3MGCylation occurs in vivo was obtained in studies with liver-specific 3-hydroxy-3-methylglutaryl (HMG) CoA lyase knockout mice. Therefore, trans-3MGC CoA is a reactive, potentially toxic metabolite, and under normal physiological conditions, lowering trans-3MGC CoA levels via AUH-mediated hydration to HMG CoA protects against aberrant non-enzymatic chemical reactions that lead to protein 3MGCylation and 3MGC acid production.
反式-3MGC CoA 是一种不稳定的分子,可通过一系列非酶化学反应生成 3MGC 酸或 3MGC 蛋白质。反式-3MGC CoA 是通过重组 3-甲基巴豆酰 CoA 羧化酶(3MCCCase)的活性生成的。酶培养后,对反应混合物进行旋流过滤以除去 3MCCCase。然后将含有反式-3MGC CoA 的回收滤液在牛血清白蛋白(BSA)存在下进行孵育。之后,对等分的样品进行α-3MGC IgG 免疫印迹分析,以检测 3MGCylated BSA。实验表明,反式-3MGC CoA 培养温度与 3MGCylated BSA 免疫印迹信号强度之间存在正相关。孵育时间与 3MGCylated BSA 免疫印迹信号强度之间也存在类似的相关性。在含有反式-3MGC CoA 和 BSA 的培养液中加入反式-3MGC CoA 水合酶(AUH)时,3MGCylated BSA 免疫印迹信号强度降低。在对肝脏特异性 3-hydroxy-3-methylglutaryl (HMG) CoA lyase 基因敲除小鼠的研究中,获得了蛋白质 3MGCylation 在体内发生的证据。因此,反式-3MGC CoA 是一种反应性、潜在毒性的代谢产物,在正常生理条件下,通过 AUH 介导的水合 HMG CoA 来降低反式-3MGC CoA 的水平,可以防止导致蛋白质 3MGCylation 和 3MGC 酸生成的非酶化学反应异常。
MetabolitesBiochemistry, Genetics and Molecular Biology-Molecular Biology
CiteScore
5.70
自引率
7.30%
发文量
1070
审稿时长
17.17 days
期刊介绍:
Metabolites (ISSN 2218-1989) is an international, peer-reviewed open access journal of metabolism and metabolomics. Metabolites publishes original research articles and review articles in all molecular aspects of metabolism relevant to the fields of metabolomics, metabolic biochemistry, computational and systems biology, biotechnology and medicine, with a particular focus on the biological roles of metabolites and small molecule biomarkers. Metabolites encourages scientists to publish their experimental and theoretical results in as much detail as possible. Therefore, there is no restriction on article length. Sufficient experimental details must be provided to enable the results to be accurately reproduced. Electronic material representing additional figures, materials and methods explanation, or supporting results and evidence can be submitted with the main manuscript as supplementary material.