Detection of S-(2-succino)cysteine (2SC) as a Biomarker of Mitochondrial Disease

描述(申请人提供)：线粒体疾病以5000名活产儿中1名的比率发生，通常在~5岁时致命。这些线粒体脑肌病通常是氧化磷酸化(OXPHOS)障碍，其中电子传输链(ETC)的一个或多个组成部分不再有效地合成ATP。尽管基因测序已经确定了线粒体或核DNA中潜在的致病突变，但遗传缺陷和神经退行性病变之间的明确生化联系仍有待阐明。因此，没有准确的疾病生物标志物，婴儿通常最初被识别为未能进行Thriv或存在严重的发育迟缓。这里提出的创新研究有望揭示OXPHOS减少与神经病理之间的一种新的代谢联系，并可能作为线粒体疾病的诊断生物标志物。此前，我们已经检测到糖尿病蛋白的一种新的化学修饰，S-(2-琥珀酸)半胱氨酸(2SC)，它是由Krebs环中间体富马酸与蛋白质中的反应性半胱氨酸残基反应形成的。在高糖培养的脂肪细胞和糖尿病小鼠脂肪组织中，富马酸和蛋白质琥珀酸化均增加。我们已经证明，增加琥珀酸化可以通过降低酶活性和干扰激素的分泌结构来显著影响蛋白质的调节。在糖尿病中，2SC修饰蛋白的增加是营养过剩、NADH积累和反馈抑制Krebs循环脱氢酶的结果，从而允许富马酸的增加。在一个新的，这些观察的横向扩展中，我们提出了类似的ETC抑制，例如由于Leigh综合征期间复合体I缺乏的结果，将导致线粒体疾病中NADH、富马酸和蛋白质琥珀酸化的增加。在初步数据中，我们证明在Leigh综合征(Ndufs4基因敲除)小鼠模型的脑干中一系列蛋白质上可以检测到蛋白质琥珀酸化增加，这与神经病理学有关。我们推测，在Leigh综合征期间，2SC水平的升高也可在血清或尿液中检测到，并且可能比目前的乳酸测量提供更有用的新生儿代谢紊乱的衡量标准。在这项研究中，我们将优化血清和尿液中2SC的测定，以确定其作为OXPHOS缺乏症生物标志物的有效性。我们还将探索一种治疗干预措施，降低NADH水平，减少富马酸，从而防止蛋白质琥珀酸化增加，为未来线粒体疾病的治疗开辟新的治疗途径。

DESCRIPTION (provided by applicant): Mitochondrial diseases occur at a rate of 1 in 5000 live births and are often fatal by ~5 years old. These mitochondrial encephalomyopathies are usually oxidative phosphorylation (OXPHOS) disorders in which one or more components of the electron transport chain (ETC) are no longer operating efficiently to synthesize ATP. Although genetic sequencing has identified >200 of the underlying causative mutations in mitochondrial or nuclear DNA, a definitive biochemical link between the genetic defect and the neurodegenerative pathology remains to be elucidated. As a result there are no accurate disease biomarkers and infants are often initially identified as a result of their failure to thriv or by the existence of severe developmental delays. The innovative studies proposed here are expected to reveal a novel metabolic link between reduced OXPHOS and neuropathology, and may have utility to act as a diagnostic biomarker of mitochondrial disease. Previously, we have detected a new chemical modification of proteins in diabetes, S-(2-succino)cysteine (2SC), which is formed by reaction of the Krebs cycle intermediate fumarate with reactive cysteine residues in protein. Both fumarate and protein succination are increased in adipocytes cultured in high glucose and in diabetic mouse adipose tissue. We have shown that increased succination can significantly affect protein regulation by lowering enzymatic activity and interfering with the secretory structure of hormones. In diabetes, the increase in 2SC-modified proteins occurs as a result of nutrient excess, accumulation of NADH, and feedback inhibition of the Krebs cycle dehydrogenases, allowing the increase in fumarate. In a novel, lateral extension of these observations we propose that a similar ETC inhibition e.g. as a consequence of Complex I deficiency during Leigh Syndrome, would result in increased NADH, fumarate and protein succination in mitochondrial disease. In Preliminary Data, we demonstrate that increased protein succination is detectable on a range of proteins in the brainstem of a mouse model of Leigh syndrome (Ndufs4 knockout) in association with neuropathology. We hypothesize that the increased 2SC levels are also detectable in serum or urine during Leigh syndrome and may provide a more useful measure of metabolic derangement in the newborn than the current lactate measurements. In this study we will optimize the measurement of 2SC in serum and urine to determine its usefulness as a biomarker of OXPHOS deficiencies. We will also explore a therapeutic intervention which should lower NADH levels, reduce fumarate and thereby prevent increased protein succination, opening novel therapeutic avenues for the future treatment of mitochondrial diseases.