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

描述（由申请人提供）：线粒体疾病的发生率为1/5000活产，通常在5岁左右死亡。这些线粒体脑肌病通常是氧化磷酸化（OXPHOS）病症，其中电子传递链（ETC）的一种或多种组分不再有效地合成ATP。尽管基因测序已经在线粒体或核DNA中鉴定出> 200种潜在的致病突变，但遗传缺陷与神经退行性病理学之间的明确生物化学联系仍有待阐明。因此，没有准确的疾病生物标志物，婴儿通常最初被确定为未能茁壮成长或存在严重发育迟缓的结果。本文提出的创新研究有望揭示减少OXPHOS和神经病理学之间的新的代谢联系，并可能用作线粒体疾病的诊断生物标志物。以前，我们已经检测到一种新的化学修饰的蛋白质在糖尿病，S-（2-琥珀酰基）半胱氨酸（2SC），这是形成的克雷布斯循环中间体富马酸与反应性半胱氨酸残基的蛋白质。富马酸盐和蛋白琥珀酸在高糖培养的脂肪细胞和糖尿病小鼠脂肪组织中均增加。我们已经表明，增加琥珀酸可以显着影响蛋白质的调节，通过降低酶的活性和干扰激素的分泌结构。在糖尿病中，2SC修饰的蛋白质的增加是由于营养过剩、NADH积累和克雷布斯循环脱氢酶的反馈抑制而发生的，从而允许富马酸的增加。在这些观察结果的新的横向延伸中，我们提出，类似的ETC抑制，例如，作为Leigh综合征期间复合物I缺乏的结果，将导致线粒体疾病中的NADH、富马酸盐和蛋白琥珀酸化增加。在初步数据中，我们证明了在与神经病理学相关的Leigh综合征（Ndufs4敲除）小鼠模型的脑干中的一系列蛋白质上可检测到蛋白质琥珀酸化增加。我们推测，在Leigh综合征期间，血清或尿液中也可检测到2SC水平的升高，这可能是一种比目前的乳酸盐测量更有用的新生儿代谢紊乱测量方法。在这项研究中，我们将优化血清和尿液中2SC的测量，以确定其作为OXPHOS缺陷的生物标志物的有用性。我们还将探索一种治疗干预措施，该措施应降低NADH水平，减少富马酸盐，从而防止蛋白琥珀酸化增加，为未来治疗线粒体疾病开辟新的治疗途径。

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.