Human serum carbonyl proteome in cardiovascular diseases

描述(由申请人提供)：查格西奇心肌病出现在30-40%感染克氏锥虫的患者中，每年造成50,000人死亡和274万残疾调整生命年的损失。到大多数患者被诊断为恰加斯病时，心脏功能障碍已经进展了很多年。现有的抗寄生虫药物治疗是有毒的，在临床疾病阶段基本上无效。我们还不知道易感患者的分子机制受到干扰，这些患者将继续发展为临床心脏病。在最近的临床前和临床研究中，我们已经证明，感染弓形虫的宿主承受着炎症和线粒体来源的氧化应激，心脏的氧化病理在导致进行性恰加斯病的心功能障碍中起着重要作用。此外，我们还发现，在急慢性心肌病动物中，不同的血浆蛋白质被氧化/硝化，这些蛋白质不同于其他原因的心肌病动物血浆中修饰的蛋白质。这些观察结果支持我们的假设，即导致患者查格性心肌病发展的病理过程将导致血液中蛋白质浓度/氧化的特征性变化，并产生可检测到的疾病特异性分子表型。在这项研究中，我们建议建立一种在萎缩症患者中的血浆氧化蛋白质组。在目标1中，我们将利用ProteomeLab PF2D液相色谱系统来鉴定失调症受试者血浆蛋白质组的变化，并将其与正常/健康受试者和其他心肌病受试者的结果进行比较。在目标2中，我们将确定查格斯病患者血浆蛋白质组中的氧化修饰。为此，我们增加了一个直列式光电二极管阵列分光光度计，从而增强了PF2D系统，使我们能够在其签名波长处可视化翻译后修饰。以疾病特异性方式差异表达/氧化的蛋白质将通过LC-MS/MS鉴定，并通过Western blotting进行验证。在这些研究完成后，我们预计将识别分子标记，这些标记将在Chagas病状态的分类和那些有发展成慢性扩张型心肌病风险的无症状个体的诊断中有用。通过对差异表达/修饰的蛋白质组数据的生物学分析，我们将识别在从无症状到有症状的临床疾病进展过程中失控的互锁通路和共调控亚网络。这些受干扰的网络将提示为什么以及何时感染者易患查加斯病，并为设计预防和治疗查格斯病以及可能还有其他心肌病的联合疗法提供新的靶点。公共相关性：该项目的研究目的是开发恰加斯病相关的血浆氧化蛋白质组。在这些研究完成后，我们将确定患者疾病状态的分子标记和患者患恰加斯病的易感性，以及在没有症状的个人中增加临床疾病发展风险的分子紊乱。这些研究将为开发新的药物疗法提供潜在的靶点。

DESCRIPTION (provided by applicant): Chagasic cardiomyopathy emerges in 30-40% of the patients infected by Trypanosoma cruzi, and accounts for >50,000 deaths and the loss of 2.74 million disability-adjusted life years per year. By the time most patients are diagnosed with Chagas disease, heart dysfunction has been progressing for many years. Available anti-parasite drug therapies are toxic and largely ineffective during the clinical disease phase. We do not yet know the molecular mechanisms that are disturbed in susceptible patients who will proceed to develop clinical cardiac disease. In recent preclinical and clinical studies, we have shown that T. cruzi-infected hosts sustain an oxidative stress of an inflammatory and mitochondrial origin, and oxidative pathology of the heart plays an important role in eliciting cardiac dysfunction during progressive Chagas disease. Further, we have found that distinct plasma proteins are oxidized/nitrated in acute and chronic chagasic animals, and these proteins are different from those modified in the plasma of animals with cardiomyopathy of other etiologies. These observations support our hypothesis that pathological processes leading to the development of chagasic cardiomyopathy in patients would cause characteristic changes in the concentration/oxidation of proteins in the blood and generate a detectable disease-specific molecular phenotype. In this study, we propose to develop a plasma oxidative proteome in chagasic patients. In aim 1, we would utilize a ProteomeLab PF2D liquid chromatography system to identify the changes in the plasma proteome of chagasic subjects and compare them to findings in normal/healthy and other cardiomyopathy subjects. In aim 2, we would identify the oxidative modifications in the plasma proteome of Chagasic patients. For this purpose, we have enhanced the PF2D system by adding an in-line photodiode array spectrophotometer that allows us to visualize the post-translational modifications at their signature wavelength. Proteins that are differentially expressed/oxidized in a disease-specific manner will be identified by LC-MS/MS and validated by western blotting. Upon completion of these studies, we anticipate identifying molecular markers that will be useful in classification of the Chagas disease state and diagnosis of those asymptomatic individuals who are at risk of developing chronic, dilated cardiomyopathy. By biological analysis of the differentially expressed/modified proteome data, we will identify the interlocking pathways and co-regulated sub-networks that are dysregulated during progression from asymptomatic to symptomatic clinical disease. These disturbed networks will suggest why and when infected individuals become susceptible to Chagas disease and yield novel targets for engineering the combination therapies for the prevention and treatment of chagasic, and possibly other cardiomyopathies. PUBLIC RELEVANCE: The studies in this project are aimed at developing the Chagas disease-associated plasma oxidative proteome. Upon completion of these studies, we will identify molecular markers of patient's disease state and patient's susceptibility to develop Chagas disease and the molecular disturbances that enhance the risk of clinical disease development in asymptomatic individuals. These studies will yield potential targets for the development of new drug therapies.