Human serum carbonyl proteome in cardiovascular diseases

描述（申请人提供）：克氏锥虫感染患者中有30-40%出现恰加斯型心肌病，每年造成50万例死亡，损失274万残疾调整生命年。当大多数患者被诊断为恰加斯病时，心功能障碍已经发展了很多年。现有的抗寄生虫药物治疗是有毒的，在临床疾病阶段大多无效。我们还不知道易感患者的分子机制受到干扰，他们将继续发展为临床心脏病。在最近的临床前和临床研究中，我们已经证明克鲁兹锥虫感染的宿主维持炎症和线粒体起源的氧化应激，心脏的氧化病理在进行性恰加斯病期间引发心功能障碍中起重要作用。此外，我们发现急性和慢性恰加斯病动物血浆中有不同的蛋白被氧化/硝化，这些蛋白与其他病因的心肌病动物血浆中的蛋白修饰不同。这些观察结果支持了我们的假设，即导致患者恰加斯型心肌病发展的病理过程会导致血液中蛋白质浓度/氧化的特征性变化，并产生可检测的疾病特异性分子表型。在这项研究中，我们建议在chagasic患者中建立血浆氧化蛋白质组。在目标1中，我们将使用ProteomeLab PF2D液相色谱系统来识别chagasic受试者血浆蛋白质组的变化，并将其与正常/健康和其他心肌病受试者的发现进行比较。在目标2中，我们将确定Chagasic患者血浆蛋白质组中的氧化修饰。为此，我们通过添加一个在线光电二极管阵列分光光度计来增强PF2D系统，使我们能够在其特征波长上可视化翻译后修饰。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验证。在完成这些研究后，我们期望确定分子标记，这将有助于对恰加斯病状态的分类和对那些有发展为慢性扩张型心肌病风险的无症状个体的诊断。通过对差异表达/修改的蛋白质组数据的生物学分析，我们将确定在从无症状到有症状的临床疾病进展过程中失调的联锁途径和共调节子网络。这些受干扰的网络将提示受感染的个体为何以及何时易受恰加斯病的影响，并为设计预防和治疗恰加斯病以及可能的其他心肌病的联合疗法提供新的靶点。公共相关性：该项目的研究旨在开发恰加斯病相关的血浆氧化蛋白质组。在完成这些研究后，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，以及在无症状个体中增加临床疾病发展风险的分子干扰。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.