Human serum carbonyl proteome in cardiovascular diseases

描述（由申请人提供）：克氏锥虫感染的患者中有30-40%出现查氏锥虫心肌病，每年造成> 50，000例死亡和274万残疾调整生命年的损失。当大多数患者被诊断患有恰加斯病时，心脏功能障碍已经进展多年。现有的抗寄生虫药物治疗是有毒的，在临床疾病阶段基本无效。我们还不知道在易受影响的患者中受到干扰的分子机制，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，我们已经表明T。Cruzi感染的宿主维持炎症和线粒体来源的氧化应激，并且心脏的氧化病理学在进行性恰加斯病期间引起心脏功能障碍中起重要作用。此外，我们已经发现，不同的血浆蛋白质被氧化/硝化在急性和慢性chagglomerate动物，这些蛋白质是不同的血浆中的修改与其他病因的心肌病的动物。这些观察结果支持了我们的假设，即导致患者发生脉络膜心肌病的病理过程会引起血液中蛋白质浓度/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，我们建议开发一个在chagglutamine患者的血浆氧化蛋白质组。在目标1中，我们将利用ProteomeLab PF 2D液相色谱系统来鉴定chagglutinin受试者的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结果进行比较。在目标2中，我们将鉴定恰古什患者血浆蛋白质组中的氧化修饰。为此，我们通过添加在线光电二极管阵列分光光度计来增强PF 2D系统，该分光光度计允许我们在其特征波长处可视化翻译后修饰。将通过LC-MS/MS鉴定以疾病特异性方式差异表达/氧化的蛋白质，并通过蛋白质印迹法进行验证。在完成这些研究后，我们预计将确定分子标记物，这将有助于分类的恰加斯病的状态和诊断那些无症状的个人谁是发展慢性扩张型心肌病的风险。通过对差异表达/修饰的蛋白质组数据的生物学分析，我们将确定在从无症状到有症状的临床疾病进展期间失调的联锁通路和共调节子网络。这些受到干扰的网络将提示为什么以及何时感染个体变得易受恰加斯病的影响，并产生新的靶点，用于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。公众相关性：本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志物和患者发展恰加斯病的易感性以及增加无症状个体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.