Protein Modification: Isoaspartic Acid

描述(由申请人提供)：我们的长期目标是更好地了解蛋白质翻译后修饰(PTM)的病因学作用。这个项目的重点是异天冬氨酸(isAsp，isod或β-Asp)，它是由天冬酰胺脱酰胺或天冬氨酸异构化自发产生的。IsoAsp存在于无数蛋白质中(例如，大脑中的β-淀粉样蛋白和眼睛中的晶体)。值得注意的是，isAsp赋予多肽骨架D-构型和β-连接；这种多肽骨架的改变非常罕见，可能会极大地改变蛋白质的结构和功能。虽然它在衰老过程中得到了很好的认识，但它在信号、调节和自身免疫方面的新角色还得到了广泛的重视。缺乏对isAsp的全面了解及其分析中的独特技术挑战阻碍了这一领域的进展。首先，isAsp的形成 是质量变化最小的Ptm(Da；isAsp与Asp具有相同的质量)。最近，通过将化学-酶标记和浓缩与质谱学相结合的新方法，这些技术障碍已经被克服。将实现四个具体目标。首先，将对具有生物化学和医学相关性的样本进行isAsp的蛋白质组学分析。其次，isAsp的紧密结合体将被设计并用作抗体，用于补充现有方法的应用。第三，将探讨修复酶蛋白异门冬氨酸甲基转移酶(PIMT)的底物专一性，主要集中在PIMT的不良底物上。异天冬氨酸多肽文库筛选、酶动力学分析、蛋白质组学分析和蛋白质结构建模将综合起来，以预测等天冬氨酸在单个蛋白质和整个系统中的命运。最后，isAsp的替代代谢途径将被发现。这项拟议的研究将导致发现人类疾病的生物标记物，以及预防、干预和治疗与年龄相关的疾病、神经退行性疾病(例如阿尔茨海默病)、自身免疫性疾病和其他等天冬氨酸相关疾病的有效方法。

DESCRIPTION (provided by applicant): Our long-term goal is to better understand the etiologic roles of protein posttranslational modifications (PTMs). This project focuses on isoaspartic acid (isoAsp, isoD or beta-Asp), which is generated spontaneously from either asparagine deamidation or aspartic acid isomerization. IsoAsp has been found in myriad proteins (e.g., beta-amyloid in the brain and crystalline in the eyes). Notably, isoAsp imparts a D-configuration and beta-linkage into the peptide backbone; such alteration of peptide backbones is exceptionally rare and may drastically alter protein structure and function. While well-recognized in aging, its emerging roles in signaling, regulation and autoimmunity have yet been widely appreciated. A lack of comprehensive knowledge of isoAsp and the unique technical challenges in its analysis hamper progress in this field. For one thing, isoAsp formation is the smallest PTM by mass change (Da; isoAsp has the same mass as Asp). Recently, these technical barriers have been overcome by novel approaches that combine chemo-enzymatic labeling and enrichment with mass spectrometry. Four specific aims will be undertaken. First, proteomic analysis of isoAsp in samples with biochemical and medical relevance will be performed. Second, tight binders to isoAsp will be engineered and used as antibodies for applications that complement existing methodologies. Third, the substrate specificity of the repair enzyme protein isoaspartate methyltransferase (PIMT) will be probed, focusing most assiduously on poor substrates of PIMT. Isoaspartyl peptide library screening, enzyme kinetic characterization, proteomic analysis and protein structural modeling will be integrated to augur the fate of isoAsp in individual proteins and whole systems. Last, alternative metabolic pathways for isoAsp will be discovered. The proposed research will lead to the discovery of biomarkers of human diseases, and effective methods of prevention, intervention and treatment for age-related conditions, neurodegenerative disorders (e.g., Alzheimer's disease), autoimmune diseases and other isoAsp-related ailments.