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）的病因作用。该项目的重点是异天冬氨酸（isoAsp，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已在无数蛋白质中发现（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，isoAsp赋予肽骨架D-构型和β-连接;这种肽骨架的改变非常罕见，可能会彻底改变蛋白质的结构和功能。虽然在衰老中得到了广泛的认可，但其在信号传导、调节和自身免疫中的新兴作用尚未得到广泛的认识。缺乏对isoAsp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，isoAsp的形成 是质量变化的最小PTM（Da; isoAsp与Asp质量相同）。最近，这些技术障碍已被克服的新方法，结合联合收割机化学-酶标记和富集与质谱。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中的isoAsp进行蛋白质组学分析。其次，将设计与isoAsp紧密结合的抗体，并将其用作补充现有方法的抗体。第三，底物特异性的修复酶蛋白质异天冬氨酸甲基转移酶（PIMT）将被探测，最刻苦地集中在穷人的PIMT基板。异天冬氨酸肽库筛选、酶动力学表征、蛋白质组学分析和蛋白质结构建模将被整合，以预测isoAsp在单个蛋白质和整个系统中的命运。最后，将发现isoAsp的替代代谢途径。拟议的研究将导致发现人类疾病的生物标志物，以及预防、干预和治疗与年龄有关的疾病、神经退行性疾病（例如，阿尔茨海默氏病），自身免疫性疾病和其他isoAsp-related疾病。

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.