Immunoglobulin Structure, Stability and Aging

描述（由申请方提供）：轻链淀粉样变性（AL）是最常见的全身性蛋白质聚集性疾病。它不仅使人衰弱，而且往往是致命的，而且由于受影响的内脏器官的多样性，也很难诊断。其分子原因是由于在免疫应答过程中积累的体细胞突变导致的特异性免疫球蛋白轻链（LC）的构象变化。特定的突变导致LC形成纤维，然后存款作为斑块。这种疾病没有直接有效的治疗方法，而且由于缺乏动物模型，这种疾病的发展变得复杂。该更新申请旨在建立在第一个授权期开发的体外原纤维生成反应，细胞模型和抑制肽的基础上。这些工具将用于确定LC原纤维生成反应中固有的分子特异性。由于我们以前表明，一些淀粉样蛋白生成LC不分泌，并形成细胞内包涵体，我们的第一个目标是测试多种淀粉样蛋白生成LC，并确定哪些序列与分泌相容，哪些LC形成包涵体。一种类型的细胞内聚集体，称为侵略者，是特别感兴趣的，因为它可能是纤维状的，基于其对抑制肽的敏感性。这一调查的LC是重要的特征LC淀粉样蛋白形成的细胞内和细胞外途径。Aim2将继续我们的观察，即一些LC可以形成两种不同类型的原纤维，另一方面，LC原纤维可以引发其他更远距离的蛋白质的聚合。突变体文库将用于确定由氧化或还原LC形成原纤维的要求。我们接下来将测试以下假设：具有希腊关键折叠的蛋白质（如LC，p2m，TTR或SOD1）的解折叠受到限制，从而导致可以产生类似原纤维的共同构象。这一假设将以两种方式进行检验：用另一种蛋白质的原纤维接种这些蛋白质中的每一种，并确定不同原纤维对抑制肽的敏感性。然后，通过确定Greek关键蛋白的共聚集程度，将体外发现的相似性应用于细胞内环境。目的3是使用转基因小鼠的一个淀粉样蛋白的LC和评估其适用性作为动物模型的AL通过操纵他们的遗传和免疫，以形成淀粉样蛋白沉积。如果成功，该模型将用于测试特异性阻断Greek Key蛋白的原纤维形成的肽作为潜在治疗方式的功效。相关性：该项目继续研究轻链淀粉样变性的分子机制，轻链淀粉样变性是一种致命的蛋白质聚集性疾病，其中抗体轻链的沉积引起斑块并导致重要器官功能障碍。

DESCRIPTION (provided by applicant): Light chain amyloidosis (AL) is the most common systemic protein aggregation disease. It is not only debilitating and often fatal, but also difficult to diagnose, because of the variety of affected internal organs. Its molecular cause is a conformational change in specific immunoglobulin light chains (LCs), due to somatic mutations accumulated during the course of immune responses. Specific mutations cause the LCs to polymerize into fibrils that then deposit as plaques. The disease has no direct and effective treatment and the development of one is complicated by the lack of an animal model. This renewal application seeks to build upon the in vitro fibrillogenesis reactions, cellular models and an inhibitory peptide that were developed in the first grant period. These tools will be used to determine the molecular specificity inherent in LC fibrillogenesis reactions. Since we previously showed that some amyloidogenic LC are not secreted and form intracellular inclusion bodies, our first Aim is to test multiple amyloidogenic LCs and determine which sequences are compatible with secretion and which LCs form inclusion bodies. One type of intracellular aggregate, termed aggresome, is of particular interest, because it is likely fibrillar, based on its sensitivity to the inhibitory peptide. This survey of LCs is important in characterizing the intracellular and extracellular pathways of LC amyloid formation. Aim2 will pursue our observations that some LCs can form two distinct types of fibrils, and on the other hand, that LC fibrils can initiate polymerization of other, more distant proteins. A library of mutants will be used to determine the requirements for formation of fibrils from either the oxidized or the reduced LC. We will next test the hypothesis that the unfolding of proteins with Greek key fold (such as LC, p2m, TTR or SOD1) is constrained and thereby leads to a common conformation that can give rise to similar fibrils. This hypothesis will be tested in two ways: seeding of each of these proteins with fibrils of another, and determining the sensitivity of different fibrils to the inhibitory peptide. The similarity uncovered in vitro will then be applied to the intracellular environment by determining the degree of co-aggregation of Greek key proteins. Aim 3 is to use mice transgenic for one amyloidogenic LC and assess their suitability as an animal model for AL by manipulating them genetically and immunologically so as to form amyloid deposits. If successful, this model will be used to test the efficacy of the peptide that specifically blocks fibrillogenesis of Greek Key proteins as a potential treatment modality. Relevance: This project continues to investigate the molecular mechanisms that underlie light chain amyloidosis, a fatal protein aggregation disease where deposits of antibody light chains cause plaques and lead to dysfunction of vital organs.