Immunoglobulin Structure, Stability and Aging

描述(申请人提供)：轻链淀粉样变性(AL)是最常见的全身性蛋白聚集性疾病。它不仅使人虚弱，而且往往是致命的，而且由于受影响的内脏多种多样，诊断也很困难。其分子原因是免疫应答过程中积累的体细胞突变导致特异性免疫球蛋白轻链(LCS)的构象变化。特定的突变会导致LC聚合成纤维，然后以斑块的形式沉积。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起来。这一更新申请寻求建立在体外纤维形成反应、细胞模型和在第一次赠款期间开发的抑制肽的基础上。这些工具将被用来确定LC纤维形成反应中固有的分子特异性。由于我们之前已经证明了一些淀粉样变性LC不能分泌并形成细胞内包涵体，所以我们的第一个目标是检测多个淀粉样变性LC，并确定哪些序列与分泌相容，哪些LC形成包涵体。有一种类型的细胞内聚集体，称为侵袭体，特别令人感兴趣，因为它可能是纤维状的，基于它对抑制肽的敏感性。LCS的这一研究对于描述LC淀粉样蛋白形成的细胞内和细胞外途径具有重要意义。AIM2将继续我们的观察，即一些LC可以形成两种不同类型的纤维，另一方面，LC纤维可以引发其他更远距离的蛋白质的聚合。突变体库将被用来确定氧化或还原的LC形成纤维的要求。接下来，我们将测试这样的假设，即带有希腊关键折叠的蛋白质(如LC、p2m、TTR或SOD1)的展开受到限制，从而导致共同的构象，从而可能产生类似的纤维。这一假设将通过两种方式进行检验：将这些蛋白质中的每一种与另一种蛋白质的纤维一起播种，并确定不同纤维对抑制肽的敏感性。在体外发现的相似性将通过确定希腊关键蛋白的共聚程度应用于细胞内环境。目的3是利用一种淀粉样变性LC转基因小鼠，通过对其进行遗传学和免疫学操作以形成淀粉样蛋白沉积，以评价其作为AL动物模型的适用性。如果成功，这个模型将被用来测试这种特定阻止希腊关键蛋白纤维形成的多肽作为一种潜在的治疗方式的有效性。相关性：该项目继续研究轻链淀粉样变性的分子机制，这是一种致命的蛋白质聚集性疾病，抗体轻链沉积会导致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.