Cloning and expression in vitro and in vivo of amyloid

该项目所基于的假设是，AL淀粉样变性中纤维形成的主要决定因素是患有这种危及生命的疾病的患者的克隆浆细胞中重排的免疫球蛋白轻链基因的核苷酸序列。该核苷酸序列决定了一级氨基酸序列，我们假设该序列是纤维形成蛋白的病理性质的关键，包括蛋白本身的折叠、其翻译后修饰、其与包括其他蛋白质和蛋白聚糖的辅助分子的缔合以及其与宿主组织的相互作用。该计划的其他项目将侧重于后者的性质;该项目将侧重于鉴定表达的mRNA的核苷酸序列，并在体外和体内研究重组蛋白。我们将从患者的浆细胞中克隆和测序轻链基因，利用这种疾病在波士顿医学中心寻求咨询的独特患者群体。将重排的淀粉样蛋白生成轻链基因序列与种系序列、非恶性轻链序列和骨髓瘤轻链序列进行比较。与波士顿大学生物医学工程研究中心的同事合作，我们将分析这些序列，模拟由它们编码的蛋白质，并确定在纤维形成轻链中过量存在的氨基酸残基，这些氨基酸残基被预测为纤维形成的结构决定因素。通过与项目2和核心合作，在体外表达野生型和信息性定点突变轻链，并检查其生物化学和生物物理特性，测定这些残基对原纤维形成的贡献。 除了这些体外试验外，还将使用转染细胞和转基因小鼠在体内表达原纤维轻链，以确定整个动物中人淀粉样蛋白轻链产生的后果（与项目3和核心A合作）。这些小鼠将作为一个整体动物模型来测试关于AL淀粉样蛋白发病机制的假设;此外，与项目4合作，它们将作为免疫治疗的临床前模型。

The hypothesis upon which this project is based is that a major determinant of fibrillogenesis in AL amyloidosis is the nucleotide sequence of the rearranged immunoglobulin light chain gene in the clonal plasma cells of patients with this life-threatening disorder. This nucleotide sequence determines the primary amino acid sequence which we postulate is key for the pathologic properties of fibrillogenic proteins, including the folding of the protein itself, its post-translational modification, its association with accessory molecules including other proteins and proteoglycans, and its interactions with the tissues of the host. Other projects in this Program will focus on the latter properties; this project will focus on identifying the nucleotide sequence of the expressed mRNA and studying recombinant proteins in vitro and in vivo. We will clone and sequence light chain genes from patients' plasma cells, taking advantage of the unique patient population with this disease seeking consultation at Boston Medical Center. The rearranged amyloidogenic light chain gene sequences will be compared to germ line sequences, non-malignant light chain sequences, and to myeloma light chain sequences. In collaboration with colleagues at the Boston University Biomedical Engineering Research Center we will analyze these sequences, model the proteins encoded by them, and identify amino acid residues over-represented in fibril-forming light chains that are predicted to sere as structural determinants of fibrillogenesis. The contribution of these residues to fibrillogenesis will be assayed by expressing wild type and informative site-directed mutant light chains in vitro and examining their biochemical and biophysical properties in collaboration with Project 2 and the Cores. In addition to these in vitro assays, fibrillogenic light chains will be expressed in vivo using transfected cells and transgenic mice to determine the consequences of human amyloidogenic light chain production in the whole animal (n collaboration with Project 3 and Core A). The mice will sere as a whole animal model for testing hypotheses about AL amyloid pathogenesis; furthermore, in collaboration with Project 4, they will serve as a preclinical model for immunotherapy.