Cloning and expression in vitro and in vivo of amyloid

这个项目所基于的假设是，AL淀粉样变性纤维形成的一个主要决定因素是这种危及生命的疾病患者克隆浆细胞中重排的免疫球蛋白轻链基因的核苷酸序列。这个核苷酸序列决定了主要的氨基酸序列，我们推测这对纤维蛋白的病理性质是关键的，包括蛋白质本身的折叠，它的翻译后修饰，它与包括其他蛋白质和蛋白多糖在内的辅助分子的结合，以及它与宿主组织的相互作用。该计划的其他项目将侧重于后一种性质；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。我们将从患者的血浆细胞中克隆轻链基因并进行测序，利用波士顿医疗中心寻求咨询的独特患者群体的优势。重排的淀粉样变性轻链基因序列将与胚系序列、非恶性轻链序列以及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，对它们编码的蛋白质进行建模，并识别在形成纤维的轻链中过度代表的氨基酸残基，这些轻链被预测为纤维形成的结构决定因素。这些残基对纤维形成的贡献将通过在体外表达野生型和信息丰富的定点突变轻链，并与项目2和核心合作检测其生化和生物物理性质来分析。除了这些体外分析外，还将使用转基因细胞和转基因小鼠在体内表达纤维样变轻链，以确定在整个动物中产生人淀粉样变轻链的后果(与项目3和核心A合作)。这些小鼠将作为一个完整的动物模型来测试AL淀粉样蛋白发病机制的假说；此外，与Project 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.