Cellular, kinetic, and structural mechanisms of toxicity in light chain amyloidosis

轻链淀粉样变性毒性的细胞、动力学和结构机制

• 批准号: 9539266
• 负责人: Marina Ramirez-Alvarado
• 金额: $ 42.51万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9539266
• 关键词: Affect; Amyloid; Amyloid Fibrils; Amyloidosis; Apoptosis; Area; Binding; Biological Assay; Blood Circulation; Bone Marrow; C-terminal; Cardiac Myocytes; Cell Culture Techniques; Cell Death; Cell Survival; Cell surface; Cellular Stress; Cessation of life; Chemicals; Complex; Data; Dependence; Deposition; Disease; Event; Fluorescence Microscopy; Foundations; Functional disorder; Future; Glycosaminoglycans; Goals; Human; In Vitro; Kinetics; Knowledge; Laboratories; Lead; Learning; Length; Ligands; Light; Light-Chain Immunoglobulins; Lipids; Membrane; Methods; Molecular Conformation; Monoclonal Antibodies; N-terminal; Organ; Organ failure; Pathway interactions; Plasma Cells; Play; Property; Proteins; Reaction; Recombinants; Reporting; Resolution; Role; Scanning; Severities; Somatic Mutation; Structure; Techniques; Testing; Therapeutic; Thermodynamics; Toxic effect; Urine; Work; amyloid formation; amyloid structure; base; cell growth; cytotoxicity; design; diagnostic biomarker; dimer; experimental study; improved; in vivo; innovation; primary amyloidosis of light chain type; protein complex; protein misfolding; small molecule; solid state nuclear magnetic resonance

Light chain (AL) amyloidosis is a devastating, incurable, systemic and complex protein misfolding disease in which immunoglobulin light chains misfold and aggregate into amyloid fibrils in vital organs, causing organ failure and death. Work by our laboratory and others have demonstrated the complex role between thermodynamic stability and amyloid formation kinetics, the role of somatic mutations in the amyloidogenicity and cellular toxicity of light chains, and the cellular internalization pathways followed by light chains. A recent report from our groups has demonstrated that AL amyloid fibrils are the most toxic species to human cardiomyocytes compared to soluble AL proteins species, shedding light into the complex mechanism of organ damage in AL amyloidosis. Preliminary data of the structure of amyloidogenic AL protein and control amyloid fibrils using solid state NMR (ssNMR) with the Rienstra laboratory has shown that there are conformational differences between the fibrils of AL proteins and controls that may be correlated with their toxicity potential. Based on these findings, the overall goal of this proposal is to understand the structural determinants that drive AL amyloidosis correlating protein stability, amyloid formation kinetics, fibril toxicity and fibril structure. For aim 1, we propose to study the thermodynamic and kinetic stability of full length AL amyloidogenic proteins. With this aim, we will learn about the role of the constant domain modulating the stability and amyloidogenic properties of these proteins and we will be able to correlate with toxic properties of full length light chains. For aim 2, we propose to dissect the events that lead to AL amyloid fibril-derived cell death by studying the early events of fibril formation in vitro in a quantitative way and moving towards studies in cell culture. These studies will help us identify key species in the fibril formation pathway, compare the reactions in vitro and in cell culture and will help us discern if amyloid fibril binding to the cell surface is enough to induce cellular stress. Finally, for aim 3, we will determine the high resolution structure of AL amyloid fibrils. We will compare the amyloid structure of different AL proteins and their germline (non-amyloidogenic) control, with full length fibrils, fibrils seeded with ex-vivo amyloid deposits and fibrils formed in the presence of lipids and other co-factors such as glycosaminoglycans. We will compare the possible differences in the amyloid structure with the differences found in the soluble proteins dimer structures, allowing us to correlate the fibril structures with their associated toxicity. Currently, there are no therapeutic strategies approved for the misfolding aspect of AL amyloidosis. Our results will provide critical new knowledge for the structural determinants of fibril-based toxicity, the possible structural differences between soluble and fibril toxic species, and the possible structural determinants of the severity of some cases. This knowledge will be essential to guide rational design of diagnostic biomarkers and therapeutics, including small molecule ligands and monoclonal antibodies, to reduce and ultimately eliminate the devastating consequences of this disease.

轻链（AL）淀粉样变性是一种毁灭性的，不可治愈的，系统性和复杂的蛋白质错误折叠疾病， 其中免疫球蛋白轻链在重要器官中错误折叠并聚集成淀粉样纤维， 失败和死亡。我们实验室和其他实验室的工作已经证明了 热力学稳定性和淀粉样蛋白形成动力学，体细胞突变在淀粉样蛋白生成中的作用 和轻链的细胞毒性，以及轻链遵循的细胞内化途径。最近的一 本课题组的一份报告表明，AL淀粉样纤维是对人体毒性最大的物质 心肌细胞相比，可溶性AL蛋白的种类，揭示了复杂的机制，器官 AL淀粉样变性中的损伤。致淀粉样变AL蛋白和对照淀粉样蛋白结构的初步数据 Rienstra实验室使用固态NMR（ssNMR）的原纤维已经表明，存在构象 AL蛋白原纤维和对照之间的差异可能与其潜在的毒性有关。 基于这些发现，本提案的总体目标是了解驱动 AL淀粉样变性与蛋白质稳定性、淀粉样蛋白形成动力学、原纤维毒性和原纤维结构相关。为宗旨 1，我们建议研究全长AL淀粉样蛋白的热力学和动力学稳定性。与 为此，我们将了解恒定结构域调节淀粉样蛋白稳定性和淀粉样蛋白生成的作用。 这些蛋白质的特性，我们将能够与全长轻链的毒性特性相关联。为 目的2，我们建议通过研究AL淀粉样蛋白纤维衍生的细胞死亡的早期过程， 以定量的方式在体外研究原纤维形成的事件，并在细胞培养中进行研究。这些研究 将帮助我们确定纤维形成途径中的关键物种，比较体外和细胞培养中的反应 并将帮助我们辨别淀粉样蛋白纤维与细胞表面的结合是否足以诱导细胞应激。最后， 对于目标3，我们将确定AL淀粉样纤维的高分辨率结构。我们将淀粉样蛋白 不同AL蛋白的结构及其种系（非淀粉样蛋白生成）控制，具有全长原纤维、原纤维 接种有在脂质和其它辅因子存在下形成的离体淀粉样蛋白沉积物和原纤维， 糖胺聚糖我们将比较淀粉样蛋白结构中可能存在的差异与 在可溶性蛋白质二聚体结构中发现，使我们能够将原纤维结构与其相关的 毒性目前，没有批准用于AL淀粉样变性的错误折叠方面的治疗策略。 我们的研究结果将为纤维毒性的结构决定因素提供关键的新知识， 可溶性和原纤维毒性物质之间可能的结构差异，以及可能的结构决定因素 一些案件的严重性。这些知识对于指导诊断性药物的合理设计至关重要。 生物标志物和治疗剂，包括小分子配体和单克隆抗体，以减少和 最终消除这种疾病的毁灭性后果。