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

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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.