The goal of the parent proposal "Mechanisms of aggregation in light chain Amyloidosis" is to dissect the molecular mechanisms that cause light chain amyloidosis (AL) deposits to form. AL is a devastating disease caused by the abnormal proliferation of plasma cells that secrete large amounts of monoclonal light chains that misfold in vital organs, causing tissue damage, organ failure and death. In aim 1 and 2 of the parent proposal we have studied the structure, thermodynamic stability and amyloid formation properties of selected light chain proteins from AL patients (AL-09, AL-12, AL-103) and their corresponding wild-type counterpart, the germline protein. We have initiated studies on aim 3 where we are doing a systematic study of the cell biology of the disease by studying the role of heavy chain/light chain pairing in the secretion of these proteins in cell culture as well as dissecting the mechanism of light chain internalization in target organs by studying a cell culture model. We had originally propose to do the internalization studies with AL-09 only. We would like to propose for this supplement to incorporate internalization studies using the full length germline protein tagged with the molecule oregon green as was initially proposed with the amyloidogenic protein AL-09. Our preliminary data on this project shows that AL-09 is being internalized into ardiomyocytes after 24 hours and the protein becomes insoluble within the cells, suggesting that the aggregation process may initiate while the protein is intracellularly. It would be very important to compare the AL-09 internalization studies with germline internalization studies since these proteins differ by only 7 amino acids, 3 of which are non-conservative Substitutions that have been extensively characterized by our laboratory. Once the germline protein has been characterized, we will perform internalization studies with the restorative mutants characterized in aims 1 and 2 (AL-09 134N, AL-09 Q42K and AL-09 H87Y). This work will increase our understanding of the mechanism of pathogenesis in amyloidosis and will help us delineate better strategies for its management and cure.