Immunoglobulin (AL) amyloidosis is characterized by extracellular deposition of fibril complexes composed of monoclonal immunoglobulin light chains (LC). This is the most common form of systemic amyloidosis and has the worst prognosis of any of the systemic forms. Median life span after tissue biopsy proven diagnosis is approximately 20 months. While some clinical response to chemotherapy has been documented in anecdotal cases there is not specific therapy for this disease. The overall objective of this proposal is to understand the pathogenesis of AL amyloidosis so that therapeutic strategies can be tested. The hypothesis to be tested is that the structure of the monoclonal immunoglobulin light chain proteins which for beta-fibrils of amyloid is a key factor in the fibril forming process. The basis for this hypothesis is the well documented observation that only certain light chain protein structures have been found to be associated with amyloid fibril formation. These observations include the predominance of lambda compared to kappa amyloid light chain proteins, the profound amyloid forming potential of lambda VI subgroup proteins, predominance of kappa I amyloid proteins within the kappa light chain series, and in vitro studies showing fibril formation from specific immunoglobulin light chain structures. Despite these findings which are all based on primary protein structure, no unifying hypothesis has been proven for involvement of protein structure in amyloid pathogenesis. The present project is designed to investigate the tertiary and quaternary structure of amyloid light chain proteins in the pathogenesis of amyloid fibril formation. To achieve this goal the variable segments of immunoglobulin light chain proteins which have been proven to be associated with amyloid fibril formation will be produced by recombinant techniques. These recombinant proteins will then be crystallized and their structures determined by X-ray diffraction. Preliminary data have already proven the feasibility of this approach. Immunoglobulin light chain proteins have been produced with this protocol and the crystal structure of one such protein determined. Once sufficient structures of amyloid light chain proteins are determined, they will be compared to structures of light chain proteins which are not associated with amyloid formation. Analysis should reveal those structural factors which are necessary for fibril formation in vivo. These proteins will also serve as substrates for enzymatic degradation studies in vitro. These studies and in vitro fibril forming potential should reveal more information on factors necessary for protein processing which leads to amyloid fibril formation. The ultimate goal will be to identify methods of interfering with amyloid fibril formation and, therefore, amelioration or prevention of this condition.
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