METABOLISM OF SERUM AMYLOID A (SAA:HDL)

Indiana Univ-Purdue Univ At Indianapolis, Indianapolis IN

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Abstract

DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate pathogenic mechanisms of reactive (AA) amyloidosis. This type of amyloidosis develops as a complication of chronic and recurrent inflammatory conditions such as rheumatoid arthritis and familial Mediterranean fever, and is characterized by extracellular fibrillar deposits of amyloid A (AA) protein, a peptide proteolytically derived from the acute phase reactant serum amyloid A (SAA). While overproduction of SAA has long been recognized as a prerequisite for amyloid formation, factors governing why and how SAA starts forming fibrils, as well as the mechanism by which nascent fibrils grow into clinically significant extracellular masses, remain largely unknown. A cell culture model employing maerophages cultured in the presence of recombinant mouse SAA1.1 will be used to ascertain critical events in amyloid fibril initiation and propagation. Amyloid formation and SAA processing will be followed intracellularly and on cell surfaces via fluorescent-labeled SAA and confocal microscopy, 125I-SAA combined with SDS-PAGE and phosphorimaging, immunocytochemistry, and Congo red staining. Focusing on the intracellular formation of an amyloid nucleus, Aim 1 will test the hypothesis that an unrelenting influx of SAA during chronic inflammation results in compromised intracellular proteolysis, which, in turn, permits fibrillization. Macrophages will be also be subjected to two regimens of SAA treatment mimicking patterns of either chronic or recurrent inflammation to gain clues about how the duration and degree of SAA elevation influence the likelihood of fibril formation. Aim 2 will determine if the initiation of amyloid fibrillogenesis requires processing of SAA to AA. Cleavage sites in the C-terminal region of SAA will be determined by mass spectroscopy analysis of macrophage-produced amyloid. Mutant SAAI.1 proteins, designed to be protease-resistant at one of the identified sites, will be produced and tested for amyloid-forming capability. Extracellular mechanisms of fibril propagation will be investigated in Aim 3. Direct incorporation of SAA onto preformed cell surface amyloid or protofibrils (AEF) followed by in situ processing will be examined as a potential step in propagation. The ability of macrophagegenerated AEF to seed, and thereby spread amyloid deposition throughout tissues, will be studied in a fibroblast model. By clarifying pathogenic mechanisms at the cellular level, these studies will help identify potential targets for drugs aimed at inhibiting amyloid formation or arresting its progression.