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Lysosome-related generation of potentially amyloidogenic serum amyloid A polypeptides in activated murine macrophages and the potential role of ubiquitin in this process

Reactive amyloidosis characterized by the tissue deposition of insoluble AA amyloid (AA), is a potentially serious complication of certain chronic infections and recurrent inflammatory diseases. AA is derived from the amyloidogenic serum amyloid A (SAA), an acute phase protein whose plasma concentration may increase up to 1000-fold in response to tissue trauma. Chronic elevation of SAA in conjugation with inflammation-related host factors is believed to induce conversion of SAA to AA. One such host factor, called amyloid enhancing factor (AEF), has recently been identified as ubiquitin (UB). However, the molecular pathogenesis of AA amyloidosis in vivo is not well understood. / Prior studies in our laboratory using alveolar hydatid cyst infected mouse model of AA amyloidosis (AHC-mouse) has demonstrated that (a) SAA and UB co-deposit in the splenic perifollicular areas prior to AA deposition, (b) both SAA and UB co-localize to endosomes-lysosomes (ELs) inflammatory macrophages (MA) and splenic reticuloendothelial (RE) cells, and (c) UB binds to the tissue AA deposits. / My working hypothesis is that EL-mediated physiological degradation of SAA may serve as the primary clearance mechanism for exogenous SAA. As a secondary consequence, incessant overloading of ELs with ubiquitin-associated SAA during chronic inflammation may alter the rate or the extent of proteolysis leading to prolonged retention of SAA derivatives and the formation of "nascent" AA fibrils in ELs. / The objectives of the present study are the following: (a) to establish intra-macrophage topographical relationship between murine SAA3, which is synthesized by activated MA and RE cells, and two precursors of AA, SAM and SAA2, that are endocytosed by MA and RE cells, (b) to immunochemically and chemically analyze the MA-derived degradation products of SAA in order to establish whether they could be potentially amyloidogenic and (c) to seek functional clues regarding the role of ubiquitin in SAA processing and in AA formation. / Both the in vivo and in vitro experiments were carried out to pursue these objectives. I have shown that (a) murine SAA1/SAA2 localize exclusively to ELs whereas SAM occupies the cytoplasmic compartment in MAs, (b) the lysates derived from MA contain at least four AA-sized potentially amyloidogenic N-terminally intact SAM and SAA2 derivatives and (c) AA amyloid purified from AHC-mice is derived from both SAA2 and SAA1. As to the role of UB in SAA processing, I have shown that (a) UB binds avidly but non-covalently to both SAA1 and SAA2, (b) UB binds to ELs containing both immature and mature AA fibrils and (c) UB dissociated and purified from the tissue AA deposits retains its AEF activity in vivo. / Collectively, these data show for the first time a direct relationship between the localization of SAA1 and SAA2 to ELs in MAs and the intracellular generation of potentially amyloidogenic N-terminally intact SAM and SAA2 derivatives which may polymerize into "nascent" AA fibrils in ELs. We believe, as has also been proposed by others, that these "nascent" AA fibrils either after exocytosis or cell death may act as nucleation sites for extracellular AA deposition. As to the role of UB in SAA processing, we suggest that non-covalent binding of UB to SAA1 and SAA2 may (a) structurally alter theses substrates, thereby rendering them susceptible to phagocytosis by MA/RE-cells, and (b) interfere with their complete degradation culminating in the formation of UB-associated AA found in the EL. Non-covalent linkage of UB has been shown to induce structural alterations in other substrate proteins rendering them susceptible to phagocytosis.

Identiferoai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.34708
Date January 1997
CreatorsChan, Sic L.
ContributorsAli-Khan, Z. (advisor)
PublisherMcGill University
Source SetsLibrary and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada
LanguageEnglish
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation
Formatapplication/pdf
CoverageDoctor of Philosophy (Department of Microbiology and Immunology.)
RightsAll items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated.
Relationalephsysno: 001601765, proquestno: NQ44381, Theses scanned by UMI/ProQuest.

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