| Popis: |
Transforming growth factor beta 1 (TGF-β1) is translated as a proprotein. The main enzyme responsible for the proteolytic processing of proTGF-β1 is furin. The aim of this thesis is to characterise the proteolytic processing of proTGF-β1 and investigate whether this step may represent an important point at which TGF-β1 activity may be regulated. I show that most cells secrete proTGF-β1, and that this can be processed by recombinant furin and then activated to yield mature TGF-β1 with biological activity. The extent of proTGF-β1 secretion differs between cell types, as does the extent of LAP glycosylation. I go on to investigate the regulation of proteolysis of proTGF-β1 in two processes in which the TGF-β1 bioavailability is increased; upon platelet degranulation and macrophage phagocytosis of apoptotic cells. I describe a series of attempts to produce a polyclonal antibody with affinity for proTGF-β1 but not processed latent TGF-β1, needed to investigate proTGF-β1 levels and localisation in vivo. The possibility of distinguishing and quantifying these species using non-specific anti-TGF-β1 antibodies and physiochemical activation, was also investigated but without success. Purified recombinant proTGF-β1 was produced and reacted with recombinant furin to investigate the kinetics of furin-mediated proTGF-β1 processing. I establish that furin is able to process proTGF-β1 in a minimal system without the need for cofactors. Finally, I performed a multiple SNP and haplotype association analysis of both the furin and TGF-β1 genes with coronary artery disease (CAD) in the MaGiCAD study. I report a candidate furin haplotype that may be protective against myocardial infarction and two TGF-β1 SNPs that were significantly associated with angiographically defined CAD and myocardial infarction respectively. |
