Abstract
Cardiovascular disease (CVD) is one of the leading causes of death in the United States. High density lipoprotein (HDL), which carries cholesterol from peripheral tissues to the liver for excretion, is associated with a lowered risk for CVD. Apolipoprotein A-I (apoA-I), the major protein in HDL, has also been correlated with lowered CVD risk, possibly due to its anti-inflammatory and anti-oxidant properties. Despite these beneficial features, apoA-I misfolded into amyloid fibrils has been found in the arterial plaque of patients suffering from CVD. There are 14 known fibril-forming mutations of apoA-I associated with amyloidosis in vivo. These mutations are found in the N-terminal helix bundle domain (1-190) between residues 26-90 (inside cluster) and residues 170-178 (outside cluster). ApoA-I is found in both lipid-free and lipid-bound forms in vivo, with about 90% of the protein existing in the lipid-bound form. It is unknown whether lipid-free, lipid-bound, or both forms contribute to the development of amyloid deposits in vivo. Recent in vitro experiments on isolated protein demonstrates significant structural effects of the inside cluster mutation G26R (Lagerstedt J.O., Cavigiolio G, Roberts L.M., Hong H.S., Jin L.W., Fitzgerald PG, Oda M.N., Voss J.C., 2007, Biochemistry, 46, 9693-9) and the outside cluster mutation L178H (Petrlova, J., Duong, T., Cochran, M. C., Axelsson, A., Morgelin M., Roberts, L.M., Lagerstedt, J.O., 2012, J. Lipid Res., 53, 390-398) on lipid-free apoA-I. In contrast, there was little effect of the inside cluster mutations G26R and W50R on lipid-binding ability (Das, M., Mei, X., Jayaraman, S. Atkinson, D, Gursky, O. 2014. FEBS Journal, 281, 2525-2542). The outside cluster mutation L170P reduced the stability of lipid-bound apoA-I but not its ability to bind lipid and form HDL (Das, M., Wilson, C.J., Mei, X., Wales, T., Engen, J.R. & Gursky, O. 2016. Journal of Molecular Biology, 428, 449–462). A full understanding of apoA-I amyloidosis in vivo requires analysis of all apoA-I amyloid mutants. The goals of this thesis research were to examine the effect of the outside cluster L178H mutation on (1) the structure of lipid-bound apoA-I and (2) the formation and stability of reconstituted HDL (rHDL). The lipid-bound structure was examined using limited proteolysis. The effect of the L178H mutation on rHDL particle size, distribution and stability was examined through native gradient gel (NDGGE). The properties of lipid-bound L178H apoA-I are very similar to those of lipid-bound WT protein. The rHDL particles formed are similar in size, ranging from 9.3-9.5 nm in diameter. The 24-hour proteolysis of rHDL samples incubated for 0, 1, or 7 days at 4°C and 37°C produced an end particle of 8.4 nm in diameter and a peptide fragment size of 24 kDa for both WT and L178H at all time and temperatures, this indicating similar stabilities. Edman sequencing of peptides produced by limited proteolysis was inconclusive for WT but revealed cleavage at L42 in L178H, a known point of cleavage in lipid-bound WT. A minor difference in rHDL prepared from the two proteins was observed in samples incubated over a seven-day period at 4°C. WT rHDL increased in diameter from 9.3 ± 0.1 nm to 9.9 ± 0.1 nm whereas L178H rHDL showed little change. The reduced ability of L178H to acquire more lipid at a lower temperature is likely due to introduction of the polar Histidine residue, which could reduce hydrophobic interactions. Overall, these results show there is little effect of the L178H mutation on lipid-bound protein structure and rHDL formation, consist with results from other amyloidogenic mutants. Thus, our research supports the conclusion that amyloid fibrils in vivo develop from lipid-free protein.