Morphology characterization of amyloid-prone apolipoprotein A-I mutants aggregates

RAMELLA, N.; FINARELLI, G. S.; PRIETO, E. D.; TRICERRI, M. A.; SCHINELLA, G; URBANO, B.; ROSU, S. A.; SANCHEZ, S. A.

La Plata

Workshop; Imaging Techniques for Biotechnology and Biomedical Applications Workshop; 2016

4Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA)

Protein aggregation is characterized by a remarkable polymorphism. Regardless of the extensive research indicating that fibrillar aggregates deposit inducing organ damage, soluble oligomers have been identified as toxic intermediates in some neurodegenerative diseases1. Amyloidosis induced by natural mutants of apolipoprotein A-I (apoA-I) depends on the protein variant and affects different organs such as heart, liver and kidney. In previous studies we have demonstrated that different apoA-I mutants show increased tendency to aggregate giving rise to different conformations depending on the incubation conditions 2,3.To get insight into the mechanism inducing protein misfolding, we constructed four natural amyloidogenic variants (Arg173Pro, Gly26Arg, Leu60Arg and Lys107-0), and compared their behavior with the protein with the wild type sequence (WT). In this goal microscopy approaches worked as powerful tools to characterize the effect of proteins stability, oxidation, mild acidification and interaction with ligands on protein conformation. All the mutants are less stable than WT at physiological pH, and show a more flexible structure, indicating a shift from the native state that could in part explain their aggregation tendency. The aggregation pattern of these variants was characterized by Atomic Force Microscopy (AFM) as small size (4-10 nm height) oligomers (Figure 1). Mild acidification induced the formation of some protofibers but the most of the protein conformation was characterized as higher size and more heterogeneous amorphous, non-amyloid species. By biophysical approaches we determined that although less soluble, these aggregates are reversible and disassemble under physiological pH. Previously, we have shown that binding of apoA-I variants to heparin (as a model of Glycosamine Glycane, GAG) is selective, and the mutant Arg173Pro forms protein-heparin complexes and binds negative lipids with high efficiency. In addition some small fibers were detected when Lys107-0 is incubated with heparin at pH 5.0 (Figure 2). As sulfate groups in the GAGs are probably involved in such interaction, we built synthetic polymers with different charge composition and tested the binding affinity of the mutant Arg173Pro respect to WT. We labeled the proteins with Fluorescein Isotiocyanate and analyzed fluorescence associated to the polymers by Confocal Spectral Biphotonic Microscopy. These data confirmed the increased binding of the mutant to matrices with higher sulfate group composition. Finally, we simulated a pro-inflammatory milieu and determined that even the WT protein when incubated with this pro-oxidant medium gives rise to longer-fiber-amyloid like structures that are clearly detected by AFM and transmission electron Microscopy (Figure 3). Altogether Microscopy helped us to get deep insight in the hypothesis supporting that amyloid conformations of apoA-I variants are obtained under post translational events that yield either full length or a proteolyzed product which causes organ damage.