Species in the aggregation pathways of human apolipoprotein A-I amyloidosis

GISONNO, R; TRICERRI, MA; PRIETO; ED; ROSU; SA; SCHINELLA, G; GADDI, GM; RAMELLA; NA

KUMAMOTO

Simposio; ISA 2018; 2018

ISA

Amyloidosis induced by natural mutants of apolipoprotein A-I is detected with different manifestations depending on the protein variant. Among other, organs affected include 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 1,2. Nevertheless, fibril-like aggregates are obtained for this and other proteins only by forcing the incubation conditions with high concentrations, incubation times or temperatures. Regardless of the extensive research indicating that fibrillar aggregates deposit inducing organ damage, soluble oligomers have been identified as toxic intermediates in some neurodegenerative and amyloid diseases3. Unlucky no information is available about the structure that could be participating in organ damage induced by apoA-I. Here we tested the hypothesis that intermediate aggregation species could be present in the misfolding event and probably participating in the pathological cascades. The Wt apoA-I and the amyloidogenic variant Lys107-0 were incubated at pH 7.4 or 5.0 at 37 0C at low protein concentrations. Soluble and aggregated species were isolated by centrifugation and their morphology studied by different biophysical approaches. Soluble, oligomer-like and aggregated species were observed for the mutant at both pH, having lower secondary structure than the monomer, and a more flexible conformation. Lys107-0 also aggregates in some extent at pH 7.4. By specific-site labeling the proteins with the Excited Stated Proton Transfer probe MFC, we were able to detect different conformations for monomer, soluble and aggregated species, with increasing non polar environment in that order. Microscopy methods as were used to characterize these species. Different aggregates were observed by Atomic Force and electron transmission Microscopy, as oligomers amorphous and proto fibrils, but highly ordered fibrillar structures were not visible under our conditions. Our results support the concept that oligomer, non amyloid species are representative of apoA-I mutants in circulation, opening the question that oligomer-like aggregates could play a role in the pathogenic pathways of this mutant.