Intermediate species in the aggregation of the human apolipoprotein A-I.

TRICERRI, M. A.; RAMELLA, N.; ROSU, S. A.; PRIETO, E. D.; JOVIN, T.; FINARELLI, G. S.

Salto

Congreso; Latin American Crosstalk in Biophysics and Physiology.; 2015

Sociedad de Biofisica

Protein aggregation is characterized by a remarkable polymorphism, where soluble amyloid oligomers, amyloid fibrils and amorphous aggregates are characterized as final products. 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 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 2,3. Here we expand our results, and tested the hypothesis that intermediate aggregation species could be present in the misfolding event. 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. [1] Uversky, V.N. FEBS J. 2010 Jul;277(14):2940-53[2] Ramella, N.A. et al. PLoS One. 2012;7(8):e43755. doi: 10.1371[3] Rosú, S.A. et al. PLoS One. 2015 May 7;10(5):e0124946. doi: 10.1371