Amyloidogenic tendency of N-terminal variants of human Apolipoprotein A-I

RAMELLA, N. A.; M. A. TRICERRI; ROSU, S. A; FINARELLI, G; GADDI, G; GISONNO, ROMINA

Buenos Aires

Congreso; XLVI Reunión Anual Sociedad Argentina de Biofísica; 2017

Sociedad Argentina de Biofísica

Amyloidosis is a heterogeneous disease involving protein´s misfolding. Among more than 20 described natural variants, some N-terminal mutated apoA-I (as Leu60Arg (L60R) and Trp50Arg (W50R) are involved in renal amyloid protein deposition. Previous studies with other mutants (Gly26Arg, Lys107-0 and Arg173Pro) suggested that structural conformational shifts determine the involvement of apoA-I in this pathology. In the present study we extended our knowledge and compared protein stability, aggregation tendency and susceptibility to proteolysis of the protein with the native sequence (Wt), with the N-terminal variants W50R and L60R. Structural parameters were analyzed under pH 7.4 and 5.0 by fluorescence, following protein chemical denaturalization, Trp arrangement by the quenching with acrilamide and hydrophobic pockets by the binding of BisAns. The aggregation tendency was evaluated measuring Thioflavin T associated fluorescence following incubation at 37°C in the presence and absence of heparin (as a glucosaminoglycan model). Susceptibly to Trypsin-induced proteolysis was determined at pH 7.4.Our results show that both mutants are less stable than Wt, especially L60R (p0.05) and showed a higher quenching of Trp residues (p0.001); this variant evidenced a loss of hydrophobic pockets. While the aggregation tendency of both variants was similar than Wt at pH 5.0, W50R showed higher ThT binding than Wt or L60R under this condition. No aggregation of the variants tested was detected at pH 7.4, either pure or combined with heparin. Susceptibly to proteolysis was increased for L60R (p0.05) respect Wt and W50R. We suggest that structural unstability of the variants induce misfolding that could expose binding sites of ligands and cleavage sites for proteases in the protein which could either increase protein catabolism or to favor an aggregation-prone conformation. Maybe induced under a pro inflammatory microenvironment. Keywords: misfolding, aggregation