The structure peturbations of N-terminal variants of human apolipoprotein A-I may induce aggregation

GADDI, GM; GISONNO, R; ROSU; SA; CURTO, ML; FINARELLI; GS; RAMELLA; NA; TRICERRI, MA

Simposio; III SIMPOSIO JÓVENES BIOFISICOS SAB; 2018

SAB

Amyloidosis is a genetic disorder involving proteins´ misfolding; more than 20 by analyzing Trp intrinsic fluorescence as a function of guanidium clorhidric concentration. Trp arrangement was compared by the quenching with acrylamide and circular dichroism in far UV and near UV. The hydrophobic pockets within the protein structure were estimated by the binding of the fluorescent probe Bis-ANS at physiological pH. The aggregation tendency was registered by Tioflavin T (ThT) fluorescence measured in single point mutations of apolipoprotein A-I (apoA-I) have been described as inducers of the disease, some associated to renal deposits such as Leu60Arg (L60R) in interstitial tubes and Trp50arg deposited mainly in glomeruli (W50R). Both variants are comprised in the so-called ?hot spot? N-terminal domain.In the present work we compared apoA-I having the native sequence (Wt) with the two mentioned amyloid variants analyzing their structural behavior, aggregation tendency in the presence of ligands, susceptibility to the proteolysis mediated by trypsin and capability to elicit the release of TNF alpha as an inflammatory marker from cultured macrophages. The structural parameters were analyzed at pH 7.4 and 5.0. Proteins stability was studied the absence or the presence of cell ligands such as heparin (as aglycosaminoglican model) and sodium dodecyl sulphate (SDS) as a model of negative lipid). Susceptibility to trypsin proteolysis was compared by SDS-PAGE. Macrophages activation mediated by apoA-I variants was detected by quantification the release of TNF alpha by ELISA. Our data suggest tha the mutants are more unstable respect to Wt, especially L60R in both pH conditions and exhibit higher relative quenching of Trp residues; this variant shows less hydrofobic pockets and lower alpha helix content in the far UV CD in comparison with the Wt; near UV CD in the range 250-280 nm indicates small structural differences among aromatic amino acid residues. W50R shows increment of ThT intensity in the presence of heparin at pH 5.0 respect to the others, indicating higher aggregation tendency under these conditions. No aggregation of the variants was detected at pH 7.4, either pure or combined with heparin. L60R binds more efficiently ThT in the presence of SDS than Wt or W50R. In addition this variant is more susceptible to proteolysis. Instead L60R induces macrophages activation in conditions where the other tested variants don´t. We suggest that small structural perturbations produce misfolding of the Wt and N-terminal variants that could favor the exposition of new sites for binding cell ligands and cleavage by proteases. These mild conformational shifts could in addition result in the initiation of a local pro inflammatory micro environment