Interaction and association with lipids of Apolipoprotein A I studied with Pyrene labelled cysteine mutants

TÁRRAGA WILSON; FALOMIR LOCKHART, LISANDRO J.; GONZALEZ MARINA CECILIA; GARDA, HORACIO A.

La Plata

Congreso; XLIX Reunión anual de la Sociedad Argentina de Biofísica (SAB); 2021

Sociedad Argentina de Biofísica SAB

Apolipoprotein A I (apoA I) is the main protein of high density lipoproteins (HDL), to which antiatherogenic properties are attributed to its role in the reverse transport of cholesterol excess from peripheral tissues to the liver for catabolism and disposal.In water solution, apoA I forms a bundle composed of several amphipathic alpha helices. Depending on the concentration, apoA I self associates to form dimers and oligomers of higher orders with poorly characterized quaternary structures. It also interacts with phospholipids to form discoidal HDL (dHDL) in alternative anti parallel helical arrangements. Previous studies from this laboratory have shown the participation of helices 5 and 10 in contact regions during certain oligomerization steps. However, these self interactions during lipid interaction and lipid association are unknown.The aim of the present study is to characterize the lipid interaction and lipid association to form dHDL with single labelled mutants of apoA I, focusing on the proximity of helices known to be important to detect dHDL arrangements.Selected cysteine mutants of apoA I were purified and labelled with pyrenyl maleimide in positions corresponding to helices 4 (K107C), 5 (K133C and L137C) and 10 (F225C and K226C). The monomer and excimer fluorescence of the labelled proteins were registered in apoA I titrations with lipid vesicles and dHDL generated in vitro by different methods. Additionally, dHDL formation with K107C and K133C were evaluated in vivo by incubation with THP 1 macrophage cells.The labelled mutants were stable in solution as indicated in a previous study and biologically active since they can form dHDL. Fluorescence emission spectra of pyrene showed lipid interaction by increased total fluorescence emission. It was also observed with P value, showing little changes except with mutant L137C. In the same way, excimer formation indicated poor or absent oligomerization especially with mutants K133C, F225C and K226C, which self associate in lipid free environments. On the other hand, the pyrene emission in dHDL generated in vitro showed poor excimer formation except with K133C, which was minimal. It was also reported in vivo with mutants K107C and K133C, suggesting a 5/5 anti parallel helical arrangement. In conclusion, we proposed that self interactions in helix 5 could be critical to form dHDL since they are observed in lipid free apoA I and dHDL, except during the association with lipids where it can be different.Acknowledgments:This work was financed by: CONICET, ANPCyT and UNIVERSIDAD NACIONAL DE LA PLATA (UNLP).