Amyloidosis due to natural apoliprotein A-I Variants. Mechanisms involved in the pathogenicity pathways.

TRICERRI, M. A.; GADDI, G. M.; PRIETO, E. D.; FINARELLI, G. S.; RAMELLA, N.; GISONNO, R.; CALABRESE G; ROSU, S. A.; SCHINELLA, G; BARIANDARÁN, A.

Kumamoto

Simposio; XVI th International Symposium on Amyloidosis; 2018

International Soc of amyloidosis

INTRODUCTION: The cascade of molecular events leading to amyloidosis is variable. Different protein conformations such as the precursor monomer, oligomeric or fibrillar structures could determine the pathology1. Components of the extracellular matrix, like glycoproteins or their glycosaminoglycan chains (GAGs) have in addition been suggested to induce the formation of amyloid aggregates2. Human apolipoprotein A-I (apoA-I)-derived amyloidosis is poorly known. About 20 naturally occurring mutations have been associated with hereditary amyloidosis causing multiple organ failure. Our previous studies strongly suggest that a decrease in apoA-I variants´ stability and changes in the microenvironment could elicit protein misfolding and tissue deposition3. Here we got deep insight into the mechanisms that could determine amyloidosis due to apoA-I. MATERIALS & METHODS:The expression and purification of apoA-I variants (Wt, Gly26Arg, Trp50Arg, Leu60Arg, Arg173Pro), was carried out following traditional Molecular Biology techniques. Protein structural characterization was achieved by biophysical approaches including fluorescence and microscopy set ups. Chemokine release was analyzed by ELISA tests and p65 levels by western blots. Macrophage and endothelial (HUVEC) cells were cultured under controlled CO2 atmosphere. Proteoglycans expression was analyzed by RT-PCR and western blot. Binding of variants to GAGs was followed by electrophoresis and by fluorescence associated to polymeric matrices. RESULTS:Monomeric conformations (Gly26Arg) induce from macrophages a specific pro-inflammatory cascade involving activation of NF-B and its translocation into the nucleus. At the N-terminus, sensibility to proteolysis is stronger for the mutant Leu60Arg than the Trp50Arg. Oligomeric conformations could be obtained under incubation at mild acidic pH but a stable fiber conformation is attained after several days incubation under physiological pH or controlled oxidation. The possibility that these species interact with component of the extracellular matrix was tested. Interaction of the mutant Arg173Pro is efficient with heparin as a model of GAG and mediated by negative charges of sulfonate groups. In addition, Wt induces from HUVEC a change in the expression of proteoglycans such as decorin, indicating a dynamic and fine interaction with these matrix components. DISCUSSION & CONCLUSIONS: Our results strongly suggest that some natural mutations in apoA-I variants elicit protein tendency to aggregate, but in addition macrophages activation may contribute to cellular stress and toxicity in hereditary amyloidosis. A specific interaction of some mutants with components of the extracellular matrix could not only increase their aggregation but it could induce a matrix composition which opens a field to explain the organ selectivity for protein deposition. We conclude that it may be a crosstalk among apoA-I variants and component of the microenvironment which determine protein aggregation and pathology. REFERENCES:1. Uversky, V.N. Mysterious oligomerization of the amyloidogenic proteins. Febs J 2010; 277: 2940-2953.2. Ramella, N.A., Schinella, G.R., Ferreira, et al. Human apolipoprotein A-I natural variants: molecular mechanisms underlying amyloidogenic propensity. PLoS One 2012; 7: e43755.3. Rosu, S.A., Rimoldi, O.J., Prieto, E.D. et al. Amyloidogenic propensity of a natural variant of human apolipoprotein A-I: stability and interaction with ligands. PLoS One 2015; 10: e0124946.