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

ROSU, S. A; CALABRESE, GRACIELA C.; RAMELLA, NAHUEL A.; GUILLERMO R. SCHINELLA; FINARELLI, G; TRICERRI, M. ALEJANDRA; EDUARDO D. PRIETO; BARANDIARAN, ALDANA

KUMAMOTO

Simposio; ISA KUMAMOTO: The XVIth International Symposium on Amyloidosis; 2018

International Society of Amyloidosis

INTRODUCTION: The cascade of molecular events leading to amyloidosis is variable. Either the precursor monomer, the oligomeric or final fibrillar protein conformations were suggested as key to determine the pathology1. Human apolipoprotein A-I (apoA-I)-derived amyloidosis is poorly known. Either a non hereditary form may occur in which wild-type (Wt) protein deposits in atherosclerotic plaques or as hereditary pathology with apoA-I variants deposited causing multiple organ failure. A number of naturally occurring mutations of this protein have been associated with hereditary amyloidoses. Our previous studies strongly suggest that changes in the microenvironment could elicit protein misfolding and tissue deposition2. Among other components of the extracellular matrix (ECM), glycoproteins and glycosaminoglycans (GAGs) have been strongly associated to the retention or misfolding of different proteins inducing the formation of deposits in amyloid diseases3. Here we got deep insight into the mechanisms that could elicit apoA-I mutants pathology. MATERIALS & METHODS:The expression and purification of protein variants (Wt, G26R, W50R, L60R, R173P), 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 HUVEC cells cultures were performed under controlled CO2 atmosphere. Proteoglycans expression was analyzed by RT-PCR and western blot. Binding to GAGs was followed by electrophoresis and by fluorescence associated to polymeric matrices. RESULTS:Monomeric conformations (Gly26Arg) induce 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 that the Trp50Arg. Oligomeric conformations could be attained 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 glycosamine glycans and mediated by negative charges of sulfonate groups. In addition, Wt induces 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 the specific interaction of different variants with macrophages may contribute to cellular stress and toxicity in hereditary amyloidosis. An specific interaction of some mutants with components of the extracellular matrix could not only increase their aggregation but in addition 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, S.T., Prieto, E.D., Vela, M.E., Ríos, J.L., Tricerri, M.A., Rimoldi, O.J. 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., Curto, L.M., Delfino, J.M., Ramella, N.A., Tricerri, M.A. Amyloidogenic propensity of a natural variant of human apolipoprotein A-I: stability and interaction with ligands. PLoS One 2015; 10 : e0124946.