23Since the early description of different human apolipoprotein A-I variants 24 associated to amyloidosis, the reason that determines its deposition inducing organ 25 failure has been under research. To shed light into the events associated to protein 26 aggregation, we studied the effect of the structural perturbations induced by the 27 replacement of a Leucine in position 60 by an Arginine as it occurs in the natural 28 amyloidogenic variant (L60R). Circular dichroism, intrinsic fluorescence measurements 29 and assays of binding to ligands indicate that L60R is more unstable, more sensitive to 30 proteolysis and interacts with sodium dodecyl sulfate (a model of negative lipids) more 31 than the protein with the native sequence and other natural variant tested, involving a 32 replacement of a Trytophan by and Arginine in the amino acid 50 (W50R). In addition, 33 the small structural rearrangement observed under physiological pH leads to the release 34 of tumor necrosis factor α and interleukin-1β from a model of macrophages. Our results 35 strongly suggest that the chronic disease may be a consequence of the loss in the native 36 conformation which alters the equilibrium among native and cytotoxic proteins 37 conformation. 38 39 40 41 42 Human apolipoprotein A-I (apoA-I) is the main protein associated to high 43 density lipoproteins (HDL). It is synthesized in liver and intestine and both the liver and 44 the kidney are the major sites of its catabolism. Its multiple functions as lipid transport, 45 endothelial homeostasis and inhibition of inflammatory pathways [1][2][3] could 46 however be counter balanced by the presence of single point mutations which could, by 47 not yet completely known pathways, induce its misfunction, increased clearance rate or 48 its tendency to aggregate [4][5]. Hereditary apoA-I amyloidosis has been described 49 since 1969, and is characterized by specific deposits of natural variant proteins within 50 organs, in a pattern which is dependent on the mutation in the protein sequence. From 51 the more than 20 known natural variants, almost half result from substitutions between 52 amino acids 26 and 107, and involve with different severity hepatic and kidney failure.53 Instead, a "hot spot" affecting residues 173-178 was described, in which variants are 54 especially associated to cardiac, skin and testis damage. The reasons for the different 55 deposit patterns are still unknown but may be the consequence of long term seeding of 56 misfolded proteins that yield a final fibrillar conformation. 57 The first identified apoA-I natural mutant was the result of a substitution of a 58 Glycine by an Arginine in position 26 from the native sequence (Gly26Arg, in the 59 abbreviated nomenclature G26R), inducing in patients peripheral neuropathy, peptic 60 ulcer, and nephropathy [6]. The next mutations described in the N terminus of apoA-I 61 were Trp50Arg (W50R) [7] and Leu60Arg (L60R) [8]. These variants show similarities 62 with G26R: in each, a neutral residue is replaced by an Arginine thu...