N-terminal mutants of human apolipoprotein A-I: structural perturbations associated to protein misfolding

Since the early description of different human apolipoprotein A-I variants associated to amyloidosis, the reason that determines its deposition inducing organ failure has been under research. To shed light into the events associated to protein aggregation, we studied the effect of the structural perturbations induced by the replacement of a Leucine in position 60 by an Arginine as it occurs in the natural amyloidogenic variant (L60R). Circular dichroism, intrinsic fluorescence measurements and assays of binding to ligands indicate that L60R is more unstable, more sensitive to proteolysis and interacts with sodium dodecyl sulfate (a model of negative lipids) more than the protein with the native sequence and other natural variant tested, involving a replacement of a Trytophan by and Arginine in the amino acid 50 (W50R). In addition, the small structural rearrangement observed under physiological pH leads to the release of tumor necrosis factor α and interleukin-lβ from a model of macrophages. Our results strongly suggest that the chronic disease may be a consequence of the loss in the native conformation which alters the equilibrium among native and cytotoxic proteins conformation.


Introduction
As a difference from other hereditary amyloidosis, in which renal failure occurs 66 mainly due to glomerular protein deposits [9][10], apoA-I associated disease is mostly 67 characterized by amyloid retention in the medullary interstitium and/or vasculature, 68 which is probably a reason for its misdiagnosis [11] [12]. Only rare exceptions were 69 observed within a few mutants including W50R, in which amyloid deposits were found  In order to further remove this N terminal peptide, an Asp-Pro sequence 114 involving amino acid residues 2 and 3 was previously generated to allow specific   was similar to the Wt, a small but significant red shift (4 nm +/-1) was observed for 293 L60R (Table 1).   unfolded) model for L60R (Fig 1A), and thus we were not able to precisely calculate 319 this parameter. Interestingly, its total intrinsic fluorescence intensity is lower than the 320 one of the Wt, even though it keeps the four native Trp residues. As long as protein is 321 unfolded by titration with GmdCl the total intensity tends to equal that of the Wt (Inset 322 Fig 1A). helical structure is observed for the W50R and L60R variants, respectively.

348
The comparison among the tertiary structures was analyzed by CD in the near 349 UV ( Fig 1D); spectra of both mutants preserve fine structure although the lower   . 386 We herein show (Fig 3), as it was previously demonstrated with the monomer ANS, that 387 W50R binds Bis-ANS with a relatively higher quantum yield than the Wt [17].  Instead, intensity of the Bis-ANS associated to L60R is lower indicating a lower 404 permeation or binding of this probe within the spatial protein arrangement.

405
Nevertheless, WMF of the Bis-ANS bound to L60R is similar to the probe associated to 406 the Wt (Fig 3 inset). In a different experiment, to evaluate whether the presence of the   (Fig 4). followed by scattering ( Fig 5A) and ThT fluorescence (Fig 5B). These data indicate that    Next, we compared the binding of the variants under study to this model of GAG 477 at pH 5.0. As shown, a higher scattering (Fig 5C) and ThT associated fluorescence (Fig   478   5D) in the case of W50R indicates its higher efficiency to bind to heparin as amyloid-479 like complexes under acid conditions while L60R behaves similar to Wt.

480
In order to answer whether the structural disruption induced by a change in the After 48 h at 37°C ThT was added to a 1:1 495 molar ratio to protein and relative fluorescence quantified as described in Figure 5D.

496
The symbol * denotes difference with respect to Wt at P<0.05.  500 It is clear that the conformational flexibility could alter the equilibrium between 501 function and toxicity, and thus we set out to test whether the structural shift detected in 502 the N terminus of L60R could affect the protein interaction with a model of membrane.

503
It is well known that the analysis of MLV clearance at the lipid transition temperature is 504 a traditional functional parameter to test efficiency of apoA-I for lipids solubilization. the Arg (Fig 9) could bring this amino acid more exposed, as it results from a higher 607 binding to SDS which is not detected for the other amyloidogenic mutant tested in this 608 work ( Fig 6); moreover, this structural disorder should allow the permeation of 609 proteases, as it is shown here. Although trypsin is not a physiological protease of apoA-610 I in circulation, it helps to get insight into protein structure. In order to compare trypsin 611 induced proteolysis, we analyzed by the expassy software (www.expasy.org) the 612 predicted sites to be substrate of this enzyme. Arg in position 60 should separate only 613 one amino acid from a predicted fragment of three residues (59-61). Nevertheless, a 614 higher efficiency of this variant´s processing is observed (Fig 2); the same tendency is 615 detected under this experimental design by using MMP-12. Altogether, the 616 rearrangement of the Trp environment in the L60R mutant, the higher accessibility to 617 proteases and the lower CD spectral signal at 255-280 nm respect to the Wt agree with a 618 spatial rearrangement with a decreased stability and an increase in protein flexibility.

619
The increased susceptibility to proteases could explain the appearance of a 10 kDa The positive charge of the Arg may thus perturb the hydrophobic bottom cluster.

642
The mutation in position 60 should in addition disrupt a Leu zipper stabilized with other 643 Leu residues in the same molecule or with a second molecule in the native dimer [47].

644
In our hands the shift among dimer and monomer conformation was not dramatically 645 modified (S. Fig 1). Moreover, this effect is not drastic enough to decrease the 646 efficiency to solubilize neutral lipids (Fig 7) as it is the case with W50R and G26R