C-terminal sequence of amyloid-resistant type F apolipoprotein A-II inhibits amyloid fibril formation of apolipoprotein A-II in mice

In murine senile amyloidosis, misfolded serum apolipoprotein (apo) A-II deposits as amyloid fibrils (AApoAII) in a process associated with aging. Mouse strains carrying type C apoA-II (APOA2C) protein exhibit a high incidence of severe systemic amyloidosis. Previously, we showed that N- and C-terminal sequences of apoA-II protein are critical for polymerization into amyloid fibrils in vitro. Here, we demonstrate that congenic mouse strains carrying type F apoA-II (APOA2F) protein, which contains four amino acid substitutions in the amyloidogenic regions of APOA2C, were absolutely resistant to amyloidosis, even after induction of amyloidosis by injection of AApoAII. In vitro fibril formation tests showed that N- and C-terminal APOA2F peptides did not polymerize into amyloid fibrils. Moreover, a C-terminal APOA2F peptide was a strong inhibitor of nucleation and extension of amyloid fibrils during polymerization. Importantly, after the induction of amyloidosis, we succeeded in suppressing amyloid deposition in senile amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We suggest that the C-terminal APOA2F peptide might inhibit further extension of amyloid fibrils by blocking the active ends of nuclei (seeds). We present a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro and believe that this system will be useful for the development of novel therapies.Amyloidosis refers to a group of protein structural disorders characterized by the extracellular deposits of insoluble amyloid fibrils resulting from abnormal conformational changes (1–5). Amyloid fibrils have a characteristic ultrastructural appearance and a β-pleated sheet core structure that consists of full-length proteins and/or fragments of either WT or mutant proteins found in familial diseases (2, 6–8). In humans, 28 amyloidogenic proteins have been identified. They are associated with prominent diseases such as Alzheimer’s disease, hemodialysis-associated amyloidosis, and familial amyloid polyneuropathy (2, 9, 10). To develop a therapeutic strategy for these disorders, it is essential to understand the mechanisms of amyloid fibril formation. Currently, the molecular and biological mechanisms that convert proteins into amyloid fibrils in vivo and in vitro remain largely unknown.Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in human and mouse plasma high-density lipoproteins (HDLs) (11) and the most important protein associated with murine senile amyloidosis because it is the precursor of amyloid fibrils (AApoAII) (12–15). Seven alleles of the apoA-II gene have been found among inbred strains of mice, with polymorphisms in 15 nucleotide positions comprising eight amino acid positions (16). Each inbred laboratory mouse strain has a single type apoA-II protein, and the pathological findings of senile amyloidosis in strains with type A, B, or C apoA-II (APOA2A, APOA2B, or APOA2C, respectively) have been investigated (13, 17, 18). C57BL/6J, ICR, and DBA/2 strains have APOA2A and exhibit a moderate incidence of mild amyloid deposits with aging (19, 20). BALB/c, C3H/He, N2B, 129/SV, and SAMR1 strains have APOA2B and exhibit a low incidence of slight amyloid deposits with aging. In contrast, the SAMP1 strain has APOA2C and spontaneously exhibits a high incidence of severe systemic amyloid deposits with aging (20–22). We previously reported a unique mechanism in which N- and C-terminal peptides of apoA-II protein associated into amyloid fibrils in vitro (23) according to the nucleation-dependent polymerization model, which can explain the general mechanisms of amyloid fibril formation (24–28). The 11-residue amino acid sequence from positions 6–16 in the N terminus of apoA-II protein is critical for polymerization into amyloid fibrils. The 18-residue amino acid sequence from positions 48–65 in the C terminus of apoA-II is also necessary for nucleation, but not for the extension phase. Both sequences are common, and there is no substitution among APOA2A, APOA2B, and APOA2C (Fig. 1).Open in a separate windowFig. 1.Amino acid sequences of mouse apoA-II and synthetic partial peptides. For types A, B, and C apoA-II proteins (APOA2A, APOA2B, and APOA2C, respectively), the two amino acid sequences indicated in the red-colored boxes at positions 6–16 at the N terminus and 48–65 at the C terminus are the essential and common sequences required for amyloid fibril formation (23). Synthetic partial peptides were used to evaluate polymerization into amyloid fibrils in vitro and suppression against amyloid deposition in mice. The bold and blue-colored letters at positions 9, 16, 54, and 62 indicate the four variant amino acids in the core sequences for types A/B/C and F apoA-II proteins. Peptides containing orange letters represent substitutions of the a48/65(N62K) peptide.We hypothesized that some amino acid substitutions in these N- and C-terminal amyloidogenic sequences of apoA-II might inhibit the polymerization of apoA-II into amyloid fibrils. In that regard, type F apoA-II (APOA2F) contains four substitutions in the N- and C-terminal peptides relative to APOA2C (16) (Fig. 1). In this study, we evaluated the in vivo incidence of amyloidosis in mice having APOA2F and compared it with those in mice having APOA2A or APOA2C. We also analyzed the ability of APOA2F peptides to polymerize into amyloid fibrils in vitro. In previous studies, we found that injection of a very small amount of AApoAII amyloid fibrils markedly accelerated amyloid deposition (13–15). We demonstrated that mice with APOA2F were absolutely resistant against senile amyloidosis, even after induction of amyloidosis by injection with type C AApoAII fibrils. Thus, we have succeeded in suppressing amyloid deposits in amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We thus demonstrate that the C-terminal sequence of APOA2F is an important inhibitor of polymerization into amyloid fibrils in vitro and in vivo. These findings provide a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro.