Amyloid Formation under Complicated Conditions in Which ß2-Microglobulin Coexists with Its Proteolytic Fragments.

Amyloid formation in vivo occurs under complicated conditions in which various amyloidogenic and non-amyloidogenic components coexist, often under crowding. Controversy surrounds the role of additional components under complicated conditions. They have been suggested to accelerate amyloid formation because molecular crowding or interactions with additives increase effective concentrations and, thus, break the supersaturation of amyloidogenic proteins. On the other hand, cellular crowding conditions with various heterogeneous components may retard or prevent amyloid formation because they impede homologous amyloidogenic associations. To elucidate the roles of these additional components, we examined the amyloid formation of ß2-microglobulin (ß2m), a protein responsible for dialysis-related amyloidosis, with a simplified model system in which intact ß2m and its proteolytic peptides coexist. Among the nine proteolytic peptides of ß2m produced in vitro with lysyl endopeptidase, the 22-residue K3 peptide is highly amyloidogenic. The amyloid formation of the K3 peptide, which occurred with a lag time of 1 h at pH 2 and 37 °C, was significantly retarded by the coexistence of ß2m or a mixture of the proteolytic digests. To identify the sites of inhibitory interactions, we performed paramagnetic relaxation enhancement measurements using spin-labeled K3 and uniformly 15N-labeled ß2m with nuclear magnetic resonance detection. The results revealed that K3 interacted weakly with a broad cluster of the hydrophobic residues of ß2m, which accommodated the residues located in some distant sequence, leading to competitive inhibition. The results showed that relatively weak and broad interactions formed a nonproductive complex, implying a role for heterogeneous interactions under complicated conditions.