Revised Manuscript Received May 21, 1992 abstract: Amyloid diseases are caused by the self-assembly of a given proteininto an insoluble cross-jS-sheet quaternary structural form which is pathogenic. An understanding of the biochemical mechanism of amyloid fibril formation should prove useful in understanding amyloid disease. Toward this end, a procedure for the conversion of the amyloidogenic protein transthyretin into amyloid fibrils under conditions which mimic the acidic environment of a lysosome has been developed. Association of a structured transthyretin denaturation intermediate is sufficient for amyloid fibril formation in vitro. The rate offibril formation is pH dependent with significant rates being observed at pHs accessible within the lysosome (3.6-4.8). Far-UV CD spectroscopic studies suggest that transthyretin retains its secondary structural features at pHs where fibrils are formed. Near-UV CD studies demonstrate that transthyretin has retained the majority of its tertiary structure during fibril formation as well. Near-UV CD analysis in combination with glutaraldehyde cross-linking studies suggests that a pH-mediated tetramer to monomer transition is operative in the pH range where fibril formation occurs. The rate of fibril formation decreases markedly at pHs below pH 3.6, consistent with denaturation to a monomeric TTR intermediate which has lost its native tertiary structure and capability to form fibrils. It is difficult to specify with certainty which quaternary structural form of transthyretin is the amyloidogenic intermediate at this time. These difficulties arise because the maximal rate offibril formation occurs at pH 3.6 where tetramer, traces of dimer, and significant amounts of monomer are observed. Aromatic UV analysis demonstrates the presence of a subtle tetramer rearrangement which is centered around pH 5.3, indicating that it is definitely not the native tetramer that associates into amyloid fibrils. Spectroscopicstudies and cross-linking experiments are consistent with either a rearranged tetramer or a monomeric intermediate as the amyloid precursor. These studies demonstrate that TTR can self-assemble into transthyretin amyloid fibrils under acidic conditions similar to those found in a lysosome, supporting the feasibility of lysosomal and/or endosomal involvement inamyloid disease.

Amyloid fibril formation refers to the abnormal self-assembly of a given protein into an insoluble cross-0-sheet quaternary structural form (Cohen et al., 1983; Stone, 1990; Kisilevsky, 1983; Castano & Frangione, 1988; Lansbury, 1992). Experimental evidence supports the cause and effect relationship between amyloid fibrils and amyloid disease; however, the pathogenic mechanism remains unclear (Benson & Wallace, 1989). Understanding how a normally soluble protein is transformed intofibrils is a critical part of understanding the mechanism of related amyloid diseases. The X-ray diffraction data for amyloid fibrils composed of different amyloidogenic proteins are nearly identical, suggesting that fibrils derived from different proteins have the same major structural features (ie, cross-/3-structure)(Glen-neretal., 1974). Thesimilarities (fibril morphology and nerve pathology) between the fibrils composed of the A4 polypeptide (responsible for Alzheimer’s disease) and transthyretin (TTR) 1 fibrils suggest that either would be acceptable choices to probe the biochemical mechanism of fibril formationin human