Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro.
Amyloid diseases are caused by the self-assembly of a given protein into an insoluble cross-beta-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 of fibril 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 of fibril formation occurs at pH 3.6 where tetramer, traces of dimer, and significant amounts of monomer are observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Colon
W
W
Department of Chemistry, Texas A&M University, College Station 77843-3255.
Kelly
J W
JW
D000143:Acids / Q000494:pharmacology; D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D005976:Glutaral; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D011228:Prealbumin / Q000737:chemistry* / Q000187:drug effects; D011487:Protein Conformation; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet
D000143:Acids; D000682:Amyloid; D003432:Cross-Linking Reagents; D011228:Prealbumin; D005976:Glutaral