Probing the mechanism of transthyretin amyloid fibril formation by site-directed mutagenesis and by in vitro inhibition of amyloid fibril formation /
The mechanism of transthyretin (TTR) amyloid fibril formation was studied by acid-induced denaturation which results in the formation of an intermediate that either undergoes further denaturation or self-assembles into amyloid fibrils. TTR acid denaturation involves a tetramer to monomer transition...
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1995.
|
| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=742145291&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The mechanism of transthyretin (TTR) amyloid fibril formation was studied by acid-induced denaturation which results in the formation of an intermediate that either undergoes further denaturation or self-assembles into amyloid fibrils. TTR acid denaturation involves a tetramer to monomer transition as discerned by biophysical studies of the acid denaturation pathway. The TTR amyloid fibril inhibitor, Z 3-14, has made it possible to monitor acid denaturation without competing amyloid fibril formation to probe the structure of the amyloidogenic intermediates. Site-directed mutagenesis using PCR methodology was used to introduce point mutations in the TTR sequence in order to express TTR variants implicated in familial amyloid polyneuropathy (FAP). Over thirty variants of TTR putatively cause the amyloid disease, FAP. We have characterized several variants and compared their biophysical properties to wild type TTR. A clear correlation between in vitro acid instability and in vivo amyloidogenicity has emerged suggesting that the quaternary instability of FAP variants is related to amyloid deposition in vivo. The order of acid stability in vitro of TTR variants which have been characterized, from most to least stable, is Thr- 1 19-Met > Val-30-Met / Thr- I 19-Met > wild type > Val-30-Met >> Leu- 55-Pro, correlating with the severity of amyloid disease associated with each variant. Wild type is less pathogenic than Val-30-Met which is less pathogenic than Leu-55-Pro. Methodology developed to effect amyloid fibril formation in vitro reveals that FAP variants can form amyloid around pH 5.5, which is the operating pH of the lysosome. Wild type TTR is nonamyloidogenic at this pH. We propose that a structured monomer of TTR is the amyloidogenic intermediate which can self-assemble via noncovalent interactions, and that FAP variants predispose TTR to amyloid deposition by decreasing the acid stability of the tetramer. |
|---|---|
| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xiv, 138 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |