Understanding transthyretin amyloid fibril formation by in vitro inhibition of amyloid fibril formation, protease sensitivity studies and alanine scanning mutagenesis /
Transthyretin (TTR) amyloid fibril formation can be induced
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1997.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=736584641&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Transthyretin (TTR) amyloid fibril formation can be induced by partial acid denaturation where soluble tetrameric TTR dissociates into a monomeric intermediate having an altered tertiary structure. In this dissertation, we demonstrate that the natural ligand of TTR, thyroxine and a non-natural ligand, 2,4,6, triiodophenol inhibit TTR fibril formation efficiently in vitro. Analytical ultracentrifugation and SDS-PAGE studies demonstrate that suppression of amyloid fibril formation is due to the stabilization of tetrameric TTR preventing the formation of the monomeric amyloidogenic intermediate under conditions where it would normally form. To further understand the mechanism of amyloid formation the structure of the monomeric amyloidogenic intermediate was evaluated by protease sensitivity studies. These studies demonstrate that there are regions in TTR that are protease labile under amyloid fibril forming conditions but are stable to proteolysis under physiological conditions where fibril formation is not possible. MALDI-TOF mass spectrometry and N-terminal sequencing of the fragments electroblotted onto a PVDF membrane have identified the C-strand-loop-D-strand and the F strand regions to be protease sensitive at acidic pH. The sensitivity of the C-strand-loop-D-strand region suggests that the region has become disordered and migrates away from the P-sheet core of the protein. This conformational change exposes strand A that can participate in intermolecular P- sheet interactions facilitating the self-assembly of TTR into a Phelical amyloid structure. Alanine scanning mutagenesis within the A strand and Hstrand were carried out to probe the critical interactions that facilitate the self assembly of the monomeric amyloidogenic intermediate. Results from this alanine scanning mutagenesis suggest single amino-acid replacement within TTR is not sufficient to generate TTR variants that are capable of forming a monomeric intermediate but do not self-assemble. In contrast, the stability of tetrameric TTR is highly sensitive that single amino acid replacements produced either highly stabilized variants that do not form amyloid or highly destabilized mutants that have increased amyloidogenecity. Finally, electrospray mass spectrometry of recombinant TTR has detected post- translational modifications in the protein that may mediate the neurotoxicity of amyloid fibrils. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xvi, 176 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 164-174. |