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|Title:||A study of the chaperone-like activity of thioredoxin||Authors:||Yu, Wang Grace||Keywords:||Hong Kong Polytechnic University -- Dissertations
|Issue Date:||2002||Publisher:||The Hong Kong Polytechnic University||Abstract:||Thioredoxin (12 kDa) is a ubiquitous and multifunctional protein widely distributed in prokaryotes and eukaryotes. It was first discovered as the hydrogen donor for ribonucleotide reductase in E. coli and later found with different functions in eukaryotes, such as regulation of transcriptional factors, apoptosis, and immunomodulation. These functions are primarily dependent on its oxidoreductase activity. Protein disulfide isomerase (PDI), a protein of the thioredoxin superfamily with thioredoxin domains, has been recently found to have chaperone functions. It would be crucial to study if thioredoxin itself possesses similar chaperone-like activity. In this study, we attempted to use porcine thioredoxin and its mutant C32S to explore the new function of thioredoxin in assisting protein refolding. Porcine wild-type thioredoxin, which was successfully cloned and purified to homogeneity, has been shown to possess oxidoreductase activity. Thioredoxin was used to regenerate protein inactivated by H₂O₂ and guanidinium hydrogen chloride. Mammalian glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the model enzyme for monitoring the processes, because there is only one cysteine residue at its active site, which cannot undergo disulfide exchange with thioredoxin. It was found that thioredoxin system (thioredoxin together with thioredoxin reductase and NADPH) was capable of regenerating H₂O₂-inactivated GAPDH. This showed that thioredoxin could restore the oxidative damaged enzyme. To further study the role of catalytic site of thioredoxin, thioredoxin was treated either by reduction or alkylation. Both the reduced and alkylated thioredoxin were found to help the renaturation of guanidinium HC1-denatured enzyme, showing that oxidoreductase activity was not involved in the protein renaturation process. Our findings suggest that thioredoxin assists the renaturation of denatured GAPDH to its active state in a way closely similar to the action of PDI. To further investigate the effect of the catalytic site on protein renaturation function, one of the catalytic positions was mutated to generate the C32S mutant thioredoxin. In the mutant, the thiol side chain of the amino acid was replaced by a more hydrophilic hydroxyl group at position 32.
The C32S thioredoxin was, however, incapable of renaturing guanidinium HC1-denatured enzyme. This could be resulted from the destabilization of thioredoxin fold. Circular dichroism analysis indicated that there were certain alterations in the structure of mutant thioredoxin. The increased exposure of β sheets to external environment might cause the destabilization of thioredoxin. This could be attributed to the change of the residue C32S in the active site. In general, the active site and its vicinity residues together form the hydrophobic pocket in order to interact with other proteins. An alteration on the pocket would affect the protein binding affinity, and hence failed to reactivate the denatured GAPDH as revealed in this study. Our notion is that the catalytic site of thioredoxin contributes to make up the stable structure of thioredoxin, and the intact thioredoxin fold can perform a similar function like PDI in renaturing denatured protein activity.
|Description:||vi, 118 leaves : ill. (some col.) ; 30 cm.
PolyU Library Call No.: [THS] LG51 .H577M ABCT 2002 Yu
|URI:||http://hdl.handle.net/10397/3667||Rights:||All rights reserved.|
|Appears in Collections:||Thesis|
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