Proteostasis in the endoplasmic reticulum (ER) is maintained, in part, through the activity of protein disulfide isomerase (PDI). This essential protein exhibits a modular domain arrangement of abb'xa'c that contributes to both its oxidoreductase and chaperone activities, with substrate binding primarily located in the b and b' domains and oxidoreductase activity located in the a and a' domains. During prolonged nitrosative stress conditions, PDI is post-translationally modified with nitric oxide at its cysteine residues in both the active site domains (a and a') and the substrate binding b' domain. This S-nitrosylation (SNO) event inactivates PDI activity by a mechanism thought to involve the reactive CGHC motifs in the a and a' domains. However, recent evidence suggests that cysteine 343 in the b' domain is stably S-nitrosylated and resistant to reversal compared to the active site cysteines. In addition, arginine 300 in the b' domain contributes to the redox-regulated conformational flexibility of PDI that allows it to act upon a wide range of substrates. Here, we used cholera toxin (CT) as a model substrate to examine the roles of C343 and R300 in PDI-substrate interactions. In the ER, PDI facilitates cholera intoxication by acting as a disaggregase to physically separate the enzymatically active CTA1 subunit from the rest of the holotoxin. The free CTA1 is then exported out of the ER to the cytosol where it alters cellular signaling through its ADP-ribosyltransferase activity. Using site-direct mutagenesis, we generated two PDI variants with single C343S or R300A substitutions. We then examined the effect of these mutations on PDI-CT interactions and the inactivation of PDI by S-nitrosylation. Although the R300A variant had a slightly altered secondary structure, neither C343S or R300A inhibited the binding or disassembly of CT by PDI. These results suggest a unique mechanism of action for PDI's disaggregase activity against CT. Current experiments are exploring if C343S is resistant to the inactivation of PDI's disaggregase activity that results from S-nitrosylation. This work also provides a possible molecular basis to understanding why SNO-PDI is linked to amyloid fibril formation in neurodegenerative diseases.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd2020-2742 |
| Date | 15 August 2023 |
| Creators | Vincent, Evie |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations, 2020- |
Page generated in 0.0174 seconds