The ovarian tumour (OTU) domain present at the N-terminus of the nairoviral L protein has been shown to deconjugate ubiquitin and interferon-stimulated gene 15 protein (ISG 15) from cellular proteins, thereby interfering with innate immune cell signalling pathways. I have confirmed and extended these findings for the Dugbe OTU domain by showing that it has deubiquitinating and deISGylating activity and is able to effectively block the TNFα/NF-KB and interferon/JAK-STAT signalling pathways even at low doses. A larger OTU expression construct that included the downstream zinc finger domain had a reduced effect on ubiquitination and on the above-mentioned signalling pathways, but was equally effective at deISGylating cellular proteins. The effect of the OTU domain on ubiquitination, ISGylation and cell signalling was confirmed by generating point mutants of the catalytic site [C40A, H 151 A and a double mutant]. These mutations completely abolished the ability of the OTU domain to deubiquitinate and deISGylate cellular proteins. Unexpectedly, when used at higher doses, the C40A mutant still had a significant inhibitory effect on the TNFa/NF-lCB pathway, and all three mutants inhibited type 1 interferon action, suggesting that mutants can still block some cellular functions by binding to their target proteins even if they are no longer enzymatically active. This effect observed with the mutants was greatly reduced when the amount of each mutant OTU plasmid transfected was reduced by 10-fold; however the C40A mutant was still able to block type I interferon action even at low doses. Interestingly, infection of cells with the virus itself resulted in deubiquitination and delSGylation of cellular proteins, but had no effect on the cell signalling pathways.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:674918 |
| Date | January 2013 |
| Creators | Bakshi, Siddharth |
| Publisher | Ulster University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.1543 seconds