Date of Award
PhD (Doctor of Philosophy)
Dr Caroline Jefferies
Lupus Erythematosus - Systemic, Tyrosine, Phosphorylation, Ribonucleoproteins
Patients suffering from Systemic Lupus Erythematous (SLE) have elevated type I interferon (IFN) levels which correlate with disease activity and severity. Ro52, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that turns off and limits type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs). To date, the mechanism regulating Ro52 activity has not been determined. In this study we have demonstrated that Ro52 is tyrosine phosphorylated following TLR stimulation, suggesting that Ro52 activity is regulated by this posttranslational modification. Using Netphos, we have identified two key tyrosines that are highly conserved between human and mouse, at residues 343 and 388, both of which have been mutated from tyrosine to phenylalanine (Y343F and Y388F). Another tyrosine residue at 393 that corresponds to serine in the murine form has also been mutated to phenylalanine (Y393F). We have identified that tyrosine phosphorylation of Ro52 occurs in the substrate interacting PRYISPRY domain and that tyrosine phosphorylation of tyr393 is essential for an interaction between Ro52 and IRF3 to occur. Our results have shown that tyrosine phosphotylation of tyr388 and tyr393 appears to be important for the activity of Ro52 in that the ability of Ro52 to inhibit IFN-P production is abrogated when tyr388 and tyr393 are not phosphorylated. Previous unpublished work in our lab has demonstrated the ability of Ro52 and Bruton's Tyrosine Kinase (Btk) to interact and here we have observed that in an in vitro nase assay Btk has a causal affect on the tyrosine phosphorylation of Ro52.
With respect to how these results pertain to disease, we have observed that whilst SLE patients have been shown to have an increased expression of Ro52, our findings herein suggest that this increase in Ro52 expression does not correlate with tyrosine phosphorylation levels and that SLE patients have a lower level of tyrosine phosphorylated Ro52. This supports our hypothesis that Ro52 activity is regulated by tyrosine phosphorylation and suggests that in SLE, activation of Ro52 may be absent or lowered thus contributing to the enhanced IFN levels observed in patients. Molecular modelling has been used to model the PRYISPRY domain of Ro52 when phosphorylated on Y388 and Y393 in order to design a strategy for enhancing the act'lvity of Ro52. This analysis shows that Y388 lies in a potential substrate binding pocket on Ro52 and our results suggest that a pharmacophore designed to fit into this site and mimic the tyrosine phosphorylation may act to lock Ro52 in an active conformation. A number of small molecules that mimic the phosphorylation of Y388 have been uncovered by in silico screening using such a pharmacophore and preliminary results have demonstrated their ability to increase Ro52 activity and hence increased inhibition of IFN-P promoter activity. This is the first report of tyrosine phosphorylation regulating the activity of Ro52 and the discovery of small molecules that mimic this event and which increase that activity could theoretically lead to the development of new small molecule therapeutics to treat the cause and not just the symptoms of SLE.
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Stacey KB. The role of tyrosine phosphorylation in the regulation of Ro52. [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2011.