PhD SCIENCE REPORT Madeleine Hipp

MADELEINE HIPP

I will finish my PhD probably in 2010.

Abstract:
Toll-like receptors (TLRs) are important receptors of the innate immune system able to respond to molecular patterns borne by microbial and viral pathogens. TLR3, TLR7 and TLR9 form a subset of TLRs that are localized intracellularly and can recognize nucleic acids to initiate antiviral immune responses. Among these TLRs, TLR7 is triggered by viral or bacterial single stranded RNA (ssRNA) and mediates responses to RNA viruses. However, self-RNA can in some instances also activate TLR7, leading to the development of autoimmune disorders. The mechanisms that allow TLR7 triggering by viral ssRNA but normally prevent activation by self-ssRNA remain incompletely understood but might be achieved solely on the basis of intracellular localization, as proposed for self/foreign DNA discrimination by TLR9 (Barton et al., 2006).

We therefore attempted to mislocalize TLR7 to different cell compartments and correlate its localization with the ability to initiate signalling. We generated a chimeric TLR7/4 receptor by fusing the ectodomain of TLR7 to the transmembrane and cytosolic domains of TLR4. We then introduced the construct into the genome of different human cell lines using a lentiviral approach. Using confocal microscopy we showed that TLR7 could be successfully misdirected to the cell surface. Furthermore the cell surface expressed TLR7 remained functionally active. However, in a different set of experiments, we detected a longer and a shorter form of TLR7, the latter of which might correspond to a cleavage product. As it has recently been shown that proteolytic cleavage of TLR9 is a prerequisite for TLR9 signalling (Park et al. 2008, Ewald et al. 2008), it is possible that TLR7 cleavage in endosomes is also necessary for receptor activation.
Thus, the results of these experiments are consistent with the notion that the endosomal localization of TLR7 is critical to allow discrimination between self and foreign RNA and provide insights into the mechanisms controlling TLR7 dependent recognition of RNA viruses.

To further investigate whether TLR7 is becoming cleaved in the endolysosomal compartment we are planning to inhibit lysosomal acidification and also a broad range of lysosomal proteases. We are also planning to do pulse chase analysis to analyse the trafficking pathway of wild type TLR7 and the chimeric TLR7/4 molecules.

To address the question whether TLR7 forms homodimers we furthermore cloned constructs containing TLR7 fused to YFP or CFP, which we will use to perform Förster Resonance Energy Transfer.

created over 14 years ago (16 April 2010)    last modified over 13 years ago (28 September 2011)   [ RDF ]   [ RelFinder ]