Projects / Programmes
Molecular mechanism of the recognition of double stranded viral RNA mediated by TLR3
| Code |
Science |
Field |
Subfield |
| 3.01.00 |
Medical sciences |
Microbiology and immunology |
|
| Code |
Science |
Field |
| B230 |
Biomedical sciences |
Microbiology, bacteriology, virology, mycology |
Toll like receptor 3; innate immunity, double-stranded RNA; viral infections; RNA silencing; molecular modeling; recognition site
Researchers (6)
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0104 |
National Institute of Chemistry |
Ljubljana |
5051592000 |
21,007 |
Abstract
Innate immune response is an essential component of our defense against the pathogenic microorganisms. Recognition of the molecules characteristic for pathogenic microorganisms (PAMPs) such as lipopolysaccharide, flagellin or viral dsRNA proceeds through binding to the ectodomain of Toll-like receptors. TLR3 is activated by the double-stranded RNA, which is characteristic for viruses and induces transcription of type I interferons.
Current model of TLR3 activation proposes that the dsRNA recognition proceeds through a binding site around the residues H539 and N541 at the C-terminal segment of TLR3 ectodomain. This model is not satisfactory since TLR3 should in this case also be activated by endogenous short siRNA molecules.
We propose in our hypothesis that the ectodomain of TLR3 contains the secondary binding site for the dsRNA, which is located at the conserved N-terminal segment of the ecto domain. According to our model dsRNA should span the distance between the two sites to cause activation, which like a “molecular ruler” elegantly eliminates the activation by short segments of dsRNA, which are shorter than the threshold of approximately 35 bp. Our molecular modeling of TLR3 ectodomain docking to the dsRNA identified the residues which we propose to be essential for the activation. This secondary binding site has similar structure as the site at the C-terminal segment, which would be expected due to the symmetric dimeric ligand.
We will test this hypothesis by preparing several point mutants of TLR3 that should inactivate the binding site and by assaying the activation of TLR3-transfected HEK293 cells by poly(I:C) and viral dsRNA sequences. Direct binding of dsRNA to the ectodomain of TLR3 will be tested and correct localization of EGFP-fused TLR3 mutants will be performed with confocal fluorescence microscopy. Detailed information on the mechanism of TLR3 recognition will represent the milestone for this area of research, since it will represent the first molecular characterization of the principle of TLR recognition and the likely common motif for the recognition of other TLRs, which recognize nucleic acids (TLR7, 8, 9).
Our project will provide the explanation for the selectivity of TLR3 for dsRNA including size and sequence requirements. This knowledge will provide the possibility to engineer better agonists and antagonists, which can be used as vaccine adjuvants, anticancer drugs (similar as imiquimod) or anti-inflammatory agents for treatment of diseases such as arthritis, systemic lupus erythematosis or atherosclerosis.
Significance for science
The project contributed significantly to the development of biological sciences, specifically with findings in the field of viral dsRNA recognition by one of the TLR receptors of innate immunity. This is important for understanding the immune response and defense against viral infections, which are one of the leading causes for modern life health problems. The biochemical basis of interactions between the dsRNA ligand and the TLR3 ectodomain will also help in exploring activation mechanisms of other TLRs that detect nucleic acids. Our findings have medical and biotechnological importance due to their possible contribution to the design of vaccines against viral infections and other diseases where the TLR3 ligand has an adjuvant function for priming of the specific immune response. The novel use of transferrin-poly-L-lysine conjugates for targeted delivery of TLR3 and TLR9 ligands will have a positive impact on finding new ways for cancer treatment. The study is interdisciplinary. It combines biochemical knowledge about intermolecular interactions with the knowledge about nucleic acid structure and thus has biotechnological and medical relevance. It also contributes to the pool of knowledge in the field of innate immunity, particularly in the field of pathogen-detection mechanisms. The results are thereby particularly important in the field of medicine with a significant impact on improving human health. The in-depth knowledge we have gained from the study will lead to better design of agonists and antagonists, which can be useful as a vaccines or cures for cancer or anti-inflammatory agents for treatment of arthritis, systemic lupus erythematosus (SLE), atherosclerosis, autoimmune encephalitis and others.
The project had also a significant educational role for PhD and graduate students, who during their work on segments of the study were able to gain very valuable knowledge in scientific methodology and laboratory practice.
Significance for the country
In our country our laboratory is a pioneer in the field of innate immunity, molecular-structure and interaction studies. We provide an important pool of knowledge for future generations of Slovenian researchers. Through training of graduate and PhD students we contribute importantly to the quality and world-wide visibility of Slovenian research. By attending courses and conferences we have passed on our findings and knowledge to a broader professional public. The knowledge we gained with our study is key to better understanding of molecular mechanism of TLR3 activation by viral dsRNA. Recognition of dsRNA by TLR3 is essential for the anti-viral innate immune response and important for priming of acquired immunity. We investigated the role of positively charged amino acid residues near the N-terminus of the TLR3 ectodomain and found they constitute a second dsRNA-binding site. We also found out that the two binding sites interact only with the phosphate backbone and that the distance between them corresponds exactly to the length of two turns of A-type dsRNA. This also explains the conformational hypothesis, which states that the only form of dsRNA to activate TLR3 is the conformational type A. We have demonstrated that TLR3 with the distance between the two binding sites accurately distinguishes dsRNA from other nucleic acids, and thus functions as a molecular ruler to separate self and non-self nucleic acids. The results of our study were published in the high profile scientific journal Nature Structural and Molecular Biology and also presented at a number of conferences with international participation. By those means we represented the high level of Slovenian science and contributed to its world-wide recognition. The results of the research project are also of economic importance, of interest to biotechnology and pharmaceutical industry and can help to improve treatment of many infectious diseases.
Most important scientific results
Annual report
2008,
2009,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2008,
2009,
final report,
complete report on dLib.si
