Filters
cris logo
-
New search Filters
Select from list
Displayed from Show more
You have reached the maximum number of displayed search results.
All results displayed
No results are available for the search performed
Displayed from
You have reached the maximum number of displayed search results.
All results displayed
Loading results
Obtaining statistical data

Projects / Programmes source: ARIS

The molecular mechanism of single stranded DNA signaling via intracellular Toll-like receptor 9

Edit
Research activity

Code Science Field Subfield
3.01.00  Medical sciences  Microbiology and immunology   

Code Science Field
B500  Biomedical sciences  Immunology, serology, transplantation 

Code Science Field
3.01  Medical and Health Sciences  Basic medicine 
Keywords
Toll like receptor TLR9, nonmethylated CpG containing single stranded DNA, innate immunity, proteolytic cleavag,
Evaluation (rules)
source: COBISS
Evaluation of bibliographic research performance indicators according to ARIS methodology
Citations Citations for bibliographic records in COBIB.SI that are linked to records in citation databases
source: WoS source: Scopus source: COBISS
Researchers (13)
no. Code Name and surname Research area Role Period No. of publications
1.  14360  PhD Mojca Benčina  Biotechnology  Head  2014 - 2017  392 
2.  35277  PhD Tina Fink  Biochemistry and molecular biology  Technical associate  2017  25 
3.  34529  PhD Vida Forstnerič  Biotechnology  Junior researcher  2014 - 2015  40 
4.  17915  PhD Helena Gradišar  Biotechnology  Researcher  2014 - 2017  130 
5.  23563  PhD Iva Hafner Bratkovič  Neurobiology  Researcher  2014 - 2017  210 
6.  10412  PhD Simon Horvat  Biotechnical sciences  Researcher  2014 - 2017  561 
7.  06628  PhD Roman Jerala  Biochemistry and molecular biology  Researcher  2014 - 2017  1,189 
8.  34069  PhD Duško Lainšček  Biotechnology  Junior researcher  2014 - 2015  148 
9.  34252  Tina Lebar  Biochemistry and molecular biology  Technical associate  2015 - 2017  67 
10.  32984  PhD Jan Lonzarić  Biochemistry and molecular biology  Technical associate  2014 - 2016  46 
11.  17917  PhD Andreja Majerle  Biotechnology  Researcher  2014 - 2017  92 
12.  29991  Alja Oblak  Biochemistry and molecular biology  Researcher  2014  60 
13.  32113  PhD Jelka Pohar  Biotechnology  Researcher  2015 - 2017  97 
Organisations (2)
no. Code Research organisation City Registration number No. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  20,957 
2.  2992  EN-FIST CENTRE OF EXCELLENCE  Ljubljana  3664830  2,835 
Abstract
Innate immune response is the first line of defense against the pathogenic microorganisms. A large range of distinct pathogen associated molecules, PAMPs are recognized by Toll-like receptors (TLRs). Activation of TLRs leads to the production of inflammatory mediators, which are required initiate immediate response and to efficiently prime the adaptive immune response. An excessive and uncontrolled response can lead to an acute or chronic inflammation. Some agonists of TLRs, such as nucleic acids, are also present in the host organism and can be released through tissue damage, activate TLRs and contribute to the development of autoimmune diseases such as systemic lupus erythematosus, inflammatory bowel disease, psoriasis, multiple sclerosis, rheumatoid arthritis or atherosclerosis. Molecular mechanism of the recognition of single stranded DNA (ssDNA) by TLR9 and the physiological role of proteolysis in TLR9 activation and signaling are poorly understood. TLR9 recognizes nonmethylated ssDNA motifs commonly found in bacteria and viruses. Synthetic oligonucleotides with CpG motif, CpG-ODNs recapitulate activation of TLR9 by microbial DNA. Point mutations at both the C- and N-terminal end of the TLR9 ectodomain inhibit TLR9 activation. Complex of TLR9 with CpG-ODN is colocalized in the endosomal vesicles. Inhibition of lysosomal proteolysis inhibits TLR9 activation by ODN and also prevents the proteolytic cleavage of TLR9 ectodomain. Although it has been proposed that proteolytic truncation of TLR9 ectodomain is essential for TLR9 activation, this is incompatible with the demonstrated role of the N-terminal residues and with activation of TLR9 when it is targeted towards the plasma membrane. We propose a model of TLR9 activation where multiple binding sites within the TLR9 ectodomain are required for the recognition of CpG-ODNs and receptor activation, and propose that the proteolysis during endocytosis is required to degrade the proteins that bind DNA, thereby making it available for binding to the TLR9 ectodomain. Proteolytic cleavage of TLR9 is therefore just a collateral event not required for the receptor activation. We aim to determine the recognition mechanism for ssDNA on the TLR9 ectodomain and the biochemical basis for the differentiation between TLR9 agonists and antagonists. We plan to test this hypothesis by investigating the effect of rationally selected point mutations within the TLR9 ectodomain on the receptor activation in mammalian cells and in direct binding assays. We plan to identify the origin of the species-specific differences of CpG-ODN recognition by the preparation and analysis of chimeric human-mouse receptors. Our unpublished results demonstrate that truncation of the mouse TLR9 at the proteolytic cleavage site almost completely and truncation of human receptor completely inhibits TLR9 activation, which is in agreement with the existence of the second binding site at the N-terminal site of ectodomain of TLR9. We plan to investigate our hypothesis on the role of proteolysis in TLR9 activation by in vitro reconstitution of endosomal interactions consisting of TLR9, ODNs, DNA-binding proteins, proteases and their inhibitors, where the direct interaction between ODNs and TLR9 will be monitored through AlphaScreen method. This hypothesis will also be investigated by the effect of proteolyticaly resistant DNA binding peptides composed of D-amino acids on TLR9 activation. We plan to perform a QSAR study of different synthetic ODNs with respect to their TLR9 stimulatory ability, which will further contribute to the understanding of the mechanism of TLR9 activation and will result in ODNs with improved therapeutic potential, which can be used as vaccine adjuvant or for therapeutic intervention in immune-mediated inflammatory disorders such as rheumatoid arthritis, systemic lupus erythematosus or psoriasis.
Significance for science
Objectives of the project are directed towards the study of molecular mechanism of receptor-ligand recognition for the development of new agonists and antagonists. Inadequate response of cells to infections could have serious consequences for human health. In contrast, an excessive immune response could initiate autoimmune diseases and chronic and acute inflammation. Therefore, understanding TLR activation as well as the mechanism of intracellular signal transduction are basic questions of immunology and medicine. Knowing the mechanism of molecular pattern recognition is indispensable for understanding the physiological processes in cells and organisms as well as a prerequisite for rational drug design. The research was designed to answer fundamental questions on structural biology and immunology. The determined new insights on TLR9 signaling will promote understanding of defense mechanisms against bacterial and viral infections and of initiating inflammatory diseases. We decifered poorly understood structural details of the ssDNA-TLR9 interaction. The structural mechanism of TLR9 will be a platform for construction of therapeutically relevant agonists, which we designed and tested. The results of the project on innate immunity activation generated new scientific knowledge and should be interesting for a broad scientific audience. Additionally, research provided new application of experimental methods, their adaptation and development for analysis of receptor-ligand interactions.
Significance for the country
During the last years the members of project group achieved excellent success in a competition of research projects among the most eminent academic competitors and thus contributed to the promotion of natural science to the general audience as well as promotion of Slovenia as a country with good science and education standards. The members of the research group promoted the public awareness of science via interviews for mass media. The aquired knowledge was available to experts as well as a wider audience via lectures and scientific publications. We intensified already established international collaborations by promoting short working exchange visits of researchers, that promoted networking and knowledge transfer. We have hosted successful exchanges of postgraduate students from foreign research centers and we increased short and longer term visits. Young researchers and undergraduate students were included in the research that increased the number of highly educated researchers. Experienced researchers were mentors for younger colleagues and graduate students. The researchers found excellent platform for carrying out cutting-edge research, sharing their experiences and knowledge as well as the possibility to train their soft skillsDue to the multidisciplinary nature of the project, the project offered great opportunities for scientists to expand their expertise. The innovative technologies and results were disseminated via publishing in internationally well established scientific journals, via presentations on conferences; both are important for relevance of the research and for the recognition of the research group and the country.
Most important scientific results Annual report 2014, 2015, final report
Most important socioeconomically and culturally relevant results Annual report 2014, 2015, final report
Confirmation required
Views history
Favourite