Projects / Programmes
Molecular mechanism of negative regulation of TLR4 signaling by RP105 and its coreceptor MD-1
| Code |
Science |
Field |
Subfield |
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| Code |
Science |
Field |
| P000 |
Natural sciences and mathematics |
|
| Code |
Science |
Field |
| 3.05 |
Medical and Health Sciences |
Other medical sciences |
MD-1, RP105, MD-2, TLR4, LPS, innate immunity, negative regulation
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
25436 |
PhD Jožica Vašl |
Natural sciences and mathematics |
Head |
2010 - 2013 |
37 |
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
Gram-negative bacterial endotoxin (i.e lipopolysaccharide; LPS) is one of the most potent stimulants of the innate immune system, which is recognized by the TLR4/MD-2 complex. TLR4 signaling has to be precisely regulated since a suboptimal response may cause the organism to succumb to infection, while an excessive response may result in sepsis or autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and other disorders. Binding of LPS triggers formation of the active homodimer TLR4/MD-2, which initiates intracellular signal transmission via the intracellular TIR domain of TLR4. RP105 is a TLR homolog lacking an intracellular TIR domain. In parallel with TLR4, whose signaling depends on the secreted extracellular protein MD-2, the full function of RP105 is dependent on co-expression of the MD-2 homolog, MD-1. MD-1, in contrast to MD-2, does not bind LPS. RP105 is a specific inhibitor of TLR4 signaling in HEK 293 cells, a function conferred by its extracellular domain. RP105 and its helper molecule, MD-1, interact directly with the TLR4 signaling complex, inhibiting its ability to fully respond to the microbial ligand. Moreover, RP105-regulated TLR4 signaling in dendritic cells, as well as endotoxin responses in vivo, label RP105 as a physiological negative regulator of TLR4 responses.
The molecular model of the active TLR4/MD-2/LPS homodimer, which was suggested by our research team and afterwards confirmed by the determination of its crystal structure, provides us with an excellent base to investigate the molecular mechanism of negative regulation of TLR4 signaling by RP105 and its co-receptor MD-1. Briefly, the lipid A moiety of monomeric LPS binds to the hydrophobic pocket of MD-2, which causes dimerization of the TLR4 membrane receptor mediated by two binding sites on the extracellular domain of TLR4. Based on the homology between the RP105/MD-1 and TLR4/MD-2 pairs and known biochemical data, we propose a hypothesis that the mentioned complexes form the RP105/MD-1:TLR4/MD-2 heterodimer in which no dimerization of TIR domains or signaling can occur. On the basis of our assumptions that there is contact between MD-1-TLR4 and MD-2-RP105 similar to the geometry of the protein units of the active (TLR4/MD-2)2 tetramer, we proposed a model of interaction for the TLR4/MD-2:RP105/MD-1 heterodimer. Our model does not foresee direct interaction between MD-2 and MD-1, as was proposed by others. We also designed the hybrid between MD-2 and MD-1, which should bind to TLR4 while not to LPS. Our preliminary results of TLR4/MD-2 signaling inhibition by the MD-2/MD-1 hybrid are consistent with our proposed model of inhibition and follow the hypothesis which predicts that the hybrid would act as a TLR4 antagonist. The MD-2/MD-1 hybrid therefore represents a good basis for introducing a new type of endotoxin inhibitor.
To confirm our hypothesis, we will perform mutagenesis studies on RP105 and MD-1 which should lead us to identify the functional parts of the molecules involved in heterodimer formation. Sites for RP105 and MD-1 mutations will be selected using molecular modeling, similar as previously used for the TLR4/MD-2/LPS complex. The hybrid will be expressed in bacteria and in mammalian cell cultures. Activation and inhibition of TLR4 signaling will be monitored at the level of phosphorylation, transcription (RT-PCR) and translation (luciferase reporter system and detection of inflammatory cytokines by ELISA). Cellular localization and extracellular accumulation of the investigated proteins and their mutants will also be monitored. Spectroscopic and other biochemical methods will be used to characterize proteins and their interactions with each other. We have a wide array of techniques available and in the area of TLRs, particularly LPS receptors and inhibitors, we have a well-established scientific record. Studies on the mouse model will be performed in the laboratory of Prof. Dr. Karp from Cincinnati.
Significance for science
Bacterial infection continues to cause major disease problems despite the availability of antibiotics. Bacteria have a certain ability to mutate. Antibiotics kill bacteria that are susceptible to their action, but this leaves the field open for mutant strains to multiply even more. It is a case of survival of the fittest. The use of antibiotics actually encourages the development of the mutant, drug-resistant super-bacteria. It seems that doctors in hospitals and clinics around the world are losing the battle against an onslaught of continuously new drug-resistant bacterial infections. That is why a detailed understanding of the molecular mechanism of the response to bacteria is valuable. Our goal in the proposed project was to determine the detailed molecular mechanism of RP105/MD-1 acting as a physiological inhibitor of the TLR4 signaling pathway. The structural, biochemical and functional determination of the inhibitory TLR4/MD-2:RP105/MD-1 complex had broaden the knowledge of TLR4 immunology. Moreover, the results provided the method of using RP105/MD-1 and its analogs as a specific inhibitor of TLR4. Our interdisciplinary approach combining expertise of structural and cell biology represents an innovative and original contribution in this very competitive field of innate immunity. Moreover, in the long-term, our results could be used in the treatment of diseases marked by excessive or inappropriate inflammatory processes, including autoimmune diseases, pathological systemic responses to a variety of injures and localized and systemic infection processes such as sepsis and meningitis. Our group also has great expertise in the design of TLR receptor inhibitors, where we have already identified and patented compounds with the potential for treatment of inflammatory diseases (inhibitors of TLR4/MD-2 activation). Describing a role of the RP105/MD-1 receptor complex in inhibition of TLR4 signaling could broaden the application of these compounds and increase the possibilities to treat these disorders.
Significance for the country
Taking advantage of and increasing knowledge is the best way of sustainable development ensuring a high added value concomitant with environmental protection. The project was opened for collaboration with other research organizations in Slovenia as well as in Europe and worldwide. The results of research could contribute to health improvement which would lead to lowering of economic and social damage due to diseases. The proposed research project comprises the tools of modern structural and cell biology. The research was based on seeking new basic knowledge in the field of medicine with concomitant possibility for applicative research in industry. The results obtained will be published in scientific journal with an impact factor and was presented in the form of poster at international conference, what was the promotion of Slovenia in the world. Apart from professional meetings, we pased our knowledge to undergraduate and PhD students .
Most important scientific results
Annual report
2011,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Final report,
complete report on dLib.si
