Projects / Programmes
Mechanism and diagnostics of prion replication
| Code |
Science |
Field |
Subfield |
| 3.03.00 |
Medical sciences |
Neurobiology |
|
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| B230 |
Biomedical sciences |
Microbiology, bacteriology, virology, mycology |
prions, conformation, replication, diagnostics, fluorescence, neuroblasoma, cell cultures
Researchers (9)
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
Transmissible spongiform encephalopaties (TSE), also called prion diseases are a unique type of diseases, where the infectious agent seems to be the changed conformation of the native protein, without of any participation of nucleic acids. In this project we intend to find an answer to one of the key questions in understanding prion diseases: what is the mechanism of conformational change in these transmissible diseases and to test the new approach of monitoring the conformational change of PrP in living cells. The inventive idea of this project is to restrict the ability of the murine PrP to adopt alternative conformation by the addition of disulfide bridges in selected parts of the structure. In this way we are going to challenge the prion hypothesis and discover which parts of the tertiary structure are converted during the pathological process and which remain unchanged. We plan to develop mutants, resistant to the pathologic conversion. We will determine the biophysical properties of the mutant proteins and determine their structure and dynamics with respect to differences with wild type PrP using high resolution NMR. The ability of mutants to support prion replication will be tested in murine neuroblastoma and PrP-knockout cell lines. Present TSE diagnostics is capable of identifying the disease at the late stage or post mortem, but having the ability to detect the disease at an early (asymptomatic) stage is necessary to stop transmission by transplantation, blood transfusion or food. Most of the current protocols for TSE determination are based on the detection of protease resistant PrP (PrPSc) at the late stage of the disease. Most sensitive are animal assays, which are time consuming and require the sacrifice of many animals. Cell-based tests in development, but the discrimination between the inoculated and nascent prion has not been possible up to now. We intend to develop the new method to following the conformational change of PrP. PrP will be tagged with a peptide (PrP-C4) to which fluorescent FlAsH reagent binds in living cells. Fluorescence of this complex depends on the protein conformation. Conformational change will be initially followed in isolated proteins and later in cell cultures, which should allow detection of prion infectivity in the inoculum. Results of this project will give new insight into the mechanism of conformational change of prion protein and protection against the TSE as well as the new platform for early stage TSE detection.
Significance for science
Despite extensive research in the field of transmissible spongiform enfephalopathies, there is still not much knowledge on the structure of pathological form of prion protein. Engineered disulfide bridges tethering selected segments of prion protein enabled us to define regions amenable to conversion. In contrast to the majority of proposed PrPSc models our results show that majority of secondary structure elements are conserved upon structural conversion. In vitro fibrilization results are further supported by cell culture models of prion disease. Defining the structure of the pathological form is important in design of possible therapies for these devastating diseases. We showed that curcumin, the active ingredient of the spice turmeric, binds specifically to pathological forms of prion protein but not to the normal protein. In addition to binding to structures with high content of beta secondary structure, curcumin also labeled a partially unfolded alpha helical form, an intermediate formed at acidic pH. This is the first report of natural non-toxic compound binding to partially unfolded alpha PrP. We also showed curcumin labels prion plaques in vCJD brain sections. In addition to its inhibitory action on formation of amyloid PrP, curcumin or its derivatives with increased stability might be useful for diagnostics of prion disease in living patients. During the project we also developed new reagents for the detection of PrP conversion. We report a new synthesis method for production of biarsenical reagents which bind specifically to tetracysteine motif. We introduced tetracysteine tag into mouse prion protein and developed protease-free protocol for detection of PrP conversion. We also showed specific labeling of tetracysteine-tagged PrP in cell culture.
Significance for the country
During the project we cooperated strongly with the best groups in the field of transmissible spongiformn encephalopathies in Europe. In addition to exchange of ideas our student visited the laboratory of dr. Ina Vorberg (TUM) where she learned how to work with cell culture models of prion disease and additionally how to prepare cell lines stably expressing gene of interest by viral transduction, which we plan to use in the future also in other fields of our research. With such experiments in mind we renovated and registered several laboratories at our department as biosafety level 2.
Most important scientific results
Annual report
2008,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2008,
final report,
complete report on dLib.si
