Final Activity Report Summary - PROTARC (Molecular prospecting for novel bioactivities and understanding cellular proteolysis: Studies on proteases and unfoldases from hyperthermophilic Archaea) Archaea's kingdom of life includes microorganisms growing in some of the most extreme environments on Earth. These extremophilic organisms produce enzymes that are functional under severe conditions and their discovery has had a great impact on the field of biocatalysis. The aim of the PROTARC project was the study of large protease, i.e. protein degrading enzymes, complexes from archaea, whose enzymology, structures and physiological roles were unknown. The objectives were to increase our knowledge regarding the proteolytic processes, which were crucial in the normal physiology of cells as well as in several pathological conditions. The project focussed on the investigation of proteases related to a new type of giant protease, TET, which was discovered and purified for the first time from halophilic, i.e. salt-loving, haloarcula in the host laboratory. There were four genes coding for TET-like proteins in the sequenced genome of the hyperthermophilic, i.e. high temperature-loving, and barophilic, pressure-loving, archaeon pyrococcus horikoshii. The substrates that these proteases could manipulate and their structures needed further research to elucidate the role of TET-like proteins in the cell. Moreover, these enzymes were intrinsically stable and active at high temperatures of around 90 degrees Celsius, hence they offered major biotechnological advantages. Additionally, hyperthermophilic enzymes could serve as model systems for use by biologists, chemists and physicists interested in understanding enzyme evolution, molecular mechanisms for protein thermostability and the upper temperature limit for enzyme function. Taking into account all these premises, the global objective was to assess the activities and the function of TET-like proteins from p. horikoshii by in vitro assays, including the study of the enzymatic and biophysical aspects of the complexes. These studies started by determining the structure and enzymatic properties of three hyperthermophilic proteins that were homologous to TET, namely PhTET1, 2 and 3. The obtained results showed that all three proteins formed dodecameric (12-subunit) tetrahedral complexes of around 450 KDa, and that PhTET1 also assembled in a 24-subunit octahedral complex of almost 900 KDa. The activity of the PhTET proteins was characterised and the results indicated differences between the three PhTETs. Therefore, it was likely that these complexes acted in a complementary manner to meet cell requirements. The fact that TET formed complex self-compartmentalised edifices suggested that they could have an important position in protein degradation routes. Stability studies performed on PhTET1 and PhTET3 showed that the structure of the complexes was maintained after prolonged incubation at high temperatures of 80 and 90 degrees Celsius, respectively. Moreover, PhTET3 activity increased with pressure and the particle structure was not affected under pressure of at least 3 000 bar even at 90 degrees Celsius. The knowledge gained through this research would be very useful in considering biotechnological applications of the TET enzymes. In fact, a possible biotechnological use, i.e. the utilisation of TET edifices as vectors for in vivo imaging and other medical purposes, such as drug carriers, was tested. PhTET1 was successfully marked with a fluorescent probe and injected in mouse. The in vivo distribution and the blood circulation time of the TET particles demonstrated that they were stable enough, i.e. detectable five days post-injection, and apparently non-toxic. Therefore, the TET particles were a promising option for creating multifunctional assembly platforms for use in medical imaging, as well as for in vivo transport and delivering of therapeutic agents. Finally, TET was used as a model for a new Nuclear magnetic resonance (NMR) methodological approach for the study of supramolecular assemblies. Those techniques were limited, by the time of the project completion, to the study of small protein complexes of less than 50 KDa. However, we obtained enough pure TET, specifically labelled on methyl groups, to observe most of the labels of the assembly and obtain high quality spectra. Consequently, these results opened a possibility for the future use of NMR techniques in structural studies of large biomolecules in solution.