Final Report Summary - ACTIVOXY (Oxygen activation in ribonucleotide reductase and multicopper oxidases proteins)
Through evolution, nature has engineered diverse molecules programmed to be the active components in 'key metabolic paths' of the oxygen metabolism in life. These molecules often contain copper and / or iron metal ions as operational engines; here, processes such as O2 binding and transport, reduction of O2 to peroxide coupled with oxidation of various types of substrates, or O2 detoxification take place. Understanding the functional factors that regulate the red-ox chemistry and hence the reactivity towards oxygen of the protein-caged metal ion(s) and or molecular cofactors were the research objectives of Dr Giorgio Zoppellaro biomedical / biophysical projects. Two main classes of proteins and their interaction with molecular oxygen were explored within the fellowship period (24 months) together with synthetic functional biomimics.
The first class of proteins utilises iron as metal cofactor (project 1, 1 - 24 months), while the second class contains copper centres as active sites both in the native proteins and in their synthetically engineered counterparts (project 2, 1 - 24 months). Both projects involved the extensive use of spectroscopy techniques but required further training of the fellow in biology and genomics. Dr Giorgio Zoppellaro, within the fellowship period, has been embedded and furthermore strongly contributed to expand the in-house international collaborations between host institution (Department of Molecular Biosciences, University of Oslo) and several leading groups from both Europe such as:
(1) Karolinska Institute, Sweden;
(2) University of Pavia, University of Padova and University of Milan, Italy;
(3) Grenoble High Magnetic Field Laboratory, CNRS, France;
and USA such as Stanford University and University of Rochester.
In the first project, Dr Giorgio Zoppellaro dissected the oxygen activation path in several iron proteins involved in the medical domain of genomic integrity (DNA synthesis, repair) or associated to electron transfer processes. These molecules included ribonucleotide reductases (project 1-RNR) from various sources, enzymes that catalyse the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides, the recently discovered ALKBH4 protein (project 1-ALKBH4), which is a member of the nine DNA encoded human homologue of AlkB (DNA repair enzyme), and a series of electron shuttling proteins, cytochrome c from bacterial sources (project 1-cyt c).
During the project 1-RNR, the fellow analysed RNR protein over-expressed from Epstein-Barr virus (EBV). The EBV viral agent belongs to the gamma subfamily of herpes viruses and represents one of the most common pathogenic viruses in humans worldwide. The virus may induce development of several diseases such as infectious mononucleosis, and is associated with neoplastic diseases, including lymphomas and carcinomas. The R2 subunit of viral RNR contains a tyrosyl radical which is generated within enzymatic turnover and controls the synthesis of the building blocks necessary for viral transfection. This tyrosyl radical specie featured unique electronic fingerprints, falling in between those observed in mammals (mouse R2) and bacteria (e.g. E. coli R2) and indicated that EBV R2 contains a more 'advanced / evolved' tyrosyl radical than those known in simple bacterial organisms, such as E. coli. Furthermore, its interaction with radical scavengers, such as hydroxy urea (HU) clinically used as inhibitors of RNR activity, and as anticancer drug since 1967, showed that the EBV active site has lower accessibility to the drug as compared to mouse RNR R2.
The importance of the work lies on the fact that the current pharmaceutical effort is devoted on the screening of small organic molecules that are thought to be potential inhibitors of herpes viral RNRs. Those can be used for the treatment of herpes viral infections by targeting the R2 tyrosyl radical site.
The first class of proteins utilises iron as metal cofactor (project 1, 1 - 24 months), while the second class contains copper centres as active sites both in the native proteins and in their synthetically engineered counterparts (project 2, 1 - 24 months). Both projects involved the extensive use of spectroscopy techniques but required further training of the fellow in biology and genomics. Dr Giorgio Zoppellaro, within the fellowship period, has been embedded and furthermore strongly contributed to expand the in-house international collaborations between host institution (Department of Molecular Biosciences, University of Oslo) and several leading groups from both Europe such as:
(1) Karolinska Institute, Sweden;
(2) University of Pavia, University of Padova and University of Milan, Italy;
(3) Grenoble High Magnetic Field Laboratory, CNRS, France;
and USA such as Stanford University and University of Rochester.
In the first project, Dr Giorgio Zoppellaro dissected the oxygen activation path in several iron proteins involved in the medical domain of genomic integrity (DNA synthesis, repair) or associated to electron transfer processes. These molecules included ribonucleotide reductases (project 1-RNR) from various sources, enzymes that catalyse the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides, the recently discovered ALKBH4 protein (project 1-ALKBH4), which is a member of the nine DNA encoded human homologue of AlkB (DNA repair enzyme), and a series of electron shuttling proteins, cytochrome c from bacterial sources (project 1-cyt c).
During the project 1-RNR, the fellow analysed RNR protein over-expressed from Epstein-Barr virus (EBV). The EBV viral agent belongs to the gamma subfamily of herpes viruses and represents one of the most common pathogenic viruses in humans worldwide. The virus may induce development of several diseases such as infectious mononucleosis, and is associated with neoplastic diseases, including lymphomas and carcinomas. The R2 subunit of viral RNR contains a tyrosyl radical which is generated within enzymatic turnover and controls the synthesis of the building blocks necessary for viral transfection. This tyrosyl radical specie featured unique electronic fingerprints, falling in between those observed in mammals (mouse R2) and bacteria (e.g. E. coli R2) and indicated that EBV R2 contains a more 'advanced / evolved' tyrosyl radical than those known in simple bacterial organisms, such as E. coli. Furthermore, its interaction with radical scavengers, such as hydroxy urea (HU) clinically used as inhibitors of RNR activity, and as anticancer drug since 1967, showed that the EBV active site has lower accessibility to the drug as compared to mouse RNR R2.
The importance of the work lies on the fact that the current pharmaceutical effort is devoted on the screening of small organic molecules that are thought to be potential inhibitors of herpes viral RNRs. Those can be used for the treatment of herpes viral infections by targeting the R2 tyrosyl radical site.