Structural studies of nucleic acids and proteins involved in nucleic acid recognition
Russo Krauss, Irene (2010) Structural studies of nucleic acids and proteins involved in nucleic acid recognition. [Tesi di dottorato] (Inedito)
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This PhD thesis focuses on the structural analysis of the protein-nucleic acid recognition. In particular the research work has been focalized on two different kinds of proteins and their nucleotide ligands. The first part concerns the structural characterization of complexes between human α-thrombin, a protein of physiological and pathological relevance, and two oligonucleotide aptamers (the so called thrombin binding aptamer and a modified version of it), which adopt a G-quadruplex fold. The high resolution crystal structures of these complexes open the door to the design of novel aptamers with improved antithrombotic activity, high stability and increased binding efficacy. The second part of the thesis, subdivided into more issues, globally deals with the study of proteins belonging to the ribonucleases family (RNases), whose cellular target is RNA. This is a class of enzymes whose functions are very wide and fall outside the limits of simple destruction of RNA molecules. A particular attention is devoted to ribonucleases that, thanks to their peculiar structural features of domain swapping, can exert antitumor activity. The results obtained through a combination of crystallographic and docking analyses have allowed to identify the structural determinants of antitumor activity in dimeric RNases and successfully convert non-cytotoxic RNase A in a dimeric cytotoxic mutant. Another topic is the characterization of the whole ribonuclease system in zebrafish organism. This is an ideal model system for vertebrate phylogenetic and evolutionary analyses and for biomedical studies. Structural determination by X-ray crystallography and homology modelling of these ribonucleases, endowed with angiogenic activity, has allowed understanding the molecular bases for their different properties and represents a good starting point to study evolution of a protein scaffold from ancestral organisms to contemporary mammals. Finally, the attention of the thesis moves to an immuno-RNase, a chimeric molecule containing a RNase moiety, endowed with specific cell-type cytotoxicity. This fully human fusion protein is highly selective toward tumour cells that overexpress the receptor ErbB2 and does not present the side effects of humanized antibodies in use for cancer treatment, such as cardiotoxicity. Since immuno-RNase properties depend on the specific bound region of ErbB2, a computational approach was used to identify its epitope. This epitope is a small ErbB2 region, different with respect to that recognized by immunoagents approved for cancer therapy. This finding on one hand could justify the lack of cardiotoxic effects, on the other hand suggests that this protein could be used in combination with other drugs to obtain more efficient antitumoral therapies.
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