Ανάπτυξη και εφαρμογή εργαλείων βιοπληροφορικής για τη φυλογενετική ανάλυση και πρόβλεψη της δομής και ρύθμισης της λειτουργίας των πρωτεϊνών

Abstract

In the present thesis, the availability of an increasing number of complete or almostcomplete genomes, including those that were completed recently, enabled the study ofthe evolutionary history of three functionally important protein families: (a) the plant DNAmethyltransferases and (b) the eukaryotic RNA methyltransferases, which are enzymesthat catalyze the transfer of a methyl group to nucleotide sequences, as well as (c) thekallikrein-related peptidases or KLKs, which are trypsin- or chymotrypsin-like serineproteases. The evolutionary relationships of the already known and the novel proteins ofthe three families that were identified here were investigated using phylogenetic trees.Moreover, the secondary and tertiary structures of the homologous proteins wereanalyzed, as well as the structure of the protein-encoding genes, and diagnostic proteinmotifs were constructed based on the sequences of the three enzyme families. Ourresults led to suggestions pertaining to the biological function of the identified novelproteins. In particular, homologous plant DNA methyltransferases and novel eukaryoticRNA methyltransferases were identified in publicly accessible sequence databases.Detailed phylogenetic analysis of plant DNA methyltransferases identified four alreadyknown families and a novel subfamily in addition (Pavlopoulou and Kossida, 2007).Moreover, five distinct eukaryotic RNA methyltransferase subfamilies were identified;apart from the three already known subfamilies (NOP2, NCL1 and YNL022C), onenovel subfamily (RCMT9) and the FMU which hitherto was considered to existexclusively in prokaryotes were also identified (Pavlopoulou and Kossida, 2009).Furthermore, protein fingerprints were constructed from the generic family of RNAmethyltransferases (and the individual subfamilies), which were deposited in thePRINTS database (http://www.bioinf.man.ac.uk/dbbrowser/PRINTS). We developed the computational program RCMTHMM, in order to discriminate/identify eukaryotic RNAmethyltransferases from other proteins. The RCMTHMM program has been madepublicly available in the URL: http://www.bioacademy.gr/bioinformatics/RCMTHMM.Finally, the evolutionary history of KLKs was reconstructed. Kallikreins are importantproteolytic enzymes which are involved in proteolytic cascade pathways and theirdysregulated expression has been associated with major human pathologies(cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, skindiseases, different cancer types). The prominent feature of the kallikrein family is that itconsists of tandemly and uninterruptedly arrayed genes on a single locus at humanchromosome 19q13.3-13.4. This unique feature was used in order to identify novelKLKs and KLK-like genes/proteins. Previous studies on the evolution of kallikreins wererestricted to mammals and the emergence of the kallikrein genes was suggestedapproximately 150 million years ago. In the present study, homologous novel kallikreinprotein sequences were detected in silico in the genomes of various species. For thefirst time, novel KLK orthologues were identified in reptiles, aves and amphibia, whichallowed us to trace the evolutionary origin of kallikreins 330 million years ago. Inaddition, apart from the 15 already known KLK genes (KLK1-15), three novel memberswere identified (orphan Klks). All the defining structural features which are related to thecatalytic activity of KLKs were found to be conserved in the novel KLK proteinsequences. Of particular interest, the synteny of the KLK-encoding genes was analyzedand it was shown that these genes are co-localized in contiguous, uninterrupted clustersmaintaining the same orientation in all species under investigation. We suggest that aseries of gene duplication and mutation events gave rise to the family of KLK enzymesand KLKs have co-evolved with their specific substrates (Pavlopoulou et al., 2010).