Search

Η μετα-μεταφραστική ρύθμιση αποτελεί ένα σημαντικό, γρήγορο και ενεργειακά αποδοτικό επίπεδο της κυτταρικής ρύθμισης, για το οποίο έχουν αναπτυχθεί πολλές ομικές τεχνολογίες μεγάλης κλίμακας. Η μετα-μεταφραστική τροποποίηση των αμινοξέων και ειδικότερα η πρωτεϊνική φωσφορυλίωση και μεθυλίωση έχουν κεντρικό ρόλο. Αυτή η διδακτορική διατριβή εστίασε σε δημοσιευμένα δεδομένα φωσφο-πρωτεωμικής και μεθυλ-πρωτεωμικής με σκοπό να αναπτύξει υπολογιστικά εργαλεία και βιοπληροφορικές μεθόδους/αναλύσεις που θα μπορούν να τα αναλύσουν και να εξάγουν γνώση για τις ιδιότητες αυτού του επιπέδου ρύθμισης. Κατά την διάρκεια αυτής της διατριβής, συλλέχθησαν, φιλτραρίστηκαν, αποθηκεύτηκαν και οργανώθηκαν δημοσιευμένα δεδομένα, με τη βοήθεια ενός υπολογιστικού εργαλείου διαχείρισης της Βιβλιογραφίας και μιας βάσης δεδομένων που ανέπτυξα. Επιπλέον, και άλλα ομικά και εξελικτικά δεδομένα ενσωματώθηκαν με σκοπό να πραγματοποιηθούν βιοπληροφορικές αναλύσεις σε βάθος. Στατιστικές αναλύσεις επέτρεψαν να εκτιμηθεί το σύνολο των πρωτεϊνών και των αμινοξέων ενός ευκαρυωτικού οργανισμού που υφίστανται φωσφορυλίωση. Mια εις βάθος βιοπληροφορική ανάλυση επέτρεψε να αποκαλυφθεί η σημασία της πρωτεϊνικής φωσφορυλίωσης στην ρύθμιση του κεντρικού μεταβολισμού του ζυμομύκητα S. cerevisiae όπως επίσης και οι θέσεις φωσφορυλίωσης με πιθανές βιοτεχνολογικές εφαρμογές, σε περίπτωση στοχευμένης μετάλλαξής τους. Επιπλέον, αναπτύχθηκαν νευρωνικά δίκτυα για την πρόβλεψη θέσεων φωσφορυλίωσης, μεθυλίωσης, καθώς επίσης και συνδυαστικών μοριακών διακοπτών. Τα εργαλεία και οι μέθοδοι/αναλύσεις που αναπτύχθηκαν/εφαρμόστηκαν κατά την πραγματοποίηση αυτής της διατριβής δύνανται να εξελιχθούν ώστε να επιτρέψουν την ενσωμάτωση επιπλέον μετα-μεταφραστικών τροποποιήσεων στο μέλλον.

Protein phosphorylation is the most frequent eukaryotic post-translational modification and can act as either a molecular switch or rheostat for protein functions. The deliberate manipulation of protein phosphorylation has great potential for regulating specific protein functions with surgical precision, rather than the gross effects gained by the over/underexpression or complete deletion of a protein-encoding gene. In order to assess the impact of phosphorylation on central metabolism, and thus its potential for biotechnological and medical exploitation, a compendium of highly confident protein phosphorylation sites (p-sites) for the model organism Saccharomyces cerevisiae has been analyzed together with two more datasets from the fungal pathogen Candida albicans. Our analysis highlights the global properties of the regulation of yeast central metabolism by protein phosphorylation, where almost half of the enzymes involved are subject to this sort of post-translational modification. These phosphorylated enzymes, compared to the nonphosphorylated ones, are more abundant, regulate more reactions, have more protein–protein interactions, and a higher fraction of them are ubiquitinated. The p-sites of metabolic enzymes are also more conserved than the background p-sites, and hundreds of them have the potential for regulating metabolite production. All this integrated information has allowed us to prioritize thousands of p-sites in terms of their potential phenotypic impact. This multi-source compendium should enable the design of future high-throughput (HTP) mutation studies to identify key molecular switches/rheostats for the manipulation of not only the metabolism of yeast, but also that of many other biotechnologically and medically important fungi and eukaryotes.

Nucleobase transporters are important for supplying the cell with purines and/or pyrimidines, for controlling the intracellular pool of nucleotides, and for obtaining exogenous nitrogen/carbon sources for metabolism. Nucleobase transporters are also evaluated as potential targets for antimicrobial therapies, since several pathogenic microorganisms rely on purine/pyrimidine salvage from their hosts. The majority of known nucleobase transporters belong to the evolutionarily conserved and ubiquitous nucleobase-ascorbate transporter/nucleobase-cation symporter-2 (NAT/NCS2) protein family. Based on a large-scale phylogenetic analysis that we performed on thousands of prokaryotic proteomes, we developed a webserver that can detect and distinguish this family of transporters from other homologous families that recognize different substrates. We can further categorize these transporters to certain evolutionary groups with distinct substrate preferences. The webserver scans whole proteomes and graphically displays which proteins are identified as NAT/NCS2, to which evolutionary groups and subgroups they belong to, and which conserved motifs they have. For key subgroups and motifs, the server displays annotated information from published crystal-structures and mutational studies pointing to key functional amino acids that may help experts assess the transport capability of the target sequences. The server is 100% accurate in detecting NAT/NCS2 family members. We also used the server to analyze 9,109 prokaryotic proteomes and identified Clostridia, Bacilli, β- and γ-Proteobacteria, Actinobacteria, and Fusobacteria as the taxa with the largest number of NAT/NCS2 transporters per proteome. An analysis of 120 representative eukaryotic proteomes also demonstrates the server's capability of correctly analyzing this major lineage, with plants emerging as the group with the highest number of NAT/NCS2 members per proteome.

Background Phosphorylation is the most frequent post-translational modification made to proteins and may regulate protein activity as either a molecular digital switch or a rheostat. Despite the cornucopia of high-throughput (HTP) phosphoproteomic data in the last decade, it remains unclear how many proteins are phosphorylated and how many phosphorylation sites (p-sites) can exist in total within a eukaryotic proteome. We present the first reliable estimates of the total number of phosphoproteins and p-sites for four eukaryotes (human, mouse, Arabidopsis, and yeast). Results In all, 187 HTP phosphoproteomic datasets were filtered, compiled, and studied along with two low-throughput (LTP) compendia. Estimates of the number of phosphoproteins and p-sites were inferred by two methods: Capture-Recapture, and fitting the saturation curve of cumulative redundant vs. cumulative non-redundant phosphoproteins/p-sites. Estimates were also adjusted for different levels of noise within the individual datasets and other confounding factors. We estimate that in total, 13 000, 11 000, and 3000 phosphoproteins and 230 000, 156 000, and 40 000 p-sites exist in human, mouse, and yeast, respectively, whereas estimates for Arabidopsis were not as reliable. Conclusions Most of the phosphoproteins have been discovered for human, mouse, and yeast, while the dataset for Arabidopsis is still far from complete. The datasets for p-sites are not as close to saturation as those for phosphoproteins. Integration of the LTP data suggests that current HTP phosphoproteomics appears to be capable of capturing 70 % to 95 % of total phosphoproteins, but only 40 % to 60 % of total p-sites.

Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of >2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived. Although AlaRS, GlyRS, LeuRS, IleRS, ValRS are the most stable members of the family, GluRS, LysRS and CysRS often have paralogs, whereas AsnRS, GlnRS, PylRS and SepRS are often absent from many genomes. In the course of this analysis, highly conserved protein motifs and domains within each of the AARS loci were identified and used to build a web-based computational tool for the genome-wide detection of AARS coding sequences. This is based on hidden Markov models (HMMs) and is available together with a cognate database that may be used for specific analyses. The bioinformatics tools that we have developed may also help to identify new antibiotic agents and targets using these essential enzymes. These tools also may help to identify organisms with alternative pathways that are involved in maintaining the fidelity of the genetic code.

During the last decade, there has been great progress in high-throughput (HTP) phosphoproteomics and hundreds or even thousands of phosphorylation sites (p-sites) can now be detected in a single experiment. This success is attributable to a combination of very sensitive Mass Spectrometry instruments, better phosphopeptide enrichment techniques and bioinformatics software that are capable of detecting peptides and localizing p-sites. These new technologies have opened up a whole new level of gene regulation to be studied, with great potential for therapeutics and synthetic biology. Nevertheless, many challenges remain to be resolved; these concern the biases and noise of these proteomic technologies, the biological noise that is present, as well as the incompleteness of the current datasets. Despite these problems, the datasets published so far appear to represent a good sample of a complete phosphoproteome of some organisms and are capable of revealing their major properties.