The aldehyde dehydrogenase AldA contributes to the hypochlorite defense and is redox-controlled by protein S-bacillithiolation in Staphylococcus aureus

Staphylococcus aureus produces bacillithiol (BSH) as major low molecular weight (LMW) thiol which functions in thiol-protection and redox-regulation by protein S-bacillithiolation under hypochlorite stress. The aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus. Here, we have studied the expression, function, redox regulation and structural changes of AldA of S. aureus. Transcription of aldA was previously shown to be regulated by the alternative sigma factor SigmaB. Northern blot analysis revealed SigmaB-independent induction of aldA transcription under formaldehyde, methylglyoxal, diamide and NaOCl stress. Deletion of aldA resulted in a NaOCl-sensitive phenotype in survival assays, suggesting an important role of AldA in the NaOCl stress defense. Purified AldA showed broad substrate specificity for oxidation of several aldehydes, including formaldehyde, methylglyoxal, acetaldehyde and glycol aldehyde. Thus, AldA could be involved in detoxification of aldehyde substrates that are elevated under NaOCl stress. Kinetic activity assays revealed that AldA is irreversibly inhibited under H2O2 treatment in vitro due to overoxidation of Cys279 in the absence of BSH. Pre-treatment of AldA with BSH prior to H2O2 exposure resulted in reversible AldA inactivation due to S-bacillithiolation as revealed by activity assays and BSH-specific Western blot analysis. Using molecular docking and molecular dynamic simulation, we further show that BSH occupies two different positions in the AldA active site depending on the AldA activation state. In conclusion, we show here that AldA is an important target for S-bacillithiolation in S. aureus that is up-regulated under NaOCl stress and functions in protection under hypochlorite stress

Current state-of-the-art of the research conducted in mapping protein cavities – binding sites of bioactive compounds, peptides or other proteins

Ο σκοπός της διπλωματικής εργασίας είναι η διερεύνηση και αποτύπωση των ερευνητικών μελετών που αφορούν στον χαρακτηρισμό μιας πρωτεϊνικής κοιλότητας – κέντρου πρόσδεσης βιοδραστικών ενώσεων, πεπτιδίων ή άλλων πρωτεϊνών. Στην παρούσα εργασία χρησιμοποιήθηκε η μέθοδος της βιβλιογραφικής επισκόπησης. Παρουσιάζονται τα κυριότερα ευρήματα προηγούμενων ερευνών που σχετίζονται με τη διαδικασία σχεδιασμού φαρμάκων και τον εντοπισμό φαρμακοφόρων με βάση ένα σύνολο προσδετών. Στη συνέχεια συγκρίνονται διαδικασίες επεξεργασίας και ανάλυσης της πρωτεϊνικής κοιλότητας προγενέστερων ερευνών με τη προσέγγιση που προτάθηκε από τους Παπαθανασίου και Φωτόπουλου το 2015. Αναδεικνύονται βασικά πλεονεκτήματα της προσέγγισης αυτής, όπως η εφαρμογή του αλγορίθμου πολυδιάστατη k-means ομαδοποίηση (multidimensional k-means clustering). Η εύρεση βιβλιογραφίας βασίστηκε σε αναζήτηση επιστημονικών άρθρων σε ξενόγλωσσα επιστημονικά περιοδικά, σε κεφάλαια βιβλίων και σε διάφορα άρθρα σε ηλεκτρονικούς ιστότοπους σχετικά με τον σχεδιασμό φαρμάκων και τις κοιλότητες που απαντώνται στις πρωτεΐνες. Στην παρούσα εργασία παρουσιάζονται εν συντομία εργαλεία που εντοπίστηκαν χρησιμοποιώντας λέξεις κλειδιά όπως για παράδειγμα δυναμική πρωτεϊνικής κοιλότητας, καταλυτικό κέντρο ενός ενζύμου, πρόσδεση, πρωτεϊνική θήκη κλπ. Στη συνέχεια συγκροτήθηκε κατάλογος με τα εργαλεία βιοπληροφορικής ανάλυσης που βρέθηκαν και ακολούθησε εκτενής αναφορά επιλεκτικά σε κάποια από αυτά. Κριτήριο επιλογής αυτών των εργαλείων αποτέλεσε η ημερομηνία δημοσίευσής τους, οι αλγόριθμοι και η μεθοδολογία που χρησιμοποιούν. Τα εργαλεία αυτά κατηγοριοποιήθηκαν με βάση τις λέξεις κλειδιά που χρησιμοποιήθηκαν για την εξόρυξη των δεδομένων από την βιβλιογραφία. Τέλος πραγματοποιήθηκε συγκριτική μελέτη αυτών αναδεικνύοντας τα πλεονεκτήματα και εστιάζοντας στην περαιτέρω αξιοποίησή τους.The aim of this thesis was to report on the current state-of-the-art of the research conducted concerning mapping of protein cavities with a potential function role as binding sites of bioactive compounds, peptides or other proteins. A literature review was performed with emphasis on the relevant tools developed during the last decade. In addition, the main research findings regarding drug design and druggable targets based on binding sites are presented. Processes performed in protein cavity detection and analysis, of previous research articles, are compared with the approach described by Anaxagoras Fotopoulos and Athanasios Papathanasiou (2015). The results showed that a competitive advantage of their approach is the multidimensional k-means algorithm for clustering. For the bibliographic review the scientific knowledgebase has been used, which includes international articles and journals, book chapters, as well as online articles regarding drug design and protein cavity. Search keywords such as protein cavity dynamics, catalytic sites of enzymes, protein pocket etc. were used to identify bioinformatics tools with text mining. A catalogue of the most recently developed tools is presented followed by a brief description of selected tools. The selection criteria imposed for preparing the catalogue and the detailed description included the publication date, as well as the algorithms and the methods they use. The tools were then classified according to the search keywords. The findings of this research are discussed, and the algorithms and methods they use are compared, highlighting the advantages of protein cavity detection

Discovery of Covalent Ligands via Noncovalent Docking by Dissecting Covalent Docking Based on a “Steric-Clashes Alleviating Receptor (SCAR)” Strategy

Covalent ligands modulating protein activities/signals have attracted unprecedented attention in recent years, but the insufficient understanding of their advantages in the early days of drug discovery has hindered their rational discovery and development. This also left us inadequate knowledge on the rational design of covalent ligands, e.g., how to balance the contribution from the covalent group and the noncovalent group, respectively. In this work, we dissected the noncovalent docking from covalent docking by creating SCARs (steric-clashes alleviating receptors). We showed that the SCAR method outperformed those specifically developed but more complicated covalent docking protocols. We furthermore provided a “proof-of-principle” example by implementing this method in the first high-throughput screening and discovery of novel covalent inhibitors of <i>S</i>-adenosylmethionine decarboxylase. This work demonstrated that noncovalent groups play a predeterminate role in the design of covalent ligands, and would be of great value in accelerating the discovery and development of covalent ligands