The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24995号 / 医博第5029号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 椛島 健治, 教授 伊藤 能永 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The activation of the innate immune system is the first line of host defence against infection. Nucleic acids can potently stimulate this response and trigger a series of signalling cascades leading to cytokine production and the establishment of an inflammatory state. Mutations in genes encoding nucleases have been identified in patients with autoimmune diseases, including Aicardi-Goutières syndrome (AGS). This rare childhood inflammatory disorder is characterised by the presence of high levels of the antiviral cytokine interferon-α in the cerebrospinal fluid and blood, which is thought to be produced as a consequence of the activation of the innate immunity by unprocessed self-nucleic acids. This thesis therefore aimed to define the role of one of the AGS nucleases, the Ribonuclease H2 (RNase H2) complex, in innate immunity, and to establish if nucleic acid substrates of this enzyme were able to induce type I interferon production in vitro. The AGS nucleases may function as components of the innate immune response to nucleic acids. Consistent with this hypothesis, RNase H2 was constitutively expressed in immune cells, however, its expression was not upregulated in response to type I interferons. RNase H2-deficient cells responded normally to a range of nucleic acid PAMPs, which implied that a role for RNase H2 as a negative regulator of the immune response was unlikely, in contrast to the reported cellular functions of two other AGS proteins, TREX1 and SAMHD1. Therefore, no clear evidence was found for the direct involvement of RNase H2 in the innate immune response to nucleic acids. An alternative model for the pathogenesis of disease hypothesises that decreased RNase H2 activity within the cell results in an accumulation of RNA:DNA hybrids. To investigate the immunostimulatory potential of such substrates, RNA:DNA hybrids with different physiochemical properties were designed and synthesised. Methods to purify the hybrids from other contaminating nucleic acid species were established and their capacity as activators of the innate immune response tested using a range of in vitro cellular systems. A GU-rich 60 bp RNA:DNA hybrid was shown to be an effective activator of a pro-inflammatory cytokine response exclusively in Flt3-L bone marrow cultures. This response was completely dependent on signalling involving MyD88 and/or Trif, however the specific receptor involved remains to be determined. Reduced cellular RNase H2 activity did not affect the ability of Flt3-L cultures to mount a cytokine response against the RNA:DNA hybrid. These in vitro studies suggested that RNA:DNA hybrids may be a novel nucleic acid PAMP. Taken together, the data in this thesis suggest that the cellular function of RNase H2 is in the suppression of substrate formation rather than as a component of the immune response pathways. Future studies to identify endogenous immunostimulatory RNA:DNA hybrids and the signalling pathways activated by them should provide a detailed understanding of the molecular mechanisms involved in the pathogenesis of AGS and related autoimmune diseases.

Disulfide bonds play important roles in establishing and stabilizing three-dimensional protein structure, and mass spectrometry (MS) has become the primary detection method to decipher their biological and pathological roles. Several experimental methods before or after MS detection have been developed to aid in disulfide bond assignment, such as tandem MS followed by database searching or modification of the disulfide bond via chemical reduction or oxidation. Despite these technological advancements, the detection and proper assignment of disulfide bonds have remained experimentally difficult. Therefore, we have developed an alternative method for disulfide bond elucidation using capillary electrophoresis-mass spectrometry (CE-MS) combined with an on-target performic acid oxidation method for matrix assisted laser desorption/ionization (MALDI) deposited samples. An information rich CE-MS method that results in distinct charge-state trends observed in two-dimensional plots of log(mu eff) versus log (MW) was developed to enhance the confidence of peptide and protein identifications. The charge-state trends provide information about the number of basic amino acid residues present within each peptide. This information can be used to develop methods to screen for posttranslationally modified peptides (e.g., phosphorylation, disulfide bonds, etc.). In the case of disulfide bonds, the highly charged peptides (i.e., 3, 4 or greater charge states) have a high probability of being disulfide-linked peptides, owing to charge contribution of both peptides forming the disulfide bridged peptide. However, intra-linked disulfide bridged peptides can also be present at lower charge states. Therefore, a chemically selective method to rapidly locate disulfide-linked peptides that have been separated by CE-MS must be developed. An on-target performic acid oxidation method was developed to provide the chemical selectivity towards disulfide bonds, i.e., converting the cystine bond to form two peptides modified with a cysteic acid (SO3H) side chain. The on-target oxidation method offers (i) no post-oxidation sample cleanup, (ii) improved throughput over solution-phase oxidation methods, and (iii) easily adapted to CE separations coupled offline with MALDI-MS. The evaluation of the on-target oxidation experimental parameters, the fragmentation behavior of cysteic acid-containing peptides and an alternative method for disulfide bond elucidation, using CE-MS combined with the ontarget oxidation method, are discussed within.

Η ριβονουκλεάση P (RNase P) είναι το ένζυμο που ευθύνεται για την ωρίμανση του 5΄ άκρου των πρόδρομων μορίων tRNA συμμετέχοντας έτσι στην πρωτεΐνοσύνθεση. H RNase P καταλύει την ενδονουκλεολυτική θραύση ενός φωσφοδιεστερικού δεσμού παρουσία ιόντων Mg2+ παράγοντας προϊόντα με 3΄ υδροξυλικά και 5΄ φωσφορικά άκρα. Η πλειοψηφία των RNase P ενζύμων που έχουν μελετηθεί μέχρι σήμερα είναι ριβονουκλεοπρωτεΐνες αποτελούμενες από μια RNA και πρωτεϊνικές υπομονάδες. Η RNA υπομονάδα της βακτηριακής RNase P είναι ένα από τα πρώτα καταλυτικά RNA που μελετήθηκαν. Η RNase P και το ριβόσωμα είναι τα μόνα γνωστά ριβοένζυμα που είναι συντηρημένα και στις τρείς φυλογεννετικές περιοχές. Δεδομένης της ζωτικής σημασίας των λεμφοκυττάρων στην ακεραιότητα του ανοσολογικού συστήματος και στην παθογένεια ευρέος φάσματος δερματολογικών και μη νοσημάτων, σκοπός της παρούσας εργασίας ήταν η ανάπτυξη μεθοδολογίας για την απομόνωση και τον καθαρισμό της RNase P από περιφερικά λεμφοκύτταρα υγιών μαρτύρων, ο προσδιορισμός της ενζυμικής της δραστικότητας, καθώς και η μελέτη της in vitro επιδράσεως δυο συνθετικών ρετινοειδών, του 13-cis ρετινοϊκού οξέος και της ασιτρετίνης, της αμινογλυκοσίδης νεομυκίνης Β, όπως και της διφωσφορικής χλωροκίνης στην δραστικότητα της λεμφοκυτταρικής RNase P. Το ένζυμο καθαρίστηκε με τη χρήση κατιοντοανταλλακτικής χρωματογραφίας φωσφοκυτταρίνης και προσδιορίστηκαν οι βέλτιστες συνθήκες δραστικότητάς του.Μετά από μελέτη της επίδρασης των συνθετικών ρετινοϊδών και της νεομυκίνης Β στην δραστικότητα της RNase P, προέκυψε ότι και τα τρία προκαλούν μια δοσοεξαρτώμενη αναστολή της δραστικότητας της RNase Ρ των ανθρωπίνων λεμφοκυττάρων. Η διφωσφορική χλωροκίνη δεν επηρεάζει τη δραστικότητα της λεμφοκυτταρικής RNase P. Λεπτομερής κινητική ανάλυση έδειξε πως η αναστολή που προκαλείται από την ασιτρετίνη είναι συναγωνιστικού τύπου, ενώ αυτή από την νεομυκίνη Β είναι μη συναγωνιστικού τύπου.Η κινητική σταθερά Km της λεμφοκυτταρικής RNase P βρέθηκε ότι ισούται με 245 nM και η Vmax με 0.42 pmol/min. Συμπερασματικά, η απομόνωση της RNase P από ανθρώπινα περιφερικά λεμφοκύτταρα καθιστά δυνατή τη μελέτη της πιθανής ανάμιξης αυτού του ριβοενζύμου στους παθογενετικούς μηχανισμούς διαφόρων αυτοάνοσων, φλεγμονωδών και νεοπλασματικών δερματοπαθειών και μπορεί να διευκολύνει τη περαιτέρω ανάπτυξη της βασιζομένης στην RNase P τεχνολογίας για τη γονιδιακή θεραπεία δερματικών και μη παθήσεων. / Ribonuclease P (RNase P) is the enzyme responsible for the 5΄ maturation of the precursor tRNA molecules, participating in tRNA biogenesis and therefore in protein synthesis. It catalyses the endonucleolytic cleavage of a phosphodiester bond in the presence of Mg2+ and results in the production of molecules that bear 3΄ hydroxyl and 5΄ phosphoric ends. Most forms of RNase P are ribonucleoproteins consisting of an essential RNA and protein subunits. The RNA component of the bacterial RNase P was one of the first identified catalytic RNAs. So far, RNase P and the ribosome are the only ribozymes known to be conserved in all kingdoms of life (bacteria, archaea and eucarya). In view of the vital importance of lymphocytes for an effective immune system, we proceeded to the RNase P isolation from human peripheral lymphocytes. The enzyme was purified with cation exchange phosphocellulose chromatography and the optimal conditions were determined. Herein, it was investigated the effect of the synthetic retinoids (cis-retinoic acid and acitretin), neomycin B, as well as chloroquine diphosphate, on the RNase P activity. Cis-retinoic acid, acitretin and neomycin B exerted a dose-dependent inhibitory effect on RNase P activity from human lymphocytes, wlile the activity was not affected in the presence of chloroquine diphosphate. A detailed kinetic analysis showed that the inhibition caused by acitretin was of competitive type, whereas that caused by neomycin B was of noncompetitive type. The kinetic constant Km of RNase P activity isolated from lymphocytes for the tRNA maturation reaction has been estimated equal to 245 nM and the Vmax value has been estimated equal to 0.42 pmol/min. Finally, the isolation of RNase P from human peripheral lymphocytes will enable the study of the possible involvement of this ribozyme in the pathogenetic mechanisms of diverse autoimmune, inflammatory and neoplastic cutaneous disorders and may facilitate the further development of RNase P-based technology for gene therapy of infectious and neoplastic dermatoses.

Στην παρούσα εργασία, εξετάσαμε λεπτομερώς την κινητική της ενεργοποίησης της δραστικότητας της RNase P του E. coli από τη σπιραμυκίνη. Τα αποτελέσματα δείχνουν ότι η σπιραμυκίνη δρα σαν μικτού τύπου «μη απαραίτητος» ενεργοποιητής . Η κινητική συμπεριφορά του ενεργοποιητή που εξετάστηκε, μπορεί να εξηγηθεί μ’ ένα κινητικό σχήμα ανάλογο αυτού που περιγράφεται στο Segel (1993). Σε κορεσμένη συγκέντρωση σπιραμυκίνης η τιμή της φαινομενικής Vmax (Vmax,app) για το ανασχηματισμένο ολοένζυμο αυξάνεται κατά 2,5 φορές και τιμή της φαινομενικής K(Ks s,app) μειώνεται κατά 7,1 φορές. Ομοίως, σε κορεσμένη συγκέντρωση σπιραμυκίνης, η τιμή της φαινομενικής Vmax για το M1 RNA αυξάνεται κατά 2,4 φορές και η τιμή της φαινομενικής K μειώνεται κατά 5 φορές. / -