Οι macro τομείς αποτελούν μία οικογένεια δομών, συντηρημένη εξελικτικά σε πολλά είδη οργανισμών. Ομόλογά τους βρίσκονται σε βακτήρια, αρχαία, ασπόνδυλα, φυτά καθώς και σε θηλαστικά. Η βιολογική τους λειτουργία δεν έχει αποσαφηνιστεί πλήρως αλλά το βασικό βιοχημικό τους χαρακτηριστικό είναι η ύπαρξη μιας θετικά φορτισμένης κοιλότητας η οποία χρησιμεύει ως σημείο πρόσδεσης της διφωσφορικής αδενικής ριβόζης (ADPr). Ο macro τομέας αποτελεί επίσης μέρος των μη δομικών πρωτεϊνών ιών, οι οποίοι φέρουν ως γενετικό υλικό νοηματικό μονόκλωνο RNA. Τέτοιου είδους ιοί είναι και οι άλφα ιοί στους οποίους ανήκει και ο ιός Mayaro. Στην παρούσα εργασία, πραγματοποιείται δομική μελέτη του macro τομέα του ιού Mayaro. Με χρήση της τεχνολογίας του ανασυνδυασμένου DNA, ο πρωτεϊνικός τομέας εκφράστηκε σε μεγάλες ποσότητες και κατέστη δυνατή η μελέτη του με τη χρήση πολυπυρηνικής/πολυδιάστατης φασματοσκοπίας πυρηνικού μαγνητικού συντονισμού. Η NMR δομή του macro τομέα του ιού Mayaro επιλύθηκε σε υψηλή διακριτικότητα (tf=1.03+/-9.7*10e-2, RMSD=0.90+/-0.15 και RMSD=1.44+/-0.14 για τα άτομα της πολυπεπτιδικής αλυσίδας και τα βαρέα άτομα αντίστοιχα) και αποκάλυψε μια αβα σάντουιτς δομή στο κέντρο της οποίας σχηματίζεται μια εκτεταμένη β-πτυχωτή επιφάνεια. Στη συνέχεια πραγματοποιήθηκαν πειράματα αλληλεπίδρασης, τα οποία αποκάλυψαν ότι το ADPr αποτελεί προσδέτη στο macro τομέα του ιού Mayaro. / Macro domains are a family of structures, evolutionarily conserved in many kinds of organisms such as bacteria, archaea, invertebrates, plants and mammals. Their biological function is not fully elucidated but their key biochemical feature seems to be the binding of the Adenosine Diphosphate ribose(ADPr). Macro domain is also found in non-structural proteins of several positive strand RNA viruses like Mayaro virus, a member of the genus of Alphavirus. In the present study, a structural analysis of the macro domain of virus Mayaro is being performed. Using the technology of recombinant DNA, the macro domain was expressed in high yield, making the analysis by multinuclear/multidimensional Nuclear Magnetic Resonance possible. The NMR solution structure of the macro domain od virus Mayaro was determined in high resolution(tf=1.03+/-9.7*10e-2, RMSD=0.90+/-0.15 and RMSD=1.44+/-0.14 for backbone and heavy atoms respectively) and revealed a well folded 3-Layer(aba)Sandwich structure in the center of which, an extended beta sheet is formed. Furthermore, interaction experiments were performed which revealed that the ADPr is a ligand for the macro domain of virus Mayaro.

Gene expression pathways exhibit many “twists and turns,” with theoretically numerous ways in which the pathways can be regulated by both negative and positive feedback mechanisms. A key step in gene expression is RNA maturation (RNA processing), which in the bacterial cell can be accomplished through RNA binding and enzymatic cleavages. The well-characterized bacterial protein Ribonuclease III (RNase III), is a conserved, double-stranded(ds)-specific ribonuclease. In the gram-negative bacterium Escherichia coli, RNase III catalytic activity is subject to both positive and negative regulation. A recent study has indicated that an E. coli protein, YmdB, may negatively regulate RNase III catalytic activity. It has been proposed that YmdB inhibition of RNase III may be part of an adaptive, post-transcriptional physiological response to cellular stress. In E. coli, the model organism in this study, YmdB protein is encoded by the single ymdB gene, and has a predicted molecular mass of ~18.8 kDa. YmdB has been classified as a macrodomain protein, as it exhibits a characteristic fold that specifically provides an ADP-ribose (ADPR) binding site. While YmdB can bind ADPR with good affinity, there may be additional ligands for the binding site. Thus, YmdB protein may interact with other components in the cell, which in turn could modulate the interaction of YmdB with RNase III. In previous research conducted within the Nicholson laboratory at Temple University, affinity-purified Escherchia coli(Ec) YmdB and Aquifex aeolicus (Aa) YmdB were found to exhibit ribonucleolytic activity. This observation initiated the long-term goal of learning how YmdB regulates RNase III, and how the ribonucleolytic activity of YmdB may be involved in this process. The specific goal of this thesis project was to further characterize the ribonucleolytic activity of Ec-YmdB through site-specific mutational analysis. Mutations were introduced into a proposed adenine-binding pocket previously identified by crystallography and by molecular modeling. The adenine-binding pocket is a region within the macrodomain fold where ADP-ribose could bind. The mutations were examined for their effect on Ec-YmdB cleavage of a model RNA, R1.1. The results of this study will contribute to the development of a model describing how the ribonucleolytic activity of YmdB is regulated. / Biology

Increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) in the β-cells triggers insulin exocytosis. Among the Ca2+ channels present in the plasma membrane, the transient receptor potential (TRP) channels receptors are currently of great interest. The mechanisms by which the extracellular adenosine diphosphate ribose (ADPr) increases the [Ca2+]i is unknown. Our aims were to study the roles of the TRP channels in the tolbutamide induced [Ca2+]i increase and to identify the surface receptor that is activated by ADPr. We used S5 cells, a highly differentiated rat insulinoma cell line, as a model for β-cells. Single cell ratiometric microfluorometry was used to measure the [Ca2+]i changes in the Fura-2 loaded cells. Tolbutamide increased [Ca2+]i in the form of oscillations. After tolbutamide increased [Ca2+]i,capsazepine, a potent blocker of the transient receptor potential vanilloid subtype 1 (TRPV1) channel was added to the β-cells, which reduced the tolbutamide-induced [Ca2+]i increase. capsazepine, N-(p-Amylcinnamoyl) anthranilic acid (ACA),  TRPM2 channel blocker, and triphenyl phosphine oxide (TPPO), TRPM5 channel blocker were tested for their effect on potassium chloride (KCl) induced [Ca2+]i response. These blockers did not inhibit the KCl induced [Ca2+]i increase.   Adenosine diphosphate ribose (ADPr) increased [Ca2+]i in the form of initial transient peak followed by an elevated plateau. Application of ADPr shortly after a prior application and washout of Adenosine diphosphate (ADP) elicited only small [Ca2+]i increase  indicating desensitization of the receptor involved. 2´deoxy-N6-methyladenosine 3´5´bis-phosphate (MRS2179), and chloro N6-methyl-(N)-methanocarba 2´deoxyadenosine 3´5´ bis-phosphate (MRS2279), two selective inhibitors of P2Y1 receptor, abolished the ADPr-induced [Ca2+]i increase. Tolbutamide closes ATP sensitive potassium (KATP) channels. Our results demonstrate that besides the closure of the KATP channels, inward cation currents carried by Ca2+through the TRPV1 channel are necessary for depolarization to the threshold for the activation of the voltage gated calcium channels (VGCC) to increase the [Ca2+]i. Our results also show that ADPr increases [Ca2+]i by activating the P2Y1 receptor.