Εξω-ριβοσωμικές λειτουργίες της S5 ριβοσωμικής πρωτεϊνης από ποντίκι
Creators: name: Ματράγκου, Χριστίνα Ν.
Publisher: name: Aristotle University of Thessaloniki
Year: 2005
Subjects: Ριβοσωμική πρωτεϊνη
Φωσφορυλίωση
Ερυθρολευχαιμικά κύτταρα
Ribosomal protein
Phorphorylation
Erythroleukemia cells

description: Κεντρικό στόχο της συγκεκριμένης διατριβής αποτελεί η διερεύνηση του ρόλου της ριβοσωμικής πρωτεϊνης S5,(rpS5) σε βασικές κυτταρικές λειτουργίες, καθώς επίσης και ο χαρακτηρισμός δομικών στοιχείων στο μόριο της πρωτεϊνης, με πιθανή λειτουργική συμμετοχή στη συμπεριφορά του μορίου ενδοκυτταρικά. Στη διατριβή αυτή η rpS5 του ποντικιού, εκφράστηκε ως πρωτεϊνη σύντηξης με τη GST, οπότε και καθορίστηκε με στήλη αγχιστείου. Προέκυψε πως η rpS5 πρωτοελύεται στο Ν-τελικό άκρο, και συγκεκριμένα στην περιοχή των 37 πρώτων αμινοξέων. Πειράματα in vitro φωσφορυλίωσης έδειξαν πως η πρωτεϊνη φωσφορυλιώνεται από την κινάση της καζεϊνης ΙΙ, (CKII). Η φωσφορυλίωση αφορά στην περιοχή των 37 πρώτων αμινοξέων όπου και είχαν βρεθεί με προγράμματα βιοπληροφορικής, τρεις πιθανοί στόχοι φωσφορυλίωσης από την CKII, η θρεονίνη στη θέση 2, Τ(2), η σερίνη στη θέση 24, S(24) και η σερίνη στη θέση 34, S(34). Η απάλειψη της Τ(2) δεν εμπόδισε την in vitro φωσφορυλίωση της πρωτεϊνης από την CKII, αντίθετα την ενίσχυσε. Πειράματα ανοσοφθορισμού με μικροσκοπία συνεστίασης έδειξαν πως η rpS5 εντοπίζεται στον πυρηνίσκο και λιγότερο στο κυτταρόπλασμα, η rpS5 38-204 βρίσκεται διάχυτη στον πυρήνα ενώ η rpS52-204 και τα μεταλλάγματα rpS5T(8) G και rpS5 Τ(14) G, εντοπίζονται στο κυτταρόπλασμα και σπάνια στους πυρηνίσκους. Επιμόλυνση ΜΕL κυττάρων με το ανασυνδυασμένο πλασμίδιο pcDNA 3.1-rpS5, είχε σαν αποτέλεσμα, παρουσία χημικών επαγωγέων, το πρόγραμμα διαφοροποίησης να παρουσιάζει υστέρηση ως προς την έναρξή του, ενώ επηρεάζονται και τα επίπεδα των CdK2 &amp; CdK4
description: Ribosomal proteins are integral components of the basal cellular machinery involved in protein synthesis, whose roles have been regarded collectively as important, but individually somewhat mundane. However, the last two to three decades, various individual ribosomal proteins from all species, have been found to play roles in regulating cell growth, differentiation, transformation and death, giving rise to increasing speculations that ribosomal proteins can act as multifunctional proteins. The main objective of this thesis encompasses the investigation of the functional role of mouse ribosomal protein S5, (rpS5), in basic cellular functions, such as differentiation and apoptosis. To this respect, several structural elements of rpS5 have been characterized and their possible implication in extra- ribosomal function has been suggested. In particular thesis, the mouse rpS5 was expressed as fusion protein with GST in E.coli, by cloning the corresponding cDNA in the plasmid vector pGEX-2T in order to make its purification easier as well as with GFP by using the vector pEGFP-C1 to facilitate its location intracellurly. It has been found that the recombinant protein is labile within its N-terminal region, the cleavage happening after the first 37aa. Furthermore, by carrying and in vitro phosphorylation assays, it has been found that the phosphorylation of rpS5 taking place by Casein Kinase II, (CKII) is localized within this region of 37aa at N-terminal. Interestingly, bioinformatic analysis of rpS5 primary structure predicted that CKII phosphorylates threonine in position 2, T(2), serine in position 24, S(24) and serine in position 34, S(34), while the target of M38PK kinase, is threonine in position 8, T(8) and threonine in position 14, T(14), is the possible target of an unpredicted kinase. The purification and isolation of mutant forms [at T(2), T(8) and T(14)] of rpS5, in fusion either with GST or GFP, was performed in order to assess in vitro phosphorylation and intracellular localisation, respectively. To this respect, it has been found that the deletion of the first two aa [T(2)] and even the mutant forms T(8) and T(14), did not prevent the in vitro phosphorylation of GST-rpS5 protein by CKII but the phosphorylation seems to be enchanced. Concerning the localization of rpS5 protein within the nucleoli, nucleus and cytoplasm, took place experiments by using confocal microscopy, after deletions of the first 37 aa, (rpS538-204), of the first two aa, (rpS53-204), as well as mutations of T(8) and T(14) to G. The produced truncated or mutated proteins were expressed as fused molecules with GFP, (Green Fluorescent Protein), by using the vector pEGFP-C1. The data obtained has shown that the wild type rpS5 is mainly localised in the nucleolus and to a lesser extent in the cytoplasm. However, the mutant protein rpS538-204 is found diffused inside the nucleus, whereas rpS53-204 and the other two mutants, are mainly localized in the cytoplasm and rarely in the nucleolus. These results suggest that the N-terminal of mouse rpS5 contains structural elements involved in protein localization within the cell, whereas additional work is needed in order to further elucidate their implication in rpS5 structure and function. In order to explore the role of rpS5 in cell differentiation, we stably transfected MEL-745A-PC4 cells with the recombinant eukaryotic vector pcDNA-3.1-rpS5 carrying the full length mouse rpS5 cDNA, before the selection of clones capable to produce high levels of exogenous rpS5 mRNA. The constitutive expression of the recombinant rpS5, does not seem to prevent the completion of the differentiation programme of MEL cells upon exposure to different inducers, (HMBA, DMSO, UDP-4), as far as they are the two sub programmes are concerned, (the one that has to do with the control of cell growth and the other that concerns the expression of terminally differentiated cell protein markers, such as haemoglobin). However, it seems that it causes a delay of 12-24h in the initiation of the differentiation programme, independently from the chemical inducer used. To this direction, by assessing the protein levels of the important cell cycle kinases, Cdk2, Cdk4 and Cdk6, it has been shown that the constitutive expression of rpS5 in MEL cells seems to influence, mainly, the expression of Cdk2 and Cdk4. This result constitutes a new interesting element for the differentiation system of MEL cells, since it involves the function of a ribosomal protein, rpS5, in the initiation of cell differentiation, with a possible result in the deregulation of the cell cycle