Η αποικοδόμηση των πρωτεϊνών είναι μια διαδικασία απαραίτητη για τη διατήρηση της ομοιόστασης του κυττάρου. Ένας από τους κύριους μηχανισμούς αποικοδόμησης των βραχύβιων πρωτεϊνών καθώς και όσων εμφανίζουν λανθασμένη αναδίπλωση, χωρεί μέσω του μονοπατιού ουβικιτίνης- πρωτεασώματος. Η ουβικιτινίωση είναι μια μετα-μεταφραστική διαδικασία, η οποία έγκειται στη σηματοδότηση των υποψήφιων για αποικοδόμηση πρωτεϊνών με ουβικιτίνη και περιλαμβάνει τρεις ενζυμικές ενεργότητες: Ε1 (εκκινητής ουβικιτίνης), Ε2(μεταφορέας ουβικιτίνης) και Ε3 (λιγάση ουβικιτίνης). Η πρωτεΐνη Arkadia (Rnf11) είναι μια Ε3 λιγάση ουβικιτίνης με συνολικό μήκος 994 αμινοξέα. Σε μοριακό επίπεδο, ενισχύει το TGF-β σηματοδοτικό μονοπάτι, διαμεσολαβώντας την εξαρτώμενη από το πρωτεάσωμα αποικοδόμηση των αρνητικών ρυθμιστών του, c-Ski και Sno-N. Η δραστικότητα Ε3 λιγάσης ουβικιτίνης εδράζεται στον C΄-τελικό RING-H2 τομέα, που σχηματίζεται από τα τελευταία 60 περίπου αμινοξέα της ακολουθίας. Η δομή και η σταθερότητα του RING τομέα εξαρτώνται από την πρόσδεση δύο ιόντων Zn μέσω ενός χαρακτηριστικού μοτίβου, που περιλαμβάνει 6 κυστεϊνικά και 2 ιστιδινικά κατάλοιπα. Στην προσπάθεια αποσαφήνισης της σχέσης δομής-δράσης της πρωτεΐνης Arkadia, ένα από τα κατάλοιπα που συναρμόζονται με Zn -συγκεκριμένα η His965- αντικαταστάθηκε από κυστεΐνη μέσω κατευθυνόμενης μεταλλαξιγένεσης. Η μετάλλαξη αυτή, με την οποία, ουσιαστικά, μετατρέψαμε τον RING-H2 σε RING-HC τομέα, μελετήθηκε με χρήση πολυπυρηνικής/πολυδιάστατης φασματοσκοπίας πυρηνικού μαγνητικού συντονισμού (NMR). H NMR δομή του RING-H2 τομέα της Η965C Arkadia επιλύθηκε σε υψηλή διακριτικότητα (tf=0.94±7.53*10-2, RMSD=0.75±0.20 και RMSD=1.45±0.24 για τα άτομα του πολυπεπτιδικού σκελετού και τα βαρέα άτομα αντίστοιχα) και αποκάλυψε μια ββαββ τοπολογία. Παράλληλα, πραγματοποιήθηκε μελέτη κινητικότητας, από την οποία προέκυψε ότι η εν λόγω μετάλλαξη υφίσταται ως μονομερές και διαθέτει έναν συμπαγή πυρήνα, που περικλείεται μεταξύ δύο ευκίνητων άκρων. / Protein degradation is necessary for the maintenance of cell homeostasis. A major mechanism for the degradation of short-lived as well as misfolded proteins involves the ubiquitin-proteasome pathway. Ubiquitination is a post translational modification, which targets the proteins to be degraded through the covalent attachment of a ubiquitin tag and consists of three enzyme activities: Ε1 (ubiquitin activator), E2 (ubiquitin carrier) and E3 (ubiquitin ligase). Arkadia (Rnf11) is a 994 amino acid protein, which acts as an E3 ubiquitin ligase. On a molecular level, Arkadia enhances TGF-β signaling by mediating the proteasome-dependent degradation of its negative regulators, c-Ski and Sno-N. Its E3 ubiquitin ligase activity lies on a C΄-terminal RING-H2 domain, formed by the last 60 residues. The structure as well as stability of the RING finger domain depend strongly on the binding of two zinc ions in a unique ΄΄cross-brace΄΄ arrangement through a defined motif of six cysteines and two histidines. Trying to elucidate the structure-activity relationship in the case of Arkadia, one of the amino acid ligands –specifically His965- was replaced by cysteine through site-directed mutagenesis. This particular mutation, which, in reality, transformed the RING-H2 to a RING-HC domain, was studied with the use of multinuclear/multidimensional nuclear magnetic resonance spectroscopy (NMR). The NMR solution structure of the H965 Arkadia RING-H2 domain was determined in high resolution (tf=0.94±7.53*10-2, RMSD=0.75±0.20 και RMSD=1.45±0.24 for backbone and heavy atoms respectively) and revealed a ββαββ topology. Furthermore, a mobility study was conducted with the following results: the mutated protein is not expected to form dimers and shows a compact core region including the four metal binding motifs flanked by two flexibly disordered termini.

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Philosophiae Doctor - PhD / Retinoblastoma-binding protein 6 (RBBP6) is a multi-domain human protein known to play a role in mRNA splicing, cell cycle control and apoptosis. The protein interacts with tumour suppressor proteins p53 and pRb and recent studies have shown that it plays a role in the ubiquitination of p53 by interacting with Hdm2, the human homologue of mouse double minute protein 2 (Mdm2), in which the RING finger domain plays an essential role. Recently, RBBP6 has been shown to ubiquitinate the mRNA-associated proteinYB-1 through its RING finger domain, causing it to be degraded in the proteasome.RING (Really Interesting New Gene) fingers are small commonly-occurring domains of approximately 70 amino acids in length which coordinate two zinc ions in a cross-brace fashion.They are characterized by a conserved pattern of eight Cysteine or Histidine residues which are involved in coordinating the zinc ions. In terms of this conserved consensus, the RING finger from RBBP6 is expected to coordinate the zinc ions through eight Cysteine residues, making it a “C4C4” RING finger similar to those identified in transcription-associated proteins CNOT4(CCR4-NOT transcription complex, subunit 4) and p44 (interferon-induced protein 44). The amino acid sequence of the domain also shares many similarities with the U-box family of domains, which have an identical three-dimensional structure despite not requiring zinc ions in order to fold. This thesis reports the bacterial expression of a fragment containing the RING finger domain from human RBBP6, and determination of its structure using heteronuclear Nuclear Magnetic Resonance (NMR) spectroscopy. Preliminary NMR analysis of the fragment revealed that the domain was folded, but that it was preceded by an unstructured region at the N-terminus. A shortened fragment was therefore expressed and used for structural studies. Isotope-enriched protein samples were generated by growing bacteria in minimal media supplemented with 15NNH4Cl and 13C-glucose and purified using a combination of glutathione agarose affinity chromatography, anion exchange and size exclusion chromatography. A complete set of heteronuclear NMR data was collected at 600 MHz from which almost complete assignment of the backbone, side-chain and aromatic resonances was achieved. By exchange of Zn2+ with 113Cd2+ we managed to confirm that the domain binds two Zn2+ ions, and confirm that they are coordinated in the expected cross-brace manner. Structural data in the form of 2-Dimensional Nuclear Overhauser Enhancement Spectroscopy (2D-NOESY), 15N-separated NOESY and 13Cseparated NOESY spectra were recorded and used to determine the structure using restrained molecular dynamics on the Combined Assignment and Dynamics Algorithm for NMR Applications (CYANA) platform.As expected, the structure contains a triple-stranded β-sheet packing against an α-helix and two zinc-stabilized loops as found in all RING fingers. However, it also contains a C-terminal helix which packs against an N-terminal loop which is similar to that found in many U-box domains.A search using the DALI server revealed that the structure is most similar to the U-box from CHIP (C-terminus of Hsp70-interacting protein), an E3 ligase that cooperates with Hsp70 to degrade unfolded proteins that cannot be refolded. Using NMR we showed that the domain dimerizes with a KD of approximately 200 Μm, which means that it is dimeric at the concentrations used for NMR structure determination. Chemical shift analysis showed the dimerization interface to be very similar to that identified in U-box domains found in C-terminus of Hsp70 interacting proteins (CHIP).The structural similarities reported here between the RING finger from RBBP6 and the U-box family lead us to conclude that RBBP6 may, like CHIP, play a role in protein quality control.

Although the RING finger protein 121 (RNF121) is a highly conserved E3 ubiquitin ligase from Caenorhabditis elegans to human, its function is poorly understood and in higher eukaryotes it has been studied only at in vitro level. RNF121 has been described to have various functions: i) it was ascribed to function as a broad regulator of NF-κB activation, ii) it was shown to control intracellular trafficking of various membrane proteins, and iii) its downregulation leads to apoptosis. Moreover, RNF121 might have a role in cancer as its expression was found to be 16.4-fold higher in patients suffering from Barrett esophagus (precancerous lesion of esophageal adenocarcinoma) and was even more increased in esophageal adenocarcinoma comparing to healthy population. In addition, RNF121 gene is localized in the candidate region containing breast cancer susceptibility genes. To gain insight into physiological functions of RNF121, Rnf121 knockout mice (Rnf121tm1b(EUCOMM)Hmgu ) were generated in the Czech Centre for Phenogenomics and further studied in our laboratory. Rnf121+ /- intercross breedings showed a prenatal lethal phenotype of Rnf121-/- embryos, which were dying prior embryonic day (E) 11.5. Preliminary experiments carried out in our laboratory showed numerous vascular defects in null mutant embryo,...

Philosophiae Doctor - PhD / Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa protein that is implicated in mRNA processing and ubiquitination functions and has been shown to be highly up-regulated in a number of cancers. In humans and mice,RBBP6 interacts with both tumour suppressors p53 and pRb, suggesting that it is involved in regulation of transcription, induction of apoptosis and cell cycle control. Knock-out of an RBBP6 homologue PACT resulted in p53 dependent cell cycle arrest and apoptosis. Although the biological functions of RBBP6 remain largely unclear, it is possible that its functions are mediated through interaction with other cellular proteins. Since it is possible to unveil novel functions of a target protein through identifying its interacting protein partners,this study aims to further characterize the functions of RBBP6 through identifying novel protein interacting partners using a yeast 2-hybrid screen.In order to identify interaction partners of RBBP6, two well characterized domains of RBBP6, the N-terminal ubiquitin-like DWNN domain and RING finger domain, were used as baits in a yeast 2-hybrid screen of a human testis cDNA library. Putative interactors were verified using in vitro and in vivo immunoprecipitation assays. The RING finger domain was shown to interact with transcriptional factors Y-Box binding protein 1 (YB-1) and zinc finger and BTB containing protein 38 (zBTB38), resulting in their ubiquitination. In the case of YB-1 ubiquitination was correlated with a decrease in the intra-cellular levels of YB-1, suggesting that ubiquitination leads to degradation in the proteosome. The DWNN domain was shown to interact with a splicing associated small nuclear ribonucleoprotein polypeptide G (snRPG) and heat shock protein 70 (Hsp70).The results of this work suggest that, at least in the case of YB-1 and zBTB38,RBBP6 plays a role in the regulation of gene expression by ubiquitination of transcription factors, causing them to be degraded in the proteosome. The study provides further evidence of RBBP6’s involvement in mRNA splicing through its interaction with snRPG. The interaction with Hsp70 suggests a possible role in protein quality control similar to that played by other E3 ligases such as Parkin and CHIP.

Philosophiae Doctor - PhD / Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa protein that is implicated in mRNA processing and ubiquitination functions and has been shown to be highly up-regulated in a number of cancers. In humans and mice, RBBP6 interacts with both tumour suppressors p53 and pRb, suggesting that it is involved in regulation of transcription, induction of apoptosis and cell cycle control. Knock-out of an RBBP6 homologue PACT resulted in p53 dependent cell cycle arrest and apoptosis. Although the biological functions of RBBP6 remain largely unclear, it is possible that its functions are mediated through interaction with other cellular proteins. Since it is possible to unveil novel functions of a target protein through identifying its interacting protein partners, this study aims to further characterize the functions of RBBP6 through identifying novel protein interacting partners using a yeast 2-hybrid screen. In order to identify interaction partners of RBBP6, two well characterized domains of RBBP6, the N-terminal ubiquitin-like DWNN domain and RING finger domain, were used as baits in a yeast 2-hybrid screen of a human testis cDNA library. Putative interactors were verified using in vitro and in vivo immunoprecipitation assays. The RING finger domain was shown to interact with transcriptional factors V-Box binding protein 1 (YB-1) and zinc finger and BTB containing protein 38 (zBTB38), resulting in their ubiquitination. In the case of YB-1 ubiquitination was correlated with a decrease in the intra-cellular levels of YB-1, suggesting that ubiquitination leads to degradation in the proteosome. The DWNN domain was shown to interact with a splicing associated small nuclear ribonucleoprotein polypeptide G (snRPG) and heat shock protein 70 (Hsp70). The results of this work suggest that, at least in the case of YB-1 and zBTB38, RBBP6 plays a role in the regulation of gene expression by ubiquitination of transcription factors, causing them to be degraded in the proteosome. The study provides further evidence of RBBP6's involvement in mRNA splicing through its interaction with snRPG. The interaction with Hsp70 suggests a possible role in protein quality control similar to that played by other E3 ligases such as Parkin and CHIP.

No description available.