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No description available.

We present a statistical mechanical approach for quantifying thermodynamic properties of proteins in mixed solvents. This approach, based on molecular dynamics simulations which incorporate all atom models and the theory of preferential binding, allows us to compute transfer free energies with experimental accuracy and does not incorporate any adjustable parameters. Specifically, we applied our approach to the model proteins RNase A and T1, and the solvent components water, glycerol, and urea. We found that the observed differences in the binding of glycerol and urea to RNase T1 and A are predominantly a consequence of density differences in the first coordination shell of the protein with the cosolvents, but the second solvation shell also contributes to the overall binding coefficients. The success of this approach in modeling preferential binding indicates that it incorporates the important underlying physics of proteins in mixed solvent systems and that the difficulty in quantitative prediction to date can be surmounted by explicitly incorporating the complex protein-solvent and solvent-solvent interactions. / Singapore-MIT Alliance (SMA)

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

RNA degradation plays a fundamental role in maintaining cellular homeostasis, along with being a part of normal regulatory mechanisms, whether it occurs as a surveillance mechanism eliminating aberrant mRNAs or during RNA processing to generate mature transcripts. 3’-5’ exoribonucleases are essential mediators of RNA decay pathways, and one such evolutionarily conserved enzyme is polynucleotide phosphorylase (PNPase). The human homologue of this fascinating enzymatic protein (hPNPaseold-35) was cloned a decade ago in the context of terminal differentiation and senescence through a novel ‘overlapping pathway screening’ approach. Since then, significant insights have been garnered about this exoribonuclease and its repertoire of expanding functions. hPNPaseold-35 has progressed a long way from being just a 3’-5’ exoribonuclease to a functionally relevant molecule implicated in a multitude of diverse and important biological effects. hPNPaseold-35 plays central roles in diverse physiological processes including growth inhibition, senescence, mtRNA import, mitochondrial homeostasis, and RNA degradation, all while primarily being localized in the mitochondrial IMS (inter membrane space). hPNPaseold-35 also holds immense promise as a therapeutic agent due to its ability to degrade specific miRNA (miR-221) and mRNA (c-myc) species, and this property can be exploited in treating malignancies that are characterized by upregulation of harmful miRNA or mRNA molecules. But apart from these two targets, little is known about any other targets hPNPaseold-35 may degrade. Thus, the primary objective of this dissertation research was to identify targets other than c-myc or miR-221 that hPNPaseold-35 could directly degrade to discover newer and biologically relevant therapeutic targets for the treatment of hPNPaseold-35 –associated disease states. In order to do this we performed extensive microarray analyses following hPNPaseold-35 overexpression and depletion in mammalian cell lines, and were able to identify transcripts that could be potentially regulated by hPNPaseold-35 directly or indirectly. Apart from this we also analyzed the 3’UTR of c-myc in order to identify any specific sequence or secondary structural elements necessary for hPNPaseold-35 mediated degradation. Lastly, we identified certain residues in hPNPaseold-35 that have been under positive natural selection through evolution.

Poly(A)-specific ribonuclease (PARN) is a deadenylase that degrades the poly(A) tail of eukaryotic mRNA. PARN also interacts with the 5’-cap structure of the mRNA. The binding of the cap structure enhances the deadenylation rate. PARN has previously been described as a dimer. We have studied PARN with size exclusion chromatography to investigate the oligomeric composition and revealed oligomeric compositions of PARN that are larger than dimeric PARN. Deadenylation assays have been used to measure the cap stimulated activity of PARN. The deadenylation assays showed that the cap stimulated activity of PARN correlated with the abundance of oligomers corresponding in size to tetrameric PARN. We present a model for tetrameric PARN and propose a mechanistic model for how the cap stimulates PARN mediated deadenylation.

Thesis (M.S.)--University of Missouri--Columbia, 2003. / "December 2003." Typescript. Includes bibliographical references (leaves 37-38). Also issued on the Internet.

Ο μυξομύκητας Dictyostelium discoideum είναι εξελικτικά κατώτερος ευκαρυωτικός οργανισμός και η RNase P από αυτόν είναι ένα εξαιρετικά ενδιαφέρον ένζυμο, δεδομένου του ότι, το ένζυμο αυτό προσφέρεται για σύγκριση, τόσο με ένζυμα RNase P ανώτερων ευκαρυωτικών οργανισμών, όσο και με τα καλύτερα χαρακτηρισμένα βακτηριακά ολοένζυμα. Η RNase P του D. discoideum έχει βρεθεί ότι είναι και αυτή ένα ριβονουκλεοπρωτεΐνικό σύμπλοκο, που αποτελείται από μία RNA υπομονάδα και από οχτώ πρωτεϊνικές υπομονάδες. Στην εργασία αυτή έγινε προσπάθεια να κλωνοποιηθεί και να χαρακτηριστεί μια επιπλέον πρωτεΐνική υπομονάδα, η DPop5, πέραν των τεσσάρων που έχουν ήδη κλωνοποιηθεί και χαρακτηριστεί από το εργαστήριο του κ. Δραΐνα (DRpp20, DRpp30, DRpp40, DRpp29), έτσι ώστε να δοθεί επιπλέον πληροφορία για την ολοκλήρωση της αρχιτεκτονικής του μακρομοριακού συμπλόκου της RNase P. Για το λόγο αυτό πραγματοποιήθηκε κλωνοποίηση του γονιδίου και υπερέκφραση αυτής μέσω ενός συστήματος έκφρασης, το οποίο βασίζεται στην παραγωγή χιμαιρικών πρωτεϊνών, αποτελούμενων από την πρωτεΐνη-στόχο και τη βακτηριακή συνοδό πρωτεΐνη, HSP70 ή DnaK. Στη συνέχεια, αφού ακολούθησε καθαρισμός της ανασυνδιασμένης πρωτεΐνης πραγματοποιήθηκε ο βιοχημικός χαρακτηρισμός της και η παρασκευή πολυκλωνικών αντισωμάτων έναντι αυτής. Ο βιοχημικός χαρακτηρισμός της καθώς και η μοντελοποίηση αυτής in silico έδειξαν ότι η πρωτεΐνη αυτή είναι αρνητικά φορτισμένη (pI 5.19) και ότι έχει την ικανότητα να αλληλεπιδρά με RNA μόρια, μέσω ενός χαρακτηριστικού μοτίβου πρόσδεσης σ’ αυτά. Περαιτέρω πειραματικές διαδικασίες βρίσκονται σε εξέλιξη έτσι ώστε να αποσαφηνιστεί πλήρως ο ρόλος της στο ολοένζυμο. / Cloning and characterization of DPop5 protein subunit from Dictyostelium discoideum ribonucleoprotein complex RNase P.

Classical molecular dynamics simulations were applied on complexes of RNA-dependent RNA-polymerase, Ribonuclease H, Argonaute and Ribonuclease L with chemically modified nucleic acids, which are studied as potential chemotherapeutic agents. Powerful graphics processing units, through which these molecular dynamics simulations were performed, enabled to acquire trajectory length from hundreds of nanoseconds to one microsecond. Molecular dynamics simulations allowed capture differences in binding of various modified nucleic acids to the above mentioned enzymes. These identified differences fitted well with experimental results. It opens the door for rational design of the structure of potential chemotherapeutic agents based on chemically modified nucleic acids.

Artificial ribonucleases (ARs) are recognised as a potential strategy to selectively target and cleave biologically significant RNA in cells. However, in order to work as true enzymes they must exhibit catalytic turnover. Many of the reported ARs incorporate metal-containing centres (e.g. dysprosium, copper) in order to induce substantial phosphodiester cleavage, which is not amenable to use in vivo. Therefore, new strategic directions employing metal-independent ARs, such as peptidyl-oligonucleotide conjugates (POCs), need to be investigated. Previous work has shown that poor or non-complementary POCs demonstrate catalytic turnover of a HIV-1 substrate; however, sequence specificity is an issue. For POCs to be useful from a therapeutic standpoint they must only cleave specific RNA molecules and do so in a catalytic fashion, therefore removing the requirement for stoichiometric drug delivery and binding. Consequently, novel POC design strategies are required that allow selective RNA targeting but promote dissociation of the POC following phosphodiester cleavage. In this research, three types of different peptidyl-oligonucleotide conjugate designs have been implemented with the attempt to find an appropriate balance between selectivity and catalytic turnover.(i) Selective targeting and quantitative cleavage (97-100%) of a tRNAPhe target was achieved through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to catalytic amphiphilic peptide sequences containing leucine, arginine and glycine. Although the half-life of tRNAPhe was less than 1 h on exposure to some of these POCs, hybridisation studies reveal that the POCs bind too tightly to their target RNA sequences and thus an excess of POC is required for efficient cleavage activity. The effect of peptide and oligonucleotide sequence variations as well as the role of enhanced conformational freedom via incorporating an abasic deoxyribose linker between the oligonucleotide targeting motif and catalytic peptide is also investigated. (ii) Most of ‘Dual’ peptidyl-oligonucleotide conjugates containing an amphiphilic RNA-cleaving peptide placed between two RNA recognition motifs directed towards the TΨC loop and 3’ acceptor stem of tRNAPhe demonstrate marked RNA binding and cleavage activities. Interestingly, those dual conjugates which showed poor or negligible binding ability in electrophoresis assays, demonstrated sufficient RNA cleavage (70%) within the vicinity of the 65GACAC61 target region. Therefore, weak POC:RNA complexes may exist which could facilitate substrate turnover. (iii) Finally, POCs were designed which induce bulge-loops in their target RNA region upon hybridisation. By introducing regions of non-complementarity into the oligonucleotide sequences, 2- to 5- membered bulges sizes were formed. Via attachment of a catalytic peptide to an internally modified oligonucleotide residue, catalytic peptides were placed directly adjacent to single-stranded RNA regions to promote cleavage by nuclease mimics. Through probing the hybridised complexes with RNase H, the presence of bulges were confirmed for all bulge-loop sizes, which will be followed by cleavage experiments to assess the possibility for reaction catalytic turnover. In conclusion, a variety of POCs have been synthesised, characterised and partially tested for their RNA cleaving and turnover activity. Based on the encouraging results presented POCs could be further developed to target disease specific RNA sequences such as micro- or messenger RNAs.