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Thesis--Auburn University, 2009. / Abstract. Vita. Includes bibliographical references (p. 224-238).

The mitochondrial tyrosyl tRNA synthetases (mtTyrRS) from certain fungii are found to be bifunctional enzymes that aid in group I intron splicing in addition to charging tRNA[superscript Tyr]. This splicing activity is conferred by several insertions that are unique to these mtTyrRS. Initial biochemical evidence suggested the similar tertiary structures of the tRNA and the intron enable binding of the protein to both. However, a recently solved co-crystal structure showed that the tRNA and intron were bound on opposite faces of the protein. The intron was bound almost exclusively by a novel surface formed by several insertions in the protein. This work presents the structure of the C-terminal domain of the A. nidulans mtTyrRS (PDB ID -- 2ktl). NMR results show that the C-terminal domain contains an S4 fold with a mixed [beta]-sheet and two anti-parallel [alpha]-helices that pack against these strands. The strands [beta]1 and [beta]5 are parallel, and [beta]2 to [beta]5 are arranged anti-parallel to each other. The C-terminal domain from A. nidulans mtTyrRS has three insertions in its sequence that make it almost twice the size of bacterial TyrRS. NMR results show that insertion 3 at the N-terminus of the domain is flexible. Insertion 4 is contained in the loop connecting [beta]2-[beta]3 and does not have a well defined structure. Insertion 5 and the C-terminal extension form two helices, [alpha]5 and [alpha]6 that fold away from the core of the protein. An extended helix ([alpha]4) between strands [beta]3 and [beta]4 was identified by NMR. Based on structural alignments with bacterial TyrRS, this helix was classified as a novel insertion 4b in the C-terminal domain. Conserved positively charged residues used to bind the tRNA are found in the turn between the anti-parallel [alpha]-helices and the turn connecting strands [beta]4-[beta]5. Based on a comparison with other TyrRS structures, the three insertions are positioned away from the tRNA binding site. The insertions form a novel RNA binding surface that could interact with the intron. Since these insertions are found in loop and termini regions, they could be a structural adaptation acquired by these splicing mtTyrRS. NMR spectra of the full length TyrRS from B. stearothermophilus and mtTyrRS from A. nidulans indicate that the motion of the C-terminal domain is coupled to that of the full length protein. This provides new information regarding the organization of the full length TyrRS. / text

Dissertation (Ph.D.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Submitted by Rosana Amâncio (rosana.amancio@ufcg.edu.br) on 2018-07-11T20:08:46Z No. of bitstreams: 1 propriedades eletronicas, ópticas e vibracionais da região C.pdf: 18345145 bytes, checksum: e90bc1985f0f75d2e3d83ecaa39d4a34 (MD5) / Made available in DSpace on 2018-07-11T20:08:46Z (GMT). No. of bitstreams: 1 propriedades eletronicas, ópticas e vibracionais da região C.pdf: 18345145 bytes, checksum: e90bc1985f0f75d2e3d83ecaa39d4a34 (MD5) Previous issue date: 2016-07-29 / Neste trabalho, apresentamos um estudo das propriedades eletrônicas, ópticas, vibracionais e termodinâmicas da região C-Terminal de Fosfolipases A2 Lisina 49 (PLA2s Lys 49). Este foi realizado por meio de cálculos quânticos através da Teoria do Funcional da Densidade (DFT), utilizando a aproximação da densidade local (Local Density Approximation - LDA) e a aproximação do gradiente generalizado (Generalized Gradient Approximation - GGA). Também foram realizados cálculos baseados no modelo tight binding. As PLA2 Lys 49 compõe um grupo de miotoxinas que apresentam pouca ou nenhuma atividade catalítica e ainda assim são capazes de atuarem na membrana celular por meio de um mecanismo alternativo propiciando a morte da célula. A região C-terminal destas proteínas, em particular a região compreendida entre os aminoácidos 115-129, é apontada como responsável pelo dano as membranas. Pouco se sabe sobre as características que propiciam a esta região tal capacidade e como acontece a sua interação com a membrana celular. Este trabalho realizou uma caracterização das propriedades físicas desta região. Buscando, portanto, estabelecer uma relação entre as propriedades físicas expressas por essa região e seu potencial de dano celular. Um resultado inicial obtido por este estudo foram as curvas corrente-voltagem (I-V ) para nove diferentes peptídeos que correspondem as regiões 115-129 de PLA2 de diferentes espécies de serpentes. As curvas I-V foram obtidas por meio do modelo tight binding. Elas demostraram que os peptídeos estudados apresentam características de semicondutores. Também apresentam semelhança com resultados experimentais obtidos por LOMONTE et al, 2003. Usando a DFT, foram realizados os cálculos da área acessível ao solvente, da densidade eletrônica, análise populacional de cargas, orbitais de fronteira, densidade de estados. Também foram realizados cálculos de propriedades vibracionais como espectro infravermelho, de propriedades ópticas e das propriedades termodinâmicas como capacidade térmica, entropia, entalpia e energia livre. As propriedades eletrônicas demostram que há a possibilidade de que a interação da região C-Terminal com a membrana seja predominantemente eletrostática. A análise populacional de carga demostrou que o aminoácido Lisina 122 possui carga igual a zero. Este fato indica que ele pode não possuir papel importante como é descrito na literatura. Os resultados obtidos para a área acessível ao solvente indicam que um peptídeo com maior área disponível para interagir com a membrana não causará maior dano. Os resultados ópticos apresentaram picos de absorção dentro da região visível. Estes resultados juntamente com os resultados vibracionais servem como uma "digital" para a identificação dos peptídeos estudados. Os resultados termodinâmicos apresentados neste trabalhos podem ser utilizados em futuras pesquisas envolvendo PLA2s Lisina 49. / In this work, we present a study of the electrical properties, optical, vibrational and thermodynamic of the C-terminal Phospholipase A2 Lysine region 49 (PLA2 Lys 49). This was done by means of quantum calculations by Density Functional Theory (DFT), using the approach of Local Density Approximation (LDA) and the approach of the Generalized Gradient Approximation (GGA). They were also made calculations based on the model tight binding. The PLA2 Lys 49 composes myotoxins group that presents a little or no catalytic activity and still are able to act on the cell membrane by providing an alternative mechanism of cell's death. The C-terminal region of these proteins, in particular the region between amino acids 115-129 is identi_ed as responsible for the damage the membranes. Little is known about the characteristics that propitiate to this region such capacity and how are their interaction with the cell membrane. This work constitutes a characterization of the physical properties of this region. Searching, therefore, establish a relationship between the physical properties expressed by this region and its potential for cell damage. An initial results obtained in this study were current-voltage curves (I-V) for nine di_erent peptides corresponding to regions 115-129 PLA2 from di_erent snake species. The (I-V) curves were obtained by the model tight binding. They demonstrate that the peptides studied have semiconductor characteristics. They also have similarity with experimental results obtained by LOMONTE et al., 2003. Using the DFT were performed the calculations of the area accessible to the solvent, the electron density, population analysis of load, orbital border, and density of states. They were also carried out calculations of vibrational properties as infrared spectrum, optical properties and thermodynamic properties such as heat capacity, entropy, enthalpy and free energy. The electronic properties show that there is a possibility the interaction of the C-terminal region with the membrane it's predominantly electrostatic. The load population analysis showed that the amino acid Lysine 122 has load zero. This indicates that it may not have important role as described in the literature. The consequences obtained for the solvent accessible area indicates that a peptide with the largest area available to interact with the membrane not cause greater damage. The optical results presented absorption peaks in the visible region. These results together with the results vibrational serve like a "digital"to identify the studied peptides. The Thermodynamic results presented in this work can be used in future research involving PLA2 Lysine 49.

La voie NOTCH est activée de manière aberrante dans de nombreux cancers. Son activation implique la liaison d’un récepteur transmembranaire NOTCH à son ligand, engendrant une série de clivages qui libèrent le domaine intracellulaire de NOTCH appelé NIC. Ce dernier transloque au noyau, s’associe à ses partenaires transcriptionnels MAML1 et CSL pour réguler l’expression génique. À prime abord, la signalisation NOTCH apparaît donc simple. Cependant, il existe certainement des mécanismes de régulation précis encore mal connus à ce jour qui permettent de réguler finement cette voie de signalisation. Bien que des études montrent que NOTCH1 est constamment internalisé, l’activation de NOTCH1 au niveau des endosomes est controversée chez les mammifères. Nous n’avons pas pu déterminer clairement si l’activation de NOTCH1 pouvait se réaliser au niveau des endosomes. Néanmoins, nous avons observé que l’inhibition de l’endocytose réduit les niveaux d’expression de NIC1 suggérant une contribution des processus endocytiques dans la régulation de NOTCH1/NIC1. De plus, nous avons découvert qu’une forme de NIC1 non phosphorylée est rapidement dégradée dans le lysosome tandis qu’une forme de NIC1 hautement phosphorylée est dégradée par le protéasome. Les mécanismes de régulation de NIC1, une fois libéré, étant peu connus, une équipe avait généré des souris transgéniques ROSANic1. Cependant, nous avons remarqué que ce Nic1 était tronqué d’une grande partie de son domaine C-terminal. Nous avons donc généré une version humaine de NIC1 similaire au Nic1 des souris ROSANic1, nommé NIC1dC. Nous avons observé que NIC1dC est beaucoup plus stable que NIC1 et qu’il n’est plus dégradé par le protéasome. Cependant, nos résultats montrent qu’une plus grande stabilité de NIC1dC ne confère ni un plus fort pouvoir transcriptionnel à NIC1dC vs. NIC1 ni une plus grande capacité aux cellules de croître en indépendance d’ancrage comparativement aux cellules qui expriment NIC1. Nos analyses de spectrométrie de masse montrent que les partenaires d’interaction de NIC1dC sont différents de NIC1. Finalement, nos résultats démontrent que la délétion du domaine C-terminal de NIC1 augmente la stabilité de son partenaire transcriptionnel MAML1 et prévient sa phosphorylation. Dans leur ensemble, notre étude montre que la signalisation induite par NIC1dC ne phénocopie pas celle de NIC1 et suggère que le domaine C-terminal de NIC1 est important pour récapituler la durée et l’amplitude du signal NOTCH1 afin de médier des réponses cellulaires appropriées.

L’Integrase du VIH est une ADN recombinase catalysant deux réactions qui permettent l'intégration de l'ADN viral dans l'ADN hôte. L’intégrase du VIH comprend 3 domaines : N-terminal impliqué dans la réaction de « 3' processing » et le transfert de brin, le domaine catalytique contenant le site actif et le domaine C-terminal liant l'ADN non-spécifiquement (CTD). Des recherches récentes mettent en évidence l'importance du CTD dans la liaison avec d'autres protéines virales comme la transcriptase inverse. Le but de la thèse était de comprendre les rôles et l'importance du domaine C-terminal de l’intégrase dans deux contextes : l'intégration dans la chromatine et la coévolution, avec l'objectif de comprendre le rôle de la multimerisation dans la fonction de l’intégrase. Globalement, les résultats de mon projet indiquent que l'IN-CTD joue un rôle important, en contribuant à la formation de multimères d'ordre supérieur importants pour la fonction de l’IN. / HIV Integrase is a DNA recombinase that catalyzes two endonucleolytic reactions that allow the viral DNA integration into host DNA for replication and subsequent viral protein production. HIV Integrase consists of 3 structural and functional domains: The N-terminal zinc domain involved in 3’ processing and strand transfer, the catalytic core domain which contains the active site, and the C-terminal domain that binds DNA non- specifically. Recent research highlights the importance of the CTD in binding with other viral proteins such as Reverse Transcriptase. The aim of the thesis was to understand the roles and importance of the C-terminal domain of HIV-1 Integrase in two contexts: chromatin integration, and co-evolution, with the overall purpose of understanding the role of multimerization in IN function. Overall, results from my project indicate that the IN-CTD plays an important role, by contributing to the formation of higher order multimers that are important for IN functionality.

Transcription of human protein-coding genes and most small nuclear RNA genes is mediated by RNA Polymerase II (Pol II). During a cycle of transcription, Pol II recruits a variety of factors that facilitate transcription elongation, RNA processing and termination, through its long, unstructured C-terminal domain (CTD). The CTD in humans comprises 52 tandem heptapeptide repeats with the consensus sequence Y₁S₂P₃T₄S₅P₆S₇. Each amino acid of the heptapeptide can be chemically modified, which influences the recruitment of other protein factors to the transcription machinery. Not all enzymes that modify the CTD have been discovered. Recent studies have identified a novel CTD phosphatase: RPAP2 in humans and its yeast homologue Rtr1, which dephosphorylate phospho-Ser5 of the heptapeptide repeats. RPAP2 has been shown to stimulate 3' end cleavage of nascent snRNAs through recruitment of the Integrator complex, and unpublished work suggests the involvement of RPAP2 in regulating vertebrate developmental programs. However, the exact mechanisms that regulate the function of human RPAP2, and thus impact on CTD modification, are not well-understood. This thesis presents a novel mechanism whereby RPAP2 recruits protein phosphatase 1 (PP1) to snRNA genes, where PP1 is postulated to activate P-TEFb to phosphorylate Ser2 of the CTD. At the same time, P-TEFb may have a role in activating the phosphatase activity of RPAP2. Furthermore, RPAP2 itself is shown to be recruited to a number of gene promoters by the RPRD1A protein, which also stimulates its phosphatase activity. RPAP2 was shown to have another role in regulating transcription termination: by recruiting the Integrator complex, which is shown here to mediate termination of snRNA genes, and by a so far unknown mechanism on a long protein-coding gene. An attempt was made to purify and crystallise the human RPAP2 to obtain a crystal structure, however the crystallisation trials were not successful. Finally, a correlation was found in human embryonic stem cells and induced pluripotent stem cells between low levels of RPAP2 and high levels of CTD Ser5P, suggesting a potential involvement of RPAP2 in regulating transcription at a key developmental stage. The results presented here contribute to the understanding of human transcriptional mechanisms and the numerous interactions within the transcription machinery. In particular, the mechanism of terminating the transcription of snRNA genes is identified. An interesting possibility is the regulation of development and stem cell differentiation by RPAP2; however the exact pathways by which this occurs are yet to be discovered.

The melanocortin 3 and 4 receptors share 58% overall amino acid identity and 76% similarity. This high level of similarity between the MC3R and the MC4R underscores the difficulties associated with developing MC3R selective ligands, and as a consequence little is known of the physiological functions of the melanocortin 3 receptor. Previous research showing the differences between endogenous non-selective ligands and melanocortin 3 receptor selective ligands are mainly within the C-terminus of the melanocortin peptide. These findings have been exploited in this research using known melanocortin 3 and 4 selective ligands modified at their respective C-termini to develop some very promising melancortin 3 selective antagonists and agonists, analog 5 ([CO(CH₂)₂CO-DNal(2')-Arg-Trp-Lys]-Gly-Lys-Pro-Val-NH₂) and analog 20 ((H-DNal(2')-c[Asp-Pr6-DPhe-Arg-Trp-Lys]-Ala-Gly-Pro-Val-NH₂) respectively. Additional studies using molecular modeling have produced further insights into the structural basis for selectivity. Finally, we have been developing a new scaffold for the melanocortin receptor using cyclic dipeptide derivatives.

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