Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Resumo: YacG é uma pequena proteína (65 resíduos de aminoácidos) ligada ao zinco codificada pelo gene yacG de Escherichia coli. Seu papel fisiológico não está bem caracterizado, porém acredita-se que ela exerça ação inibitória sobre a atividade catalítica da DNA girase, enzima responsável por alterações no estado topológico do DNA bacteriano. Com base nas informações da estrutura primária desta proteína, uma série constituída de oito seqüências peptídicas foram projetadas e sintetizadas pela metodologia da fase sólida, objetivando-se avaliar e melhor entender o efeito da coordenação do íon zinco no seu mecanismo de ação. As sequências foram projetadas de maneira a resultar em uma substituição parcial ou integral dos resíduos de cisteína da sequência nativa da YacG, por resíduos de serina, além da variação da carga efetiva da molécula, por amidação ou acetilação das extremidades C e N terminais, respectivamente. Os peptídeos obtidos e purificados foram ensaiados quanto à estequiometria de coordenação empregando titulação com íon cobalto, bem como na capacidade inibitória frente à DNA girase, empregando eletroforese em gel de agarose. YacGAG4, inibiu a atividade de superenovelamento do DNA, catalisada pela girase, somente na ausência de íons zinco em concentrações inferiores a 120 μmol.L-1. Os demais peptídeos não apresentaram capacidade inibitória, tanto na presença quanto na ausência de zinco. Ensaios de susceptibilidade bacteriana, empregando algumas espécies de bactérias da família Enterobacteriaceae, confirmaram os resultados in vitro,com exceção das sequências YacGAG1-AC e YacGAG2-AC que mostraram inibição no crescimento bacteriano, sem porém resultarem em atividade in vitro. Com base nos resultados obtidos, é possível concluir que o domínio estrutural relacionado à coordenação do zinco, bem como a presença... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: YacG is a small protein (65 amino acid residues) bounded to zinc and encoded by the Escherichia coli yacG gene. Its physiological role is not well characterized, but it is believed that YacG is an inhibitor of the catalytic activity of DNA gyrase, an enzyme responsible for changes in the topological state of bacterial DNA. Based on information from the primary structure of this protein, a series of eight peptide sequences were designed and synthesized by solid phase methodology, aiming to evaluate and better understand the effect of zinc coordination of in their mechanism of action. The sequences were designed so as to result in a partial or full replacement of the cysteine residues of the native YacG sequence by serine residues and to change the effective charge of the molecule by amidation or acetylation of C and N terminal ends, respectively. The obtained peptides were purified and tested by titration with cobalt ion (coordination stoichiometry), as well as by inhibitory effect against the DNA gyrase, using agarose gel electrophoresis. YacGAG4 inhibited DNA supercoiling activity catalyzed by gyrase only in the zinc ions absence at concentrations below of 120 μmol.L-1. The other peptides showed no inhibitory effect in both the presence and absence of zinc. Bacterial susceptibility tests, using some species of bacteria of the Enterobacteriaceae, confirmed in vitro results, with the exception of the sequences YacGAG1-AC and YacGAG2-AC that showed inhibition of bacterial growth, but no in vitro activity. Based on these results, we conclude that the structural matters related to the coordination of zinc as well as the presence of this ion, showed no significant importance in the activity of DNA gyrase inhibition. In this case, the inhibition of activity recently proposed, should be linked to any other region of the protein molecule, structurally organized when the zinc ion is bound... (Complete abstract click electronic access below) / Orientador: Reinaldo Marchetto / Coorientador: Saulo Santesso Garrido / Banca: Clarice Queico Fujimura Leite / Banca: Vani Xavier de Oliveira Junior / Mestre

Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.

Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [114]-131).

No description available.

The liver is the largest internal organ and performs a wide variety of functions to maintain organismal homeostasis. While some liver functions are carried out by all hepatocytes, other functions are restricted to certain populations of hepatocytes within the liver. This phenomenon, called zonal gene regulation or liver zonation, controls may metabolic processes within the liver including ammonia detoxification; glucose homeostasis; bile acid and glutamine synthesis; and metabolism of xenobiotics, lipids, and amino acids. The liver also expresses many genes in a developmental or sex-biased manner. Some genes are expressed at higher levels early or late in development, or alternatively, in male or female liver. Several years ago, our lab identified a transcription factor called Zinc finger and homeoboxes 2 (Zhx2) based on its ability to control the silencing of genes that are normally expressed in the fetal liver. Zhx2 belongs to a small gene family that also includes Zhx1 and Zhx3. These four exon genes have a rather unique structure in that their entire protein coding region is located on an unusually large third exon. Preliminary studies indicate that these proteins are found only in vertebrates. I have performed a comprehensive analysis of Zhx proteins across a number of chordate species to determine their relationship throughout chordate evolution. Using multiple sequence alignment and phylogenetic tree-building, my studies have found that the primordial Zhx gene is most related to Zhx3 and that this gene exists in lower chordates including lancelet, sea squirt, and sea lamprey. Additional studies from our lab showed that Zhx2 regulates numerous hepatic genes in the adult liver, including cytochrome p450 (Cyp) genes as well as other genes that exhibit sex-biased expression. Previous studies have demonstrated that female-biased expression of Cyp2a4, is controlled, in part, by Zhx2. I have extended these studies to perform a comprehensive analysis of Cyp2a4 and the highly related Cyp2a5 gene. Despite the high similarity of these two Cyp genes, my data indicate that these genes exhibit different zonal expression patterns and are differentially regulated in the regenerating liver. In the course of these studies, I discovered and characterized antisense transcripts for both Cyp2a4 and Cyp2a5. Both Cyp2a4as and Cyp2a5as have positively correlated expression patterns compared to their sense counterparts. In contrast to Cyp2a4 and Cyp2a5, Cyp2a4as and Cyp2a5as show sex-biased expression patterns earlier in development, suggesting that they might contribute to later sex-biased patterns established for Cyp2a4 and Cyp2a5.

The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 70-81).