Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 83-97).

Knowing the sequence specificities of transcription factors allows us to surmise their functions and establish their regulatory roles in genomes. The most common DNA-binding domain among eukaryotic transcription factors is the Cys2His2 zinc finger domain; however, despite their prevalence, the specificities of the majority of Cys2His2 zinc finger proteins remain unknown due to the difficulty in assaying them. My objective was to develop a new phage displayed-based assay, in which individual Cys2His2 domains are displayed on phage in an otherwise constant three-finger protein scaffold. In Chapter 2, I discuss evidence for the modularity of the Cys2His2 domain, since my assay requires that zinc fingers be modular. In Chapter 3, I describe my results on the development of this phage display-based assay. This work provides support for a new strategy to determine the specificities of individual zinc fingers, which can be used to infer specificities for multi-finger Cys2His2 proteins.

Knowing the sequence specificities of transcription factors allows us to surmise their functions and establish their regulatory roles in genomes. The most common DNA-binding domain among eukaryotic transcription factors is the Cys2His2 zinc finger domain; however, despite their prevalence, the specificities of the majority of Cys2His2 zinc finger proteins remain unknown due to the difficulty in assaying them. My objective was to develop a new phage displayed-based assay, in which individual Cys2His2 domains are displayed on phage in an otherwise constant three-finger protein scaffold. In Chapter 2, I discuss evidence for the modularity of the Cys2His2 domain, since my assay requires that zinc fingers be modular. In Chapter 3, I describe my results on the development of this phage display-based assay. This work provides support for a new strategy to determine the specificities of individual zinc fingers, which can be used to infer specificities for multi-finger Cys2His2 proteins.

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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [255]-266).

Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-03Bitstream added on 2014-06-13T19:49:45Z : No. of bitstreams: 1 garcia_a_me_araiq.pdf: 1326845 bytes, checksum: c851f7e719ab97b784e7423d4226e61b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / 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... / 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)

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The growing obesity epidemic in America carries with it numerous health risks, including diabetes, increased serum lipid levels, and excess fat accumulation in the liver. If these conditions persist or become exacerbated, they may lead to the development of cardiovascular disease, the current leading cause of death among Americans, or to nonalcoholic fatty liver disease (NAFLD) which can progress to hepatocellular carcinoma (HCC), one of the deadliest forms of cancer. Better understanding of the genes involved in these diseases can lead to improved identification of at-risk individuals and treatment strategies. Our lab previously identified zinc fingers and homeoboxes 2 (Zhx2) as a regulator of hepatic gene expression. The BALB/cJ mouse strain has a hypomorphic mutation in the Zhx2 gene, causing a 95% reduction in Zhx2 protein expression. The near ablation of Zhx2 in BALB/cJ mice confers protection from cardiovascular disease when fed a high fat diet, yet these mice show increased hepatic lipid accumulation and liver damage. Microarray data indicates Zhx2 may be involved in the regulation of numerous genes involved in lipid metabolism. Recent GWAS studies indicate ZHX2 may contribute to the risk of cardiovascular disease and liver damage in humans as well. In this dissertation, I characterize the role of Zhx2 expression in the liver and how it affects the risk of both cardiovascular disease and liver damage. I generated liver-specific Zhx2 knockout mice and confirmed Zhx2 regulates several novel targets that could contribute to the fatty liver phenotype seen in BALB/cJ mice. Further studies revealed that hepatic Zhx2 expression is necessary for proper sex-specific expression of several Cyptochrome P450 (CYP) genes and could contribute to gender differences in disease susceptibility. Lastly, I performed studies into the functional role of the Zhx2 target gene Elovl3. A mouse model of HCC revealed that Elovl3 is completely repressed in HCC tumors. Cell viability and cell cycle assays indicate that Elovl3 expression slows cell proliferation and may be important for proper cell cycle checkpoints. Together, these data indicate that Zhx2 and/or its targets could be clinically relevant in the detection, prevention, or treatment of cardiovascular disease, fatty liver, and HCC.