Thesis advisor: Clare O'Connor / Within cells, reactive oxygen species (ROS) are synthesized naturally and in response to environmental stimuli. However, ROS have deleterious effects on a wide range of cellular molecules. Oxidative stress, caused by the ROS generated by the partial reduction of oxygen, is a major cause of cell damage linked to the initiation and progression of numerous diseases. Thioredoxin peroxidase (Tpx1) plays important roles in cellular defense against ROS. Although homologous genes and their functions have been identified in other eukaryotes, the level of activity as well as the necessity of this protective enzyme in S. pombe exposed to oxidative stress has yet to be fully elucidated. To explore the role of the Tpx1 protein in oxidative stress resistance, novel strains were constructed in which the tpx1 gene was overexpressed. The polymerase chain reaction was used to amplify txp1, and the amplified sequence was cloned into the yeast overexpression plasmid, pNMT41, which allows overexpression under the control of the powerful promoter. DNA sequencing was used to determine that the sequences had been properly inserted into the vector. The plasmids were transformed into two leu- yeast strains: FWP6 and TP108-3C. Production of the Tpx1 protein was ensured using Western Blot techniques. Experimentation to test the responses of the tpx1 strain to oxidative stress will employ a variety of reactive oxygen generators, including hydrogen n peroxide, menadione, tert-butyl hydroperoxide, and paraquat. The results generally supported the proposed role of Tpx1 to confer additional resistance against the oxidative stress. In a complementary line of investigation, knockout strains are being constructed to reduce the levels of the Tpx1 in S. pombe. Gene deletion cassettes were constructed for tpx1. Currently, the strains are being analyzed for the successful replacement of the endogenous tpx1 gene by homologous recombination. If the absence of the protein results in decreased cell viability, the role of Tpx1 indicated by the overexpression experiments could be supported. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Made available in DSpace on 2016-08-17T18:39:45Z (GMT). No. of bitstreams: 1 4644.pdf: 10384990 bytes, checksum: c8ab8c109d1671b477c700f556bd68bc (MD5) Previous issue date: 2011-08-26 / Universidade Federal de Minas Gerais / The thioredoxin peroxidase (Tpx) is a group of antioxidant proteins that has been widely studied due to its role in the decomposition of different peroxides such as H2O2, peroxynitrite and organic peroxides. The ability of peroxide decomposition by Tpx is related to the presence of a conserved cysteine called peroxidatic cysteine (CysP). Most Tpx has a second cysteine (resolving cysteine - CysR) which forms a disulfide with CysP after peroxide decomposition. In addition to the peroxidase activity, some Tpx have molecular chaperone activity and are also involved in signaling of cell growth induced by hydroperoxides. It has been demonstrated that the Tpx cytosolic isoform of Schizosaccharomyces pombe is able to interact directly with MAPK (Sty1) via mixed disulfide, which is stabilized when the CysR is replaced by a serine residue. Saccharomyces cerevisiae have a nuclear isoform of Tpx (nTPx) and review of the literature shows the importance of this protein in maintaining the telomere silencing and decomposition of organic peroxides in the nucleus. Scale proteomic studies using mass spectrometry and two-hybrid indicate the nTPx association with MAP kinases. However, despite its location and participation in biological processes of relevance, works related to nTPx are scarce. Scale proteomics studies reported the physical interaction between nTPx and Mec3, Gts1, Pc1 and Dog2. These proteins are related to cell signaling or maintenance of telomeric silencing. However, no specific studies were performed to confirm these interactions and if they are established by mixed disulfides. This study aimed to evaluate the interactions previously described in the literature between nTPx and Mec3, Pcl1, Dog2 and Gts1 through the expression and purification of these proteins and in vitro evaluation of interactions as well as in vivo tests using two-hybrid. Several efforts were made with different approaches, nevertheless it was impossible overexpression of Mec3, Pcl1, Dog2, indicating a toxic effect of these proteins on the strains used. Furthermore, we found great success in overexpression of nTPx and nTpxC112S (8 mg and 10 mg per liter of cell culture) in Eschericchia coli strain BL21 (DE3) C43. This is the first time that these proteins were expressed in native form. It was also possible to overexpress the Gts1 protein in the same strain. These results could lead for new approaches in future studies in order to determine these threedimensional structures, by methods such as X-ray crystallography or nuclear magnetic resonance (NMR). Finally, the results obtainedusing the technique of two-hybrid yeast confirmed the interaction in vivo among nTPx and Mec3, Gts1, Dog2. However, opposing the results described in the literature, no interaction was detected between nTPx and PCL1, emphasizing the necessity of specific experiments in addition to the large-scale ones. / As tiorredoxinas peroxidases (TPx), constituem um grupo de proteínas antioxidantes que vêm sendo bastante estudadas pela sua atuação na decomposição de diversos tipos peróxidos, como o H2O2, peroxinitritos e peróxidos orgânicos. A capacidade de decomposição de peróxidos pelas TPx está relacionada a presença de uma cisteína conservada denominada de cisteína peroxidásica (CysP). A maioria das TPx possuem uma segunda cisteína (cisteína de resolução - CysR) a qual forma um dissulfeto com CysP após a decomposição de um peróxido. Adicionalmente, à atividade peroxidásica, algumas TPx possuem atividade de chaperona molecular e também estão envolvidas em processos de sinalização de crescimento celular induzidos por hidroperóxidos. Já foi demonstrado que a isoforma citosólica de TPx de Schizosaccharomyces pombe é capaz de interagir diretamente com uma MAPK (Sty1) através da formação de um dissulfeto misto entre as proteínas, que é estabilizado quando a CysR é substituída por um resíduo de serina. Entretanto, nenhuma interação deste tipo foi descrita para outros organismos. Em Saccharomyces cerevisiae ocorre uma isoforma de TPx no núcleo (nTPx) e a revisão da literatura demonstra a relevância desta proteína na manutenção do silenciamento dos telômeros e decomposição de peróxidos orgânicos no núcleo. Estudos em escala proteômica utilizando espectrometria de massa e duplo híbrido indicam a associação de nTPx com MAP quinases, entretanto, apesar de sua localização e participação em processos biológicos de relevância, trabalhos relacionados com nTPx são escassos. Estudos em escala proteômica relataram a interação física entre nTPx e as proteínas Mec3, Gts1, Pcl1 e Dog2 relacionadas a sinalização celular ou manutenção do silenciamento telomérico. No entanto, não foram efetuados estudos pontuais visando confirmar estas interações como também averiguar a possibilidade das interações entre nTPx e as proteínas supracitadas serem estabelecidas através de dissulfetos mistos. Este trabalho teve por objetivo a avaliação de interações previamente descritas na literatura entre nTPx e Mec3, Pcl1 e Dog2 por meio da expressão e purificação destas proteínas e avaliação in vitro de interações como também in vivo através de ensaios de duplo híbrido. Diversos esforços com diferentes abordagens foram efetuados, entretanto não foi possível a superexpressão de Mec3, Pcl1, Dog2, indicando um efeito tóxico destas proteínas sobre as linhagens utilizadas. Por outro lado, obtivemos grande sucesso na superexpressão de nTPx e nTpxC112S (8 mg e 10 mg por litro de cultura de células) em linhagens de Eschericchia coli BL21 (DE3) C43, o que representa a primeira vez que estas proteínas foram expressas sem trucamentos. Também foi possível expressar na mesma linhagem a proteína Gts1. Estes resultados abrem a possibilidade de estudos posteriores visando a determinação de suas estruturas tridimensionais, por metodologias como cristalografia de raios-X ou ressonância magnética nuclear (RMN). Por fim, os resultados de interação in vivo utilizando a técnica de duplo híbrido em levedura, confirmaram a interação entre nTPx e Mec3, Gts1 e Dog2. Entretanto ao contrario dos resultados descritos na literatura, não foi detectada interação entre nTPx e Pcl1, reforçando que experimentos pontuais são necessários em adição aos experimentos de larga escala.