Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Brian J. Bahnson, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Peroxiredoxins are a family of multifunctional enzymes that are able to protect the cell against oxidative stress. Peroxiredoxins form part of a recently discovered peroxide scavenging system along with thioredoxin, thioredoxin reductase and sulfiredoxin. This study describes the purification of a recombinant human peroxiredoxin II from human erythrocytes. The original recombinant clone contained a point mutation at the fourth residue from glycine to valine and a number of problems were encountered with aggregation during purification. Reverting back to the original amino acid sequence allowed the protein to be purified and concentrated without aggregation, as well as leading to over-expression in the same oligomeric state as the native sample from blood. This study also describes the over-expression and purification of the human peroxiredoxin II protein in the intermolecular disulfide form as well as the subsequent crystallisation and X-ray diffraction studies. The crystal structure for this form of the protein was obtained to 3.3 Å resolution revealing the peroxiredoxin to be in the decameric form. In addition conformational changes in the protein that are necessary for formation of the intermolecular disulfide between the peroxidatic (Cys52) and resolving cysteine (Cys172) have been observed. The structure also revealed that these movements did not interfere with the dimer:dimer interface as had been previously suggested. This then allows the disulfide to be seen within the decameric form of peroxiredoxin. The production of covalent complexes formed between peroxiredoxin and sulfiredoxin, and peroxiredoxin and thioredoxin was also investigated. Complexes were stabilised by using DTNB to form a covalent bond between specific cysteine residues. The complex binding results from size exclusion chromatography showed that decameric peroxiredoxin bound to sulfiredoxin in a 1:5 ratio and decameric peroxiredoxin bound to thioredoxin in a 1:10 ratio. Cloning, over-expression and purification of the selenocysteine containing enzyme thioredoxin reductase was achieved. A minimal selenocysteine insertion sequence was added to the 3’ end of the DNA sequence to drive selenocysteine insertion in place of the typical stop UGA codon. The activity of this protein was found to be low but was greatly increased when co-expressed with a plasmid containing the selA, selB and selC genes. Although the activity of this co-expressed thioredoxin reductase was ~20% of the native enzyme activity, it was comparable to the activity of other recombinant forms of the enzyme. These studies report the purification of all of the proteins necessary to reform the peroxiredoxin system and allow the production of a working assay for peroxiredoxin activity. Together with the first report for a structure of a decameric disulfide form of human peroxiredoxin II a greater insight into the peroxiredoxin system has been obtained.

Peroxiredoxin 6 (PRDX6) is a bifunctional enzyme comprising a peroxidase and a Ca2+-independent phospholipase (iPLA2) activity. This renders the enzyme capable of detoxifying reactive oxygen species (ROS) and of catalyzing the liberation of arachidonic acid (AA) from cellular membranes. Released AA can be further metabolized to bioactive lipids including eicosanoids, which are involved in inflammation, cell growth, differentiation, invasion and proliferation. Human melanoma cells are often characterized by imbalances in both ROS and lipid levels, which can be generated by oncogenic signaling, altered metabolism or UV irradiation. In previous studies, a comparative proteome analysis of the Xiphophorus fish melanoma model revealed a strong upregulation of Prdx6 in benign and malignant lesions compared to healthy skin. As the Xiphophorus melanoma model displays in many respects molecular characteristics that are similar to human melanoma, I investigated the functional role of PRDX6 in human melanoma cells. The first part of the study deals with the regulation of PRDX6 in melanocytes and human melanoma cells. I could demonstrate that the protein level of PRDX6 was strongly enhanced by the induction of the EGFR orthologue Xmrk from the Xiphophorus fish as well as the human EGFR. The upregulation of PRDX6 was further shown to be mediated in a PI3K-dependent and ROS-independent manner. The main part of the thesis comprises the investigation of the functional role of PRDX6 in human melanoma cells as well as the analysis of the underlying mechanism. I could show that knockdown of PRDX6 enhanced the oxidative stress response and led to decreased proliferation of melanoma cells. This cell growth effect was mainly mediated by the iPLA2 activity of PRDX6. Under conditions of strongly enhanced oxidative stress, the peroxidase activity became also important for cellular proliferation. Furthermore, the anti-proliferative effect in cells with lowered PRDX6 levels was the result of reduced cellular AA content and the decrease in the activation of SRC family proteins. Similarly, supplementation with AA led to regeneration of SRC family kinase activity and to an improvement in the reduced proliferation after knockdown of PRDX6. Since AA can be further processed into the prostaglandin PGE2, which has a pro-tumorigenic function in some cancer types, I further examined whether this eicosanoid is involved in the proliferative function of PRDX6. In contrast to AA, PGE2 was not consistently required for melanoma proliferation. In summary, I could demonstrate that PRDX6 plays a major role in AA-dependent lipid signaling in melanoma cells and thereby regulates proliferation. Interestingly, the proliferation relevant iPLA2 activity can be pharmacologically targeted, and melanoma cell growth was clearly blocked by the inhibitor BEL. Thus, I could identify the phospholipase activity of PRDX6 as a new therapeutically interesting target for melanoma treatment. / Peroxiredoxin 6 (PRDX6) ist ein bifunktionales Enzym, welches neben seiner Peroxidase-Aktivität auch eine Ca2+-unabhängige Phospholipase-Aktivität besitzt. Aufgrund dieser beiden Aktivitäten ist das Enzym in der Lage, sowohl oxidativen Stress zu bekämpfen als auch die Freisetzung von Arachidonsäure aus zellulären Membranen zu katalysieren. Freie Arachidonsäure (AA) dient der Generierung von bioaktiven Lipiden wie zum Beispiel Eicosanoiden, welche an Entzündungsreaktionen, Zellwachstum, Differenzierung, Invasion und Proliferation beteiligt sind. Humane Melanomzellen zeichnen sich oft durch ein gestörtes Gleichgewicht reaktiver Sauerstoffspezies und zellulärer Lipide aus. Dieses Ungleichgewicht kann durch onkogene Signalgebung, einen veränderten Metabolismus oder UV-Bestrahlung hervorgerufen werden. Eine vorangegangene Proteomanalyse des Xiphophorus-Fisch-Melanommodells zeigte, dass im Vergleich zur gesunden Haut die Menge an PRDX6 in benignen und malignen Läsionen stark erhöht ist. Da das Xiphophorus-Melanommodell in vielerlei Hinsicht die molekulare Situation des humanen Melanoms wiederspiegelt, habe ich die funktionale Rolle von PRDX6 in humanen Melanomzellen untersucht. Der erste Teil der Studie beschäftigt sich mit der Regulierung von PRDX6 in Melanozyten und humanen Melanomzellen. Ich konnte nachweisen, dass die Menge an PRDX6 Protein durch die Induktion des EGFR Orthologs Xmrk aus Xiphophorus Fischen, sowie des humanen EGFR stark erhöht wurde. Auch konnte ich zeigen, dass die Heraufregulierung von PRDX6 von der Signalgebung der PI3 Kinase, aber nicht von reaktiven Sauerstoffspezies abhängig war. Der Hauptteil der vorliegenden Forschungsarbeit befasst sich mit der Ermittlung der funktionalen Rolle von PRDX6 in humanen Melanomzellen und der Analyse des zugrundeliegenden Mechanismus. Ich konnte nachweisen, dass ein Knockdown von PRDX6 die oxidative Stress-Antwort verstärkte und die Proliferation von Melanomzellen reduzierte. Der Effekt auf das zelluläre Wachstum wurde hierbei hauptsächlich durch die iPLA2-Aktivität von PRDX6 verursacht. Bei stark erhöhtem oxidativem Stress konnte auch eine Relevanz der Peroxidase-Aktivität für die zelluläre Proliferation nachgewiesen werden. Auch ging der anti-proliferative Effekt mit einer Abnahme zellulärer AA und der Reduktion aktiver Kinasen der SRC-Familie einher. Die Zugabe von AA zu Zellen mit PRDX6-Knockdown führte zur Regeneration der SRC-Kinase-Aktivität und konnte die Proliferation wieder verbessern. Da AA zum Prostaglandin PGE2 prozessiert werden kann, welches in einigen Krebsarten pro-tumorigene Funktionen erfüllt, untersuchte ich, ob dieses Eicosanoid auch für die proliferative Funktion von PRDX6 relevant ist. Im Gegensatz zu AA wies PGE2 jedoch keine kontinuierliche pro-proliferative Funktion auf. Zusammenfassend konnte ich zeigen, dass PRDX6 eine entscheidende Rolle im AA- Stoffwechsel von Melanomzellen spielt und hierdurch die Proliferation reguliert. Interessanterweise ist die proliferationsrelevante iPLA2-Aktivität pharmakologisch hemmbar, und auch das Wachstum der Melanomzellen wurde durch den Inhibitor BEL deutlich inhibiert. Mit der Phospholipase-Aktivität von PRDX6 konnte ich somit einen neuen therapeutisch nutzbaren Angriffspunkt für das Melanom identifizieren.

The formation of large, complex structures from small building blocks through self-assembly is widely seen in proteins and provides a tool for the creation of functional nanoscale devices. However, the factors controlling protein self-assembly are complex and often poorly understood. Peroxiredoxins are a large family of proteins, many of which are able to form a variety of large structures from a small, basic unit. This assembly has been shown to be strongly influenced by the redox state of the enzyme, which functions as a switch, controlling self-assembly. This thesis uses a protein from this family, human peroxiredoxin 3 (hPrx3) as a model system to investigate whether the self-assembly properties of hPrx3 can be influenced by rational protein engineering. Three forms of hPrx3 were purified and examined. These were the wild type and two variants: a mutant (S78A) and a His-tagged form. Size exclusion chromatography showed that each form showed a different ratio of dimers and larger species. Both variants showed preference for larger species, especially in the His-tagged form. This was shown to be partially dependent on metal binding in the His-tagged form. Larger species formed from multiple rings were also identified. SAXS measurement indicated that in the wild type enzyme, higher order species were dodecameric rings. For the His-tagged variant, SAXS measurement showed that the species observed had a different structure than that of the wild type. Electron microscopy showed that higher order structures seen in both wild type hPrx3 and His-tagged hPrx3 were ring shaped, with dimensions consistent with dodecamers. A competitive assay showed that the wild type, with kcat/km values near 2 x 10⁷, consistent with published results. Both variant forms showed evidence of slightly higher activity than the wild type, indicating a link between activity and assembly. A peroxiredoxin from the thermophilic bacteria Thermus aquaticus, TaqPrx was also examined, in an attempt to investigate a peroxiredoxin capable of self-assembly at high temperatures, which would be very useful for a nanoscale device. TaqPrx was cloned, purified and examined, however, no evidence of self-assembly was observed. Protein modelling and dynamic light scattering measurement indicated that the protein purified was monomeric and had a structure. Sparse matrix crystal screening identified conditions that allowed crystal formation, although strongly diffracting crystals were not produced. A novel assay for peroxiredoxin activity was developed, and suggested that TaqPrx shows peroxiredoxin activity. This thesis shows that peroxiredoxins are a useful model system for the investigation of how protein self-assembly is controlled, and how it can be influenced by protein engineering.

Philosophiae Doctor - PhD / XvPrx2 is a 1-Cys-containing member of the Prx5 subfamily of peroxiredoxins isolated from the resurrection plant Xerophyta viscosa. It is reported to be up-regulated during periods of desiccation and to protect nucleic acids and cellular proteins from oxidative damage through scavenging of reactive oxygen species, suggesting that it may play a role the desiccation tolerance of X. viscosa (Govender, 2006). Members of the Prx5 subfamily have previously been reported to occur as non-covalent homodimers associating across an A-type interface. PrxD from Populus tremula, a close homologue of XvPrx2, forms disulphide bonds with glutathione (glutathionylation) resulting in the unfolding of the Cp-loop and α2-helix and disruption of the homodimer, on the basis of which glutathionylation has been proposed as a physiological mechanism for regeneration of all members of the Prx5 subfamily (Noguera-Mazon, et al., 2006b).

Both vertebrates and anopheline mosquitoes inhibit <i>Plasmodium</i> spp. (malaria parasite) development via induction of nitric oxide (·NO) synthase. Expression of <i>Anopheles stephensi</i> ·NO synthase (<i>AsNOS</i>) is induced in the midgut epithelium beginning at 6 h following a <i>Plasmodium berghei</i>-infected blood meal. ·NO reacts readily with other biocompounds forming a variety of reactive nitrogen intermediates (RNIs) that may impose a nitrosative stress. These RNIs are proposed to be responsible for the AsNOS-dependent inhibition of <i>Plasmodium</i> development. In my studies, I identified several RNIs that are induced in the blood-filled midgut in response to <i>Plasmodium</i> infection. Stable end products of ·NO (NO₃⁻ and NO₂⁻), measured using a modified Griess assay, are elevated in infected midguts at 24 h post-blood meal (pBM). Further studies using chemical reduction-chemiluminescence with Hg displacement showed that infected midguts contained elevated levels of potentially toxic higher oxides of nitrogen (NO<SUB>x</SUB>), but <i>S</i>-nitrosothiol (SNO) and nitrite levels did not differ between infected and uninfected midguts at 12.5 and 24 h pBM. Thus, nitrates contributed to elevated NO<SUB>x</SUB> levels. SNO-biotin switch westerns indicated that <i>S</i>-nitrosated midgut proteins change over the course of blood meal digestion, but not in response to infection. Photolysis-chemiluminescence was used to release and detect bound ·NO from compounds in blood-filled midguts dissected from 0-33 h pBM. Results showed increased ·NO levels in <i>Plasmodium</i>-infected midgut lysates beginning at 8 h, with significant increases at 12.5-13.5 h and 24-25.5 h pBM and peak levels at 20-24 h. Photolyzed ·NO is derived from SNOs and metal nitrosyls. Since SNO concentrations did not change in response to infection, I proposed that metal nitrosyls, specifically Fe nitrosyl hemoglobin (nitrosylHb) based on the concentration of hemoglobin, were elevated in the infected midgut. At 12-24 h pBM, levels of midgut RNIs in infected mosquitoes were typical of levels measured during mammalian septic inflammation. The inverse relationship between AsNOS activity and parasite abundance indicates that nitrosative stress has a detrimental effect on parasite development. However, nitrosative stress may impact mosquito tissues as well in a manner analogous to mammalian tissue damage during inflammation. Elevated levels of nitrotyrosine (NTYR), a marker for nitrosative stress in many mammalian disease states, were detected in tissues of parasite-infected <i>A. stephensi</i> at 24 h pBM. Greater nitration of tyrosine residues was detected in the blood bolus, midgut epithelium, eggs and fat body. In the midgut, Hb remained in an oxygenated state for the duration of blood digestion. The reaction between ·NO and oxyhemoglobin (oxyHb) can result in the formation of nitrate and methemoglobin (metHb). Although nitrate levels were elevated in response to parasite infection, there was little to no metHb present in the mosquito midgut. The simultaneous presence of nitrates, nitrosylHb, oxyHb, and NTYR, together with a lack of elevated nitrites and metHb, suggested that alternative reaction mechanisms involving â ¢NO had occurred in the reducing environment of the midgut. In addition, I proposed that nitroxyl and peroxynitrite participated in reactions that yielded observed midgut RNIs. To cope with the parasite-induced nitrosative stress, cellular defenses in the mosquito may be induced to minimize self damage. I proposed that peroxiredoxins (Prx), enzymes that can detoxify peroxides and peroxynitrite, may protect <i>A. stephensi</i> from nitrosative stress. Six Prx genes were identified in the <i>A. gambiae</i> genome based on homology with known <i>D. melanogaster</i> Prxs. I identified one <i>A. stephensi</i> Prx, AsPrx, that shared 78% amino acid identity with a <i>D. melanogaster</i> 2-Cys Prx known to protect fly cells against various oxidative stresses. <i>AsPrx</i> was expressed in the midgut epithelium and is encoded by a single-copy, intronless gene. Quantitative RT-PCR analyses confirmed that induction of <i>AsPrx</i> expression in the midgut was correlated with malaria parasite infection and nitrosative stress. To determine whether AsPrx could protect against RNI- and ROS-mediated cell death, transient transfection protocols were established for AsPrx overexpression in <i>D. melanogaster</i> (S2) and <i>A. stephensi</i> (MSQ43) cells and for <i>AsPrx</i> gene silencing using RNA interference in MSQ43 cells. Viability assays in MSQ43 cells showed that AsPrx conferred protection against hydrogen peroxide, ·NO, nitroxyl and peroxynitrite. These data suggested that the ·NO-mediated defense response is toxic to both host and parasite. However, AsPrx may shift the balance in favor of the mosquito. / Ph. D.

Mecanismos redox estão envolvidos em diversos processos, como sobrevivência, proliferação e diferenciação celular, pela modulação da atividade de quinases, fosfatases e fatores de transcrição, entre outros, através da modificação oxidativa e reversível de resíduos de cisteína. Neste trabalho, nós estudamos processos redox subjacentes a diferenciação osteogênica induzida por BMP2, utilizando linhagens de células MC3T3-E1. Nosso objetivo foi investigar modificações redox como possíveis moduladores do processo de diferenciação osteogênica. Para isso, nós primeiramente caracterizamos a diferenciação osteogênica nas células MC3T3-E1 após o tratamento com BMP2, através da expressão do marcador osteogênico Osteocalcina, da fosforilação do complexo Smad 1/5/8 e da deposição de matriz extracelular calcificada. Análises de expressão gênica por qPCR mostraram que o tratamento com BMP2 resultou no aumento de expressão de NOX4, o que provavelmente leva ao aumento na produção de peróxido de hidrogênio intracelular. Nós investigamos também a modulação de peroxiredoxinas nesse processo e análises de expressão gênica mostraram que não há alterações nos níveis de expressão de Prx1 e 2 durante a diferenciação, mas os ensaios de western blot redox indicam que a Prx1 pode ser oxidada após o tratamento com BMP2, de maneira dose dependente. Outras análises in vitro mostram que células expostas a N-acetilcisteína (NAC) e PEG-catalase apresentam diferenciação osteogênica prejudicada, detectada por baixos níveis de deposição de matriz extracelular calcificada, comparado com células não-tratadas. Além disso, a fosforilação de Smad 1/5/8 são reduzidas nessas condições. Nossos dados sugerem que processos redox podem modular a sinalização celular durante o processo de diferenciação osteogência / Redox mechanisms are involved in several processes, such as cell survival, proliferation and differentiation, among other ways by modulating kinases, phosphatases and transcription factors activity that can occur through reversible and oxidative modification of cysteine residues. We were interested in studying redox processes underlying osteogenic differentiation induced by BMP-2, using MC3T3-E1 cell lineage. Our objective was to investigate redox modifications as possible modulators of the osteogenic differentiation process. We first characterized osteogenic differentiation in MC3T3-E1 cells upon BMP2 treatment, through gene expression of the osteogenic marker Osteocalcin, Smad 1/5/8 (belonging to the BMP-2 pathway) protein phosphorylation and extracellular matrix calcification. Gene expression analysis by qPCR showed that BMP2 treatment resulted in NOX4 upregulation, which probably also leads to hydrogen peroxide production. We have investigated peroxiredoxin modulation in this process, and gene expression analysis shows no significant change in peroxiredoxin 1 and 2 expression levels, but redox western blotting assays indicate that Prx1 can be oxidized after BMP2 treatment, in a dose dependent manner. In vitro analysis shows that cells exposed to N-acetyl-L-cysteine (NAC) and PEG-catalase display impaired osteogenic differentiation, detected by lower levels of calcified extracellular matrix deposition compared with non-treated cells. Moreover, phosphorylation of Smad 1/5/8 complex is reduced under these redox treatments. Our data suggest that redox pathways can modulate cell signaling during the osteogenic differentiation process

Type 1 and type 2 diabetes are characterized by an excessive loss of insulin producing β-cells. β-cells are particularly susceptible to increased oxidative stress induced apoptosis due to low expression of major antioxidants. Peroxiredoxin-2 (PRDX2) belongs to a group of antioxidants with antiapoptotic roles. Preliminary data indicate PRDX2 is expressed in the β-cells. Endogenous PRDX2 in the β-cell line MIN6 is found to decrease under oxidative stress conditions. I hypothesize that PRDX2 has a role in protecting β-cells against oxidative stress induced apoptosis. Overexpression or knockdown strategies were used to examine the role of PRDX2 in insulin-secreting MIN6 cells treated with various stimuli (cytokines, palmitate, streptozotocin) to induce apoptosis. Results showed that PRDX2 overexpression decreased oxidative stress induced apoptosis markers and cell death indicators, whereas knockdown of PRDX2 exaggerated oxidative stress induced toxicity. These findings suggest that PRDX2 plays a protective role in pancreatic β-cells under oxidative stress conditions.

Type 1 and type 2 diabetes are characterized by an excessive loss of insulin producing β-cells. β-cells are particularly susceptible to increased oxidative stress induced apoptosis due to low expression of major antioxidants. Peroxiredoxin-2 (PRDX2) belongs to a group of antioxidants with antiapoptotic roles. Preliminary data indicate PRDX2 is expressed in the β-cells. Endogenous PRDX2 in the β-cell line MIN6 is found to decrease under oxidative stress conditions. I hypothesize that PRDX2 has a role in protecting β-cells against oxidative stress induced apoptosis. Overexpression or knockdown strategies were used to examine the role of PRDX2 in insulin-secreting MIN6 cells treated with various stimuli (cytokines, palmitate, streptozotocin) to induce apoptosis. Results showed that PRDX2 overexpression decreased oxidative stress induced apoptosis markers and cell death indicators, whereas knockdown of PRDX2 exaggerated oxidative stress induced toxicity. These findings suggest that PRDX2 plays a protective role in pancreatic β-cells under oxidative stress conditions.

xi, 81 p. : ill. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Post-translational modification of proteins is an important cellular method of controlling various aspects of protein activity, including protein-protein interactions, half- life, and transport. An important class of post-translational modifications involves the ubiquitin family of proteins. In these modifications, a small protein, such as ubiquitin, is conjugated to a target protein through an isopeptide bond. Conjugation by a ubiquitin family member acts as a signal to regulate the activity, function, or stability of the target protein. Urm1, a ubiquitin-like protein conserved throughout all eukaryotes, was initially identified in S. cerevisiae. Loss of Urm1 leads to the disruption of a variety of cellular processes, including oxidative stress response, filamentous growth, and temperature sensitivity. This body of work comprises efforts to identify novel targets of Urm1, the mechanism by which Urm1 is attached to target proteins, and the physiological consequences of such conjugation. To gain understanding of the function and mechanism of Urm1 conjugation, the only known conjugate of Urm1, the peroxiredoxin reductase Ahp1, was examined in an effort to identify the site of modification on Ahp1 and to evaluate the physiological consequences of urmylation of Ahp1. I then completed a series of screens--a synthetic lethal screen, a two-hybrid screen, and a protein over-expression screen--to identify novel Urm1 conjugates and cellular functions dependent on Urm1. Of particular interest were genes identified in the synthetic lethal screen, namely PTC1, which encodes a protein phosphatase, and a set of genes encoding the Elongator complex, which functions in transcriptional elongation and tRNA modification. During this time period, other groups showed that thiolation of tRNAs depends on Urm1. Thus, Urm1 does not function only in protein conjugation, but also as a sulfur carrier in the thiolation of tRNA. Interestingly, I identified Elp2, a component of the Elongator complex, as a new Urm1-conjugate. Because Elp2 is also required for tRNA modification, perhaps Urm1 plays more than one role in tRNA modification. Loss of tRNA modification may disrupt many cellular functions and could explain the variety of urm1 mutant phenotypes. I have determined that all known Urm1 dependent processes are also associated with tRNA modification. / Committee in charge: Karen Guillemin, Chairperson, Biology; George Sprague, Advisor, Biology; Alice Barkan, Member, Biology; Kenneth Prehoda, Member, Chemistry; Tom Stevens, Outside Member, Chemistry