Friedreich Ataxia (FRDA) is an autosomal recessive degenerative disorder. It is caused by an abnormal expansion of GAA trinucleotide repeats in the first intron of the gene encoding frataxin. Since rates of cell division have been linked to oxidative stress, we have examined several parameters of oxidative stress in a FRDA primary fibroblast cell line that had a dramatically different growth rate. In the FRDA fibroblasts, the high level of reactive oxygen species (ROS) indicated elevated oxidative stress. The elevated glutathione peroxidase (Gpx) activity in the ROS defense system may represent an adaptive response to the high oxidative stress. The increased mitochondrial membrane potential (MMP) likely contributed to increased oxidant production, which could be contributed by elevated ROS. This increased oxidant production might be responsible for increased rate of progression through the cell cycle.<p> Furthermore, the elevated oxidative stress is also associated with progressive neural pathology of FRDA. In FRDA, pathology is first seen in the dorsal root ganglia and the dorsal columns of the spinal cord. Due to the abnormal metal distribution seen in the FRDA spinal cord and medulla, we hypothesized that metal binding proteins were abnormally distributed in FRDA. In our FRDA samples, we observed the well established histopathology of FRDA and examined the distribution of some metal binding proteins (frataxin, ferritin and metallothionein) through immunohistochemistry. Our results showed demyelination and loss of axons in the degeneration areas of the two FRDA cases. In addition, we found that the metal binding proteins were abnormally distributed in the FRDA spinal cord and the medulla. The abnormal distributions of the metal binding proteins were characterized by low expressions of iron binding proteins, especially frataxin and cytosolic ferritin, and undetectable expression of the copper and zinc binding protein, metallothionein. In summary, the rapid cell growth is a feature of FRDA fibroblast cell lines. We also tested Gpx activity, measured oxidant levels and determined the MMP in a FRDA primary fibroblast cell line that had a dramatically fast growth rate. The FRDA histopathology studies showed the metal binding proteins including frataxin, ferritin and metallothionein were abnormally distributed in the spinal cord and the medulla.

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In our efforts to engineer a DNA binding and cleaving protein with greater sequence discrimination, we have designed dimeric proteins derived from engrailed homeodomain and calmodulin. Previous research by our group has shown that a hydrolytically active lanthanide binding site can be incorporated into a DNA binding motif. To understand protein-DNA interaction and improve the sequence selectivity of the chimeric complex, two lanthanide-binding homodimers were designed and expressed. One of the dimers, F2, is coupled together by a flexible polypeptide linker and the other, R7C, is a disulfide cross-linked cysteine mutant at the N-terminus. Studies of fluorescence of tryptophan residues document that the overall affinity for lanthanide and calcium is similar to traditional EF-hand peptides (1-10 μM). Metal titrations monitored by circular dichroism (CD) revealed that the secondary structures of the dimers contained a lower degree of -helicity than the designed monomeric protein due to additional modifications, but because of their flexibility and their two active-site domain, hydrolytic activity was several folds faster than our previously designed proteins and peptides. Unlike earlier reports on our chimeras, F2 also demonstrated the capability to hydrolyze DNA in the presence of some biological relevant metal ions suggesting different cleavage mechanisms were carried out. Extensive DNA sequencing studies on cleavage patterns with oligonucleotide duplexes confirmed the unique sequence selectivity and kinetic properties of F2. Two engrailed homeodomain target sites, TAATTA, were favored for hydrolytic activity corresponding to one domain acting as a DNA anchor on the first target site while the other was an "opportunist" at recognizing the second site. Nonetheless, the hydrolytic behavior at the phosphodiester bond on a specific dsDNA sequence is in good agreement with the behavior of restriction endonucleases. Unlike restriction enzymes, metallated F2 has not only demonstrated the ability to cleave DNA plasmid, but it also excises the entire nucleotide on a selected sequence. This homodimer is the first example of an active and selective hydrolytic artificial nuclease based on the modular turn substitution design approach that can be a potential template for genomic modification.

Orientador: Pedro de Magalhães Padilha / Resumo: Devido a sua grande concentração de nutrientes, tais como proteínas, vitaminas e minerais, o peixe é considerado um dos alimentos mais saudáveis que se pode encontrar na natureza. No entanto, a ingestão de peixes é considerada a forma predominante de via de exposição do ser humano ao mercúrio (Hg), principalmente para as populações que vivem às margens dos rios, onde o peixe constitui a principal fonte de proteína. Na tentativa de elucidar os mecanismos de toxicidade das espécies mercuriais, o teor desse metal tem sido estudado intensamente pela comunidade científica nas últimas décadas em amostras de solo, sedimentos, humanos e peixes na Amazônia brasileira. Sabe-se que as espécies mercuriais bioacumuladas nos tecidos dos seres vivos ligam-se a metaloproteínas, e quando há uma concentração alta de metal tóxico nos organismos, esses passam a expressar proteínas de defesa, denominadas metalotioneínas (MTs) responsáveis pelo transporte e eliminação de metais tóxicos. Apesar de estudos mostrarem o aumento das metalotioneínas em animais expostos a metais potencialmente tóxicos, essas proteínas não foram caracterizadas para confirmação de sua veridicidade, são analisadas por métodos indiretos, esse fato leva a necessidade de técnicas mais precisas na identificação de metalotioneínas. Levando em consideração o exposto esse estudo teve como objetivo otimizar métodos de quantificação de mercúrio e técnica de eletroforese para identificação de possíveis metalotioneínas biomarcadoras d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to its high concentration of nutrients, such as proteins, vitamins and minerals, fish is considered one of the healthiest foods that one can find in nature. However, fish intake is considered to be the predominant human exposure pathway to mercury (Hg), especially for populations living along riverbanks where fish are the main source of protein. In the attempt to elucidate the toxicity mechanisms of mercurial species, the content of this metal has been intensively studied by the scientific community in recent decades in soil, sediment, human and fish samples in the Brazilian Amazon. It is known that mercurial species bioaccumulated in the tissues of living beings bind to metalloproteins, and when there is a high concentration of toxic metal in organisms, they begin to express defense proteins, called metallothioneins (MTs) responsible for the transport and elimination of Toxic metals. Although studies have shown the increase of metallothioneins in animals exposed to potentially toxic metals, these proteins have not been characterized to confirm their veridicity, are analyzed by indirect methods, this fact leads to the need for more precise techniques in the identification of metallothioneins. Taking into account the above, this study aimed to optimize mercury quantification methods and electrophoresis technique for identification of possible mercury biomarkers metallothionein in muscular and hepatic tissue of fish of economic interest, Tucunaré (Cichla spp.), Filhote (Bra... (Complete abstract click electronic access below) / Doutor

Orientador: Marco Aurelio Zezzi Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-07T09:23:21Z (GMT). No. of bitstreams: 1 Garcia_JerusaSimone_D.pdf: 2794112 bytes, checksum: 68557f9b9cfbc996644d2a33b82031bc (MD5) Previous issue date: 2006 / Doutorado / Quimica Analitica / Doutor em Ciências

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Protein pK(a)s and metalloprotein reduction potentials are studied with computational methodologies based on an ab initio quantum mechanics (QM) description of the protein and a linearized Poisson-Boltzmann Equation (LPBE) description of the solvent. The practical applicability of the QM/LPBE method is extended to proteins by using a QM description of the ionizable residue and a molecular mechanics (MM) description of the rest of the protein. This QM/MM/LPBE method is used to predict the pKa of Lys55 in the serine protease inhibitor turkey ovomucoid third domain (OMTKY3) and the prediction of 11.0 is in good agreement with the experimental value of 11.1. This is the first time a protein pKa value has been predicted with QM/MM methods. The QM/LPBE method is used to predict and interpret the pKa values of the five carboxyl residues (Asp7, Glu10, Glu19, Asp27, and Glu43) in OMTKY3. All the predicted pKa values are within 0.5 pH units of experiment, with a root mean square deviation of 0.31 pH units. We find that the decreased pKa values observed for some of the residues are primarily due to hydrogen bonds to the carboxyl oxygens. Hydrophobic effects are also shown to be important in raising the pKa. Interactions with charged residues are shown to have relatively little effect on the carboxyl pKa values in this protein, in general agreement with experiment. The relative Cu2+/Cu+ reduction potentials of six type-1 copper sites (cucumber stellacyanin, P. aeruginosa azurin, poplar plastocyanin, C. cinereus laccase, T. ferrooxidans rusticyanin and human ceruloplasmin), which lie in a reduction potential range from 260 mV to over 1000 mV, have been studied with the QM/LPBE method. For the first time, the range and relative orderings of the reduction potentials are reproduced well compared to experimental values. The study suggests that the main interactions determing the relative reduction potentials of blue copper sites are located within 6 Å of the Cu atoms. Further analysis suggests that the reduction potential differences of type-1 copper sites are caused by axial ligand interactions, hydrogen bonding to the S(Cys), and protein constraints on the inner sphere ligand orientations.

is a functionally diverse large group of proteins sharing a jelly roll β-barrel fold. An enzymatic member 3-hydroxyanthranilate-3,4-dioxygenase (HAO) and a non-enzymatic member pirin, which is a human nuclear metalloprotein of unknown function present in all human tissues, were selected for structural and functional studies in this dissertation work. HAO is an important enzyme for tryptophan catabolism and for 2-nitrobenzoic acid biodegradation. In this work, seven catalytic intermediate were captured in HAO single crystals, enabling for the first time a nearly complete structural snapshot viewing of the entire molecular oxygen activation and insertion mechanism in an iron- and O2-depedent enzyme. The rapid catalytic turnover rate was found achieved in large part by protein dynamics that facilitates O2 binding to the catalytic iron, which is bound to the enzyme by a facile 2-His-1-carboxylate ligand motif. An iron storage and chaperon mechanism was also discovered in the bacterial source of this enzyme, which led to a proposed novel biological function of a mononuclear iron-sulfur center. Although human pirin protein shares the same structural fold with HAO, its iron ion is coordinated by a 3-His-1-carboxylate ligand motif. Pirin belongs to a subset of proteins whose members are playing regulatory functions in the superfamily. In this work, pirin is shown to act as a redox sensor for the NF-κB transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to pro-inflammatory signals which controls the expression of a vast array of genes involved in immune and stress responses.

X-ray absorption is not only element specific, but atom specific: two atoms of the same element in different states or in different neighbourhoods will have slightly different absorption characteristics. These energy dependent atomic form factors are carried over to the diffraction intensities. The atomic form factors are sensitive not only to the the energy of the X-ray but also the diffraction criteria; providing individual local physical data at different ratios in various diffractions. This process is referred to as site selectivity, it is unique to Diffraction Spectroscopy, and is achieved only when the sample is in crystal form. Through this work, a technique has been devised to site-separate two atoms of iron from within a protein, that builds on prior small unit cell Diffraction Anomalous Fine Structure experiments and harnesses the collection and processing software commonly used in large unit cell crystallography. A technique (dev + PCA) has been developed to retrieve the small signals from individual atom-labels out of the large and noisy background of real diffraction taken across a spectrum. The intensity of the diffractions are calculated by integrating over multiple images, profiling spots, merging datasets, and scaling across the whole spectrum. This thesis explores how Diffraction Spectroscopy can be used effectively on large unit cells, namely those of proteins. Site-selective absorption experiments were conducted on large unit cell crystals at a 3rd generation beamline, exclusively using existing equipment. The spectra generated were limited in scope but are an adequate proof of concept.