Biometals such as copper, cobalt and zinc are essential to life. These transition metals are used as cofactors in many enzymes. Nonetheless, these metals cause deleterious effects if their intracellular concentration exceeds the cells' requirement. Prokaryotic organisms usually employ efflux systems to maintain metals in appropriate intracellular concentrations.The Cus system of Escherichia coli plays a crucial part in the copper homeostasis of the organism. This system is a tetrapartite efflux system, which includes an additional component compared to similar efflux systems. This fourth component is a small periplasmic protein, CusF. CusF is essential for full copper resistance, yet its role within the Cus system has not been characterized. It could potentially serve in the role of a metallochaperone or as a regulator to the Cus system.To gain insight into the molecular mechanism of resistance of this system, I have structurally and biochemically characterized CusF. Using X-ray crystallography I determined the CusF structure. CusF displays a novel fold for a copper binding protein. Through multiple sequence alignment and NMR chemical shift experiments, I proposed a metal binding site in CusF, which I confirmed through determination of the structure of CusF-Ag(I). CusF displays a novel coordination of Ag(I) and Cu(I) through a Met2His motif and a cation-pi interaction between the metal ion and a tryptophan sidechain. Furthermore, I have shown that CusF binds Cu(I) and Ag(I) specifically and tightly.I investigated the role of the tryptophan at the binding site to establish its effect on metal binding and function of CusF. I have shown through competitive binding assays, NMR studies and through collaborative EXAFS studies that the tryptophan plays an essential role in CusF metal handling. The affinity of CusF for Cu(I) is influenced by this residue. Moreover, the tryptophan also caps the binding site such that oxidation of the bound metal as well access to adventitious ligands is prevented. In summary, these findings show that the structure and metal site of CusF are unique and are specifically designed to perform the function of CusF as a metallochaperone to the Cus system.

Ph.D. / Biochemistry and Molecular Biology / Selenocysteine-containing mutants of human copper chaperone for superoxide dismutase (hCCS) were constructed using intein-mediated peptide ligation. These mutants were studied with respect to their ability to transfer Cu to E,Zn superoxide dismutase (SOD1) and their Cu-binding and X-ray absorption spectroscopic (XAS) properties. Previous studies have shown that three functionally distinct polypeptide domains are present in CCS: the N-terminal domain 1 (D1, residues 1-85) contains the copper-binding MXCXXC motif, domain 2 (D2, residues 86-234) has sequence homology to residues associated with the native SOD1 dimer interface, and the C-terminal domain 3 (D3, residues 235-274) contains a CXC motif. Recent results suggest the formation of a D3- D3 cluster within a dimeric or tetrameric protein and suggest that this cluster may be an important element of the copper transfer machinery. D3 cysteine-to-selenocysteine mutants of wild-type and D1 mutants of hCCS were constructed to investigate the D3 copper cluster in more detail. These mutants display similar activity to wild-type protein. The structure of the Cu centers of selenocysteine-containing mutants as shown by Cu EXAFS is similar to that of wild-type protein, with clear indications of a Cu cluster. Cu and Se EXAFS of these constructs reveal a unique adamantane-like cluster formed between two molecules of CCS at the D3-D3 interface. These results confirm the existence of a D3-D3 copper cluster in hCCS and suggest that a unique copper cluster may exist in this protein.

Many processes in living systems occur through transient interactions among proteins. Those interactions are often weak and are driven by small changes in free energy. Due to the short-living nature of these interactions, our knowledge about driving forces, dynamics and structures of these types of protein-protein heterocomplexes are though limited. This is especially important for cellular copper (Cu) trafficking: Copper ions are essential for all eukaryotes and most bacteria. As a cofactor in many enzymes, copper is especially vital in respiration or detoxification. Since the same features that make copper useful also make it toxic, it needs to be controlled tightly. Additionally, in the reducing environment of the cytosol, Cu is present as insoluble Cu(I). To circumvent both toxicity and solubility issues, a system has evolved where copper is comforted by certain copper binding proteins, so-called Cu-chaperones. They transiently interact with each other to distribute the Cu atoms in a cell. In humans, one of them is Atox1. It binds copper with a binding site containing two thiol residues and transfers it to other binding sites, mostly those of a copper pump, ATP7B (also known as Wilsons disease protein). My work was aimed at understanding copper-mediated protein-protein interactions on a molecular and mechanistic level. Which amino acids interact with the metal? Which forces drive the transfer from one protein to the other? Using biophysical and biochemical methods such as chromatography and calorimetry on wild type and point-mutated proteins in vitro, we found that the copper is transferred via a dynamic intermediate complex that keeps the system flexible while shielding the copper against other interactions. Although similar transfer interactions can be observed in other organisms, and many conclusions in the copper field are drawn from bacterial and yeast analogs, we believe that it is important to investigate human proteins, too. Not only is their regulation different, but also only in humans we find the diseases linked to the proteins: Copper level regulation diseases are to be named first, but atypical copper levels have also been linked to tumors and amyloid dispositions. In summary, my observations and conclusions are of basic research character and can be of importance for both general copper and human medicinal research.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2018-04-19T17:09:39Z No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-23T11:47:10Z (GMT) No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-23T11:47:10Z (GMT). No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-13 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Paracoccidioidomycosis (PCM) is an important mycosis of Latin America, caused by thermodymorphic fungi of the genus Paracoccidioides. Homeostasis of metals such as copper, zinc and iron is important for the survival of fungi in the host environment. In this context, copper is an important cofactor for several enzymes, such as as superoxide dismutases and cytochrome c oxidase. The excess free copper in the cell promotes accumulation of reactive oxygen species, causing damage to nucleic acids, lipids and proteins. Thus, organisms shall to maintain cytoplasmic levels of that micronutrient at non-toxic levels, just sufficient for cell growth and vital metabolic processes. The metabolism of that metal is strictly controlled by high and low affinity uptake systems. The objective of this work is to perform heterologous expression of the Atx1 protein, described in the literature as a copper chaperone, and to analyze its cellular location, as well as the protein behavior upon copper deprivation in Paracoccidioides spp. Heterologous expression was performed on electrocompetent cells of Escherichia coli strain BL21. Identity of the recombinant protein was confirmed by LC-MS / MS. BALB/c mice were immunized to obtain anti-Atx1 polyclonal antibodies. After performing immunoblotting the produced antibodies were used in immunofluorescence assays. Analysis by qRT-PCR allowed us to evaluate the levels of the transcript encoding the Atx1 protein during copper deprivation in Paracoccidioides spp. / A paracoccidioidomicose (PCM) é uma importante micose da América Latina, causada por fungos termodimórficos do gênero Paracoccidioides. A homeostase de metais como cobre, zinco e ferro é importante para a sobrevivência dos fungos no ambiente do hospedeiro. Nesse contexto, o cobre é um importante cofator para várias enzimas, como as superóxido dismutases e a citocromo c oxidase. O excesso de cobre livre na célula promove o acúmulo de espécies reativas de oxigênio, causando danos a ácidos nucléicos, lipídeos e proteínas. Assim, os organismos devem manter concentrações citoplasmáticas desses micronutrientes em níveis não tóxicos, apenas suficiente para o crescimento celular e processos metabólicos vitais. O metabolismo desse metal é rigorosamente controlado por sistemas de captação de alta e baixa afinidade. O objetivo do trabalho é realizar a expressão heteróloga da proteína Atx1, descrita na literatura como uma chaperona citosólica de cobre e analisar a sua localização celular, bem como o comportamento diante da privação de cobre em Paracoccidioides spp. A expressão heteróloga foi realizada em células eletrocompetentes de Escherichia coli, cepa BL21. A identidade da proteína recombinante foi confirmada por LC-MS/MS. Camundongos BALB/c foram imunizados para obtenção de anticorpos policlonais anti-Atx1. Após realização de imunoblotting, os anticorpos produzidos foram utilizados para ensaios de imunofluorescência em células leveduriformes. Análise por qRT-PCR nos permitiram avaliar os níveis do transcrito codificante da proteína Atx1 durante a privação de cobre em Paracoccidioides spp.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.