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The KATP channel is a key regulator of the pancreatic β-cell, effectively linking metabolic status to electrical activity. Syntaxin-1A has been previously reported by our lab to both bind and inhibit the KATP channel via the nucleotide-binding folds (NBFs). The purpose of this thesis project was to elucidate the precise regions within the NBFs responsible for the Syn-1A- KATP interaction. In vitro binding assays revealed that Syn-1A associates with the Walker domains of both NBF1 and NBF2. Furthermore, site directed mutagenesis of the conserved lysine in Walker A of both NBFs abolishes Syn-1A affinity for this region. Electrophysiological recordings indicate that channel inhibition was mediated primarily through interaction with NBF1-Walker B and both Walkers of NBF2. Based on these results, we propose a model by which Syn-1A acts as an inhibitory clamp on the KATP channel, effectively buffering minor fluctuations in ATP/ADP concentration to prevent unnecessary channel activity.

The KATP channel is a key regulator of the pancreatic β-cell, effectively linking metabolic status to electrical activity. Syntaxin-1A has been previously reported by our lab to both bind and inhibit the KATP channel via the nucleotide-binding folds (NBFs). The purpose of this thesis project was to elucidate the precise regions within the NBFs responsible for the Syn-1A- KATP interaction. In vitro binding assays revealed that Syn-1A associates with the Walker domains of both NBF1 and NBF2. Furthermore, site directed mutagenesis of the conserved lysine in Walker A of both NBFs abolishes Syn-1A affinity for this region. Electrophysiological recordings indicate that channel inhibition was mediated primarily through interaction with NBF1-Walker B and both Walkers of NBF2. Based on these results, we propose a model by which Syn-1A acts as an inhibitory clamp on the KATP channel, effectively buffering minor fluctuations in ATP/ADP concentration to prevent unnecessary channel activity.

O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira. / The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.

O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira. / The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.

<p>Life on Earth is dependent on the oxygen produced through photosynthesis. The thylakoid membrane is the site for the light-driven reactions of photosynthesis, which oxidize water and supply energy in the form of ATP, mainly for carbon fixation. The utilization of ATP in the lumenal space of the thylakoid has not been considered in the past. In the latest years, increasing evidence for nucleotide metabolism in the thylakoid lumen of plant chloroplasts has been presented; ATP transport across the thylakoid membrane, and GTP binding to the PsbO extrinsic subunit of the water-oxidizing photosystem II (PSII) complex.</p><p>In this thesis, various methods for prediction, identification, and characterization of novel plant proteins, are described. Nucleotide-binding motifs and nucleotide-dependent processes are reviewed, and the experimental data is discussed. 1) A thylakoid ATP/ADP carrier (TAAC) in Arabidopsis thaliana was identified and functionally characterized, and 2) the spinach PsbO protein was characterized as a GTPase. The Arabidopsis At5g01500 gene product is predicted as a chloroplast protein and to be homologous to the well-studied mitochondrial ADP/ATP carrier. The putative chloroplast localization was confirmed by transient expression of a TAAC-green fluorescent protein fusion construct. Immuno detection with peptide-targeted antibodies and immunogold electron microscopy showed the thylakoid as the main localization of TAAC, with a minor fraction in the chloroplast envelope. TAAC is readily expressed in etiolated seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. It is proposed that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover. Recombinant TAAC protein was functionally integrated in the cytoplasmic membrane of Escherichia coli, and was shown to specifically transport ATP/ADP in a protonophore-sensitive manner, as also reported for mitochondrial AACs.</p><p>The PsbO protein stabilizes the oxygen-evolving complex of PSII and is ubiquitous in all oxygenic photosynthetic organisms, including cyanobacteria, green algae, and plants. So far only the 3D-structure of the cyanobacterial PsbO is available. Four GTP-binding motifs in the primary structure of spinach PsbO were predicted from comparison with classic GTP-binding proteins. These motifs were only found in the plant PsbOs, in the -barrel domain of the homologous 3D-structure. Using circular dichroism and intrinsic fluorescence spectroscopy, it was shown that MgGTP induces specific structural changes in the PsbO protein. Spinach PsbO has a low intrinsic GTPase activity, which is considerably stimulated when associated with a dimeric PSII complex. GTP stimulates the dissociation of PsbO from PSII under both inhibitory and non-inhibitory light conditions. A role for PsbO as a GTPase in the function of the oxygen-evolving complex and PSII repair is proposed.</p>

Life on Earth is dependent on the oxygen produced through photosynthesis. The thylakoid membrane is the site for the light-driven reactions of photosynthesis, which oxidize water and supply energy in the form of ATP, mainly for carbon fixation. The utilization of ATP in the lumenal space of the thylakoid has not been considered in the past. In the latest years, increasing evidence for nucleotide metabolism in the thylakoid lumen of plant chloroplasts has been presented; ATP transport across the thylakoid membrane, and GTP binding to the PsbO extrinsic subunit of the water-oxidizing photosystem II (PSII) complex. In this thesis, various methods for prediction, identification, and characterization of novel plant proteins, are described. Nucleotide-binding motifs and nucleotide-dependent processes are reviewed, and the experimental data is discussed. 1) A thylakoid ATP/ADP carrier (TAAC) in Arabidopsis thaliana was identified and functionally characterized, and 2) the spinach PsbO protein was characterized as a GTPase. The Arabidopsis At5g01500 gene product is predicted as a chloroplast protein and to be homologous to the well-studied mitochondrial ADP/ATP carrier. The putative chloroplast localization was confirmed by transient expression of a TAAC-green fluorescent protein fusion construct. Immuno detection with peptide-targeted antibodies and immunogold electron microscopy showed the thylakoid as the main localization of TAAC, with a minor fraction in the chloroplast envelope. TAAC is readily expressed in etiolated seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. It is proposed that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover. Recombinant TAAC protein was functionally integrated in the cytoplasmic membrane of Escherichia coli, and was shown to specifically transport ATP/ADP in a protonophore-sensitive manner, as also reported for mitochondrial AACs. The PsbO protein stabilizes the oxygen-evolving complex of PSII and is ubiquitous in all oxygenic photosynthetic organisms, including cyanobacteria, green algae, and plants. So far only the 3D-structure of the cyanobacterial PsbO is available. Four GTP-binding motifs in the primary structure of spinach PsbO were predicted from comparison with classic GTP-binding proteins. These motifs were only found in the plant PsbOs, in the -barrel domain of the homologous 3D-structure. Using circular dichroism and intrinsic fluorescence spectroscopy, it was shown that MgGTP induces specific structural changes in the PsbO protein. Spinach PsbO has a low intrinsic GTPase activity, which is considerably stimulated when associated with a dimeric PSII complex. GTP stimulates the dissociation of PsbO from PSII under both inhibitory and non-inhibitory light conditions. A role for PsbO as a GTPase in the function of the oxygen-evolving complex and PSII repair is proposed.

Chronic low-grade inflammation is associated the Metabolic Syndrome (MetS) and may contribute to its development. A diet high in saturated fat (SFA) has been associated with increased inflammation and development of the MetS. SFAs have been shown to elicit pro-inflammatory signaling through proteins of innate immunity, TLR4 and Nods 1 and 2. We determined whether common polymorphisms in the genes of these proteins could modify the association between fat intake and biomarkers of the MetS. Fat intake was measured using a food frequency questionnaire and genotyping was completed using real-time PCR. The TLR4 Asp299Gly (rs4986790) polymorphism was associated with decreased insulin sensitivity while an intronic polymorphism (rs5030728) modified the association between dietary SFA and HDL-cholesterol. The Nod1 Glu266Lys polymorphism modified the association between dietary SFA and HOMA-IR. These results suggest a role for innate immunity in mediating some of the effects of dietary SFAs on factors associated with the MetS.