Spectroscopic and Theoretical Study of CuI Binding to His111 in the Human Prion Protein Fragment 106-115

The ability of the cellular prion protein (PrPC) to bind copper in vivo points to a physiological role for PrPC in copper transport. Six copper binding sites have been identified in the nonstructured N-terminal region of human PrPC. Among these sites, the His111 site is unique in that it contains a MKHM motif that would confer interesting CuI and CuII binding properties. We have evaluated CuI coordination to the PrP(106-115) fragment of the human PrP protein, using NMR and X-ray absorption spectroscopies and electronic structure calculations. We find that Met109 and Met112 play an important role in anchoring this metal ion. CuI coordination to His111 is pH-dependent: at pH >8, 2N1O1S species are formed with one Met ligand; in the range of pH 5-8, both methionine (Met) residues bind to CuI, forming a 1N1O2S species, where N is from His111 and O is from a backbone carbonyl or a water molecule; at pH <5, only the two Met residues remain coordinated. Thus, even upon drastic changes in the chemical environment, such as those occurring during endocytosis of PrPC (decreased pH and a reducing potential), the two Met residues in the MKHM motif enable PrPC to maintain the bound CuI ions, consistent with a copper transport function for this protein. We also find that the physiologically relevant CuI-1N1O2S species activates dioxygen via an inner-sphere mechanism, likely involving the formation of a copper(II) superoxide complex. In this process, the Met residues are partially oxidized to sulfoxide; this ability to scavenge superoxide may play a role in the proposed antioxidant properties of PrPC. This study provides further insight into the CuI coordination properties of His111 in human PrPC and the molecular mechanism of oxygen activation by this site.Fil: Arcos López, Trinidad. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; MéxicoFil: Qayyum, Munzarin. University of Stanford; Estados UnidosFil: Rivillas Acevedo, Lina. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; MéxicoFil: Miotto, Marco César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; Argentina. Max Planck Laboratory for Structural Biology; ArgentinaFil: Grande Aztatzi, Rafael. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; MéxicoFil: Fernandez, Claudio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; Argentina. Max Planck Laboratory for Structural Biology; ArgentinaFil: Hedman, Britt. University of Stanford; Estados UnidosFil: Hodgson, Keith O.. University of Stanford; Estados UnidosFil: Vela, Alberto. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; MéxicoFil: Solomon, Edward I.. University of Stanford; Estados UnidosFil: Quintanar, Liliana. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; Méxic

Análise de tarefas para as funções espaciais em crianças escolares com diagnóstico de TDAH

Objective. Identify and detail the difficulties in children with attention deficit disorder in spatial function tasks. Method. 24 school children between 6 and 12 years old with diagnosis of attention deficit disorder were included in the study. Neuropsychological tasks used in the study were divided in graphic tasks, verbal tasks and a constructive task, all of them included in the Neuropsychological Evaluation for Children, Neuropsychological Evaluation of the Spatial Integration and Neuropsychological Evaluation of School Success assessments. Results. Analysis of the results has pointed out specific errors in graphic tasks such as disproportion, disintegration, difficulties with angles, problems with usage of space and inversions. Younger participants with complex neuropsychological syndromes committed more mistakes. The spatial difficulties were accompanied by regulatory, activation, motor and kinesthetic difficulties. The participants with higher frequencies of error were those of lower age. Conclusion. The results show that children with attention deficit disorder diagnosis may have difficulties in the development of spatial function; therefore, it is necessary to include a more profound neuropsychological analysis of the domain that allows the investigation of the nature of the difficulties and its plausible relation to the development of other functions.Objetivo. Identificar y precisar las dificultades en funciones espaciales que presentan los niños con TDAH en edad escolar. Método. Se evaluaron 24 niños, entre los 6 y los 12 años de edad, con diagnóstico de TDAH. Todos los niños fueron evaluados con tareas gráficas, tareas verbales y tareas constructivas, incluidas en las pruebas Evaluación Neuropsicológica Infantil, Evaluación Neuropsicológica de la Integración Espacial y Evaluación Neuropsicológica del Éxito Escolar. Resultados. El análisis clínico realizado al desempeño de los niños mostró dificultades no solo en el manejo de la proporción, la integración y al dibujar ángulos, sino en el uso adecuado del espacio e inversión. Las dificultades espaciales estuvieron acompañadas de otras de índole regulatorio, tono de activación, motoras y cinestésicas. Los participantes con mayor número de errores fueron aquellos de menor edad. Conclusión. Los niños con diagnóstico de TDAH pueden presentar dificultades en el desarrollo de las funciones espaciales, por lo cual se hace necesario que la evaluación neuropsicológica incluya un análisis profundo de esta dimensión cognitiva, que permita indagar la naturaleza de estas dificultades y su posible relación con el desarrollo de otras funciones. Escopo. Identificar e reconhecer as dificuldades em funções espaciais que apresentam as crianças com TDAH em idade escolar. Metodologia. Foram avaliadas 20 crianças masculinas e 4 femininas, com idades compreendidas entre os 6 e 12 anos de idade, com diagnóstico de TDAH. Todas as crianças foram avaliadas com tarefas gráficas, tarefas verbais e tarefas construtivas, incluídas nas provas Avaliação Neuropsicológica da Integração espacial e Avaliação Neuropsicológica do Sucesso Escolar. Resultados. A análise clínica realizada aos desempenhos das crianças, mostraram dificuldades de desproporção, desintegração, dificuldade para desenhar ângulos, uso inadequado do espaço e inversão. As dificuldades espaciais estiveram acompanhadas de outras de natureza regulatória, tono de ativação, motoras e cinestésicas. Os participantes com maior número de erros foram aqueles de menor idade. Conclusão. As crianças com diagnóstico de déficit de atenção podem apresentar dificuldades no desenvolvimento das funções espaciais, pelo qual é preciso que a avaliação neuropsicológica possa incluir uma análise profunda de esta dimensão cognitiva, que permita indagar a natureza de estas dificuldades e sua possível relação com o desenvolvimento de outras funções

Interaction of Cu(i) with the Met-X3-Met motif of alpha-synuclein: binding ligands, affinity and structural features

The identity of the Cu(i) binding ligands at Met-X3-Met site of AcαS and its role into the affinity and structural properties of the interaction were elucidated by NMR spectroscopy. We provide evidence that the source of ligands for Cu(i) binding to the Met-X3-Met site comes from the N-terminal acetyl group and the Met-1, Asp-2 and Met-5 residues. From the study of site-directed mutants and synthetic peptide models of αS we demonstrated the critical role played by Met-1 and Met-5 residues on the binding affinity of the Cu(i) complex, acting as the main metal anchoring residues. While having a more modest impact in the affinity features of Cu(i) binding, as compared to the Met residues, the N-terminal acetyl group and Asp-2 are important in promoting local helical conformations, contributing to the stabilization of these structures by favoring Cu(i) binding.Fil: Gentile, Iñaki. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; ArgentinaFil: Garro, Hugo Alejandro. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Delgado Ocaña, Susana. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: González, Nazareno. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Strohäker, Timo. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Schibich, Daniela. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Quintanar, Liliana. Centro de Investigación y de Estudios; MéxicoFil: Sambrotta, Luis Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Zweckstetter, Markus. Max Planck Institute For Biophysical Chemistry; Alemania. Deutsches Zentrum Für Neurodegenerative Erkrankungen; AlemaniaFil: Griesinger, Christian. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Menacho Márquez, Mauricio Ariel. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnol.conicet - Rosario. Unidad de Direccion; ArgentinaFil: Fernandez, Claudio Oscar. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; Argentina. Max Planck Institute For Biophysical Chemistry; Alemani

Neuroprotective alpha-cleavage of the human prion protein significantly impacts Cu(II) coordination at its His111 site

The cellular prion protein (PrPC) is a copper binding protein that undergoes post-translational modifications, such as endoproteolytic alpha cleavage, which occurs in the vicinity of the His111 Cu binding site. Alpha cleavage processing of PrPC is considered to be neuroprotective since the cleavage site is located in a region that is key to the conversion of PrPC into the infectious scrapie isoform (PrPSc), yielding a membrane bound C1 fragment of PrPC that still contains His111. In this work, we use hPrP(111-115) fragment as a model peptide to evaluate the impact of alpha cleavage processing of PrPC in its ability to coordinate Cu(ii) ions at His111. By using different spectroscopic techniques such as electronic absorption, circular dichroism, nuclear magnetic resonance, and electron paramagnetic resonance, this study demonstrates that Cu(ii) binding to the cleaved His111 site is highly dependent on Cu and proton concentrations. The imidazole group of His111 and its free NH2 terminus emerge as the main anchoring sites for Cu(ii) coordination, yielding very different complexes from those characterized for the intact His111 site in the full protein. Different Cu(ii) coordination modes that could form with the alpha cleaved PrPC under physiological conditions are identified and characterized. Overall, this study contributes to understand how alpha cleavage processing of PrPC impacts its Cu(ii) binding properties at His111. While the functional implications of Cu binding to the cleaved PrPC remain to be discovered, proteolytic processing of PrPC and its Cu binding features appear to be molecular events that might be strongly linked to its cellular function.Fil: Sánchez López, Carolina. Centro de Investigación y de Estudios Avanzados; MéxicoFil: Fernandez, Claudio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; ArgentinaFil: Quintanar, Liliana. Centro de Investigación y de Estudios Avanzados; Méxic

Structural and Electronic Analysis of the Octarepeat Region of Prion Protein with Four Cu(II) by Polarizable MD and QM/MM Simulations

The prion protein, located mainly in neurons, is believed to play the role of metal ion transporter. A 32-residue region of the N-terminal domain, known as octarepeat, can bind up to four Cu ions. Different coordination modes have been observed and are strongly dependent on Cu concentration. Many theoretical studies carried out so far have focused on studying the coordination modes of a single copper ion. In this work we investigate the octarepeat region coordinated with four copper ions. Molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations using the polarizable AMOEBA force field have been carried out. Results indicate that the 4Cu-octarepeat complex forms a globular structure, in agreement with experimental results. Subsequent QM/MM simulations on several snapshots suggests the system is in a high-spin quintet state, with all Cu ions bearing one single electron, and all unpaired electrons are ferromagnetically coupled

Structural Determinants of the Prion Protein N-Terminus and Its Adducts with Copper Ions

The N-terminus of the prion protein is a large intrinsically disordered region encompassing approximately 125 amino acids. In this paper, we review its structural and functional properties, with a particular emphasis on its binding to copper ions. The latter is exploited by the region’s conformational flexibility to yield a variety of biological functions. Disease-linked mutations and proteolytic processing of the protein can impact its copper-binding properties, with important structural and functional implications, both in health and disease progression

Análisis de tareas para las funciones espaciales en niños escolares con diagnóstico de TDAH

Objective. Identify and detail the difficulties in children with attention deficit disorder in spatial function tasks. Method. 24 school children between 6 and 12 years old with diagnosis of attention deficit disorder were included in the study. Neuropsychological tasks used in the study were divided in graphic tasks, verbal tasks and a constructive task, all of them included in the Neuropsychological Evaluation for Children, Neuropsychological Evaluation of the Spatial Integration and Neuropsychological Evaluation of School Success assessments. Results. Analysis of the results has pointed out specific errors in graphic tasks such as disproportion, disintegration, difficulties with angles, problems with usage of space and inversions. Younger participants with complex neuropsychological syndromes committed more mistakes. The spatial difficulties were accompanied by regulatory, activation, motor and kinesthetic difficulties. The participants with higher frequencies of error were those of lower age. Conclusion. The results show that children with attention deficit disorder diagnosis may have difficulties in the development of spatial function; therefore, it is necessary to include a more profound neuropsychological analysis of the domain that allows the investigation of the nature of the difficulties and its plausible relation to the development of other functions.Escopo. Identificar e reconhecer as dificuldades em funções espaciais que apresentam as crianças com TDAH em idade escolar. Metodologia. Foram avaliadas 20 crianças masculinas e 4 femininas, com idades compreendidas entre os 6 e 12 anos de idade, com diagnóstico de TDAH. Todas as crianças foram avaliadas com tarefas gráficas, tarefas verbais e tarefas construtivas, incluídas nas provas Avaliação Neuropsicológica da Integração espacial e Avaliação Neuropsicológica do Sucesso Escolar. Resultados. A análise clínica realizada aos desempenhos das crianças, mostraram dificuldades de desproporção, desintegração, dificuldade para desenhar ângulos, uso inadequado do espaço e inversão. As dificuldades espaciais estiveram acompanhadas de outras de natureza regulatória, tono de ativação, motoras e cinestésicas. Os participantes com maior número de erros foram aqueles de menor idade. Conclusão. As crianças com diagnóstico de déficit de atenção podem apresentar dificuldades no desenvolvimento das funções espaciais, pelo qual é preciso que a avaliação neuropsicológica possa incluir uma análise profunda de esta dimensão cognitiva, que permita indagar a natureza de estas dificuldades e sua possível relação com o desenvolvimento de outras funções.Objetivo. Identificar y precisar las dificultades en funciones espaciales que presentan los niños con TDAH en edad escolar. Método. Se evaluaron 24 niños, entre los 6 y los 12 años de edad, con diagnóstico de TDAH. Todos los niños fueron evaluados con tareas gráficas, tareas verbales y tareas constructivas, incluidas en las pruebas Evaluación Neuropsicológica Infantil, Evaluación Neuropsicológica de la Integración Espacial y Evaluación Neuropsicológica del Éxito Escolar. Resultados. El análisis clínico realizado al desempeño de los niños mostró dificultades no solo en el manejo de la proporción, la integración y al dibujar ángulos, sino en el uso adecuado del espacio e inversión. Las dificultades espaciales estuvieron acompañadas de otras de índole regulatorio, tono de activación, motoras y cinestésicas. Los participantes con mayor número de errores fueron aquellos de menor edad. Conclusión. Los niños con diagnóstico de TDAH pueden presentar dificultades en el desarrollo de las funciones espaciales, por lo cual se hace necesario que la evaluación neuropsicológica incluya un análisis profundo de esta dimensión cognitiva, que permita indagar la naturaleza de estas dificultades y su posible relación con el desarrollo de otras funciones.

An ATCUN-like copper site in B2-crystallin plays a protective role in cataract-associated aggregation

Cataracts is the leading cause of blindness worldwide and it is caused by crystallin damage and aggregation. Senile cataractous lenses have relatively high levels of metals, while some metal ions can directly induce aggregation of human -crystallins. Here we evaluated the impact of divalent metal ions in the aggregation of human B2-crystallin, one of the most abundant crystallins in the lens. Turbidity assays showed that Pb2+, Hg2+, Cu2+, and Zn2+ ions induce the aggregation of B2-crystallin. Metal-induced aggregation is partially reverted by a chelating agent, indicating formation of metal-bridged species. Our study focused on the mechanism of copper-induced aggregation of B2-crystallin, finding that it involves metal-bridging, disulfide-bridging, and loss of protein stability. Circular dichroism (CD) and electron paramagnetic resonance (EPR) revealed the presence of at least three Cu2+ binding sites in B2-crystallin; one of them with spectroscopic features typical of Cu2+ bound to an amino-terminal copper and nickel binding motif (ATCUN), a motif found in Cu transport proteins. The ATCUN-like Cu binding site is located at the unstructured N-terminus of B2-crystallin, and it could be modeled by a peptide with the first six residues in the protein sequence (NH2-ASDHQF-). Removal of the N-terminus yields an N-truncated form of B2-crystallin that is more susceptible to Cu-induced aggregation and loss of thermal stability, indicating a protective role for the ATCUN-like site. EPR and X-ray absorption spectroscopy (XAS) studies reveal the presence of a copper redox active site in B2-crystallin that is associated to metal-induced aggregation and formation of disulfide-bridged oligomers. Our study demonstrates metal-induced aggregation of cataract-related B2-crystallin and the presence of putative copper binding sites in the protein. Whether the copper-transport ATCUN-like site in B2-crystallin plays a functional/protective role or constitute a vestige from its evolution as a lens structural protein, remains to be elucidated