RESUMEN
Neuroglobin is a member of the globin superfamily expressed in vertebrate brain and retina. The protein is thought to be involved in neuronal protection from hypoxia or oxidative stress and could represent a key element of Alzheimer disease pathogenesis. Our aim was to determine whether neuroglobin could be directly associated with mitochondrial metabolism and integrity. We identified three different forms of neuroglobin in the retina, varying in their apparent molecular masses; all forms are abundant in mitochondrial fractions. This indicates that a significant fraction of the protein localizes within the organelle either in the matrix or in the matrix side of the inner membrane. Since neuroglobin was especially abundant in the ganglion cell layer, we transduced retinal ganglion cells with an anti-neuroglobin short hairpin RNA using in vivo electroporation. Neuroglobin knockdown leads to reduced activities of respiratory chain complexes I and III, degeneration of retinal ganglion cells, and impairment of visual function. The deleterious effect on cell survival was confirmed in primary retinal ganglion cells subjected to inhibition of neuroglobin expression. Hence, neuroglobin should be considered as a novel mitochondrial protein involved in respiratory chain function which is essential for retinal ganglion cell integrity.
Asunto(s)
Transporte de Electrón/fisiología , Globinas/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Células Ganglionares de la Retina/fisiología , Animales , Western Blotting , Células Cultivadas , Angiografía con Fluoresceína , Globinas/antagonistas & inhibidores , Globinas/genética , Masculino , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Neuroglobina , Neuronas/citología , Nervio Óptico/citología , Nervio Óptico/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Long-Evans , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The oligomeric state and kinetics of ligand binding were measured for wild-type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N- and C-terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.
Asunto(s)
Disulfuros/química , Globinas/química , Citoglobina , Histidina/química , Cinética , Conformación Proteica , Estructura Cuaternaria de Proteína , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Neuroglobin (Ngb) and the cellular prion protein (PrP(c)), proteins of unknown function in the nervous system, are known to be expressed in the retina and have been observed in different rat retinal cells. The retina is the site of the highest concentration for Ngb, a heme protein of similar size and conformation to myoglobin. In this study, we demonstrated by immunohistochemical analysis of retinal colocalization of Ngb and PrP(c) in the ganglion cell layer. Considering for these two a common protective role in relation to oxidative stress and a possible transient contact during migration of PrP(c) through the eye or upon neuronal degradation, we undertook in vitro studies of the interaction of the purified proteins. Mixing these two proteins leads to rapid aggregation, even at submicromolar concentrations. As observed with the use of dynamic light scattering, particles comprising both proteins evolve to hundreds of nanometers within several seconds, a first report showing that PrP(c) is able to form aggregates without major structural changes. The main effect would then appear to be a protein-protein interaction specific to the surface charge of the Ngb protein with PrP(c) N-terminal sequence. A dominant parameter is the solvent ionic force, which can significantly modify the final state of aggregation. PrP(c), normally anchored to the cell membrane, is toxic in the cytoplasm, where Ngb is present; this could suggest an Ngb function of scavenging proteins capable of forming deleterious aggregates considering a charge complementarity in the complex.
Asunto(s)
Globinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas PrPC/metabolismo , Retina/citología , Animales , Globinas/química , Globinas/genética , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuroglobina , Estrés Oxidativo , Tamaño de la Partícula , Proteínas PrPC/química , Proteínas PrPC/genética , Conformación Proteica , Multimerización de Proteína , Ratas , Ratas Long-Evans , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sales (Química)/química , Electricidad EstáticaRESUMEN
Progress in developing a blood substitute is aided by new biotechnologies and a better understanding of the circulatory system. For Hb based solutions, there is still a debate over the best set of fundamental parameters concerning the oxygen affinity which is correlated with the oxidation rate, the cooperativity, the transporter size, and of course the final source of material. Genetic engineering methods have helped discover novel globins, but not yet the quantity necessary for the high demand of blood transfusions. The expanding database of globin properties has indicated that certain individual parameters are coupled, such as the oxygen affinity and the oxidation rate, indicating that one must accept a compromise of the best parameters. After a general introduction of these basic criteria, we will focus on two strategies concerning the size of the oxygen transporter: Hb octamers, and Hb integrated within a nanoparticle.
Asunto(s)
Sustitutos Sanguíneos/uso terapéutico , Hemoglobinas/uso terapéutico , Nanopartículas/uso terapéutico , Transporte Biológico , Sustitutos Sanguíneos/síntesis química , Sustitutos Sanguíneos/química , Ingeniería Genética/métodos , Vectores Genéticos , Globinas/química , Globinas/genética , Globinas/uso terapéutico , Haptoglobinas/química , Haptoglobinas/genética , Haptoglobinas/uso terapéutico , Hemoglobinas/química , Hemoglobinas/genética , Humanos , Cinética , Preparaciones Farmacéuticas/administración & dosificación , Polisacáridos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéuticoRESUMEN
We describe a software program to help exploit a database of aligned protein sequences. In addition to the classical lists of sequences, a graphical representation is used to get a better overview of the information. As natural parameters, the type of amino acid and sequence position are used. Various plots or 3D representations are then updated. Examples are shown based on globin sequences from various species and on the abnormal human hemoglobins. The software should be of interest to protein engineers who need to know what variants are already known.
Asunto(s)
Hemoglobinas/química , Modelos Moleculares , Programas Informáticos , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Animales , Gráficos por Computador , Bases de Datos de Proteínas , Hemoglobinas/genética , Humanos , Mutación , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
The prion protein occurs as a globular domain and a leading fragment whose structure is not well-defined. For the ovine species, all of the tryptophan residues are in the initial fragment, while the globular domain is rich in tyrosine residues. Using heme as a spectroscopic probe, we have studied the recombinant prion protein before and after a temperature-induced conformational change. As for most heme proteins, the absorption spectrum of heme-CO displays a red shift upon binding to the protein, and both the Y and W fluorescence are highly quenched. Flash photolysis kinetics of the PrP-heme-CO complex shows a low yield for the bimolecular phase, indicating a pocket around the hemes. By comparing the holoprotein and the truncated sequence corresponding to the globular domain, the stoichiometry was determined to be five hemes for the globular domain and two hemes for the leading fragment. At high temperature, the hemes are released; upon cooling, only two hemes bind, and only the tryptophan fluorescence is quenched; this would indicate that the globular domain has formed a more compact structure, which is inert with respect to the hydrophobic probe. The final state of polymerization is perturbed if the synthetic peptide "N3" (PrP residues 142-166, which include the first helix) is added to the prion protein solution; the temperature cycle no longer reduces the number of heme binding sites. This would indicate that the peptide may alter or inhibit the polymer formation.