RESUMEN

High sensitivity differential scanning calorimetry (HSDSC) has been used to study the interaction of the model proteins lactate dehydrogenase (LDH) and tyrosinase with dimyristoylphosphatidylcholine (DMPC) liposomes, and relate this to the thermal and physical stability of the proteins. On heating, both LDH and tyrosinase denatured irreversibly in a time-dependent manner and modified the phase transition behaviour of DMPC liposomes at all concentrations investigated. The most marked effects occurred for the pretransition rather than the main phospholipid phase transition. The effects on the bilayer are likely to result from electrostatic interactions of the hydrophilic proteins with the head-groups of DMPC molecules, whilst due to their hydrophilic nature they do not penetrate into the bilayer. Tyrosinase is more highly ionised than LDH at the pH of the investigation, which may explain why tyrosinase has a greater effect than LDH on the HSDSC scans at mg/ml protein concentrations.

Asunto(s)

L-Lactato Deshidrogenasa/química , Liposomas/química , Monofenol Monooxigenasa/química , Rastreo Diferencial de Calorimetría , Temperatura

RESUMEN

The aim of this study was to investigate the suitability of commercial jet and ultrasonic nebulisers for effective delivery of the model hydrophilic protein lactate dehydrogenase (LDH). Two jet nebulisers (Pari LC Plus and Pari LC Star) and two ultrasonic nebulisers (Sonix 2000 and Omron U1) were used to nebulise LDH solutions and the effects on protein activity and protein concentration determined. The size distribution of the aerosols produced, measured by laser diffraction analysis, temperature changes during nebulisation, the time to atomise a 5 ml dose volume and the mass output of the four nebulisers were compared. A twin impinger (TI) was used to collect the nebulised protein, which was assayed for total and active protein content. There was a large variation in the median size and size distribution of the aerosols produced by each of the nebulisers from LDH and Sørensen's modified phosphate buffer, and in the time taken to reach the sputtering phase of aerosolisation. During use, the concentration of LDH increased in the Omron U1 nebuliser, but did not change significantly in the others. The temperature of the protein solution decreased by approximately 8 degrees C during jet nebulisation but increased by 3 and 10 degrees C in the Omron U1 and Sonix 2000 nebulisers, respectively. Denaturation of LDH within the nebuliser reservoir, occurred in the order Sonix>Pari LC Plus>Pari LC Star>Omron U1, whilst the deposition of active and total protein within the stages and throat of the TI was a function of the particle size of the aerosols generated and the specific device used.

Asunto(s)

L-Lactato Deshidrogenasa/administración & dosificación , Nebulizadores y Vaporizadores , Aerosoles , Diseño de Equipo , Soluciones

RESUMEN

The PICK1 protein interacts in neurons with the AMPA-type glutamate receptor subunit 2 (GluR2) and with several other membrane receptors via its single PDZ domain. We show that PICK1 also binds in neurons and in heterologous cells to protein kinase Calpha (PKCalpha) and that the interaction is highly dependent on the activation of the kinase. The formation of PICK1-PKCalpha complexes is strongly induced by TPA, and PICK1-PKCalpha complexes are cotargeted with PICK1-GluR2 complexes to spines, where GluR2 is found to be phosphorylated by PKC on serine 880. PICK1 also reduces the plasma membrane levels of the GluR2 subunit, consistent with a targeting function of PICK1 and a PKC-facilitated release of GluR2 from the synaptic anchoring proteins ABP and GRIP. This work indicates that PICK1 functions as a targeting and transport protein that directs the activated form of PKCalpha to GluR2 in spines, leading to the activity-dependent release of GluR2 from synaptic anchor proteins and the PICK1-dependent transport of GluR2 from the synaptic membrane.

Asunto(s)

Proteínas Portadoras/metabolismo , Hipocampo/metabolismo , Isoenzimas/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Proteína Quinasa C/metabolismo , Receptores AMPA/metabolismo , Células 3T3 , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Células Cultivadas , Cricetinae , Proteínas del Citoesqueleto , Dimerización , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Hipocampo/citología , Humanos , Isoenzimas/genética , Sustancias Macromoleculares , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Neuronas/citología , Proteínas Nucleares/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Proteína Quinasa C/genética , Proteína Quinasa C-alfa , Transporte de Proteínas/fisiología , Ratas , Agregación de Receptores , Receptores AMPA/genética , Acetato de Tetradecanoilforbol/farmacología , Transfección , Técnicas del Sistema de Dos Híbridos

RESUMEN

Excitatory synaptic currents in the mammalian brain are typically mediated by the neurotransmitter glutamate, acting at AMPA receptors. We used immunocytochemistry to investigate the distribution of AMPA receptor-binding protein (ABP) in the cerebral neocortex. ABP was most prominent in pyramidal neurons, although it was also present (at lower levels) in interneurons. ABP and its putative binding partners, the GluR2/3 subunits of the AMPA receptor, exhibited prominent cellular colocalization. Under appropriate processing conditions, colocalization could also be documented in puncta, many of which could be recognized as dendritic spines. However, a sizable minority of GluR2/3-positive puncta were immunonegative for ABP. Because glutamate receptor-interacting protein (GRIP) may also anchor GluR2, we studied the relative distribution of ABP and GRIP. There was extensive colocalization of these two antigens at the cellular level, although GRIP, unlike ABP, was strongest in nonpyramidal neurons. Different parts of a single dendrite could stain selectively for ABP or GRIP. To further characterize this heterogeneity, we investigated punctate staining of neuropil using synaptophysin and the membrane tracer DiA to identify probable synapses. Some puncta were comparably positive for both ABP and GRIP, but the majority were strongly positive for one antigen and only weakly positive or immunonegative for the other. This heterogeneity could be seen even within adjacent spines of a single dendrite. These data suggest that ABP may act as a scaffold for AMPA receptors either in concert with or independently from GRIP.

Asunto(s)

Proteínas Portadoras/metabolismo , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Contraindicaciones , Dendritas/metabolismo , Dendritas/ultraestructura , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular , Interneuronas/citología , Interneuronas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Masculino , Neocórtex/citología , Neurópilo/metabolismo , Neurópilo/ultraestructura , Especificidad de Órganos , Células Piramidales/citología , Células Piramidales/metabolismo , Compuestos de Piridinio , Ratas , Ratas Sprague-Dawley , Sinapsis/metabolismo , Sinaptofisina/metabolismo

RESUMEN

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.

Asunto(s)

Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores AMPA/metabolismo , Membranas Sinápticas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Sitios de Unión/genética , Células Cultivadas , Proteínas del Citoesqueleto , Hipocampo/citología , Hipocampo/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Mutagénesis Sitio-Dirigida , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína/genética , Proteínas Proto-Oncogénicas c-myc/genética , Ratas , Ratas Sprague-Dawley , Receptores AMPA/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Virus Sindbis/genética

RESUMEN

We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.

Asunto(s)

Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores AMPA/química , Receptores AMPA/metabolismo , Médula Espinal/metabolismo , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Fragmentos de Péptidos/química , Conformación Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Médula Espinal/citología , Médula Espinal/crecimiento & desarrollo , Sinapsis/ultraestructura , Transcripción Genética

RESUMEN

In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.

Asunto(s)

Adenosina Trifosfato/fisiología , Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Receptores AMPA/fisiología , Proteínas de Transporte Vesicular , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Animales , Precipitación Química , Dendritas/metabolismo , Interacciones Farmacológicas , Proteínas Sensibles a N-Etilmaleimida , Neuronas/metabolismo , Proteínas Qa-SNARE , Ratas , Ratas Sprague-Dawley , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Levaduras/genética

RESUMEN

The present report is an analysis of 12 cases of herpes gestationis seen in Tripoli (Libya) over a period of four years, with special emphasis on clinical features. In one case the disease occurred in alternate pregnancies.