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1.
J Biol Chem ; 287(23): 19715-24, 2012 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-22511769

RESUMEN

Amyloid ß-precursor protein (APP) is primarily cleaved by α- or ß-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr(668) and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648-695)) with Thr(P)(668) did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aß generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aß in Alzheimer disease.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Microdominios de Membrana/metabolismo , Triptófano/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/patología , Química Encefálica/genética , Microdominios de Membrana/genética , Ratones , Fosforilación , Estructura Terciaria de Proteína , Triptófano/genética
2.
Nat Cell Biol ; 7(8): 808-16, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16025105

RESUMEN

Phosphatidylserine (PS) exposure is normally associated with apoptosis and the removal of dying cells. We observed that PS is exposed constitutively at high levels on T lymphocytes that express low levels of the transmembrane tyrosine phosphatase CD45RB. CD45 was shown to be a negative regulator of PS translocation in response to various signals, including activation of the ATP receptor P2X(7). Changes in PS distribution were shown to modulate several membrane activities: Ca(2+) and Na(+) uptake through the P2X(7) cation channel itself; P2X(7)-stimulated shedding of the homing receptor CD62L; and reversal of activity of the multidrug transporter P-glycoprotein. The data identify a role for PS distribution changes in signal transduction, rapidly modulating the activities of several membrane proteins. This seems to be an all-or-none effect, coordinating the activity of most or all the molecules of a target protein in each cell. The data also suggest a new approach to circumventing multidrug resistance.


Asunto(s)
Membrana Celular/metabolismo , Antígenos Comunes de Leucocito/fisiología , Linfocitos/metabolismo , Fosfatidilserinas/metabolismo , Receptores Purinérgicos P2/fisiología , Transducción de Señal/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Anexina A5/metabolismo , Apoptosis/fisiología , Transporte Biológico/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Canales Iónicos/fisiología , Selectina L/metabolismo , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Linfocitos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Paclitaxel/farmacocinética , Agonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/fisiología
3.
J Am Soc Nephrol ; 20(6): 1275-81, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19389853

RESUMEN

The P2X7 receptor is a ligand-gated cation channel that is normally expressed by a variety of immune cells, including macrophages and lymphocytes. Because it leads to membrane blebbing, release of IL-1beta, and cell death by apoptosis or necrosis, it is a potential therapeutic target for a variety of inflammatory diseases. Although the P2X7 receptor is usually not detectable in normal renal tissue, we previously reported increased expression of both mRNA and protein in mesangial cells and macrophages infiltrating the glomeruli in animal models of antibody-mediated glomerulonephritis. In this study, we used P2X7-knockout mice in the same experimental model of glomerulonephritis and found that P2X7 deficiency was significantly renoprotective compared with wild-type controls, evidenced by better renal function, a striking reduction in proteinuria, and decreased histologic glomerular injury. In addition, the selective P2X7 antagonist A-438079 prevented the development of antibody-mediated glomerulonephritis in rats. These results support a proinflammatory role for P2X7 in immune-mediated renal injury and suggest that the P2X7 receptor is a potential therapeutic target.


Asunto(s)
Glomerulonefritis/fisiopatología , Receptores Purinérgicos P2/fisiología , Animales , Femenino , Glomerulonefritis/prevención & control , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Antagonistas del Receptor Purinérgico P2 , Piridinas/farmacología , Piridinas/uso terapéutico , Ratas , Receptores Purinérgicos P2X7 , Tetrazoles/farmacología , Tetrazoles/uso terapéutico
4.
Purinergic Signal ; 5(4): 513-20, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19507052

RESUMEN

Our knowledge and understanding of the P2 receptor signalling system in the kidney have increased significantly in the last ten years. The broad range of physiological roles proposed for this receptor system and the variety of P2 receptor subtypes found in the kidney suggest that any disturbance of function may contribute to several pathological processes. So far, most reports of a possible pathophysiological role for this system in the kidney have focussed on polycystic kidney disease, where abnormal P2 receptor signalling might be involved in cyst expansion and disease progression, and on the P2X(7) receptor, a unique P2X subtype, which when activated enhances inflammatory cytokine release and production, and also cell death. Expression of this particular receptor is upregulated in some forms of chronic renal injury and inflammatory diseases. Further studies of adenosine triphosphate signalling and P2 receptor expression in renal disorders could provide us with novel insights into the role of these receptors in both normal and abnormal kidney function.

5.
Diabetes ; 53(8): 2012-7, 2004 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15277380

RESUMEN

It has been hypothesized that type 1 diabetes is initiated by neonatal physiological pancreatic beta-cell death, indicating that the early stages of this autoimmune response may reflect a dysregulated response to immune "danger" signals. One potential danger signal is ATP, high concentrations of which stimulate the purinergic receptor P2X7 on hematopoietic cells. We compared the sensitivity of lymphocytes from model type 1 diabetic (NOD) and control (C57BL/10) mice to activation of this pathway. Stimulation of the P2X7 receptor of NOD mice resulted in more pronounced shedding of the lymphocyte homing receptor CD62L and in increased programmed cell death. Levels of major histocompatibility complex class I molecules, which have previously been reported to be poorly expressed on NOD lymphocytes, were initially normal, but the molecules were shed preferentially from NOD cells after P2X7 receptor stimulation. Thus, although NOD lymphocytes have been considered resistant to programmed cell death, they are highly sensitive to that stimulated through the P2X7 receptor. Because NOD mice express a low activation threshold allele of the P2X7 receptor and the P2X7 gene maps to a locus associated with disease, P2X7 is a good candidate susceptibility gene for NOD diabetes.


Asunto(s)
Apoptosis/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Predisposición Genética a la Enfermedad/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Linfocitos/inmunología , Receptores Purinérgicos P2/inmunología , Animales , Diabetes Mellitus Tipo 1/patología , Modelos Animales de Enfermedad , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Linfocitos/patología , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Necrosis , Receptores Purinérgicos P2X7
6.
Br J Pharmacol ; 143(7): 899-907, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15492020

RESUMEN

Multidrug transporters play a dual role in haematopoietic cells, mediating the efflux of xenobiotics and regulating cell migration. For several reasons including the lack of specific antibodies, reports of multidrug transporter distribution on lymphocytes conflict. Murine B cells have been reported to completely lack transporter activity. Through analysis of parental and 'knockout' mice we show that, contrary to previous studies, murine B and T lymphocytes possess at least three active multidrug transporters and also a hitherto unrecognised drug-specific import activity. Surprisingly, the drug specificity of P-glycoprotein appears cell type dependent. The data indicate that a range of developmentally regulated, multidrug transporters can impose a barrier to treatment of immune disorders.


Asunto(s)
Linfocitos/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Antagonistas Adrenérgicos alfa/metabolismo , Compuestos de Anilina , Animales , Antineoplásicos/metabolismo , Antineoplásicos Fitogénicos/metabolismo , Linfocitos B/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Fluoresceínas , Colorantes Fluorescentes/metabolismo , Ratones , Ratones Noqueados , Mitoxantrona/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Paclitaxel/metabolismo , Prazosina/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo , Verapamilo/metabolismo , Xantenos
8.
Nat Struct Mol Biol ; 17(5): 561-7, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20383142

RESUMEN

The amyloid-beta(1-42) (Abeta42) peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimer's disease. We show that low-temperature and low-salt conditions can stabilize disc-shaped oligomers (pentamers) that are substantially more toxic to mouse cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the beta-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C termini are protected from solvent exchange and which have a turn conformation, placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Abeta42 oligomers to fibrils involves the association of these loosely aggregated strands into beta-sheets whose individual beta-strands polymerize in a parallel, in-register orientation and are staggered at an intermonomer contact between Gln15 and Gly37.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Neuronas/citología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Supervivencia Celular , Células Cultivadas , Frío , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Estructura Secundaria de Proteína , Sales (Química)/química
10.
J Leukoc Biol ; 85(6): 978-86, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19276178

RESUMEN

The purinergic receptor P2X(7) is expressed on immune cells, and its stimulation results in the release of IL-1beta from macrophages. Its absence, as evidenced from the analysis of two independent strains of P2X(7)-deficient mice, results in reduced susceptibility to inflammatory disease, and the molecule is an important, potential therapeutic target in autoimmunity. However, P2X(7) has also been detected in several neuronal cell types, although its function and even its presence in these cells are highly contested, with anti-P2X(7) antibodies staining brain tissue from both strains of P2X(7)(-/-) mice identically to wild-type mice. It has therefore been suggested that neurons express a distinct "P2X(7)-like" protein that has similar antibody recognition epitopes to P2X(7) and some properties of the genuine receptor. In this study, we show that whereas P2X(7) activity is absent from macrophages and dendritic cells in P2X(7)(-/-) animals, T cells from one gene-deficient strain unexpectedly exhibit higher levels of P2X(7) activity than that found in cells from control, unmanipulated C57BL/6 mice. A potential mechanism for this tissue-specific P2X(7) expression in P2X(7)(-/-) animals is discussed, as is the implication that the immune and indeed neuronal functions of P2X(7) may have been underestimated.


Asunto(s)
Linfocitos/metabolismo , Receptores Purinérgicos P2/deficiencia , Alelos , Empalme Alternativo/genética , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Marcación de Gen , Linfocitos/citología , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transgenes , beta-Galactosidasa/metabolismo
11.
J Immunol ; 180(1): 300-8, 2008 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-18097031

RESUMEN

Patterns of change in cell volume and plasma membrane phospholipid distribution during cell death are regarded as diagnostic means of distinguishing apoptosis from necrosis, the former being associated with cell shrinkage and early phosphatidylserine (PS) exposure, whereas necrosis is associated with cell swelling and consequent lysis. We demonstrate that cell volume regulation during lymphocyte death stimulated via the purinergic receptor P2X7 is distinct from both. Within seconds of stimulation, murine lymphocytes undergo rapid shrinkage concomitant with, but also required for, PS exposure. However, within 2 min shrinkage is reversed and swelling ensues ending in cell rupture. P2X7-induced shrinkage and PS translocation depend upon K+ efflux via KCa3.1, but use a pathway of Cl- efflux distinct from that previously implicated in apoptosis. Thus, P2X7 stimulation activates a novel pathway of cell death that does not conform to those conventionally associated with apoptosis and necrosis. The mixed apoptotic/necrotic phenotype of P2X7-stimulated cells is consistent with a potential role for this death pathway in lupus disease.


Asunto(s)
Apoptosis , Lupus Eritematoso Sistémico/inmunología , Linfocitos/patología , Fosfatidilserinas/metabolismo , Receptores Purinérgicos P2/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Tamaño de la Célula , Cloruros/metabolismo , Conexinas , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Metabolismo de los Lípidos , Activación de Linfocitos , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Ratones Endogámicos , Proteínas del Tejido Nervioso , Fosfatidilserinas/farmacología , Agonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2X7 , Linfocitos T/inmunología , Tamoxifeno/farmacología
12.
J Immunol ; 178(6): 3474-82, 2007 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-17339442

RESUMEN

Regulatory T cells (Tregs) are relatively autoreactive yet, paradoxically, have been found to display normal sensitivity to thymic deletion. The relationship between self-avidity, apoptosis, and the selection of Tregs therefore remains unclear. We show that thymic Tregs develop efficiently, even at low self-avidity, and are moderately resistant to apoptosis in comparison to conventional thymocytes. Consistent with this, although conventional self-reactive T cell populations undergo chronic peripheral deletion, self-reactive Tregs are largely spared removal. Similarly, the distribution of Tregs among peripheral CD4(+) cells exhibits a linear inverse relationship with CD45RB expression, indicating relative apoptosis resistance of Tregs in chronic responses to environmental Ags. We also show that appropriate controls for CD45RB levels are important for comparisons of Treg and conventional T cell activity. When thus controlled, and contrary to previous reports, Tregs exhibit normal sensitivity to cell death through TCR-independent stimuli, such as the purinergic receptor, P2X(7). Finally, although absence of CD45 in gene-targeted mice results in profound T cell hyporesponsiveness, there is little or no effect on thymic Treg frequency. In summary, the data support a model in which signal strength plays little part in Treg lineage specification, though moderate resistance of self-reactive Tregs to apoptosis may result in progressive biasing of peripheral Treg TCRs toward autoreactivity in comparison to those of conventional T cells.


Asunto(s)
Apoptosis/inmunología , Autoinmunidad , Modelos Inmunológicos , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Purinérgicos P2/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos Comunes de Leucocito/inmunología , Ratones , Receptores Purinérgicos P2X7 , Transducción de Señal/inmunología , Linfocitos T Reguladores/citología
13.
Blood ; 108(5): 1611-7, 2006 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-16684961

RESUMEN

Plasma membrane lipids are usually distributed asymmetrically, with phosphatidylserine (PS) confined to the inner leaflet. PS exposure at the outer leaflet occurs early in apoptosis, but it is also constitutive on some nonapoptotic cell populations where it plays a role in cell signaling. How PS is transported ("flopped") to the cell surface is unknown. Contrary to previous reports that normal murine B lymphocytes lack lipid asymmetry, we show that PS is normally restricted to the inner leaflet of these cells. PS exposure on normal B cells did, however, occur spontaneously ex vivo. Consistent with the hypothesis that loss of PS asymmetry is regulated by CD45, PS is constitutively exposed on viable, CD45-deficient B cells. We show that calcium-stimulated PS exposure in B cells is strain variable, ABCA1 independent, and both preceded by and dependent on a decrease in lipid packing. This decrease in lipid packing is concomitant with cell shrinkage and consequent membrane distortion, both of which are potently inhibited by blockers of volume-regulatory K+ and Cl- ion channels. Thus, changes in plasma membrane organization precede PS translocation. The data suggest a model in which PS redistribution may occur by a translocase-independent mechanism at energetically favorable sites of membrane perturbation where lipid packing is decreased.


Asunto(s)
Linfocitos B/fisiología , Lípidos/fisiología , Fosfatidilinositoles/farmacología , Animales , Linfocitos B/efectos de los fármacos , Transporte Biológico , Citometría de Flujo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositoles/metabolismo
14.
Biochemistry ; 45(17): 5503-16, 2006 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-16634632

RESUMEN

Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.


Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Oligopéptidos/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Amiloide/efectos de los fármacos , Amiloide/ultraestructura , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/farmacología , Péptidos beta-Amiloides/toxicidad , Animales , Benzotiazoles , Colorantes Fluorescentes , Glicina/química , Humanos , Metionina/química , Microscopía Electrónica , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Espectrometría de Fluorescencia , Tiazoles
15.
Arthritis Res Ther ; 7(3): R468-75, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15899033

RESUMEN

Systemic lupus erythematosus and its murine equivalent, modelled in the New Zealand Black and New Zealand White (NZB x NZW)F1 hybrid strain, are polygenic inflammatory diseases, probably reflecting an autoimmune response to debris from cells undergoing programmed cell death. Several human and murine loci contributing to disease have been defined. The present study asks whether the proinflammatory purinergic receptor P2X7, an initiator of a form of programmed cell death known as aponecrosis, is a candidate product of murine and human lupus susceptibility loci. One such locus in (NZB x NZW)F1 mice is lbw3, which is situated at the distal end of NZW chromosome 5. We first assess whether NZB mice and NZW mice carry distinct alleles of the P2RX7 gene as expressed by common laboratory strains, which differ in sensitivity to ATP stimulation. We then compare the responses of NZB lymphocytes, NZW lymphocytes and (NZB x NZW)F1 lymphocytes to P2X7 stimulation. NZB and NZW parental strains express the distinct P2X7-L and P2X7-P alleles of P2RX7, respectively, while lymphocytes from these and (NZB x NZW)F1 mice differ markedly in their responses to P2X7 receptor stimulation. NZB mice and NZW mice express functionally distinct alleles of the proinflammatory receptor, P2X7. We show that current mapping suggests that murine and human P2RX7 receptor genes lie within lupus susceptibility loci lbw3 and SLEB4, and we argue that these encode a product with the functional characteristics consistent with a role in lupus. Furthermore, we argue that aponecrosis as induced by P2X7 is a cell death mechanism with characteristics that potentially have particular relevance to disease pathogenesis.


Asunto(s)
Alelos , Predisposición Genética a la Enfermedad/genética , Lupus Eritematoso Sistémico/genética , Sitios de Carácter Cuantitativo/genética , Receptores Purinérgicos P2/genética , Animales , Humanos , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones , Ratones Endogámicos NZB , Polimorfismo Genético/genética , Polimorfismo Genético/inmunología , Sitios de Carácter Cuantitativo/inmunología , Receptores Purinérgicos P2/inmunología , Receptores Purinérgicos P2/fisiología , Receptores Purinérgicos P2X7
16.
Biochemistry ; 44(9): 3591-7, 2005 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-15736968

RESUMEN

Amyloid fibrils associated with diseases such as Alzheimer's are often derived from the transmembrane helices of membrane proteins. It is known that the fibrils have a cross-beta-sheet structure where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. However, the structural basis for how the membrane-spanning helix is converted into a beta-sheet or how protofibrils associate into fibrils is not known. Here, we use a model peptide corresponding to a portion of the single transmembrane helix of glycophorin A to investigate the structural role of glycine in amyloid-like fibrils formed from transmembrane helices. Glycophorin A contains a GxxxG motif that is found in many transmembrane sequences including that of the amyloid precursor protein and prion protein. We propose that glycine, which mediates helix interactions in membrane proteins, also provides key packing motifs when it occurs in beta-sheets. We show that glycines in the glycophorin A transmembrane helix promote extended beta-strand formation when the helix partitions into aqueous environments and stabilize the packing of beta-sheets in the formation of amyloid-like fibrils. We demonstrate that fibrillization can be disrupted with a new class of inhibitors that target the molecular grooves created by glycine.


Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Glicina/química , Glicina/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/ultraestructura , Diseño de Fármacos , Glicoforinas/síntesis química , Glicoforinas/metabolismo , Glicoforinas/ultraestructura , Proteínas de la Membrana/síntesis química , Proteínas de la Membrana/ultraestructura , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Estructura Secundaria de Proteína
17.
Blood ; 106(2): 542-9, 2005 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-15790791

RESUMEN

Scott syndrome (SS) is a bleeding disorder characterized by a failure to expose phosphatidylserine (PS) to the outer leaflet of the platelet plasma membrane. Because the adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) is implicated in the exofacial translocation of PS, we assessed its role in the pathophysiology of a patient with SS. Substantially reduced levels of ABCA1 mRNA were found in the patient's leukocytes, compared with controls. The SS patient was heterozygous for a novel missense mutation c.6064G>A (ABCA1 R1925Q), absent from unaffected family members and controls. Both mutant and wild-type alleles were reduced in mRNA expression, and no causative mutation for this phenomenon was identified in the ABCA1 gene or its proximal promoter, suggesting a putative second mutation in a trans-acting regulatory gene may also be involved in the disorder in this patient. In vitro expression studies showed impaired trafficking of ABCA1 R1925Q to the plasma membrane. Overexpression of wild-type ABCA1 in SS lymphocytes complemented the Ca2+-dependent PS exposure at the cell surface. These data identify a mutation in ABCA1 that contributes to the defective PS translocation phenotype in our patient with SS.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/sangre , Transportadoras de Casetes de Unión a ATP/genética , Trastornos de la Coagulación Sanguínea Heredados/sangre , Trastornos de la Coagulación Sanguínea Heredados/genética , Mutación Missense , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Transporte Biológico Activo/efectos de los fármacos , Trastornos de la Coagulación Sanguínea Heredados/metabolismo , Calcio/farmacología , Línea Celular , ADN/genética , Femenino , Expresión Génica , Prueba de Complementación Genética , Humanos , Técnicas In Vitro , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Fosfatidilserinas/sangre , Fosfatidilserinas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido
18.
EMBO Rep ; 4(2): 189-94, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12612610

RESUMEN

Apoptotic cell volume decrease (AVD) and exposure of phosphatidylserine (PtdSer) at the cell surface are early events in apoptosis. However, the ion channels responsible for AVD, and their relationship to PtdSer translocation and cell death are poorly understood. Real-time analysis of calcium-induced apoptosis in lymphocytes and thymocytes showed that AVD occurs rapidly, and precedes PtdSer translocation. Blockers of the K(+) channel IKCa1 completely inhibited AVD. Blockade of IKCa1, and hence AVD, also completely prevented PtdSer translocation and cell death. Thus, IKCa1-mediated AVD is the earliest-defined essential step in calcium-induced apoptosis, required for both PtdSer translocation and cell death.


Asunto(s)
Apoptosis/fisiología , Linfocitos T CD4-Positivos/metabolismo , Fosfatidilserinas/metabolismo , Canales de Potasio/metabolismo , Animales , Transporte Biológico Activo , Linfocitos T CD4-Positivos/citología , Calcimicina/metabolismo , Calcio/metabolismo , Tamaño de la Célula , Canales de Potasio de Conductancia Intermedia Activados por el Calcio , Cinética , Ganglios Linfáticos/citología , Ganglios Linfáticos/fisiología , Ratones
19.
Magn Reson Med ; 50(2): 293-302, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12876705

RESUMEN

The presence of amyloid-beta (Abeta) plaques in the brain is a hallmark pathological feature of Alzheimer's disease (AD). Transgenic mice overexpressing mutant amyloid precursor protein (APP), or both mutant APP and presenilin-1 (APP/PS1), develop Abeta plaques similar to those in AD patients, and have been proposed as animal models in which to test experimental therapeutic approaches for the clearance of Abeta. However, at present there is no in vivo whole-brain imaging method to detect Abeta plaques in mice or men. A novel method is presented to detect Abeta plaques in the brains of transgenic mice by magnetic resonance microimaging (muMRI). This method uses Abeta1-40 peptide, known for its high binding affinity to Abeta, magnetically labeled with either gadolinium (Gd) or monocrystalline iron oxide nanoparticles (MION). Intraarterial injection of magnetically labeled Abeta1-40, with mannitol to transiently open the blood-brain barrier (BBB), enabled the detection of many Abeta plaques. Furthermore, the numerical density of Abeta plaques detected by muMRI and by immunohistochemistry showed excellent correlation. This approach provides an in vivo method to detect Abeta in AD transgenic mice, and suggests that diagnostic MRI methods to detect Abeta in AD patients may ultimately be feasible.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/análisis , Química Encefálica , Imagen por Resonancia Magnética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/farmacocinética , Animales , Encéfalo/patología , Medios de Contraste , Óxido Ferrosoférrico , Gadolinio DTPA , Inmunohistoquímica , Hierro , Proteínas de la Membrana/análisis , Ratones , Ratones Transgénicos , Óxidos , Fragmentos de Péptidos/farmacocinética , Placa Amiloide/patología , Presenilina-1
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