RESUMEN
Pro-endothelial monocyte-activating polypeptide II (EMAP II), one component of the multi-aminoacyl tRNA synthetase complex, plays multiple roles in physiological and pathological processes of protein translation, signal transduction, immunity, lung development, and tumor growth. Recent studies have determined that pro-EMAP II has an essential role in maintaining axon integrity in central and peripheral neural systems where deletion of the C terminus of pro-EMAP II has been reported in a consanguineous Israeli Bedouin kindred suffering from Pelizaeus-Merzbacher-like disease. We hypothesized that the N terminus of pro-EMAP II has an important role in the regulation of protein-protein interactions. Using a GFP reporter system, we defined a putative leucine zipper in the N terminus of human pro-EMAP II protein (amino acid residues 1-70) that can form specific strip-like punctate structures. Through GFP punctum analysis, we uncovered that the pro-EMAP II C terminus (amino acids 147-312) can repress GFP punctum formation. Pulldown assays confirmed that the binding between the pro-EMAP II N terminus and its C terminus is mediated by a putative leucine zipper. Furthermore, the pro-EMAP II 1-70 amino acid region was identified as the binding partner of arginyl-tRNA synthetase, a polypeptide of the multi-aminoacyl tRNA synthetase complex. We also determined that the punctate GFP pro-EMAP II 1-70 amino acid aggregate colocalizes and binds to the neurofilament light subunit protein that is associated with pathologic neurofilament network disorganization and degeneration of motor neurons. These findings indicate the structure and binding interaction of pro-EMAP II protein and suggest a role of this protein in pathological neurodegenerative diseases.
Asunto(s)
Arginino-ARNt Ligasa/metabolismo , Citocinas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neurofilamentos/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Citocinas/química , Citocinas/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Immunoblotting , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Agregado de Proteínas , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Homología de Secuencia de AminoácidoRESUMEN
Aberrant elevation in the levels of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) contributes to neuroinflammatory diseases. Blood-brain barrier (BBB) dysfunction is a hallmark phenotype of neuroinflammation. It is known that IL-1ß directly induces BBB hyperpermeability but the mechanisms remain unclear. Claudin-5 (Cldn5) is a tight junction protein found at endothelial cell-cell contacts that are crucial for maintaining brain microvascular endothelial cell (BMVEC) integrity. Transcriptional regulation of Cldn5 has been attributed to the transcription factors ß-catenin and forkhead box protein O1 (FoxO1), and the signaling molecules regulating their nuclear translocation. Non-muscle myosin light chain kinase (nmMlck, encoded by the Mylk gene) is a key regulator involved in endothelial hyperpermeability, and IL-1ß has been shown to mediate nmMlck-dependent barrier dysfunction in epithelia. Considering these factors, we tested the hypothesis that nmMlck modulates IL-1ß-mediated downregulation of Cldn5 in BMVECs in a manner that depends on transcriptional repression mediated by ß-catenin and FoxO1. We found that treating BMVECs with IL-1ß induced barrier dysfunction concomitantly with the nuclear translocation of ß-catenin and FoxO1 and the repression of Cldn5. Most importantly, using primary BMVECs isolated from mice null for nmMlck, we identified that Cldn5 repression caused by ß-catenin and FoxO1 in IL-1ß-mediated barrier dysfunction was dependent on nmMlck.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Claudina-5/genética , Células Endoteliales/fisiología , Factores de Transcripción Forkhead/fisiología , Interleucina-1beta/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , beta Catenina/fisiología , Animales , Antígenos CD/metabolismo , Encéfalo/irrigación sanguínea , Cadherinas/metabolismo , Células Cultivadas , Claudina-5/metabolismo , Regulación hacia Abajo , Endotelio Vascular/fisiopatología , Proteína Forkhead Box O1 , Ratones , Microvasos/patología , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Activación TranscripcionalRESUMEN
Reciprocity of inflammation, oxidative stress and neovascularization is emerging as an important mechanism underlying numerous processes from tissue healing and remodelling to cancer progression. Whereas the mechanism of hypoxia-driven angiogenesis is well understood, the link between inflammation-induced oxidation and de novo blood vessel growth remains obscure. Here we show that the end products of lipid oxidation, ω-(2-carboxyethyl)pyrrole (CEP) and other related pyrroles, are generated during inflammation and wound healing and accumulate at high levels in ageing tissues in mice and in highly vascularized tumours in both murine and human melanoma. The molecular patterns of carboxyalkylpyrroles are recognized by Toll-like receptor 2 (TLR2), but not TLR4 or scavenger receptors on endothelial cells, leading to an angiogenic response that is independent of vascular endothelial growth factor. CEP promoted angiogenesis in hindlimb ischaemia and wound healing models through MyD88-dependent TLR2 signalling. Neutralization of endogenous carboxyalkylpyrroles impaired wound healing and tissue revascularization and diminished tumour angiogenesis. Both TLR2 and MyD88 are required for CEP-induced stimulation of Rac1 and endothelial migration. Taken together, these findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.
Asunto(s)
Neovascularización Patológica/metabolismo , Neovascularización Fisiológica , Estrés Oxidativo/fisiología , Pirroles/metabolismo , Receptor Toll-Like 2/metabolismo , Envejecimiento/metabolismo , Animales , Aorta/citología , Aorta/efectos de los fármacos , Línea Celular , Movimiento Celular , Células Endoteliales/metabolismo , Miembro Posterior/metabolismo , Humanos , Inmunidad Innata/inmunología , Inflamación/metabolismo , Isquemia/metabolismo , Ligandos , Melanoma/irrigación sanguínea , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Oxidación-Reducción , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Propionatos , Pirroles/química , Pirroles/farmacología , Receptores Depuradores/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 4/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiología , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Integrin activation on hematopoietic cells is essential for platelet aggregation, leukocyte adhesion, and transmigration through endothelium and extracellular matrix into inflamed tissues. To migrate through matrix, leukocyte integrin adhesion complexes undergo dynamic changes. Here we show that Kindlin-3, a main activator and binding partner of integrins in hematopoietic cells, can be cleaved by calpain in an activation-dependent manner. This calpain-mediated cleavage occurs in platelets and leukocytes as well as in endothelial cells. We determined the calpain I cleavage site in Kindlin-3 at tyrosine 373 in the N-terminal part of Kindlin-3 pleckstrin homology domain. Expression of the calpain-resistant Y373N mutant of Kindlin-3 promotes stronger cell adhesion to extracellular matrix under flow as well as to activated endothelium. In contrast, Y373N mutation in Kindlin-3 hinders cell migration. Mechanistically, calpain-resistant Y373N mutant of Kindlin-3 exhibited an activation-independent association with ß integrin cytoplasm domain. Thus, cleavage of Kindlin-3 by calpain controls the dynamics of integrin-Kindlin-3 interaction and as a result, integrin-dependent adhesion and migration of hematopoietic cells. This represents a novel mechanism regulating reversibility of integrin adhesion complexes in leukocytes, which, in turn, is critical for their successful transmigration through the extracellular matrix.
Asunto(s)
Calpaína/metabolismo , Movimiento Celular/fisiología , Matriz Extracelular/metabolismo , Células Madre Hematopoyéticas/metabolismo , Leucocitos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteolisis , Sustitución de Aminoácidos , Calpaína/genética , Adhesión Celular/fisiología , Matriz Extracelular/genética , Femenino , Células HEK293 , Células HL-60 , Células Madre Hematopoyéticas/citología , Humanos , Integrinas/genética , Integrinas/metabolismo , Células K562 , Leucocitos/citología , Masculino , Proteínas de la Membrana/genética , Mutación Missense , Proteínas de Neoplasias/genéticaRESUMEN
PURPOSE OF REVIEW: In the current review, we summarize recent progress on vasculature-specific function and regulation of integrins and integrin-associated proteins, including advances in our understanding of inside-out integrin activation. The studies on regulation of integrin activation received new impulse in 2009 with the identification of kindlin protein family members as crucial mediators of integrin inside-out signaling. In the current review, we outline the recent findings on the role of kindlins in the vascular system, as well as new studies that have begun shaping the mechanistic model of kindlins' function. RECENT FINDINGS: Several tissue-specific knockout models for integrins and genes associated with the integrin functions have been recently presented, including smooth muscle-specific integrin-linked kinase and endothelial-specific focal adhesion kinase and talin-1 ablation. In the heterozygous animal knockout model, kindlin-2 has been demonstrated as a crucial modulator of angiogenesis and vascular permeability. As a number of articles have advanced our understanding of kindlin function, they are reviewed and discussed in further detail. New findings include an additional lipid-binding site within the kindlin molecule and preferential binding of the nonphosphorylated form of ß-integrins. SUMMARY: The role of integrins in angiogenesis has been demonstrated to include, in addition to cell adhesion and mechanotransduction, specific signaling functions. The importance of integrin inside-out pathway in vascular physiology has been unequivocally proven, and endothelial permeability is directly regulated by this process. Inhibition of kindlin-dependent steps in the inside-out pathway as an approach to block platelet aggregation should be paralog-specific, as it may have adverse effects on vascular permeability.
Asunto(s)
Integrinas/fisiología , Neovascularización Fisiológica/fisiología , Animales , Técnicas de Inactivación de Genes , Humanos , Proteínas de la Membrana/fisiologíaRESUMEN
The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. The 3 members of this family, Kindlin-1, -2, and -3, perform an essential role in activation of integrin adhesion receptors, and expression of at least 1 Kindlin paralog is required to enable integrin activation in physiologically relevant settings. In humans, deficiencies in Kindlin-3 lead to a number of abnormalities affecting hemostasis, the immune system, and bone function, whereas the lack of Kindlin-1 causes profound skin defects. The importance of Kindlins is underscored by the results of animal knockout studies, which clearly show the indispensable and nonredundant functions of all 3 Kindlins in development and normal physiology. This review discusses recent progress in the studies of Kindlin protein family, emphasizing newly identified functions and potential mechanisms underlying differential activities of the family members.
Asunto(s)
Adhesión Celular/fisiología , Integrinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/fisiología , Animales , Complejo GPIb-IX de Glicoproteína PlaquetariaRESUMEN
Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Edición Génica/métodos , Genoma Humano , Reacción en Cadena de la Polimerasa/métodos , ARN Guía de Kinetoplastida/genética , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cartilla de ADN/síntesis química , Cartilla de ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/química , Electroporación/métodos , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Cultivo Primario de Células , ARN Guía de Kinetoplastida/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
Current methods can illuminate the genome-wide activity of CRISPR-Cas9 nucleases, but are not easily scalable to the throughput needed to fully understand the principles that govern Cas9 specificity. Here we describe 'circularization for high-throughput analysis of nuclease genome-wide effects by sequencing' (CHANGE-seq), a scalable, automatable tagmentation-based method for measuring the genome-wide activity of Cas9 in vitro. We applied CHANGE-seq to 110 single guide RNA targets across 13 therapeutically relevant loci in human primary T cells and identified 201,934 off-target sites, enabling the training of a machine learning model to predict off-target activity. Comparing matched genome-wide off-target, chromatin modification and accessibility, and transcriptional data, we found that cellular off-target activity was two to four times more likely to occur near active promoters, enhancers and transcribed regions. Finally, CHANGE-seq analysis of six targets across eight individual genomes revealed that human single-nucleotide variation had significant effects on activity at ~15.2% of off-target sites analyzed. CHANGE-seq is a simplified, sensitive and scalable approach to understanding the specificity of genome editors.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Epigénesis Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Secuencia de Bases , Línea Celular , Cromatina/genética , Edición Génica , Variación Genética , Genoma Humano , Humanos , Aprendizaje AutomáticoRESUMEN
The cross-talk between receptor tyrosine kinases and integrin receptors are known to be crucial for a number of cellular functions. On endothelial cells, an interaction between integrin alphavbeta3 and VEGFR2 seems to be particularly important process during vascularization. Importantly, the functional association between VEGFR2 and integrin alphavbeta3 is of reciprocal nature since each receptor is able to promote activation of its counterpart. This mutually beneficial relationship regulates a number of cellular activities involved in angiogenesis, including endothelial cell migration, survival and tube formation, and hematopoietic cell functions within vasculature. This article discusses several possible mechanisms reported by different labs which mediate formation of the complex between VEGFR-2 and alphavbeta3 on endothelial cells. The pathological consequences and regulatory events involved in this receptor cross-talk are also presented.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Neovascularización Fisiológica , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Humanos , Integrina alfaVbeta3/química , Unión Proteica , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
The intricacy of multiple feedback loops in the pathways downstream of Akt allows this kinase to control multiple cellular processes in the cardiovascular system and precludes inferring consequences of its activation in specific pathological conditions. Akt1, the major Akt isoform in the heart and vasculature, has a protective role in the endothelium during atherosclerosis. However, Akt1 activation may also have detrimental consequences in the cardiovascular system. Mice lacking both the high-density lipoprotein receptor SR-BI (scavenger receptor class B type I) and ApoE (apolipoprotein E), which promotes clearance of remnant lipoproteins, are a model of severe dyslipidemia and spontaneous myocardial infarction. We found that Akt1 was activated in these mice, and this activation correlated with cardiac dysfunction, hypertrophy, and fibrosis; increased infarct area; cholesterol accumulation in macrophages and atherosclerosis; and reduced life span. Akt1 activation was associated with inflammation, oxidative stress, accumulation of oxidized lipids, and increased abundance of CD36, a major sensor of oxidative stress, and these events created a positive feedback loop that exacerbated the consequences of oxidative stress. Genetic deletion of Akt1 in this mouse model resulted in decreased mortality, alleviation of multiple complications of heart disease, and reduced occurrence of spontaneous myocardial infarction. Thus, interference with Akt1 signaling in vivo could be protective and improve survival under dyslipidemic conditions by reducing oxidative stress and responses to oxidized lipids.
Asunto(s)
Infarto del Miocardio/enzimología , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Antígenos CD36/genética , Antígenos CD36/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática/genética , Ratones , Ratones Noqueados , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/prevención & control , Proteínas Proto-Oncogénicas c-akt/genética , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismoRESUMEN
Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between ß(3) integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of ß(3) and VEGFR2. Specifically, the membrane-proximal motif around (801)YLSI in VEGFR2 mediates its binding to non-phosphorylated ß(3)CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y(747) phosphorylation of ß(3) enhances the above interaction. To demonstrate the importance of ß(3) phosphorylation in endothelial cell functions, we synthesized ß(3)CT-mimicking Y(747) phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y(747) but not F(747) significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y(747) peptide exhibits inhibitory effect only in WT but not in ß(3) integrin knock-out or ß(3) integrin knock-in cells expressing ß(3) with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2.
Asunto(s)
Integrina beta3/metabolismo , Fosfotirosina/metabolismo , Receptor Cross-Talk , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Técnicas In Vitro , Integrina beta3/química , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neovascularización Fisiológica , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Receptor Cross-Talk/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/químicaRESUMEN
Multiple biological consequences of oxidative stress are known to contribute to aging and aging-related pathologies. It was recently shown that (carboxyalkyl)pyrroles (CAPs), the end products of phospholipid oxidation serve as a novel class of endogenous ligands for Toll-like receptors (TLRs) and promote the process of angiogenesis. In this review, we discuss implications of these findings in the context of age-related pathologies, including tumorigenesis. Accumulation of oxidation products in tissues of aging organisms might create conditions for uncontrolled pathological angiogenesis as seen in patients with age related macular degeneration. CAPs and their receptors, TLRs might also promote the progression of atherosclerotic lesions. Importantly, besides their role in a number of pathologies, oxidative products of phospholipids contribute to tissue repair processes thereby antagonizing the destructive effects of oxidation.
Asunto(s)
Envejecimiento/fisiología , Estrés Oxidativo , Animales , Humanos , Neovascularización Fisiológica , Oxidación-Reducción , Fosfolípidos/metabolismo , Pirroles/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Monogenic deficiency diseases provide unique opportunities to define the contributions of individual molecules to human physiology and to identify pathologies arising from their dysfunction. Here we describe a deficiency disease in two human siblings that presented with severe bleeding, frequent infections and osteopetrosis at an early age. These symptoms are consistent with but more severe than those reported for people with leukocyte adhesion deficiency III (LAD-III). Mechanistically, these symptoms arose from an inability to activate the integrins expressed on hematopoietic cells, including platelets and leukocytes. Immortalized lymphocyte cell lines isolated from the two individuals showed integrin activation defects. Several proteins previously implicated in integrin activation, including Ras-associated protein-1 (RAP1) and calcium and diacylglycerol-regulated guanine nucleotide exchange factor-1 (CALDAG-GEF1), were present and functional in these cell lines. The genetic basis for this disease was traced to a point mutation in the coding region of the KINDLIN3 (official gene symbol FERMT3) gene. When wild-type KINDLIN-3 was expressed in the immortalized lymphocytes, their integrins became responsive to activation signals. These results identify a genetic disease that severely compromises the health of the affected individuals and establish an essential role of KINDLIN-3 in integrin activation in humans. Furthermore, allogeneic bone marrow transplantation was shown to alleviate the symptoms of the disease.
Asunto(s)
Integrinas/fisiología , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/fisiología , Mutación Puntual , Trasplante de Médula Ósea , Línea Celular Transformada , Humanos , Síndrome de Deficiencia de Adhesión del Leucocito/genética , Síndrome de Deficiencia de Adhesión del Leucocito/cirugía , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genéticaRESUMEN
Pathological hallmarks of Alzheimer's disease are the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles, and neurodegeneration. The principal component of amyloid plaques is the amyloid-beta peptide (Abeta). Accumulating evidence indicates that Abeta may play a causal role in Alzheimer's disease. In this report, we demonstrate that Abeta deposition and neurotoxicity in human cortical primary neurons are mediated through alpha2beta1 and alphaVbeta1 integrins using specific integrin-blocking antibodies. An aberrant integrin signaling pathway causing the neurotoxicity is mediated through Pyk2. The role of alpha2beta1 and alphaVbeta1 integrins can be extended to another amyloidosis using an amylin in vitro neurotoxicity model. These results indicate that the alpha2beta1 and alphaVbeta1 integrin signaling pathway may be critical components of neurodegeneration in Alzheimer's disease and that integrins may recognize and be activated by a shared structural motif of polymerizing amyloidogenic proteins.
Asunto(s)
Péptidos beta-Amiloides/administración & dosificación , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Integrina alfa2beta1/metabolismo , Neuronas/metabolismo , Neuronas/patología , Transducción de Señal/efectos de los fármacos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Apoptosis/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Neuronas/efectos de los fármacos , Neurotoxinas/administración & dosificaciónRESUMEN
Based on a variety of genetic, biochemical, and neuropathological evidence, amyloid-beta peptide (Abeta) has been suggested to be causal in Alzheimer's disease (AD). Abeta has been shown to mediate neurodegenerative and inflammatory changes associated with amyloid plaques, as well as exert direct neurotoxicity through oligomeric forms of Abeta. The mechanism of Abeta toxicity, however, remains largely unknown. In this work, we show that an early event after exposure of postmitotic neurons to Abeta is tyrosine phosphorylation of FISH adapter protein. FISH binds to and potentially regulates certain ADAM family members. We present evidence that FISH and ADAM12 mediate the neurotoxic effect of Abeta. Expression of an ADAM12 protease-deficient mutant (ADAM12DeltaMP) blocks Abeta-induced neuronal death, and expression of an N-terminal fragment of FISH reduces Abeta toxicity. The C-terminal fragment of FISH containing the ADAMs binding region is found to be sufficient for induction of neuronal death, which is prevented by coexpression of the ADAM12DeltaMP. Abeta treatment, as well as expression of the C-terminal toxic FISH fragment, induces accumulation of ADAM12 N-terminal cleavage product in conditioned medium, demonstrating activation of the ADAM metalloprotease/sheddase activity. ADAM12 protein is reduced in AD brains, pointing to a possible increase in ADAM12 proteolytic activity. These data suggest that Abeta toxicity is mediated by FISH and ADAM12 and may provide insights into therapeutic strategies for AD treatment.