RESUMEN
AIMS/HYPOTHESIS: We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2ß. We also identified miR-153 target genes that correlated with IA-2ß localisation and function. METHODS: A bioinformatics approach was used to identify miR-153's genomic organisation. To analyse the co-regulation of miR-153 and IA-2ß, quantitative PCR analysis of miR-153 and Ia-2ß (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2ß single knockout and Ia-2/Ia-2ß double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out. RESULTS: Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2ß, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2ß in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2ß knockout and Ia-2/Ia-2ß double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2ß expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels. CONCLUSIONS/INTERPRETATION: This study suggests the involvement of miR-153, IA-2ß and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.
Asunto(s)
Encéfalo/metabolismo , MicroARNs/genética , Páncreas/metabolismo , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Femenino , Masculino , Ratones , Ratones Noqueados , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Regulated intramembrane proteolysis by gamma-secretase cleaves proteins in their transmembrane domain and is involved in important signaling pathways. At least four different gamma-secretase complexes have been identified, but little is known about their biological role and specificity. Previous work has demonstrated the involvement of the Aph1A-gamma-secretase complex in Notch signaling, but no specific function could be assigned to Aph1B/C-gamma-secretase. We demonstrate here that the Aph1B/C-gamma-secretase complex is expressed in brain areas relevant to schizophrenia pathogenesis and that Aph1B/C deficiency causes pharmacological and behavioral abnormalities that can be reversed by antipsychotic drugs. At the molecular level we find accumulation of Nrg1 fragments in the brain of Aph1BC(-/-) mice. Our observations gain clinical relevance by the demonstration that a Val-to-Leu mutation in the Nrg1 transmembrane domain, associated with increased risk for schizophrenia, affects gamma-secretase cleavage of Nrg1. This finding suggests that dysregulation of intramembrane proteolysis of Nrg1 could increase risk for schizophrenia and related disorders.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/deficiencia , Antipsicóticos/farmacología , Endopeptidasas/deficiencia , Trastornos Neurológicos de la Marcha/etiología , Neurregulina-1/metabolismo , Subunidades de Proteína/deficiencia , Animales , Antipsicóticos/uso terapéutico , Proteínas de la Membrana , Ratones , Ratones Noqueados , Mutación Missense/fisiología , Esquizofrenia/etiologíaRESUMEN
BACKGROUND: Preclinical models to determine blood to brain transport ability of therapeutics are often ambiguous. In this study a method is developed that relies on CNS target-engagement and is able to rank brain-penetrating capacities. This method led to the discovery of an anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via receptor-mediated transcytosis. METHODS: Various nanobodies against the mouse transferrin receptor were fused to neurotensin and injected peripherally in mice. Neurotensin is a neuropeptide that causes hypothermia when present in the brain but is unable to reach the brain from the periphery. Continuous body temperature measurements were used as a readout for brain penetration of nanobody-neurotensin fusions after its peripheral administration. Full temperature curves were analyzed using two-way ANOVA with Dunnett multiple comparisons tests. RESULTS: One anti-transferrin receptor nanobody coupled to neurotensin elicited a drop in body temperature following intravenous injection. Epitope binning indicated that this nanobody bound a distinct transferrin receptor epitope compared to the non-crossing nanobodies. This brain-penetrating nanobody was used to characterize the in vivo hypothermia model. The hypothermic effect caused by neurotensin is dose-dependent and could be used to directly compare peripheral administration routes and various nanobodies in terms of brain exposure. CONCLUSION: This method led to the discovery of an anti-transferrin receptor nanobody that can reach the brain via receptor-mediated transcytosis after peripheral administration. This method could be used to assess novel proteins for brain-penetrating capabilities using a target-engaging readout.
Asunto(s)
Temperatura Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Neurotensina/farmacología , Receptores de Transferrina/inmunología , Anticuerpos de Dominio Único/farmacología , Transcitosis/fisiología , Animales , Camélidos del Nuevo Mundo , Femenino , Masculino , Ratones , Neurotensina/administración & dosificación , Receptores de Neurotensina/efectos de los fármacos , Anticuerpos de Dominio Único/administración & dosificaciónRESUMEN
Amyloid precursor protein (APP) secretase plays a pivotal role in the processing of APP since its activity precludes the formation of amyloid peptide in Alzheimer's Disease. The identity and the subcellular localization of this enzyme are at this moment unknown. It is also unclear how APP escapes the activity of this enzyme when amyloid is formed. We have previously shown that APP-secretase activity is not inhibited by exogenously added proteinase inhibitors of different specificity (De Strooper, B., F. Van Leuven, and H. Van Den Berghe. 1992. FEBS (Fed. Eur. Biochem. Soc.) Lett. 308:50-53). We show here that the primary amine methylamine inhibits the secretion of APP into the medium. Furthermore, we show that a truncated form of APP, devoid of the cytoplasmic domain, is more efficiently cleaved and secreted than wild-type APP, which together with the methylamine block, shows that APP-secretase is located in a late compartment of the default constitutional secretion pathway. The sorting signals in the cytoplasmic domain of APP are therefore important in the deviation of APP from the secretase pathway. Finally we show that mutation of Arg609 to Asp in combination with Lys612 to Glu makes APP a less efficiently cleaved substrate for APP-secretase. The results are discussed in the context of recent findings on the targeting of APP and a parallel is drawn with some lysosomal glycoproteins that follow similar pathways.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/análisis , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/biosíntesis , Precursor de Proteína beta-Amiloide/química , Animales , Arginina , Asparagina , Ácido Aspártico Endopeptidasas , Secuencia de Bases , Compartimento Celular , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Metilaminas/farmacología , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
We investigated the relationship between PS1 and gamma-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no gamma-secretase processing was observed when holo-APP or APP-C99, a direct substrate for gamma-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent gamma-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the gamma-cleavage of APP-C99. In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment. Our data confirm the "spatial paradox" and raise several questions regarding the PS1 is gamma-secretase hypothesis.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Compartimento Celular , Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/aislamiento & purificación , Animales , Ácido Aspártico Endopeptidasas , Células Cultivadas , Endopeptidasas/aislamiento & purificación , Retículo Endoplásmico , Aparato de Golgi , Proteínas de la Membrana/aislamiento & purificación , Ratones , Mutación , Neuronas/citología , Neuronas/ultraestructura , Presenilina-1 , Transporte de Proteínas/genéticaRESUMEN
Mutations of presenilin 1 (PS1) causing Alzheimer's disease selectively increase the secretion of the amyloidogenic betaA4(1-42), whereas knocking out the gene results in decreased production of both betaA4(1-40) and (1-42) amyloid peptides (De Strooper et al. 1998). Therefore, PS1 function is closely linked to the gamma-secretase processing of the amyloid precursor protein (APP). Given the ongoing controversy on the subcellular localization of PS1, it remains unclear at what level of the secretory and endocytic pathways PS1 exerts its activity on APP and on the APP carboxy-terminal fragments that are the direct substrates for gamma-secretase. Therefore, we have reinvestigated the subcellular localization of endogenously expressed PS1 in neurons in vitro and in vivo using confocal microscopy and fine-tuned subcellular fractionation. We show that uncleaved PS1 holoprotein is recovered in the nuclear envelope fraction, whereas the cleaved PS fragments are found mainly in post-ER membranes including the intermediate compartment (IC). PS1 is concentrated in discrete sec23p- and p58/ERGIC-53-positive patches, suggesting its localization in subdomains involved in ER export. PS1 is not found to significant amounts beyond the cis-Golgi. Surprisingly, we found that APP carboxy-terminal fragments also coenrich in the pre-Golgi membrane fractions, consistent with the idea that these fragments are the real substrates for gamma-secretase. Functional evidence that PS1 exerts its effects on gamma-secretase processing of APP in the ER/IC was obtained using a series of APP trafficking mutants. These mutants were investigated in hippocampal neurons derived from transgenic mice expressing PS1wt or PS1 containing clinical mutations (PS1(M146L) and PS1(L286V)) at physiologically relevant levels. We demonstrate that the APP-London and PS1 mutations have additive effects on the increased secretion of betaA4(1-42) relative to betaA4(1-40), indicating that both mutations operate independently. Overall, our data clearly establish that PS1 controls gamma(42)-secretase activity in pre-Golgi compartments. We discuss models that reconcile this conclusion with the effects of PS1 deficiency on the generation of betaA4(1-40) peptide in the late biosynthetic and endocytic pathways.
Asunto(s)
Precursor de Proteína beta-Amiloide/fisiología , Endopeptidasas/metabolismo , Hipocampo/fisiología , Proteínas de la Membrana/fisiología , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Aparato de Golgi/fisiología , Aparato de Golgi/ultraestructura , Hipocampo/ultraestructura , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Neuronas/fisiología , Neuronas/ultraestructura , Presenilina-1 , Procesamiento Proteico-PostraduccionalRESUMEN
Alzheimer's disease (AD) research has shown that patients with an inherited form of the disease carry mutations in the presenilin proteins or the amyloid precursor protein (APP). These disease-linked mutations result in increased production of the longer form of amyloid-beta (the primary component of the amyloid deposits found in AD brains). However, it is not clear how the presenilins contribute to this increase. New findings now show that the presenilins affect APP processing through their effects on gamma-secretase, an enzyme that cleaves APP. Also, it is known that the presenilins are involved in the cleavage of the Notch receptor, hinting that they either directly regulate gamma-secretase activity or themselves are protease enzymes. These findings suggest that the presenilins may prove to be valuable molecular targets for the development of drugs to combat AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Humanos , Inhibidores de Proteasas/uso terapéutico , Procesamiento Proteico-Postraduccional , Receptores Notch , Transducción de SeñalRESUMEN
The carboxyl terminus of presenilin 1 and 2 (PS1 and PS2) binds to the neuron-specific cell adhesion molecule telencephalin (TLN) in the brain. PS1 deficiency results in the abnormal accumulation of TLN in a yet unidentified intracellular compartment. The first transmembrane domain and carboxyl terminus of PS1 form a binding pocket with the transmembrane domain of TLN. Remarkably, APP binds to the same regions via part of its transmembrane domain encompassing the critical residues mutated in familial Alzheimer's disease. Our data surprisingly indicate a spatial dissociation between the binding site and the proposed catalytic site near the critical aspartates in PSs. They provide important experimental evidence to support a ring structure model for PS.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/metabolismo , Precursor de Proteína beta-Amiloide/química , Animales , Sitios de Unión , Diferenciación Celular , Expresión Génica , Hipocampo/citología , Glicoproteínas de Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/química , Neuronas/citología , Neuronas/metabolismo , Presenilina-1 , Presenilina-2 , Estructura Terciaria de Proteína , Técnicas del Sistema de Dos HíbridosRESUMEN
The apoptosis-inducing Fas ligand (FasL) is a type II transmembrane protein that is involved in the downregulation of immune reactions by activation-induced cell death (AICD) as well as in T cell-mediated cytotoxicity. Proteolytic cleavage leads to the generation of membrane-bound N-terminal fragments and a soluble FasL (sFasL) ectodomain. sFasL can be detected in the serum of patients with dysregulated inflammatory diseases and is discussed to affect Fas-FasL-mediated apoptosis. Using pharmacological approaches in 293T cells, in vitro cleavage assays as well as loss and gain of function studies in murine embryonic fibroblasts (MEFs), we demonstrate that the disintegrin and metalloprotease ADAM10 is critically involved in the shedding of FasL. In primary human T cells, FasL shedding is significantly reduced after inhibition of ADAM10. The resulting elevated FasL surface expression is associated with increased killing capacity and an increase of T cells undergoing AICD. Overall, our findings suggest that ADAM10 represents an important molecular modulator of FasL-mediated cell death.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteína Ligando Fas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Muerte Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteína Ligando Fas/química , Humanos , Células Jurkat , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Solubilidad/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Presenilin-1 (PS1) is a transmembrane protein that is located in the endoplasmic reticulum and the cis Golgi apparatus. Missense mutations of PS1 that modify gamma-secretase function, leading to a pathologic processing of amyloid precursor protein, are an important cause of familial Alzheimer's disease. Physiologically, the presenilins are involved in the Notch and Wnt-beta-catenin signaling pathways. RESULTS: PS1-deficient mice develop a cortical dysplasia resembling human type 2 lissencephaly, with leptomeningeal fibrosis and migration of cortical-plate neurons beyond their normal position into the marginal zone and subarachnoid space. This disorder of neuronal migration is associated with the disappearance of the majority of the cells of the marginal zone, notably most of the Cajal-Retzius pioneer neurons, between embryonic days E14 and E18, and is preceded and accompanied by disorganization of Notch-1 immunoreactivity on the neuronal cell membranes. The marginal zone also becomes depleted of the extracellular matrix protein reelin and chondroitin sulfate proteoglycans. At that stage PS1 is transiently expressed in leptomeningeal fibroblasts, which are mandatory for the trophic support of Cajal-Retzius neurons. CONCLUSIONS: In agreement with models in which neuronal migration disorders have been linked to a defect in Cajal-Retzius cells, the loss of most of these cells in PS1-deficient mice leads to cortical dysplasia. Because PS1 is normally expressed in the leptomeninges, and these become fibrotic in the PS1-knockout mice, we favor the hypothesis that the loss of Cajal-Retzius cells is caused by a defective trophic interaction with leptomeningeal cells, possibly involving disruption of Notch signaling.
Asunto(s)
Corteza Cerebral/anomalías , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Neuronas/fisiología , Receptores de Superficie Celular , Factores de Transcripción , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Moléculas de Adhesión Celular Neuronal/metabolismo , Movimiento Celular , Corteza Cerebral/anatomía & histología , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/embriología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Endopeptidasas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Meninges/metabolismo , Ratones , Ratones Noqueados , Mutación Missense , Proteínas del Tejido Nervioso , Presenilina-1 , Receptor Notch1 , Proteína Reelina , Serina Endopeptidasas , Factores de TiempoRESUMEN
The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites). Notably, Nanobodies generally bind conformational epitopes making them very interesting tools to stabilize, dissect, and characterize specific protein conformations. In the clinic, several Nanobodies are under evaluation either as potential drug candidates or as diagnostic tools. In recent years, we have successfully generated high-affinity, conformation-sensitive anti-γ-secretase Nanobodies. γ-Secretase is a multimeric membrane protease involved in processing of the amyloid precursor protein with high clinical relevance as mutations in its catalytic subunit (Presenilin) cause early-onset Alzheimer's disease. Advancing our knowledge on the mechanisms governing γ-secretase intramembrane proteolysis through various strategies may lead to novel therapeutic avenues for Alzheimer's disease. In this chapter, we present the strategies we have developed and applied for the screening and characterization of anti-γ-secretase Nanobodies. These protocols could be of help in the generation of Nanobodies targeting other membrane proteins.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide/química , Biología Molecular/métodos , Presenilinas/química , Anticuerpos de Dominio Único/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/uso terapéutico , Dominio Catalítico/genética , Epítopos/inmunología , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mutación , Presenilinas/genética , Presenilinas/inmunología , Presenilinas/uso terapéutico , Conformación Proteica , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéutico , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/uso terapéutico , Relación Estructura-ActividadRESUMEN
Since the first report showing that Alzheimer disease (AD) might be caused by mutations in the amyloid precursor protein gene (APP), 20 different missense mutations have been reported. The majority of early-onset AD mutations alter processing of APP increasing relative levels of Abeta42 peptide, either by increasing Abeta42 or decreasing Abeta40 peptide levels or both. In a diagnostic setting using direct sequence analysis, we identified in one patient with familial early-onset AD a novel mutation in APP (c.2172G>C), predicting a K724N substitution in the intracytosolic fragment. The mutation is located downstream of the epsilon-cleavage site of APP and is the furthermost C-terminal mutation reported to date. In vitro expression of APP K724N cDNA showed an increase in Abeta42 and a decrease in Abeta40 levels resulting in a near three-fold increase of the Abeta42/Abeta40 ratio. Further, in vivo amyloid positron emission tomography (PET) imaging revealed significantly increased cortical amyloid deposits, supporting that in human this novel APP mutation is likely causing disease.
Asunto(s)
Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Mutación Missense , Anciano , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/diagnóstico por imagen , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Bélgica , Encéfalo/diagnóstico por imagen , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Linaje , Tomografía de Emisión de Positrones , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Análisis de Secuencia de ProteínaRESUMEN
Mis-sense mutations of presenilin 1 increase the release of amyloidogenic peptide from amyloid precursor protein (APP) and are a major cause of familial Alzheimer's Disease. Loss-of-function mutations of presenilins in the mouse, Caenorhabditis elegans and Drosophila result in severe developmental defects caused by disturbed Notch signalling. Recent studies suggest that the diverse biological roles of presenilin 1 can be explained at the molecular level by its role in the proteolytic cleavage of the integral membrane domains of Notch and APP. This cleavage is a central switch in Notch signalling, while, for APP, its physiological role remains elusive. Evidence that presenilin 1 itself has catalytic properties could explain many of the biological and biochemical alterations caused by presenilin-1 deficiency or clinical mutations in presenilin 1. However, as presenilins reside in the endoplasmic reticulum and the cleavage of Notch and APP is believed to occur close to the cell membrane, the scientific field now faces a 'spatial paradox'.
Asunto(s)
Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/enzimología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación Missense , Presenilina-1 , Presenilina-2RESUMEN
It is widely believed that the pathogenesis of Alzheimer's disease (AD) is intimately, if not causatively, associated with the deposition of approximately 4 kDa beta-amyloid (A beta) peptides in the cerebral cortex and hippocampus of affected individuals. A beta peptides are liberated from transmembrane proteins, termed beta-amyloid precursor proteins (APP), by the concerted action of beta- and gamma-secretase(s). Whereas the identity of beta-secretase is no longer in question, the identity of gamma-secretase, which is responsible for the intramembranous processing of APP, has never been more enigmatic. Considerable evidence has accrued to impugn the presenilins (PS) as the executioners of intramembranous processing of APP. Here, we summarize these observations and review recent evidence that argues against the prevailing hypothesis that PS function as gamma-secretases.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Receptores de Superficie Celular , Factores de Transcripción , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Receptor Notch1Asunto(s)
Endopeptidasas/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/fisiología , Células Madre/enzimología , Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas , Activación Enzimática , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Mutación , Presenilina-1 , Presenilina-2 , Células Madre/metabolismoRESUMEN
The cDNA sequence for amyloid precursor protein and the two alternatively spliced forms were determined. Seven nucleotide differences resulting in six amino acid differences compared to the previously published mouse sequence were found. Moreover, the 3' non-coding end was completely different. Part of these findings were confirmed in three different mouse strains. The resulting mouse cDNA resembles the human APP more closely than originally reported.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Homología de Secuencia de Ácido Nucleico , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Procesamiento Postranscripcional del ARN/genéticaRESUMEN
Proteolytic cleavage of the amyloid precursor protein (APP) has previously been shown to release its extracellular domain into the medium. The identification of the responsible proteinase(s), termed secretase(s), is a high priority in ongoing Alzheimer research. This is hampered by the unusual characteristics of these enzyme(s) and by the fact that they cleave only membrane associated APP. We report here, using a vaccinia virus based expression system, that pig kidney PK(15) cells express full-length, membrane bound APP695, but that secretion of APP is low. This heterologous expression system allows to assay candidate secretases in a cellular context by simple co-transfection of the APP and candidate secretase cDNA containing plasmids. Eight different members of the mouse and human furin family of proprotein processing enzymes were tested in this assay, but none of them enhanced the secretion of APP. Secretion of von Willebrand's factor was used as a positive control.
Asunto(s)
Amiloide/metabolismo , Riñón/enzimología , Proteínas de la Membrana , Precursores de Proteínas/metabolismo , Subtilisinas/metabolismo , Amiloide/biosíntesis , Animales , Células Cultivadas , Endopeptidasas/metabolismo , Furina , Humanos , Ratones , Plásmidos , Proteínas Priónicas , Priones , Precursores de Proteínas/biosíntesis , Porcinos , Transfección , Virus Vaccinia/enzimologíaRESUMEN
We have molecularly cloned and sequenced the mouse alpha-2-macroglobulin receptor cDNA. The cDNA contained 14849 bases with one large open reading frame of 4545 codons which is one more than in the corresponding human cDNA. Comparison of the predicted mouse and human receptor proteins revealed the very conserved nature of this receptor with an overall amino acid identity of more than 97%. A dramatic example of this is the presence of 331 cysteine residues predicted in the mouse protein, of which 327 are positionally conserved relative to human.
Asunto(s)
Receptores Inmunológicos/biosíntesis , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/análisis , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Señales de Clasificación de Proteína/química , Receptores Inmunológicos/química , Receptores Inmunológicos/genéticaRESUMEN
PINK1 (phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced kinase 1), a Parkinson's disease-associated gene, was identified originally because of its induction by the tumor-suppressor PTEN. PINK1 promotes cell survival and potentially metastatic functions and protects against cell stressors including chemotherapeutic agents. However, the mechanisms underlying PINK1 function in cancer cell biology are unclear. Here, using several model systems, we show that PINK1 deletion significantly reduced cancer-associated phenotypes including cell proliferation, colony formation and invasiveness, which were restored by human PINK1 overexpression. Results show that PINK1 deletion causes major defects in cell cycle progression in immortalized mouse embryonic fibroblasts (MEFs) from PINK1(-/-) mice, and in BE(2)-M17 cells stably transduced with short hairpin RNA against PINK1. Detailed cell cycle analyses of MEF cell lines from several PINK1(-/-) mice demonstrate an increased proportion of cells in G2/M and decreased number of cells in G1 following release from nocodazole block. This was concomitant with increased double and multi-nucleated cells, a reduced ability to undergo cytokinesis and to re-enter G1, and significant alterations in cell cycle markers, including failure to increase cyclin D1, all indicative of mitotic arrest. PINK1(-/-) cells also demonstrated ineffective cell cycle exit following serum deprivation. Cell cycle defects associated with PINK1 deficiency occur at points critical for cell division, growth and stress resistance in cancer cells were rescued by ectopic expression of human PINK1 and demonstrated PINK1 kinase dependence. The importance of PINK1 for cell cycle control is further supported by results showing that cell cycle deficits induced by PINK1 deletion were linked mechanistically to aberrant mitochondrial fission and its regulation by dynamin-related protein-1 (Drp1), known to be critical for progression of mitosis. Our data indicate that PINK1 has tumor-promoting properties and demonstrates a new function for PINK1 as a regulator of the cell cycle.
Asunto(s)
División Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Puntos de Control de la Fase M del Ciclo Celular/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular/genética , Ciclina D1/biosíntesis , Citocinesis/genética , Dinaminas , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Células MCF-7 , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/genética , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Invasividad Neoplásica/genética , Enfermedad de Parkinson/genética , Proteínas Quinasas/biosíntesis , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Because it has been suggested that alpha 2M could be involved in the generation of amyloid peptide, attention was given to a possible association of alpha 2M expression and amyloid accumulation in the brain. Therefore, we investigated the presence of the proteinase inhibitor alpha 2-macroglobulin (alpha 2M) in the cerebra of 4 patients with Alzheimer's Disease (AD). One case of a patient with Down's syndrome, 2 cases of patients with Dementia of the Lewy Body type (DLB), 1 case of an aged, clinically nondemented person who displayed many amyloid plaques, and 3 normal aged control brains were also studied. The results obtained by immunocytochemistry with monoclonal antibodies directed against two different epitopes of human alpha 2M showed an association of alpha 2M, only with neuritic-type plaques in patients with AD. No alpha 2M immunoreactivity was found in either preamyloid-type plaques or burned out-type plaques in AD, DLB, or aged nondemented controls. The results do not support a direct role of this proteinase inhibitor in the formation of amyloid. Because alpha 2M is observed to be associated with reactive microglia in the outer border of the neuritic plaques, the data suggest that alpha 2M could be a marker for an inflammatory cellular process in these neuritic plaques.