RESUMEN
Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular ß-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.
Asunto(s)
Presenilina-1/fisiología , Presenilina-2/fisiología , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Sistemas CRISPR-Cas , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Hidrólisis , Ratones , Presenilina-1/genética , Presenilina-2/genética , Especificidad por SustratoRESUMEN
Alzheimer's disease is the most common form of age-related dementia. At least 15 mutations in the human gene PRESENILIN 2 (PSEN2) have been found to cause familial Alzheimer's disease (fAD). Zebrafish possess an orthologous gene, psen2, and present opportunities for investigation of PRESENILIN function related to Alzheimer's disease. The most prevalent and best characterized fAD mutation in PSEN2 is N141I. The equivalent codon in zebrafish psen2 is N140. We used genome editing technology in zebrafish to target generation of mutations to the N140 codon. We isolated two mutations: psen2N140fs, (hereafter "N140fs"), causing truncation of the coding sequence, and psen2T141_L142delinsMISLISV, (hereafter "T141_L142delinsMISLISV"), that deletes the two codons immediately downstream of N140 and replaces them with seven codons coding for amino acid residues MISLISV. Thus, like almost every fAD mutation in the PRESENILIN genes, this latter mutation does not truncate the gene's open reading frame. Both mutations are homozygous viable although N140fs transcripts are subject to nonsense-mediated decay and lack any possibility of coding for an active γ-secretase enzyme. N140fs homozygous larvae initially show grossly normal melanotic skin pigmentation but subsequently lose this as they grow while retaining pigmentation in the retinal pigmented epithelium. T141_L142delinsMISLISV homozygotes retain faint skin melanotic pigmentation as adults, most likely indicating that the protein encoded by this allele retains weak γ-secretase activity. Null mutations in the human PRESENILIN genes do not cause Alzheimer's disease so these two mutations may be useful for future investigation of the differential effects of null and fAD-like PRESENILIN mutations on brain aging.
Asunto(s)
Melanocitos/metabolismo , Presenilina-2/genética , Pigmentación de la Piel/genética , Proteínas de Pez Cebra/genética , Pez Cebra/fisiología , Enfermedad de Alzheimer/genética , Animales , Animales Modificados Genéticamente , Embrión no Mamífero , Humanos , Melanocitos/fisiología , Presenilina-2/fisiología , Epitelio Pigmentado de la Retina/fisiología , Homología de Secuencia , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/fisiologíaRESUMEN
Alzheimer's disease (AD) is the most common form of dementia and causes impairments of memory, cognition and behavior. Remarkably, most AD patients exhibit personality changes that often precede other early clinical manifestations. Conditional presenilin1 (PS1) and presenilin2 (PS2) double knockout (DKO) mice have age-related forebrain atrophy, tau hyperphosphorylation, synaptic dysfunction, cognitive deficits and increased inflammatory responses in both the periphery and the brain. Whether these mice have age-related emotional changes have not yet been investigated. In the present study, we used 2-, 6- and 11-month-old DKO and littermate control (CON) mice to examine their age-related emotional conditions. Our results indicate that DKO mice have observable age-related neuropsychiatric symptoms, such as anxiety, irritability, depression, apathy, aggressivity, anhedonia and aberrant motor behavior when compared with other AD-like mouse models. In summary, our results not only indicate that DKO mice may be a valuable model for probing age-related AD diagnoses but also suggest a new pathogenesis of neurodegenerative diseases that is worth further investigation.
Asunto(s)
Síntomas Conductuales/fisiopatología , Encéfalo/fisiopatología , Presenilina-1/fisiología , Presenilina-2/fisiología , Factores de Edad , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Presenilina-1/genética , Presenilina-2/genéticaRESUMEN
Studies on the identification of the genetic basis for sexual dimorphism in peak bone mass are obviously important for providing novel therapeutic approaches to prevent or treat metabolic bone diseases. Our goal in this study was to identify the bone microstructure that could lead to differences in volumetric bone mineral density (vBMD) and new candidate genes that regulate the gender effect on bone. We used a congenic line of mice that carry the BMD1-4 locus from CAST/EiJ (CAST) mice in a C57BL/6J (B6) background and show greater vBMD in female, but not male, congenics compared to age- and gender-matched B6 mice. To assess the vBMD variations between the two lines of mice, we performed µCT measurements and found no difference in cortical bone volume by tissue volume (BV/TV) between congenics and B6 mice. However, trabecular BV/TV was significantly greater in female, but not male, congenics compared to corresponding B6 mice, which was due to increased trabecular thickness but not reduced trabecular separation, suggesting that bone formation, but not bone resorption, is responsible for the trabecular bone phenotype observed in the female, but not male, congenics. To identify the gender candidate genes, we determined the polymorphisms between B6 and CAST within the BMD1-4 locus and performed gene expression profiling. We identified EF-hand calcium binding domain (Efcab2), consortin, connexin sorting protein (Cnst), and presenilin 2 (Psen2) as potential candidate genes that regulate bone mass by influencing trabecular thickness in a gender-specific manner.
Asunto(s)
Densidad Ósea/genética , Huesos/citología , Cromosomas/genética , Genes Reguladores/genética , Polimorfismo de Nucleótido Simple/genética , Caracteres Sexuales , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Femenino , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Fenotipo , Presenilina-2/genética , Presenilina-2/fisiologíaRESUMEN
Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER-mitochondrial communication-as measured by cholesteryl ester and phospholipid synthesis, respectively-are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER-mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Embrión de Mamíferos/patología , Fibroblastos/patología , Microdominios de Membrana/patología , Mitocondrias/patología , Membranas Mitocondriales/patología , Presenilina-1/fisiología , Presenilina-2/fisiología , Enfermedad de Alzheimer/metabolismo , Animales , Western Blotting , Células Cultivadas , Embrión de Mamíferos/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Fibroblastos/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Presenilina-1/antagonistas & inhibidores , Presenilina-2/antagonistas & inhibidores , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones SubcelularesRESUMEN
Presenilins 1 and 2 (PS1 and PS2) are the catalytic subunits of the γ-secretase complex, and genes encoding mutant PS1 and PS2 variants cause familial forms of Alzheimer's disease. Lee et al. (2010) recently reported that loss of PS1 activity lead to impairments in autophagosomal function as a consequence of lysosomal alkalinization, caused by failed maturation of the proton translocating V0a1 subunit of the vacuolar (H+)-ATPase and targeting to the lysosome. We have reexamined these issues in mammalian cells and in brains of mice lacking PS (PScdko) and have been unable to find evidence that the turnover of autophagic substrates, vesicle pH, V0a1 maturation, or lysosome function is altered compared with wild-type counterparts. Collectively, our studies fail to document a role for presenilins in regulating cellular autophagosomal function. On the other hand, our transcriptome studies of PScdko mouse brains reveal, for the first time, a role for PS in regulating lysosomal biogenesis.
Asunto(s)
Autofagia/fisiología , Lisosomas/metabolismo , Presenilina-1/fisiología , Presenilina-2/fisiología , Presenilinas/fisiología , Animales , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Blastocisto/metabolismo , Western Blotting , Catepsina D/metabolismo , Línea Celular Tumoral , Células Cultivadas , Cartilla de ADN , Expresión Génica/fisiología , Humanos , Concentración de Iones de Hidrógeno , Ratones , Ratones Noqueados , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Presenilina-1/genética , Presenilina-2/genética , Presenilinas/genética , ARN/biosíntesis , ARN/genética , ARN Interferente Pequeño/farmacología , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
The presenilin complex is composed of four core proteins (presenilin 1 or presenilin 2, APH1, nicastrin, and PEN2). Several endogenous proteins have been reported to selectively modulate the function of the presenilin complexes; these include transmembrane trafficking protein, 21-KD (TMP21), CD147 antigen (basigin), the γ-secretase-activating protein (gSAP), and the orphan G-protein-coupled receptor 3. Because the structure and assembly of these complexes underlies their activity, this review will discuss current work on the assembly of the complex and on presenilin-interacting proteins that regulate secretase activity.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Presenilina-1/química , Presenilina-2/química , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/fisiología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Animales , Endopeptidasas , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Péptido Hidrolasas/química , Péptido Hidrolasas/fisiología , Presenilina-1/fisiología , Presenilina-2/fisiologíaRESUMEN
Alzheimer's disease causing mutations in the amyloid precursor protein (APP) or in the Presenilin 1 (PS1) or Presenilin 2 (PS2) genes increase the production of amyloid peptides (Aß) that precipitate in amyloid plaques. Since amyloid plaques are also a prominent feature of sporadic Alzheimer's disease (AD), abnormal proteolysis of APP and the generation of amyloid beta (Aß) are key events in the pathogenesis of AD. The proteases (secretases) that cleave APP are therefore important therapeutic targets, both for the rare familial forms but likely also for the sporadic forms of AD. The identification and understanding of the (neuro)biological functions of the α-, ß-, and presenilin/γ-secretase (complexes) is important for the development of drugs and the delineation of their associated side effects. The potential impact of this type of research exceeds the AD field since the function of these secretases are also linked to cellular pathways like ectodomain shedding of growth factors and regulated intramembrane proteolysis of receptors in developmental biology, tissue homeostasis, and tumorigenesis. The generation of mice deficient in presenilin 1, presenilin 2, the α-secretase ADAM10, and the ß-secretases BACE1 and BACE2 were instrumental for the elucidation of the physiological functions of these proteases. Using these mouse models understanding how these secretases regulate amyloid peptide formation and how they exert their diverse biological functions could be significantly increased. This review attempts to summarize selected aspects of the current view of the multiple roles such proteases play in health and disease.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/fisiología , Proteínas ADAM/fisiología , Proteína ADAM10 , Animales , Ácido Aspártico Endopeptidasas/fisiología , Humanos , Proteínas de la Membrana/fisiología , Ratones , Modelos Neurológicos , Vías Nerviosas/fisiología , Presenilina-1/fisiología , Presenilina-2/fisiologíaRESUMEN
Presenilins are ubiquitous, intramembrane proteins that function in Alzheimer's disease (AD) as the catalytic component of the γ-secretase complex. Familial AD mutations in presenilin are known to exacerbate lysosomal pathology. Hence, we sought to elucidate the function endogenous, wild-type presenilins play in autophagy-mediated protein degradation. We report the finding that genetic deletion or knockdown of presenilins alters many autophagy-related proteins demonstrating a buildup of autophagosomes, indicative of dysfunction in the system. Presenilin-deficient cells inefficiently clear long-lived proteins and fail to build up autophagosomes when challenged with lysosomal inhibitors. Our studies further show that γ-secretase inhibitors do not adversely impact autophagy, indicating that the role of presenilins in autophagy is independent of γ-secretase activity. Based on our findings, we conclude that endogenous, wild-type presenilins are necessary for proper protein degradation through the autophagosome-lysosome system by functioning at the lysosomal level. The role of presenilins in autophagy has many implications for its function in neurological diseases such as AD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/fisiología , Autofagia/genética , Lisosomas/metabolismo , Presenilina-1/fisiología , Presenilina-2/fisiología , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Células Cultivadas , Lisosomas/enzimología , Lisosomas/patología , Ratones , Neuronas/enzimología , Neuronas/metabolismo , Neuronas/patología , Presenilina-1/deficiencia , Presenilina-2/deficiencia , Proteínas/metabolismoRESUMEN
It has recently become clear that proteins associated with neurodegenerative disorders can be selectively incorporated into intraluminal vesicles of multivesicular bodies and subsequently released within exosomes. Multiple lines of research support a neuroprotective role for cystatin C in Alzheimer's disease (AD). Herein we demonstrate that cystatin C, a protein targeted to the classical secretory pathway by its signal peptide sequence, is also secreted by mouse primary neurons in association with exosomes. Immunoproteomic analysis using SELDI-TOF MS revealed the presence in exosomes of at least 9 different cystatin C glycoforms. Moreover, the over-expression of familial AD-associated presenilin 2 mutations (PS2 M239I and PS2 T122R) resulted in reduced levels of all cystatin C forms (native and glycosylated) and of amyloid-ß precursor protein (APP) metabolites within exosomes. A better understanding of the mechanisms involved in exosomal processing and release may have important implications for the fight against AD and other neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Comunicación Celular/fisiología , Cistatina C/metabolismo , Exosomas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células Cultivadas , Cistatina C/antagonistas & inhibidores , Cistatina C/química , Glicosilación , Humanos , Ratones , Presenilina-2/genética , Presenilina-2/fisiología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
This study was conducted to identify genes expressed during adipocyte differentiation of bovine intramuscular fibroblast-like cells using differential display reverse-transcriptase polymerase chain reaction. The presenilin enhancer-2 (PSENEN) gene was found to be down-regulated during adipocyte differentiation of bovine intramuscular fibroblast-like cells. The ectopic expression of bovine PSENEN in 3T3-L1 reduced adipogenesis and the inhibition of endogenous PSENEN by siRNA induced adipogenesis on d 4 of adipocyte differentiation of 3T3-L1 cells. Interestingly, the expression of gamma-secretase complex gene-related Notch signaling was decreased at d 2 and d 4 during adipocyte differentiation. In addition, expression of the Notch-signaling genes (Notch-1, Hes-1, Pref-1, adipsin) was regulated during adipocyte differentiation by regulation of PSENEN expression. These results suggest that PSENEN plays an important role in adipocyte differentiation and that Notch signaling is involved in adipogenesis.
Asunto(s)
Adipocitos/citología , Tejido Adiposo/citología , Bovinos/fisiología , Diferenciación Celular/fisiología , Presenilina-2/fisiología , Adipocitos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Presenilina-2/genética , Alineación de SecuenciaRESUMEN
To address the potential impact of presenilin mutations on the prostaglandin metabolism in a neurodegenerative model of glutamatergic excitotoxicity, we injected kainic acid intraperitoneally (30mg/kg body weight) into mice over-expressing the human N141I mutation of presenilin-2, which is known to cause an early-onset form of Alzheimer's disease. We compared the seizure activity as well as seizure lethality in 2- and 6-month-old mice, transgenic for the above-mentioned point mutation, and their wildtype littermates and found that mice harboring the hN141I mutation showed a relative resistance to excitotoxic treatment. This was associated with a constituitively reduced expression of the cyclooxygenases COX-1 and COX-2 in the hippocampus of N141I presenilin-2 mice and a reduced induction of COX-2 expression post-kainate injection. In the past, clinical trials have suggested that both non-steroidal anti-inflammatory drugs, which impact upon a cell's prostaglandin metabolism, and glutamatergic antagonists might be of benefit to patients suffering from Alzheimer's-type dementias. Yet, the exact mechanism by which these drugs are beneficial remains unclear, although it seems possible that presenilins might be implicated in the process, at least in the case of early-onset forms. The data presented here strongly support the notion of an implication of presenilins in the alterations in the prostaglandin system, which have been observed in Alzheimer's disease and may contribute to the underlying pathogenesis of the disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Agonistas de Aminoácidos Excitadores/toxicidad , Ácido Kaínico/toxicidad , Presenilina-2/genética , Prostaglandinas/metabolismo , Animales , Western Blotting , Ciclooxigenasa 1/biosíntesis , Ciclooxigenasa 1/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Inyecciones Intraperitoneales , Ratones , Ratones Transgénicos , Presenilina-2/fisiología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Prostaglandina E/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Convulsiones/inducido químicamente , Convulsiones/fisiopatologíaRESUMEN
The gamma-secretase, composed of presenilin-1 (PS1) or presenilin-2 (PS2), nicastrin (NCT), anterior pharynx-defective phenotype 1 (APH-1), and PEN-2, is critical for the development of Alzheimer's disease (AD). PSs are autoproteolytically cleaved, producing an N-terminal fragment (NTF) and a hydrophilic loop domain-containing C-terminal fragment. However, the role of the loop domain in the gamma-secretase complex assembly remains unknown. Here, we report a novel PS2 isoform generated by alternative splicing, named PS2beta, which is composed of an NTF with a hydrophilic loop domain. PS2beta disturbed the interaction between NCT and APH-1, resulting in the inhibition of amyloid-beta production. We concluded that PS2beta may inhibit gamma-secretase activity by affecting the gamma-secretase complex assembly.
Asunto(s)
Empalme Alternativo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores Enzimáticos , Presenilina-2/fisiología , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Presenilina-2/química , Presenilina-2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
gamma-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Abeta42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of gamma-secretase, invariably increase the Abeta42:Abeta40 ratio. However, the mechanism by which these mutations affect gamma-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Abeta42:Abeta40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active gamma-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Abeta42:Abeta40 ratios. These data suggest that perturbations to gamma-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Abeta42:Abeta40 ratio.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/fisiología , Proteínas de la Membrana/fisiología , Presenilina-1/fisiología , Presenilina-2/fisiología , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Biotina/metabolismo , Línea Celular , Flavina-Adenina Dinucleótido/genética , Flavina-Adenina Dinucleótido/fisiología , Técnicas de Sustitución del Gen , Células HeLa , Humanos , Ratones , Presenilina-1/metabolismo , Presenilina-2/metabolismoRESUMEN
Presenilins (PSs) play important roles in modulating the trafficking and maturation of several membrane proteins. However, the target membrane proteins whose trafficking and maturation are regulated by PS are largely unknown. By characterizing PS-deficient fibroblasts, we found that integrin beta1 maturation is promoted markedly in PS1 and PS2 double-deficient fibroblasts and moderately in PS1- or PS2-deficient fibroblasts; in contrast, nicastrin maturation is completely inhibited in PS1 and PS2 double-deficient fibroblasts. Subcellular fractionation analysis demonstrated that integrin beta1 maturation is promoted in the Golgi apparatus. The mature integrin beta1 with an increased expression level was delivered to the cell surface, which resulted in an increased cell surface expression level of mature integrin beta1 in PS1 and PS2 double-deficient fibroblasts. PS1 and PS2 double-deficient fibroblasts exhibited an enhanced ability to adhere to culture dishes coated with integrin beta1 ligands, namely, fibronectin and laminin. The inhibition of gamma-secretase activity enhances neither integrin beta1 maturation nor the adhesion of wild-type cells. Moreover, PS deficiency also promoted the maturation of integrins alpha3 and alpha5 and the cell surface expression of integrin alpha3. Integrins alpha3 and alpha5 were coimmunoprecipitated with integrin beta1, suggesting the formation of the functional heterodimers integrins alpha3beta1 and alpha5beta1. Note that integrin beta1 exhibited features opposite those of nicastrin in terms of maturation and trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus in PS1 and PS2 double-deficient fibroblasts. Our results therefore suggest that PS regulates the maturation of membrane proteins in opposite directions and cell adhesion by modulating integrin maturation.
Asunto(s)
Integrina beta1/metabolismo , Presenilina-1/fisiología , Presenilina-2/fisiología , Animales , Transporte Biológico Activo/genética , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Humanos , Integrina beta1/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Presenilina-1/deficiencia , Presenilina-2/deficiencia , Transporte de Proteínas/genéticaRESUMEN
The presenilin-containing gamma-secretase complex is an unusual membrane-embedded protease that processes a wide variety of integral membrane proteins, clearing protein stubs from the lipid bilayer and participating in critical signaling pathways. The protease is also central to Alzheimer's disease and certain cancers and is therefore an important therapeutic target. Here we highlight recent progress in deciphering the role of presenilin/gamma-secretase in biology and medicine and pose key questions for future study.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/fisiología , Presenilina-1/fisiología , Presenilina-2/fisiología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/química , Animales , Humanos , Presenilina-1/química , Presenilina-2/química , Conformación Proteica , Transducción de SeñalRESUMEN
Circadian rhythms are generated by a molecular clock composed of clock genes and their protein products. Other genes are regulated in a rhythmic way by this molecular clockwork, but are not themselves constituents of the clock. This study shows that one of these clock-controlled genes encodes the signalling protein Presenilin-2. Indeed, evidence is presented that the promoter of the mouse Presenilin-2 gene is bound and activated by CLOCK and BMAL1, transcription factors of the mammalian circadian clock. Quantification of Presenilin-2 RNA shows that its expression is non-rhythmic in many peripheral tissues (heart, muscle, kidney, spleen, and thymus). Note, though, that careful analysis of the liver data shows that Presenilin-2 RNA exists in distinct isoforms in this tissue, and that rhythmicity is restricted to only a subset of these RNA isoforms. These data indicate a unique mode of regulation of Presenilin-2 transcripts, the circadian control of which appears to happen at the transcriptional and post-transcriptional levels.