RESUMEN
Amyloidoses (misfolded polypeptide accumulation) are among the most debilitating diseases our aging societies face. Amyloidogenesis can be catalyzed by hydrophobic-hydrophilic interfaces (e.g., air-water interface in vitro [AWI]). We recently demonstrated hydrogelation of the amyloidogenic type II diabetes-associated islet amyloid polypeptide (IAPP), a hydrophobic-hydrophilic interface-dependent process with complex kinetics. We demonstrate that human IAPP undergoes AWI-catalyzed liquid-liquid phase separation (LLPS), which initiates hydrogelation and aggregation. Insulin modulates these processes but does not prevent them. Using nonamyloidogenic rat IAPP, we show that, whereas LLPS does not require the amyloidogenic sequence, hydrogelation and aggregation do. Interestingly, both insulin and rat sequence delayed IAPP LLPS, which may reflect physiology. By developing an experimental setup and analysis tools, we show that, within the whole system (beyond the droplet stage), macroscopic interconnected aggregate clusters form, grow, fuse, and evolve via internal rearrangement, leading to overall hydrogelation. As the AWI-adsorbed gelled layer matures, its microviscosity increases. LLPS-driven aggregation may be a common amyloid feature and integral to pathology.
Asunto(s)
Amiloidosis/patología , Diabetes Mellitus Tipo 2/patología , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Amiloide/fisiología , Proteínas Amiloidogénicas/metabolismo , Animales , Hidrogeles , Interacciones Hidrofóbicas e Hidrofílicas , Insulina/metabolismo , Agregado de Proteínas/fisiología , RatasRESUMEN
Many protein misfolding diseases (e.g. type II diabetes and Alzheimer's disease) are characterised by amyloid deposition. Human islet amyloid polypeptide (hIAPP, involved in type II diabetes) spontaneously undergoes liquid-liquid phase separation (LLPS) and a kinetically complex hydrogelation, both catalysed by hydrophobic-hydrophilic interfaces (e.g. air-water interface and/or phospholipids-water interfaces). Gelation of hIAPP phase-separated liquid droplets initiates amyloid aggregation and the formation of clusters of interconnected aggregates, which grow and fuse to eventually percolate the whole system. Droplet maturation into irreversible hydrogels via amyloid aggregation is thought to be behind the pathology of several diseases. Biological fluids contain a high volume fraction of macromolecules, leading to macromolecular crowding. Despite crowding agent addition in in vitro studies playing a significant role in changing protein phase diagrams, the mechanism underlying enhanced LLPS, and the effect(s) on stages beyond LLPS remain poorly or not characterised.We investigated the effect of macromolecular crowding and increased viscosity on the kinetics of hIAPP hydrogelation using rheology and the evolution of the system beyond LLPS by microscopy. We demonstrate that increased viscosity exacerbated the kinetic variability of hydrogelation and of the phase separated-aggregated system, whereas macromolecular crowding abolished heterogeneity. Increased viscosity also strengthened the gel meshwork and accelerated aggregate cluster fusion. In contrast, crowding either delayed cluster fusion onset (dextran) or promoted it (Ficoll). Our study highlights that an in vivo crowded environment would critically influence amyloid stages beyond LLPS and pathogenesis.
Asunto(s)
Amiloide/química , Proteínas Amiloidogénicas/química , Hidrogeles/química , Polipéptido Amiloide de los Islotes Pancreáticos/química , Agua/química , Enfermedad de Alzheimer/metabolismo , Dextranos/química , Diabetes Mellitus Tipo 2/metabolismo , Ficoll/química , Glicerol/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Fosfolípidos/química , Agregado de Proteínas , Agregación Patológica de Proteínas , Factores de Tiempo , ViscosidadRESUMEN
Many neurodegenerative diseases are characterized by amyloid deposition. In Alzheimer's disease (AD), ß-amyloid (Aß) peptides accumulate extracellularly in senile plaques. The AD amyloid cascade hypothesis proposes that Aß production or reduced clearance leads to toxicity. In contrast, the cholinergic hypothesis argues for a specific pathology of brain cholinergic pathways. However, neither hypothesis in isolation explains the pattern of AD pathogenesis. Evidence suggests that a connection exists between these two scenarios: the synaptic form of human acetylcholinesterase (hAChE-S) associates with plaques in AD brains; among hAChE variants, only hAChE-S enhances Aß fibrillization in vitro and Aß deposition and toxicity in vivo Only hAChE-S contains an amphiphilic C-terminal domain (T40, AChE575-614), with AChE586-599 homologous to Aß and forming amyloid fibrils, which implicates T40 in AD pathology. We previously showed that the amyloid scavenger, insulin-degrading enzyme (IDE), generates T40-derived amyloidogenic species that, as a peptide mixture, seed Aß fibrillization. Here, we characterized 11 peptides from a T40-IDE digest for ß-sheet conformation, surfactant activity, fibrillization, and seeding capability. We identified residues important for amyloidogenicity and raised polyclonal antibodies against the most amyloidogenic peptide. These new antisera, alongside other specific antibodies, labeled sections from control, hAChE-S, hAPPswe, and hAChE-S/hAPPswe transgenic mice. We observed that hAChE-S ß-sheet species co-localized with Aß in mature plaque cores, surrounded by hAChE-S α-helical species. This observation provides the first in vivo evidence of the conformation of hAChE-S species within plaques. Our results may explain the role of hAChE-S in Aß deposition and aggregation, as amyloidogenic hAChE-S ß-sheet species might seed Aß aggregation.
Asunto(s)
Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Péptidos/metabolismo , Acetilcolinesterasa/química , Acetilcolinesterasa/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Animales , Encéfalo/patología , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Ratones , Ratones Transgénicos , Péptidos/química , Péptidos/genética , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Deposition of misfolded amyloid polypeptides, associated with cell death, is the hallmark of many degenerative diseases (e.g. type II diabetes mellitus and Alzheimer's disease). In vivo, cellular and extracellular spaces are occupied by a high volume fraction of macromolecules. The resulting macromolecular crowding energetically affects reactions. Amyloidogenesis can either be promoted by macromolecular crowding through the excluded volume effect or inhibited due to a viscosity increase reducing kinetics. Macromolecular crowding can be mimicked in vitro by the addition of non-specific polymers, e.g. Ficoll, dextran and polyvinyl pyrrolidone (PVP), the latter being rarely used to study amyloid systems. We investigated the effect of PVP on amyloidogenesis of full-length human islet amyloid polypeptide (involved in type II diabetes) using fibrillisation and surface activity assays, ELISA, immunoblot and microscale thermophoresis. We demonstrate that high molecular mass PVP360 promotes amyloidogenesis due to volume exclusion and increase in effective amyloidogenic monomer concentration, like other crowders, but without the confounding effects of viscosity and surface activity. Interestingly, we also show that low molecular mass PVP10 has unique inhibitory properties as inhibition of fibril elongation occurs mainly in the bulk solution and is due to PVP10 directly and strongly interacting with amyloid species rather than the increase in viscosity typically associated with macromolecular crowding. In vivo, amyloidogenesis might be affected by the properties and proximity of endogenous macromolecular crowders, which could contribute to changes in associated pathogenesis. More generally, the PVP10 molecular backbone could be used to design small compounds as potential inhibitors of toxic species formation.
Asunto(s)
Amiloide/química , Sustancias Macromoleculares/química , Polímeros/química , Polivinilos/química , Pirrolidinonas/química , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Amiloidosis/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Cinética , Sustancias Macromoleculares/metabolismo , Polímeros/metabolismo , Polivinilos/metabolismo , Pirrolidinonas/metabolismo , ViscosidadRESUMEN
The nuclei of cells may exhibit invaginations of the nuclear envelope under a variety of conditions. These invaginations form a branched network termed the nucleoplasmic reticulum (NR), which may be found in cells in pathological and physiological conditions. While an extensive NR is a hallmark of cellular senescence and shows associations with some cancers, very little is known about the formation of NR in physiological conditions, despite the presence of extensive nuclear invaginations in some cell types such as endometrial cells. Here we show that in these cells the NR is formed in response to reproductive hormones. We demonstrate that oestrogen and progesterone are sufficient to induce NR formation and that this process is reversible without cell division upon removal of the hormonal stimulus. Nascent lamins and phospholipids are incorporated into the invaginations suggesting that there is a dedicated machinery for its formation. The induction of NR in endometrial cells offers a new model to study NR formation and function in physiological conditions.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Endometrio/citología , Estrógenos/farmacología , Línea Celular Tumoral , Núcleo Celular/ultraestructura , Células Endoteliales/efectos de los fármacos , Células Endoteliales/ultraestructura , Femenino , HumanosRESUMEN
Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects whose complex multigenic causation has hampered efforts to delineate their molecular basis. The effect of putative modifier genes in determining NTD susceptibility may be investigated in mouse models, particularly those that display partial penetrance such as curly tail, a strain in which NTDs result from a hypomorphic allele of the grainyhead-like-3 gene. Through proteomic analysis, we found that the curly tail genetic background harbours a polymorphic variant of lamin B1, lacking one of a series of nine glutamic acid residues. Lamins are intermediate filament proteins of the nuclear lamina with multiple functions that influence nuclear structure, cell cycle properties, and transcriptional regulation. Fluorescence loss in photobleaching showed that the variant lamin B1 exhibited reduced stability in the nuclear lamina. Genetic analysis demonstrated that the variant also affects neural tube closure: the frequency of spina bifida and anencephaly was reduced three-fold when wild-type lamin B1 was bred into the curly tail strain background. Cultured fibroblasts expressing variant lamin B1 show significantly increased nuclear dysmorphology and diminished proliferative capacity, as well as premature senescence, associated with reduced expression of cyclins and Smc2, and increased expression of p16. The cellular basis of spinal NTDs in curly tail embryos involves a proliferation defect localised to the hindgut epithelium, and S-phase progression was diminished in the hindgut of embryos expressing variant lamin B1. These observations indicate a mechanistic link between altered lamin B1 function, exacerbation of the Grhl3-mediated cell proliferation defect, and enhanced susceptibility to NTDs. We conclude that lamin B1 is a modifier gene of major effect for NTDs resulting from loss of Grhl3 function, a role that is likely mediated via the key function of lamin B1 in maintaining integrity of the nuclear envelope and ensuring normal cell cycle progression.
Asunto(s)
Ciclo Celular , Lamina Tipo B , Defectos del Tubo Neural , Membrana Nuclear , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular , División Celular , Proliferación Celular , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Ratones , Mutación , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Polimorfismo Genético , Proteómica , Disrafia Espinal/genética , Disrafia Espinal/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Understanding mechanisms of information processing in cellular signaling networks requires quantitative measurements of protein activities in living cells. Biosensors are molecular probes that have been developed to directly track the activity of specific signaling proteins and their use is revolutionizing our understanding of signal transduction. The use of biosensors relies on the assumption that their activity is linearly proportional to the activity of the signaling protein they have been engineered to track. We use mechanistic mathematical models of common biosensor architectures (single-chain FRET-based biosensors), which include both intramolecular and intermolecular reactions, to study the validity of the linearity assumption. As a result of the classic mechanism of zero-order ultrasensitivity, we find that biosensor activity can be highly nonlinear so that small changes in signaling protein activity can give rise to large changes in biosensor activity and vice versa. This nonlinearity is abolished in architectures that favor the formation of biosensor oligomers, but oligomeric biosensors produce complicated FRET states. Based on this finding, we show that high-fidelity reporting is possible when a single-chain intermolecular biosensor is used that cannot undergo intramolecular reactions and is restricted to forming dimers. We provide phase diagrams that compare various trade-offs, including observer effects, which further highlight the utility of biosensor architectures that favor intermolecular over intramolecular binding. We discuss challenges in calibrating and constructing biosensors and highlight the utility of mathematical models in designing novel probes for cellular signaling.
Asunto(s)
Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Transducción de Señal , Técnicas Biosensibles/normas , Interpretación Estadística de Datos , Transferencia Resonante de Energía de Fluorescencia/normas , Modelos Biológicos , Variaciones Dependientes del Observador , Fosfoproteínas Fosfatasas/metabolismo , Unión Proteica , Proteínas Quinasas/metabolismo , Sensibilidad y EspecificidadRESUMEN
Amyloid formation is a hallmark of protein misfolding diseases (e.g. Type II diabetes mellitus). The energetically unfavourable nucleation step of amyloidogenesis can be accelerated by seeding, during which pre-formed aggregates act as templates for monomer recruitment. Hydrophobic-hydrophilic interfaces [e.g. AWI (air-water interface)] can also catalyse amyloidogenesis due to the surfactant properties of amyloidogenic polypeptides. Using thioflavin T fluorescence and electron microscopy, we demonstrate that the outcome of seeding on human islet amyloid polypeptide amyloidogenesis is dependent upon whether the AWI is present or absent and is dictated by seed type. Seeding significantly inhibits (with AWI) or promotes (without AWI) plateau height compared with seedless controls; with short fibrils being more efficient seeds than their longer counterparts. Moreover, promotion of nucleation by increasing monomer concentrations can only be observed in the absence of an AWI. Using biophysical modelling, we suggest that a possible explanation for our results may reside in lateral interactions between seeds and monomers determining the fibril mass formed in seeded reactions at steady-state. Our results suggest that in vivo hydrophobic-hydrophilic interfaces (e.g. the presence of membranes and their turnover rate) may dictate the outcome of seeding during amyloidogenesis and that factors affecting the size of the pre-aggregate may be important.
Asunto(s)
Aire , Polipéptido Amiloide de los Islotes Pancreáticos/química , Agua/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos BiológicosRESUMEN
The nuclear envelope (NE) surrounds the nucleus and separates it from the cytoplasm. The NE is not a passive structural component, but rather contributes to various cellular processes such as genome organization, transcription, signaling, and stress responses. Although the NE is mostly a smooth surface, it also forms invaginations that can reach deep into the nucleoplasm and may even traverse the nucleus completely. Cancer cells are generally characterized by irregularities and invaginations of the NE that are of diagnostic and prognostic significance. In the current chapter, we describe the link between nuclear invaginations and irregularities with cancer and explore possible mechanistic roles they might have in tumorigenesis.
Asunto(s)
Membrana Nuclear/fisiología , Transformación Celular Neoplásica , Humanos , Neoplasias/diagnóstico , Neoplasias/patología , Neoplasias/fisiopatología , PronósticoRESUMEN
Amyloid formation and accumulation is a hallmark of protein misfolding diseases and is associated with diverse pathologies including type II diabetes and Alzheimer's disease (AD). In vitro, amyloidogenesis is widely studied in conditions that do not simulate the crowded and viscous in vivo environment. A high volume fraction of most biological fluids is occupied by various macromolecules, a phenomenon known as macromolecular crowding. For some amyloid systems (e.g. α-synuclein) and under shaking condition, the excluded volume effect of macromolecular crowding favors aggregation, whereas increased viscosity reduces the kinetics of these reactions. Amyloidogenesis can also be catalyzed by hydrophobic-hydrophilic interfaces, represented by the air-water interface in vitro and diverse heterogeneous interfaces in vivo (e.g. membranes). In this study, we investigated the effects of two different crowding polymers (dextran and Ficoll) and two different experimental conditions (with and without shaking) on the fibrilization of amyloid-ß peptide, a major player in AD pathogenesis. Specifically, we demonstrate that, during macromolecular crowding, viscosity dominates over the excluded volume effect only when the system is spatially non homogeneous (i.e. an air-water interface is present). We also show that the surfactant activity of the crowding agents can critically influence the outcome of macromolecular crowding and that the structure of the amyloid species formed may depend on the polymer used. This suggests that, in vivo, the outcome of amyloidogenesis may be affected by both macromolecular crowding and spatial heterogeneity (e.g. membrane turn-over). More generally, our work suggests that any factors causing changes in crowding may be susceptibility factors in AD.
Asunto(s)
Amiloide/química , Interacciones Hidrofóbicas e Hidrofílicas , Sustancias Macromoleculares/química , Vibración , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Amiloide/ultraestructura , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Dextranos/química , Diabetes Mellitus Tipo 2/metabolismo , Ficoll/química , Humanos , Microscopía Electrónica , Soluciones/química , ViscosidadRESUMEN
The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.
Asunto(s)
Compartimento Celular , Laminas/metabolismo , Membrana Nuclear/patología , Animales , Proteínas Bacterianas/metabolismo , División Celular , Dextranos/metabolismo , Regulación de la Expresión Génica , Humanos , Lamina Tipo A/metabolismo , Proteínas Luminiscentes/metabolismo , Sustancias Macromoleculares/metabolismo , Ratones , Peso Molecular , Membrana Nuclear/ultraestructura , Señales de Localización Nuclear , Transportador 1 de Catión Orgánico/metabolismo , Transporte de ProteínasRESUMEN
Farnesylated prelamin A accumulates when the final endoproteolytic maturation of the protein fails to occur and causes a dysmorphic nuclear phenotype; however, the morphology and mechanisms of biogenesis of these changes remain unclear. We show here that acute prelamin A accumulation after reduction in the activity of the ZMPSTE24 endoprotease by short interfering RNA knockdown, results in the generation of a complex nucleoplasmic reticulum that depends for its formation on the enzyme CTP:phosphocholine-cytidylyltransferase-α (CCT-α, also known as choline-phosphate cytidylyltransferase A). This structure can form during interphase, confirming that it is independent of mitosis and therefore not a consequence of disordered nuclear envelope assembly. Serial-section dual-axis electron tomography reveals that these invaginations can take two forms: one in which the inner nuclear membrane infolds alone with an inter membrane space interior, and the other in which an invagination of both nuclear membranes occurs, enclosing a cytoplasmic core. Both types of invagination can co-exist in one nucleus and both are frequently studded with nuclear pore complexes (NPC), which reduces NPC abundance on the nuclear surface.
Asunto(s)
Citidililtransferasa de Colina-Fosfato/fisiología , Membrana Nuclear/ultraestructura , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Animales , Núcleo Celular/ultraestructura , Células Cultivadas , Citidililtransferasa de Colina-Fosfato/análisis , Citidililtransferasa de Colina-Fosfato/antagonistas & inhibidores , Lamina Tipo A , Lamina Tipo B/análisis , Proteínas de la Membrana/antagonistas & inhibidores , Metaloendopeptidasas/antagonistas & inhibidores , Ratones , Mitosis , Membrana Nuclear/química , Membrana Nuclear/enzimología , Poro Nuclear/ultraestructura , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Prenilación , Precursores de Proteínas/análisis , Precursores de Proteínas/químicaRESUMEN
Regulatory T (Treg) cells are critically important for the maintenance of immunological tolerance. Both centrally arising natural nTreg cells and those emerging in the periphery in response to TGF-ß, iTreg cells, play a role in the control of unwanted immune responses. Treg cells adopt multiple mechanisms to inhibit effector T cells, yet it is unclear whether these mechanisms are shared by nTreg cells and iTreg cells alike. Here, we show that iTreg cells, like nTreg cells, are able to out-compete naïve T cells in clustering around dendritic cells (DCs). However, using both a tamoxifen-responsive inducible Foxp3 retroviral construct and TGF-ß-induced iTreg cells from hCD2-Foxp3 knock in reporter mice, we show that it is prior antigen-induced activation rather than Foxp3 expression per se that determines the ability of iTreg cells to competitively cluster around DCs. We found no difference in the capacity of iTreg cells to displace naïve T cells around DCs to that of Tr1, Th1, Th2, or Th9 cells. An important difference was, however, that clustering of iTreg cells around DCs, just as for naïve T cells, did not effectively activate DCs.
Asunto(s)
Células Dendríticas/inmunología , Factores de Transcripción Forkhead/análisis , Activación de Linfocitos , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/farmacología , Secuencia de Aminoácidos , Animales , Agregación Celular , Polaridad Celular , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz-sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked α-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans. Cytochemical analysis demonstrates that high-mannose-containing glycoproteins are predominantly localized to the ER and the early secretory pathway. Indirect immunofluorescence microscopy revealed that GNA colocalizes with the ER marker protein disulfide isomerase (PDI) and the COPI coat protein ß-COP. In situ competition with concanavalin A (ConA), another high-mannose specific lectin, and subsequent GNA lectin histochemistry refined the localization of N-glyans containing nonreducing mannosyl groups, accentuating the GNA vesicular staining. Using GNA and treatments that perturb ER-Golgi transport, we demonstrate that lectins can be used to detect changes in membrane trafficking pathways histochemically. Overall, we find that conjugated plant lectins are effective tools for combinatory biochemical and cytological analysis of membrane trafficking of glycoproteins.
Asunto(s)
Técnicas Citológicas , Glicoproteínas/química , Glicoproteínas/metabolismo , Membranas Intracelulares/metabolismo , Oligosacáridos/análisis , Oligosacáridos/metabolismo , Lectinas de Plantas/química , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Galanthus/química , Glicoproteínas/análisis , Aparato de Golgi/química , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/química , Yohexol , Lens (Planta)/química , Oligosacáridos/química , Pisum sativum/química , Transporte de Proteínas , SacarosaRESUMEN
BACKGROUND: Lamins are intermediate filament proteins that form a major component of the nuclear lamina, a protein complex at the surface of the inner nuclear membrane. Numerous clinically diverse conditions, termed laminopathies, have been found to result from mutation of LMNA. In contrast, coding or loss of function mutations of LMNB1, encoding lamin B1, have not been identified in human disease. In mice, polymorphism in Lmnb1 has been shown to modify risk of neural tube defects (NTDs), malformations of the central nervous system that result from incomplete closure of the neural folds. METHODS: Mutation analysis by DNA sequencing was performed on all exons of LMNB1 in 239 samples from patients with NTDs from the United Kingdom, Sweden, and United States. Possible functional effects of missense variants were analyzed by bioinformatics prediction and fluorescence in photobleaching. RESULTS: In NTD patients, we identified two unique missense variants that were predicted to disrupt protein structure/function and represent putative contributory mutations. Fluorescence loss in photobleaching analysis showed that the A436T variant compromised stability of lamin B1 interaction within the lamina. CONCLUSION: The genetic basis of human NTDs appears highly heterogenous with possible involvement of multiple predisposing genes. We hypothesize that rare variants of LMNB1 may contribute to susceptibility to NTDs.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Lamina Tipo B/genética , Defectos del Tubo Neural/genética , Estudios de Cohortes , Biología Computacional , Análisis Mutacional de ADN , Exones/genética , Fluorescencia , Humanos , Lamina Tipo B/metabolismo , Mutación Missense/genética , Lámina Nuclear/metabolismo , Fotoblanqueo , Suecia , Reino Unido , Estados UnidosRESUMEN
The aggregation of proteins or peptides into amyloid fibrils is a hallmark of protein misfolding diseases (e.g., Alzheimer's disease) and is under intense investigation. Many of the experiments performed are in vitro in nature and the samples under study are ordinarily exposed to diverse interfaces, e.g., the container wall and air. This naturally raises the question of how important interfacial effects are to amyloidogenesis. Indeed, it has already been recognized that many amyloid-forming peptides are surface-active. Moreover, it has recently been demonstrated that the presence of a hydrophobic interface can promote amyloid fibrillization, although the underlying mechanism is still unclear. Here, we combine theory, surface property measurements, and amyloid fibrillogenesis assays on islet amyloid polypeptide and amyloid-ß peptide to demonstrate why, at experimentally relevant concentrations, the surface activity of the amyloid-forming peptides leads to enriched fibrillization at an air-water interface. Our findings indicate that the key that links these two seemingly different phenomena is the surface-active nature of the amyloid-forming species, which renders the surface concentration much higher than the corresponding critical fibrillar concentration. This subsequently leads to a substantial increase in fibrillization.
Asunto(s)
Aire , Péptidos beta-Amiloides/química , Polipéptido Amiloide de los Islotes Pancreáticos/química , Fragmentos de Péptidos/química , Multimerización de Proteína , Agua/química , Adsorción , Humanos , Modelos Moleculares , Estructura Secundaria de Proteína , SolucionesRESUMEN
Lipoprotein lipase (LPL) is secreted into the interstitial spaces by adipocytes and myocytes but then must be transported to the capillary lumen by GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells. The mechanism by which GPIHBP1 and LPL move across endothelial cells remains unclear. We asked whether the transport of GPIHBP1 and LPL across endothelial cells was uni- or bidirectional. We also asked whether GPIHBP1 and LPL are transported across cells in vesicles and whether this transport process requires caveolin-1. The movement of GPIHBP1 and LPL across cultured endothelial cells was bidirectional. Also, GPIHBP1 moved bidirectionally across capillary endothelial cells in live mice. The transport of LPL across endothelial cells was inhibited by dynasore and genistein, consistent with a vesicular transport process. Also, transmission electron microscopy (EM) and dual-axis EM tomography revealed GPIHBP1 and LPL in invaginations of the plasma membrane and in vesicles. The movement of GPIHBP1 across capillary endothelial cells was efficient in the absence of caveolin-1, and there was no defect in the internalization of LPL by caveolin-1-deficient endothelial cells in culture. Our studies show that GPIHBP1 and LPL move bidirectionally across endothelial cells in vesicles and that transport is efficient even when caveolin-1 is absent.
Asunto(s)
Células Endoteliales/metabolismo , Lipoproteína Lipasa/metabolismo , Receptores de Lipoproteína/metabolismo , Animales , Células CHO , Cricetinae , Células Endoteliales/química , Células Endoteliales/enzimología , Genisteína/farmacología , Humanos , Hidrazonas/farmacología , Lipoproteína Lipasa/antagonistas & inhibidores , Ratones , Ratones Noqueados , Ratas , Receptores de Lipoproteína/deficiencia , Relación Estructura-ActividadRESUMEN
Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.
Asunto(s)
Cromosomas de los Mamíferos , Regulación de la Expresión Génica , Interfase/genética , Lamina Tipo B/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Animales , Compartimento Celular , Endopeptidasas/metabolismo , Perfilación de la Expresión Génica , Interfase/fisiología , Lamina Tipo B/genética , Ratones , Lámina Nuclear/genética , Lámina Nuclear/metabolismo , Lámina Nuclear/ultraestructura , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The general segregation of a molecular class, lipids, from the pathways of cellular communication, via endo-membranes, has resulted in the over-simplification and misconceptions in deciphering cell signalling mechanisms. Mechanisms in signal transduction and protein activation require targeting of proteins to membranous compartments with a specific localised morphology and dynamics that are dependent on their lipid composition. Many posttranslational events define cellular behaviours and without the active role of membranous compartments these events lead to various dysregulations of the signalling pathways. We summarise the key findings, using tools such as the rapalogue dimerisation, in the structural roles and signalling of the inter-related phosphoinositide lipids and their derivative, diacylglycerol, in the regulation of nuclear envelope biogenesis and other subcellular compartments such as the nucleoplasmic reticulum.
Asunto(s)
Comunicación Celular/genética , Lípidos de la Membrana/genética , Fosfatidilinositoles/genética , Transducción de Señal/genética , Membrana Nuclear/genéticaRESUMEN
The nuclear envelope is not only important for the structural integrity of the nucleus, but also involved in a number of cellular functions. It has been shown to be important for maintaining and controlling chromatin organization, sequestering transcription factors, replication, transcription and signalling. The nuclear envelope is thus important for development and differentiation, and some of its components are essential for cell viability. Among the many functions which are emerging for the nuclear envelope is its involvement in protecting the cell against different types of cellular stress. In the present paper, we review key findings which describe the roles of nuclear envelope components in responses to common types of stress conditions.