RESUMEN
The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer's disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response. Ivermectin was reported as one of the positive allosteric modulators, since the binding of Ivermectin to the channel enhances acetylcholine-evoked α7 currents. One research has suggested that tilting motions of the nicotinic acetylcholine receptor are responsible for channel opening and activation. To verify this hypothesis applies to α7 nicotinic acetylcholine receptor, we utilized a diffracted X-ray tracking method to monitor the stable twisting and tilting motion of nAChR α7 without a ligand, with acetylcholine, with Ivermectin, and with both of them. The results show that the α7 nicotinic acetylcholine receptor twists counterclockwise with the channel transiently opening, transitioning to a desensitized state in the presence of acetylcholine and clockwise without the channel opening in the presence of Ivermectin. We propose that the conformational transition of ACh-bound nAChR α7 may be due to the collective twisting of the five α7 subunits, resulting in the compression and movement, either downward or upward, of one or more subunits, thus manifesting tilting motions. These tilting motions possibly represent the transition from the resting state to channel opening and potentially to the desensitized state.
Asunto(s)
Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa 7 , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Acetilcolina/química , Acetilcolina/metabolismo , Ligandos , Ivermectina/farmacología , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Regulación AlostéricaRESUMEN
Antifreeze proteins (AFPs) are multifunctional polypeptides that adsorb onto ice crystals to inhibit their growth and onto cells to protect them from nonfreezing hypothermic damage. However, the mechanism by which AFP exerts its hypothermic cell protective (HCP) function remains uncertain. Here, we assessed the HCP function of three types of fish-derived AFPs (type I, II, and III AFPs) against human T-lymphoblastic lymphoma by measuring the survival rate (%) of the cells after preservation at 4 °C for 24 h. All AFPs improved the survival rate in a concentration-dependent manner, although the HCP efficiency was inferior for type III AFP compared to other AFPs. In addition, after point mutations were introduced into the ice-binding site (IBS) of a type III AFP, HCP activity was dramatically increased, suggesting that the IBS of AFP is involved in cell adsorption. Significantly, high HCP activity was observed for a mutant that exhibited poorer antifreeze activity, indicating that AFP exerts HCP- and ice-binding functions through a different mechanism. We next incubated the cells in an AFP-containing solution, replaced it with pure EC solution, and then preserved the cells, showing that no significant reduction in the cell survival rate occurred for type I and II AFPs even after replacement. Thus, these AFPs irreversibly bind to the cells at 4 °C, and only tightly adsorbed AFP molecules contribute towards the cell-protection function.
Asunto(s)
Hielo , alfa-Fetoproteínas , Animales , Humanos , Sitios de Unión , Proteínas Anticongelantes/genética , Proteínas Anticongelantes/química , Proteínas Anticongelantes/metabolismo , Fenómenos Biofísicos , Proteínas de Peces/genéticaRESUMEN
The CCT/TRiC complex is a type II chaperonin that undergoes ATP-driven conformational changes during its functional cycle. Structural studies have provided valuable insights into the mechanism of this process, but real-time dynamics analyses of mammalian type II chaperonins are still scarce. We used diffracted X-ray tracking (DXT) to investigate the intramolecular dynamics of the CCT complex. We focused on three surface-exposed loop regions of the CCT1 subunit: the loop regions of the equatorial domain (E domain), the E and intermediate domain (I domain) juncture near the ATP-binding region, and the apical domain (A domain). Our results showed that the CCT1 subunit predominantly displayed rotational motion, with larger mean square displacement (MSD) values for twist (χ) angles compared with tilt (θ) angles. Nucleotide binding had a significant impact on the dynamics. In the absence of nucleotides, the region between the E and I domain juncture could act as a pivotal axis, allowing for greater motion of the E domain and A domain. In the presence of nucleotides, the nucleotides could wedge into the ATP-binding region, weakening the role of the region between the E and I domain juncture as the rotational axis and causing the CCT complex to adopt a more compact structure. This led to less expanded MSD curves for the E domain and A domain compared with nucleotide-absent conditions. This change may help to stabilize the functional conformation during substrate binding. This study is the first to use DXT to probe the real-time molecular dynamics of mammalian type II chaperonins at the millisecond level. Our findings provide new insights into the complex dynamics of chaperonins and their role in the functional folding cycle.
Asunto(s)
Simulación de Dinámica Molecular , Pliegue de Proteína , Animales , Rayos X , Chaperoninas del Grupo II/química , Chaperoninas del Grupo II/metabolismo , Chaperoninas/metabolismo , Adenosina Trifosfato/metabolismo , Nucleótidos , Chaperonina con TCP-1/química , Conformación Proteica , Mamíferos/metabolismoRESUMEN
Understanding the cellular environment as molecular crowding that supports the structure-specific functional expression of biomolecules has recently attracted much attention. Time-resolved X-ray observations have the remarkable capability to capture the structural dynamics of biomolecules with subnanometre precision. Nevertheless, the measurement of the intracellular dynamics within live organisms remains a challenge. Here, we explore the potential of utilizing crystallized proteins that spontaneously form intracellular crystals to investigate their intracellular dynamics via time-resolved X-ray observations. We generated transgenic Caenorhabditis elegans specifically expressing the crystallized protein in cells and observed the formation of the protein aggregates within the animal cells. From the toxic-effect observations, the aggregates had minimal toxic effects on living animals. Fluorescence observations showed a significant suppression of the translational diffusion movements in molecules constituting the aggregates. Moreover, X-ray diffraction measurements provided diffraction signals originating from these molecules. We also observed the blinking behaviour of the diffraction spots, indicating the rotational motion of these crystals within the animal cells. A diffracted X-ray blinking (DXB) analysis estimated the rotational motion of the protein crystals on the subnanometre scale. Our results provide a time-resolved X-ray diffraction technique for the monitoring of intracellular dynamics.
Asunto(s)
Caenorhabditis elegans , Proteínas , Animales , Rayos X , Difracción de Rayos X , Radiografía , Cristalografía por Rayos XRESUMEN
X-ray crystallography has revolutionized our understanding of biological macromolecules by elucidating their three-dimensional structures. However, the use of X-rays in this technique raises concerns about potential damage to the protein crystals, which results in a quality degradation of the diffraction data even at very low temperatures. Since such damage can occur on the micro- to millisecond timescale, a development in its real-time measurement has been expected. Here, we introduce diffracted X-ray blinking (DXB), which was originally proposed as a method to analyze the intensity fluctuations of diffraction of crystalline particles, to small-angle X-ray scattering (SAXS) of a lysozyme single-crystal. This novel technique, called the small-angle X-ray blinking (SAXB) method, analyzes the fluctuation in SAXS intensity reflecting the domain fluctuation in the protein crystal caused by the X-ray irradiation, which could be correlated with the X-ray-induced damage on the crystal. There was no change in the protein crystal's domain dynamics between the first and second X-ray exposures at 95K, each of which lasted 0.7 s. On the other hand, its dynamics at 295K increased remarkably. The SAXB method further showed a dramatic increase in domain fluctuations with an increasing dose of X-ray radiation, indicating the significance of this method.
Asunto(s)
Parpadeo , Proteínas , Difracción de Rayos X , Rayos X , Dispersión del Ángulo Pequeño , Proteínas/química , Cristalografía por Rayos XRESUMEN
A cryoprotectant known as ice-binding protein (IBP) is thought to facilitate the cold survival of plants, insects, and fungi. Here, we prepared a genetically modified Caenorhabditis elegans strain to synthesize fish-derived IBPs in its body wall muscles and examined whether the antifreeze activity modification of this IBP by point mutation affects the cold tolerance of this worm. We chose a 65-residue IBP identified from notched-fin eelpout, for which the replacement of the 20th alanine residue (A20) modifies its antifreeze activity. These mutant proteins are denoted A20L, A20G, A20T, A20V, and A20I along with the wild-type (WT) protein. We evaluated the survival rate (%) of the transgenic C. elegans that synthesized each IBP mutant following 24 h of preservation at -5, +2, and +5 °C. Significantly, a dramatic improvement in the survival rate was detected for the worms synthesizing the activity-enhanced mutants (A20T and A20I), especially at +2 °C. In contrast, the rate was not improved by the expression of the defective mutants (A20L, A20G, WT and A20V). The survival rate (%) probably correlates with the antifreeze activity of the IBP. These data suggest that IBP protects the cell membrane by employing its ice-binding mechanism, which ultimately improves the cold tolerance of an IBP-containing animal.
Asunto(s)
Proteínas Anticongelantes , Hielo , Animales , Alanina/genética , Proteínas Anticongelantes/química , Proteínas Anticongelantes/genética , Proteínas Anticongelantes/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Peces/genética , Congelación , Proteínas Mutantes/metabolismo , MutaciónRESUMEN
Membrane proteins change their conformations in response to chemical and physical stimuli and transmit extracellular signals inside cells. Several approaches have been developed for solving the structures of proteins. However, few techniques can monitor real-time protein dynamics. The diffracted X-ray tracking method (DXT) is an X-ray-based single-molecule technique that monitors the internal motion of biomolecules in an aqueous solution. DXT analyzes trajectories of Laue spots generated from the attached gold nanocrystals with a two-dimensional axis by tilting (θ) and twisting (χ). Furthermore, high-intensity X-rays from synchrotron radiation facilities enable measurements with microsecond-timescale and picometer-spatial-scale intramolecular information. The technique has been applied to various membrane proteins due to its superior spatiotemporal resolution. In this review, we introduce basic principles of DXT, reviewing its recent and extended applications to membrane proteins and living cells, respectively.
Asunto(s)
Oro , Proteínas de la Membrana , Oro/química , Movimiento (Física) , Nanotecnología , Difracción de Rayos X , Rayos XRESUMEN
Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.
Asunto(s)
Proteínas de la Membrana , Nanotecnología , Microscopía por Crioelectrón , Cinética , Microscopía de Fuerza Atómica/métodosRESUMEN
Interleukin 15 receptor (IL-15R) is a transmembrane signalling protein consisting of 3 subsets: α, ß (IL-15Rß), and γ (γc). IL-2 and IL-15 share the signalling domains IL-15Rß and γc, although they bind to intrinsic α-subsets and non-signalling domains. Additionally, IL-2 and IL-15 play different roles; therefore, there have been many observations of the dynamic behaviours of IL-15R, which are linked to physiological functions. For more practical discrimination between IL-2 and IL-15, a study was designed and carried out in which α-subsets were removed and a cytoplasmic inhibitor was applied to create a simplified environment in which secondary signalling molecules were reduced. We also applied a new measurement method, diffracted X-ray blinking (DXB), to achieve higher accuracy (<0.01 Å). The dynamics of IL-2 binding (confined motion, max range = 0.71 Å) and IL-15 binding (normal motion) in live natural killer cells were different. We also confirmed. that DXB was a suitable method to quantitatively evaluate the transmembrane protein dynamics of inner/outer live cell membranes by labeling the extracellular domain since the measurements were dependent on the cytosolic environment.
Asunto(s)
Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Receptores de Interleucina-15/análisis , Receptores de Interleucina-15/metabolismo , Difracción de Rayos X/métodos , Supervivencia Celular , Difusión , Humanos , Hidroxibenzoatos , Interleucina-15/metabolismo , Interleucina-2/metabolismo , Simulación de Dinámica Molecular , Nitrofuranos , Dominios Proteicos , Especificidad por SustratoRESUMEN
Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HT2AR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HT2ARs have been well studied, little has been known about their real-time dynamics. In this study, we analyzed the intramolecular motion of the 5-HT2AR in living cells using the diffracted X-ray tracking (DXT) technique. The DXT is a very precise single-molecular analytical technique, which tracks diffraction spots from the gold nanocrystals labeled on the protein surface. Trajectory analysis provides insight into protein dynamics. The 5-HT2ARs were transiently expressed in HEK 293 cells, and the gold nanocrystals were attached to the N-terminal introduced FLAG-tag via anti-FLAG antibodies. The motions were recorded with a frame rate of 100 µs per frame. A lifetime filtering technique demonstrated that the unliganded receptors contain high mobility population with clockwise twisting. This rotation was, however, abolished by either a full agonist α-methylserotonin or an inverse agonist ketanserin. Mutation analysis revealed that the "ionic lock" between the DRY motif in the third transmembrane segment and a negatively charged residue of the sixth transmembrane segment is essential for the torsional motion at the N-terminus of the receptor.
Asunto(s)
Receptor de Serotonina 5-HT2A/metabolismo , Receptor de Serotonina 5-HT2A/fisiología , Imagen Individual de Molécula/métodos , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X/métodos , Oro , Células HEK293 , Humanos , Iones/metabolismo , Ligandos , Nanotecnología/métodos , Receptores de Serotonina/metabolismo , Receptores de Serotonina/fisiología , Difracción de Rayos X/métodos , Rayos XRESUMEN
G protein-coupled receptors (GPCRs) are seven-transmembrane proteins, which transmit extracellular signals inside cells via activating G proteins. GPCRs are involved in a wide variety of physiological functions, such as signal sensing, immune system processes, and neurotransmission. Although the structures and functions of GPCRs have been well studied, little has been known about their real-time dynamics on live cells. In this study, we used Diffracted X-ray Tracking (DXT) and Diffracted X-ray Blinking (DXB) techniques for analysis. These methods are very precise single-molecular analytical techniques that elucidate protein dynamics by analyzing the diffraction spots from the gold nanocrystals labeled on the protein surface. DXT tracks diffraction spot movements, whereas DXB analyzes continuation of signals by calculating the autocorrelation function of each pixel from the recorded data. Serotonin receptor subtype 2A (5-HT2A receptors) were transiently expressed on HEK 293 cells, and the gold nanocrystals were attached to the N-terminally introduced FLAG-tag via anti-FLAG antibodies. Fast- and mid-range motions were recorded by DXT with 100µs and 1.25 ms/frame rate, respectively. Slow-range motion was obtained using the DXB method with 100 ms/frame rate. An agonist interestingly suppressed the fluctuations of 5-HT2A receptors at the microsecond-ranged fast measurement. On the contrary, the motion was enhanced by the agonist in the hundred-millisecond-ranged slow time scale. These dual-natured data may suggest that we succeeded in extracting different modes of receptor's motion on live cells; microsecond ranged fluctuation on the cell membrane, and millisecond-ranged dynamic movement comprising interactions with intracellular signaling molecules.
Asunto(s)
Receptor de Serotonina 5-HT2A/análisis , Diseño de Equipo , Células HEK293 , Humanos , Cinética , Movimiento (Física) , Imagen Individual de Molécula/instrumentación , Imagen Individual de Molécula/métodos , Difracción de Rayos X/instrumentación , Difracción de Rayos X/métodosRESUMEN
The C-terminal Ig-domain of lamin A plays critical roles in cell function via interaction with proteins, DNA, and chromatin. Mutations in this domain are known to cause various diseases including Emery-Dreifuss muscular dystrophy (EDMD) and familial partial lipodystrophy (FPLD). Here we examined the biophysical and biochemical properties of mutant Ig-domains identified in patients with EDMD and FPLD. EDMD-related mutant Ig-domain showed decreased stability to heat and denaturant. This result was also confirmed by experiments using full-length mutant lamin A, although the decrease in melting temperature was much less than that of the mutant Ig-domain alone. The unstable EDMD Ig-domain disrupted the proper assembly of lamin A, resulting in abnormal paracrystal formation and decreased viscosity. In contrast, FPLD-related mutant Ig-domains were thermally stable, although they lost DNA binding function. Alanine substitution experiments revealed a functional domain of DNA binding in the Ig-domain. Thus, the overall biophysical property of Ig-domains is closely associated with clinical phenotype.
Asunto(s)
Lamina Tipo A/química , Distrofia Muscular de Emery-Dreifuss/metabolismo , Sustitución de Aminoácidos , Fenómenos Biofísicos , ADN/química , ADN/metabolismo , Humanos , Técnicas In Vitro , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia Parcial Familiar/genética , Lipodistrofia Parcial Familiar/metabolismo , Microscopía Electrónica de Transmisión , Modelos Moleculares , Distrofia Muscular de Emery-Dreifuss/genética , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios Proteicos , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
HLA-G is a natural tolerogenic molecule and has the following unique features: seven isoforms (HLA-G1 to HLA-G7), formation of disulfide-linked homodimers, and ß2-microglobulin (ß2m)-free forms. Interestingly, individuals null for the major isoform, HLA-G1, are healthy and expressed the α2 domain-deleted isoform, HLA-G2, which presumably compensates for HLA-G1 function. However, the molecular characteristics of HLA-G2 are largely unknown. In this study, we unexpectedly found that HLA-G2 naturally forms a ß2m-free and nondisulfide-linked homodimer, which is in contrast to the disulfide-bonded ß2m-associated HLA-G1 homodimer. Furthermore, single-particle analysis, using electron microscopy, revealed that the overall structure and domain organization of the HLA-G2 homodimer resemble those of the HLA class II heterodimer. The HLA-G2 homodimer binds to leukocyte Ig-like receptor B2 with slow dissociation and a significant avidity effect. These findings provide novel insights into leukocyte Ig-like receptor B2-mediated immune regulation by the HLA-G2 isoform, as well as the gene evolution of HLA classes.
Asunto(s)
Antígenos HLA-G/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes/metabolismo , Dimerización , Evolución Molecular , Femenino , Antígenos HLA-G/química , Antígenos HLA-G/genética , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunomodulación , Intercambio Materno-Fetal , Embarazo , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Microglobulina beta-2/metabolismoRESUMEN
LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-κB pathway through linear polyubiquitination of NEMO (NF-κB essential modulator, also known as IKKγ) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-κB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-κB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70-1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IκB kinase (IKK), which results in NF-κB suppression. These findings provide new insight into the regulation of immune and inflammatory responses.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Dedos de Zinc/fisiología , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Enzima Desubiquitinante CYLD , Células HEK293 , Humanos , Quinasa I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Linfoma de Células B/genética , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Poliubiquitina/biosíntesis , Unión Proteica/genética , Conformación Proteica , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina/química , Ubiquitina/metabolismoRESUMEN
TRPA1 is a member of the transient receptor potential (TRP) cation channel family that is expressed primarily on sensory neurons. This chemosensor is activated through covalent modification of multiple cysteine residues with a wide range of reactive compounds including allyl isothiocyanate (AITC), a spicy component of wasabi. The present study reports on potent and selective agonists of TRPA1, discovered through screening 1657 electrophilic molecules. In an effort to validate the mode of action of hit molecules, we noted a new TRPA1-selective agonist, JT010 (molecule 1), which opens the TRPA1 channel by covalently and site-selectively binding to Cys621 (EC50 = 0.65 nM). The results suggest that a single modification of Cys621 is sufficient to open the TRPA1 channel. The TRPA1-selective probe described herein might be useful for further mechanistic studies of TRPA1 activation.
Asunto(s)
Proteínas del Tejido Nervioso/agonistas , Canales de Potencial de Receptor Transitorio/agonistas , Canales de Calcio , Células HEK293 , Humanos , Canal Catiónico TRPA1RESUMEN
The adhesion of circulating lymphocytes to the surface of vascular endothelial cells is important for their recruitment from blood to secondary lymphoid organs and to inflammatory sites. CD44 is a key adhesion molecule for this interaction and its ligand-binding ability is tightly regulated. Here we show that the hyaluronan-binding ability of CD44 in T cells is upregulated by the depletion of membrane cholesterol with methyl-ß-cyclodextrin (MßCD), which disintegrates lipid rafts, i.e. cholesterol- and sphingolipid-enriched membrane microdomains. Increasing concentrations of MßCD led to a dose-dependent decrease in cellular cholesterol content and to upregulation of hyaluronan binding. Additionally, a cholesterol-binding agent filipin also increased hyaluronan binding. Cholesterol depletion caused CD44 to be dispersed from cholesterol-enriched membrane microdomains. Cholesterol depletion also increased the number of cells undergoing rolling adhesion under physiological flow conditions. Our results suggest that the ligand-binding ability of CD44 is governed by its cholesterol-dependent allocation to membrane microdomains at the cell surface. These findings provide novel insight into the regulation of T cell adhesion under blood flow.
Asunto(s)
Colesterol/metabolismo , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Linfocitos T/metabolismo , Animales , Adhesión Celular/fisiología , Línea Celular , Membrana Celular/metabolismo , Ratones , Ratones Endogámicos C57BL , Linfocitos T/citologíaRESUMEN
Mutation of the lamin A gene (LMNA) causes a diverse range of diseases referred to as laminopathies. Because most laminopathies have a dominant inheritance pattern and progress gradually, cultured cells stably expressing mutant lamin A at the same level as endogenous wild-type cells are required for chronological analysis. In this study, we showed that an expression system involving a lentiviral vector that carries the human metallothionein gene basal promoter ensures stable and basal-level expression of proteins and is thus suitable for investigating the properties of lamin A mutants. The small ubiquitin-related modifier (SUMO) modification (SUMOylation)-defective E203G mutant that is associated with familial dilated cardiomyopathy exhibited abnormal subnuclear distribution and inhibited normal localization of WT lamin A in a dominant-negative manner. Low-level and long-term expression of the E203G mutant resulted in multinucleated giant cells, aberrant lipid droplet accumulation in the cytoplasm and premature senescence. Expression of another SUMOylation-defective mutant (K201R) did not induce any phenotypes observed in cells expressing E203G. These results indicate that the E203G mutant may inhibit the normal functions of wild-type lamin A in a dominant-negative manner, but a defect in SUMOylation itself may not be involved in disease pathogenesis.
Asunto(s)
Cardiomiopatía Dilatada/patología , Lamina Tipo A/metabolismo , Cardiomiopatía Dilatada/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular , Senescencia Celular , Células Gigantes/metabolismo , Células Gigantes/patología , Células HEK293 , Células HeLa , Heterocromatina/metabolismo , Humanos , Lamina Tipo B/metabolismo , Metabolismo de los Lípidos , Mutación , SumoilaciónRESUMEN
The Sec translocon facilitates transportation of newly synthesized polypeptides from the cytoplasm to the lumen/periplasm across the phospholipid membrane. Although the polypeptide-conducting machinery is formed by the SecYEG-SecA complex in bacteria, its transportation efficiency is markedly enhanced by SecDF. A previous study suggested that SecDF assumes at least two conformations differing by a 120° rotation in the spatial orientation of the P1 head subdomain to the rigid base, and that the conformational dynamics plays a critical role in polypeptide translocation. Here we addressed this hypothesis by analyzing the 3D structure of SecDF using electron tomography and single particle reconstruction. Reconstruction of wt SecDF showed two major conformations; one resembles the crystal structure of full-length SecDF (F-form structure), while the other is similar to the hypothetical structural variant based on the crystal structure of the isolated P1 domain (I-form structure). The transmembrane domain of the I-form structure has a scissor like cleft open to the periplasmic side. We also report the structure of a double cysteine mutant designed to constrain SecDF to the I-form. This reconstruction has a protrusion at the periplasmic end that nicely fits the orientation of P1 in the I-from. These results provide firm evidence for the occurrence of the I-form in solution and support the proposed F- to I-transition of wt SecDF during polypeptide translocation.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Proteínas de la Membrana/ultraestructura , Proteínas de Transporte de Membrana/ultraestructura , Thermus thermophilus/genética , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Tomografía con Microscopio Electrónico , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Mutación , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
BACKGROUND: It has been demonstrated that the hepatitis E virus (HEV) can be transmitted via blood transfusion, and the risk of HEV transmission via transfusion has become a major global concern. An HEV culture system for blood-derived HEV has been sought to obtain valuable knowledge of the virus and the risk of HEV infection through blood products. STUDY DESIGN AND METHODS: We endeavored to establish an HEV culture system using RNA-positive blood specimens for Genotypes (G) 3 and 4 and applied this system to evaluate tissue culture infectious dose (TCID). We applied this method to investigate the potential of the Mirasol pathogen reduction technology (PRT) system (Terumo BCT) to inactivate live HEV in contaminated platelet samples (PLTs). PLTs were spiked with cultured HEV G3 or G4 and then treated with the Mirasol PRT system. PLTs were examined before and after the treatment for HEV load using TCID titration. RESULTS: We successfully established two strains for HEV production: the JRC-HE3 strain for G3 and the UA1 strain for G4. The Mirasol PRT system expressed more than 3 log inactivation for JRC-HE3 and more than 2 log inactivation for UA1. CONCLUSION: The Mirasol PRT system inactivated greater than 2 to 3 logs of live HEV in PLTs and can potentially be used to lower the possibility of blood-borne HEV transmission. The G3 and G4 HEV inocula identified in this study and the hepatoma cell culture system provide a new means to assess HEV infectious titer and to evaluate other pathogen reduction strategies.
Asunto(s)
Genotipo , Virus de la Hepatitis E/crecimiento & desarrollo , Inactivación de Virus , Línea Celular Tumoral , Virus de la Hepatitis E/aislamiento & purificación , HumanosRESUMEN
Protein dynamics play important roles in biological functions, which accompany allosteric structure changes. Diffracted X-ray blinking (DXB) uses monochromatic X-rays and nanocrystal probes. The intramolecular motion of target proteins is analyzed from the intensity changes in detector signals at the diffraction rings. In contrast, diffracted X-ray tracking (DXT) elucidates molecular dynamics by analyzing the trajectories of Laue spots. In this study, we have developed a dual-labeling technique for DXB and DXT, allowing the simultaneous observation of motions at different domains in proteins. We identified zinc oxide (ZnO) crystals as promising candidates for the second labeling probes due to their excellent diffraction patterns, high chemical stability, and favorable binding properties with proteins. The diffraction spots from the ZnO crystals are sufficiently separated from those of gold, enabling independent motion analysis at different domains. Dual-labeling DXB was employed for the motion analysis of the 5-HT2A receptor in living cells. Simultaneous motion recording of the N-terminus and the second extracellular loop demonstrated ligand-induced motion suppression at both domains. The dual-labeling DXT technique demonstrated a capsaicin-induced peak shift in the two-dimensional motion maps at the N-terminus of the TRPV1 protein, but the peak shift was not obvious in the C-terminus. The capsaicin-induced motion modulation was recovered by the addition of the competitive inhibitor AMG9810.