RESUMEN
BACKGROUND: Steroid-resistant nephrotic syndrome is the second leading cause of chronic kidney disease among patients < 25 years of age. Through exome sequencing, identification of > 65 monogenic causes has revealed insights into disease mechanisms of nephrotic syndrome (NS). METHODS: To elucidate novel monogenic causes of NS, we combined homozygosity mapping with exome sequencing in a worldwide cohort of 1649 pediatric patients with NS. RESULTS: We identified homozygous missense variants in MYO1C in two unrelated children with NS (c.292C > T, p.R98W; c.2273 A > T, p.K758M). We evaluated publicly available kidney single-cell RNA sequencing datasets and found MYO1C to be predominantly expressed in podocytes. We then performed structural modeling for the identified variants in PyMol using aligned shared regions from two available partial structures of MYO1C (4byf and 4r8g). In both structures, calmodulin, a common regulator of myosin activity, is shown to bind to the IQ motif. At both residue sites (K758; R98), there are ion-ion interactions stabilizing intradomain and ligand interactions: R98 binds to nearby D220 within the myosin motor domain and K758 binds to E14 on a calmodulin molecule. Variants of these charged residues to non-charged amino acids could ablate these ionic interactions, weakening protein structure and function establishing the impact of these variants. CONCLUSION: We here identified recessive variants in MYO1C as a potential novel cause of NS in children.
Asunto(s)
Secuenciación del Exoma , Mutación Missense , Miosina Tipo I , Síndrome Nefrótico , Humanos , Miosina Tipo I/genética , Miosina Tipo I/química , Síndrome Nefrótico/genética , Masculino , Femenino , Niño , Homocigoto , Proteinuria/genética , Genes Recesivos , Preescolar , Adolescente , Podocitos/metabolismo , Modelos MolecularesRESUMEN
Myosin-1D (myo1D) is important for Drosophila left-right asymmetry, and its effects are modulated by myosin-1C (myo1C). De novo expression of these myosins in nonchiral Drosophila tissues promotes cell and tissue chirality, with handedness depending on the paralog expressed. Remarkably, the identity of the motor domain determines the direction of organ chirality, rather than the regulatory or tail domains. Myo1D, but not myo1C, propels actin filaments in leftward circles in in vitro experiments, but it is not known if this property contributes to establishing cell and organ chirality. To further explore if there are differences in the mechanochemistry of these motors, we determined the ATPase mechanisms of myo1C and myo1D. We found that myo1D has a 12.5-fold higher actin-activated steady-state ATPase rate, and transient kinetic experiments revealed myo1D has an 8-fold higher MgADP release rate compared to myo1C. Actin-activated phosphate release is rate limiting for myo1C, whereas MgADP release is the rate-limiting step for myo1D. Notably, both myosins have among the tightest MgADP affinities measured for any myosin. Consistent with ATPase kinetics, myo1D propels actin filaments at higher speeds compared to myo1C in in vitro gliding assays. Finally, we tested the ability of both paralogs to transport 50 nm unilamellar vesicles along immobilized actin filaments and found robust transport by myo1D and actin binding but no transport by myo1C. Our findings support a model where myo1C is a slow transporter with long-lived actin attachments, whereas myo1D has kinetic properties associated with a transport motor.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Lateralidad Funcional , Miosina Tipo I , Animales , Actinas/metabolismo , Cinética , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Dominios Proteicos , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/enzimologíaRESUMEN
In Covid-19 and autoimmune patients, there are several similarities revealed in the immune responses (Liu et al., 2021; Woodruff et al., 2020). Earlier, we firstly detected a truncated (48 kDa) form of the unconventional Myosin 1C (48/Myo1C) in a fraction of proteins soluble in 10% 2,2,2-trichloroacetic acid (TCA). These proteins were obtained from blood serum of patients with autoimmune diseases, such as multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis (Kit et al., 2018). Here, we demonstrated that content of 48/Myo1C was also elevated in blood serum of the severe Covid-19 patients. Whereas in blood of 28 clinically healthy human individuals regularly tested for Covid-19 infection, the amount of this protein was undetectable or very low, in blood of 16 of 28 patients hospitalized with severe course of this disease, its amount was significantly increased. Dexamethasone, steroid hormone which is widely used for treatment of severe Covid-19 patients, induced time-dependent elevation of the 48/Myo1C in blood of such patients. The 48/Myo1C dose-dependently suppressed the viability of anti-CD3-activated lymphocytes of human peripheral blood. Recently, we used affinity chromatography on the magnetic poly(glycidyl-methacrylate) (mag-PGMA-NH2) microparticles functionalized with Myo1C and MALDI-TOF mass spectrometry with molecular modeling in silico in order to identify potential molecular partners of the 48/Myo1C. It was found that 48/Myo1C might bind to component 3 of the complement system and the anti-thrombin-III (Starykovych et al., 2021). Thus, the mechanisms of the pathogenic action of truncated form of Myo1C in severe COVID-19 patients may involve a suppression of the immune cells, as well as modulation of complement and coagulation cascades.
Asunto(s)
Enfermedades Autoinmunes , COVID-19 , Esclerosis Múltiple , Humanos , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Suero/metabolismo , COVID-19/diagnósticoRESUMEN
Myosin-1C is a single-headed, short-tailed member of the myosin class I subfamily that supports a variety of actin-based functions in the cytosol and nucleus. In vertebrates, alternative splicing of the MYO1C gene leads to the production of three isoforms, myosin-1C0, myosin-1C16, and myosin-1C35, that carry N-terminal extensions of different lengths. However, it is not clear how these extensions affect the chemomechanical coupling of human myosin-1C isoforms. Here, we report on the motor activity of the different myosin-1C isoforms measuring the unloaded velocities of constructs lacking the C-terminal lipid-binding domain on nitrocellulose-coated glass surfaces and full-length constructs on reconstituted, supported lipid bilayers. The higher yields of purified proteins obtained with constructs lacking the lipid-binding domain allowed a detailed characterization of the individual kinetic steps of human myosin-1C isoforms in their productive interaction with nucleotides and filamentous actin. Isoform-specific differences include 18-fold changes in the maximum power output per myosin-1C motor and 4-fold changes in the velocity and the resistive force at which maximum power output occurs. Our results support a model in which the isoform-specific N-terminal extensions affect chemomechanical coupling by combined steric and allosteric effects, thereby reducing both the length of the working stroke and the rate of ADP release in the absence of external loads by a factor of 2 for myosin-1C35. As the large change in maximum power output shows, the functional differences between the isoforms are further amplified by the presence of external loads.
Asunto(s)
Actinas/metabolismo , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Nucleótidos/metabolismo , Fenómenos Biomecánicos , Humanos , Cinética , Unión Proteica , Isoformas de ProteínasRESUMEN
We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isolated it from blood serum of multiple sclerosis patients. The amount of p48/Myo1C in human blood serum correlated with some autoimmune, hemato-oncological and neurodegenerative diseases and thus may serve as a potential molecular biomarker. The biological functions of this protein in human blood remain unknown. Previously, we used the monodisperse magnetic poly (glycidyl methacrylate)(mag-PGMA-NH2 ) microspheres with immobilized 48/Myo1C and western-blot analysis, which allowed us to identify IgM and IgG immunoglobulins presenting an affinity to this protein. Here, we used mass spectrometry followed by the western blotting in order to identify other blood serum proteins with affinity to 48/Myo1C. The obtained data demonstrate that 48/Myo1C binds to component 3 of the complement and the antithrombin-III proteins. A combination of magnetic microparticle-based affinity chromatography with MALDI-TOF mass spectrometry and an in silico analysis provided an opportunity to identify the partners of interaction of 48/Myo1C with other proteins, in particular those participating in complement and coagulation cascades.
Asunto(s)
Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/metabolismo , Cromatografía de Afinidad/métodos , Miosina Tipo I/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteínas Sanguíneas/química , Western Blotting , Humanos , Imanes , Microesferas , Modelos Moleculares , Esclerosis Múltiple/sangre , Miosina Tipo I/química , Pronóstico , Unión ProteicaRESUMEN
Fusarium is a genus of filamentous fungi that includes species that cause devastating diseases in major staple crops, such as wheat, maize, rice, and barley, resulting in severe yield losses and mycotoxin contamination of infected grains. Phenamacril is a novel fungicide that is considered environmentally benign due to its exceptional specificity; it inhibits the ATPase activity of the sole class I myosin of only a subset of Fusarium species including the major plant pathogens F. graminearum, F. asiaticum and F. fujikuroi. To understand the underlying mechanisms of inhibition, species specificity, and resistance mutations, we have determined the crystal structure of phenamacril-bound F. graminearum myosin I. Phenamacril binds in the actin-binding cleft in a new allosteric pocket that contains the central residue of the regulatory Switch 2 loop and that is collapsed in the structure of a myosin with closed actin-binding cleft, suggesting that pocket occupancy blocks cleft closure. We have further identified a single, transferable phenamacril-binding residue found exclusively in phenamacril-sensitive myosins to confer phenamacril selectivity.
Asunto(s)
Cianoacrilatos/química , Proteínas Fúngicas/química , Fungicidas Industriales/química , Fusarium/enzimología , Miosina Tipo I/química , Cianoacrilatos/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungicidas Industriales/farmacología , Fusarium/química , Fusarium/efectos de los fármacos , Fusarium/genética , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Enfermedades de las Plantas/microbiología , Triticum/microbiología , Zea mays/microbiologíaRESUMEN
Myosin-IC (Myo1c) has been proposed to function in delivery of glucose transporter type 4 (GLUT4)-containing vesicles to the plasma membrane in response to insulin stimulation. Current evidence suggests that, upon insulin stimulation, Myo1c is phosphorylated at Ser701, leading to binding of the signaling protein 14-3-3ß. Biochemical and functional details of the Myo1c-14-3-3ß interaction have yet to be described. Using recombinantly expressed proteins and mass spectrometry-based analyses to monitor Myo1c phosphorylation, along with pulldown, fluorescence binding, and additional biochemical assays, we show here that 14-3-3ß is a dimer and, consistent with previous work, that it binds to Myo1c in the presence of calcium. This interaction was associated with dissociation of calmodulin (CaM) from the IQ motif in Myo1c. Surprisingly, we found that 14-3-3ß binds to Myo1c independent of Ser701 phosphorylation in vitro Additionally, in contrast to previous reports, we did not observe Myo1c Ser701 phosphorylation by Ca2+/CaM-dependent protein kinase II (CaMKII), although CaMKII phosphorylated four other Myo1c sites. The presence of 14-3-3ß had little effect on the actin-activated ATPase or motile activities of Myo1c. Given these results, it is unlikely that 14-3-3ß acts as a cargo adaptor for Myo1c-powered transport; rather, we propose that 14-3-3ß binds Myo1c in the presence of calcium and stabilizes the calmodulin-dissociated, nonmotile myosin.
Asunto(s)
Proteínas 14-3-3/metabolismo , Miosina Tipo I/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Secuencias de Aminoácidos , Calcio/química , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Dimerización , Ácido Egtácico/química , Humanos , Espectrometría de Masas , Miosina Tipo I/química , Miosina Tipo I/genética , Fosforilación , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , UltracentrifugaciónRESUMEN
The emergence of asymmetry from an initially symmetrical state is a universal transition in nature. Living organisms show asymmetries at the molecular, cellular, tissular, and organismal level. However, whether and how multilevel asymmetries are related remains unclear. In this study, we show that Drosophila myosin 1D (Myo1D) and myosin 1C (Myo1C) are sufficient to generate de novo directional twisting of cells, single organs, or the whole body in opposite directions. Directionality lies in the myosins' motor domain and is swappable between Myo1D and Myo1C. In addition, Myo1D drives gliding of actin filaments in circular, counterclockwise paths in vitro. Altogether, our results reveal the molecular motor Myo1D as a chiral determinant that is sufficient to break symmetry at all biological scales through chiral interaction with the actin cytoskeleton.
Asunto(s)
Citoesqueleto de Actina/química , Proteínas de Drosophila/química , Modelos Moleculares , Miosina Tipo I/química , Animales , Proteínas de Drosophila/antagonistas & inhibidores , Drosophila melanogaster/crecimiento & desarrollo , Isomerismo , Larva , Miosina Tipo I/antagonistas & inhibidores , Miosina Tipo V/química , Dominios ProteicosRESUMEN
Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-Fe2O3) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 µm in diameter, contain 0.9 mM · g-1 of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients. Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.
Asunto(s)
Autoanticuerpos/química , Autoanticuerpos/aislamiento & purificación , Enfermedades Autoinmunes/inmunología , Microesferas , Esclerosis Múltiple/inmunología , Miosina Tipo I/inmunología , Ácidos Polimetacrílicos/química , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Humanos , Imanes/química , Peso Molecular , Miosina Tipo I/químicaRESUMEN
Familial aggregation is a significant risk factor for the development of thyroid cancer and familial non-medullary thyroid cancer (FNMTC) accounts for 5-7% of all NMTC. Whole exome sequencing analysis in the family affected by FNMTC with oncocytic features where our group previously identified a predisposing locus on chromosome 19p13.2, revealed a novel heterozygous mutation (c.400G > A, NM_012335; p.Gly134Ser) in exon 5 of MYO1F, mapping to the linkage locus. In the thyroid FRTL-5 cell model stably expressing the mutant MYO1F p.Gly134Ser protein, we observed an altered mitochondrial network, with increased mitochondrial mass and a significant increase in both intracellular and extracellular reactive oxygen species, compared to cells expressing the wild-type (wt) protein or carrying the empty vector. The mutation conferred a significant advantage in colony formation, invasion and anchorage-independent growth. These data were corroborated by in vivo studies in zebrafish, since we demonstrated that the mutant MYO1F p.Gly134Ser, when overexpressed, can induce proliferation in whole vertebrate embryos, compared to the wt one. MYO1F screening in additional 192 FNMTC families identified another variant in exon 7, which leads to exon skipping, and is predicted to alter the ATP-binding domain in MYO1F. Our study identified for the first time a role for MYO1F in NMTC.
Asunto(s)
Proliferación Celular , Embrión no Mamífero/patología , Mitocondrias/patología , Mutación , Miosina Tipo I/genética , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Células Cultivadas , Niño , Cromosomas Humanos Par 19 , Embrión no Mamífero/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/genética , Mitocondrias/metabolismo , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Consumo de Oxígeno , Linaje , Conformación Proteica , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Adulto Joven , Pez CebraRESUMEN
Single-headed myosin 1 has been identified in neurons, but its function in these cells is still unclear. We demonstrate that depletion of myosin 1b (Myo1b), inhibition of its motor activity, or its binding to phosphoinositides impairs the formation of the axon, whereas overexpression of Myo1b increases the number of axon-like structures. Myo1b is associated with growth cones and actin waves, two major contributors to neuronal symmetry breaking. We show that Myo1b controls the dynamics of the growth cones and the anterograde propagation of the actin waves. By coupling the membrane to the actin cytoskeleton, Myo1b regulates the size of the actin network as well as the stability and size of filopodia in the growth cones. Our data provide the first evidence that a myosin 1 plays a major role in neuronal symmetry breaking and argue for a mechanical control of the actin cytoskeleton both in actin waves and in the growth cones by this myosin.
Asunto(s)
Actinas/metabolismo , Axones/metabolismo , Conos de Crecimiento/metabolismo , Miosina Tipo I/metabolismo , Secuencias de Aminoácidos , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Cinesinas/metabolismo , Ratones , Actividad Motora , Miosina Tipo I/química , Neuritas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Seudópodos/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion-retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms.
Asunto(s)
Miosina Tipo I/metabolismo , Fosfatidilinositoles/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Citoplasma/metabolismo , Expresión Génica , Genes Reporteros , Humanos , Inmunohistoquímica , Ratones , Miosina Tipo I/química , Miosina Tipo I/genética , Señales de Localización Nuclear/química , Unión Proteica , Proteínas Recombinantes de FusiónRESUMEN
Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.
Asunto(s)
Actinas/química , Actinas/metabolismo , Microscopía por Crioelectrón/métodos , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Actomiosina/química , Actomiosina/metabolismo , Adenosina Difosfato/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Simulación de Dinámica Molecular , Faloidina/química , Faloidina/metabolismo , Conformación ProteicaRESUMEN
Retinitis pigmentosa 2 (RP2) is the causative gene for a form of X-linked retinal degeneration. RP2 was previously shown to have GTPase-activating protein (GAP) activity towards the small GTPase ARL3 via its N-terminus, but the function of the C-terminus remains elusive. Here, we report a novel interaction between RP2 and osteoclast-stimulating factor 1 (OSTF1), an intracellular protein that indirectly enhances osteoclast formation and activity and is a negative regulator of cell motility. Moreover, this interaction is abolished by a human pathogenic mutation in RP2. We utilized a structure-based approach to pinpoint the binding interface to a strictly conserved cluster of residues on the surface of RP2 that spans both the C- and N-terminal domains of the protein, and which is structurally distinct from the ARL3-binding site. In addition, we show that RP2 is a positive regulator of cell motility in vitro, recruiting OSTF1 to the cell membrane and preventing its interaction with the migration regulator Myo1E.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Actinas/genética , Proteínas del Ojo/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteínas/genética , Retinitis Pigmentosa/genética , Factores de Ribosilacion-ADP/química , Actinas/química , Sitios de Unión/genética , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Movimiento Celular/genética , Cilios/genética , Cilios/metabolismo , Proteínas del Ojo/química , Proteínas de Unión al GTP , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Simulación del Acoplamiento Molecular , Miosina Tipo I/química , Miosina Tipo I/genética , Unión Proteica/genética , Conformación Proteica , Dominios Proteicos/genética , Estructura Terciaria de Proteína , Proteínas/química , Retina/metabolismo , Retina/patología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patologíaRESUMEN
Nuclear myosin 1c (NM1) associates with RNA polymerases and is a partner in the chromatin remodeling complex B-WICH. This complex, which also contains WSTF and SNF2h proteins, is involved in transcriptional regulation. We report herein that papillomavirus protein E2 binds to NM1 and co-precipitates with the WSTF and SNF2h proteins. Our data suggest that E2 associates with the cellular B-WICH complex through binding to NM1. E2 and NM1 associate via their N-terminal domains and this interaction is ATP dependent. The cellular multifunctional protein Brd4 and beta-actin are also present in the NM1-E2 complex. NM1 downregulation by siRNA increases the replication of the BPV1 and HPV5 genomes but does not affect HPV18 genome replication. These results suggest that the B-WICH complex may play a role in the papillomavirus life cycle through NM1 and E2 protein interaction.
Asunto(s)
Betapapillomavirus/metabolismo , Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 18/metabolismo , Miosina Tipo I/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/metabolismo , Replicación Viral , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Betapapillomavirus/genética , Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Interacciones Huésped-Patógeno , Papillomavirus Humano 18/química , Papillomavirus Humano 18/genética , Humanos , Miosina Tipo I/química , Miosina Tipo I/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , Unión Proteica , Dominios Proteicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The MYO1C gene produces three alternatively spliced isoforms, differing only in their N-terminal regions (NTRs). These isoforms, which exhibit both specific and overlapping nuclear and cytoplasmic functions, have different expression levels and nuclear-cytoplasmic partitioning. To investigate the effect of NTR extensions on the enzymatic behavior of individual isoforms, we overexpressed and purified the three full-length human isoforms from suspension-adapted HEK cells. MYO1CC favored the actomyosin closed state (AMC), MYO1C16 populated the actomyosin open state (AMO) and AMC equally, and MYO1C35 favored the AMO state. Moreover, the full-length constructs isomerized before ADP release, which has not been observed previously in truncated MYO1CC constructs. Furthermore, global numerical simulation analysis predicted that MYO1C35 populated the actomyosin·ADP closed state (AMDC) 5-fold more than the actomyosin·ADP open state (AMDO) and to a greater degree than MYO1CC and MYO1C16 (4- and 2-fold, respectively). On the basis of a homology model of the 35-amino acid NTR of MYO1C35 (NTR35) docked to the X-ray structure of MYO1CC, we predicted that MYO1C35 NTR residue Arg-21 would engage in a specific interaction with post-relay helix residue Glu-469, which affects the mechanics of the myosin power stroke. In addition, we found that adding the NTR35 peptide to MYO1CC yielded a protein that transiently mimics MYO1C35 kinetic behavior. By contrast, NTR35, which harbors the R21G mutation, was unable to confer MYO1C35-like kinetic behavior. Thus, the NTRs affect the specific nucleotide-binding properties of MYO1C isoforms, adding to their kinetic diversity. We propose that this level of fine-tuning within MYO1C broadens its adaptability within cells.
Asunto(s)
Empalme Alternativo , Miosina Tipo I , Actomiosina/química , Actomiosina/genética , Actomiosina/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/genética , Adenosina Difosfato/metabolismo , Sustitución de Aminoácidos , Cristalografía por Rayos X , Células HEK293 , Humanos , Isoenzimas , Mutación Missense , Miosina Tipo I/química , Miosina Tipo I/genética , Miosina Tipo I/metabolismoRESUMEN
The versatility in the recognition of various interacting proteins by the SH3 domain drives a variety of cellular functions. Here, the crystal structure of the C-terminal SH3 domain of myosin IB from Entamoeba histolytica (EhMySH3) is reported at a resolution of 1.7â Å in native and PEG-bound states. Comparisons with other structures indicated that the PEG molecules occupy protein-protein interaction pockets similar to those occupied by the peptides in other peptide-bound SH3-domain structures. Also, analysis of the PEG-bound EhMySH3 structure led to the recognition of two additional pockets, apart from the conventional polyproline and specificity pockets, that are important for ligand interaction. Molecular-docking studies combined with various comparisons revealed structural similarity between EhMySH3 and the SH3 domain of ß-Pix, and this similarity led to the prediction that EhMySH3 preferentially binds targets containing type II-like PXXP motifs. These studies expand the understanding of the EhMySH3 domain and provide extensive structural knowledge, which is expected to help in predicting the interacting partners which function together with myosin IB during phagocytosis in E. histolytica infections.
Asunto(s)
Entamoeba histolytica/metabolismo , Miosina Tipo I/metabolismo , Polietilenglicoles/metabolismo , Proteínas Protozoarias/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Cristalografía por Rayos X , Entamoeba histolytica/química , Entamebiasis/parasitología , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Miosina Tipo I/química , Polietilenglicoles/química , Unión Proteica , Multimerización de Proteína , Proteínas Protozoarias/química , Alineación de SecuenciaRESUMEN
The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing â¼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.
Asunto(s)
Proteínas Inmovilizadas/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Esclerosis Múltiple/diagnóstico , Miosina Tipo I/inmunología , Polihidroxietil Metacrilato/química , Animales , Humanos , Proteínas Inmovilizadas/química , Magnetismo/métodos , Imanes/química , Ratones , Microesferas , Esclerosis Múltiple/inmunología , Miosina Tipo I/química , Isoformas de Proteínas/química , Isoformas de Proteínas/inmunologíaRESUMEN
Several key processes in the cell, such as vesicle transport and spindle positioning, are mediated by the motor protein cytoplasmic dynein, which produces force on the microtubule. For the functions that require movement of the centrosome and the associated nuclear material, dynein needs to have a stable attachment at the cell cortex. In fission yeast, Mcp5 is the anchor protein of dynein and is required for the oscillations of the horsetail nucleus during meiotic prophase. Although the role of Mcp5 in anchoring dynein to the cortex has been identified, it is unknown how Mcp5 associates with the membrane as well as the importance of the underlying attachment to the nuclear oscillations. Here, we set out to quantify Mcp5 organization and identify the binding partner of Mcp5 at the membrane. We used confocal and total internal reflection fluorescence microscopy to count the number of Mcp5 foci and the number of Mcp5 molecules in an individual focus. Further, we quantified the localization pattern of Mcp5 in fission yeast zygotes and show by perturbation of phosphatidylinositol 4-phosphate 5-kinase that Mcp5 binds to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Remarkably, we discovered that the myosin I protein in fission yeast, Myo1, which is required for organization of sterol-rich domains in the cell membrane, facilitates the localization of Mcp5 and that of cytoplasmic dynein on the membrane. Finally, we demonstrate that Myo1-facilitated association of Mcp5 and dynein to the membrane determines the dynamics of nuclear oscillations and, in essence, dynein activity.
Asunto(s)
Dineínas Citoplasmáticas/metabolismo , Proteínas Fúngicas/fisiología , Miosina Tipo I/fisiología , Sitios de Unión , Citoplasma/metabolismo , Proteínas Fúngicas/análisis , Proteínas Fúngicas/química , Miosina Tipo I/análisis , Miosina Tipo I/química , SchizosaccharomycesRESUMEN
Real-time imaging was used to study the effects of a novel Fusarium-specific cyanoacrylate fungicide (JS399-19) on growth and morphology of four Fusarium sp. This fungicide targets the motor domain of type I myosin. Fusarium graminearum PH-1, Fusarium solani f. sp. pisi 77-13-4, Fusarium avenaceum IBT8464, and Fusarium avenaceum 05001, which has a K216Q amino-acid substitution at the resistance-implicated site in its myosin type I motor domain, were analyzed. Real-time imaging shows that JS399-19 inhibits fungal growth but not to the extent previously reported. The fungicide causes the hypha to become entangled and unable to extend vertically. This implies that type I myosin in Fusarium is essential for hyphal and mycelia propagation. The K216Q substitution correlates with reduced susceptibility in F. avenaceum.