RESUMEN
The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.
Asunto(s)
Adenosina Trifosfato , Pruebas de Enzimas , Miosina Tipo IIA no Muscular , Porcinos , ortoaminobenzoatos , Animales , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Bencilaminas/farmacología , Pruebas de Enzimas/métodos , Pruebas de Enzimas/normas , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/enzimología , Ventrículos Cardíacos/metabolismo , Contracción Miocárdica , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/metabolismo , ortoaminobenzoatos/metabolismo , Uracilo/análogos & derivados , Uracilo/farmacologíaRESUMEN
Recent genomic studies reported that 90 to 95% of human genes can undergo alternative splicing, by which multiple isoforms of proteins are synthesized. However, the functional consequences of most of the isoforms are largely unknown. Here, we report a novel alternatively spliced isoform of nonmuscle myosin IIA (NM IIA), called NM IIA2, which is generated by the inclusion of 21 amino acids near the actin-binding region (loop 2) of the head domain of heavy chains. Expression of NM IIA2 is found exclusively in the brain tissue, where it reaches a maximum level at 24 h during the circadian rhythm. The actin-dependent Mg2+-ATPase activity and in vitro motility assays reveal that NM IIA2 lacks its motor activities but localizes with actin filaments in cells. Interestingly, NM IIA2 can also make heterofilaments with NM IIA0 (noninserted isoform of NM IIA) and can retard the in vitro motility of NM IIA, when the two are mixed. Altogether, our findings provide the functional importance of a previously unknown alternatively spliced isoform, NM IIA2, and its potential physiological role in regulating NM IIA activity in the brain.
Asunto(s)
Empalme Alternativo , Encéfalo , Miosina Tipo IIA no Muscular , Humanos , Actinas/metabolismo , Encéfalo/metabolismo , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ritmo Circadiano , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Especificidad de ÓrganosRESUMEN
Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.
Asunto(s)
Células Endoteliales/citología , Miosina Tipo IIA no Muscular/genética , Seudópodos/genética , Proteína de Unión al GTP rac1/genética , Complejo 2-3 Proteico Relacionado con la Actina/química , Complejo 2-3 Proteico Relacionado con la Actina/genética , Animales , Extensiones de la Superficie Celular , Células Endoteliales/metabolismo , Ratones , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Miosina Tipo IIA no Muscular/química , Activación Transcripcional/genéticaRESUMEN
To identify hepatitis B virus (HBV)-related lncRNA(s), we previously examined the transcription profiles of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells by RNA deep sequencing and identified 38 upregulated long noncoding RNAs (lncRNAs). In the present study, the lncRNA MAFG-AS1 is investigated in detail because its gene is located adjacent to the MAFG gene, which is an important transcription factor involved in cell proliferation. The level of MAFG-AS1 is significantly higher in HCC tissue than in nontumor tissues. TCGA data show that the expression level of MAFG-AS1 is negatively correlated with survival of HCC patients. GEO cohort data show that compared with healthy tissues, the expression level of MAFG-AS1 is significantly higher in HBV-infected liver tissues. Real-time PCR and luciferase reporter assay data show that HBx can enhance the transcription of MAFG-AS1. Gain-of-function and loss-of-function experiments indicate that MAFG-AS1 promotes proliferation, migration, and invasion of HCC cells. Tumor formation assay results demonstrate that knockdown of MAFG-AS1 significantly inhibits cell proliferation in nude mice. Furthermore, MAFG-AS1 enhances the transcription of adjacent MAFG via E2F1. Additionally, MAFG-AS1 interacts with three subunits (MYH9, MYL12B, and MYL6) of nonmuscle myosin IIA (NM IIA). Knockdown of MAFG-AS1 inhibits ATPase activity of MYH9, interaction of NM IIA subunits, and cell cycle progression. Thus, the lncRNA MAFG-AS1 is upregulated by HBV and promotes proliferation and migration of HCC cells. Our findings suggest that MAFG-AS1 is a potential oncogene that may contribute to HBV-related HCC development.
Asunto(s)
Carcinoma Hepatocelular/patología , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Factor de Transcripción MafG/metabolismo , Miosina Tipo IIA no Muscular/química , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Factor de Transcripción MafG/antagonistas & inhibidores , Factor de Transcripción MafG/genética , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Oligonucleótidos Antisentido/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Parasites of the phylum Apicomplexa are responsible for significant morbidity and mortality on a global scale. Central to the virulence of these pathogens are the phylum-specific, unconventional class XIV myosins that power the essential processes of parasite motility and host cell invasion. Notably, class XIV myosins differ from human myosins in key functional regions, yet they are capable of fast movement along actin filaments with kinetics rivaling previously studied myosins. Toward establishing a detailed molecular mechanism of class XIV motility, we determined the 2.6-Å resolution crystal structure of the Toxoplasma gondii MyoA (TgMyoA) motor domain. Structural analysis reveals intriguing strategies for force transduction and chemomechanical coupling that rely on a divergent SH1/SH2 region, the class-defining "HYAG"-site polymorphism, and the actin-binding surface. In vitro motility assays and hydrogen-deuterium exchange coupled with MS further reveal the mechanistic underpinnings of phosphorylation-dependent modulation of TgMyoA motility whereby localized regions of increased stability and order correlate with enhanced motility. Analysis of solvent-accessible pockets reveals striking differences between apicomplexan class XIV and human myosins. Extending these analyses to high-confidence homology models of Plasmodium and Cryptosporidium MyoA motor domains supports the intriguing potential of designing class-specific, yet broadly active, apicomplexan myosin inhibitors. The successful expression of the functional TgMyoA complex combined with our crystal structure of the motor domain provides a strong foundation in support of detailed structure-function studies and enables the development of small-molecule inhibitors targeting these devastating global pathogens.
Asunto(s)
Miosina Tipo IIA no Muscular/química , Toxoplasma/metabolismo , Secuencia de Aminoácidos , Antiprotozoarios/química , Antiprotozoarios/farmacología , Sitios de Unión , Diseño de Fármacos , Imitación Molecular , Mutación , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Homología de Secuencia de Aminoácido , Toxoplasma/efectos de los fármacosRESUMEN
Apicomplexan parasites, through their motor machinery, produce the required propulsive force critical for host cell-entry. The conserved components of this so-called glideosome machinery are myosin A and myosin A Tail Interacting Protein (MTIP). MTIP tethers myosin A to the inner membrane complex of the parasite through 20 amino acid-long C-terminal end of myosin A that makes direct contacts with MTIP, allowing the invasion of Plasmodium falciparum in erythrocytes. Here, we discovered through screening a peptide library, a de-novo peptide ZA1 that binds the myosin A tail domain. We demonstrated that ZA1 bound strongly to myosin A tail and was able to disrupt the native myosin A tail MTIP complex both in vitro and in vivo. We then showed that a shortened peptide derived from ZA1, named ZA1S, was able to bind myosin A and block parasite invasion. Overall, our study identified a novel anti-malarial peptide that could be used in combination with other antimalarials for blocking the invasion of Plasmodium falciparum.
Asunto(s)
Antimaláricos/farmacología , Proteínas de la Membrana/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Péptidos/farmacología , Plasmodium falciparum/crecimiento & desarrollo , Secuencias de Aminoácidos , Antimaláricos/química , Sitios de Unión , Evaluación Preclínica de Medicamentos , Eritrocitos/parasitología , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas de la Membrana/química , Modelos Moleculares , Complejos Multiproteicos/efectos de los fármacos , Miosina Tipo IIA no Muscular/química , Biblioteca de Péptidos , Péptidos/química , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
Nonmuscle myosin 2 (NM2) has three paralogs in mammals, NM2A, NM2B, and NM2C, which have both unique and overlapping functions in cell migration, formation of cell-cell adhesions, and cell polarity. Their assembly into homo- and heterotypic bipolar filaments in living cells is primarily regulated by phosphorylation of the N-terminally bound regulatory light chain. Here, we present evidence that the equilibrium between these filaments and single NM2A and NM2B molecules can be controlled via S100 calcium-binding protein interactions and phosphorylation at the C-terminal end of the heavy chains. Furthermore, we show that in addition to S100A4, other members of the S100 family can also mediate disassembly of homotypic NM2A filaments. Importantly, these proteins can selectively remove NM2A molecules from heterotypic filaments. We also found that tail phosphorylation (at Ser-1956 and Ser-1975) of NM2B by casein kinase 2, as well as phosphomimetic substitutions at sites targeted by protein kinase C (PKC) and transient receptor potential cation channel subfamily M member 7 (TRPM7), down-regulates filament assembly in an additive fashion. Tail phosphorylation of NM2A had a comparatively minor effect on filament stability. S100 binding and tail phosphorylation therefore preferentially disassemble NM2A and NM2B, respectively. These two distinct mechanisms are likely to contribute to the temporal and spatial sorting of the two NM2 paralogs within heterotypic filaments. The existence of multiple NM2A-depolymerizing S100 paralogs offers the potential for diverse regulatory inputs modulating NM2A filament disassembly in cells and provides functional redundancy under both physiological and pathological conditions.
Asunto(s)
Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas S100/metabolismo , Animales , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Citoesqueleto/metabolismo , Proteínas Fluorescentes Verdes/genética , Humanos , Mutación , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIB no Muscular/química , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Células Sf9 , Canales Catiónicos TRPM/metabolismoRESUMEN
Apicomplexan parasites such as Toxoplasma gondii rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA-light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775-818) using a combination of hydrogen-deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm Kd measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a Kd of 12 nm). Using the full-length MyoA motor (residues 1-831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction.
Asunto(s)
Miosina Tipo IIA no Muscular/química , Proteínas Protozoarias/química , Toxoplasma/metabolismo , Animales , Calcio/metabolismo , Movimiento Celular , Cristalografía por Rayos X , Miosina Tipo IIA no Muscular/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Protozoarias/metabolismo , Toxoplasma/crecimiento & desarrolloRESUMEN
Nonmuscle myosin II is an actin-based motor that executes numerous mechanical tasks in cells including spatiotemporal organization of the actin cytoskeleton, adhesion, migration, cytokinesis, tissue remodeling, and membrane trafficking. Nonmuscle myosin II is ubiquitously expressed in mammalian cells as a tissue-specific combination of three paralogs. Recent studies reveal novel specific aspects of their kinetics, intracellular regulation and functions. On the other hand, the three paralogs also can copolymerize and cooperate in cells. Here we review the recent advances from the prospective of how distinct features of the three myosin II paralogs adapt them to perform specialized and joint tasks in the cell.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/química , Matriz Extracelular/química , Cadenas Pesadas de Miosina/química , Miosina Tipo II/química , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIB no Muscular/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestructura , Actinas/genética , Actinas/metabolismo , Animales , Fenómenos Biomecánicos , Adhesión Celular , Movimiento Celular , Citocinesis/genética , Células Eucariotas/metabolismo , Células Eucariotas/ultraestructura , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Expresión Génica , Humanos , Mamíferos , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/metabolismo , Multimerización de ProteínaRESUMEN
Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.
Asunto(s)
Miosina Tipo IIB no Muscular/química , Plasmodium berghei/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium knowlesi/metabolismo , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Calmodulina/química , Dicroismo Circular , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes/química , Datos de Secuencia Molecular , Cadenas Ligeras de Miosina/química , Miosina Tipo IIA no Muscular/química , Péptidos/química , Unión Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Dysregulation of Ca2+ -binding S100 proteins plays important role in various diseases. The asymmetric complex of Ca2+ -bound S100A4 with nonmuscle myosinâ IIA has high stability and highly increased Ca2+ affinity. Here we investigated the possible causes of this allosteric effect by NMR spectroscopy. Chemical shift-based secondary-structure analysis did not show substantial changes for the complex. Backbone dynamics revealed slow-timescale local motions in the H1 helices of homodimeric S100A4; these were less pronounced in the complex form and might be accompanied by an increase in dimer stability. Different mobilities in the Ca2+ -coordinating EF-hand sites indicate that they communicate by an allosteric mechanism operating through changes in protein dynamics; this must be responsible for the elevated Ca2+ affinity. These multilevel changes in protein dynamics as conformational adaptation allow S100A4 fine-tuning of its protein-protein interactions inside the cell during Ca2+ signaling.
Asunto(s)
Calcio/metabolismo , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/metabolismo , Proteína de Unión al Calcio S100A4/química , Proteína de Unión al Calcio S100A4/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Resonancia Magnética Nuclear BiomolecularRESUMEN
Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization.
Asunto(s)
Megacariocitos/citología , Cadenas Pesadas de Miosina/análisis , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo IIB no Muscular/análisis , Miosina Tipo IIB no Muscular/metabolismo , Animales , Diferenciación Celular , Megacariocitos/metabolismo , Ratones , Ratones Transgénicos , Mitosis , Cadenas Pesadas de Miosina/genética , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/genética , Poliploidía , Estructura Terciaria de ProteínaRESUMEN
S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4-NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4-NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly α-helical myosin chain is wrapped around the Ca(2+)-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4-NMIIA interaction.
Asunto(s)
Miosina Tipo IIA no Muscular/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas S100/química , Sitios de Unión , Calorimetría , Dicroismo Circular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación , Miosina Tipo IIA no Muscular/metabolismo , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Proteína de Unión al Calcio S100A4 , Proteínas S100/genética , Proteínas S100/metabolismoRESUMEN
Nonmuscle myosin II forms a folded conformation (10S form) in the inactivated state; however, the physiological importance of the 10S form is still unclear. To investigate the role of 10S form, we generated a chimeric mutant of nonmuscle myosin IIB (IIB-SK1·2), in which S1462-R1490 and L1551-E1577 were replaced with the corresponding portions of skeletal muscle myosin heavy chain. The IIB-SK1·2 mutant did not fold into a 10S form under physiological condition in vitro. IIB-SK1·2 was less dynamic by stabilizing the filamentous form and accumulated in the posterior region of migrating cells. IIB-SK1·2 functioned properly in cytokinesis but altered migratory properties; the rate and directional persistence were increased by IIB-SK1·2 expression. Surprisingly, endogenous nonmuscle myosin IIA was excluded from the posterior region of migrating cells expressing IIB-SK1·2, which may underlie the change of the cellular migratory properties. These results suggest that the 10S form is necessary for maintaining nonmuscle myosin II in an unassembled state and for recruitment of nonmuscle myosin II to a specific region of the cell.
Asunto(s)
Espacio Intracelular/metabolismo , Miosina Tipo IIB no Muscular/química , Miosina Tipo IIB no Muscular/metabolismo , Pliegue de Proteína , Línea Celular , Citocinesis/fisiología , Humanos , Miosina Tipo II/química , Miosina Tipo II/metabolismo , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/genética , Conformación Proteica , Estabilidad Proteica , Transporte de Proteínas , Fracciones SubcelularesRESUMEN
The interaction between the C-terminal tail of myosin A (MyoA) and its light chain, myosin A tail domain interacting protein (MTIP), is an essential feature of the conserved molecular machinery required for gliding motility and cell invasion by apicomplexan parasites. Recent data indicate that MTIP Ser-107 and/or Ser-108 are targeted for intracellular phosphorylation. Using an optimized MyoA tail peptide to reconstitute the complex, we show that this region of MTIP is an interaction hotspot using x-ray crystallography and NMR, and S107E and S108E mutants were generated to mimic the effect of phosphorylation. NMR relaxation experiments and other biophysical measurements indicate that the S108E mutation serves to break the tight clamp around the MyoA tail, whereas S107E has a smaller but measurable impact. These data are consistent with physical interactions observed between recombinant MTIP and native MyoA from Plasmodium falciparum lysates. Taken together these data support the notion that the conserved interactions between MTIP and MyoA may be specifically modulated by this post-translational modification.
Asunto(s)
Proteínas del Citoesqueleto/química , Miosina Tipo IIA no Muscular/química , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Sustitución de Aminoácidos , Células Cultivadas , Cristalografía por Rayos X , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Análisis Diferencial Térmico , Eritrocitos/parasitología , Fluorometría , Humanos , Modelos Moleculares , Miosina Tipo IIA no Muscular/metabolismo , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Termodinámica , VolumetríaRESUMEN
BACKGROUND: S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood. RESULTS: We determined the X-ray crystal structure of the S100A4Δ8C/MIIA(1908-1923) peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4Δ8C binds the MIIA(1908-1923) peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site. CONCLUSION: The structure of the S100A4/MIIA(1908-1923) peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908-1923 of the myosin-IIA chain heavy represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.
Asunto(s)
Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/metabolismo , Proteínas S100/química , Proteínas S100/metabolismo , Sitios de Unión , Dicroismo Circular , Cristalografía por Rayos X , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Miosinas/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteína de Unión al Calcio S100A4RESUMEN
Natural killer (NK) cells are innate immune lymphocytes that provide critical defense against virally infected and transformed cells. NK-cell cytotoxicity requires the formation of an F-actin rich immunologic synapse (IS), as well as the polarization of perforin-containing lytic granules to the IS and secretion of their contents at the IS. It was reported previously that NK-cell cytotoxicity requires nonmuscle myosin IIA function and that granule-associated myosin IIA mediates the interaction of granules with F-actin at the IS. In the present study, we evaluate the nature of the association of myosin IIA with lytic granules. Using NK cells from patients with mutations in myosin IIA, we found that the nonhelical tailpiece is required for NK-cell cytotoxicity and for the phosphorylation of granule-associated myosin IIA. Ultra-resolution imaging techniques demonstrated that single myosin IIA molecules associate with NK-cell lytic granules via the nonhelical tailpiece. Phosphorylation of myosin IIA at residue serine 1943 (S1943) in the tailpiece is needed for this linkage. This defines a novel mechanism for myosin II function, in which myosin IIA can act as a single-molecule actin motor, claiming granules as cargo through tail-dependent phosphorylation for the execution of a pre-final step in human NK-cell cytotoxicity.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Citotoxicidad Inmunológica , Células Asesinas Naturales/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Línea Celular , Citotoxicidad Inmunológica/fisiología , Pérdida Auditiva/genética , Pérdida Auditiva/inmunología , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/inmunología , Humanos , Células K562 , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/fisiología , Activación de Linfocitos/fisiología , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Mutación Missense/fisiología , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Nefritis Hereditaria/genética , Nefritis Hereditaria/inmunología , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/genética , Fosforilación/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Trombocitopenia/genética , Trombocitopenia/inmunologíaRESUMEN
Repair of injury to the plasma membrane is an essential mechanism for maintenance of cellular homeostasis and integrity that involves coordinated movement of intracellular vesicles to membrane injury sites to facilitate patch formation. We have previously identified MG53 as an essential component of the cell membrane repair machinery. In order for MG53 and intracellular vesicles to translocate to membrane injury sites, motor proteins must be involved. Here, we show that nonmuscle myosin type IIA (NM-IIA) interacts with MG53 to regulate vesicle trafficking during cell membrane repair. In cells that are deficient for NM-IIA expression, MG53 cannot translocate to acute injury sites, whereas rescue of NM-IIA expression in these cells can restore MG53-mediated membrane repair. Compromised cell membrane repair is observed in cells with RNAi-mediated knockdown of NM-IIA expression, or following pharmacological alteration of NM-IIA motor function. Together, our data reveal NM-IIA as a key cytoskeleton motor protein that facilitates vesicle trafficking during MG53-mediated cell membrane repair.
Asunto(s)
Proteínas Portadoras/fisiología , Miosina Tipo IIA no Muscular/fisiología , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Membrana Celular/fisiología , Chlorocebus aethiops , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/genética , Transporte de Proteínas , Interferencia de ARN , Proteínas de Motivos TripartitosRESUMEN
Post-translational modifications (PTMs) including phosphorylation and palmitoylation have emerged as crucial biomolecular events that govern many cellular processes including functioning of motility- and invasion-associated proteins during Plasmodium falciparum invasion. However, no study has ever focused on understanding the possibility of a crosstalk between these two molecular events and its direct impact on preinvasion- and invasion-associated protein-protein interaction (PPI) network-based molecular machinery. Here, we used an integrated in silico analysis to enrich two different catalogues of proteins: (i) the first group defines the cumulative pool of phosphorylated and palmitoylated proteins, and (ii) the second group represents a common set of proteins predicted to have both phosphorylation and palmitoylation. Subsequent PPI analysis identified an important protein cluster comprising myosin A tail interacting protein (MTIP) as one of the hub proteins of the glideosome motor complex in P. falciparum, predicted to have dual modification with the possibility of a crosstalk between the same. Our findings suggested that blocking palmitoylation led to reduced phosphorylation and blocking phosphorylation led to abrogated palmitoylation of MTIP. As a result of the crosstalk between these biomolecular events, MTIP's interaction with myosin A was found to be abrogated. Next, the crosstalk between phosphorylation and palmitoylation was confirmed at a global proteome level by click chemistry and the phenotypic effect of this crosstalk was observed via synergistic inhibition in P. falciparum invasion using checkerboard assay and isobologram method. Overall, our findings revealed, for the first time, an interdependence between two PTM types, their possible crosstalk, and its direct impact on MTIP-mediated invasion via glideosome assembly protein myosin A in P. falciparum. These insights can be exploited for futuristic drug discovery platforms targeting parasite molecular machinery for developing novel antimalarial therapeutics.
Asunto(s)
Antimaláricos , Proteínas del Citoesqueleto/metabolismo , Malaria Falciparum , Proteínas de la Membrana/metabolismo , Miosina Tipo IIA no Muscular , Humanos , Lipoilación , Malaria Falciparum/parasitología , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/metabolismo , Fosforilación , Plasmodium falciparum , Proteoma/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
We propose that the in vivo functions of NM II (non-muscle myosin II) can be divided between those that depend on the N-terminal globular motor domain and those less dependent on motor activity but more dependent on the C-terminal domain. The former, being more dependent on the kinetic properties of NM II to translocate actin filaments, are less amenable to substitution by different NM II isoforms, whereas the in vivo functions of the latter, which involve the structural properties of NM II to cross-link actin filaments, are more amenable to substitution. In light of this hypothesis, we examine the ability of NM II-A, as well as a motor-compromised form of NM II-B, to replace NM II-B and rescue neuroepithelial cell-cell adhesion defects and hydrocephalus in the brain of NM II-B-depleted mice. We also examine the ability of NM II-B as well as chimaeric forms of NM II (II-A head and II-B tail and vice versa) to substitute for NM II-A in cell-cell adhesions in II-A-ablated mice. However, we also show that certain functions, such as neuronal cell migration in the developing brain and vascularization of the mouse embryo and placenta, specifically require NM II-B and II-A respectively.