RESUMEN
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.
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Actinas/metabolismo , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Nucleótidos/metabolismo , Fenómenos Biomecánicos , Humanos , Cinética , Unión Proteica , Isoformas de ProteínasRESUMEN
KEY POINTS: Direct binding of rumenic acid to the cardiac myosin-2 motor domain increases the release rate for orthophosphate and increases the Ca2+ responsiveness of cardiac muscle at low load. Physiological cellular concentrations of rumenic acid affect the ATP turnover rates of the super-relaxed and disordered relaxed states of ß-cardiac myosin, leading to a net increase in myocardial metabolic load. In Ca2+ -activated trabeculae, rumenic acid exerts a direct inhibitory effect on the force-generating mechanism without affecting the number of force-generating motors. In the presence of saturating actin concentrations rumenic acid binds to the ß-cardiac myosin-2 motor domain with an EC50 of 200 nM. Molecular docking studies provide information about the binding site, the mode of binding, and associated allosteric communication pathways. Free rumenic acid may exceed thresholds in cardiomyocytes above which contractile efficiency is reduced and interference with small molecule therapeutics, targeting cardiac myosin, occurs. ABSTRACT: Based on experiments using purified myosin motor domains, reconstituted actomyosin complexes and rat heart ventricular trabeculae, we demonstrate direct binding of rumenic acid, the cis-delta-9-trans-delta-11 isomer of conjugated linoleic acid, to an allosteric site located in motor domain of mammalian cardiac myosin-2 isoforms. In the case of porcine ß-cardiac myosin, the EC50 for rumenic acid varies from 10.5 µM in the absence of actin to 200 nM in the presence of saturating concentrations of actin. Saturating concentrations of rumenic acid increase the maximum turnover of basal and actin-activated ATPase activity of ß-cardiac myosin approximately 2-fold but decrease the force output per motor by 23% during isometric contraction. The increase in ATP turnover is linked to an acceleration of the release of the hydrolysis product orthophosphate. In the presence of 5 µM rumenic acid, the difference in the rate of ATP turnover by the super-relaxed and disordered relaxed states of cardiac myosin increases from 4-fold to 20-fold. The equilibrium between the two functional myosin states is not affected by rumenic acid. Calcium responsiveness is increased under zero-load conditions but unchanged under load. Molecular docking studies provide information about the rumenic acid binding site, the mode of binding, and associated allosteric communication pathways. They show how the isoform-specific replacement of residues in the binding cleft induces a different mode of rumenic acid binding in the case of non-muscle myosin-2C and blocks binding to skeletal muscle and smooth muscle myosin-2 isoforms.
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Ácidos Linoleicos Conjugados , Actinas/metabolismo , Adenosina Trifosfato , Animales , Miosinas Cardíacas , Cinética , Simulación del Acoplamiento Molecular , Ratas , PorcinosRESUMEN
KEY POINTS: A nanomachine made of an ensemble of seven heavy-meromyosin (HMM) fragments of muscle myosin interacting with an actin filament is able to mimic the half-sarcomere generating steady force and constant-velocity shortening. To preserve Ca2+ as a free parameter, the Ca2+ -insensitive gelsolin fragment TL40 is used to attach the correctly oriented actin filament to the laser-trapped bead acting as a force transducer. The new method reveals that the performance of the nanomachine powered by myosin from frog hind-limb muscles depends on [Ca2+ ], an effect mediated by a Ca2+ -binding site in the regulatory light chain of HMM. The Ca2+ -sensitivity is class-specific because the performance of the nanomachine powered by mammalian skeletal muscle myosin is Ca2+ independent. A model simulation is able to interface the nanomachine performance with that of the muscle of origin and provides a molecular explanation of the functional diversity of muscles with different orthologue isoforms of myosin. ABSTRACT: An ensemble of seven heavy-meromyosin (HMM) fragments of myosin-II purified from the hindlimb muscles of the frog (Rana esculenta) is used to drive a synthetic nanomachine that pulls an actin filament in the absence of confounding effects of other sarcomeric proteins. In the present version of the nanomachine the +end of the actin filament is attached to the laser trapped bead via the Ca2+ -insensitive gelsolin fragment TL40, making [Ca2+ ] a free parameter. Frog myosin performance in 2 mm ATP is affected by Ca2+ : in 0.1 mm Ca2+ , the isometric steady force (F0 , 15.25 pN) is increased by 50% (P = 0.004) with respect to that in Ca2+ -free solution, the maximum shortening velocity (V0 , 4.6 µm s-1 ) is reduced by 27% (P = 0.46) and the maximum power (Pmax , 7.6 aW) is increased by 21% (P = 0.17). V0 reduction is not significant for the paucity of data at low force, although it is solidified by a similar decrease (33%, P < 0.0001) in the velocity of actin sliding as indicated by an in vitro motility assay (Vf ). The rate of ATP-hydrolysis in solution (φ) exhibits a similar calcium dependence. Ca2+ titration curves for Vf and φ give Kd values of â¼30 µm. All the above mechanical and kinetic parameters are independent of Ca2+ when HMM from rabbit psoas myosin is used, indicating that the Ca2+ -sensitivity is a class-specific property of muscle myosin. A unique multiscale model allows interfacing of the nanomachine performance to that of the muscle of origin and identifies the kinetic steps responsible for the Ca2+ -sensitivity of frog myosin.
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Contracción Muscular , Miosinas , Actinas , Animales , Músculo Esquelético , Miosina Tipo II , Isoformas de Proteínas , ConejosRESUMEN
Mutations in the gene encoding cardiac myosin-binding protein-C (MyBPC), a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function, are a common cause for the development of hypertrophic cardiomyopathy. About 10% of carriers of the Δ25bp variant of MYBPC3, which is common in individuals from South Asia, are also carriers of the D389V variant on the same allele. Compared with noncarriers and those with MYBPC3Δ25bp alone, indicators for the development of hypertrophic cardiomyopathy occur with increased frequency in MYBPC3Δ25bp/D389V carriers. Residue D389 lies in the IgI-like C2 domain that is part of the N-terminal region of MyBPC. To probe the effects of mutation D389V on structure, thermostability, and protein-protein interactions, we produced and characterized wild-type and mutant constructs corresponding to the isolated 10 kDa C2 domain and a 52 kDa N-terminal fragment that includes subdomains C0 to C2. Our results show marked reductions in the melting temperatures of D389V mutant constructs. Interactions of construct C0-C2 D389V with the cardiac isoforms of myosin-2 and actin remain unchanged. Molecular dynamics simulations reveal changes in the stiffness and conformer dynamics of domain C2 caused by mutation D389V. Our results suggest a pathomechanism for the development of HCM based on the toxic buildup of misfolded protein in young MYBPC3Δ25bp/D389V carriers that is supplanted and enhanced by C-zone haploinsufficiency at older ages.
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Dominios C2 , Cardiomiopatía Hipertrófica/patología , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Mutación , Dominios y Motivos de Interacción de Proteínas , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Proteínas Portadoras/genética , Humanos , TermodinámicaRESUMEN
A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN- molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.
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Colorantes/química , Dictyostelium/enzimología , Hemo/química , Peróxido de Hidrógeno/química , Peroxidasa/química , Peroxidasa/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Hemo/metabolismo , Enlace de Hidrógeno , Oxidación-ReducciónRESUMEN
The cyanoacrylate compound phenamacril (also known as JS399-19) is a recently identified fungicide that exerts its antifungal effect on susceptible Fusarium species by inhibiting the ATPase activity of their myosin class I motor domains. Although much is known about the antifungal spectrum of phenamacril, the exact mechanism behind the phenamacril-mediated inhibition remains to be resolved. Here, we describe the characterization of the effect of phenamacril on purified myosin motor constructs from the model plant pathogen and phenamacril-susceptible species Fusarium graminearum, phenamacril-resistant Fusarium species, and the mycetozoan model organism Dictyostelium discoideum Our results show that phenamacril potently (IC50 â¼360 nm), reversibly, and noncompetitively inhibits ATP turnover, actin binding during ATP turnover, and motor activity of F. graminearum myosin-1. Phenamacril also inhibits the ATPase activity of Fusarium avenaceum myosin-1 but has little or no inhibitory effect on the motor activity of Fusarium solani myosin-1, human myosin-1c, and D. discoideum myosin isoforms 1B, 1E, and 2. Our findings indicate that phenamacril is a species-specific, noncompetitive inhibitor of class I myosin in susceptible Fusarium sp.
Asunto(s)
Fungicidas Industriales/farmacología , Fusarium/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Miosina Tipo I/antagonistas & inhibidores , Fusarium/crecimiento & desarrollo , Fusarium/metabolismo , Conformación Proteica , Especificidad de la EspecieRESUMEN
Class XVIII myosins represent a branch of the myosin family tree characterized by the presence of large N- and C-terminal extensions flanking a generic myosin core. These myosins display the highest sequence similarity to conventional class II muscle myosins and are compatible with but not restricted to myosin-2 contractile structures. Instead, they fulfill their functions at diverse localities, such as lamella, actomyosin bundles, the Golgi apparatus, focal adhesions, the cell membrane, and within sarcomeres. Sequence comparison of active-site residues and biochemical data available thus far indicate that this myosin class lacks active ATPase-driven motor activity, suggesting that its members function as structural myosins. An emerging body of evidence indicates that this structural capability is essential for the organization, maturation, and regulation of the contractile machinery in both muscle and nonmuscle cells. This is supported by the clear association of myosin-18A (Myo18A) and myosin-18B (Myo18B) dysregulation with diseases such as cancer and various myopathies.
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Miosinas , Citoesqueleto de Actina , Actinas , Actomiosina , Humanos , Contracción MuscularRESUMEN
The metazoan actin cytoskeleton supports a wide range of contractile and transport processes. Recent studies have shown how the dynamic association with specific tropomyosin isoforms generates actin filament populations with distinct functional properties. However, critical details of the associated molecular interactions remain unclear. Here, we report the properties of actomyosin-tropomyosin complexes containing filamentous ß-actin, nonmuscle myosin-2B (NM-2B) constructs, and either tropomyosin isoform Tpm1.8cy (b.-.b.d), Tpm1.12br (b.-.b.c), or Tpm3.1cy (b.-.a.d). Our results show the extent to which the association of filamentous ß-actin with these different tropomyosin cofilaments affects the actin-mediated activation of NM-2B and the release of the ATP hydrolysis products ADP and phosphate from the active site. Phosphate release gates a transition from weak to strong F-actin-binding states. The release of ADP has the opposite effect. These changes in dominant rate-limiting steps have a direct effect on the duty ratio, the fraction of time that NM-2B spends in strongly F-actin-bound states during ATP turnover. The duty ratio is increased â¼3-fold in the presence of Tpm1.12 and 5-fold for both Tpm1.8 and Tpm3.1. The presence of Tpm1.12 extends the time required per ATP hydrolysis cycle 3.7-fold, whereas it is shortened by 27 and 63% in the presence of Tpm1.8 and Tpm3.1, respectively. The resulting Tpm isoform-specific changes in the frequency, duration, and efficiency of actomyosin interactions establish a molecular basis for the ability of these complexes to support cellular processes with widely divergent demands in regard to force production, capacity to move processively, and speed of movement.
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Actinas/metabolismo , Miosinas/metabolismo , Isoformas de Proteínas/metabolismo , Tropomiosina/metabolismo , Animales , Línea Celular Tumoral , Humanos , Cinética , Neuroblastoma/metabolismo , Unión ProteicaRESUMEN
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.
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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
Molecular motors of the myosin superfamily share a generic motor domain region. They commonly bind actin in an ATP-sensitive manner, exhibit actin-activated ATPase activity, and generate force and movement in this interaction. Class-18 myosins form heavy chain dimers and contain protein interaction domains located at their unique N-terminal extension. Here, we characterized human myosin-18A molecular function in the interaction with nucleotides, F-actin, and its putative binding partner, the Golgi-associated phosphoprotein GOLPH3. We show that myosin-18A comprises two actin binding sites. One is located in the KE-rich region at the start of the N-terminal extension and appears to mediate ATP-independent binding to F-actin. The second actin-binding site resides in the generic motor domain and is regulated by nucleotide binding in the absence of intrinsic ATP hydrolysis competence. This core motor domain displays its highest actin affinity in the ADP state. Electron micrographs of myosin-18A motor domain-decorated F-actin filaments show a periodic binding pattern independent of the nucleotide state. We show that the PDZ module mediates direct binding of myosin-18A to GOLPH3, and this interaction in turn modulates the actin binding properties of the N-terminal extension. Thus, myosin-18A can act as an actin cross-linker with multiple regulatory modulators that targets interacting proteins or complexes to the actin-based cytoskeleton.
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Actinas/química , Proteínas de la Membrana/química , Miosinas/química , Actinas/genética , Actinas/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Drosophila melanogaster , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Miosinas/genética , Miosinas/metabolismo , Unión Proteica , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Familial hypertrophic cardiomyopathy (HCM) affects .2% of the world's population and is inherited in an autosomal dominant manner. Mutations in cardiac α-actin are the cause in 1%-5% of all observed cases. Here, we describe the recombinant production, purification, and characterization of the HCM-linked cardiac α-actin variants p.A21V and p.D26N. Mass spectrometric analysis of the initially purified recombinant cardiac α-actin variants and wild-type protein revealed improper N-terminal processing in the Spodoptera frugiperda (Sf-9) insect cell system, compromising the labeling of the protein with fluorescent probes for biochemical studies. Therefore, we produced N-terminal deletion mutants lacking the N-terminal cysteine (ΔC2). The ΔC2 wild-type construct behaved similar to porcine cardiac α-actin purified from native Sus scrofa heart tissue and all ΔC2 constructs showed improved fluorescent labeling. Further analysis of untruncated and ΔC2 constructs showed that while neither the A21V nor the D26N mutation affects nucleotide binding, they cause a similar slowing of the rate of filament formation as well as a reduction in the thermal stability of monomeric and filamentous cardiac α-actin. In vitro motility assays and transient-kinetic studies probing the interaction of the actin variants with cardiac ß-myosin revealed perturbed actomyosin interactions and a reduced motile activity for the p.D26N variant. Addition of the small molecule effector EMD 57033, which targets cardiac ß-myosin, rescued the approximately 40% drop in velocity observed with the p.D26N constructs and activated the motile activity of wild-type and p.D26N to the same level of 1100 nm s-1 .
RESUMEN
Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin ß4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.
Asunto(s)
Factores Despolimerizantes de la Actina , Actinas , Miosinas , Humanos , Citoesqueleto de Actina , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Mutación , Miosinas/genética , Miosinas/metabolismoRESUMEN
The mitotic spindle in eukaryotic cells is composed of a bipolar array of microtubules (MTs) and associated proteins that are required during mitosis for the correct partitioning of the two sets of chromosomes to the daughter cells. In addition to the well-established functions of MT-associated proteins (MAPs) and MT-based motors in cell division, there is increasing evidence that the F-actin-based myosin motors are important mediators of F-actin-MT interactions during mitosis. Here, we report the functional characterization of the long-tailed class-1 myosin myosin-1C from Dictyostelium discoideum during mitosis. Our data reveal that myosin-1C binds to MTs and has a role in maintenance of spindle stability for accurate chromosome separation. Both myosin-1C motor function and tail-domain-mediated MT-F-actin interactions are required for the cell-cycle-dependent relocalization of the protein from the cell periphery to the spindle. We show that the association of myosin-1C with MTs is mediated through the tail domain. The myosin-1C tail can inhibit kinesin motor activity, increase the stability of MTs, and form crosslinks between MTs and F-actin. These data illustrate that myosin-1C is involved in the regulation of MT function during mitosis in D. discoideum.
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Dictyostelium/citología , Dictyostelium/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Huso Acromático/metabolismo , Actinas/genética , Actinas/metabolismo , División Celular/genética , División Celular/fisiología , Dictyostelium/genética , Microscopía Fluorescente , Miosinas/genética , Unión Proteica , Huso Acromático/genéticaRESUMEN
Heparosan is an acidic polysaccharide expressed as a capsule polymer by pathogenic and commensal bacteria, e.g. by E. coli K5. As a precursor in the biosynthesis of heparan sulfate and heparin, heparosan has a high biocompatibility and is thus of interest for pharmaceutical applications. However, due to its low immunogenicity, developing antibodies against heparosan and detecting the polymer in biological samples has been challenging. In this study, we exploited the enzyme repertoire of E. coli K5 and the E. coli K5-specific bacteriophage ΦK5B for the controlled synthesis and depolymerization of heparosan. A fluorescently labeled heparosan nonamer was used as a priming acceptor to study the elongation mechanism of the E. coli K5 heparosan polymerases KfiA and KfiC. We could demonstrate that the enzymes act in a distributive manner, producing labeled heparosan of low dispersity. The enzymatically synthesized heparosan was a useful tool to identify the tailspike protein KflB of ΦK5B as heparosan lyase and to characterize its endolytic depolymerization mechanism. Most importantly, using site-directed mutagenesis and rational construct design, we generated an inactive version of KflB for the detection of heparosan in ELISA-based assays, on blots, and on bacterial and mammalian cells.
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Proteínas de Escherichia coli , Escherichia coli , Polimerizacion , Disacáridos , Polímeros/metabolismo , Glicosiltransferasas/metabolismo , Proteínas de Escherichia coli/metabolismo , N-AcetilglucosaminiltransferasasRESUMEN
Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 µm for mammalian class-1 myosins and greater than 90 µm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.
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Dictyostelium/enzimología , Hidrocarburos Clorados/química , Modelos Moleculares , Miosinas/antagonistas & inhibidores , Miosinas/química , Pirroles/química , Regulación Alostérica , Animales , Pollos , Conejos , RatasRESUMEN
Heterozygous dominant mutations in the ubiquitously produced cytoskeletal ß-actin isoform lead to a broad range of human disease phenotypes, which are currently classified as three distinct clinical entities termed Baraitser-Winter-Cerebrofrontofacial syndrome (BWCFF), ACTB-associated pleiotropic malformation syndrome with intellectual disability (ACTB-PMSID), and ACTB-associated syndromic thrombocytopenia (ACTB-AST). The latter two are distinguishable from BWCFF by the presence of milder craniofacial features and less pronounced developmental abnormalities, or the absence of craniofacial features in combination with a characteristic thrombocytopenia with platelet anisotropy. Production and correct function of ß-actin is required for multiple essential processes in all types of cells. Directed cell migration, cytokinesis and morphogenesis are amongst the functions that are supported by ß-actin. Here we report the recombinant production and biochemical characterization of the ACTB-AST mutant p.S368fs, resulting in an altered sequence in the C-terminal region of ß-actin that includes a replacement of the last 8 residues and an elongation of the molecule by 4 residues. The mutation affects a region important for actin polymerization and actin-profilin interaction. Accordingly, we measured markedly reduced rates of nucleation and polymerization during spontaneous actin assembly and lower affinity of p.S368fs for human profilin-1. The reduced affinity is also reflected in the lower propensity of profilin-1 to extend the nucleation phase of p.S368fs. While localized in close proximity to actin-cofilin and actin-myosin interfaces, we determined only minor effects of the mutation on the interaction of mutant filaments with cofilin and myosin family members. However, allosteric effects on sites distant from the mutation manifest themselves in a 7.9 °C reduction in thermal denaturation temperature, a 2-fold increase in the observed IC50 for DNase-I, and changes in nucleotide exchange kinetics. Our results support a disease mechanism involving impaired actin dynamics and function through disruption of actin-profilin interactions and further exacerbated by allosteric perturbations.
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Actinas , Mutación del Sistema de Lectura , Síndrome , Trombocitopenia , Factores Despolimerizantes de la Actina/genética , Actinas/genética , Anomalías Craneofaciales , Epilepsia , Facies , Humanos , Discapacidad Intelectual , Lisencefalia , Mutación , Miosinas/genética , Profilinas/genética , Trombocitopenia/genéticaRESUMEN
The effects of N-terminal acetylation of the high molecular weight tropomyosin isoforms Tpm1.6 and Tpm2.1 and the low molecular weight isoforms Tpm1.12, Tpm3.1, and Tpm4.2 on the actin affinity and the thermal stability of actin-tropomyosin cofilaments are described. Furthermore, we show how the exchange of cytoskeletal tropomyosin isoforms and their N-terminal acetylation affects the kinetic and chemomechanical properties of cytoskeletal actin-tropomyosin-myosin complexes. Our results reveal the extent to which the different actin-tropomyosin-myosin complexes differ in their kinetic and functional properties. The maximum sliding velocity of the actin filament as well as the optimal motor density for continuous unidirectional movement, parameters that were previously considered to be unique and invariant properties of each myosin isoform, are shown to be influenced by the exchange of the tropomyosin isoform and the N-terminal acetylation of tropomyosin.
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Effective tissue repair after myocardial infarction entails a vigorous angiogenic response, guided by incompletely defined immune cell-endothelial cell interactions. We identify the monocyte- and macrophage-derived cytokine METRNL (meteorin-like) as a driver of postinfarction angiogenesis and high-affinity ligand for the stem cell factor receptor KIT (KIT receptor tyrosine kinase). METRNL mediated angiogenic effects in cultured human endothelial cells through KIT-dependent signaling pathways. In a mouse model of myocardial infarction, METRNL promoted infarct repair by selectively expanding the KIT-expressing endothelial cell population in the infarct border zone. Metrnl-deficient mice failed to mount this KIT-dependent angiogenic response and developed severe postinfarction heart failure. Our data establish METRNL as a KIT receptor ligand in the context of ischemic tissue repair.
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Adipoquinas , Citocinas , Infarto del Miocardio , Neovascularización Fisiológica , Factores de Crecimiento Nervioso , Proteínas Proto-Oncogénicas c-kit , Animales , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Células Endoteliales/metabolismo , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/genética , Ligandos , Macrófagos/metabolismo , Ratones , Ratones Mutantes , Infarto del Miocardio/complicaciones , Infarto del Miocardio/fisiopatología , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismoRESUMEN
MYO18B loss-of-function mutations and depletion significantly compromise the structural integrity of striated muscle sarcomeres. The molecular function of the encoded protein, myosin-18B (M18B), within the developing muscle is unknown. Here, we demonstrate that recombinant M18B lacks motor ATPase activity and harbors previously uncharacterized N-terminal actin-binding domains, properties that make M18B an efficient actin cross-linker and molecular brake capable of regulating muscle myosin-2 contractile forces. Spatiotemporal analysis of M18B throughout cardiomyogenesis and myofibrillogenesis reveals that this structural myosin undergoes nuclear-cytoplasmic redistribution during myogenic differentiation, where its incorporation within muscle stress fibers coincides with actin striation onset. Furthermore, this analysis shows that M18B is directly integrated within the muscle myosin thick filament during myofibril maturation. Altogether, our data suggest that M18B has evolved specific biochemical properties that allow it to define and maintain sarcomeric organization from within the thick filament via its dual actin cross-linking and motor modulating capabilities.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Miocitos Cardíacos/metabolismo , Miosinas/metabolismo , Sarcómeros/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Humanos , Dominios Proteicos , Proteínas Recombinantes/metabolismoRESUMEN
Filamentous fungi belonging to the genus Fusarium are notorious plant-pathogens that infect, damage and contaminate a wide variety of important crops. Phenamacril is the first member of a novel class of single-site acting cyanoacrylate fungicides which has proven highly effective against important members of the genus Fusarium. However, the recent emergence of field-resistant strains exhibiting qualitative resistance poses a major obstacle for the continued use of phenamacril. In this study, we synthesized novel cyanoacrylate compounds based on the phenamacril-scaffold to test their growth-inhibitory potential against wild-type Fusarium and phenamacril-resistant strains. Our findings show that most chemical modifications to the phenamacril-scaffold are associated with almost complete loss of fungicidal activity and in vitro inhibition of myosin motor domain ATPase activity.