RESUMEN
We report on the basis of DFT computations a plausible and detailed reaction mechanism for the first Pd-catalyzed decarbonylative phosphorylation of amides forming C-P bonds, which reveals, among other things, crucial events in the second coordination sphere, including ion pair and hydrogen bonding interactions as well as proton transfer.
RESUMEN
Tropomyosin (Tpm) binds along actin filaments and regulates myosin binding to control muscle contraction. Tropomodulin binds to the pointed end of a filament and regulates actin dynamics, which maintains the length of a thin filament. To define the structural determinants of these Tpm functions, we examined the effects of two congenital myopathy mutations, A4V and R91C, in the Tpm gene, TPM3, which encodes the Tpm3.12 isoform, specific for slow-twitch muscle fibers. Mutation A4V is located in the tropomodulin-binding, N-terminal region of Tpm3.12. R91C is located in the actin-binding period 3 and directly interacts with actin. The A4V and R91C mutations resulted in a 2.5-fold reduced affinity of Tpm3.12 homodimers for F-actin in the absence and presence of troponin, and a two-fold decrease in actomyosin ATPase activation in the presence of Ca2+ . Actomyosin ATPase inhibition in the absence of Ca2+ was not affected. The Ca2+ sensitivity of ATPase activity was decreased by R91C, but not by A4V. In vitro, R91C altered the ability of tropomodulin 1 (Tmod1) to inhibit actin polymerization at the pointed end of the filaments, which correlated with the reduced affinity of Tpm3.12-R91C for Tmod1. Molecular dynamics simulations of Tpm3.12 in complex with F-actin suggested that both mutations reduce the affinity of Tpm3.12 for F-actin binding by perturbing the van der Waals energy, which may be attributable to two different molecular mechanisms-a reduced flexibility of Tpm3.12-R91C and an increased flexibility of Tpm3.12-A4V.
Asunto(s)
Actinas/metabolismo , Enfermedades Musculares/congénito , Mutación Puntual , Tropomodulina/metabolismo , Tropomiosina/genética , Citoesqueleto de Actina/metabolismo , Adenosina Trifosfatasas/metabolismo , Calcio/metabolismo , Humanos , Simulación de Dinámica Molecular , Enfermedades Musculares/genética , Polimerizacion , Tropomiosina/metabolismoRESUMEN
Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin's thin-filament pointed-end assembly but does not affect tropomodulin's assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin's affinity for F-actin and affects leiomodin's function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin's function.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Cardiomiopatías/genética , Mutación/genética , Tropomiosina/genética , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Calcio/metabolismo , Humanos , Hidrólisis , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patologíaRESUMEN
There are many unanswered questions about the roles of the actin pointed end capping and actin nucleation by tropomodulins (Tmod) in regulating neural morphology. Previous studies indicate that Tmod1 and Tmod2 regulate morphology of the dendritic arbor and spines. Tmod3, which is expressed in the brain, had only a minor influence on morphology. Although these studies established a defined role of Tmod in regulating dendritic and synaptic morphology, the mechanisms by which Tmods exert these effects are unknown. Here, we overexpressed a series of mutated forms of Tmod1 and Tmod2 with disrupted actin-binding sites in hippocampal neurons and found that Tmod1 and Tmod2 require both of their actin-binding sites to regulate dendritic morphology and dendritic spine shape. Proximity ligation assays (PLAs) indicate that these mutations impact the interaction of Tmod1 and Tmod2 with tropomyosins Tpm3.1 and Tpm3.2. This impact on Tmod/Tpm interaction may contribute to the morphological changes observed. Finally, we use molecular dynamics simulations (MDS) to characterize the structural changes, caused by mutations in the C-terminal helix of the leucine-rich repeat (LRR) domain of Tmod1 and Tmod2 alone and when bound onto actin monomers. Our results expand our understanding of how neurons utilize the different Tmod isoforms in development.
RESUMEN
Piperine, an alkaloid from black pepper, was found to inhibit the super-relaxed state (SRX) of myosin in fast-twitch skeletal muscle fibers. In this work we report that the piperine molecule binds heavy meromyosin (HMM), whereas it does not interact with the regulatory light chain (RLC)-free subfragment-1 (S1) or with control proteins from the same muscle molecular machinery, G-actin and tropomyosin. To further narrow down the location of piperine binding, we studied interactions between piperine and a fragment of skeletal myosin consisting of the full-length RLC and a fragment of the heavy chain (HCF). The sequence of HCF was designed to bind RLC and to dimerize via formation of a stable coiled coil, thus producing a well-folded isolated fragment of the myosin neck. Both chains were co-expressed in Escherichia coli, the RLC/HCF complex was purified and tested for stability, composition and binding to piperine. RLC and HCF chains formed a stable heterotetrameric complex (RLC/HCF)2 which was found to bind piperine. The piperine molecule was also found to bind isolated RLC. Piperine binding to RLC in (RLC/HCF)2 altered the compactness of the complex, suggesting that the mechanism of SRX inhibition by piperine is based on changing conformation of the myosin.
Asunto(s)
Alcaloides/metabolismo , Alcaloides/farmacología , Benzodioxoles/metabolismo , Benzodioxoles/farmacología , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/metabolismo , Piperidinas/metabolismo , Piperidinas/farmacología , Alcamidas Poliinsaturadas/metabolismo , Alcamidas Poliinsaturadas/farmacología , Secuencia de Aminoácidos , Animales , Ratones , Modelos Moleculares , Mutación , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Cadenas Ligeras de Miosina/química , Unión Proteica , Conformación Proteica , Estabilidad Proteica/efectos de los fármacosRESUMEN
This work illustrates the reductive coupling of electron-rich aryl halides with tertiary alkyl halides under Ni-catalyzed cross-electrophile coupling conditions, which offers an efficient protocol for the construction of all carbon quaternary stereogenic centers. The mild and easy-to-operate reaction tolerates a wide range of functional groups. The utility of this method is manifested by the preparation of cyclotryptamine derivatives, wherein successful incorporation of 7-indolyl moieties is of particular interest as numerous naturally occurring products are composed of these key scaffolds. DFT calculations have been carried out to investigate the proposed radical chain and double oxidative addition pathways, which provide useful mechanistic insights into the part of the reaction that takes place in solution.
RESUMEN
The missense mutation R21H in striated muscle tropomyosin is associated with hypertrophic cardiomyopathy, a genetic cardiac disease and a leading cause of sudden cardiac death in young people. Tropomyosin adopts conformation of a coiled coil which is critical for regulation of muscle contraction. In this study, we investigated the effects of the R21H mutation on the coiled-coil structure of tropomyosin and its interactions with its binding partners, tropomodulin and leiomodin. Using circular dichroism and isothermal titration calorimetry, we found that the mutation profoundly destabilized the structural integrity of αTM1a1-28 Zip, a chimeric peptide containing the first 28 residues of tropomyosin. The mutated αTM1a1-28 Zip was still able to interact with tropomodulin and leiomodin. However, the mutation resulted in a â¼30-fold decrease of αTM1a1-28 Zip's binding affinity to leiomodin. We used a crystal structure of αTM1a1-28 Zip that we solved at 1.5 Å resolution to study the mutation's effect in silico by means of molecular dynamics simulation. The simulation data indicated that while the mutation disrupted αTM1a1-28 Zip's coiled-coil structure, most notably from residue Ala18 to residue His31, it may not affect the N-terminal end of tropomyosin. The drastic decrease of αTM1a1-28 Zip's affinity to leiomodin caused by the mutation may lead to changes in the dynamics at the pointed end of thin filaments. Therefore, the R21H mutation is likely interfering with the regulation of the normal thin filament length essential for proper muscle contraction.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Mutación Missense , Tropomiosina/química , Tropomiosina/genética , Sitios de Unión , Dicroismo Circular , Cristalografía por Rayos X , Humanos , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Proteínas Musculares/metabolismo , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Tropomodulina/metabolismo , Tropomiosina/metabolismoRESUMEN
Correct assembly of thin filaments composed of actin and actin-binding proteins is of crucial importance for properly functioning muscle cells. Tropomyosin (Tpm) mediates the binding of tropomodulin (Tmod) and leiomodin (Lmod) at the slow-growing, or pointed, ends of the thin filaments. Together these proteins regulate thin filament lengths and actin dynamics in cardiac muscle. The K15N mutation in the TPM1 gene is associated with familial dilated cardiomyopathy (DCM) but the effect of this mutation on Tpm's function is unknown. In this study, we introduced the K15N mutation in striated muscle α-Tpm (Tpm1.1) and investigated its interaction with actin, Tmod and Lmod. The mutation caused a â¼3-fold decrease in the affinity of Tpm1.1 for actin. The binding of Lmod and Tmod to Tpm1.1-covered actin filaments also decreased in the presence of the K15N mutation. Furthermore, the K15N mutation in Tpm1.1 disrupted the inhibition of actin polymerization and affected the competition between Tmod1 and Lmod2 for binding at the pointed ends. Our data demonstrate that the K15N mutation alters pointed end dynamics by affecting molecular interactions between Tpm1.1, Lmod2 and Tmod1.
Asunto(s)
Cardiomiopatía Dilatada/genética , Mutación Missense , Tropomiosina/química , Tropomiosina/genética , Sustitución de Aminoácidos , Cardiomiopatía Dilatada/metabolismo , Tropomodulina/química , Tropomodulina/genética , Tropomodulina/metabolismo , Tropomiosina/metabolismoRESUMEN
The epiglottis plays an important role in preventing food of different consistencies from entering the airway during swallowing. Calcification of epiglottis can, potentially, alter and limit its movement causing aspiration amongst other swallowing problems. Isolated calcification of the epiglottis and its clinical presentation remains a poorly understood entity for radiologists as well as clinicians. Therefore, it is important to recognize the imaging features of epiglottic calcification, and it's known clinical presentations to help clinicians with early diagnosis and management.
Asunto(s)
Calcinosis/diagnóstico por imagen , Epiglotis/diagnóstico por imagen , Enfermedades de la Laringe/diagnóstico por imagen , Epiglotis/patología , Cuerpos Extraños/diagnóstico por imagen , Humanos , Enfermedades de la Laringe/etiología , Enfermedades de la Laringe/patología , Masculino , Persona de Mediana Edad , SensaciónRESUMEN
Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.