RESUMEN
Neuromuscular junctions (NMJs) are specialized synapses that connect motor neurons to skeletal muscle fibers and orchestrate proper signal transmission from the nervous system to muscles. The efficient formation and maintenance of the postsynaptic machinery that contains acetylcholine receptors (AChR) are indispensable for proper NMJ function. Abnormalities in the organization of synaptic components often cause severe neuromuscular disorders, such as muscular dystrophy. The dystrophin-associated glycoprotein complex (DGC) was shown to play an important role in NMJ development. We recently identified liprin-α-1 as a novel binding partner for one of the cytoplasmic DGC components, α-dystrobrevin-1. In the present study, we performed a detailed analysis of localization and function of liprin-α-1 at the murine NMJ. We showed that liprin-α-1 localizes to both pre- and postsynaptic compartments at the NMJ, and its synaptic enrichment depends on the presence of the nerve. Using cultured muscle cells, we found that liprin-α-1 plays an important role in AChR clustering and the organization of cortical microtubules. Our studies provide novel insights into the function of liprin-α-1 at vertebrate neuromuscular synapses.
Asunto(s)
Unión Neuromuscular/metabolismo , Proteínas/metabolismo , Transmisión Sináptica , Proteínas Adaptadoras Transductoras de Señales , Animales , Línea Celular , Expresión Génica , Ratones , Microtúbulos/metabolismo , Músculos/metabolismo , Transporte de Proteínas , Proteínas/genética , Receptores Colinérgicos/metabolismoRESUMEN
Deprivation for the single amino acid arginine is a rapidly developing metabolic anticancer therapy, which allows growth control in a number of highly malignant tumors. Here we report that one of the responses of human solid cancer cells to arginine starvation is the induction of prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Systematic study of two colorectal carcinoma HCT-116 and HT29, glioblastoma U251 MG and ovarian carcinoma SKOV3 cell lines revealed, however, that the ER stress triggered by the absence of arginine does not result in massive apoptosis despite a profound upregulation of the proapoptotic gene CHOP. Instead, Akt- and MAPK-dependent pathways were activated which may counteract proapoptotic signaling. Treatment with DMSO as a disaggregating agent or with cycloheximide to block protein synthesis reduced ER stress evoked by arginine deprivation. On the other hand, ER stress and apoptosis induction in arginine-starved cells could be critically augmented by the arginine analog of plant origin canavanine, but not by the classic ER stress inducer tunicamycin. Our data suggest that canavanine treatment applied under the lack of arginine may enhance the efficacy of arginine deprivation-based anticancer therapy.
Asunto(s)
Arginina/deficiencia , Medios de Cultivo/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Canavanina/farmacología , Línea Celular Tumoral , Cicloheximida/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimetilsulfóxido/farmacología , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/genética , Células HCT116 , Células HT29 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
We searched for protein markers present in blood serum of multiple sclerosis (MS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients in comparison to healthy human individuals. We used precipitation/extraction methods and MALDI TOF/TOF mass spectrometry, and identified a protein with Mr ~46 kDa as a fragment of human unconventional myosin IC isoform b (Myo1C). Western blotting with specific anti-human Myo1C antibodies confirmed the identity. Screening of blood serum samples from different autoimmune patients for the presence of Myo1c revealed its high level in MS and RA patients, relatively low level in SLE patients, and undetected in healthy donors. These data are suggesting that the level of p46 Myo1C in blood serum is a potential marker for testing of autoimmune diseases.
RESUMEN
Endocannabinoid signaling has been implicated in modulating insulin release from ß cells of the endocrine pancreas. ß Cells express CB1 cannabinoid receptors (CB1Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB1R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB1Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB1Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.
Asunto(s)
Exocitosis , Quinasa 1 de Adhesión Focal/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptor Cannabinoide CB1/metabolismo , Vesículas Secretoras/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Línea Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Endocannabinoides/genética , Endocannabinoides/metabolismo , Endocannabinoides/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Quinasa 1 de Adhesión Focal/genética , Glicéridos/farmacología , Humanos , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patología , Insulina/genética , Secreción de Insulina , Ratones , Ratones Noqueados , Alcamidas Poliinsaturadas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/genética , Vesículas Secretoras/genéticaRESUMEN
CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.
Asunto(s)
Actinas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Citoesqueleto/metabolismo , Tubulina (Proteína)/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Western Blotting , Adhesión Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Citoesqueleto/ultraestructura , Cinética , Ratones , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Células 3T3 NIH , Polímeros/metabolismo , Unión ProteicaRESUMEN
OBJECTIVE: Mechanism of denervation atrophy remains poorly understood. In particular, the question about irreversibility of the late atrophy is still open. Therefore, in the present study, we investigated whether and how a passive movement can affect a progress of atrophy in rat soleus muscle. To address this issue, a locomotor training on a treadmill was applied to rats with their right hindlimb muscles denervated. METHODS: The hindlimb muscles were denervated by cutting the sciatic nerve. Starting either 7 days or 1 month after the surgery, the animals were trained on a treadmill. Two months after denervation, the soleus muscle was investigated using light and electron microscopy and biochemical methods. Control soleus muscles were obtained from non-trained animals: the untreated and the 2-month denervated. RESULTS: Locomotor training caused slight increase in denervated rat soleus muscle weight and significant increase in its fiber diameter. The training positively affected some of the factors that were believed to be the reasons of atrophy irreversibility, because of significant increase in the number of capillary blood vessels and muscle fiber nuclei with the concomitant decrease in the number of severely damaged muscle fibers and amount of collagen. Morphology of the contractile apparatus was also improved as more regular organization of sarcomeres and the hexagonal arrangement of myosin filaments was evident. Moreover, the amount of myosin heavy chains (MHC) significantly increased after training. The effects were more evident in the animals with longer training. CONCLUSION: Passive movement seems to attenuate some of the pathologic processes within the denervated muscle.
Asunto(s)
Locomoción/fisiología , Desnervación Muscular , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Animales , Recuento de Células , Femenino , Microscopía Electrónica de Transmisión/métodos , Músculo Esquelético/química , Músculo Esquelético/ultraestructura , Miofibrillas/patología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Various lines of evidence suggest that communication between tropomyosin and myosin in the regulation of vertebrate-striated muscle contraction involves yet unknown changes in actin conformation. Possible participation of loop 38-52 in this communication has recently been questioned based on unimpaired Ca(2+) regulation of myosin interaction, in the presence of the tropomyosin-troponin complex, with actin cleaved by subtilisin between Met(47) and Gly(48). We have compared the effects of actin cleavage by subtilisin and by protease ECP32, between Gly(42) and Val(43), on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin. Both individual modifications reduced activation of S1 ATPase by actin to a similar extent. The effect of ECP cleavage, but not of subtilisin cleavage, was partially reversed by stabilization of interprotomer contacts with phalloidin, indicating different pathways of signal transmission from the N- and C-terminal parts of loop 38-52 to myosin binding sites. ECP cleavage diminished the affinity to tropomyosin and reduced its inhibition of acto-S1 ATPase at low S1 concentrations, but increased the tropomyosin-mediated cooperative enhancement of the ATPase by S1 binding to actin. These effects were reversed by phalloidin. Subtilisin-cleaved actin more closely resembled unmodified actin than the ECP-modified actin. Limited proteolysis of the modified and unmodified F-actins revealed an allosteric effect of ECP cleavage on the conformation of the actin subdomain 4 region that is presumably involved in tropomyosin binding. Our results point to a possible role of the N-terminal part of loop 38-52 of actin in communication between tropomyosin and myosin through changes in actin structure.