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1.
iScience ; 25(11): 105446, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36388955

RESUMO

Transmembrane protein 16A (TMEM16A) localizes at plasma membrane and controls chloride influx in various type of cells. We here showed an intracellular localization pattern of TMEM16A molecules. In myoblasts, TMEM16A was primarily localized to the cytosolic compartment and partially co-localized with intracellular organelles. The global deletion of TMEM16A led to severe skeletal muscle developmental defect. In vitro observation showed that the proliferation of Tmem16a-/- myoblasts was significantly promoted along with activated ERK1/2 and Cyclin D expression; the myogenic differentiation was impaired accompanied by the enhanced caspase 12/3 activation, implying enhanced endoplasmic reticulum (ER) stress. Interestingly, the bradykinin-induced Ca2+ release from ER calcium store was significantly enhanced after TMEM16A deletion. This suggested a suppressing role of intracellular TMEM16A in ER calcium release whereby regulating the flux of chloride ion across the ER membrane. Our findings reveal a unique location pattern of TMEM16A in undifferentiated myoblasts and its role in myogenesis.

2.
Proc Natl Acad Sci U S A ; 119(26): e2121513119, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35737832

RESUMO

Both chronic obstructive pulmonary disease (COPD) and asthma are severe respiratory diseases. Bitter receptor-mediated bronchodilation is a potential therapy for asthma, but the mechanism underlying the agonistic relaxation of airway smooth muscle (ASM) is not well defined. By exploring the ASM relaxation mechanism of bitter substances, we observed that pretreatment with the bitter substances nearly abolished the methacholine (MCh)-induced increase in the ASM cell (ASMC) calcium concentration, thereby suppressing the calcium-induced contraction release. The ASM relaxation was significantly inhibited by simultaneous deletion of three Gαt proteins, suggesting an interaction between Tas2R and AChR signaling cascades in the relaxation process. Biochemically, the Gαt released by Tas2R activation complexes with AChR and blocks the Gαq cycling of AChR signal transduction. More importantly, a bitter substance, kudinoside A, not only attenuates airway constriction but also significantly inhibits pulmonary inflammation and tissue remodeling in COPD rats, indicating its modulation of additional Gαq-associated pathological processes. Thus, our results suggest that Tas2R activation may be an ideal strategy for halting multiple pathological processes of COPD.


Assuntos
Asma , Músculo Liso , Doença Pulmonar Obstrutiva Crônica , Receptores Acoplados a Proteínas G , Ativação Transcricional , Animais , Asma/genética , Asma/metabolismo , Asma/fisiopatologia , Broncodilatadores/farmacologia , Cálcio/metabolismo , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
3.
J Clin Invest ; 132(14)2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35617029

RESUMO

Intractable functional constipation (IFC) is the most severe form of constipation, but its etiology has long been unknown. We hypothesized that IFC is caused by refractory infection by a pathogenic bacterium. Here, we isolated from patients with IFC a Shigella species - peristaltic contraction-inhibiting bacterium (PIB) - that significantly inhibited peristaltic contraction of the colon by production of docosapentenoic acid (DPA). PIB colonized mice for at least 6 months. Oral administration of PIB was sufficient to induce constipation, which was reversed by PIB-specific phages. A mutated PIB with reduced DPA was incapable of inhibiting colonic function and inducing constipation, suggesting that DPA produced by PIB was the key mediator of the genesis of constipation. PIBs were detected in stools of 56% (38 of 68) of the IFC patients, but not in those of non-IFC or healthy individuals (0 of 180). DPA levels in stools were elevated in 44.12% (30 of 68) of the IFC patients but none of the healthy volunteers (0 of 97). Our results suggest that Shigella sp. PIB may be the critical causative pathogen for IFC, and detection of fecal PIB plus DPA may be a reliable method for IFC diagnosis and classification.


Assuntos
Motilidade Gastrointestinal , Shigella , Animais , Colo , Constipação Intestinal/diagnóstico , Constipação Intestinal/genética , Fezes , Humanos , Camundongos , Shigella/genética
4.
J Biol Chem ; 298(1): 101516, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34942145

RESUMO

The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2'-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7highMyoDlowEdUpos) and activated satellite cells (Pax7highMyoDhighEdUpos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.


Assuntos
Músculo Esquelético , Regeneração , Células Satélites de Músculo Esquelético , Timo , Animais , Diferenciação Celular , Proliferação de Células , Camundongos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Timo/metabolismo , Cicatrização
5.
J Biol Chem ; 295(26): 8656-8667, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32354746

RESUMO

Mutations in the myotubularin 1 (MTM1) gene can cause the fatal disease X-linked centronuclear myopathy (XLCNM), but the underlying mechanism is incompletely understood. In this report, using an Mtm1-/y disease model, we found that expression of the intragenic microRNA miR-199a-1 is up-regulated along with that of its host gene, dynamin 2 (Dnm2), in XLCNM skeletal muscle. To assess the role of miR-199a-1 in XLCNM, we crossed miR-199a-1-/- with Mtm1-/y mice and found that the resultant miR-199a-1-Mtm1 double-knockout mice display markers of improved health, as evidenced by lifespans prolonged by 30% and improved muscle strength and histology. Mechanistic analyses showed that miR-199a-1 directly targets nonmuscle myosin IIA (NM IIA) expression and, hence, inhibits muscle postnatal development as well as muscle maturation. Further analysis revealed that increased expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) up-regulates Dnm2/miR-199a-1 expression in XLCNM muscle. Our results suggest that miR-199a-1 has a critical role in XLCNM pathology and imply that this microRNA could be targeted in therapies to manage XLCNM.


Assuntos
Dinamina II/genética , MicroRNAs/genética , Miopatias Congênitas Estruturais/genética , Animais , Sistemas CRISPR-Cas , Dinamina II/análise , Feminino , Longevidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/análise , Força Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miopatias Congênitas Estruturais/patologia
6.
J Allergy Clin Immunol ; 141(4): 1259-1268.e11, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28754608

RESUMO

BACKGROUND: Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness. OBJECTIVE: We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness. METHODS: We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model. RESULTS: Inflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D4, are capable of inducing Ca2+-activated Cl- currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity. CONCLUSIONS: A G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli.


Assuntos
Anoctamina-1/metabolismo , Asma/metabolismo , Hiper-Reatividade Brônquica/metabolismo , Canais de Cálcio/metabolismo , Contração Muscular , Músculo Liso/fisiopatologia , Transdução de Sinais , Animais , Asma/fisiopatologia , Biomarcadores/metabolismo , Hiper-Reatividade Brônquica/fisiopatologia , Fenômenos Eletrofisiológicos , Feminino , Cobaias , Masculino , Camundongos , Camundongos Knockout , Fenótipo
7.
Proc Natl Acad Sci U S A ; 112(44): 13627-32, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26487685

RESUMO

Inheritance of the callipyge phenotype in sheep is an example of polar overdominance inheritance, an unusual mode of inheritance. To investigate the underlying molecular mechanism, we profiled the expression of the genes located in the Delta-like 1 homolog (Dlk1)-type III iodothyronine deiodinase (Dio3) imprinting region in mice. We found that the transcripts of the microRNA (miR) 379/miR-544 cluster were highly expressed in neonatal muscle and paralleled the expression of the Dlk1. We then determined the in vivo role of the miR-379/miR-544 cluster by establishing a mouse line in which the cluster was ablated. The maternal heterozygotes of young mutant mice displayed a hypertrophic tibialis anterior muscle, extensor digitorum longus muscle, gastrocnemius muscle, and gluteus maximus muscle and elevated expression of the DLK1 protein. Reduced expression of DLK1 was mediated by miR-329, a member of this cluster. Our results suggest that maternal expression of the imprinted miR-379/miR-544 cluster regulates paternal expression of the Dlk1 gene in mice. We therefore propose a miR-based molecular working model for polar overdominance inheritance.


Assuntos
Impressão Genômica , Peptídeos e Proteínas de Sinalização Intercelular/genética , MicroRNAs/genética , Animais , Proteínas de Ligação ao Cálcio , Feminino , Camundongos , Família Multigênica
8.
J Biol Chem ; 289(41): 28478-88, 2014 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-25122766

RESUMO

Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Jejuno/metabolismo , Miócitos de Músculo Liso/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/genética , Adenoviridae/genética , Motivos de Aminoácidos , Animais , Membrana Celular/química , Movimento Celular , Regulação da Expressão Gênica , Vetores Genéticos , Jejuno/citologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Miócitos de Músculo Liso/citologia , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/genética , Fosforilação , Cultura Primária de Células , Ligação Proteica , Transdução de Sinais , Tensão Superficial , Transfecção
9.
FEBS J ; 279(8): 1485-94, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22353598

RESUMO

There is a current view that myosin light chain kinase (MLCK) plays a critical role in endothelial permeability. To investigate the functions of MLCK in endothelial cells in vivo, we generated a mouse model in which MLCK was selectively deleted by crossing Mylk1 floxed mice with Tie2/cre transgenic mice. Knocking out Mylk1 from endothelial cells had no effect on the global phenotype of the mice, including body weight and blood pressure. Lipopolysaccharide (LPS)-mediated septic death was also not altered in the knockout (KO) mice. Consistently, LPS-induced inflammatory injury and the increase in microvascular permeability in the main organs, including the lung and the kidney, was not significantly attenuated in KO mice as compared with wild-type mice. However, the LPS-induced microvascular hyperpermeability of the esophagus and the eyeballs was attenuated in KO mice. We also found that the LPS-mediated increase in the number of caveolae in the endothelial cells of the esophagus was significantly reduced in KO mice. Our results do not support a role for endothelial cell MLCK in the pathogenesis of inflammatory diseases.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Inflamação/enzimologia , Lipopolissacarídeos/farmacologia , Quinase de Cadeia Leve de Miosina/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Esôfago/citologia , Esôfago/metabolismo , Olho/citologia , Olho/metabolismo , Feminino , Inflamação/etiologia , Inflamação/mortalidade , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor TIE-2 , Taxa de Sobrevida
10.
Am J Pathol ; 179(6): 2740-50, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21983072

RESUMO

Cigarette smoke activates the extracellular signal-regulated kinase (ERK) 1/2 mitogen activated-protein kinase pathway, which, in turn, is responsible for early growth response gene-1 (EGR-1) activation. Here we provide evidence that EGR-1 activation can also reactivate ERK 1/2 mitogen activated-protein kinase through a positive feedback loop through its target gene (geranylgeranyl diphosphate synthase) GGPPS. For the first time, the GGPPS gene is identified as a target of EGR-1, as EGR-1 can directly bind to the predicted consensus-binding site in the GGPPS promoter and regulate its transcription. Long-term observations show that there are two ERK 1/2 phosphorylation peaks after cigarette smoke extract stimulation in human lung epithelial Beas-2B cells. The first peak (at 10 minutes) is responsible for EGR-1 accumulation, and the second (at 4 hours) is diminished after the disruption of EGR-1 transcriptional activity. EGR-1 overexpression enhances Ras prenylation and membrane association in a GGPPS-dependent manner, and it augments ERK 1/2 activation. Likewise, a great reduction of the second peak of ERK 1/2 phosphorylation is observed during long-term cigarette smoke extract stimulation in cells where GGPPS is disrupted. Thus, we have uncovered an intricate positive feedback loop in which ERK 1/2-activated EGR-1 promotes ERK 1/2 reactivation through promoting GGPPS transcription, which might affect cigarette smoke-related lung pathological processes.


Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Farnesiltranstransferase/genética , Sistema de Sinalização das MAP Quinases/genética , Prenilação/genética , Fumar/genética , Proteínas ras/metabolismo , Animais , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Retroalimentação Fisiológica , Células HEK293 , Humanos , Camundongos , Camundongos Mutantes , Pneumonia/etiologia , RNA Interferente Pequeno/farmacologia , Fumaça , Fumar/efeitos adversos , Transcrição Gênica
11.
Am J Pathol ; 178(1): 110-8, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21224049

RESUMO

Early growth response 1 (EGR-1) contributes to the development of chronic obstructive pulmonary disease in the lungs of smokers by mediating pulmonary inflammatory responses, but the direct downstream genes of EGR-1 that regulate this process remain unknown. We show that a new EGR-1 target gene, geranylgeranyl diphosphate synthase (GGPPS), which controls protein prenylation, can regulate the proinflammatory function of EGR-1 by activating MAPK signaling. When C57BL/6 mice were exposed to cigarette smoke, EGR-1 and GGPPS levels increased in their lungs, and the inflammatory responses were augmented, whereas these effects could be reversed by the down-regulation of EGR-1 transcription activity. The accumulation of EGR-1 and GGPPS was induced by MAPK/ERK pathway activation when Beas-2B human bronchial epithelial cells were exposed to cigarette smoke extract (CSE). Further examination showed that EGR-1 in turn regulated Erk1/2 activity because inhibition of EGR-1 transcription activity decreased CSE-induced Erk1/2 phosphorylation. Furthermore, EGR-1-promoted Erk1/2 activation was dependent on GGPPS transcription. Knockdown of GGPPS expression with small-interfering RNA abolished the EGR-1-activated Erk1/2 activity. Both EGR-1 transcription inhibition and GGPPS expression knockdown decreased the inflammatory response induced by CSE in Beas-2B cells. Our results reveal a new EGR-1/GGPPS/MAPK signaling pathway that controls cigarette smoke-induced pulmonary inflammation, and this may shed light on our understanding of the mechanism of cigarette smoke-related pulmonary diseases such as chronic obstructive pulmonary disease.


Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Farnesiltranstransferase/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nicotiana/efeitos adversos , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/efeitos adversos , Animais , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Doença Pulmonar Obstrutiva Crônica/patologia , Transdução de Sinais
12.
Gastroenterology ; 135(2): 610-20, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18586037

RESUMO

BACKGROUND & AIMS: Smooth muscle is essential for maintaining homeostasis for many body functions and provides adaptive responses to stresses imposed by pathologic disorders. Identified cell signaling networks have defined many potential mechanisms for initiating smooth muscle contraction with or without myosin regulatory light chain (RLC) phosphorylation by myosin light chain kinase (MLCK). We generated tamoxifen-inducible and smooth muscle-specific MLCK knockout (KO) mice and provide direct loss-of-function evidence that shows the primary importance of MLCK in phasic smooth muscle contractions. METHODS: We used the Cre-loxP system to establish Mlck floxed mice in which exons 23, 24, and 25 were flanked by 2 loxP sites. Smooth muscle-specific MLCK KO mice were generated by crossing Mlck floxed mice with SM-CreER(T2) (ki) mice followed by tamoxifen treatment. The phenotype was assessed by histologic, biochemical, molecular, cell biological, and physiologic analyses. RESULTS: Targeted deletion of MLCK in adult mouse smooth muscle resulted in severe gut dysmotility characterized by weak peristalsis, dilation of the digestive tract, and reduction of feces excretion and food intake. There was also abnormal urinary bladder function and lower blood pressure. Isolated muscles showed a loss of RLC phosphorylation and force development induced by K(+)-depolarization. The kinase knockout also markedly reduced RLC phosphorylation and force development with acetylcholine which activates Ca(2+)-sensitizing signaling pathways. CONCLUSIONS: MLCK and its phosphorylation of RLC are required physiologically for smooth muscle contraction and are essential for normal gastrointestinal motility.


Assuntos
Motilidade Gastrointestinal , Intestinos/enzimologia , Contração Muscular , Músculo Liso/enzimologia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Animais , Pressão Sanguínea , Cálcio/metabolismo , Defecação , Ingestão de Alimentos , Feminino , Genótipo , Intestinos/patologia , Intestinos/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Força Muscular , Músculo Liso/patologia , Músculo Liso/fisiopatologia , Quinase de Cadeia Leve de Miosina/genética , Peristaltismo , Fenótipo , Fosforilação , Potássio/metabolismo , Fatores de Tempo , Transdução Genética , Bexiga Urinária/fisiopatologia
13.
Mol Cells ; 25(4): 531-7, 2008 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-18460899

RESUMO

Abnormal activation of nuclear factor kappa B (NF-kappaB) probably plays an important role in the pathogenesis of Duchenne's muscular dystrophy (DMD). In this report, we evaluated the efficacy of curcumin, a potent NF-kappaB inhibitor, in mdx mice, a mouse model of DMD. We found that it improved sarcolemmic integrity and enhanced muscle strength after intraperitoneal (i.p.) injection. Histological analysis revealed that the structural defects of myofibrils were reduced, and biochemical analysis showed that creatine kinase (CK) activity was decreased. We also found that levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and inducible nitric oxide synthase (iNOS) in the mdx mice were decreased by curcumin administration. EMSA analysis showed that NF-kappaB activity was also inhibited. We thus conclude that curcumin is effective in the therapy of muscular dystrophy in mdx mice, and that the mechanism may involve inhibition of NF-kappaB activity. Since curcumin is a non-toxic compound derived from plants, we propose that it may be useful for DMD therapy.


Assuntos
Força Muscular/efeitos dos fármacos , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Fitoterapia , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Creatina Quinase/antagonistas & inibidores , Creatina Quinase/sangue , Curcumina/administração & dosagem , Humanos , Injeções Intraperitoneais , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Força Muscular/fisiologia , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/sangue
14.
FEBS J ; 275(10): 2489-500, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18400030

RESUMO

The functions of long smooth muscle myosin light chain kinase (L-MLCK), a molecule with multiple domains, are poorly understood. To examine the existence of further potentially functional domains in this molecule, we analyzed its amino acid sequence with a tango program and found a putative aggregation domain located at the 4Ig domain of the N-terminal extension. To verify its aggregation capability in vitro, expressible truncated L-MLCK variants driven by a cytomegalovirus promoter were transfected into cells. As anticipated, only the overexpression of the 4Ig fragment led to particle formation in Colon26 cells. These particles contained 4Ig polymers and actin. Analysis with detergents demonstrated that the particles shared features in common with aggregates. Thus, we conclude that the 4Ig domain has a potent aggregation ability. To further examine this aggregation domain in vivo, eight transgenic mouse lines expressing the 4Ig domain (4Ig lines) were generated. The results showed that the transgenic mice had typical aggregation in the thigh and diaphragm muscles. Histological examination showed that 7.70 +/- 1.86% of extensor digitorum longus myofibrils displayed aggregates with a 36.44% reduction in myofibril diameter, whereas 65.13 +/- 3.42% of diaphragm myofibrils displayed aggregates and the myofibril diameter was reduced by 43.08%. Electron microscopy examination suggested that the aggregates were deposited at the mitochondria, resulting in structural impairment. As a consequence, the oxygen consumption of mitochondria in the affected muscles was also reduced. Macrophenotypic analysis showed the presence of muscular degeneration characterized by a reduction in force development, faster fatigue, decreased myofibril diameters, and structural alterations. In summary, our study revealed the existence of a novel aggregation domain in L-MLCK and provided a direct link between L-MLCK and aggregation. The possible significance and mechanism underlying the aggregation-based pathological processes mediated by L-MLCK are also discussed.


Assuntos
Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , Diafragma/citologia , Diafragma/metabolismo , Diafragma/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Contração Muscular , Fadiga Muscular , Subfragmentos de Miosina/química , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de Proteína
15.
Cell Res ; 16(4): 367-76, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16617332

RESUMO

Myosin light chain kinases (MLCK) phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase (S-MLCK) to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase (L-MLCK) has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules (2Ig) in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased "spike"-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Actinas/metabolismo , Motivos de Aminoácidos , Animais , Células COS , Extensões da Superfície Celular/ultraestrutura , Galinhas , Chlorocebus aethiops , Isoenzimas/metabolismo , Camundongos , Proteínas dos Microfilamentos/ultraestrutura , Microscopia Confocal , Microscopia de Fluorescência , Músculo Esquelético , Células NIH 3T3/imunologia , Células NIH 3T3/ultraestrutura , Ligação Proteica , Pseudópodes/ultraestrutura , Proteínas Recombinantes/genética
16.
Immunol Lett ; 92(3): 237-43, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15081618

RESUMO

Induction of effective cytotoxic T lymphocyte (CTL) and/or a specific antibody against conserved viral proteins may be essential to the development of a safe and effective severe acute respiratory syndrome coronavirus (SARS-Cov) vaccine. DNA vaccination represents a new strategy for induction of humoral and cellular immune response. To determine the ability of SARS-Cov nucleoprotein (N protein) to induce antiviral immunity, in this report, we established a stable C2C12 line expressing SARS-Cov N protein, which was used as a target for specific CTL assay. We also expressed recombinant N proteins in Escherichia coli and prepared N protein-specific polyclonal antibodies. C3H/He mice were immunized with N protein-expressible pcDN-fn vector by intramuscular injections. We found that the DNA vaccination induced both N protein-specific antibody and specific CTL activity to the target. When C3H/He mice were immunized by three separate injections, high antibody titre (1:3200-1:6400, average titre is 1:4580) and high CTL activity (67.4+/-8.4% (E:T = 25:1), 69.6+/-6.7% (E:T = 50:1) and 71.8+/-6.2% (E:T = 100:1)) were observed. In the case of two vaccine injections, CTL activity was also high (56.6+/-12.7% (E:T = 25:1), 57.4+/-11.7% (E:T = 50:1) and 63.0+/-6.3% (E:T = 100:1)) However, antibody titres were much lower (1:200-1:3200, average titre is 1:980). Our results suggest that SARS-Cov nucleocapsid gene might be a candidate gene for SARS DNA vaccination.


Assuntos
Nucleoproteínas/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Vacinas de DNA/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos/imunologia , Feminino , Camundongos , Coelhos , Síndrome Respiratória Aguda Grave/imunologia , Linfócitos T Citotóxicos/imunologia
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