RESUMO
Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.
Assuntos
Autofagia , Glicoproteínas/metabolismo , Vírus Sin Nombre/fisiologia , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Adenina/análogos & derivados , Adenina/farmacologia , Androstadienos/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia , Linhagem Celular , Chlorocebus aethiops , Cromonas/farmacologia , Células HeLa , Humanos , Morfolinas/farmacologia , Proteólise , Interferência de RNA , RNA Interferente Pequeno , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Células Vero , WortmaninaRESUMO
The extracellular matrix at the neuromuscular junction plays many roles. The matrix plays a structural role in that it maintains the spatial relationship between the muscle cell, Schwann cell, and presynaptic motor neuron. The matrix also plays a role in cell-to-cell signaling. The most studied member of this group is the heparan sulfate proteoglycan, agrin. Agrin is an integral member of the synaptic matrix, and it plays the pivotal role of instructing the muscle cell to aggregate acetylcholine receptors (AChRs) to the synapse. Agrin is released by the motor neuron, where it binds stably to the extracellular matrix. Agrin interacts with the muscle-specific tyrosine kinase (MuSK). Mice that lack agrin, or MuSK, fail to form neuromuscular junctions. Thus, the extracellular matrix is critical to both the structure and function of the neuromuscular junction. Remodeling of the extracellular matrix at the neuromuscular junction is needed to maintain stability, to allow growth, or to destabilize and remove synapses. Matrix metalloproteinases are key regulators of the extracellular matrix. In particular, matrix metalloproteinase 3 (MMP3) has been implicated in regulation of synaptic structure. MMP3 cleaves agrin. Antibodies to MMP3 recognize molecules concentrated at the synapses of frog neuromuscular junctions. Neuromuscular junctions in MMP3 null mutant mice have increased junctional folds, and AChR aggregates. Changes in synaptic activity will alter the activity of MMP3 at the synapse. Thus, the extracellular matrix is critical to the formation of the synapse, and synaptic activity controls the structure and function of the molecules in the extracellular matrix.
Assuntos
Comunicação Celular , Matriz Extracelular/metabolismo , Junção Neuromuscular/metabolismo , Transdução de Sinais , Animais , Humanos , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismoRESUMO
Rapsyn is a synapse-specific protein that is required for clustering acetylcholine receptors at the neuromuscular junction. Analysis of the rapsyn promoter revealed a consensus site for the transcription factor Kaiso within a region that is mutated in a subset of patients with congenital myasthenic syndrome. Kaiso is a POZ-zinc finger family transcription factor which recognizes the specific core consensus sequence CTGCNA (where N is any nucleotide). Previously, the only known binding partner for Kaiso was the cell adhesion cofactor, p120 catenin. Here we show that delta-catenin, a brain-specific member of the p120 catenin subfamily, forms a complex with Kaiso. Antibodies against Kaiso and delta-catenin recognize proteins in the nuclei of C2C12 myocytes and at the postsynaptic domain of the mouse neuromuscular junction. Endogenous Kaiso in C2C12 cells coprecipitates with the rapsyn promoter in vivo as shown by chromatin immunoprecipitation assay. Minimal promoter assays demonstrated that the rapsyn promoter can be activated by Kaiso and delta-catenin; this activation is apparently muscle specific. These results provide the first experimental evidence that rapsyn is a direct sequence-specific target of Kaiso and delta-catenin. We propose a new model of synapse-specific transcription that involves the interaction of Kaiso, delta-catenin, and myogenic transcription factors at the neuromuscular junction.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Proteínas Musculares/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas do Domínio Armadillo , Sequência de Bases , Cateninas , Moléculas de Adesão Celular , Linhagem Celular , Galinhas , Ácidos Graxos Insaturados/farmacologia , Genes Reporter , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/metabolismo , Junção Neuromuscular/fisiologia , Fosfoproteínas , Alinhamento de Sequência , delta CateninaRESUMO
Duchenne muscular dystrophy is a fatal childhood disease caused by mutations that abolish the expression of dystrophin in muscle. Utrophin is a paralogue of dystrophin and can functionally replace it in skeletal muscle. A potential therapeutic approach is to increase utrophin levels in muscle. One way to achieve this aim is to increase the expression of the utrophin gene at a transcriptional level via promoter activation. In this study, we have shown that utrophin A mRNA levels can be induced by okadaic acid in murine myogenic C2C12 cells. We have found that a utrophin A promoter reporter can be induced by Sp1 in C2C12 myoblasts, but not in myotubes. This activation can be enhanced by okadaic acid treatment. Our data suggest that this induction is due to Sp1 phosphorylation during myogenesis and thus, utrophin expression in muscle could be regulated by treatment with phosphatase inhibitors. Control of utrophin promoter activation could then be used to increase the expression of utrophin, and thus ameliorate the symptoms of Duchenne muscular dystrophy.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas de Membrana/genética , Mioblastos Esqueléticos/efeitos dos fármacos , Ácido Okadáico/farmacologia , Regiões Promotoras Genéticas/genética , Regulação para Cima/efeitos dos fármacos , Animais , Sequência de Bases/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Mioblastos Esqueléticos/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/farmacologia , Regulação para Cima/genética , UtrofinaRESUMO
Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, beta2 laminins, and collagen alpha(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (alpha1/2) promote clustering of synaptic vesicles as nerve terminals form. beta2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (alpha3-6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.
Assuntos
Colágeno Tipo IV/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Laminina/metabolismo , Neurônios Motores/citologia , Junção Neuromuscular/embriologia , Junção Neuromuscular/metabolismo , Animais , Autoantígenos/metabolismo , Células Cultivadas , Embrião de Galinha , Técnicas de Cocultura , Colágeno Tipo IV/genética , Humanos , Laminina/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Previously, it was demonstrated that agrin acting through the gaseous, signaling molecule, nitric oxide (NO), induces the formation of AChR aggregates on myotubes in culture. Soluble guanylyl cyclase (sGC), which is present at the neuromuscular junction, is a common target of NO. Therefore, we hypothesized that sGC and cGMP are involved in the agrin signaling cascade. Inhibition of sGC hindered AChR aggregation in both agrin- and NO donor-treated cultured myotubes; whereas, a cGMP analogue was able to induce the formation of AChR aggregates on naïve muscle cells. Due to the presence of cyclic GMP-dependent protein kinase (PKG) at the neuromuscular junction, we tested the ability of a PKG inhibitor to alter the agrin signaling cascade. PKG inhibition did not prevent nascent AChR aggregate formation; however, these aggregates were diffuse and composed of numerous microaggregates consistent with incomplete maturation. Thus, we conclude that cGMP is important for the initiation of AChR aggregation, while PKG is involved in the maturation of AChR aggregates.
Assuntos
Agrina/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Junção Neuromuscular/enzimologia , Agregação de Receptores/fisiologia , Receptores Colinérgicos/metabolismo , Agrina/farmacologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Galinha , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase , Músculo Esquelético/inervação , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/embriologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Agregação de Receptores/efeitos dos fármacos , Receptores Colinérgicos/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/fisiologia , Guanilil Ciclase SolúvelRESUMO
Agrin is a heparan sulfate proteoglycan, which plays an essential role in the development and maintenance of the neuromuscular junction. Agrin is a stable component of the synaptic basal lamina and strong evidence supports the hypothesis that agrin directs the formation of the postsynaptic apparatus, including aggregates of AChRs, and junctional folds. Changes in the distribution of agrin during synaptic remodeling, denervation and reinnervation reveal that agrin can be quickly and efficiently removed from the synaptic basal lamina in a regulated manner. In order to fully understand this mechanism we sought to identify those molecules that were responsible for the removal of agrin. Matrix Metalloproteinases (MMPs) were the most likely molecules since MMPs are involved in the regulation of the pericellular space, including the cleavage of matrix proteins. In particular, MMP3 has been shown to be effective in cleaving heparan sulfate proteoglycans. Antibodies to MMP3 recognize molecules concentrated in the extracellular matrix of perisynaptic Schwann cells. MMP3 specific phylogenic compounds reveal that active MMP3 is localized to the neuromuscular junction. Purified recombinant MMP3 can directly cleave agrin, and it can also remove agrin from synaptic basal lamina. MMP3 activity is itself regulated as activation of MMP3 is lost in denervated muscles. MMP3 null mutant mice have altered neuromuscular junction structure and function, with increased AChRs, junctional folds and agrin immunoreactivity. Altogether these results support the hypothesis that synaptic activity induces the activation of MMP3, and the activated MMP3 removes agrin from the synaptic basal lamina.
Assuntos
Potenciais de Ação/fisiologia , Agrina/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Sinapses/metabolismo , Agrina/genética , Animais , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Humanos , Metaloproteinase 3 da Matriz/genética , Sinapses/genética , Sinapses/ultraestruturaRESUMO
The simian-human immunodeficiency virus (SHIV)/ macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of Vpu in lentivirus pathogenesis. In this report, we have mutated the two phosphorylated serine residues of the HIV-1 Vpu to glycine residues and have reconstructed a SHIV expressing this nonphosphorylated Vpu (SHIV(S52,56G)). Expression studies revealed that this protein was localized to the same intracellular compartment as wild-type Vpu. To determine if this virus was pathogenic, four pig-tailed macaques were inoculated with SHIV(S52,56G) and virus burdens and circulating CD4(+) T cells monitored up to 1 year. Our results indicate that SHIV(S52,56G) caused rapid loss in the circulating CD4(+) T cells within 3 weeks of inoculation in one macaque (CC8X), while the other three macaques developed no or gradual numbers of CD4(+) T cells and a wasting syndrome. Histological examination of tissues revealed that macaque CC8X had lesions in lymphoid tissues (spleen, lymph nodes, and thymus) that were typical for macaques inoculated with pathogenic parental SHIV(KU-1bMC33) and had no lesions within the CNS. To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and/or compensating mutations occurred in other genes associated with CD4 down-regulation, sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy. Sequence analysis revealed a reversion of the glycine residues back to serine residues in this macaque. The other macaques maintained low virus burdens, with one macaque (P003) developing a wasting syndrome between months 9 and 11. Histological examination of tissues from this macaque revealed a thymus with severe atrophy that was similar to that of a previously reported macaque inoculated with a SHIV lacking vpu (Virology 293, 2002, 252). Sequence analysis revealed no reversion of the glycine residues in the vpu sequences isolated from this macaque. These results contrast with those from four macaques inoculated with the parental pathogenic SHIV(KU-1bMC33), all of which developed severe CD4(+) T cell loss within 1 month after inoculation. Taken together, these results indicate that casein kinase II phosphorylation sites of Vpu contributes to the pathogenicity of the SHIV(KU-1bMC33) and suggest that the SHIV(KU-1bMC33)/pig-tailed macaque model will be useful in analyzing amino acids/domains of Vpu that contribute to the pathogenesis of HIV-1.