RESUMO
Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration due to mutations in the dystrophin gene. In spite of great advances in the design of curative treatments, most patients currently receive palliative therapies with steroid molecules such as prednisone or deflazacort thought to act through their immunosuppressive properties. These molecules only slightly slow down the progression of the disease and lead to severe side effects. Fundamental research is still needed to reveal the mechanisms involved in the disease that could be exploited as therapeutic targets. By studying a Caenorhabditis elegans model for DMD, we show here that dystrophin-dependent muscle degeneration is likely to be cell autonomous and affects the muscle cells the most involved in locomotion. We demonstrate that muscle degeneration is dependent on exercise and force production. Exhaustive studies by electron microscopy allowed establishing for the first time the chronology of subcellular events occurring during the entire process of muscle degeneration. This chronology highlighted the crucial role for dystrophin in stabilizing sarcomeric anchoring structures and the sarcolemma. Our results suggest that the disruption of sarcomeric anchoring structures and sarcolemma integrity, observed at the onset of the muscle degeneration process, triggers subcellular consequences that lead to muscle cell death. An ultra-structural analysis of muscle biopsies from DMD patients suggested that the chronology of subcellular events established in C. elegans models the pathogenesis in human. Finally, we found that the loss of sarcolemma integrity was greatly reduced after prednisone treatment suggesting a role for this molecule in plasma membrane stabilization.
Assuntos
Distrofia Muscular de Duchenne/patologia , Sarcolema/ultraestrutura , Sarcômeros/patologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Distrofina/genética , Distrofina/metabolismo , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Mutação , Sarcolema/metabolismo , Sarcolema/patologia , Sarcômeros/metabolismo , Sarcômeros/ultraestruturaRESUMO
Several human small heat shock proteins (sHsps) are phosphorylated oligomeric chaperones that enhance stress resistance. They are characterized by their ability to interact and form polydispersed hetero-oligomeric complexes. We have analyzed the cellular consequences of the stable expression of either wild type HspB5 or its cataracts and myopathies inducing R120G mutant in growing and oxidative stress treated HeLa cells that originally express only HspB1. Here, we describe that wild type and mutant HspB5 induce drastic and opposite effects on cell morphology and oxidative stress resistance. The cellular distribution and phosphorylation of these polypeptides as well as the oligomerization profile of the resulting hetero-oligomeric complexes formed by HspB1 with the two types of exogenous polypeptides revealed the dominant effects induced by HspB5 polypeptides towards HspB1. The R120G mutation enhanced the native size and salt resistance of HspB1-HspB5 complex. However, in oxidative conditions the interaction between HspB1 and mutant HspB5 was drastically modified resulting in the aggregation of both partners. The mutation also induced the redistribution of HspB1 phosphorylated at serine 15, originally observed at the level of the small oligomers that do not interact with wild type HspB5, to the large oligomeric complex formed with mutant HspB5. This phosphorylation stabilized the interaction of HspB1 with mutant HspB5. A dominant negative effect towards HspB1 appears therefore as an important event in the cellular sensitivity to oxidative stress mediated by mutated HspB5 expression. These observations provide novel data that describe how a mutated sHsp can alter the protective activity of another member of this family of chaperones.
Assuntos
Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Mutação , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo , Expressão Gênica , Proteínas de Choque Térmico HSP27/química , Células HeLa , Humanos , Estresse Oxidativo/genética , Fosforilação , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Cadeia B de alfa-Cristalina/químicaRESUMO
In vertebrates, zyxin is a LIM-domain protein belonging to a family composed of seven members. We show that the nematode Caenorhabditis elegans has a unique zyxin-like protein, ZYX-1, which is the orthologue of the vertebrate zyxin subfamily composed of zyxin, migfilin, TRIP6, and LPP. The ZYX-1 protein is expressed in the striated body-wall muscles and localizes at dense bodies/Z-discs and M-lines, as well as in the nucleus. In yeast two-hybrid assays ZYX-1 interacts with several known dense body and M-line proteins, including DEB-1 (vinculin) and ATN-1 (α-actinin). ZYX-1 is mainly localized in the middle region of the dense body/Z-disk, overlapping the apical and basal regions containing, respectively, ATN-1 and DEB-1. The localization and dynamics of ZYX-1 at dense bodies depend on the presence of ATN-1. Fluorescence recovery after photobleaching experiments revealed a high mobility of the ZYX-1 protein within muscle cells, in particular at dense bodies and M-lines, indicating a peripheral and dynamic association of ZYX-1 at these muscle adhesion structures. A portion of the ZYX-1 protein shuttles from the cytoplasm into the nucleus, suggesting a role for ZYX-1 in signal transduction. We provide evidence that the zyx-1 gene encodes two different isoforms, ZYX-1a and ZYX-1b, which exhibit different roles in dystrophin-dependent muscle degeneration occurring in a C. elegans model of Duchenne muscular dystrophy.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/metabolismo , Distrofina/metabolismo , Músculos/metabolismo , Zixina/fisiologia , Actinina/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/química , Expressão Gênica , Dados de Sequência Molecular , Músculos/citologia , Especificidade de Órgãos , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Transporte Proteico , Homologia de Sequência de Aminoácidos , Zixina/químicaRESUMO
Actin and small heat shock proteins (sHsps) are ubiquitous and multifaceted proteins that exist in 2 reversible forms, monomers and multimers, ie, the microfilament of the cytoskeleton and oligomers of the sHsps, generally, supposed to be in a spherical and hollow form. Two situations are described in the literature, where the properties of actin are modulated by sHsps; the actin polymerization is inhibited in vitro by some sHsps acting as capping proteins, and the actin cytoskeleton is protected by some sHsps against the disruption induced by various stressful conditions. We propose that a direct actin-sHsp interaction occurs to inhibit actin polymerization and to participate in the in vivo regulation of actin filament dynamics. Protection of the actin cytoskeleton would result from an F-actin-sHsp interaction in which microfilaments would be coated by small oligomers of phosphorylated sHsps. Both proteins share common structural motives suggesting direct binding sites, but they remain to be demonstrated. Some sHsps would behave with the actin cytoskeleton as actin-binding proteins capable of either capping a microfilament when present as a nonphosphorylated monomer or stabilizing and protecting the microfilament when organized in small, phosphorylated oligomers.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas de Choque Térmico/metabolismo , Animais , Proteínas de Choque Térmico HSP27 , Humanos , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismoAssuntos
Apoptose/fisiologia , Proteínas de Choque Térmico/fisiologia , Animais , Transformação Celular Neoplásica , Grupo dos Citocromos c/metabolismo , Citoesqueleto/metabolismo , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/química , Temperatura Alta , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , Chaperonas Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Freshly deposited eggs ofBombyx mori were microinjected with supercoiled plasmid DNA which carried the ß-galactosidase coding sequence ofEscherichia coli inserted in place of the coding sequence of theB. mori cytoplasmic actin A3 gene. Transient expression of this fusion gene in the embryo was determined by in situ histochemical detection of enzyme activity. After injection of the plasmid at different stages of embryonic development, ß-galactosidase activity depending on the injected DNA was only detected in the vitellophages. This indicates the presence of active transactivators of the actin A3 gene promoter in this cell type. Tissue specificity of the fusion gene expression could be related to the early polyploidization of vitellophages, a process which would favour the stability of the nuclear pool of injected plasmids. The activity of the transgene in vitellophages was detectable at 24-33 h of egg development, the stage presumed for the onset of zygotic gene expression, up to the end of embryogenesis. This gene transfer system is thus promising to analyse thecis regulatory sequences of the actin A3 gene and could be utilized for other ubiquitous genes.