RESUMO
The tight skin (Tsk/+) mouse is a model for fibrotic disorders. The genetic defect in the Tsk/+ is an in-frame duplication between exons 17 and 40 of the fibrillin-1 gene which gives rise to a large transcript and protein. Mice homozygous for the mutation die in utero, whereas heterozygotes survive and spontaneously develop connective tissue disease. In this study, we generated hammerhead ribozymes directed against the mutant fibrillin-1 transcript. A partially mispairing ribozyme was the most effective vehicle to cleave the mutant transcript without undesired cleavage of wild type transcripts, as shown by cell-free RNA cleavage and cleavage in cell lines harboring the ribozyme, by RT-PCR, Northern and Western Blotting. Global gene expression profiling using oligonucleotide microarrays showed the expected reduction in fibrillin-1 mRNA, and down-regulation of several gene cohorts in ribozyme harboring TskR1 cells compared to Tsk/+ cells. Two of the functional clusters included genes regulating extracellular matrix such as connective tissue growth factor, serpine-1 (plasminogen activator inhibitor-1) and TIMP-1 and TIMP-3, and those involved in cytoskeletal organization and myofibroblast formation including calponins and transgelin. Ribozyme-mediated inhibition was confirmed by Western Blot and functional analysis using cell-reporter systems and remodeling of three dimensional collagen gels. Our results underline the therapeutic potential of hammerhead ribozymes in dominant negative defects and suggest that changes in microfibril architecture brought about by fibrillin-1 mutation lead to a complex disease phenotype.
Assuntos
Inativação Gênica , Proteínas dos Microfilamentos/genética , Mutação/genética , RNA Catalítico/metabolismo , Animais , Western Blotting , Células COS , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/genética , Chlorocebus aethiops , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo , Proteínas do Citoesqueleto/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibrilina-1 , Fibrilinas , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Perfilação da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Mutantes , Proteínas dos Microfilamentos/fisiologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Catalítico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1RESUMO
Co-translational modification of eukaryotic proteins by N-myristoylation aids subcellular targeting and protein-protein interactions. The enzyme that catalyzes this process, N-myristoyltransferase (NMT), has been characterized in the kinetoplastid protozoan parasites, Leishmania and Trypanosoma brucei. In Leishmania major, the single copy NMT gene is constitutively expressed in all parasite stages as a 48.5-kDa protein that localizes to both membrane and cytoplasmic fractions. Leishmania NMT myristoylates the target acylated Leishmania protein, HASPA, when both are co-expressed in Escherichia coli. Gene targeting experiments have shown that NMT activity is essential for viability in Leishmania. In addition, overexpression of NMT causes gross changes in parasite morphology, including the subcellular accumulation of lipids, leading to cell death. This phenotype is more extreme than that observed in Saccharomyces cerevisiae, in which overexpression of NMT activity has no obvious effects on growth kinetics or cell morphology. RNA interference assays in T. brucei have confirmed that NMT is also an essential protein in both life cycle stages of this second kinetoplastid species, suggesting that this enzyme may be an appropriate target for the development of anti-parasitic agents.