RESUMO
Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.
Assuntos
DNA Antissenso/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Aminas/química , Animais , Cátions/química , DNA Antissenso/química , DNA Antissenso/genética , DNA Antissenso/farmacocinética , Dissulfetos/química , Inativação Gênica , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos ICR , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , Transfecção/métodosRESUMO
Development of intracellular delivery methods for antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor-ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10â minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide-modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of antisense DNA and siRNA.
Assuntos
Citosol/metabolismo , DNA Antissenso/metabolismo , Dissulfetos/metabolismo , RNA Interferente Pequeno/metabolismo , Transporte Biológico , HumanosRESUMO
BACKGROUND: The Huntingtin-interacting protein HYPK possesses chaperone-like activity. We hypothesized that the expression of HYPK could be regulated by heat shock factor HSF1, a transcriptional regulator of chaperone genes. METHODS: HYPK expression in HeLa cells was assessed by RT-PCR and Western blot analysis. In vivo binding of HSF1 to the HYPK promoter was analyzed by chromatin immunoprecipitation assays. The requirement for HYPK in heat-shocked cells was examined using HYPK-knockdown cells. RESULTS: Levels of HYPK mRNA were slightly increased by heat treatment; however, the levels decreased in HSF1-silenced cells. The HYPK promoter was bound by HSF1 in a heat-inducible manner; however, its core promoter activity was notably suppressed upon heat shock. When cells were exposed to heat shock, silencing HYPK caused a decrease in cell viability. CONCLUSIONS: HYPK is a novel target gene of HSF1. HSF1 maintains HYPK expression in heat-shocked cells. GENERAL SIGNIFICANCE: The maintenance of HYPK expression by HSF1 is necessary for the survival of cells under thermal stress conditions.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Regulação da Expressão Gênica , Células HeLa , Fatores de Transcrição de Choque Térmico , Temperatura Alta , Humanos , Regiões Promotoras Genéticas , Estresse FisiológicoRESUMO
The transcription factor activator protein-1 (AP-1) participates in many aspects of cell physiology, such as cellular proliferation, transformation, and death. AP-1 is a dimeric complex that primarily contains Jun and Fos family members. Here, we report that JUN is a target of heat shock transcription factor HSF1. HSF1 is the master regulator of genes encoding molecular chaperones, and is involved in cellular processes such as the stress response, cell differentiation, aging and carcinogenesis. In HeLa cells, JUN transcription was rapidly induced by heat treatment. We found that HSF1 bound to the JUN promoter and was necessary for its efficient response to heat shock. In heat-shocked cells, c-Jun-mediated gene expression was induced slowly following accumulation of c-Jun protein. Forced expression of active HSF1 in cells resulted in an increase in c-Jun expression and activation of c-Jun target genes. These results show that HSF1 regulates JUN expression, thereby modulating AP-1 activity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-jun/genética , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Western Blotting , Proliferação de Células , Imunoprecipitação da Cromatina , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Fatores de Transcrição de Choque Térmico , Resposta ao Choque Térmico , Temperatura Alta , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Luciferases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
Heat shock transcription factor (HSF) regulates the expression of genes encoding molecular chaperones and stress-responsive proteins. Conversion of HSF from a monomer to a homotrimer or heterotrimer is essential for its binding to heat shock elements (HSEs) comprised of inverted repeats of the pentamer nGAAn. Here, we constructed various human HSF1 derivatives and analyzed their transcriptional activity through the continuously and discontinuously arranged nGAAn units. We identified a short stretch of amino acids that inhibits the activation ability of HSF1, especially through discontinuous HSEs. This stretch is conserved in HSFs of various organisms, interacts with the hydrophobic repeat regions that mediate HSF oligomerization, and impedes homotrimer formation and DNA binding. This conserved domain plays an important role in maintaining HSF in an inactive monomeric form.
Assuntos
Evolução Biológica , Biopolímeros/química , Proteínas de Ligação a DNA/fisiologia , DNA/metabolismo , Fatores de Transcrição/fisiologia , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição de Choque Térmico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Expression of the RGS2 gene modulates RGS2 activity toward G protein-coupled signaling in diverse cellular processes. In this study, RGS2 transcription was induced in HeLa and rat aorta smooth muscle cells by exposure to febrile temperatures or proteotoxic stress. The promoter region of RGS2 contained a binding sequence of HSF1, which is an activator of the heat shock protein gene, and was inducibly bound by stress-activated HSF1. A single nucleotide change identified in the RGS2 promoter of hypertensive patients abolished HSF1-regulated expression of RGS2, suggesting that activated HSF1 is involved in blood pressure regulation via modulation of RGS2 expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas RGS/genética , Estresse Fisiológico/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Proteínas de Ligação a DNA/genética , Células HeLa , Fatores de Transcrição de Choque Térmico , Humanos , Miócitos de Músculo Liso/metabolismo , Regiões Promotoras Genéticas , Proteínas RGS/metabolismo , Ratos , Fatores de Transcrição/genéticaRESUMO
Heat shock transcription factor (HSF), a key regulator in the expression of heat shock protein (HSP) chaperones, is involved in the maintenance of protein homeostasis. However, the impact of HSF-mediated transcription of each HSP gene on this process is not fully understood. We show that Saccharomyces cerevisiae cells containing mutations in the HSF-binding sequences of chromosomal HSP90 promoters exhibit various phenotypes, including slow growth, proteotoxic stress sensitivity, and reduced chronological lifespan. Similar phenotypes were observed when HSF-binding sequences in five mitochondrial HSP promoters were mutated. Therefore, HSF-regulated changes in expression of these chaperone genes are necessary to maintain cell viability under various growth conditions.
