RESUMO
We have identified a particularly clear case of compensatory transcriptional regulation among ER chaperones in Caenorhabditis elegans using a GFP reporter transgene that is under the control of the promoter of hsp-4, a C. elegans homolog of GRP-78/BiP. Knockdown by RNA interference of 9 known or predicted ER chaperones induced hsp-4 upregulation via the ire-1/xbp-1 signaling cascade employed in the unfolded protein response. We show that this compensatory regulation is specific for ER chaperones, not dependent on RNA interference, and required for maintaining viability in worms containing a deletion of the hsp-3 gene.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/genética , Chaperonas Moleculares/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reporter/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Mutação/genética , Interferência de RNA , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
We have engineered transgenic Caenorhabditis elegans animals to inducibly express the human beta amyloid peptide (Abeta). Gene expression changes resulting from Abeta induction have been monitored by cDNA hybridization to glass slide microarrays containing probes for almost all known or predicted C. elegans genes. Using statistical criteria, we have identified 67 up-regulated and 240 down-regulated genes. Subsets of these regulated genes have been tested and confirmed by quantitative RT-PCR. To investigate whether genes identified in this model system also show gene expression changes in Alzheimer's disease (AD) brain, we have also used quantitative RT-PCR to examine in post-mortem AD brain tissue transcript levels of alphaB-crystallin (CRYAB) and tumor necrosis factor-induced protein 1 (TNFAIP1), human homologs of genes found to be robustly induced in the transgenic C. elegans model. Both CRYAB and TNFAIP1 show increased transcript levels in AD brains, supporting the validity of this approach.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Química Encefálica/genética , Caenorhabditis elegans/genética , Organismos Geneticamente Modificados/genética , Proteínas/análise , Fator de Necrose Tumoral alfa/análise , Cadeia B de alfa-Cristalina/análise , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/análise , Animais , Modelos Animais de Doenças , Regulação para Baixo/genética , Regulação da Expressão Gênica , Genoma , Proteínas de Choque Térmico/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção/métodos , Regulação para Cima/genéticaRESUMO
Expression of the human beta amyloid peptide (A beta) in transgenic Caenorhabditis elegans animals can lead to the formation of intracellular immunoreactive deposits as well as the formation of intracellular amyloid. We have used this model to identify proteins that interact with intracellular A beta in vivo. Mass spectrometry analysis of proteins that specifically coimmunoprecipitate with A beta has identified six likely chaperone proteins: two members of the HSP70 family, three alpha B-crystallin-related small heat shock proteins (HSP-16s), and a putative ortholog of a mammalian small glutamine-rich tetratricopeptide repeat-containing protein proposed to regulate HSP70 function. Quantitative reverse transcription-PCR analysis shows that the small heat shock proteins are also transcriptionally induced by A beta expression. Immunohistochemistry demonstrates that HSP-16 protein closely colocalizes with intracellular A beta in this model. Transgenic animals expressing a nonaggregating A beta variant, a single-chain A beta dimer, show an altered pattern of coimmunoprecipitating proteins and an altered cellular distribution of HSP-16. Double-stranded RNA inhibition of R05F9.10, the putative C. elegans ortholog of the human small glutamine-rich tetratricopeptide-repeat-containing protein (SGT), results in suppression of toxicity associated with A beta expression. These results suggest that chaperone function can play a role in modulating intracellular A beta metabolism and toxicity.
