摘要
目的:研究mfn-1 RNAi对线粒体未折叠蛋白反应的激活作用及其机制。方法:分别对线虫SJ4058和SJ4100(指示线粒体未折叠蛋白反应)、TJ375(指示细胞质未折叠蛋白反应)、SJ4005(指示内质网未折叠蛋白反应)进行mfn-1 RNAi处理,观察后代中能否激活相应分子伴侣表达;将SJ4058线虫分别与haf-1基因功能缺失线虫RB867及atfs-1基因功能缺失线虫VC3201杂交,然后对获得的纯合子线虫进行mfn-1RNAi,观测后代中是否有荧光激活,即验证分子伴侣的激活是否依赖于HAF-1及ATFS-1。结果:Mfn-1 RNAi特异性激活线粒体分子伴侣HSP-6及HSP-60的表达,而没有激活细胞质分子伴侣HSP-16.2及内质网分子伴侣HSP-4;通过杂交获得基因型为hsp-60::gfp/haf-1(ok705)IV和hsp-60::gfp/atfs-1(gk3094)V的线虫并经过PCR验证。进一步对这两种基因型的线虫进行mfn-1 RNAi处理发现,线粒体分子伴侣HSP-60的激活依赖于ATFS-1而不依赖于HAF-1。结论:Mfn-1 RNAi特异性激活线粒体而非细胞质及内质网未折叠蛋白反应,提示MFN-1蛋白的表达降低对线粒体蛋白稳态有影响,而对细胞质及内质网蛋白质稳态影响不大;Mfn-1 RNAi对线粒体未折叠蛋白反应的激活依赖于转录因子ATFS-1而不依赖于线粒体膜蛋白HAF-1。
Objective： To research the effect ofmfn-1 RNAi on mitochondrial unfolded protein response and its mechanism.Methods： Worm strains SJ4058 and SJ4100（mitochondrial unfolded protein response re-porter）, TJ375 （cytosolic unfolded protein response reporter）, and SJ4005 （endoplasmic reticulum unfolded protein response reporter） were treated bymfn-1 RNAi, and to observe whether the corresponding chaperones were activated or not; crossing strain SJ4058 intohaf-1 gene loss of function strain RB867 andatfs-1gene loss of function strain VC3201 respectively, the obtained homozygous animals were treated bymfn-1 RNAi, and their progenies were observed for lfuorescence to explore whether the activation of chaperones was relied on HAF-1 or ATFS-1.Results：Mfn-1 RNAi speciifcally activated the mitochondria speciifc chaperones HSP-6 and HSP-60, but not cytosol speciifc chaperon HSP-16.2 and endoplasmic reticulum speciifc chaperon HSP-4; We obtained thehsp-60：：gfp/atfs-1（gk3094） V andhsp-60：：gfp/haf-1（ok705）IV strains by crossing and their genotypes were conifrmed by PCR. The two strains were treated bymfn-1 RNAi, and further studies showed that the activation of chaperon HSP-60 was relied on ATFS-1 rather than HAF-1.Conclusion：Mfn-1 RNAi treatment speciifcally activated mitochondrial rather than cytosolic and endoplasmic reticulum unfolded protein response, suggesting the mitochondria proteostasis is affected, but that of cytosol and endoplasmic reticulum are not; the activation of mitochondrial unfolded protein response bymfn-1 RNAi is dependent on the transcription factor ATFS-1, not the mitochondria membrane protein HAF-1.
出处
《温州医学院学报》
CAS
2014年第6期391-394,400,共5页
Journal of Wenzhou Medical College
基金
国家自然科学基金资助项目(81200253)
浙江省大学生科技创新活动计划暨新苗人才计划项目(2013R413059)
