温州医科大学学报
溫州醫科大學學報
온주의과대학학보
Journal of Wenzhou Medical University
2014年
6期
391-394,400
,共5页
姚秀萍%陈思希%任亚光%周怀彬%吕建新
姚秀萍%陳思希%任亞光%週懷彬%呂建新
요수평%진사희%임아광%주부빈%려건신
秀丽线虫%mfn-1%线粒体未折叠蛋白反应%HAF-1%ATFS-1
秀麗線蟲%mfn-1%線粒體未摺疊蛋白反應%HAF-1%ATFS-1
수려선충%mfn-1%선립체미절첩단백반응%HAF-1%ATFS-1
C. elegans%mfn-1%mitochondrial unfolded protein response%HAF-1%ATFS-1
目的：研究mfn-1RNAi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-1RNAi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-1RNAi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-1RNAi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-1RNAi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-1RNAi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。
目的：研究mfn-1RNAi對線粒體未摺疊蛋白反應的激活作用及其機製。方法：分彆對線蟲SJ4058和SJ4100（指示線粒體未摺疊蛋白反應）、TJ375（指示細胞質未摺疊蛋白反應）、SJ4005（指示內質網未摺疊蛋白反應）進行mfn-1RNAi處理，觀察後代中能否激活相應分子伴侶錶達；將SJ4058線蟲分彆與haf-1基因功能缺失線蟲RB867及atfs-1基因功能缺失線蟲VC3201雜交，然後對穫得的純閤子線蟲進行mfn-1RNAi，觀測後代中是否有熒光激活，即驗證分子伴侶的激活是否依賴于HAF-1及ATFS-1。結果：Mfn-1RNAi特異性激活線粒體分子伴侶HSP-6及HSP-60的錶達，而沒有激活細胞質分子伴侶HSP-16.2及內質網分子伴侶HSP-4；通過雜交穫得基因型為hsp-60：gfp/haf-1（ok705）IV和hsp-60：gfp/atfs-1（gk3094）V的線蟲併經過PCR驗證。進一步對這兩種基因型的線蟲進行mfn-1RNAi處理髮現，線粒體分子伴侶HSP-60的激活依賴于ATFS-1而不依賴于HAF-1。結論：Mfn-1RNAi特異性激活線粒體而非細胞質及內質網未摺疊蛋白反應，提示MFN-1蛋白的錶達降低對線粒體蛋白穩態有影響，而對細胞質及內質網蛋白質穩態影響不大；Mfn-1RNAi對線粒體未摺疊蛋白反應的激活依賴于轉錄因子ATFS-1而不依賴于線粒體膜蛋白HAF-1。
목적：연구mfn-1RNAi대선립체미절첩단백반응적격활작용급기궤제。방법：분별대선충SJ4058화SJ4100（지시선립체미절첩단백반응）、TJ375（지시세포질미절첩단백반응）、SJ4005（지시내질망미절첩단백반응）진행mfn-1RNAi처리，관찰후대중능부격활상응분자반려표체；장SJ4058선충분별여haf-1기인공능결실선충RB867급atfs-1기인공능결실선충VC3201잡교，연후대획득적순합자선충진행mfn-1RNAi，관측후대중시부유형광격활，즉험증분자반려적격활시부의뢰우HAF-1급ATFS-1。결과：Mfn-1RNAi특이성격활선립체분자반려HSP-6급HSP-60적표체，이몰유격활세포질분자반려HSP-16.2급내질망분자반려HSP-4；통과잡교획득기인형위hsp-60：gfp/haf-1（ok705）IV화hsp-60：gfp/atfs-1（gk3094）V적선충병경과PCR험증。진일보대저량충기인형적선충진행mfn-1RNAi처리발현，선립체분자반려HSP-60적격활의뢰우ATFS-1이불의뢰우HAF-1。결론：Mfn-1RNAi특이성격활선립체이비세포질급내질망미절첩단백반응，제시MFN-1단백적표체강저대선립체단백은태유영향，이대세포질급내질망단백질은태영향불대；Mfn-1RNAi대선립체미절첩단백반응적격활의뢰우전록인자ATFS-1이불의뢰우선립체막단백HAF-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.
