Smg1 knockdown和泛素—蛋白酶体抑制剂导致Upf1和Upf2表达的增加
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摘要
第一部分Smg1 konckdown导致Upf1和Upf2表达的升高
背景及目的
mRNA的降解是基因表达过程中一个很重要的调控方式。目前已知真核细胞mRNA的降解有三种途径,其中无义介导的mRNA降解(nonsense-mediated mRNA decay, NMD)是近年发现的一种在真核生物中广泛存在的高度保守的RNA监控(RNA surveillance)机制。NMD能够选择性地降解含有提前终止翻译密码子(premature termination condon, PTC,又称无义密码子)的mRNA,从而阻止异常的截短蛋白(truncated protein)的产生。这种截短蛋白不仅不具有正常的生理功能,而且能与正常蛋白发生竞争,从而影响细胞的正常生理功能。
NMD途径需要有剪接、翻译、磷酸化过程以及特异蛋白质组分的参与。作为剪接的结果,在mRNA的外显子-外显子连接(exon-exon junction,EEJ)的上游20-24个核苷酸处装配有外显子连接复合物(exon junction complex,EJC),对于NMD是必需的。Upf(up-frameshift mutant)是目前研究较多的NMD反式作用因子,包括Upf1、Upf2和Upf3。当前体mRNA发生剪接后,EJC与核浆穿梭蛋白upf3和TAP结合,介导mRNA出核。出核后,Upf2通过与Upf3结合而补充到mRNA上,mRNA与核糖体结合并开始翻译。正常情况下,核糖体会沿着mRNA滑动将所有EJC清除,直至遇到正常终止密码子后终止翻译。如果核糖体在距EJC上游>50-55 nt处遇到PTC,翻译将提前终止,由Smg1、Upf1、eRF等组成的SURF(SMG1-Upf1-eRF)复合物与Upf2结合,最终导致mRNA的脱帽和降解。
Smg1是NMD组分中一个很重要的因子,它作为一个PI-3激酶,与另一NMD组分Upf1相结合并对其进行磷酸化,同时形成SURF复合物。后者与Upf2结合并最终导致无义mRNA的降解。Smg1和Upf1不仅作为NMD的重要组分,而且还参与DNA代谢过程。有研究报道,抑制Upf1基因的表达可以导致S早期捕获和DNA损伤反应。Smg1也被报道参与细胞周期的运行和DNA损伤的关键步骤。这些报道说明NMD组分通过多途径参与基因信息稳定性的维持。然而,目前对NMD各组分的定量调节机制还所知甚少。
方法
为了探讨Smg1对Upf1及Upf2含量的影响,本研究用RNAi方法使Hela细胞Smg1 knockdown,然后利用western blot观察其对Upf1和Upf2的蛋白表达的影响,用免疫荧光观察Upf1和Upf2在细胞中的定位及其荧光强度的变化,用realtime-PCR方法观察其对Upf1和Upf2的mRNA水平的影响;并利用不同的细胞系和不同序列的RNAi片段,对实验结果加以证实。
结果
(1) Western blotting结果显示,和Luciferase SiRNA转染的细胞相比,用SMG1 5032 SiRNA转染的Hela细胞中,Smg1的蛋白含量明显降低,说明Smg1被成功地knockdown,基因受到抑制;
(2)在Smg1 5032、2999、5202几种SiRNA转染的细胞中,Upf1和Upf2的蛋白表达明显增高,说明Smg1 knockdown能够导致Upf1和Upf2蛋白的表达增加。
(3) Smg1 SiRNA同样能够导致A549和HE-4细胞中Upf1和Upf2的蛋白表达升高,说明Smg1 knockdown对Upf1和Upf2蛋白的上调作用在培养的人类细胞系中是一个普遍现象。
(4)经过Smg1 5032 SiRNA和Smg1 2999 SiRNA转染的细胞,分别提取细胞核和细胞浆蛋白,western blotting显示Upf1和Upf2蛋白表达的增高同时出现于胞浆蛋白和胞核蛋白中。结果说明,Smg1 knockdown能够导致胞浆和胞核中的Upf1和Upf2蛋白同时增加。
(5)免疫荧光染色结果显示,Smg1主要位于胞浆内,在胞核内也有少量表达;Upf1蛋白为胞浆蛋白,主要位于细胞浆内;Upf2则主要位于围绕核膜的核周区,荧光染色显示一条绿色的细线。与Luciferase SiRNA转染的细胞相比,Smg1 knockdown的细胞中Smg1的荧光强度明显降低,而Upf1和Upf2的荧光强度均明显升高。此结果与前面的结果一致,从形态学上进一步证实了Smg1 knockdown能够导致Upf1和Upf2蛋白表达的升高。
(6)经过Smg1 knockdown的细胞,其细胞中Smg1的mRNA水平明显降低,证实了Smg1 SiRNA的基因抑制作用;同时,细胞中Upf1和Upf2的mRNA水平没有显著升高或降低,说明Smg1 knockdown不能影响细胞中Upf1和Upf2的mRNA水平,其所导致的Upf1和Upf2表达增高可能是翻译后水平的升高。
结论
本研究首次阐明了Upf1和Upf2的定量调节机制,提示Smg1 knockdown导致人细胞系中Upf1和Upf2蛋白的表达升高,亦即Smg1参与Upf1和Upf2蛋白的下调。
第二部分泛素-蛋白酶体抑制剂导致Upf1和Upf2表达的升高
背景及目的
泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS)是真核细胞内主要的蛋白水解酶体系,由泛素(ubiquitin,Ub)、泛素活化酶(ubiquitin-activating enzyme,E1)、泛素耦联酶(ubiquitin-conjugating enzymes,E2s)、泛素一蛋白连接酶(ubiquitin-protein ligating enzymes,E3s)、26S蛋白酶体(26S proteasome)以及泛素再循环酶(ubiquitin recycling enzymes)等组成。泛素分子主要通过泛素活化酶、泛素结合酶和泛素-蛋白连接酶与靶蛋白结合形成一条多泛素链,将底物蛋白泛素化,使靶蛋白被26S蛋白酶体所识别和降解。泛素一蛋白酶体系统是细胞内三磷酸腺苷(adeno-sine triphosphate,ATP)依赖的非溶酶体蛋白降解机制,可高效并高选择性地降解细胞内蛋白质,参与细胞的多种生理活动过程,比如细胞凋亡、MHC I类抗原的递呈、细胞周期以及细胞内信号传导等,对维持细胞正常的生理功能具有十分重要的意义。
UPS对蛋白质的降解包括两个连续的步骤:(1)在多种蛋白酶的作用下,多个Ub分子与底物蛋白结合,从而标记底物蛋白;(2)标记了的底物蛋白,被26S蛋白酶体识别而降解。在第一步Ub标记底物蛋白的过程中,目前认为至少需要3个接连反应来完成:首先,Ub与E1结合,形成高能硫脂键,使Ub活化;而后通过转脂作用,Ub从El转移到E2s上;活化的Ub再从E2s转移到E3s,并与底物结合,形成底物一E3s复合物,使底物发生Ub化,即多个Ub分之通过异肽键与底物蛋白链接,形成多聚泛素链。第二步:首先由26S蛋白酶体两端的19s调节亚基识别、结合并展平Ub化的底物蛋白,然后.由26S蛋白酶体的催化核心20S亚基,将蛋白质水解为多肽;最后,多聚Ub链在泛素再循环酶的作用下,释放出Ub,以重新利用,从而形成Ub的再循环途径。
为了探讨泛素-蛋白酶体系统这一被机体广泛应用的蛋白降解机制是否也适用于NMD系统,本研究采用泛素-蛋白酶体抑制剂MG132和lactacystin作用于Hela细胞,观察其对Upf1和Upf2蛋白表达的影响。
方法
利用泛素-蛋白酶体抑制剂MG132和lactacystin作用于Hela细胞,用western blot观察其对Upf1和Upf2蛋白表达的影响,用免疫荧光观察Upf1和Upf2在细胞中荧光强度的变化,用realtime-PCR方法观察其对Upf1和Upf2的mRNA水平的影响,同时用免疫共沉淀方法检测Upf1和泛素分子之间的相互作用。为了探讨Smg1和泛素-蛋白酶体系统对Upf1和Upf2作用之间的关系,本研究同时利用SMG1 knockdown和泛素-蛋白酶体抑制剂作用于Hela细胞,观察Upf1和Upf2的表达是否有所改变。
结果
(1)Western blotting结果显示,利用泛素-蛋白酶体抑制剂MG132和Lactacystin,Hela细胞中的Upf1和Upf2蛋白表达明显增加;免疫荧光染色结果中,二者的荧光强度也相应增加;但是,通过realtime-PCR的检测,我们不能观察到二者mRNA水平的变化。
(2)用泛素-蛋白酶体抑制剂MG132作用于Hela细胞,收集细胞后分别用anti Upf1抗体和anti ubiquitin抗体进行免疫沉淀检测,实验结果显示,与DMSO组细胞相比,MG132处理后的细胞与Upf1共沉淀时,Upf1的蛋白表达明显升高,但是不能观察到Upf1的条带迁移,而且也不能检测到ubiquitin的存在;在与ubiquitin共沉淀时,我们也不能观察到与Upf1分子量大小相当的条带出现。结果表明,泛素-蛋白酶体系统抑制剂不能直接导致Upf1分子的多泛素化。
(3)将泛素-蛋白酶体抑制剂MG132作用于经过SMG1 knockdown的Hela细胞中,结果发现,随着抑制剂浓度的增加,Upf1和Upf2的蛋白表达也随之增高,说明SMG1 knockdown和泛素-蛋白酶体抑制剂对Upf1和Upf2的调节具有累加作用,虽然并不排除有部分的重叠。
结论
泛素-蛋白酶体系统抑制剂导致Upf1和Upf2蛋白表达的增加,亦即泛素-蛋白酶体系统对Upf1和Upf2蛋白有下调作用。但是,Upf1分子不能被直接泛素化,推测泛素-蛋白酶体系统可能是通过其靶分子来间接地导致Upf1的降解。
Smg1 knockdown和泛素-蛋白酶体抑制剂的同时作用使Upf1和Upf2的蛋白升高具有累加效应,说明Smg1和泛素-蛋白酶体系统是通过不同的机制和通路对Upf1和Upf2进行调节的。
Part I Smg1 knockdown caused accumulation of Upf1 and Upf2 proteins in human cells
Gene expression is regulated by various mechanisms in which RNA decay pathway is one of the most important regulators. Nonsense mediated mRNA decay (NMD) is a highly conserved pathway which degrades the nonsense mutation (also called premature termination codon) containing mRNA selectively. NMD suppresses the expression of nonsense mutation derived truncated proteins which would be deleterious by competing with activity of full-length normal proteins and thus plays an important protective role for heterozygous carriers. In addition to nonsense-mutation in genome, transcription error, alternative splicing and RNA editing can also bear targets of NMD. It is thought to function as an important member of quality control in genetic information.
NMD activity requires various RNA metabolic mechanisms such as splicing, capping, and translation. As a result of splicing, exon junction complex (EJC) is assembled 20–24 nt upstream of the exon–exon junction on the mRNA, which is essential for NMD. Upf3, a member of NMD factors, is actually a component of EJC; and Upf2, another NMD factor, is recruited onto mRNA molecule by binding to Upf3. When ribosome stops at premature termination codon present at least 50–55 nt upstream of an EJC, a complex named Smg1-Upf1-eRF (SURF) binds to Upf2. Smg1 is a member of PI-3 kinases and is also an essential component for NMD. It phosphorylates Upf1 helicase during NMD reaction. Finally, this interaction causes decapping of mRNA followed by degradation.
Upf1 and Smg1 are also known to participate in DNA metabolism. Actually, knockdown of Upf1 in human cells causes arrest in early S phase and DNA-damage response by ATR. Smg1 is also known to work in cell cycle progression and DNA damage checkpoint pathway. These reports showed the multi-function of NMD components which are required for the stability of genomic information. However, their quantitative regulation of each component is largely unknown.
Hela cells were transfected with 5032 siRNA against Smg1 mRNA and their lysates were analyzed by western blotting with specific antibodies. Smg1 was down-regulated by siRNA transfection successfully as compared to control cells transfected with anti-Luciferase siRNA. We observed that both Upf1 and Upf2 proteins increased significantly in Smg1 knockdown cells. Next, we tested various Smg1 siRNA molecules with different target sequences. All of them caused similar accumulations of Upf1 and Upf2 proteins. From these results, we concluded Smg1 knockdown could cause the accumulation of Upf1 and Upf2 proteins in human cells. Similar up-regulation of Upf1 and Upf2 proteins in Smg1 knockdown cells was also observed in A549 and skin derived normal fibroblast HE-4 cells. Then we concluded that this up-regulation might be a general phenomenon in culturing human cells.
To determine the cellular fraction where Upf1 and Upf2 accumulated, western blotting was performed with fractionated cellular lysates. By Smg1 siRNA transfection, both Upf1 and Upf2 proteins increased in nuclear and cytoplasmic fractions. Then, it seemed that Smg1 could down-regulate the amounts of Upf1 and Upf2 proteins in nuclear and cytoplasmic fraction. However, we could not detect significant augmentation in mRNAs of Upf1 and Upf2 from our real-time PCR results.
To get the morphologic proofs of our results, we preformed immunofluorescence staining to test the location of Upf1 and Upf2 proteins. By using specific antibodies, we observed that Upf1 was evenly distributed throughout the cytoplasm, while Upf2 showed strong perinuclear staining. With Smg1 SiRNA knockdown, the fluorescence signals of Smg1 in cells weakened significantly, which suggested the successful knockdown of Smg1. At the same time, we found the fluorescence signals of Upf1 and Upf2 increased respectively, which confirmed our results.
Conclusion:Smg1-knockdown caused accumulation of Upf1 and Upf2 proteins in human cells.
PartⅡUbiquitin-proteasome inhibitors caused accumulation of Upf1 and Upf2 proteins in human cells
The generation and degradation of intracellular proteins must keep dynamic balance, which playsa key role to maintain cellular stable and normal functions. The ubiquitin proteasome system(UPS)is an important pathway for the intracellular proteins target degradation.Polymers of ubiquitin can be covalently attached to protein targets by a three-step(ubiquitin-activating enzymes,ubiquitin conjugating enzymes,ubiquitin-protein ligases)conjugated cascade responses . The resulting ubiquitylated proteins are then recognized and degraded by the 26S proteasome.UPS can degrade intracellular proteins with high effeciency and selectivity,especially for cell cycle regulated proteins, oncoproteins,tumor suppressor proteins,and denaturalized proteins.UPS is assential for many cellular processes,including apoptosis,MHC class I antigen presentation,cell cycle and intracellular signal transferring,which has a closed relationship with the cellular physiology and pathology.
Ubiquitin-proteasome system is a principal mechanism for protein catabolism in the mammalian cytosol and nucleus. It plays an important role in a wide variety of cellular processes including cell cycle, differentiation, DNA repair, transcriptional regulation, response to stress, and many other basic cellular processes. MG132, a kind of peptide aldehyde, is a more potent and selective proteasome inhibitor. In our present studies, MG132 treatment caused the accumulation of Upf1 and Upf2 proteins, indicating that ubiquitin-proteasome might participate in the down-regulation of the amount of Upf1 and Upf2.
These results prompted us to speculate that Upf1 and Upf2 would be target molecules of ubiquitination. To investigate this possibility, we tried detection of higher band shift caused by polyubiquitination of Upf1 proteins. However, we detected no band shift of either unpurified Upf1 or immuno-precipitated Upf1, and no signal of ubiquitin conjugation was detected even in immuno-purified Upf1. At least, the accumulation of Upf1 could not be accounted by the fraction of very little band shift in MG132 treated cells. In conclusion, our experiments provided no clear evidence for polyubiquitination of Upf1 and proposed that the target molecule(s) of ubiquitin-proteasome might down-regulate the amount of Upf1 directly or indirectly.
To test whether Smg1 knockdown and ubiquitin-proteasome inhibitors share the same pathway to cause the accumulation of Upf1 and Upf2, we treated Smg1-knockdowned cells with proteasome inhibitors and the lysates for immunoblot were obtained. If they affect on the increments in Upf1 and Upf2 independently, an additional accumulation should be expected. Smg1 knockdown and proteasome inhibitor MG132 caused accumulation of Upf1 and Upf2 respectively. Moreover, in Smg1 knockdowned cells,we detected additive effects of Upf1 and Upf2 accumulation with proteasome inhibitors treatment, which suggested the possibility that SMG1 and ubiquitin-protesome system shared a different, if not all, pathway that regulated the amounts of Upf1 and Upf2.
Conclusion:Ubiquitin-proteasome inhibitors caused accumulation of Upf1 and Upf2 proteins in human cells, which shared a different pathway with Smg1 knockdown.
背景及目的
mRNA的降解是基因表达过程中一个很重要的调控方式。目前已知真核细胞mRNA的降解有三种途径,其中无义介导的mRNA降解(nonsense-mediated mRNA decay, NMD)是近年发现的一种在真核生物中广泛存在的高度保守的RNA监控(RNA surveillance)机制。NMD能够选择性地降解含有提前终止翻译密码子(premature termination condon, PTC,又称无义密码子)的mRNA,从而阻止异常的截短蛋白(truncated protein)的产生。这种截短蛋白不仅不具有正常的生理功能,而且能与正常蛋白发生竞争,从而影响细胞的正常生理功能。
NMD途径需要有剪接、翻译、磷酸化过程以及特异蛋白质组分的参与。作为剪接的结果,在mRNA的外显子-外显子连接(exon-exon junction,EEJ)的上游20-24个核苷酸处装配有外显子连接复合物(exon junction complex,EJC),对于NMD是必需的。Upf(up-frameshift mutant)是目前研究较多的NMD反式作用因子,包括Upf1、Upf2和Upf3。当前体mRNA发生剪接后,EJC与核浆穿梭蛋白upf3和TAP结合,介导mRNA出核。出核后,Upf2通过与Upf3结合而补充到mRNA上,mRNA与核糖体结合并开始翻译。正常情况下,核糖体会沿着mRNA滑动将所有EJC清除,直至遇到正常终止密码子后终止翻译。如果核糖体在距EJC上游>50-55 nt处遇到PTC,翻译将提前终止,由Smg1、Upf1、eRF等组成的SURF(SMG1-Upf1-eRF)复合物与Upf2结合,最终导致mRNA的脱帽和降解。
Smg1是NMD组分中一个很重要的因子,它作为一个PI-3激酶,与另一NMD组分Upf1相结合并对其进行磷酸化,同时形成SURF复合物。后者与Upf2结合并最终导致无义mRNA的降解。Smg1和Upf1不仅作为NMD的重要组分,而且还参与DNA代谢过程。有研究报道,抑制Upf1基因的表达可以导致S早期捕获和DNA损伤反应。Smg1也被报道参与细胞周期的运行和DNA损伤的关键步骤。这些报道说明NMD组分通过多途径参与基因信息稳定性的维持。然而,目前对NMD各组分的定量调节机制还所知甚少。
方法
为了探讨Smg1对Upf1及Upf2含量的影响,本研究用RNAi方法使Hela细胞Smg1 knockdown,然后利用western blot观察其对Upf1和Upf2的蛋白表达的影响,用免疫荧光观察Upf1和Upf2在细胞中的定位及其荧光强度的变化,用realtime-PCR方法观察其对Upf1和Upf2的mRNA水平的影响;并利用不同的细胞系和不同序列的RNAi片段,对实验结果加以证实。
结果
(1) Western blotting结果显示,和Luciferase SiRNA转染的细胞相比,用SMG1 5032 SiRNA转染的Hela细胞中,Smg1的蛋白含量明显降低,说明Smg1被成功地knockdown,基因受到抑制;
(2)在Smg1 5032、2999、5202几种SiRNA转染的细胞中,Upf1和Upf2的蛋白表达明显增高,说明Smg1 knockdown能够导致Upf1和Upf2蛋白的表达增加。
(3) Smg1 SiRNA同样能够导致A549和HE-4细胞中Upf1和Upf2的蛋白表达升高,说明Smg1 knockdown对Upf1和Upf2蛋白的上调作用在培养的人类细胞系中是一个普遍现象。
(4)经过Smg1 5032 SiRNA和Smg1 2999 SiRNA转染的细胞,分别提取细胞核和细胞浆蛋白,western blotting显示Upf1和Upf2蛋白表达的增高同时出现于胞浆蛋白和胞核蛋白中。结果说明,Smg1 knockdown能够导致胞浆和胞核中的Upf1和Upf2蛋白同时增加。
(5)免疫荧光染色结果显示,Smg1主要位于胞浆内,在胞核内也有少量表达;Upf1蛋白为胞浆蛋白,主要位于细胞浆内;Upf2则主要位于围绕核膜的核周区,荧光染色显示一条绿色的细线。与Luciferase SiRNA转染的细胞相比,Smg1 knockdown的细胞中Smg1的荧光强度明显降低,而Upf1和Upf2的荧光强度均明显升高。此结果与前面的结果一致,从形态学上进一步证实了Smg1 knockdown能够导致Upf1和Upf2蛋白表达的升高。
(6)经过Smg1 knockdown的细胞,其细胞中Smg1的mRNA水平明显降低,证实了Smg1 SiRNA的基因抑制作用;同时,细胞中Upf1和Upf2的mRNA水平没有显著升高或降低,说明Smg1 knockdown不能影响细胞中Upf1和Upf2的mRNA水平,其所导致的Upf1和Upf2表达增高可能是翻译后水平的升高。
结论
本研究首次阐明了Upf1和Upf2的定量调节机制,提示Smg1 knockdown导致人细胞系中Upf1和Upf2蛋白的表达升高,亦即Smg1参与Upf1和Upf2蛋白的下调。
第二部分泛素-蛋白酶体抑制剂导致Upf1和Upf2表达的升高
背景及目的
泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS)是真核细胞内主要的蛋白水解酶体系,由泛素(ubiquitin,Ub)、泛素活化酶(ubiquitin-activating enzyme,E1)、泛素耦联酶(ubiquitin-conjugating enzymes,E2s)、泛素一蛋白连接酶(ubiquitin-protein ligating enzymes,E3s)、26S蛋白酶体(26S proteasome)以及泛素再循环酶(ubiquitin recycling enzymes)等组成。泛素分子主要通过泛素活化酶、泛素结合酶和泛素-蛋白连接酶与靶蛋白结合形成一条多泛素链,将底物蛋白泛素化,使靶蛋白被26S蛋白酶体所识别和降解。泛素一蛋白酶体系统是细胞内三磷酸腺苷(adeno-sine triphosphate,ATP)依赖的非溶酶体蛋白降解机制,可高效并高选择性地降解细胞内蛋白质,参与细胞的多种生理活动过程,比如细胞凋亡、MHC I类抗原的递呈、细胞周期以及细胞内信号传导等,对维持细胞正常的生理功能具有十分重要的意义。
UPS对蛋白质的降解包括两个连续的步骤:(1)在多种蛋白酶的作用下,多个Ub分子与底物蛋白结合,从而标记底物蛋白;(2)标记了的底物蛋白,被26S蛋白酶体识别而降解。在第一步Ub标记底物蛋白的过程中,目前认为至少需要3个接连反应来完成:首先,Ub与E1结合,形成高能硫脂键,使Ub活化;而后通过转脂作用,Ub从El转移到E2s上;活化的Ub再从E2s转移到E3s,并与底物结合,形成底物一E3s复合物,使底物发生Ub化,即多个Ub分之通过异肽键与底物蛋白链接,形成多聚泛素链。第二步:首先由26S蛋白酶体两端的19s调节亚基识别、结合并展平Ub化的底物蛋白,然后.由26S蛋白酶体的催化核心20S亚基,将蛋白质水解为多肽;最后,多聚Ub链在泛素再循环酶的作用下,释放出Ub,以重新利用,从而形成Ub的再循环途径。
为了探讨泛素-蛋白酶体系统这一被机体广泛应用的蛋白降解机制是否也适用于NMD系统,本研究采用泛素-蛋白酶体抑制剂MG132和lactacystin作用于Hela细胞,观察其对Upf1和Upf2蛋白表达的影响。
方法
利用泛素-蛋白酶体抑制剂MG132和lactacystin作用于Hela细胞,用western blot观察其对Upf1和Upf2蛋白表达的影响,用免疫荧光观察Upf1和Upf2在细胞中荧光强度的变化,用realtime-PCR方法观察其对Upf1和Upf2的mRNA水平的影响,同时用免疫共沉淀方法检测Upf1和泛素分子之间的相互作用。为了探讨Smg1和泛素-蛋白酶体系统对Upf1和Upf2作用之间的关系,本研究同时利用SMG1 knockdown和泛素-蛋白酶体抑制剂作用于Hela细胞,观察Upf1和Upf2的表达是否有所改变。
结果
(1)Western blotting结果显示,利用泛素-蛋白酶体抑制剂MG132和Lactacystin,Hela细胞中的Upf1和Upf2蛋白表达明显增加;免疫荧光染色结果中,二者的荧光强度也相应增加;但是,通过realtime-PCR的检测,我们不能观察到二者mRNA水平的变化。
(2)用泛素-蛋白酶体抑制剂MG132作用于Hela细胞,收集细胞后分别用anti Upf1抗体和anti ubiquitin抗体进行免疫沉淀检测,实验结果显示,与DMSO组细胞相比,MG132处理后的细胞与Upf1共沉淀时,Upf1的蛋白表达明显升高,但是不能观察到Upf1的条带迁移,而且也不能检测到ubiquitin的存在;在与ubiquitin共沉淀时,我们也不能观察到与Upf1分子量大小相当的条带出现。结果表明,泛素-蛋白酶体系统抑制剂不能直接导致Upf1分子的多泛素化。
(3)将泛素-蛋白酶体抑制剂MG132作用于经过SMG1 knockdown的Hela细胞中,结果发现,随着抑制剂浓度的增加,Upf1和Upf2的蛋白表达也随之增高,说明SMG1 knockdown和泛素-蛋白酶体抑制剂对Upf1和Upf2的调节具有累加作用,虽然并不排除有部分的重叠。
结论
泛素-蛋白酶体系统抑制剂导致Upf1和Upf2蛋白表达的增加,亦即泛素-蛋白酶体系统对Upf1和Upf2蛋白有下调作用。但是,Upf1分子不能被直接泛素化,推测泛素-蛋白酶体系统可能是通过其靶分子来间接地导致Upf1的降解。
Smg1 knockdown和泛素-蛋白酶体抑制剂的同时作用使Upf1和Upf2的蛋白升高具有累加效应,说明Smg1和泛素-蛋白酶体系统是通过不同的机制和通路对Upf1和Upf2进行调节的。
Part I Smg1 knockdown caused accumulation of Upf1 and Upf2 proteins in human cells
Gene expression is regulated by various mechanisms in which RNA decay pathway is one of the most important regulators. Nonsense mediated mRNA decay (NMD) is a highly conserved pathway which degrades the nonsense mutation (also called premature termination codon) containing mRNA selectively. NMD suppresses the expression of nonsense mutation derived truncated proteins which would be deleterious by competing with activity of full-length normal proteins and thus plays an important protective role for heterozygous carriers. In addition to nonsense-mutation in genome, transcription error, alternative splicing and RNA editing can also bear targets of NMD. It is thought to function as an important member of quality control in genetic information.
NMD activity requires various RNA metabolic mechanisms such as splicing, capping, and translation. As a result of splicing, exon junction complex (EJC) is assembled 20–24 nt upstream of the exon–exon junction on the mRNA, which is essential for NMD. Upf3, a member of NMD factors, is actually a component of EJC; and Upf2, another NMD factor, is recruited onto mRNA molecule by binding to Upf3. When ribosome stops at premature termination codon present at least 50–55 nt upstream of an EJC, a complex named Smg1-Upf1-eRF (SURF) binds to Upf2. Smg1 is a member of PI-3 kinases and is also an essential component for NMD. It phosphorylates Upf1 helicase during NMD reaction. Finally, this interaction causes decapping of mRNA followed by degradation.
Upf1 and Smg1 are also known to participate in DNA metabolism. Actually, knockdown of Upf1 in human cells causes arrest in early S phase and DNA-damage response by ATR. Smg1 is also known to work in cell cycle progression and DNA damage checkpoint pathway. These reports showed the multi-function of NMD components which are required for the stability of genomic information. However, their quantitative regulation of each component is largely unknown.
Hela cells were transfected with 5032 siRNA against Smg1 mRNA and their lysates were analyzed by western blotting with specific antibodies. Smg1 was down-regulated by siRNA transfection successfully as compared to control cells transfected with anti-Luciferase siRNA. We observed that both Upf1 and Upf2 proteins increased significantly in Smg1 knockdown cells. Next, we tested various Smg1 siRNA molecules with different target sequences. All of them caused similar accumulations of Upf1 and Upf2 proteins. From these results, we concluded Smg1 knockdown could cause the accumulation of Upf1 and Upf2 proteins in human cells. Similar up-regulation of Upf1 and Upf2 proteins in Smg1 knockdown cells was also observed in A549 and skin derived normal fibroblast HE-4 cells. Then we concluded that this up-regulation might be a general phenomenon in culturing human cells.
To determine the cellular fraction where Upf1 and Upf2 accumulated, western blotting was performed with fractionated cellular lysates. By Smg1 siRNA transfection, both Upf1 and Upf2 proteins increased in nuclear and cytoplasmic fractions. Then, it seemed that Smg1 could down-regulate the amounts of Upf1 and Upf2 proteins in nuclear and cytoplasmic fraction. However, we could not detect significant augmentation in mRNAs of Upf1 and Upf2 from our real-time PCR results.
To get the morphologic proofs of our results, we preformed immunofluorescence staining to test the location of Upf1 and Upf2 proteins. By using specific antibodies, we observed that Upf1 was evenly distributed throughout the cytoplasm, while Upf2 showed strong perinuclear staining. With Smg1 SiRNA knockdown, the fluorescence signals of Smg1 in cells weakened significantly, which suggested the successful knockdown of Smg1. At the same time, we found the fluorescence signals of Upf1 and Upf2 increased respectively, which confirmed our results.
Conclusion:Smg1-knockdown caused accumulation of Upf1 and Upf2 proteins in human cells.
PartⅡUbiquitin-proteasome inhibitors caused accumulation of Upf1 and Upf2 proteins in human cells
The generation and degradation of intracellular proteins must keep dynamic balance, which playsa key role to maintain cellular stable and normal functions. The ubiquitin proteasome system(UPS)is an important pathway for the intracellular proteins target degradation.Polymers of ubiquitin can be covalently attached to protein targets by a three-step(ubiquitin-activating enzymes,ubiquitin conjugating enzymes,ubiquitin-protein ligases)conjugated cascade responses . The resulting ubiquitylated proteins are then recognized and degraded by the 26S proteasome.UPS can degrade intracellular proteins with high effeciency and selectivity,especially for cell cycle regulated proteins, oncoproteins,tumor suppressor proteins,and denaturalized proteins.UPS is assential for many cellular processes,including apoptosis,MHC class I antigen presentation,cell cycle and intracellular signal transferring,which has a closed relationship with the cellular physiology and pathology.
Ubiquitin-proteasome system is a principal mechanism for protein catabolism in the mammalian cytosol and nucleus. It plays an important role in a wide variety of cellular processes including cell cycle, differentiation, DNA repair, transcriptional regulation, response to stress, and many other basic cellular processes. MG132, a kind of peptide aldehyde, is a more potent and selective proteasome inhibitor. In our present studies, MG132 treatment caused the accumulation of Upf1 and Upf2 proteins, indicating that ubiquitin-proteasome might participate in the down-regulation of the amount of Upf1 and Upf2.
These results prompted us to speculate that Upf1 and Upf2 would be target molecules of ubiquitination. To investigate this possibility, we tried detection of higher band shift caused by polyubiquitination of Upf1 proteins. However, we detected no band shift of either unpurified Upf1 or immuno-precipitated Upf1, and no signal of ubiquitin conjugation was detected even in immuno-purified Upf1. At least, the accumulation of Upf1 could not be accounted by the fraction of very little band shift in MG132 treated cells. In conclusion, our experiments provided no clear evidence for polyubiquitination of Upf1 and proposed that the target molecule(s) of ubiquitin-proteasome might down-regulate the amount of Upf1 directly or indirectly.
To test whether Smg1 knockdown and ubiquitin-proteasome inhibitors share the same pathway to cause the accumulation of Upf1 and Upf2, we treated Smg1-knockdowned cells with proteasome inhibitors and the lysates for immunoblot were obtained. If they affect on the increments in Upf1 and Upf2 independently, an additional accumulation should be expected. Smg1 knockdown and proteasome inhibitor MG132 caused accumulation of Upf1 and Upf2 respectively. Moreover, in Smg1 knockdowned cells,we detected additive effects of Upf1 and Upf2 accumulation with proteasome inhibitors treatment, which suggested the possibility that SMG1 and ubiquitin-protesome system shared a different, if not all, pathway that regulated the amounts of Upf1 and Upf2.
Conclusion:Ubiquitin-proteasome inhibitors caused accumulation of Upf1 and Upf2 proteins in human cells, which shared a different pathway with Smg1 knockdown.
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