节点文献

端粒相关蛋白TRF1和PinX1的翻译后修饰研究及功能解析

The Mechanism About Post Translational-modification and Functional Dissection Study of TRF1 and PinX1

【作者】 王冲

【导师】 黄河;

【作者基本信息】 浙江大学 , 内科学, 2011, 博士

【摘要】 端粒是一种特殊的由富含G的重复DNA序列和相关蛋白质组成的复合体,位于真核细胞染色体末端,起到保护末端染色体不被降解以及防止染色体末端融合的重要作用。端粒功能的缺失将导致细胞基因组的不稳定,从而大大增加正常细胞转化为癌细胞的风险。在大多数肿瘤细胞中,端粒长度主要由一种特殊的RNA逆转录酶即端粒酶以及端粒相关蛋白来调节,端粒相关蛋白通过直接或间接与端粒DNA相互作用来维持端粒结构和功能的稳定。近年来,越来越多的证据表明:传统的端粒蛋白不仅仅在端粒末端起到保护端粒结构的作用,他们还直接参与了细胞有丝分裂进程的复杂调控,例如端粒相关蛋白TRF1,TRF1的蛋白表达量在整个细胞周期中呈现动态变化,此外,TRF1能够和微管,微管相关蛋白EB1以及一些重要的纺锤体检验点蛋白比如Mad1,Nek2以及Plk1等有相互作用,这些结果表明:TRF1及其相互作用蛋白可能在细胞周期的调控中起到重要作用。由于DNA末端复制问题的存在(即细胞每分裂一次端粒就丢失数十至数百个不等的碱基),大部分肿瘤细胞和永生化细胞株例如睾丸细胞,均通过端粒酶维持端粒长度的稳定,但有一小部分肿瘤细胞,是依靠端粒延伸替代机制的激活来维持自身端粒长度的稳定,而在ALT机制维持端粒长度的调节过程中,ALT相关PML小体起到至关重要的作用。研究表明,ALT机制的核心是端粒染色体的互换重组,这个互换重组的过程在APB发生且受到精确调控,之前有研究表明TRF1的SUMO化影响了APB的形成。我们课题组此前的研究亦表明,TRF1受PML3招募上PML小体,且PML3在维持APB的形成中起到关键作用,但关于TRF1定位于APB的具体调控机制还不清楚。泛素-蛋白酶体途径是真核生物体内重要的蛋白降解途径之一。针对目标蛋白的泛素化降解是一个多步骤过程,Ub首先由E1激活酶激活,进而被转移到E2接合酶的活化胱氨酸上,再由一个特异的E3泛素连接酶将Ub从接合酶转移到靶蛋白上。其中,含有HECT结构域的E3连接酶能够和泛素形成硫酯中间体,进而直接催化靶蛋白的泛素化。而含有锌指环结构的E3连接酶则作为支架蛋白将E2接合酶与底物招募到一起,将Ub直接转移到底物上,其本身并不直接参与靶蛋白的泛素化。SCFβ-TrCP即是一种含有锌指结构的E3连接酶复合体,由四种核心亚基组成:Skp1、Cull,β-TrCP和Rbxl。其中Rbxl负责募集E2接合酶并催化亚基,Cull作为分子支架在其氨基端与Skpl结合,在羧基端和Rbxl以及E2接合酶相互作用。Rbx1和其他亚基之间的支架参与底物的选择,β-TrCP通过其F-box结构域与Skpl相互结合,并通过其WD40重复性序列特异性识别底物,多数情况下β-TrCP能够识别底物上的一个特殊的磷酸化氨基酸序列,但亦有报道称β-TrCP也能够识别非磷酸化蛋白。本课题组通过体内免疫沉淀结合蛋白质质谱鉴定的方法,从稳定表达Flag-TRF1的Hela细胞中分离出了8个可能的TRF1相互作用蛋白,序列比对发现,β-TrCP有可能是一个新的TRFl结合蛋白质,我们通过一系列体内实验证实了β-TrCP能够与TRF1在体内相互作用,外源性过量表达β-TrCP能够导致TRF1的表达量降低,进一步研究显示,β-TrCP通过促进TRF1的泛素化降解调控TRF1的蛋白表达水平,并且这一调控功能依赖于β-TrCP的F-box结构域。令人意外的是,我们发现在端粒酶阴性细胞中,PML3可以保护TRF1不受β-TrCP的降解,过量表达β-TrCP能够调控APB的形成,而利用siRNA抑制内源性β-TrCP的表达,则使得APB数量明显减少,提示β-TrCP在调控APB的形成中起到重要作用。本次研究首次发现了TRF1是一个新的β-TrCP底物蛋白,并且证明了β-TrCP在APB形成的调控中起到重要作用,为进一步探讨APB的形成及调控机制提供了新的佐证及线索。PinX1最初是作为一个新的TRF1相互作用蛋白,由Lu等在2001年首次报道,PinX1含有两个特殊的结构域:通常存在于RNA结合蛋白中的G-patch结构域以及端粒酶抑制结构域(TID)。PinX1是迄今为止发现的最强力的端粒酶抑制因子,PinX1的端粒酶抑制作用通过其本身与hTERT和hTR直接作用来实现,尽管如此,PinX1在酵母中的同源物Gnolp是否具有端粒酶抑制作用目前还存在争议。除了端粒定位以及端粒酶抑制作用外,PinX1还被发现在端粒酶阳性细胞株中能够定位于核仁,并且能够增强TRF1在核仁的聚集,表明PinX1还具有其他功能。我们课题组在前期的研究中,意外的发现在有丝分裂期,PinX1能够定位于染色体周边以及动点外层,在有丝分裂期,SiRNA敲除PinX1够导致滞后染色体的出现,在间期,敲除PinX1能够导致小核细胞的增多,但关于PinX1具体的调控机制仍不清楚。在前期工作的基础上,本课题组利用酵母双杂交的方法来寻找新的PinX1的相互作用蛋白,我们成功的从人睾丸cDNA文库中筛选到了19个阳性克隆,通过序列比对,我们发现Plk1可能是一个新的PinX1的相互作用蛋白。通过体内体外实验,进一步证实了PinX1与Plk1之间的相互作用。免疫荧光实验显示:PinXl与Plk1在有丝分裂期共定位于纺锤体上。PinX1通过其92-254段与Plk1的激酶结构域直接结合。此外,我们通过体外磷酸化的方法鉴定出PinX1是一个新的Plk1的磷酸化底物蛋白。通过蛋白质磷酸化质谱技术,我们得到了潜在的PinX1的磷酸化位点,我们构建了PinX1的非磷酸化突变体和模拟磷酸化突变体,并在体外磷酸化实验中得到了验证。进一步的研究表明,PinX1在体内能发生泛素化修饰,Plk1对PinX1的磷酸化调控PinX1的蛋白表达量变化。通过流式细胞术和免疫荧光的方法,我们发现外源转染PinX1的非磷酸化突变体可导致细胞阻滞到有丝分裂期,并且导致错误染色体的排列。综上,本部分研究证实了PinX1是一个新的Plk1的底物蛋白,Plk1通过磷酸化PinX1促进PinX1的泛素化降解,更进一步,我们发现了PinX1的磷酸化对于细胞的有丝分裂正常进行是必须的。通过以上研究,我们阐明了PinX1的磷酸化修饰的具体机制和功能,为深入阐明细胞有丝分裂过程的调控机制提供了新的实验证据和思路。综上所述,本项研究分为二个部分,第一部分,我们首次发现了β-TrCP与TRF1之间的相互作用,β-TrCP是一个新的负责TRF1泛素化降解的泛素-蛋白质连接酶,在端粒酶阴性细胞株中,PML3能够特异性的保护TRF1不受β-TrCP的降解,且β-TrCP在调控APB的形成中起重要作用。为深入探讨APB的形成及调控机制提供了新的佐证和线索。本课题的第二部分研究,首次发现了PinX1与Plk1的相互作用,PinX1是Plkl的磷酸化底物蛋白,Plk1通过磷酸化PinX1促进PinX1的泛素化降解,并且PinX1的磷酸化对于细胞有丝分裂的正常进行是必须的,这为深入研究细胞有丝分裂的调控机制提供了新的实验证据和思路。

【Abstract】 Telomere is a specialized nucleoprotein complex which contains repetitive G-rich DNA sequences and partner proteins to protect the chromosomal ends from degradation and to prevent the chromosomal end-to-end fusion. Dysfunction of telomere leads to genome instability and higher incidence of cancer in the aged cells. Although the maintenance of telomere length is mainly performed by a specific reverse transcriptase named telomerase in most malignant cancer cells, telomere-associated proteins are essential for adequate maintenance of telomeric DNA. Telomere-associated proteins directly and indirectly interact with telomeric DNA and contribute to telomere structure and function. Moreover, mounting evidence suggests that telomeric associated proteins may play important roles in cell cycle progression. For examble, expression level of TRF1 is regulated during the cell cycle, besides, it is reported that TRF1 can interact with microtubule, EB1 and some spindle checkpoint proteins such as Madl and Nek2. These results indicate that TRF1 and its partner telomeric interacting proteins may function as important signals in cell cycle.The ubiquitin-proteasome system, the most important protein degradation pathway in eukaryotic cells, regulates a host of critical cellular functions such as cell cycle progression and apoptosis through mediating the selective and time-dependent degradation of short-lived regulatory proteins. Previous studies reported that TRF1 can be ubiquitiated by some E3 ligases such as FBX4 and RLIM, indicating the importance of ubiquitin-proteasome system in regulating telomeric associated proteins.Telomeres can be shorten by 50-150 base pairs (bp) per cell division owing to the end-replication problem of the chromosome, which leads to replicative senescence when the telomere length reaches a critical point in normal somatic cells. Most cancer cells elongate there telomeres by activating telomerase. However, there are some cancer cells cannot activate telomerase and use telomere homologous recombination to elongate telomeres, a mechanism termed alternative lengthening of telomeres (ALT). The presence of ALT-associated PML bodies (APBs) is a hallmark of ALT cells. It has been suggested that APBs may have an integral role in the ALT mechanism, but the precise mechanism of APBs formation remains to be elucidated. Previous studies revealed that TRFl and its sumoylation is essential for the formation of APBs. Our recent study also showed that PML3 can assist the recruitment of TRF1 to APBs and is essential for APBs formation, but the detailed mechanism of the APBs formation is unknown.The ubiquitin-proteasome system is one of the most important protein degradation pathway in eukaryotic cells. Ubiquitination of target proteins is a multistep process. After activation by E1 enzyme, ubiqui tin is transferred to an active cystine of a ubiquitin-conjugating enzyme (E2). A ubiquitin ligase (E3) then transfers ubiquitin from the E2 ubiquitin-conjugating enzyme to the target protein either by forming an E3-ubiquitin thioester intermediate in the case of HECT E3 ubiquitin ligases or by facilitating the transfer of ubiquitin directly from the E2 to the substrate for RING finger E3 ubiquitin ligases. The cullin subunit Cull functions as a molecular scaffold that interacts at the amino terminus with the adaptor subunit Skpl (S-phase kinase-associated protein 1) and at the carboxyl terminus with a RING-finger protein Rbxl and a specific E2 enzyme or ubiquitin conjugating enzyme. The F-box protein β-TrCP function as the variable component that binds Skp1, through the F-box domain, and the substrate, through its WD40 motif.Here, we show thatβ-TrCP interacts with TRF1 and promotes its ubiquition. We first identified 8 novel TRF1 interacting proteins from Hela cell lysates which stably express Flag-TRF1 using immunoprecipitation and protein mass spectrometry. We validated that TRF1 interacts withβ-TrCP in vivo. Overexpression ofβ-TrCP can decrease the expression of TRF1. Moreover, we carried out in vivo ubiquitination assay and the results showed thatβ-TrCP can promote ubiquitination of TRF1 in vivo. Further studies showed that overexpression ofβ-TrCP but not itsΔF-box mutant enhances the ubiquitination of TRF1 and promotes the turnover of endogenous TRF1, whereas depletion ofβ-TrCP decreases TRF1 degradation. Interestingly, we found in telomerase-negative cells, PML3 can specially protect TRF1 from degradation caused byβ-TrCP, andβ-TrCP can negatively regulate the APBs formation through interacting with TRF1. These findings indicate thatβ-TrCP is a novel E3 ligase for TRF1 and may facilitate APBs formation via regulating ubiquitination and degradation of TRF1.Human PinX1 was originally identified as a novel TRF1 interacting protein. PinXl is a widely expressed protein which notably contains two special domains: G-patch domain, which usually in RNA-binding proteins, and the telomerase inhibitor domain (TID). Suppression of telomerase activity by PinX1 is mediated by its direct interaction with hTERT and hTR. However, whether Gno1p, the yeast homolog of human PinX1, can suppress the telomerase ability is controversial.In addition to its telomeric localization and telomerase inhibitory function, PinX1 can be found in the nucleoli of human telomerase positive cells and enhance the accumulation of TRF1 in nucleolus. Moreover, our recent studies show that PinX1 can localize to chromosome periphery and outer plate of kinetochores during mitosis. Depletion of PinX1 results in lagging chromosome in mitosis and micronuclei in interphase. However, the precise regulatory mechanism of PinX1 remains elusive. Here we show that Plkl is a novel interacting protein of PinX1. We performed a yeast two-hybrid assay to search its interacting protein using full-length human PinX1 cDNA as bait. In all total 1×106 clones, we identified 19 positive clones from human testis cDNA library. Nucleotide sequencing revealed that one of these interactors encodes the N terminus of Plkl (21-217 aa). Our biochemical experiments demonstrate that Plkl can interact with and phosphorylate PinX1 in vitro and in vivo. Indirect immunofluorescence staining shows that both Plkl and PinX1 can localize to spindle in mitosis. PinX1 binds to the N-terminal domain of Plk1 through its 92-254 domain. In vitro phosphorylation assay showed that PinX1 is a novel phosphorylation substrate of Plk1. The phosphopeptide of full-length PinX1 was analyzed by MALDI-MS, and three serine and two threonine residues were identified. We generated PinX1 5A mutant which all five serines and threonines were replaced by alanine and phospho-mimicking PinXl mutant which five serines and threonines were replaced by asparagine. Overexpression of wild-type Plkl but not its kinase-defective mutant interrupts the stability of PinX1 through regulating ubiquitin-associated proteolytic degradation of PinX1. Depletion of Plkl by siRNA increases the protein level of PinX1. We validated that Plkl-associated phosphorylation of PinX1 is essential for Plkl-mediated degradation of PinX1. Moreover, expression of GFP-PinX1 resulted in a significant increase in cells bearing misaligned chromosomes. Our studies demonstrate that Plkl interacts with PinX1 and regulates its stability by phosphorylation and such phosphorylation of PinX1 by Plk1 is essential for faithful chromosome congression.

  • 【网络出版投稿人】 浙江大学
  • 【网络出版年期】2011年 07期
节点文献中: 

本文链接的文献网络图示:

本文的引文网络