【作者】 余建；

【导师】 黄河；

【作者基本信息】 浙江大学 ， 内科学， 2008， 博士

【摘要】 端粒是存在于真核生物线性染色体末端由核酸DNA重复序列和蛋白质组成的特殊保护性结构,端粒功能异常将导致细胞衰老和染色体不稳定性,是人类肿瘤发生的机制之一。中心体是高等真核生物细胞的主要微管组织中心,在细胞有丝分裂过程中起着至关重要的作用,与双极纺锤体的形成、纺锤体的定向和胞质分裂直接相关。中心体异常将损害细胞分裂的忠实性并导致染色体不稳定性。从低等生物到人都有实验证据证明端粒与细胞周期和有丝分裂之间存在密切联系,但目前具体介导端粒与细胞分裂之间相互关系的信号分子及功能尚不是很清楚。近年来研究发现TRF1在有丝分裂中期转位于纺锤体极点/中心体部位,而过量表达TRF1将促进细胞进入有丝分裂,提示TRF1可能是介导端粒和细胞分裂之间关系的信号分子。在前期研究中,本课题组分离鉴定了一个新的TRF1相互作用蛋白并对该蛋白的细胞生物学功能进行了研究。结果显示该蛋白在细胞有丝分裂期定位于中心体,而siRNA基因干扰该蛋白会导致多极纺锤体形成,同时影响TRF1及Tankyrase1在中心体上的定位。根据该蛋白在有丝分裂期细胞的中心体定位以及siRNA敲除后对双极纺锤体形成的影响,我们将该蛋白命名为端粒相关的中心体蛋白1(Telomere associated centrosomal protein 1,TACP1)。为更清楚的阐明TACP1在联系端粒和细胞有丝分裂中扮演的角色,本课题第一部分研究以TACP1全长为诱饵进行酵母双杂交筛库,试图寻找TACP1新的相互作用蛋白。通过酵母双杂交,我们得到23个可能的TACP1相互作用蛋白,生物信息学分析显示其中KIAA0649和HECTD3具有进一步研究的意义。进一步的酵母共转及体外Pull-down实验证实TACP1能和HECTD3 1-75相互作用。泛素—蛋白酶体途径是目前已知的所有真核生物体内具有高度选择性的最为重要的蛋白降解途径,它通过选择性的降解蛋白,精确调控细胞内蛋白质的量,进而对许多重要的细胞生理功能(如细胞周期调控、凋亡等)起到调节作用。许多中心体蛋白表达水平随细胞周期进展而变化,其功能受到它们表达和降解的调控,而泛素—蛋白质酶体途径作为细胞内最主要的蛋白质降解途径,细胞周期相关蛋白多数通过这条途径降解。前期我们的实验发现TACP1作为一个新的端粒相关中心体蛋白,是一个细胞周期性调节蛋白。为明确TACP1降解机制,本课题第二部分研究应用免疫共沉淀结合Western免疫印迹技术检测TACP1在体内是否发生泛素化修饰。结果显示TACP1在体内能发生泛素化修饰,而加入蛋白酶体抑制剂MG132后,TACP1泛素化修饰条带更为明显,说明TACP1在体内通过泛素—蛋白酶体途径降解。泛素—蛋白酶体途径最重要的特征就是底物的多样性和选择性,而这一功能是由泛素—蛋白连接酶E3s决定的。鉴于生物信息学分析显示HECTD3是一个含有HECT结构域的泛素—蛋白连接酶,其很可能是负责TACP1泛素化修饰的泛素—蛋白连接酶。为证实这个假设,本课题组设法得到HECTD3的全长,深入研究HECTD3对TACP1泛素化降解及功能的影响。酵母共转及生化实验证实TACP1和HECTD3存在相互作用,TACP1通过羧基端(332-476)和HECTD3相互作用,而HECTD3通过N端和TACP1相互作用;进一步研究显示HECTD3具有促进TACP1泛素化修饰的作用;HECTD3对体内TACP1蛋白的量具有负调控的作用,且该作用和其泛素—蛋白连接酶活性有关;而利用siRNA基因干扰抑制HECTD3蛋白表达将导致多极纺锤体的出现。综上所述,本部分研究证明TACP1通过泛素—蛋白酶体途径降解,而HECTD3作为负责TACP1泛素化修饰的泛素—蛋白连接酶,通过调控TACP1降解进而影响TACP1的功能,共同参与双极纺锤体的形成。通过上述研究,基本阐明了TACP1降解的具体机制,为深入理解TACP1在细胞有丝分裂特别是维持中心体稳定性中的作用提供新的实验佐证。由于DNA聚合酶末端复制问题,每次细胞分裂端粒就会丢失50-150 bp,当端粒缩短至一定长度时(约4～5kb),细胞将不可逆地退出细胞周期进入复制性衰老继而死亡,因此端粒被认为是细胞的“有丝分裂钟”,其长度直接反映了细胞的增殖分裂潜能。为获得永生化的能力,绝大多数肿瘤细胞和永生化细胞系通过激活端粒酶来维持端粒长度,但部分神经上皮或间充质来源的肿瘤细胞如骨肉瘤、星形细胞瘤、里—费综合症,其端粒酶检测阴性。这些肿瘤细胞通过激活端粒延伸替代机制(Alternative lengthening of telomeres,ALT)从而弥补随细胞分裂而逐渐缩短的端粒长度。在ALT细胞中,存在一种特异性的ALT相关的PML小体(ALT-associated PML bodies,APBs)。ALT途径通过染色体重组来维持端粒长度,而APBs又是端粒重组的场所,故完整的APBs结构在ALT途径维持肿瘤细胞端粒长度,保持细胞永生化中扮演着重要角色,但目前有关APBs形成的具体机制尚不清楚。最近研究发现TRF1以及TRF1的SUMO化修饰和APBs的形成有关,但TRF1究竟是如何定位于APBs上,尚不清楚。PML蛋白作为PML核小体(PML nuclear bodies,PNBs)最主要的组份,在PML核小体的形成中起到重要作用。根据PML基因外显子剪接方式的不同,PML共有7个拼接异构体PML1～7。目前有关PML各个异构体的研究尚不多,主要集中在PML3和PML4这两个异构体。研究显示,PML3能够将P53招募到PML核小体上进而促进P53的转录活性。鉴于PML3的作用,我们设想在ALT细胞中TRF1通过和PML3的相互作用而被招募到PML核小体上。为证明这个假设,在本课题第三部分研究中,我们首先通过生化实验证实PML3和TRF1在体内外均有相互作用。进一步免疫荧光实验显示在ALT细胞中,TRF1和PML3共定位于APBs;TRF1在APBs上的定位和细胞周期密切相关,以G2/M期最高;外源性过表达PML3能招募TRF1上PML核小体;而利用siRNA基因干扰抑制内源性PML3蛋白表达,不仅会抑制TRF1在APBs上的定位,对TRF2在APBs上的定位也有影响,提示PML3对于APBs的形成至关重要。本研究首次发现PML3具有招募TRF1上PML核小体的作用,且PML3对于APBs的形成至关重要,为阐明ALT相关PML小体形成的分子机制提供新的实验证据。综上所述,本研究可分为两个部分。在第一、二部分研究中,我们首次发现TACP1通过泛素—蛋白酶体途径降解,而HECTD3作为负责TACP1泛素化修饰的泛素—蛋白连接酶,通过调控TACP1降解进而影响TACP1的功能,共同参与双极纺锤体的形成。通过研究,基本阐明了TACP1降解的具体机制,为深入理解TACP1在细胞有丝分裂特别是维持中心体稳定性中的作用提供新的实验佐证。而本课题第三部分研究首次发现PML3具有招募TRF1上PML核小体的作用,且PML3对于APBs的形成至关重要,为阐明ALT相关PML小体形成的分子机制提供新的实验证据。更多还原

【Abstract】 Telomeres are specialized protective structures at the extreme ends of linearly eukaryotic chromosomes.Telomere dysfunction is associated with chromosomal instability which is implicated in ageing and carcinogenesis.Centrosomes,the principal microtubule organizing center(MTOC)in nearly all higher eukaryotic cells, have a pivotal role in regulating cell division in mitotic cells.During mitosis, centrosomes govern assembly and orientation of the bipolar mitotic spindle that is essential for correct chromosome segregation and cytokinesis.Centrosome aberrations may compromise the fidelity of cell division and cause chromosomal instability.There is compelling evidence to suggest a strong link between telomere maintenance and cell cycle control,especially mitotic regulation.However,little is known about the identity and function of the signaling molecule(s)connecting telomere maintenance and mitotic regulation.Recent studies showed that TRF1 translocates to centrosome and/or spindle pole during mitosis and TRF1 overexpression induces mitotic entry in cells with short telomeres,suggesting the role of TRF1 in mitotic regulation by providing a functional link between telomere and centrosome.In our previous study,a novel TRF1 interacting protein was identified from mitotic HeLa cell lysates by employing immunoaffinity isolatin and mass spectrometry(MS).We refer to the protein as Telomere Associated Centrosomal Protein 1(TACP1)since it distinguishes from other TRF1 binding proteins and traffics toward centrosome during mitosis.Further studies revealed that depletion of TACP1 by small interfering RNA leads to multipolar spindle formation and abolishes the centrosomal localization of TRF1 and Tankyrase 1.To further elucidate the role of TACP1 in connecting telomere maintenance and mitotic regulation,we performed a yeast two-hybrid screen with TACP1 as a bait and identified 23 novel TACP1-binding proteins including KIAA0649 and HECTD3. Further yeast co-transformation assay and pull-dwon assay confirmed the interaction between TACP1 and HECTD3 1-75.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.Many centrosomal proteins are regulated in a cell cycle-dependent fashion,and most of them are degraded through ubiqultin-proteasome pathway.Our previous studies showed that TACP1,as a novel telomere associated centrosomal protein,is a cell cycle-regulated protein.To clarify the degradation mechanism of TACP1,we carded out in vivo ubiquitination assay to determine whether TACP1 is ubiquitinated in vivo.The results showed that TACP1 is ubiquitinated in vivo and treatment of cells with the proteasome inhibitor MG132 causes a robust increase of TACP1 ubuiquitination,suggesting that TACP1 is the substrate for proteasome.In the ubiquitin-proteasome system,the selectivity of substrate appears to be determined through interaction between specific ubiquitin-protein ligase(E3)and substrate.According to computational analysis,we hypothesized that HECTD3,a putative member of HECT E3 ubiquitin ligases,is a specific E3 ligase for TACP1.To confirm this hypothesis,we first validated that TACP1 interacts with HECTD3 in vitro and in vivo.Further studies showed that HECTD3 overexpression enhances the ubiquitination of TACP1 and promotes the turnover of TACP1,where depletion of HECTD3 decreases TACP1 degradation. More notably,depletion of TACP1 leads to multipolar spindle formation.All these findings suggest that HECTD3 is a specific E3 ligase for TACP1 and may facilitate cell cycle progression via regulation ubiquitination and degradation of TACP1.In normal somatic human cells,telomeres shorten by 50-150 base pairs(bp)per cell division owing to the end-replication problem of the lagging strand,which ultimately leads to replicative senescence when the telomere length reaches a critical point(4～5 kb).Therefore,telomeres have been proposed to act as a counting mechanism for cellular proliferation.To achieve unlimited replicative potential,most cancer cells activate telomerase to elongate telomeres.However,some cancer cells, including sarcomas,astrocytomas,and tumors of Li-Fraumeni syndrome,cannot activate telomerase and use telomere homologous recombination to elongate telomeres,a mechanism termed altemative lengthening of telomeres(ALT).A hallmark of ALT cells is the presence of ALT-associated PML bodies(APBs).It has been suggested that APBs may have an integral role in the ALT mechanism,but the exact mechanism of APBs formation remains unclear.Recent studies revealed that TRF1 and its sumoylation is essential for the formation of APBs,but the detailed mechanism of the recruitment of TRF1 to APBs is unknown.PML,as the most prominent component of PML nuclear bodies(PNBs),has a fundamental role in assembly of PML nuclear bodies.According to C-terminal sequence variation,seven PML isoforms have been identified.Up to date,most studies on these isoforms concentrate on PML3,which has been shown to recruit P53 to PML nuclear bodies and increase its transcriptional activity.Taking the function of PML3,we hypothesized that PML3 may assist the recruitment of TRF1 to APBs.To confirmed this,we first validated the interaction between PML3 and TRF1 by co-immunoprecipitation and GST pull-down assays. Immunofluorescence studies showed that TRF1 colocalizes with PML3 at APBs in a subset of ALT cells and the percentage of cells with co-localization of TRF1 with APBs greatly increased in cells enriched in G2/M.Further studies revealed that overexpression of PML3 strengthens the localization of TRF1 to PML nuclear bodies, while depletion of PML3 inhibits the recruitment of TRF1 and TRF2 to APBs.Taken together,our results suggest that PML3 interacts with TRF1 and is essential for APBs formation.更多还原