Subcellular localization and control of activity of telomerase

由于端粒酶活性在原代细胞中几乎检测不到，但在癌细胞中可以明显检测到，因此端粒酶被认为是癌症治疗的强有力的候选靶点。在本财年，我们集中进行了几项实验，包括重组人端粒酶复合物的纯化和表征，以及从稳定表达FLAG-hTERT的HeLa细胞系中恢复的核蛋白与hTERT特异性相互作用的生物学功能（Murakami）。hTERT永生化人原代细胞的分析（Honda），以及在细胞增殖和分化中起关键作用的受体类型的酪氨酸激酶（Yoshioka）。实验的主要结论如下：1)部分纯化的活性端粒酶复合物包括复合物I（约680 kDa）和复合物II（约400 kDa）两种不同的复杂性，它们与共表达FLAG-hTERT和hTERC的昆虫细胞可以回收的复合物非常相似。com . More复合体I不含Hsp90，对Hsp90抑制剂有耐药性，而复合体II含有Hsp90，对Hsp90抑制剂敏感。人TEAT复合物II在vim和ring中孵育期间相当不稳定。这种不稳定的性质在配合物i中没有观察到，因为MG132是一种特异性蛋白酶体抑制剂，而阴性对照MG133没有稳定配合物Z中的hTERT，这强烈表明If中的hTERT在蛋白酶体的控制下处于降解途径。人类端粒酶的两种不同的复杂性似乎暗示了端粒酶活性的两种不同实体在不同的亚细胞定位或其不同的功能。2)建立了稳定表达hTERT的人原代成纤维细胞（BJ细胞）细胞系，并比较了经MERE永生化的RI细胞和KJ细胞的表达谱，其中一些与细胞周期进程和细胞增殖有关的基因在永生化的131-hTERT中的表达明显高于原代细胞。3)核仁蛋白是hTERT特异性相互作用伙伴之一，可结合HCV依赖RNA复制的丙型肝炎病毒（HCV） NS5B。我们评估了核仁蛋白和NS5B特异性相互作用在HCV复制中的作用。我们发现，在HepG2 ml/s的瞬时HCV复制系统中，含有丙氨酸替代突变或在NS5B中两个关键序列之一的残基突变的HCV亚复制子根本无法复制，这两个突变都是核蛋白结合所必需的。尽管野生复制子可能支持高效HCV。通过引入核仁蛋白的瞬时RNAi，将核仁蛋白的表达下调了一半到三分之一，显著降低了HCV的表达，利用HCV亚复制子系统降低了HCV的复制。综上所述，核仁蛋白和NS5B的特异性相互作用对HCV复制至关重要。少

Since telomerase activity is barely detectable in primary cells but clearly detected in cancerous cells, telomerase has been considered to be a strong candidate of the targets to cancer treatment In the fiscal year, we concentrated in several experiments including purification and characterization of recombinant human telomerase complex, and biological function of the specific interaction of nucleolin and hTERT recovered from a HeLa cell line stably expressing FLAG-hTERT (Murakami), analyses of human primary cells immortalized by hTERT (Honda), and the tyrosine kinases of receptor type which play critical odes in cell proliferation and differentiation (Yoshioka). The followings are the main conclusions of the experiments.1) The partially purified active telomerase complexes comprise two different complexities, complex I (around 680 kDa), and complex II (around 400 kDa), that are quite alike to those that can be recovered firm the insect cells co-expressing FLAG-hTERT and hTERC. The com … More plex I does not contain Hsp90 and is resistant to Hsp90 inhibitors, whereas complex II contains Hsp90 and is sensitive to Hsp90 inhibitors. Human TEAT in the complex II is rather unstable during incubation in vim and in ring. Such unstable property was not observed with the complex I. Since MG132, a specific proteasome inhibitor, but not MG133, a negative control, stabilized hTERT in the complex Z strongly suggesting that hTERT in the complex If is under degradation pathway under the control of proteasome. The two different complexities of human telomerase seem to imply two different entities of active telomerase in different subcellular localization or its different functions. 2) We have established a cell line of the human primary fibroblasts(BJ cells) stably expressing hTERT, and we compared expression profiles of RI cells and the KJ cells immortalized by MERE Some genes involving in cell cycle progression and cell proliferation were evidently expressed higher in the immortalized 131-hTERT than these in in the primary cells. 3) Nucleolin, one of the specific interacting partners of hTERT can bind to Hepatitis C Virus (HCV) NS5B, RNA-dependent RNA replication of HCV. We evaluated the role of the specific interaction of nucleolin and NS5B in HCV replication. The HCV subreplicon system has been applied for the address We found that the HCV subreplicons harboring alanine substitution mutations or mutation at the residue(s) either one of two critical sequences within NS5B both of which are necessary for the nucleolin-binding could not replicate at all in a transient HCV replication system in HepG2 ml/s., although the wild replicon could support efficient HCV. By introducing transiently RNAi of nucleolin, that down regulated expression of nucleolin by half to one third, reduced HCV evidently reduced HCV replication using the HCV subreplicon system. Taken together, the specific interaction of nucleolin and NS5B is critical for HCV replication. Less