BRD7核定位信号的结构分析和功能鉴定

目的:对BRD7的核定位信号进行预测、结构分析和功能鉴定,并考察其对BRD7亚细胞定位的影响。方法:通过生物信息学对BRD7的核定位信号进行预测和结构分析,然后利用绿色荧光蛋白(GFP)介导的直接荧光和间接免疫荧光定位方法分别对核定位信号的功能进行鉴定,并考察其对BRD7亚细胞定位的影响。结果:BRD7的65～96位氨基酸残基具有潜在核定位信号(NLS)的结构特征,该核定位信号包含3簇碱性氨基酸残基,可视为由2个紧密相邻、部分重叠的双向核靶序列NLS1和NLS2组成;并发现NLS及其构成上的NLS1和NLS2均具有介导异源蛋白GFP胞核定位的功能,从而证实BRD7的65～96位残基为BRD7功能性核定位信号所在区域,且单簇碱性氨基酸残基的缺失不足以破坏其核定位信号的功能;同时发现野生型BRD7呈胞核分布,而核定位信号缺失型BRD7主要呈胞浆分布。结论:BRD7的65～96位氨基酸残基为BRD7功能性核定位信号所在区域,在BRD7胞核分布模式中发挥了十分重要的作用。     

进入细胞核之门：核移位机制研究进展

细胞中DNA复制和RNA生物形成发生在细胞核,而蛋白质的合成场所位于细胞质,这些生命活动的整合依赖于功能蛋白等在两个亚空间尺度的选择性穿梭.这是一个信号介导的过程,需要能量和可溶性因子如穿梭载体的参与.通过介绍功能蛋白受控核移位机制研究进展,拓宽了其潜在的医学应用,该领域的深入研究,将有力推动抗病毒治疗和基因治疗载体的设计研究.     

Nuclear localization signal-dependent and -independent movements of Drosophila melanogaster dUTPase isoforms during nuclear cleavage

Two dUTPase isoforms (23 kDa and 21 kDa) are present in the fruitfly with the sole difference of an N-terminal extension. In Drosophila embryo, both isoforms are detected inside the nucleus. Here, we investigated the function of the N-terminal segment using eYFP-dUTPase constructs. In Schneider 2 cells, only the 23 kDa construct showed nuclear localization arguing that it may contain a nuclear localization signal (NLS). Sequence comparisons identified a lysine-rich nonapeptide with similarity to the human c-myc NLS. In Drosophila embryos during nuclear cleavages, the 23 kDa isoform showed the expected localization shifts. Contrariwise, although the 21 kDa isoform was excluded from the nuclei during interphase, it was shifted to the nucleus during prophase and forthcoming mitotic steps. The observed dynamic localization character showed strict timing to the nuclear cleavage phases and explained how both isoforms can be present within the nuclear microenvironment, although at different stages of cell cycle.     

Caspase-3细胞定位信号的检测与分析

Caspase-3是凋亡过程中的重要作用蛋白。凋亡过程中,胞质定位的Caspase3被激活并进入细胞核中执行功能,但该定位变化的分子机制至今仍不清楚。分析caspase3中的细胞定位信号可以为深入了解该过程提供重要的线索。我们通过构建一系列含Caspase-3不同区段的截短突变体,与GFP融合表达,观察这些突变体在细胞中的定位,以鉴定Caspase-3中的核外运信号NES（Nuclear Export Signal）和核定位信号NLS（Nuclear Localization Signal）。Caspase-3中不存在明显的核定位信号NLS,但存在一个明显的核外运信号NES,该NES信号定位在caspase-3小亚基的C端（氨基酸220-245）。     

Predicting the nuclear localization signals of 107 types of HPV L1 proteins by bioinformatic analysis

In this study, 107 types of human papillomavirus （HPV） L1 protein sequences were obtained from available databases, and the nuclear localization signals （NLSs） of these HPV L1 proteins were analyzed and predicted by bioinformatic analysis. Out of the 107 types, the NLSs of 39 types were predicted by PredictNLS software （35 types of bipartite NLSs and 4 types of monopartite NLSs）. The NLSs of the remaining HPV types were predicted according to the characteristics and the homology of the already predicted NLSs as well as the general rule of NLSs. According to the result, the NLSs of 107 types of HPV L1 proteins were classified into 15 categories. The different types of HPV L1 proteins in the same NLS category could share the similar or the same nucleocytoplasmic transport pathway. They might be used as the same target to prevent and treat different types of HPV infection. The results also showed that bioinformatic technology could be used to analyze and predict NLSs of proteins.     

The Nucleocytoplasmic Transport of Viral Proteins

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex (NPC), while most proteins are transported by energy driven transport mechanisms. Active transport of viral proteins is mediated by nuclear localization signals (NLS), which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a lar...     

核定位信号肽修饰的抗肿瘤纳米载药体系

纳米载药体系作为一类具有可控性和靶向性的药物递送工具, 可以保护生物分子药物免于细胞内快速酶促降解、免于快速血液清除,确保将生物分子药物安全递送至作用部位,从而有效改善药物的生物利用度,提高药物疗效并降低毒副作用,在生物医学领域具有广阔的应用前景,在功能材料研究和肿瘤靶向治疗研究中受到广泛关注。近年来,通过使用功能性生物分子对纳米载药体系表面进行功能化修饰,进一步改善其生物相容性和药物的生物活性是纳米医学研究的热点。细胞核是很多抗肿瘤物质的主要作用位点,而核定位信号(nuclear localization signal, NLS)肽,作为一类能够识别并定位于细胞核的功能化多肽,能够穿透生物膜并靶向细胞核,被认为是构建纳米载药体系的通用工具。将核定位信号肽用于构建具有核靶向能力的功能化纳米载药体系,在抗肿瘤治疗领域具有重要的应用价值。尽管目前已经开发出多种核靶向功能化纳米载药体系的合成工艺,但是由于制备成本高和合成工艺复杂,将核靶向纳米载体从实验阶段转化到临床阶段还有一段漫长的研究过程。本文重点从核靶向功能化纳米载药体系的组成与构建入手进行阐述,对其入核方式和入核条件进行分析,并针对抗肿瘤纳米载药体系现存的问题对今后发展的方向作出展望。     

Characterization of nuclear localization and nuclear export signals of yeast actin-binding protein Pan1

Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.     

碱性Krppel样因子的亚细胞定位

为进一步加强对碱性Kr櫣ｐｐｅｌ样因子 (basicKr櫣ｐｐｅｌ likefactor,BKLF)的认识 ,用直接荧光和间接荧光两种观察方法证明hBKLF (humanbasicKr櫣ｐｐｅｌ likefactor)定位在细胞核内 ,并且呈点状均匀分布在核质中 ,而在核仁内没有分布。这与许多转录因子 (但不是全部 )的定位方式相似。为确定影响核定位的特异性序列 ,通过观察系列GFP/hBKLF缺失体在细胞内的定位发现 :hBKLF分子的 3个锌指结构和除去这部分的N端区域都有核定位信号的功能 ;hBKLF分子的亚核定位结构位于N端区域 ,包括CtBP结合序列和脯氨酸富含区。这些结果为进一步对BKLF功能的研究提供了基础     

BRD7 suppresses the growth of Nasopharyngeal Carcinoma cells (HNE1) through negatively regulating β-catenin and ERK pathways

BRD7 is a novel gene which involved NPC in our lab. Our previous studies showed that BRD7 was expressed at high level in normal nasopharyngeal epithelial tissues, but at low level in nasopharyngeal carcinoma biopsies and cell lines. In these papers, we found that ectopic expression of BRD7 can decrease cell proliferation and capability to form colonies in soft agar. FCM (Flow cytometry) assay indicated that the cell cycle progression from G1 to S phase was inhibited and the expression of cyclinD1 was significantly decreased after being transfected with BRD7 in HNE1 cells (NPC cells). To further investigate the molecular mechanism of BRD7 suppression of NPC cells growth, the cDNA microarray was performed to detect difference in gene expression profile induced by BRD7. The results indicated that 21 genes expression were changed after being transfected with BRD7 and the differentially expressed gene including α-catenin, cyclinD1, E2F3 was confirmed by western-blot. Next, we found that even though no obvious changes of the total expression of β-catenin were observed, the accumulation of β-catenin in nucleus was blocked. In addition, it was found that the expression of β-catenin was up-regulated in the complex composed of β-catenin and α-catenin in HNE1 cells induction of BRD7. So, we concluded that over-expression of BRD7 increased the expression of α-catenin which “hold” β-catenin in the complex and inhibited its accumulating in nucleus. At last, we demonstrated the c-jun, p-MEK, and p-ERK1/2 expression were down-regulated, and the Ap-1 promoter activity was inactive after being transfected with BRD7. We also found that over-expression of BRD7 can inactivate the c-jun and p-ERK1/2 after being treated with EGF in HNE1 cells. These results indicated that BRD7 played a negative role in ERK1/2 pathway. Taken together, our present results provide new insights for BRD7 function to inhibit NPC cells growth through negative regulating β-catenin and ERK1/2 pathways.     

Nuclear localization of Chfr is crucial for its checkpoint function

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257–259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.