人乳头瘤病毒58型E6蛋白对p53的作用研究

目的 研究HPV58 E6蛋白与p53蛋白的作用关系，以初步探讨其致癌机理。方法 先通过GST沉降试验和体外降解试验，体外研究HPV58 E6结合降解p53蛋白的作用，进而将表达质粒导人SAOS-2细胞，在细胞内研究HPV58 E6对p53诱导细胞凋亡活性的影响。结果 HPV58 E6能够有效结合p53蛋白并进一步诱导其降解，而且在细胞内，HPV58 E6具有抑制p53蛋白的诱导凋亡的功能。结论 p53蛋白的降解影响其细胞周期调控和诱导凋亡的功能，导致细胞发生恶性转化引发子宫颈癌等肿瘤。     

人p100蛋白:可增强STAT6基因转录活性的新因子

目的：建立可稳定表达人类p100蛋白的细胞株，研究细胞内p100蛋白参与STAT6介导的基因转录调控的机制。方法：利用免疫共沉淀法检测蛋白与蛋白问结合，荧光素酶法测定STAT6基因转录活性。结果：p100既可与STAT6结合，亦可与RNA多聚酶Ⅱ结合，并能增强STAT6介导的基因转录活性。结论：p100蛋白是STAT6共激活因子，可桥连STAT6和基本转录调控元件，促进STAT6介导的基因转录活性。     

HPV58 E6体外降解p53蛋白作用的研究

通过PCR方法从有乳头瘤病毒58型（HPV58）全基因克隆中扩增出HPV58E6基因片段，克隆至真核表达质粒pcDNA3的T7启动子下游，并在体外转录成mRNA后，在源自网织红细胞的翻译缓冲液中成功表达了含有生物素标记的HPV58E6蛋白，并在体外成功发现HPV58E6能够降解p53蛋白，从而推断结合并降解p53蛋白是其致癌作用的关键环节，这为日后在细胞内对其致癌机理行进一步研究以及针对其进行治疗奠定下坚实的基础。     

E4orf4蛋白诱导肿瘤细胞凋亡的研究与应用

 腺病毒早期基因编码产物在病毒感染宿主细胞后，广泛参与对宿主细胞周期的调控，以利于病毒自身的复制及释放。其中，腺病毒基因早期转录区4第4开放读码框（Adenovirus early region 4 open reading frame 4，E4orf4）编码的E4orf4蛋白，近几年来逐渐引起人们的重视。E4orf4蛋白是由114个氨基酸组成的小分子蛋白，实验证明，通过基因转染在哺乳动物细胞内表达的E4orf4蛋白在细胞质、细胞骨架和细胞核均有聚集。E4orf4蛋白作为一种多功能蛋白，可以下调病毒诱导的细胞信号传导、在转录和转录后水平调节宿主细胞及病毒蛋白的表达并诱导不依赖于p53的细胞凋亡。目前，对E4orf4蛋白诱导细胞凋亡的作用机制的研究，主要有3个方面：①通过结合和激活蛋白磷酸酶2A（protein phosphatase 2 A，PP2A）来引起细胞凋亡；②通过对Src家族激酶的调节激活胞质凋亡信号；③对肿瘤细胞选择性诱导凋亡的作用。本文主要对E4orf4蛋白诱导细胞凋亡作用的研究进展，以及这种诱导作用对肿瘤细胞的特异性进行综述。......     

e6-ap与蛋白降解

e6-ap基因是HECT E3蛋白家族成员,根据N端的不同分为3个转录本,编码的3种蛋白均具有降解蛋白的功能. E6-AP的功能复杂,主要包括:①降解抑癌基因如p53、pml、tsc2、pdz家族基因的功能;②诱导htert基因启动子激活、提高端粒酶活性的功能;③与C型肝炎病毒的核心蛋白结合,参与rb基因的降解;④双向调节固醇类激素受体的表达;⑤调节ANNEXIN A1蛋白的表达、促进其降解. E6-AP的蛋白表达受泛素及E6的调控,在肿瘤发病机制中起着重要作用.     

周期蛋白G家族——细胞周期的负调节因子

周期蛋白通过与周期蛋白依赖的蛋白激酶结合而调节细胞周期进程，而且多为促进细胞分裂。最近对周期蛋白G1和G2的研究却显示出它们对细胞生长的负调节作用，进一步的研究发现二者都能与丝氨酸，苏氨酸磷酸酶PP2A结合，并与p53/mdm2/ARF功能通路作用相关。     

STAT蛋白结构,功能及其在信号传导中的作用

细胞因子与细胞因子受体诱导的细胞内信号传导调控着一系列细胞的功能,其中ＳＴＡＴ蛋白ＪＡＫ－ＳＴＡＴ信号通路中起着关键作用,ＳＴＡＴ蛋白激活,二聚体化,向核内转移后,与特异的ＤＮＡ序列结合,指导基因的转录。近年来对ＳＴＡＴ蛋白结构,功能其在信号传导的作用有了较深入的研究,本就这些方面的研究进展加以综述。     

ARF蛋白——p53蛋白的重要调控因子

ARF蛋白是INK4a基因位点编码产物之一。ARF能抑制MDM2介导的p5 3降解和p5 3转录下调 ,某些癌基因和调节因子能通过ARF蛋白调节p5 3蛋白的功能 ,从而对细胞周期进程产生影响。在黑色素瘤、血液系统肿瘤、肺癌、肠道肿瘤中均发现有ARF的突变或缺失 ,ARF蛋白在肿瘤发生发展中可能有重要作用。     

多功能蛋白p62调节细胞自噬及相关疾病的研究进展

Sequestosome 1 （p62/SQSTM1）是一种由应激诱导产生的细胞内蛋白并作用于选择性自噬的多功能蛋白.p62包含多种蛋白作用域,可结合聚泛素化蛋白并将其进一步降解,在信号转导过程中发挥重要作用.研究表明,p62参与多种疾病的病理过程,如神经退行性疾病、糖尿病、肥胖甚至是癌症.因而从p62的结构和功能这两方面阐述其在相关疾病中的作用很有必要.     

周期蛋白G家族--细胞周期的负调节因子

周期蛋白通过与周期蛋白依赖的蛋白激酶结合而调节细胞周期进程,而且多为促进细胞分裂.最近对周期蛋白G1和G2的研究却显示出它们对细胞生长的负调节作用,进一步的研究发现二者都能与丝氨酸/苏氨酸磷酸酶PP2A结合,并与p53/mdm2/ARF功能通路作用相关.     

Papillomavirus-associated inductions of cellular proteins in mouse C127 cells: correlation with the presence of open reading frame E2

Bovine papillomavirus type 1 (BPV-1) readily transforms mouse C127 cells, conferring the ability to grow in soft agar and to form tumors in athymic (nu/nu) mice. Electrophoresis of total cellular proteins from these BPV-transformed lines on ultra-high resolution, giant two-dimensional gels displays the presence of novel, papillomavirus-related protein phenotypes. Analysis of the established BPV-1-transformed C127 cell lines, ID13 and ID14, reveals a set of six proteins which are either absent or synthesized at extremely low levels in the parental cell line. One of these proteins is also present in v-ras-transformed C127 cells, but none of the others are found in cells transformed by a variety of viral oncogenes, including the polyomavirus middle T, v-mos, or v-fes. The genome of BPV-1 contains two separate open reading frames (ORFs), E5 and E6, which can act independently to transform C127 cells. In addition, trans-activator and repressor proteins encoded respectively by the full-length and carboxy-terminal E2 ORF regulate the level of expression of other BPV-1 genes. We examined 34 cell lines transformed by intact and subgenomic recombinant DNAs of BPV-1. Cells harboring BPV-1 DNAs engineered to eliminate the expression of ORFs E4, E5, E6, or E7 display five of the PV-associated proteins, but these proteins are not seen in lines lacking the full E2 ORF. Moreover, G418-selected nontransformed cells expressing E2 cDNA from an SV40 promoter exhibit these proteins at high levels. Surprisingly, these proteins are also present in cells containing BPV-1 DNAs with amino-terminal E2 deletions, suggesting that these PV-associated proteins represent novel cellular responses to a factor encoded within the E2-C gene region.     

Are transforming properties of the bovine papillomavirus E5 protein shared by E5 from high-risk human papillomavirus type 16?

The E5 proteins of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 16 (HPV-16) are small (44-83 amino acids), hydrophobic polypeptides that localize to membranes of the Golgi apparatus and endoplasmic reticulum, respectively. While the oncogenic properties of BPV-1 E5 have been characterized in detail, less is known about HPV-16 E5 due to its low expression in mammalian cells. Using codon-optimized HPV-16 E5 DNA, we have generated stable fibroblast cell lines that express equivalent levels of epitope-tagged BPV-1 and HPV-16 E5 proteins. In contrast to BPV-1 E5, HPV-16 E5 does not activate growth factor receptors, phosphoinositide 3-kinase or c-Src, and fails to induce focus formation, although it does promote anchorage-independent growth in soft agar. These variant activities are apparently unrelated to differences in intracellular localization of the E5 proteins since retargeting HPV-16 E5 to the Golgi apparatus does not induce focus formation.     

Competitive binding to a charged leucine motif represses transformation by a papillomavirus E6 oncoprotein

E6 oncoproteins from HPV-16 and bovine papillomavirus type 1 (BPV-1) bind to similar leucine-rich peptides termed charged leucine motifs found on the cellular focal adhesion protein paxillin and the E3 ubiquitin ligase E6AP. BPV-1 E6 (BE6) mutants that do not bind to paxillin are defective at inducing cellular transformation. It is possible, however, that BE6 mutants that do not bind paxillin are defective for transformation for an unrelated reason than the ability to bind to charged leucine motifs. To address the role of BE6 interaction with charged leucine motifs, we fused a BE6-binding charged leucine motif to the amino terminus of BE6, thereby creating an autoinhibitory binding domain. We found that the fusion protein failed to bind to paxillin or transform murine C127 cells. Mutation of the amino terminal binding motif in the fusion protein restored both interaction with paxillin and transformation. This demonstrates that BE6 transformation requires binding to charged leucine motifs on particular cellular proteins and that transformation by papillomavirus oncoproteins can be repressed by competitive interactions with charged leucine motifs.     

Genus specific features of bovine papillomavirus E6, E7, E5 and E8 proteins

Six bovine papillomavirus (BPV) types, BPV-1 to -6, have been classified in genera Delta-papillomavirus (BPV-1 and -2), Epsilon-papillomavirus (BPV-5) and Xi-papillomavirus (BPV-3, -4 and -6). In addition, 16 unclassified putative BPV types have been reported. In the present study, we characterized genus specific features of E6, E7, E5 (formerly E8) and E8 proteins of seven putative BPV types, BAPV-1, -2, -3, -4 and -10, BAA-5 and BPV-3c. These putative BPV types were classified in genera Epsilon- or Xi-papillomavirus. The E6 proteins of BPV and putative BPV types in Epsilon-papillomavirus showed high sequence similarities, and contained two typical zinc-binding domains. However, E7 proteins contained atypical zinc-binding domains, and lacked the canonical retinoblastoma tumor suppressor protein (pRB)-binding motif. BPV and putative BPV types in Xi-papillomavirus contained E5 or E8 open reading frame (ORF) in the E6 position. The E5 ORFs encoded proteins consist of 42-amino acid with a hydrophobic transmembrane and a hydrophilic C-terminal domain. But the E8 ORFs encoded protein which have two transmembrane domains. Our results demonstrated that E5, E8, E6, E7 proteins of the putative BPV types, which are presumably classified in genera Epsilon- or Xi-papillomavirus, retained the some genus specific features.     

Similarities and differences between the E5 oncoproteins of bovine papillomaviruses type 1 and type 4: cytoskeleton, motility and invasiveness in E5-transformed bovine and mouse cells

Bovine papillomaviruses (BPVs) are oncogenic viruses. In cattle, BPV-1/2 is associated with urinary bladder cancer and BPV-4 with upper GI tract cancer. BPV E5 is a small hydrophobic protein localised in the endoplasmic reticulum (ER) and Golgi apparatus (GA). E5 is the major transforming protein of BPVs, capable of inducing cell transformation in cultured mouse fibroblasts and, in cooperation with E7, in primary bovine cells. E5-induced cell transformation is accompanied by activation of several cellular protein kinases, including growth factor receptors, and alkalinisation of endosomes and GA. We have reported that BPV E5 causes swelling and fragmentation of the GA and extensive vacuolisation of the cytoplasm. We now show that E5 from both BPV-1 and BPV-4 disturbs the actin cytoskeleton and focal adhesions in transformed bovine cells, where these morphological and behavioural characteristics are accompanied by hyperphosphorylation of the cellular phosphotyrosine kinase c-src. Both BPV-1 and BPV-4 E5 increase the motility of transformed mouse cells, but only BPV-1 E5 causes transformed mouse cells to penetrate a matrigel matrix. BPV-1 transformed mouse cells, but not BPV-4 transformed mouse cells, have hyperhpsphorylated c-src.     

The E5 Protein of HPV-6, but Not HPV-16, Associates Efficiently with Cellular Growth Factor Receptors

The transforming activity of the prototype E5 protein of bovine papillomavirus type 1 (BPV-1) is associated with its binding to, and activation of, both the platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. The E5 proteins of human papillomavirus types 6 and 16 (HPV-6, HPV-16) also transform rodent cells in the presence of the EGF receptor. In this study we examined whether epitope-tagged HPV E5 proteins could associate with three different tyrosine kinase-containing growth factor receptors: the EGF receptor, the erbB2 receptor, and the PDGF receptor. The HPV-6 E5 protein was found to associate efficiently with all three of these growth factor receptors, while the HPV-16 E5 protein did not. These findings suggest either that the in vitro transforming activities of HPV-6 and HPV-16 E5 proteins involve a similar mechanism unrelated to receptor binding (e.g., binding to the 16-kDa membrane pore protein) or that they proceed along distinct pathways, with receptor binding being important for HPV-6. Regardless of the ultimate mechanisms, the differences between the HPV-6 and HPV-16 E5 proteins in binding to growth factor receptors may potentially contribute to the distinctive morphologies of their respective neoplastic lesions.     

Complete genomes and phylogenetic positions of bovine papillomavirus type 8 and a variant type from a European bison

The DNA of bovine papillomavirus (BPV) type 8 was extracted from papillomas on cattle kept in Japan, and DNA of bovine papillomavirus BPV-8-EB was extracted from a European bison (Bison bonasus) born in Italy and released into the wild in Slovakia. The DNA genomes of these BPVs were amplified using multiply primed rolling circle amplification and polymerase chain reaction, then characterized by direct sequencing method. The BPV-8 and BPV-8-EB genomes consisted of 7,791 base pairs (bp) and 7,773 bp, respectively (GenBank accession numbers DQ098913 and DQ098917). The nucleotide sequence similarity of these BPVs indicated that BPV-8-EB was a variant of BPV-8. In the genome of BPV-8-EB, one nucleotide substitution was found in the E2 and E5 open reading frame (ORF) and upstream regulatory region (URR), and a short deletion and addition were found in the URR. The high similarity of sequences between the BPV-8 to BPV-5 in total genome (70%) and L1 ORF (75%) as well as a phylogenetic analysis were the bases for classifying BPV-8 in the genus Epsilon papillomavirus. The BPV-8 E6 and E7 ORFs/proteins also showed some characteristic features of genus Epsilon papillomavirus. However, BPV-8 contained E4 ORF, which was not found in BPV-5. In addition, the secondary structure of E5 proteins of BPV-5 and BPV-8 suggested that these proteins may have cell-transforming ability.