Papillomavirus E6 PDZ Interactions Can Be Replaced by Repression of p53 To Promote Episomal Human Papillomavirus Genome Maintenance

Cancer-associated human papillomaviruses (HPVs) express E6 oncoproteins that target the degradation of p53 and have a carboxy-terminal PDZ ligand that is required for stable episomal maintenance of the HPV genome. We find that the E6 PDZ ligand can be deleted and the HPV genome stably maintained if cellular p53 is inactivated. This indicates that the E6-PDZ interaction promotes HPV genome maintenance at least in part by neutralization of an activity that can arise from residual undegraded p53.     

Peptide Interactions Stabilize and Restructure Human Papillomavirus Type 16 E6 To Interact with p53

Human papillomavirus type 16 (HPV-16) E6 (16E6) binds the E3 ubiquitin ligase E6AP and p53, thereby targeting degradation of p53 (M. Scheffner, B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley, Cell 63:1129–1136, 1990). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule.     

VX-680 Inhibits Aurora A and Aurora B Kinase Activity in Human Cells

VX-680, also known as MK-0457, is a member of a diverse group of small molecules that inhibit the Aurora kinases, and has shown significant potential as an anti-cancer agent. In keeping with many protein kinase inhibitors, this compound is not a monospecific agent, and its cellular specificity remains largely unknown. In cells, VX-680 blocks mitotic Histone H3 phosphorylation and induces polyploidy and apoptosis, consistent with inhibition of the mitotic protein kinase Aurora B. In this study, we have investigated the effects of VX-680 in proliferating human cancer cells, and demonstrate that it blocks the phosphorylation and activation of both Aurora A and B. Additionally, VX-680 suppresses the phosphorylation of specific substrates of each enzyme, including the Aurora A target TACC3 on Ser558. Exposure to VX-680 induces a monopolar spindle phenotype, delays mitotic progression and rapidly overrides the spindle assembly checkpoint in the presence of spindle poisons. VX-680 also exhibits potent cytotoxicity when compared to the well documented Aurora B inhibitor ZM447439. Taken together, these data identify Aurora A and Aurora B as dual intracellular targets of VX-680.     

The Human Papillomavirus Type 16 E6 Gene Alone Is Sufficient To Induce Carcinomas in Transgenic Animals

High-risk human papillomaviruses (HPVs) are the causative agents of certain human cancers. HPV type 16 (HPV16) is the papillomavirus most frequently associated with cervical cancer in women. The E6 and E7 genes of HPV are expressed in cells derived from these cancers and can transform cells in tissue culture. Animal experiments have demonstrated that E6 and E7 together cause tumors. We showed previously that E6 and E7 together or E7 alone could induce skin tumors in mice when these genes were expressed in the basal epithelia of the skin. In this study, we investigated the role that the E6 gene plays in carcinogenesis. We generated K14E6 transgenic mice, in which the HPV16 E6 gene was directed in its expression by the human keratin 14 promoter (hK14) to the basal layer of the epidermis. We found that E6 induced cellular hyperproliferation and epidermal hyperplasia and caused skin tumors in adult mice. Interestingly, the tumors derived from E6 were mostly malignant, as opposed to the tumors from E7 mice, which were mostly benign. This result leads us to hypothesize that E6 may contribute differently than E7 to HPV-associated carcinogenesis; whereas E7 primarily contributes to the early stages of carcinogenesis that lead to the formation of benign tumors, E6 primarily contributes to the late stages of carcinogenesis that lead to malignancy.     

The Role of Protein Kinase A Regulation of the E6 PDZ-Binding Domain during the Differentiation-Dependent Life Cycle of Human Papillomavirus Type 18

Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.     

Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with Proapoptotic Kinase Mst1 to Promote Cardiomyocyte Apoptosis

Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.     

shRNA抑制宫颈癌Hela细胞株HPV18 E6、E7基因表达的研究

应用短发夹RNA(Short hairpin RNA,shRNA)表达载体抑制宫颈癌Hela细胞株HPV18 E6、E7基因的表达。应用已鉴定的shRNA表达载体pHPV1、pHPV2转染Hela细胞,G418筛选阳性细胞,建立稳定转染细胞株;倒置荧光显微镜检测转染情况;提取细胞内总RNA,RT-PCR方法检测HPV18 E6、E7 mRNA;WesternBlot检测HPV18 E6、E7蛋白表达的变化;采用灰度分析软件对PCR扩增条带与蛋白质条带进行灰度分析。pHPV1实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的31%、38%,E6、E7蛋白分别为阴性对照组的37%、31%;pHPV2实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的54%、77%,E6、E7蛋白分别为阴性对照组的52%、83%。pHPV1、pHPV2表达载体能抑制Hela细胞HPV18 E6、E7的表达,针对外显子区434-452的pHPV1抑制作用更强。     

Human Papillomavirus Oncoproteins E6 and E7 Independently Abrogate the Mitotic Spindle Checkpoint

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G₁/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.     

Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival

The Syk protein tyrosine kinase, a well-characterized regulator of immune cell function, plays an increasingly recognized role in tumorigenesis as a promoter of cell survival in both hematological and nonhematological malignancies. We show here that the expression of Syk in MCF7 or MDA-MB-231 breast cancer cells or in DG75 B-lymphoma cells protects cells from apoptosis induced by oxidative or genotoxic stress by stabilizing the mRNA for Bcl-x_L, an antiapoptotic protein. Syk binds robustly to nucleolin and phosphorylates it on tyrosine, enhancing its ability to bind the Bcl-x_L mRNA. Consequently, reducing the level of nucleolin by RNA interference attenuates the ability of Syk to protect cells from stress-induced cell death.     

The Putative Coupling Protein TcpA Interacts with Other pCW3-Encoded Proteins To Form an Essential Part of the Conjugation Complex

Conjugative plasmids encode antibiotic resistance determinants or toxin genes in the anaerobic pathogen Clostridium perfringens. The paradigm conjugative plasmid in this bacterium is pCW3, a 47-kb tetracycline resistance plasmid that encodes the unique tcp transfer locus. The tcp locus consists of 11 genes, intP and tcpA-tcpJ, at least three of which, tcpA, tcpF, and tcpH, are essential for the conjugative transfer of pCW3. In this study we examined protein-protein interactions involving TcpA, the putative coupling protein. Use of a bacterial two-hybrid system identified interactions between TcpA and TcpC, TcpG, and TcpH. This analysis also demonstrated TcpA, TcpC, and TcpG self-interactions, which were confirmed by chemical cross-linking studies. Examination of a series of deletion and site-directed derivatives of TcpA identified the domains and motifs required for these interactions. Based on these results, we have constructed a model for this unique conjugative transfer apparatus.Conjugation systems are important contributors to the dissemination of antibiotic resistance determinants and virulence factors. Extensive analysis of conjugative plasmids from gram-negative bacteria has led to the elucidation of a general mechanism of conjugative transfer (10, 22). In this process, the transferred DNA is processed by components of a relaxosome complex. Specifically, the DNA is nicked at the origin of transfer (oriT) by a relaxase, which remains covalently coupled to the transferred DNA strand. The single-stranded DNA complex then interacts with the coupling protein, a DNA-dependent ATPase that provides the energy to actively pump the DNA through the mating pair formation (Mpf) complex into the recipient cell (36). The coupling protein interacts with both DNA processing proteins and components of the Mpf complex (1, 4, 12, 35, 38). These interactions have been demonstrated using bacterial and yeast two-hybrid approaches as well as gel filtration, pull-down, and coimmunoprecipitation studies.The mechanism of conjugative transfer has yet to be precisely determined for conjugative plasmids from gram-positive bacteria although bioinformatics analysis has identified similar gene arrangements and conservation of gene sequences within the transfer regions encoded on conjugative plasmids identified from strains of streptococcal, staphylococcal, enterococcal, and lactococcal origin (15). It was proposed that gram-positive and gram-negative conjugation systems utilize a similar transfer mechanism (15).In the anaerobic pathogen Clostridium perfringens conjugative plasmids have been shown to encode antibiotic resistance genes or extracellular toxins (3, 8, 9, 18). Although the contribution of conjugation to disease dissemination has not been systematically evaluated, it has been proposed that transfer of the C. perfringens enterotoxin plasmid pCPF4969 to normal flora isolates of C. perfringens may contribute to the severity of disease caused by non-food-borne isolates of C. perfringens (9).The prototype conjugative plasmid in C. perfringens is the 47-kb tetracycline resistance plasmid, pCW3. The complete sequence of pCW3 has been determined, and its unique replication protein and conjugation locus have been identified (8). Bioinformatics analysis of this C. perfringens tcp conjugation locus identified several proteins with limited similarity to proteins encoded within the transfer region of the conjugative transposon, Tn916 (8). The role of the tcp locus in the transfer of pCW3 has been confirmed by isolation of independent tcpA, tcpF, and tcpH mutants and subsequent complementation studies (8, 29). Since the region that encompasses the tcp locus is conserved in all conjugative plasmids from C. perfringens (2, 3, 8, 9, 18, 27) and since divergent tcpA homologues can complement a pCW3tcpA mutant (29), it appears that the conjugative transfer of both antibiotic resistance and toxin plasmids from this bacterium utilizes a common but poorly understood mechanism. Note that the C. perfringens tcp conjugation locus is different from the transfer regions of conjugative plasmids from other gram-positive bacteria.We have recently shown that the essential conjugation protein TcpH, a putative membrane-associated Mpf complex component, is localized to the poles of C. perfringens cells, as is another essential conjugation protein, TcpF (37). TcpH has also been shown to interact with itself and with the pCW3-encoded TcpC protein (37). In this study we have focused on the essential conjugation protein TcpA. Since TcpA encodes an FtsK/SpoIIIE domain found in DNA translocases (8), it is proposed that TcpA is involved in the movement of DNA during conjugative transfer, fulfilling a role equivalent to that of coupling proteins in other conjugation systems. Like such proteins, TcpA encodes two N-terminal transmembrane domains (TMDs) and a C-terminal cytoplasmic region that contains three motifs predicted to be involved in ATP binding and hydrolysis (8). Our previous studies revealed that the conserved motifs, motif I (Walker A box), motif II (Walker B box), and motif III (RAAG box), are essential for the function of TcpA. The C-terminal 61 amino acids (aa), though not essential for TcpA function, were shown to be important for efficient transfer of pCW3, as were the putative TMDs (29).To further investigate pCW3 transfer and the role of TcpA in this process, we have used bacterial two-hybrid analysis to examine protein-protein interactions involving TcpA. Using this system, interactions were observed between TcpA and itself, TcpC, TcpG, and TcpH. In addition, TcpC and TcpG were also found to self-interact. By combining these data with other data generated in this laboratory (37), we have constructed a model for the conjugative transfer of pCW3.     

Comprehensive Analysis of Host Cellular Interactions with Human Papillomavirus E6 Proteins Identifies New E6 Binding Partners and Reflects Viral Diversity

We have begun to define the human papillomavirus (HPV)-associated proteome for a subset of the more than 120 HPV types that have been identified to date. Our approach uses a mass spectrometry-based platform for the systematic identification of interactions between human papillomavirus and host cellular proteins, and here we report a proteomic analysis of the E6 proteins from 16 different HPV types. The viruses included represent high-risk, low-risk, and non-cancer-associated types from genus alpha as well as viruses from four different species in genus beta. The E6 interaction data set consists of 153 cellular proteins, including several previously reported HPV E6 interactors such as p53, E6AP, MAML1, and p300/CBP and proteins containing PDZ domains. We report the genus-specific binding of E6s to either E6AP or MAML1, define the specific HPV E6s that bind to p300, and demonstrate several new features of interactions involving beta HPV E6s. In particular, we report that several beta HPV E6s bind to proteins containing PDZ domains and that at least two beta HPV E6s bind to p53. Finally, we report the newly discovered interaction of proteins of E6 of beta genus, species 2, with the Ccr4-Not complex, the first report of a viral protein binding to this complex. This data set represents a comprehensive survey of E6 binding partners that provides a resource for the HPV field and will allow continued studies on the diverse biology of the human papillomaviruses.