Identifizierung und Charakterisierung zellulärer Proteine als Interaktionspartner des Neben-Kapsidproteins L2 Humaner Papillomviren

Papillomviren sind DNA-Viren, die in einer Vielzahl von Vertebratenspezies gefunden wurden. Während die meisten Infektionen inapparent verlaufen, gibt es einige humane Papillomvirus-Typen (HPV) mit onkogenem Potential. Von besonderer Bedeutung ist dabei die Assoziation mit dem Zervixkarzinom, der zweithäufigsten Krebsart bei Frauen. Das Kapsid der Papillomviren ist aus zwei verschiedenen Kapsidproteinen aufgebaut, dem Haupt-Kapsidprotein L1 sowie dem Neben-Kapsidprotein. Die Kapsidproteine schützen nicht nur die DNA in der extrazellulären Phase des Virus und ermöglichen die Bindung des Virus an die Wirtszelle - L2 scheint auch regulatorische Funktionen zu haben. L2 spielt möglicherweise eine entscheidende Rolle bei der DNA-Verpackung und der Reifung infektiöser Virionen. In der hier vorgelegten Arbeit wurden zelluläre Interaktionspartner von L2 identifiziert. Zu diesem Zweck wurde eine humane Keratinozyten-cDNA-Bibliothek mittels des Yeast Two Hybrid-Verfahrens nach Interaktionspartnern von HPV 11 L2 durchsucht. Auf diese Weise gefundene potentielle Interaktionspartner wurden auf eine Wechselwirkung mit den L2-Proteinen anderer HPV-Typen untersucht (HPV1, HPV16). Proteine, die dabei mit mehr als einem L2-Protein interagierten, wurden auf die Bindung von HPV 16 L2 in vitro getestet und ihre subzelluläre Lokalisation analysiert. Ein mit L2 in punktartigen Kerndomänen kolokalisierendes Protein, PLINP benannt, wurde u.a. durch die Herstellung von Deletionsmutanten eingehender untersucht. Es konnte so die L2-bindende Domäne sowie der für die Lokalisation in Kerndomänen verantwortliche Bereich eingegrenzt werden. Da für BPV 1 L2 eine Lokalisation in PODs, einer ebenfalls in einem Punktmuster vorliegenden Kerndomäne, beschrieben war, wurde die Lokalisation von L2 und PLINP im Verhältnis zu PODs untersucht. Eine Assoziation dieser beiden Proteine mit PODs scheint zwar vorzuliegen, nicht aber eine ausgeprägte Kolokalisation wie zuvor für BPV 1 L2 beschrieben

The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets

Polycomb group (PcG) proteins are key regulators of stem-cell and cancer biology. They mainly act as repressors of differentiation and tumor-suppressor genes. One key silencing step involves the trimethylation of histone H3 on Lys27 (H3K27) by EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2). The mechanism underlying the initial recruitment of mammalian PRC2 complexes is not well understood. Here, we show that NIPP1, a regulator of protein Ser/Thr phosphatase-1 (PP1), forms a complex with PP1 and PRC2 components on chromatin. The knockdown of NIPP1 or PP1 reduced the association of EZH2 with a subset of its target genes, whereas the overexpression of NIPP1 resulted in a retargeting of EZH2 from fully repressed to partially active PcG targets. However, the expression of a PP1-binding mutant of NIPP1 (NIPP1m) did not cause a redistribution of EZH2. Moreover, mapping of the chromatin binding sites with the DamID technique revealed that NIPP1 was associated with multiple PcG target genes, including the Homeobox A cluster, whereas NIPP1m showed a deficient binding at these loci. We propose that NIPP1 associates with a subset of PcG targets in a PP1-dependent manner and thereby contributes to the recruitment of the PRC2 complex

Interaction of Human Papillomavirus Type 16 L2 with Cellular Proteins: Identification of Novel Nuclear Body-Associated Proteins

AbstractTwo structural proteins form the Papillomavirus (PV) capsids. While the functions of the major structural protein L1 are well established, the exact functions for the minor structural protein L2 are much less well defined, except for some information on a role in viral entry and maturation of infectious virions. To gain more insight in the function of L2 we used the yeast two hybrid system with the Human Papillomavirus (HPV) 11 L2 and HPV16 L2 as bait proteins to isolate putative cellular interaction partners. We identified four proteins interacting with L2 proteins of at least two different HPV types and this interaction was confirmed in vitro by pull-down assays. Further evidence for this interaction was obtained by in vivo localization studies. Two of the proteins, the previously described PATZ and a novel protein, designated PLINP, were localized in discrete nuclear domains and colocalized with L2. The third protein, designated PMSP, is a newly identified cytoplasmic protein which was recruited to nuclear dots when coexpressed with L2. The fourth protein interacting with HPV16, 11 and 1 L2, the tubular-nephritis antigen related protein (TIN-Ag-RP), shows a cytoplasmic as well as a membrane bound subcellular distribution. Taken together, our data indicate that L2 of HPVs with different phenotypes interacts with several cellular host proteins, recruits one of them to the nucleus, and is complexed with at least three cellular proteins in specific nuclear domains. These findings suggest an HPV type-independent modulatory function of L2 on host-cell functions that involves discrete nuclear domains and alteration of the subcellular distribution of cellular proteins. The interacting cellular proteins identified may play a role in the viral life cycle and establishment of viral persistence

Cotranscriptional spliceosome assembly and splicing are independent of the Prp40p WW domain.

Complex cellular functions involve large networks of interactions. Pre-mRNA splicing and transcription are thought to be coupled by the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). In yeast, the U1 snRNP subunit Prp40 was proposed to mediate cotranscriptional recruitment of early splicing factors through binding of its WW domains to the Pol II CTD. Here we investigate the role of Prp40 in splicing with an emphasis on the role of the WW domains, which might confer protein-protein interactions among the splicing and transcriptional machineries. Affinity purification revealed that Prp40 and Snu71 form a stable heterodimer that stably associates with the U1 snRNP only in the presence of Nam8, a known regulator of 5' splice site recognition. However, the Prp40 WW domains were dispensable for yeast viability. In their absence, no defect in splicing in vivo, U1 or U2 snRNP recruitment in vivo, or early splicing complex assembly in vitro was detected. We conclude that the WW domains of Prp40 do not mediate essential coupling between U1 snRNP and Pol II. Instead, delays in cotranscriptional U5 snRNP and Prp19 recruitment and altered spliceosome formation in vitro suggest that Prp40 WW domains assist in late steps of spliceosome assembly

Cartoon showing the basic organization of the cervical epithelium and a mechanistic model to explain how PP1/NIPP1 may contribute to invasiveness of tumour cells.

<p>Cervical and vaginal epithelia have lumen potentials of about −25 to −50 mV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Boskey1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Szatkowski1" target="_blank">[66]</a>. Such a lumen potential would correspond to a transepithelial voltage gradients of 1.7 V/cm (170 mV/mm). In these electrophysiological conditions cervical epithelial cells would migrate towards the lumen as they turn over the epithelial lining layer (green arrow). Upregulation of NIPP1 and its recruitment to PP1 would reverse migration into the lumen, encouraging invasion of the surrounding tissue (red arrow).</p

Effect of pharmacological inhibition of Cdc42-GTPase on the HTO cells.

<p><b>A.</b> Effect of ML141 on Cdc42 GTPase activity in unstimulated cells cultured in complete medium and in EF-stimulated HTO cells overexpressing the FLAG-NIPP1 protein variants. Levels of Cdc42-GTP determined by G-LISA in parental, W.T-NIPP1, ΔC-NIPP1 and mRATA cells in the absence or presence of DC EF and in cells pre-treated with 10 µM of ML141 before electrical stimulation. <i>p</i> values parental to W.T-NIPP1 and parental to ΔC-NIPP1 in complete medium were 0.1 and 0.01, respectively; <i>p</i> values comparing samples in the absence and presence of ML141 were in all cases <0.01. <b>B.</b> Cdc42 inhibition rescues cathodal polarisation and this correlates with centrosome positioning. Directedness values for the migration of EF-treated cells incubated with ML141. Cdc42 inhibition rescues the positive cell directedness decreased by W.T-NIPP1 overexpression. The strongly negative directedness value displayed by ΔC-NIPP1 cells becomes closer to 0 when cells are pretreated with Cdc42 inhibitor. For simplification directedness values in the absence of EF of the parental, W.T-NIPP1, ΔC-NIPP1, and mNIPP1 with and without ML141 have not been included in the diagram. These were, without ML141, −0.07±0.04; 0.05±0.09; −0.08±0.05 and −0.01±0.04, respectively; with ML141 were −0.07±0.04; 0.09±0.05; −0.07±0.05 and −0.01±0.04, respectively. In the absence of EF values were in all cases very close to 0 and differences between the four lines were not statistically significant in any of the cases. Data was quantified from at least three experiments. Error bars are S.E.M. <i>p</i> values for significant differences in directedness are shown. Polarisation index of centrosomes calculated as explained in materials and methods. Polarisation index of W.T-NIPP1 and ΔC-NIPP1 cells becomes similar to the polarisation index of parental cells when cells are treated with the Cdc42 inhibitor ML141.</p

PP1 loss impairs electrotaxis in HeLa cells.

<p><b>A.</b> Treatment of parental HeLa Tet-Off (HTO) cells with siRNA strongly depletes PP1 levels 48 h post transfection. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms. <b>B.</b> Plot diagrams show that loss of PP1 impairs the ability of cells to migrate towards the cathode. Each line represents the migration trajectory of a single cell. The starting point for each cell migration track is at the origin. Cell tracks with end positions to the right appear in red (“C”, cathode) and those to the left appear in black (“A”, anode). EF-untreated cells were assayed as controls. Control siRNA cells migrate strongly towards the cathode; PP1 siRNA treated cells are unable to migrate in response to a DC EF. Scales show distance migrated in µm. <b>C.</b> PP1 depletion strongly reduces distance migrated, speed, and directedness in response to physiological DC EF. Error bars are S.E.M. <i>p</i> values for significant differences in distance, speed and directedness are shown. <b>D.</b> Localization of endogenous PP1 and distribution of filamentous-actin in control and PP1 depleted cells treated with DC EF. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms (green) and polymerised actin was detected using rhodamine phalloidin (red). The nuclei have been stained with DAPI (blue). Arrows mark cells with a strong decrease in PP1 levels which correlate with defects in the formation of actin rich protrusions. Representative images are shown. Scale bar is 50 µm. <b>E.</b> Numbers of cells with filopodia were quantified by counting 100 cells. Error bars are S.E.M. <i>p</i> values for significant differences are shown. Images show a detail of cell protrusions in control siRNA and PP1 siRNA cells. Arrows mark numerous filopodia in control cells and outline areas with a major lack of filopodia at the cell edges in PP1 siRNA cells.</p

List of genes from the Cdc42 pathway that are significantly upregulated by the overexpression of W.T-NIPP1 (WT) or ΔC-NIPP1 (ΔC), but not by mNIPP1 (m), in the HTO cells and compared to parental HTO cells.

<p>List of genes from the Cdc42 pathway that are significantly upregulated by the overexpression of W.T-NIPP1 (WT) or ΔC-NIPP1 (ΔC), but not by mNIPP1 (m), in the HTO cells and compared to parental HTO cells.</p