Das E6-Onkoprotein humaner Papillomviren und seine Wechselwirkung mit E6AP als Zielstruktur für eine molekulare Therapie zervikaler Karzinome

Bestimmte Typen humaner Papillomviren (´high risk´ HPVs) spielen bei der Entstehung des Zervixkarzinoms eine kausale Rolle. Dabei scheint die Expression der viralen Proteine E6 und E7 in allen Stadien der Karzinogenese essentiell zu sein. E6 besitzt anti-apoptotisches Potential, das vermutlich zur Kompensation einer E7-vermittelten Apoptose-Sensitivität infizierter Zellen benötigt wird. Für E6 sind potentielle Wechselwirkungen mit einer Vielzahl von zellulären Proteinen beschrieben, wobei die physiologische Relevanz für die meisten Interaktionen weitestgehend unbekannt ist. In dieser Arbeit wurden mutierte Formen von E6 durch gezielte Mutagenese generiert und jeweils in ihren Wechselwirkungen mit dem Tumorsuppressor p53, der Protein-Ubiquitin-Ligase E6AP und den PDZ-Proteinen Scribble und Dlg charakterisiert. Dabei wurden Beobachtungen gemacht, die auf einen E6AP-vermittelten Abbau von Dlg durch E6 hinweisen und somit im Widerspruch zu publizierten Daten stehen. Dies ist aber vermutlich dadurch zu erklären, dass in vorherigen Studien ein Abbau von Dlg durch Formen von E6, die sich von ´low risk´ E6 Proteinen ableiten, beobachtet wurde. Da diese E6 Formen nicht in der Lage sind, E6AP in vitro zu kopräzipitieren, wurde geschlussfolgert, dass sie nicht mit E6AP interagieren. Durch die Verwendung von E6AP -/- Mausfibroblasten konnten in dieser Arbeit allerdings deutliche Hinweise für eine Interaktion von E6AP mit dem E6 Protein des ´low risk´ HPV-Typen 11 gefunden werden. Zusätzlich wurde das onkogene Potential der generierten E6 Mutanten in Zellkulturstudien untersucht. Vorläufige Ergebnisse zeigen an, dass dem extremen C-Terminus von ´high risk´ E6 Proteinen eine wichtige Rolle bei der Immortalisierung von Zellen zukommt. Um diesen Einfluss des C-Terminus näher zu spezifizieren, ist die Generierung weiterer E6 Mutanten notwendig. Um zu untersuchen, ob die Interaktion von E6 mit E6AP eine potentielle Zielstruktur für molekulare Ansätze bei der Behandlung des Zervixkarzinoms darstellt, wurde die Methode der Proteintransduktion etabliert. Dazu wurde das von E6AP-abgeleitete E6-Bindepeptid e6bp1 an ein zellpenetrierendes Peptid (im vorliegenden Fall ´ARG7´) fusioniert und es konnten alle wesentlichen Eigenschaften von zellpenetrierenden Peptiden beobachtet werden. Allerdings wurden alle Untersuchungen unter Bedingungen durchgeführt, für die kürzlich die Möglichkeit der Erzeugung von Artefakten beschrieben wurde. Daher sind weitere Untersuchungen nötig, um Aussagen über einen Einfluss von e6bp1 auf das Wachstum von HPV-positiven Zellen treffen zu können

ATG5 regulates plasma cell differentiation

Autophagy is a conserved homeostatic process in which cytoplasmic contents are degraded and recycled. Two ubiquitin-like conjugation pathways are required for the generation of autophagosomes, and ATG5 is necessary for both of these processes. Studies of mice deficient in ATG5 reveal a key role for autophagy in T lymphocyte function, as well as in B cell development and B-1a B cell maintenance. However, the role of autophagy genes in B cell function and antibody production has not been described. Using mice in which Atg5 is conditionally deleted in B lymphocytes, we showed here that this autophagy gene is essential for plasma cell homeostasis. In the absence of B cell ATG5 expression, antibody responses were significantly diminished during antigen-specific immunization, parasitic infection and mucosal inflammation. Atg5-deficient B cells maintained the ability to produce immunoglobulin and undergo class-switch recombination, yet had impaired SDC1 expression, significantly decreased antibody secretion in response to toll-like receptor ligands, and an inability to upregulate plasma cell transcription factors. These results build upon previous data demonstrating a role for ATG5 in early B cell development, illustrating its importance in late B cell activation and subsequent plasma cell differentiation

Impaired Autophagy of an Intracellular Pathogen Induced by a Crohn's Disease Associated ATG16L1 Variant

The genetic risk factors predisposing individuals to the development of inflammatory bowel disease are beginning to be deciphered by genome-wide association studies. Surprisingly, these new data point towards a critical role of autophagy in the pathogenesis of Crohn's disease. A single common coding variant in the autophagy protein ATG16L1 predisposes individuals to the development of Crohn's disease: while ATG16L1 encoding threonine at amino acid position 300 (ATG16L1*300T) confers protection, ATG16L1 encoding for alanine instead of threonine (ATG16L1*300A, also known as T300A) mediates risk towards the development of Crohn's disease. Here we report that, in human epithelial cells, the Crohn's disease-associated ATG16L1 coding variant shows impairment in the capture of internalized Salmonella within autophagosomes. Thus, we propose that the association of ATG16L1*300A with increased risk of Crohn's disease is due to impaired bacterial handling and lowered rates of bacterial capture by autophagy

Discovery of a Small-Molecule Probe for V-ATPase Function

Lysosomes perform a critical cellular function as a site of degradation for diverse cargoes including proteins, organelles, and pathogens delivered through distinct pathways, and defects in lysosomal function have been implicated in a number of diseases. Recent studies have elucidated roles for the lysosome in the regulation of protein synthesis, metabolism, membrane integrity, and other processes involved in homeostasis. Complex small-molecule natural products have greatly contributed to the investigation of lysosomal function in cellular physiology. Here we report the discovery of a novel, small-molecule modulator of lysosomal acidification derived from diversity-oriented synthesis through high-content screening

The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins

The E6 oncoprotein of human papillomaviruses associated with cervical cancer targets the tumor suppressor p53 and several other cellular proteins including the human homologs of Dlg and Scribble for degradation via the ubiquitin-proteasome system. Similar to p53 degradation, E6-induced degradation of Scribble is mediated by the ubiquitin ligase E6-AP. In contrast, degradation of Dlg in vitro and within cells has been reported to be independent of E6-AP, suggesting that the E6 oncoprotein has the ability to interact with ubiquitin ligases other than E6-AP. Furthermore, the ability of the E6 oncoprotein to interact with these yet unidentified ubiquitin ligases may be shared by the E6 protein of so-called low risk human papillomaviruses that are not associated with cervical cancer. In this study, we used the RNA interference technology and mouse embryo fibroblasts derived from E6-AP-deficient mice to obtain information about the identity of the ubiquitin ligase(s) involved in E6-mediated degradation of Dlg. We report that, within cells, E6-mediated degradation of Dlg depends on the presence of functional E6-AP and provide evidence that the E6 protein of low risk human papillomaviruses functionally interacts with E6-AP. Based on these data, we propose that, in general, the proteolytic properties of human papillomavirus E6 proteins are mediated by interaction with E6-AP

DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell-cell contact

PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination. © 2007 Elsevier Inc. All rights reserved

Induction of heat shock protein HSPA6 (HSP70B′) upon HSP90 inhibition in cancer cell lines

AbstractGenome-wide transcript profiling to elucidate responses to HSP90 inhibition revealed strong induction of HSPA6 in MCF-7 cells treated with 17-AAG. Time- and dose dependent induction of HSPA6 (confirmed by qPCR and Western Blots) occurred also upon treatment with Radicicol, another HSP90 inhibitor. HSPA6 was not detectable in untreated cells or cells treated with toxins that do not inhibit HSP90, or upon applying oxidative stress. Thus, HSPA6 induction is not a general response to cytotoxic insults. Modulation of HSPA6 levels by siRNA-mediated inhibition or recombinant expression did not influence 17-AAG mediated cell death. HSPA6 induction as a consequence of HSP90 inhibition occurs in various (but not all) cell lines and may be a more specific marker for HSP90 inhibition than induction of other HSP70 proteins

Growth Suppression Induced by Downregulation of E6-AP Expression in Human Papillomavirus-Positive Cancer Cell Lines Depends on p53

The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells. These data indicate that the anti-growth-suppressive properties of E6-AP in HPV-positive cells depend on its ability to induce p53 degradation