HPV-18 E6 enhances the interaction between EMILIN2 and SNX27 to promote WNT signaling.

Oncogenic HPV E6 proteins have a PDZ-binding motif (PBM) which plays important roles in both the viral life cycle and tumor development. The PBM confers interaction with a large number of different PDZ domain-containing substrates, one of which is Sorting Nexin 27. This protein is part of the retromer complex and plays an important role in endocytic sorting pathways. It has been shown that at least two SNX27 interacting partners, GLUT1 and TANC2, are aberrantly trafficked due to the E6 PBM-dependent interaction with SNX27. To investigate further which other components of the endocytic trafficking pathway might be affected by the SNX27-HPV E6 interaction, we analyzed the SNX27 proteome interaction profile in a previously described HeLa cell line expressing GFP-SNX27, both in the presence and absence of the HPV-18 E6 oncoprotein. In this study, we identify a novel interacting partner of SNX27, secreted glycoprotein EMILIN2, whose release is blocked by HPV18 E6 in a PBM-dependent manner. Mechanistically, E6 can block EMILIN2 interaction with the WNT1 ligand, thereby enhancing WNT1 signaling and promoting cell proliferation. IMPORTANCE: This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.

Identification and characterisation of novel potential phospho-acceptor sites in HPV-16 E7.

Several studies have described functional regulation of high-risk human papillomaviruses (HPVs), E6 and E7 oncoproteins via posttranslational modifications (PTMs). However, how these PTMs modulate the activity of E6 and E7, particularly in their targeting of cellular proteins, is not completely understood. In this study, we show that HPV16 E7 can be phosphorylated by casein kinase I (CKI) and glycogen synthase kinase 3 (GSK3). This principal phosphorylation occurs at threonine residues 5 and 7 with a more minor role for residues 19-20 in the N-terminal region of 16 E7. Intriguingly, whilst mutational analyses suggest that residues 5 and 7 may be dispensable for the transformation of primary baby rat kidney cells by E7, intact residues 19 and 20 are required. Furthermore, negative charges at these residues (TT19-20DD) enhance the pRb-E7 interaction and cells display increased proliferation and invasion capacities. Using a proteomic approach with a phosphorylated peptide spanning the TT19-20 region of HPV16 E7, we have identified a panel of new, phospho-specific E7 interacting partners. These results shed new light on the complexity of N-terminal phosphorylation of E7 and how this can contribute towards expanding the repertoire of E7 targeted pathways.

Intracellular cathepsin C levels determine sensitivity of cells to leucyl-leucine methyl ester-triggered apoptosis.

L-leucyl-leucine methyl ester (LLOMe) is a lysosomotropic detergent, which was evaluated in clinical trials in graft-vs-host disease because it very efficiently killed monocytic cell lines. It was also shown to efficiently trigger apoptosis in cancer cells, suggesting that the drug might have potential in anticancer therapy. Using U-937 and THP-1 promonocytes as models for monocytic cells, U-87-MG and HeLa cells as models for cancer cells, and noncancerous HEK293 cells, we show that the drug triggers rapid cathepsin C-dependent lysosomal membrane permeabilization, followed by the release of other cysteine cathepsins into the cytosol and subsequent apoptosis. However, monocytes were found to be far more sensitive to the drug than the cancer and noncancer cells, which is most likely a consequence of the much higher intracellular levels of cathepsin C-the most upstream molecule in the pathway-in monocytic cell lines as compared to cancer cells. Overexpression of cathepsin C in HEK293 cells substantially enhances their sensitivity to the drug, consistent with the crucial role of cathepsin C. Major involvement of cysteine cathepsins B, S, and L in the downstream signaling pathway to mitochondrial cell death was confirmed in two gene ablation models, including the ablation of the major cytosolic inhibitor of cysteine cathepsins, stefin B, in primary mouse cancer cells, and simultaneous ablation of two major cathepsins, B and L, in mouse embryonic fibroblasts (MEFs). Deletion of stefin B resulted in sensitizing primary murine breast cancer cells to cell death without affecting the release of cathepsins, whereas simultaneous ablation of cathepsins B and L largely protected MEFs against cell death. However, due to the extreme sensitivity of monocytes to LLOMe, it appears that the drug may not be suitable for anticancer therapy due to risk of systemic toxicity.

Comparative study of the differential cell death protecting effect of various ROS scavengers.

Oxidative stress has for a long time been associated with cell death, especially classical necrosis, however, its role in other cell death pathways is less clear. Here, we evaluated in a comparative way, the effect of four different reactive oxygen species (ROS) scavengers, N-acetyl-L-cysteine (NAC), α-tocopherol and two superoxide dismutase mimetics, n(III)tetrakis(4-benzoic acid)porphyrin chloride, and 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol), in four different cell death models, including menadione-triggered necrosis, staurosporine-induced apoptosis and tumor necrosis factor (TNF)-induced apoptosis and necroptosis. While menadione-triggered necrosis was completely prevented by the classical ROS scavenger NAC and to a substantial amount by the other scavengers, ROS targeting was found to have a marginal effect on the other cell death modalities investigated. Despite its side-effects at higher concentrations, Tempol was able to substantially prevent TNF-induced apoptosis and to a somewhat lesser extent TNF-induced necroptosis. However, this seems to be separated from its ROS-scavenging function.

Lysosomes in programmed cell death pathways: from initiators to amplifiers.

Lysosome is the central organelle for intracellular degradation of biological macromolecules and organelles. The material destined for degradation enters the lysosomes primarily via endocytosis, autophagy and phagocytosis, and is degraded through the concerted action of more than 50 lysosomal hydrolases. However, lysosomes are also linked with numerous other processes, including cell death, inflammasome activation and immune response, as well as with lysosomal secretion and cholesterol recycling. Among them programmed cell death pathways including apoptosis have received major attention. In most of these pathways, cell death was accompanied by lysosomal membrane permeabilization and release of lysosomal constituents with an involvement of lysosomal hydrolases, including the cathepsins. However, it is less clear, whether lysosomal membrane permeabilization is really critical for the initiation of cell death programme(s). Therefore, the role of lysosomal membrane permeabilization in various programmed cell death pathways is reviewed, as well as the mechanisms leading to it.