Regulation of HPV 16-E7 by E2, Phosphorylation and the Proteasome

In order to ensure a productive life cycle, Human Papillomaviruses (HPVs) require fine regulation of their gene products. Uncontrolled activity of the viral oncoproteins, E6 and E7, results in the immortalisation of the infected epithelial cells and thus prevents the production of mature virions. Here, we investigate the regulation of HPV-16 E7 activities through its interaction with both viral and cellular gene products. First, we show that HPV-16 E7 and E2 can interact directly and the region mediating this interaction is defined on each protein. The expression of E2 inhibits some of E7 oncogenic activities including primary cell transformation, induction of centrosome abnormalities and pRB degradation. In addition, E2 can stabilise E7 and redirect its localisation where it can associate with some of E2’s activities such as transcriptional activation and mitotic chromosome binding. Secondly, we provide evidence that E7 can be phosphorylated by CDK2 in vitro preferentially on its N-terminal domain, and we hypothesise that this occurs on more than one residue on E7. In vivo, we show that the activity of CDK2, as well as CKII, is necessary for the stability of E7. Finally, we identified an interaction between HPV-16 E2 and E7 with the cellular oncoprotein, Mdm2. Mdm2 appears to destabilise E7 targeting it to proteasome-mediated degradation at PML bodies. The stability of E7 in cells that have reduced expression of Mdm2 is markedly increased indicating that the expression of Mdm2 indeed destabilises E7. In the case of the Mdm2 interaction with E2, we observe that E2 inhibits Mdm2 mediated degradation of p53 and pRB and that the expression of Mdm2 enhances E2’s transcriptional activity and induces its re-localisation at specific structures within the nucleus. Overall, our findings expand our knowledge of the regulation of virally encoded proteins both through direct protein-protein interactions between themselves and through their interactions with cellular proteins

Autophagy and autophagy-related pathways in cancer

Maintenance of protein homeostasis and organelle integrity and function is critical for cellular homeostasis and cell viability. Autophagy is the principal mechanism that mediates the delivery of various cellular cargoes to lysosomes for degradation and recycling. A myriad of studies demonstrate important protective roles for autophagy against disease. However, in cancer, seemingly opposing roles of autophagy are observed in the prevention of early tumour development versus the maintenance and metabolic adaptation of established and metastasizing tumours. Recent studies have addressed not only the tumour cell intrinsic functions of autophagy, but also the roles of autophagy in the tumour microenvironment and associated immune cells. In addition, various autophagy-related pathways have been described, which are distinct from classical autophagy, that utilize parts of the autophagic machinery and can potentially contribute to malignant disease. Growing evidence on how autophagy and related processes affect cancer development and progression has helped guide efforts to design anticancer treatments based on inhibition or promotion of autophagy. In this Review, we discuss and dissect these different functions of autophagy and autophagy-related processes during tumour development, maintenance and progression. We outline recent findings regarding the role of these processes in both the tumour cells and the tumour microenvironment and describe advances in therapy aimed at autophagy processes in cancer

Targeting of Early Endosomes by Autophagy Facilitates EGFR Recycling and Signalling

Despite recently uncovered connections between autophagy and the endocytic pathway, the role of autophagy in regulating endosomal function remains incompletely understood. Here, we find that the ablation of autophagy-essential players disrupts EGFinduced endocytic trafficking of EGFR. Cells lacking ATG7 or ATG16L1 exhibit increased levels of phosphatidylinositol-3-phosphate (PI(3)P), a key determinant of early endosome maturation. Increased PI(3)P levels are associated with an accumulation of EEA1-positive endosomes where EGFR trafficking is stalled. Aberrant early endosomes are recognised by the autophagy machinery in a TBK1- and Gal8-dependent manner and are delivered to LAMP2-positive lysosomes. Preventing this homeostatic regulation of early endosomes by autophagy reduces EGFR recycling to the plasma membrane and compromises downstream signalling and cell survival. Our findings uncover a novel role for the autophagy machinery in maintaining early endosome function and growth factor sensing

Autophagy supports PDGFRA-dependent brain tumour development by modulating oncogenic signalling

Autophagy is a highly conserved catabolic process which sequesters intracellular substrates for lysosomal degradation. Whilst autophagy-related proteins have been shown to regulate the signalling and trafficking of some receptor tyrosine kinases, the relevance of this during cancer development is unclear. Here, we uncover a novel role for autophagy in regulating PDGFRA signalling and levels. We find that PDGFRA can be targeted to autophagic degradation through the activity of the autophagy cargo receptor, p62. As a result, short term autophagy inhibition leads to elevated intracellular levels of PDGFRA but an unexpected defect in PDGFA-mediated downstream signalling due to perturbed trafficking of the receptor. Defective PDGFRA signalling led to a reduction in receptor levels during prolonged autophagy inhibition suggesting that cells adapt to autophagy inhibition by downregulating receptor expression. Importantly, PDGFA-driven gliomagenesis in mice was disrupted when autophagy was inhibited. Activation of PI3K/AKT signalling through Pten mutation overrides the need for autophagy during tumour development upon PDGFRA activation highlighting a genotype-specific role for autophagy during tumourigenesis. In summary, our data provide a novel mechanism by which cells require autophagy for optimal oncogenic signalling that drives tumour formation

The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD. Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery.This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub. This work was funded by Cancer Research UK (C47718/A16337, O.F.), the Medical Research Council (RG89611, R.B.) and the BBSRC Institute Strategic Programme Gut Health and Food Safety (BB/J004529/1)

The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation

Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosoliccomponents. The final degradation step is essential for clearing autophagic cargo and recyclingmacromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conservedtransmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation