Tumour cells can escape antiproliferative pressure by interferon-ß through immunoediting of interferon receptor expression.

Type I interferons (IFNs) play a central role not only in innate immunity against viral infection, but also in the antitumour response, e.g. through a direct impact on cell proliferation. Particularly for cancer arising in the context of chronic inflammation, constant exposure to IFNs may constitute a strong selective pressure during tumour evolution. Expansion of neoplastic subclones resistant to the antiproliferative effects of IFNs may contribute to immunoediting of tumours, leading to more aggressive disease. Experimental evidence for this development of IFN-insensitivity has been scarce and its molecular mechanism is unclear. In this study we demonstrate that six weeks exposure of cells to IFN-ß in vitro reduces their sensitivity to its antiproliferative effects, and that this phenotype was stable for up to four weeks. Furthermore, we observed substantial differences in cellular sensitivity to growth inhibition by IFN-ß in a panel of ten different liver cancer cell lines, most prominently in a pair of highly dedifferentiated cell lines, and least in cells from well-differentiated tumours. In both, long-term IFN selection and in dedifferentiated tumour cell lines, we found IFNAR2 expression to be substantially reduced, suggesting the receptor complex to be a sensitive target amenable to immunoediting. Beyond new insights into possible molecular processes in tumour evolution, these findings might prove valuable for the development of biomarkers allowing to stratify tumours for their sensitivity to IFN treatment in the context of patient tailored therapies.

Generation and Validation of Monoclonal Antibodies Suitable for Detecting and Monitoring Parvovirus Infections.

For many applications it is necessary to detect target proteins in living cells. This is particularly the case when monitoring viral infections, in which the presence (or absence) of distinct target polypeptides potentially provides vital information about the pathology caused by the agent. To obtain suitable tools with which to monitor parvoviral infections, we thus generated monoclonal antibodies (mAbs) in order to detect the major non-structural protein NS1 in the intracellular environment and tested them for sensitivity and specificity, as well as for cross-reactivity towards related species. Using different immunogens and screening approaches based on indirect immunofluorescence, we describe here a panel of mAbs suitable for monitoring active infections with various parvovirus species by targeting the major non-structural protein NS1. In addition to mAbs detecting the NS1 of parvovirus H-1 (H-1PV) (belonging to the Rodent protoparvovirus 1 species, which is currently under validation as an anti-cancer agent), we generated tools with which to monitor infections by human cutavirus (CuV) and B19 virus (B19V) (belonging to the Primate protoparvovirus 3 and the Primate erythroparvovirus 1 species, respectively, which were both found to persistently infect human tissues). As well as mAbs able to detect NS1 from a broad range of parvoviruses, we obtained entities specific for either (distinct) members of the Rodent protoparvovirus 1 species, human CuV, or human B19V.

Reg1 and Snf1 regulate stress-induced relocalization of protein phosphatase-1 to cytoplasmic granules.

The compartmentalization of cellular function is achieved largely through the existence of membrane-bound organelles. However, recent work suggests a novel mechanism of compartmentalization mediated by membraneless structures that have liquid droplet-like properties and arise through phase separation. Cytoplasmic stress granules (SGs) are the best characterized and are induced by various stressors including arsenite, heat shock, and glucose deprivation. Current models suggest that SGs play an important role in protein homeostasis by mediating reversible translation attenuation. Protein phosphatase-1 (PP1) is a central cellular regulator responsible for most serine/threonine dephosphorylation. Here, we show that upon arsenite stress, PP1's catalytic subunit Glc7 relocalizes to punctate cytoplasmic granules. This altered localization requires PP1's recently described maturation pathway mediated by the multifunctional ATPase Cdc48 and PP1's regulatory subunit Ypi1. Glc7 relocalization is mediated by its regulatory subunit Reg1 and its target Snf1, the AMP-dependent protein kinase. Surprisingly, Glc7 granules are highly specific to arsenite and appear distinct from canonical SGs. Arsenite induces potent translational inhibition, and translational recovery is strongly dependent on Glc7, but independent of Glc7's well-established role in regulating eIF2α. These results suggest a novel form of stress-induced cytoplasmic granule and a new mode of translational control by Glc7.

Persistent Innate Immune Stimulation Results in IRF3-Mediated but Caspase-Independent Cytostasis.

Persistent virus infection continuously produces non-self nucleic acids that activate cell-intrinsic immune responses. However, the antiviral defense evolved as a transient, acute phase response and the effects of persistently ongoing stimulation onto cellular homeostasis are not well understood. To study the consequences of long-term innate immune activation, we expressed the NS5B polymerase of Hepatitis C virus (HCV), which in absence of viral genomes continuously produces immune-stimulatory RNAs. Surprisingly, within 3 weeks, NS5B expression declined and the innate immune response ceased. Proteomics and functional analyses indicated a reduced proliferation of those cells most strongly stimulated, which was independent of interferon signaling but required mitochondrial antiviral signaling protein (MAVS) and interferon regulatory factor 3 (IRF3). Depletion of MAVS or IRF3, or overexpression of the MAVS-inactivating HCV NS3/4A protease not only blocked interferon responses but also restored cell growth in NS5B expressing cells. However, pan-caspase inhibition could not rescue the NS5B-induced cytostasis. Our results underline an active counter selection of cells with prolonged innate immune activation, which likely constitutes a cellular strategy to prevent persistent virus infections.

Regulation of the unfolded protein response in yeast by oxidative stress.

In the unfolded protein response (UPR), Ire1 activates Hac1 to coordinate the transcription of hundreds of genes to mitigate ER stress. Recent work in Caenorhabditis elegans suggests that oxidative stress inhibits this canonical Ire1 signalling pathway, activating instead an antioxidant stress response. We sought to determine whether this novel mode of UPR function also existed in yeast, where Ire1 has been best characterized. We show that the yeast UPR is also subject to inhibition by oxidative stress. Inhibition is mediated by a single evolutionarily conserved cysteine, and affects both luminal and membrane pathways of Ire1 activation. In yeast, Ire1 appears dispensable for resistance to oxidative stress and, therefore, the physiological significance of this pathway remains to be demonstrated.

Phospholipase Lpl1 links lipid droplet function with quality control protein degradation.

Protein misfolding is toxic to cells and is believed to underlie many human diseases, including many neurodegenerative diseases. Accordingly, cells have developed stress responses to deal with misfolded proteins. The transcription factor Rpn4 mediates one such response and is best known for regulating the abundance of the proteasome, the complex multisubunit protease that destroys proteins. Here we identify Lpl1 as an unexpected target of the Rpn4 response. Lpl1 is a phospholipase and a component of the lipid droplet. Lpl1 has dual functions: it is required for both efficient proteasome-mediated protein degradation and the dynamic regulation of lipid droplets. Lpl1 shows a synthetic genetic interaction with Hac1, the master regulator of a second proteotoxic stress response, the unfolded protein response (UPR). The UPR has long been known to regulate phospholipid metabolism, and Lpl1's relationship with Hac1 appears to reflect Hac1's role in stimulating phospholipid synthesis under stress. Thus two distinct proteotoxic stress responses control phospholipid metabolism. Furthermore, these results provide a direct link between the lipid droplet and proteasomal protein degradation and suggest that dynamic regulation of lipid droplets is a key aspect of some proteotoxic stress responses.