Influenza A Virus Induces Autophagosomal Targeting of Ribosomal Proteins.

Seasonal epidemics of influenza A virus are a major cause of severe illness and are of high socio-economic relevance. For the design of effective antiviral therapies, a detailed knowledge of pathways perturbed by virus infection is critical. We performed comprehensive expression and organellar proteomics experiments to study the cellular consequences of influenza A virus infection using three human epithelial cell lines derived from human lung carcinomas: A549, Calu-1 and NCI-H1299. As a common response, the type I interferon pathway was up-regulated upon infection. Interestingly, influenza A virus infection led to numerous cell line-specific responses affecting both protein abundance as well as subcellular localization. In A549 cells, the vesicular compartment appeared expanded after virus infection. The composition of autophagsomes was altered by targeting of ribosomes, viral mRNA and proteins to these double membrane vesicles. Thus, autophagy may support viral protein translation by promoting the clustering of the respective molecular machinery in autophagosomes in a cell line-dependent manner.

Vectorial Import via a Metastable Disulfide-Linked Complex Allows for a Quality Control Step and Import by the Mitochondrial Disulfide Relay.

Disulfide formation in the mitochondrial intermembrane space (IMS) is an essential process. It is catalyzed by the disulfide relay machinery, which couples substrate import and oxidation. The machinery relies on the oxidoreductase and chaperone CHCHD4-Mia40. Here, we report on the driving force for IMS import and on a redox quality control mechanism. We demonstrate that unfolded reduced proteins, upon translocation into the IMS, initiate formation of a metastable disulfide-linked complex with CHCHD4. If this interaction does not result in productive oxidation, then substrates are released to the cytosol and degraded by the proteasome. Based on these data, we propose a redox quality control step at the level of the disulfide-linked intermediate that relies on the vectorial nature of IMS import. Our findings also provide the mechanistic framework to explain failures in import of numerous human disease mutants in CHCHD4 substrates.

Systematic analysis of synergistic proteome modulations in a drug combination of cisplatin and MLN4924.

Chemotherapeutic treatment regimens often take advantage of synergistic effects of drug combinations. Anticipating that synergistic effects on the cell biological level likely manifest on the proteome level, the analysis of proteome modulations represents an appropriate strategy to study drug combinations on a molecular level. More specifically, the detection of single proteins exhibiting synergistic abundance changes could be helpful to shed light on key molecules, which contribute in mechanisms facilitating the synergistic interaction and therefore represent potential targets for specific therapeutic approaches. In the reported study we aimed to provide evidence for this assumption and investigated the drug combination of cisplatin and the neddylation inhibitor MLN4924 in HCT-116 cells via cell biological analyses and mass spectrometry-based quantitative proteomics. From 1789 proteins quantified with two unique peptides, activated RNA polymerase II transcriptional coactivator p15 (SUB1) was highlighted as the most synergistically regulated protein using a synergistic scoring approach. Western blotting and analyses of cellular processes associated with this protein (DNA damage, oxidative stress and apoptosis) revealed supporting evidence for the synergistic regulation. Whereas the distinct role of SUB1 in the investigated drug combination needs to be elucidated in future studies, the presented results demonstrated the benefit and feasibility of synergistic scoring of proteome alterations to highlight proteins that likely contribute to the underlying molecular mechanisms of synergistic effects. Data are available via ProteomeXchange with identifier PXD009185.