Covalent Probes To Capture Legionella pneumophila Dup Effector Enzymes.

Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cell's cytoplasm that hijack a plethora of cellular activities, including the host ubiquitination pathways. Effectors belonging to the SidE-family are involved in noncanonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires' disease. This dynamic process is reversed by effectors called Dups that hydrolyze the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and X-ray crystallography approaches were used to identify the site of covalent cross-linking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.

Modified influenza M158-66 peptide vaccination induces non-relevant T-cells and may enhance pathology after challenge.

CD8 + T cells are promising targets for vaccination against influenza A virus (IAV) infection. Their induction via peptide vaccination is not trivial, because peptides are weakly immunogenic. One strategy to overcome this is by vaccination with chemically enhanced altered peptide ligands (CPLs), which have improved MHC-binding and immunogenicity. It remains unknown how peptide-modification affects the resulting immune response. We studied the effect of CPLs derived from the influenza M158-66 epitope (GILGFVFTL) on the T-cell response. In HLA-A2*0201 transgenic mice, CPL-vaccination led to higher T-cell frequencies, but only a small percentage of the induced T cells recognized the GILG-wildtype (WT) peptide. CPL-vaccination resulted in a lower richness of the GILG-WT-specific T-cell repertoire and no improved protection against IAV-infection compared to GILG-WT peptide-vaccination. One CPL even appeared to enhance pathology after IAV-challenge. CPL-vaccination thus induces T cells not targeting the original peptide, which may lead to potential unwanted side effects.

Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes.

Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.

Global non-covalent SUMO interaction networks reveal SUMO-dependent stabilization of the non-homologous end joining complex.

In contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify interactors of different SUMO isoforms-monomeric SUMO1, monomeric SUMO2, or linear trimeric SUMO2 chains-using a mass spectrometry-based proteomics approach. We identify 379 proteins that bind to different SUMO isoforms, mainly in a preferential manner. Interestingly, XRCC4 is the only DNA repair protein in our screen with a preference for SUMO2 trimers over mono-SUMO2, as well as the only protein in our screen that belongs to the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. A SUMO interaction motif (SIM) in XRCC4 regulates its recruitment to sites of DNA damage and phosphorylation of S320 by DNA-PKcs. Our data highlight the importance of non-covalent and covalent sumoylation for DNA double-strand break repair via the NHEJ pathway and provide a resource of SUMO isoform interactors.