SummaryAntigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal “clickable” antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer.

Peptide‐mediated membrane fusion is frequently studied with in vitro bulk leaflet mixing assays based on Förster resonance energy transfer (FRET). In these, customized liposomes with fusogenic peptides are equipped with lipids which are labeled with fluorophores that form a FRET pair. Since FRET is dependent on distance and membrane fusion comes along with lipid mixing, the assays allow for conclusions on the membrane fusion process. The experimental outcome of these assays, however, greatly depends on the applied parameters. In the present study, the influence of the peptides, the size of liposomes, their lipid composition and the liposome stoichiometry on the fusogenicity of liposomes are evaluated. As fusogenic peptides, soluble N‐ethylmaleimide‐sensitive‐factor attachment receptor (SNARE) protein analogues featuring artificial recognition units attached to the native SNARE transmembrane domains are used. The work shows that it is important to control these parameters in order to be able to properly investigate the fusion process and to prevent undesired effects of aggregation.

Peptide‐protein interactions (PPIs) are facilitated by the well‐defined three‐dimensional structure of bioactive peptides, interesting compounds for the development of new therapeutic agents. Their secondary structure and thus their propensity to engage in PPIs can be influenced by the introduction of peptide staples on the side chains. In particular, light‐controlled staples based on azobenzene photoswitches and their structural influence on helical peptides have been studied extensively. In contrast, photolabile staples bearing photocages as a structural key motif, have mainly been used to block supramolecular interactions. Their influence on the secondary structure of the target peptide is under‐investigated. Thus, in this study we use a combination of spectroscopic techniques and in silico simulations to systematically study a series of helical peptides with varying length of the photo‐labile staple to obtain a detailed insight into the structure‐property relationship in such photoresponsive biomolecules.

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