Biology subjects

Lenz, C.

Publications and source records attributed to Lenz, C..

22 records · Page 2Linked to original sources

bioRxiv2020

Dipeptidyl peptidase 9 triggers BRCA2 degradation by the N-degron pathway to promote DNA-damage repair

Dipeptidyl peptidase 9 (DPP9) is a serine protease cleaving N-terminal dipeptides preferentially post-proline with (patho)physiological roles in the immune system and cancer. Only few DPP9 substrates are known. Here we identify an association of human DPP9 with the tumour suppressor BRCA2, a key player in repair of DNA double-strand breaks that promotes the formation of RAD51 filaments. This interaction is triggered by DNA-damage and requires access to the DPP9 active-site. We present crystallographic structures documenting the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in the DPP9 active-site. Mechanistically, DPP9 targets BRCA2 for degradation by the N-degron pathway, and promotes RAD51 foci formation. Both processes are phenocopied by BRCA2 N-terminal truncation mutants, indicating that DPP9 regulates both stability and the cellular stoichiometric interactome of BRCA2. Consistently, DPP9-deprived cells are hypersensitive to DNA-damage. Together, we identify DPP9 as a regulator of BRCA2, providing a possible explanation for DPP9 involvement in cancer development.

biochemistry↗

bioRxiv2020

SugarQuant: a streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

Site-specific regulation of protein N-glycosylation is essential in human cells. However, accurate quantification of glycosylation sites and their individual glycan moieties in a cell-wide manner is still technically challenging. Here, we introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising fast protein aggregation capture (PAC)-based sample preparation, optimized multi-notch MS3 LC-MS acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that allows for confident, global identification and quantification of intact glycopeptides in complex biological samples. PAC greatly reduces the overall samplehandling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and significantly lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search which increases the identification rates of intact glycopeptides by up to 22% when compared with one-step database search strategies. SugarQuant was applied to identify and quantify more than 5,000 unique glycoforms in Burkitts lymphoma cells, and determined complex site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

biochemistry↗

bioRxiv2019

PKCα-dependent interaction of otoferlin and calbindin: evidence for regulation of endocytosis in inner hair cells

Otoferlin is essential for the fast and indefatigable release of synaptic vesicles at auditory inner hair cell (IHC) ribbon synapses, being involved in exocytic, endocytic and regenerative steps of the synaptic vesicle cycle. Serving diverse functions at this highly dynamic synapse implies that this multi-C2 domain protein is precisely regulated. Here we found protein kinase C (PKC) and otoferlin to colocalize in endocytic recycling compartments upon IHC depolarization and to interact in an activity-dependent manner. In vitro assays confirmed that PKC can phosphorylate otoferlin at five serine residues, which correlates with increased serine phosphorylation in <40 nm proximity to otoferlin in murine IHCs that can be fully blocked by combining PKC and CaMKII inhibitors. Moreover, otoferlin interacts with calbindin-D28k in stimulated IHCs, which was precluded when PKC was inhibited. Similarly, the activity-dependent increase in otoferlin-myosin VI interaction depends on PKC activation. We propose that upon strong hair cell depolarization, PKC phosphorylates otoferlin, thereby enabling it to interact with calbindin-D28k and myosin VI, building a Ca2+-dependent signaling complex that possibly regulates different modes of endocytosis.

neuroscience↗

bioRxiv2018

Understanding RNP remodelling uncovers RBPs functionally required for viral replication

The compendium of RNA-binding proteins (RBPome) has been greatly expanded by the development of RNA-interactome capture (RNA-IC). However, it remains unknown how responsive is the RBPome and whether these responses are biologically relevant. To answer these questions, we created comparative RNA-IC to analyse cells challenged with an RNA virus, called sindbis (SINV). Strikingly, the virus altered the activity of 245 RBPs, many of which were newly discovered by RNA-IC. Mechanistically, alterations in RNA binding upon SINV infection are caused by changes in the subcellular localisation of RBPs and RNA availability. Moreover, RBPome responses are crucial, as perturbation of dynamic RBPs modulates the capacity of the virus to infect the cell. For example, ablation of XRN1 causes cells to be refractory to infection, while GEMIN5 moonlights as a novel antiviral factor. Therefore, RBPome remodelling provides a mechanism by which cells can extensively rewire gene expression in response to physiological cues.\n\nHIGHLIGHTSO_LIA quarter of the RBPome remodels upon SINV infection.\nC_LIO_LIThe remodelling is caused by changes in protein localisation and RNA availability.\nC_LIO_LIRewiring of the RBPome is crucial for viral infection efficacy.\nC_LIO_LIWe discover RBPs with previously unknown anti- or pro-viral activity.\nC_LI

molecular biology↗