@article{YoungClementsLangetal.2014, author = {Young, Joanna C. and Clements, Abigail and Lang, Alexander E. and Garnett, James A. and Munera, Diana and Arbeloa, Ana and Pearson, Jaclyn and Hartland, Elizabeth L. and Matthews, Stephen J. and Mousnier, Aurelie and Barry, David J. and Way, Michael and Schlosser, Andreas and Aktories, Klaus and Frankel, Gad}, title = {The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation}, series = {Nature Communications}, volume = {5}, journal = {Nature Communications}, number = {5887}, doi = {10.1038/ncomms6887}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121157}, year = {2014}, abstract = {The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck-WIP-N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose.}, language = {en} } @article{WeigandRonchiVanselowetal.2021, author = {Weigand, Isabel and Ronchi, Cristina L. and Vanselow, Jens T. and Bathon, Kerstin and Lenz, Kerstin and Herterich, Sabine and Schlosser, Andreas and Kroiss, Matthias and Fassnacht, Martin and Calebiro, Davide and Sbiera, Silviu}, title = {PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser\(^{114}\) phosphorylation}, series = {Science Advances}, volume = {7}, journal = {Science Advances}, number = {8}, doi = {10.1126/sciadv.abd4176}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270445}, year = {2021}, abstract = {Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing's syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser\(^{114}\) phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing's syndrome.}, language = {en} } @article{SiegelVasquezHonetal.2014, author = {Siegel, T. Nicolai and Vasquez, Juan-Jos{\´e} and Hon, Chung-Chau and Vanselow, Jens T. and Schlosser, Andreas}, title = {Comparative ribosome profiling reveals extensive translational complexity in different Trypanosoma brucei life cycle stages}, doi = {10.1093/nar/gkt1386}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112657}, year = {2014}, abstract = {While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5' UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei.}, subject = {Ribosom}, language = {en} } @article{SchwanLangSchlosseretal.2022, author = {Schwan, Carsten and Lang, Alexander E. and Schlosser, Andreas and Fujita-Becker, Setsuko and AlHaj, Abdulatif and Schr{\"o}der, Rasmus R. and Faix, Jan and Aktories, Klaus and Mannherz, Hans Georg}, title = {Inhibition of Arp2/3 complex after ADP-ribosylation of Arp2 by binary Clostridioides toxins}, series = {Cells}, volume = {11}, journal = {Cells}, number = {22}, issn = {2073-4409}, doi = {10.3390/cells11223661}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-297454}, year = {2022}, abstract = {Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases.}, language = {en} } @article{PiteauPapatheodorouSchwanetal.2014, author = {Piteau, Marianne and Papatheodorou, Panagiotis and Schwan, Carsten and Schlosser, Andreas and Aktories, Klaus and Schmidt, Gudula}, title = {Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)}, series = {PLoS Pathogens}, volume = {10}, journal = {PLoS Pathogens}, number = {1}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1003884}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117987}, pages = {e1003884}, year = {2014}, abstract = {The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions. Author Summary We study a crucial virulence factor produced by pathogenic Escherichia coli strains, the Cytotoxic Necrotizing Factor 1 (CNF1). More than 80\% of urinary tract infections (UTIs), which are counted among the most common bacterial infections of humans, are caused by Uropathogenic Escherichia coli (UPEC) strains. We and others elucidated the molecular mechanism of the E. coli toxin CNF1. It constitutively activates Rho GTPases by a direct covalent modification. The toxin enters mammalian cells by receptor-mediated endocytosis. Here, we identified the protein receptor for CNF1 by co-precipitation of cell surface molecules with the tagged toxin and subsequent Maldi-TOF analysis. We identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as receptor for CNF1 and located its interaction site to the C-terminal part of the toxin. We performed direct protein-protein interaction analysis and competition studies. Moreover, cells deficient in Lu/BCAM could not bind labeled CNF1. The identification of a toxin's cellular receptor and receptor binding region is an important task for understanding the pathogenic function of the toxin and, moreover, to make the toxin accessible for its use as a cellbiological and pharmacological tool, for example for the generation of immunotoxins.}, language = {en} } @article{PaulsHamaratTrufasuetal.2019, author = {Pauls, Dennis and Hamarat, Yasmin and Trufasu, Luisa and Schendzielorz, Tim M. and Gramlich, Gertrud and Kahnt, J{\"o}rg and Vanselow, Jens and Schlosser, Andreas and Wegener, Christian}, title = {Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate}, series = {European Journal of Neuroscience}, volume = {50}, journal = {European Journal of Neuroscience}, number = {9}, doi = {10.1111/ejn.14516}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204863}, pages = {3502-3519}, year = {2019}, abstract = {Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well-characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE ), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of carboxypeptidase D (dCPD ) in global neuropeptide processing and selected peptide-regulated behaviours in Drosophila . We found that a deficiency in dCPD results in C-terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD -encoding gene silver in the larva causes lethality, and leads to deficits in starvation-induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing and peptide-regulated behaviour in Drosophila . dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE.}, language = {en} } @article{MietrachSchlosserGeibel2019, author = {Mietrach, Nicole and Schlosser, Andreas and Geibel, Sebastian}, title = {An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization}, series = {Acta Crystallographica Section F}, volume = {75}, journal = {Acta Crystallographica Section F}, number = {12}, doi = {10.1107/S2053230X1901495X}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213681}, pages = {725-730}, year = {2019}, abstract = {The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α-helices with a melting point of 34.5°C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16\% PEG 3350 using the hanging-drop vapor-diffusion method. Diffraction data were analyzed to 4.0 {\AA} resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit-cell parameters a = 197.5, b = 197.5, c = 368.3 {\AA}, α = β = γ = 90°.}, language = {en} } @article{MartinSchlosserFurtwaengleretal.2021, author = {Mart{\´i}n, Ovidio Jim{\´e}nez and Schlosser, Andreas and Furtw{\"a}ngler, Rhoikos and Wegert, Jenny and Gessler, Manfred}, title = {MYCN and MAX alterations in Wilms tumor and identification of novel N-MYC interaction partners as biomarker candidates}, series = {Cancer Cell International}, volume = {21}, journal = {Cancer Cell International}, doi = {10.1186/s12935-021-02259-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265542}, year = {2021}, abstract = {Background Wilms tumor (WT) is the most common renal tumor in childhood. Among others, MYCN copy number gain and MYCN P44L and MAX R60Q mutations have been identified in WT. MYCN encodes a transcription factor that requires dimerization with MAX to activate transcription of numerous target genes. MYCN gain has been associated with adverse prognosis in different childhood tumors including WT. The MYCN P44L and MAX R60Q mutations, located in either the transactivating or basic helix-loop-helix domain, respectively, are predicted to be damaging by different pathogenicity prediction tools, but the functional consequences remain to be characterized. Methods We screened a large cohort of unselected WTs for MYCN and MAX alterations. Wild-type and mutant protein function were characterized biochemically, and we analyzed the N-MYC protein interactome by mass spectrometric analysis of N-MYC containing protein complexes. Results Mutation screening revealed mutation frequencies of 3\% for MYCN P44L and 0.9\% for MAX R60Q that are associated with a higher risk of relapse. Biochemical characterization identified a reduced transcriptional activation potential for MAX R60Q, while the MYCN P44L mutation did not change activation potential or protein stability. The protein interactome of N-MYC-P44L was likewise not altered as shown by mass spectrometric analyses of purified N-MYC complexes. Nevertheless, we could identify a number of novel N-MYC partner proteins, e.g. PEG10, YEATS2, FOXK1, CBLL1 and MCRS1, whose expression is correlated with MYCN in WT samples and several of these are known for their own oncogenic potential. Conclusions The strongly elevated risk of relapse associated with mutant MYCN and MAX or elevated MYCN expression corroborates their role in WT oncogenesis. Together with the newly identified co-expressed interactors they expand the range of potential biomarkers for WT stratification and targeting, especially for high-risk WT.}, language = {en} } @article{MarcuSchlosserKeuppetal.2021, author = {Marcu, Ana and Schlosser, Andreas and Keupp, Anne and Trautwein, Nico and Johann, Pascal and W{\"o}lfl, Matthias and Lager, Johanna and Monoranu, Camelia Maria and Walz, Juliane S and Henkel, Lisa M and Krauß, J{\"u}rgen and Ebinger, Martin and Schuhmann, Martin and Thomale, Ulrich Wilhelm and Pietsch, Torsten and Klinker, Erdwine and Schlegel, Paul G and Oyen, Florian and Reisner, Yair and Rammensee, Hans-Georg and Eyrich, Matthias}, title = {Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors}, series = {Journal for ImmunoTherapy of Cancer}, volume = {9}, journal = {Journal for ImmunoTherapy of Cancer}, doi = {10.1136/jitc-2021-003404}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-370214}, year = {2021}, abstract = {Background Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells. Methods Here, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM. Results Comparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80\% of AT/RT exclusive peptides were able to successfully prime CD8+ T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50\% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors. Conclusions These findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion.}, language = {en} } @article{MarcuBichmannKuchenbeckeretal.2021, author = {Marcu, Ana and Bichmann, Leon and Kuchenbecker, Leon and Kowalewski, Daniel Johannes and Freudenmann, Lena Katharina and Backert, Linus and M{\"u}hlenbruch, Lena and Szolek, Andr{\´a}s and L{\"u}bke, Maren and Wagner, Philipp and Engler, Tobias and Matovina, Sabine and Wang, Jian and Hauri-Hohl, Mathias and Martin, Roland and Kapolou, Konstantina and Walz, Juliane Sarah and Velz, Julia and Moch, Holger and Regli, Luca and Silginer, Manuela and Weller, Michael and L{\"o}ffler, Markus W. and Erhard, Florian and Schlosser, Andreas and Kohlbacher, Oliver and Stevanović, Stefan and Rammensee, Hans-Georg and Neidert, Marian Christoph}, title = {HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy}, series = {Journal for ImmunoTherapy of Cancer}, volume = {9}, journal = {Journal for ImmunoTherapy of Cancer}, doi = {10.1136/jitc-2020-002071}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-370160}, year = {2021}, abstract = {Background The human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign tissues, if available, normal tissue adjacent to the tumor has been used as a benign surrogate when defining tumor-associated antigens. However, this comparison has proven to be insufficient and even resulted in lethal outcomes. In order to match the tumor immunopeptidome with an equivalent counterpart, we created the HLA Ligand Atlas, the first extensive collection of paired HLA-I and HLA-II immunopeptidomes from 227 benign human tissue samples. This dataset facilitates a balanced comparison between tumor and benign tissues on HLA ligand level. Methods Human tissue samples were obtained from 16 subjects at autopsy, five thymus samples and two ovary samples originating from living donors. HLA ligands were isolated via immunoaffinity purification and analyzed in over 1200 liquid chromatography mass spectrometry runs. Experimentally and computationally reproducible protocols were employed for data acquisition and processing. Results The initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 90,428 HLA-I- and 142,625 HLA-II ligands. The HLA allotypes are representative for the world population. We observe that immunopeptidomes differ considerably between tissues and individuals on source protein and HLA-ligand level. Moreover, we discover 1407 HLA-I ligands from non-canonical genomic regions. Such peptides were previously described in tumors, peripheral blood mononuclear cells (PBMCs), healthy lung tissues and cell lines. In a case study in glioblastoma, we show that potential on-target off-tumor adverse events in immunotherapy can be avoided by comparing tumor immunopeptidomes to the provided multi-tissue reference. Conclusion Given that T-cell-based immunotherapies, such as CAR-T cells, affinity-enhanced T cell transfer, cancer vaccines and immune checkpoint inhibition, have significant side effects, the HLA Ligand Atlas is the first step toward defining tumor-associated targets with an improved safety profile. The resource provides insights into basic and applied immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy and autoimmunity. It is publicly available and can be browsed in an easy-to-use web interface at https://hla-ligand-atlas.org .}, language = {en} }