Publications • Freund Group - Protein Biochemistry • Department of Biology, Chemistry, Pharmacy

Target Recognition in Tandem WW Domains: Complex Structures for Parallel and Antiparallel Ligand Orientation in h-FBP21 Tandem WW

Wenz MT, Bertazzon M, Sticht J, Aleksić S, Gjorgjevikj D, Freund C, Keller BG

Appeared in: Journal of Chemical Information and Modeling - 2022 (ahead of print)

Protein–protein interactions often rely on specialized recognition domains, such as WW domains, which bind to specific proline-rich sequences. The specificity of these protein–protein interactions can be increased by tandem repeats, i.e., two WW domains connected by a linker. With a flexible linker, the WW domains can move freely with respect to each other. Additionally, the tandem WW domains can bind in two different orientations to their target sequences. This makes the elucidation of complex structures of tandem WW domains extremely challenging. Here, we identify and characterize two complex structures of the tandem WW domain of human formin-binding protein 21 and a peptide sequence from its natural binding partner, the core-splicing protein SmB/B′. The two structures differ in the ligand orientation and, consequently, also in the relative orientation of the two WW domains. We analyze and probe the interactions in the complexes by molecular simulations and NMR experiments. The workflow to identify the complex structures uses molecular simulations, density-based clustering, and peptide docking. It is designed to systematically generate possible complex structures for repeats of recognition domains. These structures will help us to understand the synergistic and multivalency effects that generate the astonishing versatility and specificity of protein–protein interactions.

The synaptic scaffold protein MPP2 interacts with GABAA receptors at the periphery of the postsynaptic density of glutamatergic synapses

Schmerl, B Gimber N, Kuropka B, Stumpf A, Rentsch J, Kunde SA, von Sivers J, Ewers H, Schmitz D, Freund C, Schmoranzer J, Rademacher N, Shoichet SA

Appeared in: Public Library of Science Biology - Volume 20(3) - 2022

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor–associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that—like SynCAM 1—MPP2 is situated at the periphery of the postsynaptic density (PSD). In order to explore MPP2-associated protein complexes, we used a quantitative comparative proteomics approach and identified multiple γ-aminobutyric acid (GABA)A receptor subunits among novel synaptic MPP2 interactors. In line with a scaffold function for MPP2 in the assembly and/or modulation of intact GABAA receptors, manipulating MPP2 expression had effects on inhibitory synaptic transmission. We further show that GABAA receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold that serves as an adaptor molecule, linking peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses.

The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling

Bergfort A, Hilal T, Kuropka B, Ilik İA, Weber G, Aktaş T, Freund C, Wahl MC

Appeared in: Nucleic Acids Research - Volume 50, Issue 5, pp. 2938-2958 - 2022

Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4’s intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling.

A multi-factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing

Bergfort A, Preußner M, Kuropka B, Ilik İA, Hilal T, Weber G, Freund C, Aktaş T, Heyd F, Wahl MC

Appeared in: Nature Communications 13, 1132 - 2022

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. We find that C9ORF78 tightly interacts with the spliceosome remodeling factor, BRR2, in vitro. Affinity purification/mass spectrometry and RNA UV-crosslinking analyses identify additional C9ORF78 interactors in spliceosomes. Cryogenic electron microscopy structures reveal how C9ORF78 and the spliceosomal B complex protein, FBP21, wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock-down of C9ORF78 leads to alternative NAGNAG 3′-splice site usage and exon skipping, the latter dependent on BRR2. Inspection of spliceosome structures shows that C9ORF78 could contact several detected spliceosome interactors when bound to BRR2, including the suggested 3′-splice site regulating helicase, PRPF22. Together, our data establish C9ORF78 as a late-stage splicing regulatory protein that takes advantage of a multi-factor trafficking site on BRR2, providing one explanation for suggested roles of BRR2 during splicing catalysis and alternative splicing.

Sortase-Mediated Multi-Fragment Assemblies by Ligation Site Switching

Bierlmeier J, Álvaro-Benito M, Scheffler M, Sturm K, Rehkopf L, Freund C, Schwarzer D

Appeared in: Angewandte Chemie International Edition - Volume 61, Issue 5 - 2022

Sortase-mediatedligation(SML) is a powerfultoolof proteinchemistryallowingthe ligationof peptidescontainingLPxTGsortingmotifs and N-terminalglycinenucleophiles.The installationof a sortingmotif into theproductprohibitsthe assemblyof multiplefragmentsbySML. Here we report multi-fragmentSML based on switch-able sortasesubstrates.Substitutionof the Leu residuebydisulfide-containingCys(StBu)results in active sorting motifs,which are inactivatableby reduction.In combinationwith aphoto-protectedN-Gly nucleophile,multi-fragmentSML isenabledby repetitivecycles of SML and ligationsite switch-ing. The feasibilityof this approachwas demonstratedby aproof-of-conceptfour-fragmentligation,the assemblyofpeptideprobesfor bivalentchromatinbindingproteinsandoligomerizationof peptideantigens.Biochemicaland immu-no-assaysdemonstratedfunctionalityof these probes render-ing them promisingtools for immunologyand chromatinbiochemistry.

Exchange catalysis by tapasin exploits conserved and allele-specific features of MHC-I molecules

Lan, Huan, Abualrous, Esam T., Sticht, Jana, Fernandez, Laura Maria Arroyo, Werk, Tamina, Weise, Christoph, Ballaschk, Martin, Schmieder, Peter, Loll, Bernhard and Freund, Christian

Appeared in: Nature Communications - Volume 12, Issue 1

The repertoire of peptides presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface is tailored by the ER-resident peptide loading complex (PLC), which contains the exchange catalyst tapasin. Tapasin stabilizes MHC-I molecules and promotes the formation of stable peptide-MHC-I (pMHC-I) complexes that serve as T cell antigens. Exchange of suboptimal by high-affinity ligands is catalyzed by tapasin, but the underlying mechanism is still elusive. Here we analyze the tapasin-induced changes in MHC-I dynamics, and find the catalyst to exploit two essential features of MHC-I. First, tapasin recognizes a conserved allosteric site underneath the ?2-1-helix of MHC-I, ‘loosening’ the MHC-I F-pocket region that accomodates the C-terminus of the peptide. Second, the scoop loop11–20 of tapasin relies on residue L18 to target the MHC-I F-pocket, enabling peptide exchange. Meanwhile, tapasin residue K16 plays an accessory role in catalysis of MHC-I allotypes bearing an acidic F-pocket. Thus, our results provide an explanation for the observed allele-specificity of catalyzed peptide exchange.

The Multiple Roles of the Cytosolic Adapter Proteins ADAP, SKAP1 and SKAP2 for TCR/CD3 -Mediated Signaling Events

Dadwal, Nirdosh, Mix, Charlie, Reinhold, Annegret, Witte, Amelie, Freund, Christian, Schraven, Burkhart and Kliche, Stefanie

Appeared in: Frontiers in Immunology - Volume 12, 2021, pages 2671

T cells are the key players of the adaptive immune response. They coordinate the activation of other immune cells and kill malignant and virus-infected cells. For full activation T cells require at least two signals. Signal 1 is induced after recognition of MHC/peptide complexes presented on antigen presenting cells (APCs) by the clonotypic TCR (T-cell receptor)/CD3 complex whereas Signal 2 is mediated via the co-stimulatory receptor CD28, which binds to CD80/CD86 molecules that are present on APCs. These signaling events control the activation, proliferation and differentiation of T cells. In addition, triggering of the TCR/CD3 complex induces the activation of the integrin LFA-1 (leukocyte function associated antigen 1) leading to increased ligand binding (affinity regulation) and LFA-1 clustering (avidity regulation). This process is termed “inside-out signaling”. Subsequently, ligand bound LFA-1 transmits a signal into the T cells (“outside-in signaling”) which enhances T-cell interaction with APCs (adhesion), T-cell activation and T-cell proliferation. After triggering of signal transducing receptors, adapter proteins organize the proper processing of membrane proximal and intracellular signals as well as the activation of downstream effector molecules. Adapter proteins are molecules that lack enzymatic or transcriptional activity and are composed of protein-protein and protein-lipid interacting domains/motifs. They organize and assemble macromolecular complexes (signalosomes) in space and time. Here, we review recent findings regarding three cytosolic adapter proteins, ADAP (Adhesion and Degranulation-promoting Adapter Protein), SKAP1 and SKAP2 (Src Kinase Associated Protein 1 and 2) with respect to their role in TCR/CD3-mediated activation, proliferation and integrin regulation.

NS1-mediated upregulation of ZDHHC22 acyltransferase in influenza a virus infected cells

Gadalla M R, Morrison E, Serebryakova M V, Han X, Wolff T, Freund C, Kordyukova L, Veit M

Appeared in: Cellular Microbiology - Volume 6 - 2021

Influenza A viruses contain two S-acylated proteins, the ion channel M2 and the glycoprotein hemagglutinin (HA). Acylation of the latter is essential for virus replication. Here we analysed the expression of each of the 23 members of the family of ZDHHC acyltransferases in human airway cells, the site of virus replication. RT-PCR revealed that every ZDHHC acyltransferase (except ZDHHC19) is expressed in A549 and Calu cells. Interestingly, expression of one ZDHHC, ZDHHC22, is upregulated in virus-infected cells; this effect is more pronounced after infection with an avian compared to a human virus strain. The viral protein NS1 triggers ZDHHC22 expression in transfected cells, whereas recombinant viruses lacking a functional NS1 gene did not cause ZDHHC22 upregulation. CRISPR/Cas9 technology was then used to knock-out the ZDHHC22 gene in A549 cells. However, acylation of M2 and HA was not reduced, as analysed for intracellular HA and M2 and the stoichiometry of S-acylation of HA incorporated into virus particles did not change according to MALDI-TOF mass spectrometry analysis. Comparative mass spectrometry of palmitoylated proteins in wt and ΔZDHHC22 cells identified 25 potential substrates of ZDHHC22 which might be involved in virus replication.

Major histocompatibility complex (MHC) class I and class II proteins: impact of polymorphism on antigen presentation

Abualrous, Esam T., Sticht, Jana and Freund, Christian

Appeared in: Current Opinion in Immunology - Volume 70, 2021, pages 95-104

The major histocompatibility complex (MHC) loci are amongst the most polymorphic regions in the genomes of vertebrates. In the human population, thousands of MHC gene variants (alleles) exist that translate into distinct allotypes equipped with overlapping but unique peptide binding profiles. Understanding the differential structural and dynamic properties of MHC alleles and their interaction with critical regulators of peptide exchange bears the potential for more personalized strategies of immune modulation in the context of HLA-associated diseases.

Revisiting nonclassical HLA II functions in antigen presentation: Peptide editing and its modulation.

Álvaro-Benito, Miguel and Freund, Christian

Appeared in: HLA: Immune Response Genetics - Volume 96, Issue 4, pages 413-557

The nonclassical major histocompatibility complex of class II molecules (ncMHCII) HLA-DM (DM) and HLA-DO (DO) feature essential functions for the selection of the peptides that are displayed by classical MHCII proteins (MHCII) for CD4+ Th cell surveillance. Thus, although the binding groove of classical MHCII dictates the main features of the peptides displayed, ncMHCII function defines the preferential loading of peptides from specific cellular compartments and the extent to which they are presented. DM acts as a chaperone for classical MHCII molecules facilitating peptide exchange and thereby favoring the binding of peptide-MHCII complexes of high kinetic stability mostly in late endosomal compartments. DO on the other hand binds to DM blocking its peptide-editing function in B cells and thymic epithelial cells, limiting DM activity in these cellular subsets. DM and DO distinct expression patterns therefore define specific antigen presentation profiles that select unique peptide pools for each set of antigen presenting cell. We have come a long way understanding the mechanistic underpinnings of such distinct editing profiles and start to grasp the implications for ncMHCII biological function. DM acts as filter for the selection of immunodominant, pathogen-derived epitopes while DO blocks DM activity under certain physiological conditions to promote tolerance to self. Interestingly, recent findings have shown that the unexplored and neglected ncMHCII genetic diversity modulates retroviral infection in mouse, and affects human ncMHCII function. This review aims at highlighting the importance of ncMHCII function for CD4+ Th cell responses while integrating and evaluating what could be the impact of distinct editing profiles because of natural genetic variations.

Human Hepatitis B Viral Infection Outcomes Are Linked to Naturally Occurring Variants of HLA-DOA That Have Altered Function.

Graves, Austin M., Virdis, Francesca, Morrison, Eliot, Álvaro-Benito, Miguel, Khan, Aly A., Freund, Christian, Golovkina, Tatyana V. and Denzin, Lisa K.

Appeared in: Journal of Immunology - Volume 207, Issue 3, pages 923-935

HLA molecules of the MHC class II (MHCII) bind and present pathogen-derived peptides for CD4 T cell activation. Peptide loading of MHCII in the endosomes of cells is controlled by the interplay of the nonclassical MHCII molecules, HLA-DM (DM) and HLA-DO (DO). DM catalyzes peptide loading, whereas DO, an MHCII substrate mimic, prevents DM from interacting with MHCII, resulting in an altered MHCII–peptide repertoire and increased MHCII–CLIP. Although the two genes encoding DO (DOA and DOB) are considered nonpolymorphic, there are rare natural variants. Our previous work identified DOB variants that altered DO function. In this study, we show that natural variation in the DOA gene also impacts DO function. Using the 1000 Genomes Project database, we show that ?98% of individuals express the canonical DOA*0101 allele, and the remaining individuals mostly express DOA*0102, which we found was a gain-of-function allele. Analysis of 25 natural occurring DO? variants, which included the common alleles, identified three null variants and one variant with reduced and nine with increased ability to modulate DM activity. Unexpectedly, several of the variants produced reduced DO protein levels yet efficiently inhibited DM activity. Finally, analysis of associated single-nucleotide polymorphisms genetically linked the DOA*0102 common allele, a gain-of-function variant, with human hepatitis B viral persistence. In contrast, we found that the DO? F114L null allele was linked with viral clearance. Collectively, these studies show that natural variation occurring in the human DOA gene impacts DO function and can be linked to specific outcomes of viral infections.

Dynamic palmitoylation events following T-cell receptor signaling

Morrison, Eliot, Wegner, Tatjana, Zucchetti,,res Ernesto and Álvaro-Benito, Miguel

Appeared in: Nature Communications Biology - Volume 3, Issue 1, art. Num. 368

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. The reversible nature of this modification makes it a prime candidate as a mechanism for regulating signal transduction in T-cell receptor signaling. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi and to localize to the immune synapse upon T cell activation.

RIM-binding protein couples synaptic vesicle recruitment to release sites.

Petzoldt, Astrid G., Götz, Torsten W.B., Driller, Jan Heiner, Lützkendorf, Janine, Reddy-Alla, Suneel, Matkovic-Rachid, Tanja, Liu, Sunbin, Knoche, Elena, Mertel, Sara, Ugorets Vladimir, Lehmann, Martin, Ramesh, Niraja, Beuschel, Christine Brigitte,

Appeared in: Journal of Cell Biology - Volume 219, Issue 7,

"At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. We here show in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a ""hinge"" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites."

Epilepsy-causing STX1B mutations translate altered protein functions into distinct phenotypes in mouse neurons

Vardar, Gülçin, Gerth, Fabian, Schmitt, Xiao Jakob, Rautenstrauch, Pia, Trimbuch, Thorsten, Schubert, Julian, Lerche, Holger, Rosenmund, Christian and Freund, Christian

Appeared in: Brain - Volume 143, Issue7, pages 2119-2138

Syntaxin 1B (STX1B) is a core component of the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that is critical for the exocytosis of synaptic vesicles in the presynapse. SNARE-mediated vesicle fusion is assisted by Munc18-1, which recruits STX1B in the auto-inhibited conformation, while Munc13 catalyses the fast and efficient pairing of helices during SNARE complex formation. Mutations within the STX1B gene are associated with epilepsy. Here we analysed three STX1B mutations by biochemical and electrophysiological means. These three paradigmatic mutations cause epilepsy syndromes of different severity, from benign fever-associated seizures in childhood to severe epileptic encephalopathies. An insertion/deletion (K45/RMCIE, L46M) mutation (STX1BInDel), causing mild epilepsy and located in the early helical Habc domain, leads to an unfolded protein unable to sustain neurotransmission. STX1BG226R, causing epileptic encephalopathies, strongly compromises the interaction with Munc18-1 and reduces expression of both proteins, the size of the readily releasable pool of vesicles, and Ca2+-triggered neurotransmitter release when expressed in STX1-null neurons. The mutation STX1BV216E, also causing epileptic encephalopathies, only slightly diminishes Munc18-1 and Munc13 interactions, but leads to enhanced fusogenicity and increased vesicular release probability, also in STX1-null neurons. Even though the synaptic output remained unchanged in excitatory hippocampal STX1B+/- neurons exogenously expressing STX1B mutants, the manifestation of clear and distinct molecular disease mechanisms by these mutants suggest that certain forms of epilepsies can be conceptualized by assigning mutations to structurally sensitive regions of the STX1B-Munc18-1 interface, translating into distinct neurophysiological phenotypes.

The Na,K-ATPase acts upstream of phosphoinositide PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2

Appeared in: Nature Communications Biology - Valume 3, Issue 1, art.Num. 141

FGF2 is a tumor cell survival factor that is exported from cells by an ER/Golgi-independent secretory pathway. This unconventional mechanism of protein secretion is based on direct translocation of FGF2 across the plasma membrane. The Na,K-ATPase has previously been shown to play a role in this process, however, the underlying mechanism has remained elusive. Here, we define structural elements that are critical for a direct physical interaction between FGF2 and the ?1 subunit of the Na,K-ATPase. In intact cells, corresponding FGF2 mutant forms were impaired regarding both recruitment at the inner plasma membrane leaflet and secretion. Ouabain, a drug that inhibits both the Na,K-ATPase and FGF2 secretion, was found to impair the interaction of FGF2 with the Na,K-ATPase in cells. Our findings reveal the Na,K-ATPase as the initial recruitment factor for FGF2 at the inner plasma membrane leaflet being required for efficient membrane translocation of FGF2 to cell surfaces.

CD4+ Th immunogenicity of the Ascaris spp. secreted products

Ebner, Friederike, Morrison, Eliot, Bertazzon, Miriam, Midha, Ankur, Hartmann, Susanne, Freund, Christian and Álvaro-Benito, Miguel

Appeared in: NPJ Vaccines - Volume 5, art.Num. 25

Ascaris spp. is a major health problem of humans and animals alike, and understanding the immunogenicity of its antigens is required for developing urgently needed vaccines. The parasite-secreted products represent the most relevant, yet complex (>250 proteins) antigens of Ascaris spp. as defining the pathogen-host interplay. We applied an in vitro antigen processing system coupled to quantitative proteomics to identify potential CD4+ Th cell epitopes in Ascaris-secreted products. This approach considerably restricts the theoretical list of epitopes using conventional CD4+ Th cell epitope prediction tools. We demonstrate the specificity and utility of our approach on two sets of candidate lists, allowing us identifying hits excluded by either one or both computational methods. More importantly, one of the candidates identified experimentally, clearly demonstrates the presence of pathogen-reactive T cells in healthy human individuals against these antigens. Thus, our work pipeline identifies the first human T cell epitope against Ascaris spp. and represents an easily adaptable platform for characterization of complex antigens, in particular for those pathogens that are not easily amenable for in vivo experimental validation.

Intersectin-Mediated Clearance of SNARE Complexes Is Required for Fast Neurotransmission.

Jäpel, Maria, Gerth, Fabian, Sakaba, Takeshi, Bacetic, Jelena, Yao, Lijun, Koo, Seong-Joo, Maritzen, Tanja, Freund, Christian and Haucke, Volker

Appeared in: Cell Reports - Volume 30, Issue 2, 2020, pages 409-420

The rapid replenishment of release-ready synaptic vesicles (SVs) at a limiting number of presynaptic release sites is required to sustain high-frequency neurotransmission in CNS neurons. Failure to clear release sites from previously exocytosed material has been shown to impair vesicle replenishment and, therefore, fast neurotransmission. The identity of this material and the machinery that removes it from release sites have remained enigmatic. Here we show that the endocytic scaffold protein intersectin 1 clears release sites by direct SH3 domain-mediated association with a non-canonical proline-rich segment of synaptobrevin assembled into the SNARE complex for neuroexocytosis. Acute structure-based or sustained genetic interference with SNARE complex recognition by intersectin 1 causes a rapid stimulation frequency-dependent depression of neurotransmission due to impaired replenishment of release-ready SVs. These findings identify a key molecular mechanism that underlies exo-endocytic coupling during fast neurotransmitter release at central synapses.

A Missing Switch in Peptide Exchange for MHC Class II Molecules

Freund, Christian and Höfer, Thomas

Appeared in: Frontiers in Immunology - Volume 10, 2019, pages 2513

Introduction: Antigen processing and loading of peptides onto MHC class II molecules is a multistep process that involves vesicular transport of the MHCII molecules along the secretory pathway, where they eventually merge with antigen-containing endocytic vesicles or phagosomes (1). It is within these late endosomal or lysosomal compartments that protein antigens become degraded by proteases, most prominently by cathepsins, and where catalyzed peptide exchange by HLA-DM fulfills its role in the efficient replacement of the invariant chain-derived peptide CLIP by high-affinity pathogen- or host cell-derived peptides. Protease action may be limited by protein antigen abundance and redox conditions, while HLA-DM is regulated at several stages, including by expression levels, pH, or the co-expression of the competitive inhibitor HLA-DO. HLA-DM activity leads to significant changes in the immunopeptidome of antigen-presenting cells, thereby tailoring T cell responses and often shifting antigenicity toward high-affinity immunodominant epitopes (2). Control of DM activity by DO has been described to be of prime importance in thymic epithelial cells, in a subset of dendritic cells, and in B cells when entering the germinal centers for affinity maturation and class switching (3, 4). In all of these cases, the switch from a broader, self-peptide (CLIP) dominated immunopeptidome to a more focused repertoire is necessitated by the requirement for more stringent antigen presentation, often preceding more intense T cell reactivity and proliferation. Here, we review data on this cellular switch in the functionality of antigen presentation and propose that it is promoted by an as yet poorly understood molecular switch. Acknowledging that general biophysical parameters such as pH and redox are important for antigen processing in general, an elusive DM-DO switch is postulated that would allow rapid and strong shifts in immunopeptidomes. We capitalize on theoretical considerations to back our opinion that a regulatable switch would have the advantage of allowing for a rapid and possibly signal-dependent change in the peptide selection process, as might be required in the context of rapidly changing immunological conditions.

Blood Flow Suppresses Vascular Anomalies in a Zebrafish Model of Cerebral Cavernous Malformations.

Rödel, Claudia Jasmin, Otten, Cécile, Donat, Stefan, Lourenço, Fischer, Dorothea, Kuropka, Benno, Paolini, Alessio, Freund, Christian and Abdelilah-Seyfried, Salim

Appeared in: Circulation Research - Volume 125 - Issue 10

RATIONALE:Pathological biomechanical signaling induces vascular anomalies including cerebral cavernous malformations (CCM), which are caused by a clonal loss of CCM1/KRIT1 (Krev interaction trapped protein 1), CCM2/MGC4607, or CCM3/PDCD10. Why patients typically experience lesions only in lowly perfused venous capillaries of the cerebrovasculature is completely unknown. OBJECTIVE:In contrast, animal models with a complete loss of CCM proteins lack a functional heart and blood flow and exhibit vascular anomalies within major blood vessels as well. This finding raises the possibility that hemodynamics may play a role in the context of this vascular pathology. METHODS AND RESULTS:Here, we used a genetic approach to restore cardiac function and blood flow in a zebrafish model of CCM1. We find that blood flow prevents cardiovascular anomalies including a hyperplastic expansion within a large Ccm1-deficient vascular bed, the lateral dorsal aorta. CONCLUSIONS:This study identifies blood flow as an important physiological factor that is protective in the cause of this devastating vascular pathology.

Exon Inclusion Modulates Conformational Plasticity and Autoinhibition of the Intersectin 1 SH3A Domain

Gerth, Fabian, Jäpel, Maria, Sticht, Jana, Kuropka, Benno, Schmitt, Xiao Jakob, Driller, Jan H., Loll, Bernhard, Wahl, Markus C., Pagel, Kevin, Haucke, Volker and Freund, Christian

Appeared in: Structure - Volume 27, Issue 6, 2019, pages 977-987

The scaffolding protein intersectin 1 plays important roles in clathrin-mediated endocytosis and in the replenishment of release-ready synaptic vesicles (SV). Two splice variants of intersectin's SH3A domain are expressed in the brain, and association of the neuron-specific variant with synapsin I has been shown to enable sustained neurotransmission and to be regulated by an adjacent C-terminal motif. Here, we demonstrate that the ubiquitously expressed short SH3A variant of intersectin 1 interacts with an N-terminal intramolecular sequence that operates synergistically with the C-terminal motif. NMR spectroscopic investigations show that the five-amino acid insertion into the ? strand 2 of the neuronal SH3A variant introduces conformational plasticity incompatible with binding of the N-terminal sequence. The difference in the autoregulatory mechanism of the domain's variants differentially affects its synaptic binding partners, thereby establishing alternative splicing in conjunction with autoinhibitory motif variation as a mechanism to regulate protein interaction networks.

Phosphorylation of the Bruchpilot N-terminus in Drosophila unlocks axonal transport of active zone building blocks.

Driller, Jan H., Lützkendorf, Janine, Depner, Herald, Siebert, Matthias, Kuropka, Benno, Weise, Christoph, Piao, Chengji, Petzoldt, Astrig G., Lehmann, Martin, Stelzl, Ulrich, Zahedi, René, Sickmann, Albert, Freund, Christian, Sigrist, Stephan J and

Appeared in: Journal of Cell Science - Volume 132, Issue 6

Protein scaffolds at presynaptic active zone membranes control information transfer at synapses. For scaffold biogenesis and maintenance, scaffold components must be safely transported along axons. A spectrum of kinases has been suggested to control transport of scaffold components, but direct kinase-substrate relationships and operational principles steering phosphorylation-dependent active zone protein transport are presently unknown. Here, we show that extensive phosphorylation of a 150-residue unstructured region at the N-terminus of the highly elongated Bruchpilot (BRP) active zone protein is crucial for ordered active zone precursor transport in Drosophila Point mutations that block SRPK79D kinase-mediated phosphorylation of the BRP N-terminus interfered with axonal transport, leading to BRP-positive axonal aggregates that also contain additional active zone scaffold proteins. Axonal aggregates formed only in the presence of non-phosphorylatable BRP isoforms containing the SRPK79D-targeted N-terminal stretch. We assume that specific active zone proteins are pre-assembled in transport packages and are thus co-transported as functional scaffold building blocks. Our results suggest that transient post-translational modification of a discrete unstructured domain of the master scaffold component BRP blocks oligomerization of these building blocks during their long-range transport.

Intramolecular domain dynamics regulate synaptic MAGUK protein interactions

Rademacher, Nils, Kuropka, Benno, Kunde, Stella-Amrei, Wahl, Markus C., Freund, Christian and Shoichet, Sarah A.

Appeared in: eLife Sciences Publications - Volume 8, 2019

PSD-95 MAGUK family scaffold proteins are multi-domain organisers of synaptic transmission that contain three PDZ domains followed by an SH3-GK domain tandem. This domain architecture allows coordinated assembly of protein complexes composed of neurotransmitter receptors, synaptic adhesion molecules and downstream signalling effectors. Here we show that binding of monomeric CRIPT-derived PDZ3 ligands to the third PDZ domain of PSD-95 induces functional changes in the intramolecular SH3-GK domain assembly that influence subsequent homotypic and heterotypic complex formation. We identify PSD-95 interactors that differentially bind to the SH3-GK domain tandem depending on its conformational state. Among these interactors, we further establish the heterotrimeric G protein subunit Gnb5 as a PSD-95 complex partner at dendritic spines of rat hippocampal neurons. The PSD-95 GK domain binds to Gnb5, and this interaction is triggered by CRIPT-derived PDZ3 ligands binding to the third PDZ domain of PSD-95, unraveling a hierarchical binding mechanism of PSD-95 complex formation.

FBP21's C-Terminal Domain Remains Dynamic When Wrapped around the c-Sec63 Unit of Brr2 Helicase.

Sticht, Jana, Bertazzon, Miriam, Henning, Lisa M., Licha, Jan R., Abualrous, Esam T. and Freund, Christian

Appeared in: Biophysical journal - Volume 116 - Issue 3

Based on our recent finding that FBP21 regulates human Brr2 helicase activity involved in the activation of the spliceosomal B-complex, we investigated the structural and dynamic contribution of FBP21 to the interaction. By using NMR spectroscopy, we could show that the 50 C-terminal residues of FBP21 (FBP21326-376), which are sufficient to fully form the interaction with the C-terminal Sec63 unit of Brr2 (Brr2C-Sec63), adopt a random-coil conformation in their unbound state. Upon interaction with Brr2C-Sec63, 42 residues of FBP21326-376 cover the large binding site on Brr2C-Sec63 in an extended conformation. Short charged motifs are steering complex formation, still allowing the bound state to retain dynamics. Based on fragment docking in combination with experimental restraints, we present models of the complex structure. The FBP21326-376/Brr2C-Sec63 interaction thus presents an example of an intrinsically disordered protein/ordered-protein interaction in which a large binding site provides high specificity and, in combination with conformational disorder, displays a relatively high affinity.

Structure-Function Relationship of XCL1 Used for in vivo Targeting of Antigen Into XCR1+ Dendritic Cells

Kroczek, Arthur L. and Hartung, Evelyn and Gurka, Stephanie and Becker, Martina and Reeg, Nele and Mages, Hans W. and Voigt, Sebastian and Freund, Christian and Kroczek, Richard A.

Appeared in: Frontiers of Immunology - Volume 9, 2018

XCL1 is the ligand for XCR1, a chemokine receptor uniquely expressed on cross-presenting dendritic cells (DC) in mouse and man. We are interested in establishing therapeutic vaccines based on XCL1-mediated targeting of peptides or proteins into these DC. Therefore, we have functionally analyzed various XCL1 domains in highly relevant settings in vitro and in vivo. Murine XCL1 fused to ovalbumin (XCL1-OVA) was compared to an N-terminal deletion variant lacking the first seven N-terminal amino acids and to several C-terminal (deletion) variants. Binding studies with primary XCR1+ DC revealed that the N-terminal region stabilizes the binding of XCL1 to its receptor, as is known for other chemokines. Deviating from the established paradigm for chemokines, the N-terminus does not contain critical elements for inducing chemotaxis. On the contrary, this region appears to limit the chemotactic action of XCL1 at higher concentrations. A participation of the XCL1 C-terminus in receptor binding or chemotaxis could be excluded in a series of experiments. Binding studies with apoptotic and necrotic XCR1-negative cells suggested a second function for XCL1: marking of stressed cells for uptake into cross-presenting DC. In vivo studies using CD8+ T cell proliferation and cytotoxicity as readouts confirmed the critical role of the N-terminus for antigen targeting, and excluded any involvement of the C-terminus in the uptake, processing, and presentation of the fused OVA antigen. Together, these studies provide basic data on the function of the various XCL1 domains as well as relevant information on XCL1 as an antigen carrier in therapeutic vaccines.

Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

Waldt, Natalie and Seifert, Anke and Demiray, Yunus Emre and Devroe, Eric and Turk, Benjamin E. and Reichardt, Peter and Mix, Charlie and Reinhold, Annegret and Freund, Christian and M�ller, Andreas J. and Schraven, Burkhart and Stork, Oliver and Kliche, Stefanie

The integrin LFA-1 (CD11a/CD18) plays a critical role in the interaction of T cells with antigen presenting cells (APCs) to promote lymphocyte differentiation and proliferation. This integrin can be present either in a closed or in an open active conformation and its activation upon T-cell receptor (TCR) stimulation is a critical step to allow interaction with APCs. In this study we demonstrate that the serine/threonine kinase Ndr2 is critically involved in the initiation of TCR-mediated LFA-1 activation (open conformation) in T cells. Ndr2 itself becomes activated upon TCR stimulation and phosphorylates the intracellular integrin binding partner Filamin A (FLNa) at serine 2152. This phosphorylation promotes the dissociation of FLNa from LFA-1, allowing for a subsequent association of Talin and Kindlin-3 which both stabilize the open conformation of LFA-1. Our data suggest that Ndr2 activation is a crucial step to initiate TCR-mediated LFA-1 activation in T cells.

Distinct editing functions of natural HLA-DM allotypes impact antigen presentation and CD4+ T cell activation

Alvaro-Benito, Miguel and Morrison, Eliot and Ebner, Friederike and Abualrous, Esam T. and Urbicht, Marie and Wieczorek, Marek and Freund, Christian

Classical human leukocyte antigen (HLA) molecules of the major histocompatibility class II (MHCII) complex present peptides for the development, surveillance and activation of CD4+ T cells. The nonclassical MHCII-like protein HLA-DM (DM) catalyzes the exchange and loading of peptides onto MHCII molecules, thereby shaping MHCII immunopeptidomes. Natural variations of DM in both chains of the protein (DMA and DMB) have been hypothesized to impact peptide presentation, but no evidence for altered function has been reported. Here we define the presence of DM allotypes in human populations covered by the 1000 Genomes Project and probe their activity. The functional properties of several allotypes are investigated and show strong enhancement of peptide-induced T cell activation for a particular combination of DMA and DMB. Biochemical evidence suggests a broader pH activity profile for the new variant relative to that of the most commonly expressed DM allotype. Immunopeptidome analysis indicates that the compartmental activity of the new DM heterodimer extends beyond the late endosome and suggests that the natural variation of DM has profound effects on adaptive immunity when antigens bypass the canonical processing pathway.

The GTPase ARFRP1 affects lipid droplet protein composition and triglyceride release

Werno MW and Wilhelmi I and Kuropka B and Ebert F and Freund C and Schuermann A

Intestinal release of dietary triglycerides via chylomicrons is the major contributor to elevated postprandial triglyceride levels. Dietary lipids can be transiently stored in cytosolic lipid droplets (LDs) located in intestinal enterocytes for later release. ADP ribosylation factor-related protein 1 (ARFRP1) participates in processes of LD growth in adipocytes and in lipidation of lipoproteins in liver and intestine. This study aims to explore the impact of ARFRP1 on LD organization and its interplay with chylomicron-mediated triglyceride release in intestinal-like Caco-2 cells. Suppression of Arfrp1 reduced release of intracellularly derived triglycerides (0.69-fold) and increased the abundance of transitional endoplasmic reticulum ATPase TERA/VCP, fatty acid synthase-associated factor 2 (FAF2) and perilipin 2 (Plin2) at the LD surface. Furthermore, TERA/VCP and FAF2 co-occurred more frequently with ATGL at LDs, suggesting a reduced adipocyte triglyceride lipase (ATGL)-mediated lipolysis. Accordingly, inhibition of lipolysis reduced lipid release from intracellular storage pools by the same magnitude as Arfrp1 depletion. Thus, the lack of Arfrp1 increases the abundance of lipolysis-modulating enzymes TERA/VCP, FAF2 and Plin2 at LDs, which might decrease lipolysis and reduce availability of fatty acids for triglyceride synthesis and their release via chylomicrons.

Understanding the elusive protein corona of thermoresponsive nanogels

Miceli, Enrico and Kuropka, Benno and Rosenauer, Christine and Osorio Blanco, Ernesto R and Theune, Loryn E and Kar, Mrityunjoy and Weise, Christoph and Morsbach, Svenja and Freund, Christian and Calderón, Marcelo

Aim: We analyzed the protein corona of thermoresponsive, poly(N-isopropylacrylamide)- or poly(N-isopropylmethacrylamide)-based nanogels. Materials & methods: Traces of protein corona detected after incubation in human serum were characterized by proteomics and dynamic light scattering in undiluted serum. Results: Apolipoprotein B-100 and albumin were the main components of the protein coronae. For dendritic polyglycerol-poly(N-isopropylacrylamide) nanogels at 37�C, an increase in adsorbed immunoglobulin light chains was detected, followed by partially reversible nanogel aggregation. All nanogels in their hydrophilic state are colloidally stable in serum and bear a dysopsonin-rich protein corona. Conclusion: We observed strong changes in NG stability upon slight alterations in the composition of the protein coronae according to nanogel solvation state. Nanogels in their hydrophilic state possess safe protein coronae.

Quantification of HLA-DM-Dependent Major Histocompatibility Complex of Class II Immunopeptidomes by the Peptide Landscape Antigenic Epitope Alignment Utility

Alvaro-Benito, Miguel and Morrison, Eliot and Abualrous, Esam T. and Kuropka, Benno and Freund, Christian

The major histocompatibility complex of class II (MHCII) immunopeptidome represents the repertoire of antigenic peptides with the potential to activate CD4+ T cells. An understanding of how the relative abundance of specific antigenic epitopes affects the outcome of T cell responses is an important aspect of adaptive immunity and offers a venue to more rationally tailor T cell activation in the context of disease. Recent advances in mass spectrometric instrumentation, computational power, labeling strategies, and software analysis have enabled an increasing number of stratified studies on HLA ligandomes, in the context of both basic and translational research. A key challenge in the case of MHCII immunopeptidomes, often determined for different samples at distinct conditions, is to derive quantitative information on consensus epitopes from antigenic peptides of variable lengths. Here, we present the design and benchmarking of a new algorithm [peptide landscape antigenic epitope alignment utility (PLAtEAU)] allowing the identification and label-free quantification (LFQ) of shared consensus epitopes arising from series of nested peptides. The algorithm simplifies the complexity of the dataset while allowing the identification of nested peptides within relatively short segments of protein sequences. Moreover, we apply this algorithm to the comparison of the ligandomes of cell lines with two different expression levels of the peptide-exchange catalyst HLA-DM. Direct comparison of LFQ intensities determined at the peptide level is inconclusive, as most of the peptides are not significantly enriched due to poor sampling. Applying the PLAtEAU algorithm for grouping of the peptides into consensus epitopes shows that more than half of the total number of epitopes is preferentially and significantly enriched for each condition. This simplification and deconvolution of the complex and ambiguous peptide-level dataset highlights the value of the PLAtEAU algorithm in facilitating robust and accessible quantitative analysis of immunopeptidomes across cellular contexts. In silico analysis of the peptides enriched for each HLA-DM expression conditions suggests a higher affinity of the pool of peptides isolated from the high DM expression samples. Interestingly, our analysis reveals that while for certain autoimmune-relevant epitopes their presentation increases upon DM expression others are clearly edited out from the peptidome.

Intrinsically Disordered Protein Ntr2 Modulates the Spliceosomal RNA Helicase Brr2

Wollenhaupt, Jan and Henning, Lisa M. and Sticht, Jana and Becke, Christian and Freund, Christian and Santos, Karine F. and Wahl, Markus C.

Precursor messenger RNA splicing is mediated by the spliceosome, a large and dynamic molecular machine composed of five small nuclear RNAs and numerous proteins. Many spliceosomal proteins are predicted to be intrinsically disordered or contain large disordered regions, but experimental validation of these predictions is scarce, and the precise functions of these proteins are often unclear. Here, we show via circular dichroism spectroscopy, dynamic light scattering, and NMR spectroscopy that the yeast spliceosomal disassembly factor Ntr2 is largely intrinsically disordered. Peptide SPOT analyses, analytical size-exclusion chromatography, and surface plasmon resonance measurements revealed that Ntr2 uses an N-terminal region to bind the C-terminal helicase unit of the Brr2 RNA helicase, an enzyme involved in spliceosome activation and implicated in splicing catalysis and spliceosome disassembly. NMR analyses suggested that Ntr2 does not adopt a tertiary structure and likely remains disordered upon complex formation. RNA binding and unwinding studies showed that Ntr2 downregulates Brr2 helicase activity in vitro by modulating the fraction of helicase molecules productively bound to the RNA substrate. Our data clarify the nature of a physical link between Brr2 and Ntr2, and point to the possibility of a functional Ntr2-Brr2 interplay during splicing.

A new role for FBP21 as regulator of Brr2 helicase activity

Henning L M, Santos K F, Sticht J, Jehle S, Chung-Tien L, Wittwer M, Urlaub H, Stelzl U, Wahl M C, Freund C

Appeared in: Nucleic Acids Research - Volume 45, Issue 13 - 2017

Splicing of eukaryotic pre-mRNA is carried out by the spliceosome, which assembles stepwise on each splicing substrate. This requires the concerted action of snRNPs and non-snRNP accessory proteins, the functions of which are often not well understood. Of special interest are B complex factors that enter the spliceosome prior to catalytic activation and may alter splicing kinetics and splice site selection. One of these proteins is FBP21, for which we identified several spliceosomal binding partners in a yeast-two-hybrid screen, among them the RNA helicase Brr2. Biochemical and biophysical analyses revealed that an intrinsically disordered region of FBP21 binds to an extended surface of the C-terminal Sec63 unit of Brr2. Additional contacts in the C-terminal helicase cassette are required for allosteric inhibition of Brr2 helicase activity. Furthermore, the direct interaction between FBP21 and the U4/U6 di-snRNA was found to reduce the pool of unwound U4/U6 di-snRNA. Our results suggest FBP21 as a novel key player in the regulation of Brr2.