434 results on '"Heinrich Sticht"'
Search Results
2. Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection
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Irshad Sharafutdinov, Aileen Harrer, Mathias Müsken, Klemens Rottner, Heinrich Sticht, Christian Täger, Michael Naumann, Nicole Tegtmeyer, and Steffen Backert
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CagA ,T4SS ,Cortactin ,Par1b ,ZO-1 ,ASPP2 ,Biology (General) ,QH301-705.5 ,Medicine (General) ,R5-920 - Abstract
Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.
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- 2024
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3. Understanding the Cytomegalovirus Cyclin-Dependent Kinase Ortholog pUL97 as a Multifaceted Regulator and an Antiviral Drug Target
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Manfred Marschall, Martin Schütz, Markus Wild, Eileen Socher, Christina Wangen, Kishore Dhotre, William D. Rawlinson, and Heinrich Sticht
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herpesviral protein kinases ,human cytomegalovirus ,cytomegaloviral vCDK/pUL97 ,regulation of viral replication ,impact of vCDK/pUL97 ,cyclin H and CDK7 ,Cytology ,QH573-671 - Abstract
Herpesviral protein kinases, such as the therapy-relevant pUL97 of human cytomegalovirus (HCMV), are important for viral replication efficiency as well as pathogenesis, and represent key antiviral drug targets. HCMV pUL97 is a viral cyclin-dependent kinase (CDK) ortholog, as it shares functional and structural properties with human CDKs. Recently, the formation of vCDK/pUL97–cyclin complexes and the phosphorylation of a variety of viral and cellular substrate proteins has been demonstrated. Genetic mapping and structural modeling approaches helped to define two pUL97 interfaces, IF1 and IF2, responsible for cyclin binding. In particular, the regulatory importance of interactions between vCDK/pUL97 and host cyclins as well as CDKs has been highlighted, both as determinants of virus replication and as a novel drug-targeting option. This aspect was substantiated by the finding that virus replication was impaired upon cyclin type H knock-down, and that such host-directed interference also affected viruses resistant to existing therapies. Beyond the formation of binary interactive complexes, a ternary pUL97–cyclin H–CDK7 complex has also been described, and in light of this, an experimental trans-stimulation of CDK7 activity by pUL97 appeared crucial for virus–host coregulation. In accordance with this understanding, several novel antiviral targeting options have emerged. These include kinase inhibitors directed to pUL97, to host CDKs, and to the pUL97–cyclin H interactive complexes. Importantly, a statistically significant drug synergy has recently been reported for antiviral treatment schemes using combinations of pharmacologically relevant CDK7 and vCDK/pUL97 inhibitors, including maribavir. Combined, such findings provide increased options for anti-HCMV control. This review focuses on regulatory interactions of vCDK/pUL97 with the host cyclin–CDK apparatus, and it addresses the functional relevance of these key effector complexes for viral replication and pathogenesis. On this basis, novel strategies of antiviral drug targeting are defined.
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- 2024
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4. Light-dependent regulation of neurotransmitter release from rod photoreceptor ribbon synapses involves an interplay of Complexin 4 and Transducin with the SNARE complex
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Uwe Thorsten Lux, Jutta Meyer, Olaf Jahn, Adam Davison, Norbert Babai, Andreas Gießl, Anna Wartenberg, Heinrich Sticht, Nils Brose, Kerstin Reim, and Johann Helmut Brandstätter
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mouse retina ,G protein ,ribbon synapses ,neurotransmitter release ,rod photoreceptor ,light adaptation ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Adaptation of photoreceptor sensitivity to varying light intensities is a fundamental requirement for retinal function and vision. Adaptive mechanisms in signal transduction are well described, but little is known about the mechanisms that adapt the photoreceptor synapse to changing light intensities. The SNARE complex regulators Complexin 3 and Complexin 4 have been proposed to be involved in synaptic light adaptation by limiting synaptic vesicle recruitment and fusion. How this Complexin effect is exerted is unknown. Focusing on rod photoreceptors, we established Complexin 4 as the predominant Complexin in the light-dependent regulation of neurotransmitter release. The number of readily releasable synaptic vesicles is significantly smaller in light than in dark at wildtype compared to Complexin 4 deficient rod photoreceptor ribbon synapses. Electrophysiology indicates that Complexin 4 reduces or clamps Ca2+-dependent sustained synaptic vesicle release, thereby enhancing light signaling at the synapse. Complexin 4 deficiency increased synaptic vesicle release and desensitized light signaling. In a quantitative proteomic screen, we identified Transducin as an interactor of the Complexin 4-SNARE complex. Our results provide evidence for a presynaptic interplay of both Complexin 4 and Transducin with the SNARE complex, an interplay that may facilitate the adaptation of synaptic transmission to light at rod photoreceptor ribbon synapses.
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- 2024
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5. Patch-clamp studies and cell viability assays suggest a distinct site for viroporin inhibitors on the E protein of SARS-CoV-2
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Ulrike Breitinger, Christine Adel Sedky, Heinrich Sticht, and Hans-Georg Breitinger
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SARS-CoV-2 ,E protein ,Viroporins ,Cell viability assay ,Patch-clamp electrophysiology ,Viroporin inhibitors ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background SARS-CoV-2 has caused a worldwide pandemic since December 2019 and the search for pharmaceutical targets against COVID-19 remains an important challenge. Here, we studied the envelope protein E of SARS-CoV and SARS-CoV-2, a highly conserved 75–76 amino acid viroporin that is crucial for virus assembly and release. E protein channels were recombinantly expressed in HEK293 cells, a membrane-directing signal peptide ensured transfer to the plasma membrane. Methods Viroporin channel activity of both E proteins was investigated using patch-clamp electrophysiology in combination with a cell viability assay. We verified inhibition by classical viroporin inhibitors amantadine, rimantadine and 5-(N,N-hexamethylene)-amiloride, and tested four ivermectin derivatives. Results Classical inhibitors showed potent activity in patch-clamp recordings and viability assays. In contrast, ivermectin and milbemycin inhibited the E channel in patch-clamp recordings but displayed only moderate activity on the E protein in the cell viability assay, which is also sensitive to general cytotoxic activity of the tested compounds. Nemadectin and ivermectin aglycon were inactive. All ivermectin derivatives were cytotoxic at concentrations > 5 µM, i.e. below the level required for E protein inhibition. Conclusions This study demonstrates direct inhibition of the SARS-CoV-2 E protein by classical viroporin inhibitors. Ivermectin and milbemycin inhibit the E protein channel but their cytotoxicity argues against clinical application.
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- 2023
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6. Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency
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Martin Schütz, Christina Wangen, Mona Sommerer, Melanie Kögler, Jan Eickhoff, Carsten Degenhart, Bert Klebl, Zin Naing, Ece Egilmezer, Stuart T. Hamilton, William D. Rawlinson, Heinrich Sticht, and Manfred Marschall
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Human cytomegalovirus ,Viral cyclin-dependent kinase (CDK) ortholog ,vCDK/pUL97–cyclin binding ,Functional importance of cyclin H ,Modulation of vCDK/pUL97 activity in vitro ,Cyclin H knock-down ,Microbiology ,QR1-502 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs.
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- 2023
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7. In Silico Study of Camptothecin-Based Pro-Drugs Binding to Human Carboxylesterase 2
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Frank Beierlein, Anselm H. C. Horn, Heinrich Sticht, Andriy Mokhir, and Petra Imhof
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pro-drug ,camptothecin ,human carboxylesterase ,homology model ,docking ,molecular dynamics simulations ,Microbiology ,QR1-502 - Abstract
Pro-drugs, which ideally release their active compound only at the site of action, i.e., in a cancer cell, are a promising approach towards an increased specificity and hence reduced side effects in chemotherapy. A popular form of pro-drugs is esters, which are activated upon their hydrolysis. Since carboxylesterases that catalyse such a hydrolysis reaction are also abundant in normal tissue, it is of great interest whether a putative pro-drug is a probable substrate of such an enzyme and hence bears the danger of being activated not just in the target environment, i.e., in cancer cells. In this work, we study the binding mode of carboxylesters of the drug molecule camptothecin, which is an inhibitor of topoisomerase I, of varying size to human carboxylesterase 2 (HCE2) by molecular docking and molecular dynamics simulations. A comparison to irinotecan, known to be a substrate of HCE2, shows that all three pro-drugs analysed in this work can bind to the HCE2 protein, but not in a pose that is well suited for subsequent hydrolysis. Our data suggest, moreover, that for the irinotecan substrate, a reactant-competent pose is stabilised once the initial proton transfer from the putative nucleophile Ser202 to the His431 of the catalytic triad has already occurred. Our simulation work also shows that it is important to go beyond the static models obtained from molecular docking and include the flexibility of enzyme–ligand complexes in solvents and at a finite temperature. Under such conditions, the pro-drugs studied in this work are unlikely to be hydrolysed by the HCE2 enzyme, indicating a low risk of undesired drug release in normal tissue.
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- 2024
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8. Assessing clinical utility of preconception expanded carrier screening regarding residual risk for neurodevelopmental disorders
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Paranchai Boonsawat, Anselm H. C. Horn, Katharina Steindl, Alessandra Baumer, Pascal Joset, Dennis Kraemer, Angela Bahr, Ivan Ivanovski, Elena M. Cabello, Michael Papik, Markus Zweier, Beatrice Oneda, Pietro Sirleto, Tilo Burkhardt, Heinrich Sticht, and Anita Rauch
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Medicine ,Genetics ,QH426-470 - Abstract
Abstract The magnitude of clinical utility of preconception expanded carrier screening (ECS) concerning its potential to reduce the risk of affected offspring is unknown. Since neurodevelopmental disorders (NDDs) in their offspring is a major concern of parents-to-be, we addressed the question of residual risk by assessing the risk-reduction potential for NDDs in a retrospective study investigating ECS with different criteria for gene selection and definition of pathogenicity. We used exome sequencing data from 700 parents of children with NDDs and blindly screened for carrier-alleles in up to 3046 recessive/X-linked genes. Depending on variant pathogenicity thresholds and gene content, NDD-risk-reduction potential was up to 43.5% in consanguineous, and 5.1% in nonconsanguineous couples. The risk-reduction-potential was compromised by underestimation of pathogenicity of missense variants (false-negative-rate 4.6%), inherited copy-number variants and compound heterozygosity of one inherited and one de novo variant (0.9% each). Adherence to the ACMG recommendations of restricting ECS to high-frequency genes in nonconsanguineous couples would more than halve the detectable inherited NDD-risk. Thus, for optimized clinical utility of ECS, screening in recessive/X-linked genes regardless of their frequency (ACMG Tier-4) and sensible pathogenicity thresholds should be considered for all couples seeking ECS.
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- 2022
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9. A frequent SNP in TRIM5α strongly enhances the innate immune response against LINE-1 elements
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Justine Lagisquet, Marcus Conrad, Sabine Wittmann, Bianca Volkmann, Hannah Weissinger, Heinrich Sticht, and Thomas Gramberg
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TRIM5α ,LINE-1 ,retroelements ,NF-κB ,AP-1 ,restriction factor ,Immunologic diseases. Allergy ,RC581-607 - Abstract
The intracellular restriction factor TRIM5α inhibits endogenous LINE-1 retroelements. It induces innate immune signaling cascades upon sensing of cytoplasmic LINE-1 complexes, thereby underlining its importance for protecting the human genome from harmful retrotransposition events. Here, we show that a frequent SNP within the RING domain of TRIM5α, resulting in the variant H43Y, blocks LINE-1 retrotransposition with higher efficiency compared to TRIM5α WT. Upon sensing of LINE-1 complexes in the cytoplasm, TRIM5α H43Y activates both NF-κB and AP-1 signaling pathways more potently than TRIM5α WT, triggering a strong block of the LINE-1 promoter. Interestingly, the H43Y allele lost its antiviral function suggesting that its enhanced activity against endogenous LINE-1 elements is the driving force behind its maintenance within the population. Thus, our study suggests that the H43Y variant of the restriction factor and sensor TRIM5α persists within the human population since it preserves our genome from uncontrolled LINE-1 retrotransposition with higher efficiency.
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- 2023
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10. The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7–Cyclin H Determines Individual Patterns of Transcription in Infected Cells
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Martin Schütz, Arne Cordsmeier, Christina Wangen, Anselm H. C. Horn, Emanuel Wyler, Armin Ensser, Heinrich Sticht, and Manfred Marschall
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human cytomegalovirus ,cyclin-dependent kinases (CDKs) ,viral CDK ortholog (vCDK/pUL97) ,vCDK/pUL97–cyclin binding ,functional complexation with host CDK7 ,impact on RNA polymerase (RNAP II) in infected cells ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The infection of human cytomegalovirus (HCMV) is strongly determined by the host–cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus–host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97–cyclin H–CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.
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- 2023
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11. A novel D-amino acid peptide with therapeutic potential (ISAD1) inhibits aggregation of neurotoxic disease-relevant mutant Tau and prevents Tau toxicity in vitro
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Isabelle Aillaud, Senthilvelrajan Kaniyappan, Ram Reddy Chandupatla, Lisa Marie Ramirez, Sewar Alkhashrom, Jutta Eichler, Anselm H. C. Horn, Markus Zweckstetter, Eckhard Mandelkow, Heinrich Sticht, and Susanne Aileen Funke
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Alzheimer’s disease ,Tau aggregation inhibitors ,Phage display ,D-amino acid peptides ,Therapy ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Abstract Background Alzheimer’s disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. Methods We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. Results While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive β-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. Conclusions ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity.
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- 2022
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12. A newly identified secreted larval antigen elicits basophil-dependent protective immunity against N. brasiliensis infection
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Natalie Thuma, Daniela Döhler, Dirk Mielenz, Heinrich Sticht, Daniel Radtke, Lena Reimann, Bettina Warscheid, and David Voehringer
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hookworms ,basophils ,SCP/TAPS protein ,CAP domain ,immunization ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Hookworms infect more that 400 million people and cause significant socio-economic burden on endemic countries. The lack of efficient vaccines and the emergence of anthelminthic drug resistance are of major concern. Free-living hookworm larvae infect their hosts via the skin and live as adult worms in the small intestine where they feed on host tissue and blood. Excretory/secretory (E/S) products, released by helminths as they migrate through their host, are thought to play a key role in facilitating infection and successful establishment of parasitism. However, E/S products can also elicit protective immune responses that might be harnessed for vaccine development. By performing Western blots with serum of Nippostrongylus brasiliensis (Nb) infected mice as a model for human hookworm infection, we identified a largely overlapping set of IgG1- and IgE-reactive antigens in E/S from infective L3 stage larvae. Mass spectrometry analysis led to the identification of a new protein family with 6 paralogues in the Nb genome which we termed Nb-LSA1 for “Nippostrongylus brasiliensis larval secreted protein 1”. The recombinantly expressed 17 kDa family member Nb-LSA1a was recognized by antibodies in the serum of Nb immune mice. Immunization of mice with Nb-LSA1a in alum elicited a strong IgG1 response but no detectable antigen-specific IgE. Most importantly, immunized mice were largely protected against a challenge Nb infection. This effect was dependent on the presence of basophils and occurred before the parasites reached the intestine. Therefore, basophils appear to play a critical role for rapid control of infection with L3 stage larvae in mice immunized with a single secreted larval protein. A better understanding of basophil-mediated protective immunity and identification of potent larval antigens of human hookworms could help to develop promising vaccination strategies.
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- 2022
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13. Computational Characterization of the Binding Properties of the HIV1-Neutralizing Antibody PG16 and Design of PG16-Derived CDRH3 Peptides
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Manuel Deubler, Lucas Weißenborn, Simon Leukel, Anselm H. C. Horn, Jutta Eichler, and Heinrich Sticht
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antibody ,PG16 ,HIV-1 ,peptides ,antibody mimetic peptides ,molecular dynamics ,Biology (General) ,QH301-705.5 - Abstract
PG16 is a broadly neutralizing antibody that binds to the gp120 subunit of the HIV-1 Env protein. The major interaction site is formed by the unusually long complementarity determining region (CDR) H3. The CDRH3 residue Tyr100H is known to represent a tyrosine sulfation site; however, this modification is not present in the experimental complex structure of PG16 with full-length HIV-1 Env. To investigate the role of sulfation for this complex, we modeled the sulfation of Tyr100H and compared the dynamics and energetics of the modified and unmodified complex by molecular dynamics simulations at the atomic level. Our results show that sulfation does not affect the overall conformation of CDRH3, but still enhances gp120 interactions both at the site of modification and for the neighboring residues. This stabilization affects not only protein–protein contacts, but also the interactions between PG16 and the gp120 glycan shield. Furthermore, we also investigated whether PG16-CDRH3 is a suitable template for the development of peptide mimetics. For a peptide spanning residues 93-105 of PG16, we obtained an experimental EC50 value of 3nm for the binding of gp120 to the peptide. This affinity can be enhanced by almost one order of magnitude by artificial disulfide bonding between residues 99 and 100F. In contrast, any truncation results in significantly lower affinity, suggesting that the entire peptide segment is involved in gp120 recognition. Given their high affinity, it should be possible to further optimize the PG16-derived peptides as potential inhibitors of HIV invasion.
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- 2023
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14. Computational Analysis of Histamine Protonation Effects on H1R Binding
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Marcus Conrad, Anselm H. C. Horn, and Heinrich Sticht
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H1R ,histamine ,GPCR ,tautomers ,Gq ,protonation ,Organic chemistry ,QD241-441 - Abstract
Despite numerous studies investigating histamine and its receptors, the impact of histamine protonation states on binding to the histamine H1-receptor (H1R) has remained elusive. Therefore, we assessed the influence of different histamine tautomers (τ-tautomer, π-tautomer) and charge states (mono- vs. dicationic) on the interaction with the ternary histamine-H1R-Gq complex. In atomistic molecular dynamics simulations, the τ-tautomer formed stable interactions with the receptor, while the π-tautomer induced a rotation of the histamine ring by 180° and formed only weaker hydrogen bonding interactions. This suggests that the τ-tautomer is more relevant for stabilization of the active ternary histamine-H1R-Gq complex. In addition to the two monocationic tautomers, the binding of dicationic histamine was investigated, whose interaction with the H1R had been observed in a previous experimental study. Our simulations showed that the dication is less compatible with the ternary histamine-H1R-Gq complex and rather induces an inactive conformation in the absence of the Gq protein. Our data thus indicate that the charge state of histamine critically affects its interactions with the H1R. Ultimately these findings might have implications for the future development of new ligands that stabilize distinct H1R activation states.
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- 2023
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15. A proline-rich motif in the large intracellular loop of the glycine receptor α1 subunit interacts with the Pleckstrin homology domain of collybistin
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Ulrike Breitinger, Kristina Weinländer, Yvonne Pechmann, Georg Langlhofer, Ralf Enz, Cord-Michael Becker, Heinrich Sticht, Matthias Kneussel, Carmen Villmann, and Hans-Georg Breitinger
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Ion channel receptors ,Synaptic anchoring ,Protein-protein interaction ,Glycine receptor alpha1 subunit ,Polyproline II helix ,Collybistin ,Medicine (General) ,R5-920 ,Science (General) ,Q1-390 - Abstract
Introduction: The inhibitory glycine receptor (GlyR), a mediator of fast synaptic inhibition, is located and held at neuronal synapses through the anchoring proteins gephyrin and collybistin. Stable localization of neurotransmitter receptors is essential for synaptic function. In case of GlyRs, only beta subunits were known until now to mediate synaptic anchoring. Objectives: We identified a poly-proline II helix (PPII) in position 365–373 of the intra-cellular TM3-4 loop of the human GlyRα1 subunit as a novel potential synaptic anchoring site. The potential role of the PPII helix as synaptic anchoring site was tested. Methods: Glycine receptors and collybistin variants were generated and recombinantly expressed in HEK293 cells and cultured neurons. Receptor function was assessed using patch-clamp electrophysiology, protein-protein interaction was studied using co-immuno-precipitation and pulldown experiments. Results: Recombinantly expressed collybistin bound to isolated GlyRα1 TM3-4 loops in GST-pulldown assays. When the five proline residues P365A, P366A, P367A, P369A, P373A (GlyRα1P1-5A) located in the GlyRα1-PPII helix were replaced by alanines, the PPII secondary structure was disrupted. Recombinant GlyRα1P1-5A mutant subunits displayed normal cell surface expression and wildtype-like ion channel function, but binding to collybistin was abolished. The GlyRα1-collybistin interaction was independently confirmed by o-immunoprecipitation assays using full-length GlyRα1 subunits. Surprisingly, the interaction was not mediated by the SH3 domain of collybistin, but by its Pleckstrin homology (PH) domain. The mutation GlyRα1P366L, identified in a hyperekplexia patient, is also disrupting the PPII helix, and caused reduced collybistin binding. Conclusion: Our data suggest a novel interaction between α1 GlyR subunits and collybistin, which is physiologically relevant in vitro and in vivo and may contribute to postsynaptic anchoring of glycine receptors.
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- 2021
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16. T4SS-dependent TLR5 activation by Helicobacter pylori infection
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Suneesh Kumar Pachathundikandi, Nicole Tegtmeyer, Isabelle Catherine Arnold, Judith Lind, Matthias Neddermann, Christina Falkeis-Veits, Sujay Chattopadhyay, Mark Brönstrup, Werner Tegge, Minsun Hong, Heinrich Sticht, Michael Vieth, Anne Müller, and Steffen Backert
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Science - Abstract
Toll-like receptor TLR5 recognizes a domain, D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori. Here, the authors show that TLR5 can be activated independently of flagellin by a component of the H. pylori type IV secretion system that contains a D1-like motif.
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- 2019
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17. ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis
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Josephine Lösing, Sigrun Häge, Martin Schütz, Sabrina Wagner, Julia Wardin, Heinrich Sticht, and Manfred Marschall
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herpesviruses ,human cytomegalovirus ,nuclear egress complex (NEC) ,cytomegalovirus core NEC pUL50–pUL53 ,hook-into-groove-interaction ,yeast two-hybrid screening ,Cytology ,QH573-671 - Abstract
Herpesviruses replicate their genomes and assemble their capsids in the host cell nucleus. To progress towards morphogenesis in the cytoplasm, herpesviruses evolved the strategy of nuclear egress as a highly regulated process of nucleo-cytoplasmic capsid transition. The process is conserved among α-, β- and γ-herpesviruses and involves the formation of a core and multicomponent nuclear egress complex (NEC). Core NEC is assembled by the interaction between the nucleoplasmic hook protein, i.e., pUL53 (human cytomegalovirus, HCMV), and the integral membrane-associated groove protein, i.e., pUL50. Our study aimed at the question of whether a panherpesviral NEC scaffold may enable hook-into-groove interaction across herpesviral subfamilies. For this purpose, NEC constructs were generated for members of all three subfamilies and analyzed for multi-ligand interaction using a yeast two-hybrid (Y2H) approach with randomized pUL53 mutagenesis libraries. The screening identified ten library clones displaying cross-viral shared hook-into-groove interaction. Interestingly, a slightly modified Y2H screening strategy provided thirteen further changed-hook pUL53 clones having lost parental pUL50 interaction but gained homolog interaction. In addition, we designed a sequence-predicted hybrid construct based on HCMV and Epstein-Barr virus (EBV) core NEC proteins and identified a cross-viral interaction phenotype. Confirmation was provided by applying protein–protein interaction analyses in human cells, such as coimmunoprecipitation settings, confocal nuclear rim colocalization assays, and HCMV ΔUL53 infection experiments with pUL53-complementing cells. Combined, the study provided the first examples of cross-viral NEC interaction patterns and revealed a higher yield of human cell-confirmed binding clones using a library exchange rate of 3.4 than 2.7. Thus, the study provides improved insights into herpesviral NEC protein binding specificities of core NEC formation. This novel information might be exploited to gain a potential target scaffold for the development of broadly acting NEC-directed inhibitory small molecules.
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- 2022
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18. Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H
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Martin Schütz, Regina Müller, Eileen Socher, Christina Wangen, Florian Full, Emanuel Wyler, Diana Wong, Myriam Scherer, Thomas Stamminger, Sunwen Chou, William D. Rawlinson, Stuart T. Hamilton, Heinrich Sticht, and Manfred Marschall
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human cytomegalovirus ,viral CDK-like kinase ,pUL97/vCDK ,human cyclin complexes ,pUL97–cyclin interaction ,functional relevance ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97–cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97–cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97–cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97–cyclin interaction. High selective pressure on the formation of pUL97–cyclin complexes was identified by the use of clinically relevant mutants.
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- 2022
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19. Inhibition of SARS CoV Envelope Protein by Flavonoids and Classical Viroporin Inhibitors
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Ulrike Breitinger, Nourhan K. M. Ali, Heinrich Sticht, and Hans-Georg Breitinger
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SARS CoV-1 ,E protein ,viroporins ,cell viability assay ,patch-clamp electrophysiology ,ion channel inhibitors ,Microbiology ,QR1-502 - Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV), an enveloped single-stranded positive-sense RNA virus, is a member of the genus Betacoronavirus, family Coronaviridae. The SARS-CoV envelope protein E is a small (∼8.4 kDa) channel-forming membrane protein whose sequence is highly conserved between SARS-CoV and SARS-CoV-2. As a viroporin, it is involved in various aspects of the virus life cycle including assembly, budding, envelope formation, virus release, and inflammasome activation. Here, SARS-CoV E protein was recombinantly expressed in HEK293 cells and channel activity and the effects of viroporin inhibitors studied using patch-clamp electrophysiology and a cell viability assay. We introduced a membrane-directing signal peptide to ensure transfer of recombinant E protein to the plasma membrane. E protein expression induced transmembrane currents that were blocked by various inhibitors. In an ion-reduced buffer system, currents were proton-dependent and blocked by viroporin inhibitors rimantadine and amantadine. I-V relationships of recombinant E protein were not pH-dependent in a classical buffer system with high extracellular Na+ and high intracellular K+. E-protein mediated currents were inhibited by amantadine and rimantadine, as well as 5-(N,N-hexamethylene)amiloride (HMA). We tested a total of 10 flavonoids, finding inhibitory activity of varying potency. Epigallocatechin and quercetin were most effective, with IC50 values of 1.5 ± 0.1 and 3.7 ± 0.2 nM, respectively, similar to the potency of rimantadine (IC50 = 1.7 ± 0.6 nM). Patch-clamp results were independently verified using a modified cell viability assay for viroporin inhibitors. These results contribute to the development of novel antiviral drugs that suppress virus activity and proliferation.
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- 2021
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20. Characterization of SARS-CoV-2 Escape Mutants to a Pair of Neutralizing Antibodies Targeting the RBD and the NTD
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Antonia Sophia Peter, Eva Grüner, Eileen Socher, Kirsten Fraedrich, Elie Richel, Sandra Mueller-Schmucker, Arne Cordsmeier, Armin Ensser, Heinrich Sticht, and Klaus Überla
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SARS-CoV-2 ,spike protein ,NTD ,RBD ,variants of concern ,SARS-CoV-2 escape mutations ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.
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- 2022
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21. Further corroboration of distinct functional features in SCN2A variants causing intellectual disability or epileptic phenotypes
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Anaïs Begemann, Mario A. Acuña, Markus Zweier, Marie Vincent, Katharina Steindl, Ruxandra Bachmann-Gagescu, Annette Hackenberg, Lucia Abela, Barbara Plecko, Judith Kroell-Seger, Alessandra Baumer, Kazuhiro Yamakawa, Yushi Inoue, Reza Asadollahi, Heinrich Sticht, Hanns Ulrich Zeilhofer, and Anita Rauch
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SCN2A ,Nav1.2 ,Channelopathy ,Patch-clamp ,Epilepsy ,Epileptic encephalopathy ,Therapeutics. Pharmacology ,RM1-950 ,Biochemistry ,QD415-436 - Abstract
Abstract Background Deleterious variants in the voltage-gated sodium channel type 2 (Nav1.2) lead to a broad spectrum of phenotypes ranging from benign familial neonatal-infantile epilepsy (BFNIE), severe developmental and epileptic encephalopathy (DEE) and intellectual disability (ID) to autism spectrum disorders (ASD). Yet, the underlying mechanisms are still incompletely understood. Methods To further elucidate the genotype-phenotype correlation of SCN2A variants we investigated the functional effects of six variants representing the phenotypic spectrum by whole-cell patch-clamp studies in transfected HEK293T cells and in-silico structural modeling. Results The two variants p.L1342P and p.E1803G detected in patients with early onset epileptic encephalopathy (EE) showed profound and complex changes in channel gating, whereas the BFNIE variant p.L1563V exhibited only a small gain of channel function. The three variants identified in ID patients without seizures, p.R937C, p.L611Vfs*35 and p.W1716*, did not produce measurable currents. Homology modeling of the missense variants predicted structural impairments consistent with the electrophysiological findings. Conclusions Our findings support the hypothesis that complete loss-of-function variants lead to ID without seizures, small gain-of-function variants cause BFNIE and EE variants exhibit variable but profound Nav1.2 gating changes. Moreover, structural modeling was able to predict the severity of the variant impact, supporting a potential role of structural modeling as a prognostic tool. Our study on the functional consequences of SCN2A variants causing the distinct phenotypes of EE, BFNIE and ID contributes to the elucidation of mechanisms underlying the broad phenotypic variability reported for SCN2A variants.
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- 2019
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22. Campylobacter jejuni enters gut epithelial cells and impairs intestinal barrier function through cleavage of occludin by serine protease HtrA
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Aileen Harrer, Roland Bücker, Manja Boehm, Urszula Zarzecka, Nicole Tegtmeyer, Heinrich Sticht, Jörg D. Schulzke, and Steffen Backert
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Occludin ,Tight junction ,E-cadherin ,Campylobacter ,Protease ,HtrA ,Diseases of the digestive system. Gastroenterology ,RC799-869 - Abstract
Abstract Campylobacter jejuni secretes HtrA (high temperature requirement protein A), a serine protease that is involved in virulence. Here, we investigated the interaction of HtrA with the host protein occludin, a tight junction strand component. Immunofluorescence studies demonstrated that infection of polarized intestinal Caco-2 cells with C. jejuni strain 81–176 resulted in a redistribution of occludin away from the tight junctions into the cytoplasm, an effect that was also observed in human biopsies during acute campylobacteriosis. Occludin knockout Caco-2 cells were generated by CRISPR/Cas9 technology. Inactivation of this gene affected the polarization of the cells in monolayers and transepithelial electrical resistance (TER) was reduced, compared to wild-type Caco-2 cells. Although tight junctions were still being formed, occludin deficiency resulted in a slight decrease of the tight junction plaque protein ZO-1, which was redistributed off the tight junction into the lateral plasma membrane. Adherence of C. jejuni to Caco-2 cell monolayers was similar between the occludin knockout compared to wild-type cells, but invasion was enhanced, indicating that deletion of occludin allowed larger numbers of bacteria to pass the tight junctions and to reach basal membranes to target the fibronectin receptor followed by cell entry. Finally, we discovered that purified C. jejuni HtrA cleaves recombinant occludin in vitro to release a 37 kDa carboxy-terminal fragment. The same cleavage fragment was observed in Western blots upon infection of polarized Caco-2 cells with wild-type C. jejuni, but not with isogenic ΔhtrA mutants. HtrA cleavage was mapped to the second extracellular loop of occludin, and a putative cleavage site was identified. In conclusion, HtrA functions as a secreted protease targeting the tight junctions, which enables the bacteria by cleaving occludin and subcellular redistribution of other tight junction proteins to transmigrate using a paracellular mechanism and subsequently invade epithelial cells.
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- 2019
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23. Smaller, Stronger, More Stable: Peptide Variants of a SARS-CoV-2 Neutralizing Miniprotein
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Lucas Weißenborn, Elie Richel, Helena Hüseman, Julia Welzer, Silvan Beck, Simon Schäfer, Heinrich Sticht, Klaus Überla, and Jutta Eichler
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SARS-CoV-2 ,virus ,peptides ,structure-based design ,synthetic peptides ,peptide mimics ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Based on the structure of a de novo designed miniprotein (LCB1) in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, we have generated and characterized truncated peptide variants of LCB1, which present only two of the three LCB1 helices, and which fully retained the virus neutralizing potency against different SARS-CoV-2 variants of concern (VOC). This antiviral activity was even 10-fold stronger for a cyclic variant of the two-helix peptides, as compared to the full-length peptide. Furthermore, the proteolytic stability of the cyclic peptide was substantially improved, rendering it a better potential candidate for SARS-CoV-2 therapy. In a more mechanistic approach, the peptides also served as tools to dissect the role of individual mutations in the RBD for the susceptibility of the resulting virus variants to neutralization by the peptides. As the peptides reported here were generated through chemical synthesis, rather than recombinant protein expression, they are amenable to further chemical modification, including the incorporation of a wide range of non-proteinogenic amino acids, with the aim to further stabilize the peptides against proteolytic degradation, as well as to improve the strength, as well the breadth, of their virus neutralizing capacity.
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- 2022
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24. The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors
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Jintawee Kicuntod, Sigrun Häge, Friedrich Hahn, Heinrich Sticht, and Manfred Marschall
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human cytomegalovirus ,viral nucleocytoplasmic egress ,nuclear egress complex (NEC) ,core NEC pUL50-pUL53 ,oligomerization ,cross-linking ,Microbiology ,QR1-502 - Abstract
The nucleo-cytoplasmic capsid egress of herpesviruses is a unique regulated process that ensures the efficiency of viral replication and release. For human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50–pUL53 heterodimer that is able to oligomerize and thus to build hexameric lattices. These structures determine capsid binding and multicomponent protein interaction including NEC-associated host factors. The underlying characteristic of the core NEC formation is based on the N-terminal hook structure of pUL53 that binds into an alpha-helical groove of pUL50, and is thus described as a hook-into-groove interaction. This central regulatory element has recently been validated as a target of antiviral strategies, and first NEC-targeted prototypes of inhibitory small molecules were reported by our previous study. Here, we further analyzed the oligomerization properties of the viral NEC through an approach of chemical protein cross-linking. Findings were as follows: (i) a cross-link approach demonstrated the oligomeric state of the HCMV core NEC using material from HCMV-infected or plasmid-transfected cells, (ii) a Western blot-based identification of NEC-associated kinases using the cross-linked multicomponent NECs was successful, and (iii) we demonstrated the NEC-inhibitory and antiviral activity of specific inhibitors directed to these target kinases. Combined, the results strongly underline the functional importance of the oligomerization of the HCMV-specific NEC that is both phosphorylation-dependent and sensitive to antiviral kinase inhibitors.
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- 2022
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25. Campylobacter jejuni Serine Protease HtrA Cleaves the Tight Junction Component Claudin-8
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Irshad Sharafutdinov, Delara Soltan Esmaeili, Aileen Harrer, Nicole Tegtmeyer, Heinrich Sticht, and Steffen Backert
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claudin-8 ,occludin ,tight junction ,E-cadherin ,Campylobacter ,protease ,Microbiology ,QR1-502 - Abstract
Campylobacter jejuni express the high temperature requirement protein A (HtrA), a secreted serine protease, which is implicated in virulence properties of the pathogen. Previous studies have shown that C. jejuni HtrA can cleave the epithelial transmembrane proteins occludin and E-cadherin in the tight and adherens junctions, respectively. In the present report, we studied the interaction of HtrA with another human tight junction protein, claudin-8. Confocal immunofluorescence experiments have shown that C. jejuni infection of the intestinal polarized epithelial cells in vitro leads to a relocation of claudin-8. Wild-type C. jejuni induced the downregulation of claudin-8 signals in the tight junctions and an accumulation of claudin-8 agglomerates in the cytoplasm, which were not seen during infection with isogenic ΔhtrA knockout deletion or protease-inactive S197A point mutants. Western blotting of protein samples from infected vs. uninfected cells revealed that an 18-kDa carboxy-terminal fragment is cleaved-off from the 26-kDa full-length claudin-8 protein, but not during infection with the isogenic ΔhtrA mutant. These results were confirmed by in vitro cleavage assays using the purified recombinant C. jejuni HtrA and human claudin-8 proteins. Recombinant HtrA cleaved purified claudin-8 in vitro giving rise to the same 18-kDa sized carboxy-terminal cleavage product. Mapping studies revealed that HtrA cleavage occurs in the first extracellular loop of claudin-8. Three-dimensional modeling of the claudin-8 structure identified an exposed HtrA cleavage site between the amino acids alanine 58 and asparagine 59, which is in well agreement with the mapping studies. Taken together, HtrA operates as a secreted virulence factor targeting multiple proteins both in the tight and adherens junctions. This strategy may help the bacteria to open the cell-to-cell junctions, and to transmigrate across the intestinal epithelium by a paracellular mechanism and establish an acute infection.
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- 2020
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26. Effect of Ions and Sequence Variants on the Antagonist Binding Properties of the Histamine H1 Receptor
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Marcus Conrad, Christian A. Söldner, and Heinrich Sticht
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receptor-ligand interactions ,G protein-coupled receptors (GPCRs) ,molecular dynamics simulations ,metadynamics ,sodium binding ,sodium pocket ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The histamine H1 receptor (H1R) is a G protein-coupled receptor (GPCR) and represents a main target in the treatment of allergic reactions as well as inflammatory reactions and depressions. Although the overall effect of antagonists on H1 function has been extensively investigated, rather little is known about the potential modulatory effect of ions or sequence variants on antagonist binding. We investigated the dynamics of a phosphate ion present in the crystal structure and of a sodium ion, for which we determined the position in the allosteric pocket by metadynamics simulations. Both types of ions exhibit significant dynamics within their binding site; however, some key contacts remain stable over the simulation time, which might be exploited to develop more potent drugs targeting these sites. The dynamics of the ions is almost unaffected by the presence or absence of doxepin, as also reflected in their small effect (less than 1 kcal·mol−1) on doxepin binding affinity. We also examined the effect of four H1R sequence variants observed in the human population on doxepin binding. These variants cause a reduction in doxepin affinity of up to 2.5 kcal·mol−1, indicating that personalized medical treatments that take into account individual mutation patterns could increase precision in the dosage of GPCR-targeting drugs.
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- 2022
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27. Transfer of HTLV-1 p8 and Gag to target T-cells depends on VASP, a novel interaction partner of p8.
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Norbert Donhauser, Eileen Socher, Sebastian Millen, Stefanie Heym, Heinrich Sticht, and Andrea K Thoma-Kress
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24-45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26-37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells.
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- 2020
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28. The CD83 Molecule – An Important Immune Checkpoint
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Linda Grosche, Ilka Knippertz, Christina König, Dmytro Royzman, Andreas B. Wild, Elisabeth Zinser, Heinrich Sticht, Yves A. Muller, Alexander Steinkasserer, and Matthias Lechmann
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CD83 ,immune tolerance ,autoimmunity ,viral escape mechanism ,Treg cells ,Immunologic diseases. Allergy ,RC581-607 - Abstract
The CD83 molecule has been identified to be expressed on numerous activated immune cells, including B and T lymphocytes, monocytes, dendritic cells, microglia, and neutrophils. Both isoforms of CD83, the membrane-bound as well as its soluble form are topic of intensive research investigations. Several studies revealed that CD83 is not a typical co-stimulatory molecule, but rather plays a critical role in controlling and resolving immune responses. Moreover, CD83 is an essential factor during the differentiation of T and B lymphocytes, and the development and maintenance of tolerance. The identification of its interaction partners as well as signaling pathways have been an enigma for the last decades. Here, we report the latest data on the expression, structure, and the signaling partners of CD83. In addition, we review the regulatory functions of CD83, including its striking modulatory potential to maintain the balance between tolerance versus inflammation during homeostasis or pathologies. These immunomodulatory properties of CD83 emphasize its exceptional therapeutic potential, which has been documented in specific preclinical disease models.
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- 2020
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29. The C-terminal coiled-coil domain of Corynebacterium diphtheriae DIP0733 is crucial for interaction with epithelial cells and pathogenicity in invertebrate animal model systems
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Dulanthi Weerasekera, Franziska Stengel, Heinrich Sticht, Ana Luíza de Mattos Guaraldi, Andreas Burkovski, and Camila Azevedo Antunes
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Caenorhabditis elegans ,Corynebacteria ,Galleria mellonella ,Host pathogen interaction ,Microbiology ,QR1-502 - Abstract
Abstract Background Corynebacterium diphtheriae is the etiologic agent of diphtheria and different systemic infections. The bacterium has been classically described as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of C. diphtheriae still remains unclear. Recently, the DIP0733 transmembrane protein was found to play an important role in the interaction with matrix proteins and cell surfaces, nematode colonization, cellular internalization and induction of cell death. Results In this study, we identified a number of short linear motifs and structural elements of DIP0733 with putative importance in virulence, using bioinformatic approaches. A C-terminal coiled-coil region of the protein was considered particularly important, since it was found only in DIP0733 homologs in pathogenic Corynebacterium species but not in non-pathogenic corynebacteria. Infections of epithelial cells and transepithelial resistance assays revealed that bacteria expressing the truncated form of C. diphtheriae DIP0733 and C. glutamicum DIP0733 homolog are less virulent, while the fusion of the coiled-coil sequence to the DIP0733 homolog from C. glutamicum resulted in increased pathogenicity. These results were supported by nematode killing assays and experiments using wax moth larvae as invertebrate model systems. Conclusions Our data indicate that the coil-coiled domain of DIP0733 is crucial for interaction with epithelial cells and pathogenicity in invertebrate animal model systems.
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- 2018
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30. Characterizing the Interaction between the HTLV-1 Transactivator Tax-1 with Transcription Elongation Factor ELL2 and Its Impact on Viral Transactivation
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Stephan Kohrt, Sarah Strobel, Melanie C. Mann, Heinrich Sticht, Bernhard Fleckenstein, and Andrea K. Thoma-Kress
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human T-cell leukemia virus type 1 ,HTLV-1 ,ELL2 ,transcription elongation factor for RNA polymerase II ,Tax-1 ,Tax-2 ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The human T-cell leukemia virus type 1 (HTLV-1)-encoded transactivator and oncoprotein Tax-1 is essential for HTLV-1 replication. We recently found that Tax-1 interacts with transcription elongation factor for RNA polymerase II 2, ELL2, which enhances Tax-1-mediated transactivation of the HTLV-1 promotor. Here, we characterize the Tax-1:ELL2 interaction and its impact on viral transactivation by confocal imaging, co-immunoprecipitation, and luciferase assays. We found that Tax-1 and ELL2 not only co-precipitate, but also co-localize in dot-like structures in the nucleus. Tax-1:ELL2 complex formation occurred independently of Tax-1 point mutations, which are crucial for post translational modifications (PTMs) of Tax-1, suggesting that these PTMs are irrelevant for Tax-1:ELL2 interaction. In contrast, Tax-1 deletion mutants lacking either N-terminal (aa 1–37) or C-terminal regions (aa 150–353) of Tax-1 were impaired in interacting with ELL2. Contrary to Tax-1, the related, non-oncogenic Tax-2B from HTLV-2B did not interact with ELL2. Finally, we found that ELL2-R1 (aa 1–353), which carries an RNA polymerase II binding domain, and ELL2-R3 (aa 515–640) are sufficient to interact with Tax-1; however, only ELL2-truncations expressing R1 could enhance Tax-1-mediated transactivation of the HTLV-1 promoter. Together, this study identifies domains in Tax-1 and ELL2 being required for Tax-1:ELL2 complex formation and for viral transactivation.
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- 2021
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31. Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and Accelerated Molecular Dynamics Simulations
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Carina Höring, Marcus Conrad, Christian A. Söldner, Jinan Wang, Heinrich Sticht, Andrea Strasser, and Yinglong Miao
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GPCR–G protein coupling profiles ,Gaussian accelerated molecular dynamics (GaMD) ,split-luciferase complementation assay ,histamine signaling ,histamine H2 receptor ,histamine H4 receptor ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H2 and H4 receptors (H2R and H4R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H2R and H4R, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the H2R and H4R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H2R and H4R from the mini-G protein recruitment assays using HEK293T cells. Compared to H2R–mGs expressing cells, histamine responses were weaker (pEC50, Emax) for H2R–mGsi and –mGsq. By contrast, the H4R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H2R to mGs and H4R to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H4R complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied H2R function in the brain, as well as of the pharmacological potential of H4R selective drugs.
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- 2021
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32. Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97
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Martin Schütz, Mirjam Steingruber, Eileen Socher, Regina Müller, Sabrina Wagner, Merle Kögel, Heinrich Sticht, and Manfred Marschall
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human cytomegalovirus ,virus-encoded kinase pUL97 ,viral CDK ortholog ,interaction with human cyclin T1 ,replication-impaired viral mutants ,defects in pUL97 activities ,Microbiology ,QR1-502 - Abstract
The replication of human cytomegalovirus (HCMV) is characterized by a complex network of virus–host interaction. This involves the regulatory viral protein kinase pUL97, which represents a viral cyclin-dependent kinase ortholog (vCDK) combining typical structural and functional features of host CDKs. Notably, pUL97 interacts with the three human cyclin types T1, H and B1, whereby the binding region of cyclin T1 and the region conferring oligomerization of pUL97 were both assigned to amino acids 231–280. Here, we addressed the question of whether recombinant HCMVs harboring deletions in this region were impaired in cyclin interaction, kinase functionality or viral replication. To this end, recombinant HCMVs were generated by traceless BACmid mutagenesis and were phenotypically characterized using a methodological platform based on qPCR, coimmunoprecipitation, in vitro kinase assay (IVKA), Phos-tag Western blot and confocal imaging analysis. Combined data illustrate the following: (i) infection kinetics of all three recombinant HCMVs, i.e., ORF-UL97 ∆231–255, ∆256–280 and ∆231–280, showed impaired replication efficiency compared to the wild type, amongst which the largest deletion exhibited the most pronounced defect; (ii) specifically, this mutant ∆231–280 showed a loss of interaction with cyclin T1, as demonstrated by CoIP and confocal imaging; (iii) IVKA and Phos-tag analyses revealed strongly affected kinase activity for ∆231–280, with strong impairment of both autophosphorylation and substrate phosphorylation, but less pronounced impairments for ∆231–255 and ∆256–280; and (iv) a bioinformatic assessment of the pUL97–cyclin T1 complex led to the refinement of our current binding model. Thus, the results provide initial evidence for the functional importance of the pUL97–cyclin interaction concerning kinase activity and viral replication fitness.
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- 2021
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33. Mutations in the B.1.1.7 SARS-CoV-2 Spike Protein Reduce Receptor-Binding Affinity and Induce a Flexible Link to the Fusion Peptide
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Eileen Socher, Marcus Conrad, Lukas Heger, Friedrich Paulsen, Heinrich Sticht, Friederike Zunke, and Philipp Arnold
- Subjects
COVID-19 ,SARS-CoV-2 ,B.1.1.7 ,molecular dynamics simulation ,ACE2 ,receptor binding ,Biology (General) ,QH301-705.5 - Abstract
The B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. Amino acid exchanges within the SARS-CoV-2 spike protein variant of B.1.1.7 affect inter-monomeric contact sites within the trimer (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability or ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. Furthermore, we found increased flexibility in direct spatial proximity of the fusion peptide due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7. This study also implies a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD–ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2. Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD–ACE2 interface. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7.
- Published
- 2021
- Full Text
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34. AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability
- Author
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Aline Brechet, Rebecca Buchert, Jochen Schwenk, Sami Boudkkazi, Gerd Zolles, Karine Siquier-Pernet, Irene Schaber, Wolfgang Bildl, Abdelkrim Saadi, Christine Bole-Feysot, Patrick Nitschke, Andre Reis, Heinrich Sticht, Nouriya Al-Sanna’a, Arndt Rolfs, Akos Kulik, Uwe Schulte, Laurence Colleaux, Rami Abou Jamra, and Bernd Fakler
- Subjects
Science - Abstract
The biogenesis of AMPA-type glutamate receptor (AMPAR) complexes is only partially understood. Here the authors identify transient assemblies of GluA1-4 proteins and proteins FRRS1l/CPT1c that drive formation of mature AMPAR complexes in the ER. Mutations in FRRS1l are associated with intellectual disability and epilepsy in three families.
- Published
- 2017
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- View/download PDF
35. Properties of Oligomeric Interaction of the Cytomegalovirus Core Nuclear Egress Complex (NEC) and Its Sensitivity to an NEC Inhibitory Small Molecule
- Author
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Jintawee Kicuntod, Sewar Alkhashrom, Sigrun Häge, Benedikt Diewald, Regina Müller, Friedrich Hahn, Peter Lischka, Heinrich Sticht, Jutta Eichler, and Manfred Marschall
- Subjects
human cytomegalovirus ,core nuclear egress complex (NEC) ,in vitro NEC assembly assay ,core NEC coimmunoprecipitation ,oligomeric interaction properties ,associated cellular factors ,Microbiology ,QR1-502 - Abstract
Herpesviral nuclear egress is a regulated process shared by all family members, ensuring the efficient cytoplasmic release of viral capsids. In the case of human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that builds hexameric lattices for capsid binding and multicomponent interaction, including NEC-associated host factors. A characteristic feature of NEC interaction is the N-terminal hook structure of pUL53 that binds to an alpha-helical groove of pUL50, thus termed as hook-into-groove interaction. This central regulatory element is essential for viral replication and shows structural–functional conservation, which has been postulated as a next-generation target of antiviral strategies. However, a solid validation of this concept has been missing. In the present study, we focused on the properties of oligomeric HCMV core NEC interaction and the antiviral activity of specifically targeted prototype inhibitors. Our data suggest the following: (i) transiently expressed, variably tagged versions of HCMV NEC proteins exert hook-into-groove complexes, putatively in oligomeric assemblies that are distinguishable from heterodimers, as shown by in vitro assembly and coimmunoprecipitation approaches; (ii) this postulated oligomeric binding pattern was further supported by the use of a pUL50::pUL53 fusion construct also showing a pronounced multi-interaction potency; (iii) using confocal imaging cellular NEC-associated proteins were found partly colocalized with the tagged core NECs; (iv) a small inhibitory molecule, recently identified by an in vitro binding inhibition assay, was likewise active in blocking pUL50–pUL53 oligomeric assembly and in exerting antiviral activity in HCMV-infected fibroblasts. In summary, the findings refine the previous concept of HCMV core NEC formation and nominate this drug-accessible complex as a validated antiviral drug target.
- Published
- 2021
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36. A modeling strategy for G-protein coupled receptors
- Author
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Anna Kahler and Heinrich Sticht
- Subjects
GPCR ,MD simulation ,homology modeling ,HCMV ,transmembrane helix ,Biology (General) ,QH301-705.5 ,Biotechnology ,TP248.13-248.65 - Abstract
Cell responses can be triggered via G-protein coupled receptors (GPCRs) that interact with small molecules, peptides or proteins and transmit the signal over the membrane via structural changes to activate intracellular pathways. GPCRs are characterized by a rather low sequence similarity and exhibit structural differences even for functionally closely related GPCRs. An accurate structure prediction for GPCRs is therefore not straightforward. We propose a computational approach that relies on the generation of several independent models based on different template structures, which are subsequently refined by molecular dynamics simulations. A comparison of their conformational stability and the agreement with GPCR-typical structural features is then used to select a favorable model. This strategy was applied to predict the structure of the herpesviral chemokine receptor US28 by generating three independent models based on the known structures of the chemokine receptors CXCR1, CXCR4, and CCR5. Model refinement and evaluation suggested that the model based on CCR5 exhibits the most favorable structural properties. In particular, the GPCR-typical structural features, such as a conserved water cluster or conserved non-covalent contacts, are present to a larger extent in the model based on CCR5 compared to the other models. A final model validation based on the recently published US28 crystal structure confirms that the CCR5-based model is the most accurate and exhibits 80.8% correctly modeled residues within the transmembrane helices. The structural agreement between the selected model and the crystal structure suggests that our modeling strategy may also be more generally applicable to other GPCRs of unknown structure.
- Published
- 2016
- Full Text
- View/download PDF
37. Agonist Binding and G Protein Coupling in Histamine H2 Receptor: A Molecular Dynamics Study
- Author
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Marcus Conrad, Christian A. Söldner, Yinglong Miao, and Heinrich Sticht
- Subjects
receptor–ligand interactions ,G protein-coupled receptors (GPCRs) ,Gs protein ,ternary complex ,molecular dynamics simulations ,metadynamics ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The histamine H2 receptor (H2R) plays an important role in the regulation of gastric acid secretion. Therefore, it is a main drug target for the treatment of gastroesophageal reflux or peptic ulcer disease. However, there is as of yet no 3D-structural information available hampering a mechanistic understanding of H2R. Therefore, we created a model of the histamine-H2R-Gs complex based on the structure of the ternary complex of the β2-adrenoceptor and investigated the conformational stability of this active GPCR conformation. Since the physiologically relevant motions with respect to ligand binding and conformational changes of GPCRs can only partly be assessed on the timescale of conventional MD (cMD) simulations, we also applied metadynamics and Gaussian accelerated molecular dynamics (GaMD) simulations. A multiple walker metadynamics simulation in combination with cMD was applied for the determination of the histamine binding mode. The preferential binding pose detected is in good agreement with previous data from site directed mutagenesis and provides a basis for rational ligand design. Inspection of the H2R-Gs interface reveals a network of polar interactions that may contribute to H2R coupling selectivity. The cMD and GaMD simulations demonstrate that the active conformation is retained on a μs-timescale in the ternary histamine-H2R-Gs complex and in a truncated complex that contains only Gs helix α5 instead of the entire G protein. In contrast, histamine alone is unable to stabilize the active conformation, which is in line with previous studies of other GPCRs.
- Published
- 2020
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38. Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties
- Author
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Manfred Marschall, Sigrun Häge, Marcus Conrad, Sewar Alkhashrom, Jintawee Kicuntod, Johannes Schweininger, Mark Kriegel, Josephine Lösing, Julia Tillmanns, Frank Neipel, Jutta Eichler, Yves A. Muller, and Heinrich Sticht
- Subjects
human cytomegalovirus (HCMV) ,nuclear egress complex (NEC) ,core NEC crystal structures ,α-, β-, γ-herpesviral NECs ,sequence coevolution ,highly conserved structures ,Microbiology ,QR1-502 - Abstract
Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric nuclear egress complex (core NEC). These core NECs serve as hexameric lattice-structured platforms for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina as well as membrane-rearranging functions (multicomponent NEC). The regulation of nuclear egress has been profoundly analyzed for murine and human cytomegaloviruses (CMVs) on a mechanistic basis, followed by the description of core NEC crystal structures, first for HCMV, then HSV-1, PRV and EBV. Interestingly, the highly conserved structural domains of these proteins stand in contrast to a very limited sequence conservation of the key amino acids within core NEC-binding interfaces. Even more surprising, although a high functional consistency was found when regarding the basic role of NECs in nuclear egress, a clear specification was identified regarding the limited, subfamily-spanning binding properties of core NEC pairs and NEC multicomponent proteins. This review summarizes the evolving picture of the relationship between sequence coevolution, structural conservation and properties of NEC interaction, comparing HCMV to α-, β- and γ-herpesviruses. Since NECs represent substantially important elements of herpesviral replication that are considered as drug-accessible targets, their putative translational use for antiviral strategies is discussed.
- Published
- 2020
- Full Text
- View/download PDF
39. Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation
- Author
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Nicole Albrecht, Nicole Tegtmeyer, Heinrich Sticht, Joanna Skórko-Glonek, and Steffen Backert
- Subjects
C. jejuni ,H. pylori ,HtrA ,secretion ,chaperone ,E-cadherin ,Microbiology ,QR1-502 - Abstract
The HtrA family of serine proteases is found in most bacteria, and plays an essential role in the virulence of the gastric pathogen Helicobacter pylori. Secreted H. pylori HtrA (HtrAHp) cleaves various junctional proteins such as E-cadherin disrupting the epithelial barrier, which is crucial for bacterial transmigration across the polarized epithelium. Recent studies indicated the presence of two characteristic HtrAHp forms of 55 and 52 kDa (termed p55 and p52, respectively), in worldwide strains. In addition, p55 and p52 were produced by recombinant HtrAHp, indicating auto-cleavage. However, the cleavage sites and their functional importance are yet unclear. Here, we determined the amino-terminal ends of p55 and p52 by Edman sequencing. Two proteolytic cleavage sites were identified (H46/D47 and K50/D51). Remarkably, the cleavage site sequences are conserved in HtrAHp from worldwide isolates, but not in other Gram-negative pathogens, suggesting a highly specific assignment in H. pylori. We analyzed the role of the amino-terminal cleavage sites on activity, secretion and function of HtrAHp. Three-dimensional modeling suggested a trimeric structure and a role of amino-terminal processing in oligomerization and regulation of proteolytic activity of HtrAHp. Furthermore, point and deletion mutants of these processing sites were generated in the recently reported Campylobacter jejuni ΔhtrA/htrAHp genetic complementation system and the minimal sequence requirements for processing were determined. Polarized Caco-2 epithelial cells were infected with these strains and analyzed by immunofluorescence microscopy. The results indicated that HtrAHp processing strongly affected the ability of the protease to disrupt the E-cadherin-based cell-to-cell junctions. Casein zymography confirmed that the amino-terminal region is required for maintaining the proteolytic activity of HtrAHp. Furthermore, we demonstrated that this cleavage influences the secretion of HtrAHp in the extracellular space as an important prerequisite for its virulence activity. Taken together, our data demonstrate that amino-terminal cleavage of HtrAHp is conserved in this pathogen and affects oligomerization and thus, secretion and regulatory activities, suggesting an important role in the pathogenesis of H. pylori.
- Published
- 2018
- Full Text
- View/download PDF
40. Probing the role of intercalating protein sidechains for kink formation in DNA.
- Author
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Achim Sandmann and Heinrich Sticht
- Subjects
Medicine ,Science - Abstract
Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD) simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP) and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP), one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d) each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future.
- Published
- 2018
- Full Text
- View/download PDF
41. The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain
- Author
-
Mirjam Steingruber, Eileen Socher, Corina Hutterer, Rike Webel, Tim Bergbrede, Tihana Lenac, Heinrich Sticht, and Manfred Marschall
- Subjects
human cytomegalovirus ,protein kinase pUL97 ,CDK ortholog ,cyclin B ,protein-protein interaction ,kinase activity ,mode of kinase-cyclin interaction ,active conformation ,Microbiology ,QR1-502 - Abstract
Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.
- Published
- 2015
- Full Text
- View/download PDF
42. Protective capacity of neutralizing and non-neutralizing antibodies against glycoprotein B of cytomegalovirus.
- Author
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Anna Bootz, Astrid Karbach, Johannes Spindler, Barbara Kropff, Nina Reuter, Heinrich Sticht, Thomas H Winkler, William J Britt, and Michael Mach
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in immunodeficient hosts. When present before infection, both neutralizing and non-neutralizing anti-gB exhibited protective capacity.
- Published
- 2017
- Full Text
- View/download PDF
43. Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica.
- Author
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Britta Peters, Johanna Stein, Stefan Klingl, Nathalie Sander, Achim Sandmann, Nicola Taccardi, Heinrich Sticht, Roman G Gerlach, Yves A Muller, and Michael Hensel
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca2+-binding sites. BIg domains of SiiE contain distinct Ca2+-binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca2+-binding sites in SiiE, as well as the importance of Ca2+-binding sites in various positions of SiiE. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion.
- Published
- 2017
- Full Text
- View/download PDF
44. Probing the potential of CnaB-type domains for the design of tag/catcher systems.
- Author
-
Marlene Pröschel, Max E Kraner, Anselm H C Horn, Lena Schäfer, Uwe Sonnewald, and Heinrich Sticht
- Subjects
Medicine ,Science - Abstract
Building proteins into larger, post-translational assemblies in a defined and stable way is still a challenging task. A promising approach relies on so-called tag/catcher systems that are fused to the proteins of interest and allow a durable linkage via covalent intermolecular bonds. Tags and catchers are generated by splitting protein domains that contain intramolecular isopeptide or ester bonds that form autocatalytically under physiological conditions. There are already numerous biotechnological and medical applications that demonstrate the usefulness of covalent linkages mediated by these systems. Additional covalent tag/catcher systems would allow creating more complex and ultra-stable protein architectures and networks. Two of the presently available tag/catcher systems were derived from closely related CnaB-domains of Streptococcus pyogenes and Streptococcus dysgalactiae proteins. However, it is unclear whether domain splitting is generally tolerated within the CnaB-family or only by a small subset of these domains. To address this point, we have selected a set of four CnaB domains of low sequence similarity and characterized the resulting tag/catcher systems by computational and experimental methods. Experimental testing for intermolecular isopeptide bond formation demonstrated two of the four systems to be functional. For these two systems length and sequence variations of the peptide tags were investigated revealing only a relatively small effect on the efficiency of the reaction. Our study suggests that splitting into tag and catcher moieties is tolerated by a significant portion of the naturally occurring CnaB-domains, thus providing a large reservoir for the design of novel tag/catcher systems.
- Published
- 2017
- Full Text
- View/download PDF
45. Role of the N-terminus for the stability of an amyloid-β fibril with three-fold symmetry.
- Author
-
Christian A Söldner, Heinrich Sticht, and Anselm H C Horn
- Subjects
Medicine ,Science - Abstract
A key player in Alzheimer's disease is the peptide amyloid-beta (Aβ), whose aggregation into small soluble oligomers, protofilaments, and fibrils finally leads to plaque deposits in human brains. The aggregation behavior of Aβ is strongly modulated by the nature and composition of the peptide's environment and by its primary sequence properties. The N-terminal residues of Aβ play an important role, because they are known to change the peptide's aggregation propensity. Since these residues are for the first time completely resolved at the molecular level in a three-fold symmetric fibril structure derived from a patient, we chose that system as template for a systematic investigation of the influence of the N-terminus upon structural stability. Using atomistic molecular dynamics simulations, we examined several fibrillar systems comprising three, six, twelve and an infinite number of layers, both with and without the first eight residues. First, we found that three layers are not sufficient to stabilize the respective Aβ topology. Second, we observed a clear stabilizing effect of the N-terminal residues upon the overall fibril fold: truncated Aβ systems were less stable than their full-length counterparts. The N-terminal residues Arg5, Asp7, and Ser8 were found to form important interfilament contacts stabilizing the overall fibril structure of three-fold symmetry. Finally, similar structural rearrangements of the truncated Aβ species in different simulations prompted us to suggest a potential mechanism involved in the formation of amyloid fibrils with three-fold symmetry.
- Published
- 2017
- Full Text
- View/download PDF
46. A Metadynamics-Based Protocol for the Determination of GPCR-Ligand Binding Modes
- Author
-
Christian A. Söldner, Anselm H. C. Horn, and Heinrich Sticht
- Subjects
receptor-ligand interactions ,G protein-coupled receptors (GPCRs) ,molecular dynamics simulations ,metadynamics simulations ,ligand binding modes ,clustering ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
G protein-coupled receptors (GPCRs) are a main drug target and therefore a hot topic in pharmaceutical research. One important prerequisite to understand how a certain ligand affects a GPCR is precise knowledge about its binding mode and the specific underlying interactions. If no crystal structure of the respective complex is available, computational methods can be used to deduce the binding site. One of them are metadynamics simulations which have the advantage of an enhanced sampling compared to conventional molecular dynamics simulations. However, the enhanced sampling of higher-energy states hampers identification of the preferred binding mode. Here, we present a novel protocol based on clustering of multiple walker metadynamics simulations which allows identifying the preferential binding mode from such conformational ensembles. We tested this strategy for three different model systems namely the histamine H1 receptor in combination with its physiological ligand histamine, as well as the β 2 adrenoceptor with its agonist adrenaline and its antagonist alprenolol. For all three systems, the proposed protocol was able to reproduce the correct binding mode known from the literature suggesting that the approach can more generally be applied to the prediction of GPCR ligand binding in future.
- Published
- 2019
- Full Text
- View/download PDF
47. The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins
- Author
-
Laura Graf, Rike Webel, Sabrina Wagner, Stuart T. Hamilton, William D. Rawlinson, Heinrich Sticht, and Manfred Marschall
- Subjects
human cytomegalovirus ,protein kinase pUL97 ,cyclins T1, B1 and A ,protein-protein interaction ,substrate phosphorylation ,interaction-mediated regulation ,Microbiology ,QR1-502 - Abstract
The human cytomegalovirus (HCMV)-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK) ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.
- Published
- 2013
- Full Text
- View/download PDF
48. Peptides presenting the binding site of human CD4 for the HIV-1 envelope glycoprotein gp120
- Author
-
Julia Meier, Kristin Kassler, Heinrich Sticht, and Jutta Eichler
- Subjects
biomimetic synthesis ,CD4 ,HIV entry ,peptide ,protein binding site ,Science ,Organic chemistry ,QD241-441 - Abstract
Based on the structure of the HIV-1 glycoprotein gp120 in complex with its cellular receptor CD4, we have designed and synthesized peptides that mimic the binding site of CD4 for gp120. The ability of these peptides to bind to gp120 can be strongly enhanced by increasing their conformational stability through cyclization, as evidenced by binding assays, as well as through molecular-dynamics simulations of peptide–gp120 complexes. The specificity of the peptide–gp120 interaction was demonstrated by using peptide variants, in which key residues for the interaction with gp120 were replaced by alanine or D-amino acids.
- Published
- 2012
- Full Text
- View/download PDF
49. The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.
- Author
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Jens Milbradt, Corina Hutterer, Hanife Bahsi, Sabrina Wagner, Eric Sonntag, Anselm H C Horn, Benedikt B Kaufer, Yasuko Mori, Heinrich Sticht, Torgils Fossen, and Manfred Marschall
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids.
- Published
- 2016
- Full Text
- View/download PDF
50. Selection and Characterization of Tau Binding ᴅ-Enantiomeric Peptides with Potential for Therapy of Alzheimer Disease.
- Author
-
Christina Dammers, Deniz Yolcu, Laura Kukuk, Dieter Willbold, Marcus Pickhardt, Eckhard Mandelkow, Anselm H C Horn, Heinrich Sticht, Marwa Nidal Malhis, Nadja Will, Judith Schuster, and Susanne Aileen Funke
- Subjects
Medicine ,Science - Abstract
A variety of neurodegenerative disorders, including Alzheimer disease (AD), are associated with neurofibrillary tangles composed of the tau protein, as well as toxic tau oligomers. Inhibitors of pathological tau aggregation, interrupting tau self-assembly, might be useful for the development of therapeutics. Employing mirror image phage display with a large peptide library (over 109 different peptides), we have identified tau fibril binding peptides consisting of d-enantiomeric amino acids. d-enantiomeric peptides are extremely protease stable and not or less immunogenic than l-peptides, and the suitability of d-peptides for in vivo applications have already been demonstrated. Phage display selections were performed using fibrils of the d-enantiomeric hexapeptide VQIVYK, representing residues 306 to 311 of the tau protein, as a target. VQIVYK has been demonstrated to be important for fibril formation of the full lengths protein and forms fibrils by itself. Here, we report on d-enantiomeric peptides, which bind to VQIVYK, tau isoforms like tau3RD (K19) as well as to full lengths tau fibrils, and modulate the aggregation of the respective tau form. The peptides are able to penetrate cells and might be interesting for therapeutic and diagnostic applications in AD research.
- Published
- 2016
- Full Text
- View/download PDF
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