Stefan Hannus
Max Planck Society
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Featured researches published by Stefan Hannus.
Nucleic Acids Research | 2014
Michael Hannus; Michaela Beitzinger; Julia C. Engelmann; Marie-Theresa Weickert; Rainer Spang; Stefan Hannus; Gunter Meister
Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools—referred to as siPools—containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects.
The EMBO Journal | 2001
Gunter Meister; Stefan Hannus; Oliver Plöttner; Tonie Luise Baars; Enno Hartmann; Stanislav Fakan; Bernhard Laggerbauer; Utz Fischer
SMNrp, also termed SPF30, has recently been identified in spliceosomes assembled in vitro. We have functionally characterized this protein and show that it is an essential splicing factor. We show that SMNrp is a 17S U2 snRNP‐associated protein that appears in the pre‐spliceosome (complex A) and the mature spliceosome (complex B) during splicing. Immunodepletion of SMNrp from nuclear extract inhibits the first step of pre‐mRNA splicing by preventing the formation of complex B. Re‐addition of recombinant SMNrp to immunodepleted extract reconstitutes both spliceosome formation and splicing. Mutations in two domains of SMNrp, although similarly deleterious for splicing, differed in their consequences on U2 snRNP binding, suggesting that SMNrp may also engage in interactions with splicing factors other than the U2 snRNP. In agreement with this, we present evidence for an additional interaction between SMNrp and the [U4/U6·U5] tri‐snRNP. A candidate that may mediate this interaction, namely the U4/U6‐90 kDa protein, has been identified. We suggest that SMNrp, as a U2 snRNP‐associated protein, facilitates the recruitment of the [U4/U6·U5] tri‐snRNP to the pre‐spliceosome.
Nucleic Acids Research | 2015
Daniel Schraivogel; Susann G. Schindler; Johannes Danner; Elisabeth Kremmer; Janina Pfaff; Stefan Hannus; Reinhard Depping; Gunter Meister
MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago – TNRC6 levels.
British Journal of Pharmacology | 2010
Heike Glauner; Ivo R. Ruttekolk; Kerrin Hansen; Ben Steemers; Yi-Da Chung; Frank Becker; Stefan Hannus; Roland Brock
Background and purpose: In vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off‐target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off‐target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross‐correlation spectroscopy (FCCS) as a solution to this problem.
Molecular Immunology | 2009
Kerrin Hansen; Ivo R. Ruttekolk; Heike Glauner; Frank Becker; Roland Brock; Stefan Hannus
Antibodies of the IgG4 subclass, directed against cell surface antigens have received attention as therapeutic molecules due to their poor induction of the complement system. The MHC class II-directed IgG4 antibody 1D09C3 has been explored for the treatment of lymphomas. The mechanism-of-action is still controversial. Apoptosis induction following HLA-DR engagement has been proposed. However, the validity of these results has been questioned by the observation that antibodies may induce formation of cell aggregates and cell death is induced upon dispersion of these aggregates prior to the quantification of cell death by flow cytometry. Here we address the capacity of 1D09C3 to induce apoptosis in vitro, also taking account of the recently reported Fab arm exchange of IgG4 antibodies. 1D09C3 induces formation of tight cellular aggregates that can only be dispersed at the expense of massive cell damage and death. Using dual color fluorescence cross-correlation spectroscopy (FCCS) we demonstrate that also this antibody undergoes Fab arm exchange in the presence of IgG4. FCCS is a powerful technique to investigate the molecular mechanism of Fab arm exchange using minute amounts of reagents. Following exchange, the functionally monovalent 1D09C3 chimeras loose their ability to induce aggregate formation of HLA-DR-positive cells. Neither functionally monovalent nor bivalent 1D09C3 antibodies induce cell death or apoptosis in myeloma target cells, when microscopy instead of flow cytometry is employed as the analytical technique. Our results indicate that the activity of 1D09C3 in vitro may have been a consequence of assay design rather than an ability to induce HLA-DR-dependent cell death.
RNA | 2016
Leonhard Jakob; Thomas Treiber; Nora Treiber; Alexander Gust; Kevin Kramm; Kerrin Hansen; Mathias Stotz; Ludwig Wankerl; Franz Herzog; Stefan Hannus; Dina Grohmann; Gunter Meister
In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Loqs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing.
Analytical Biochemistry | 2016
Thomas Antoine; David Ott; Katharina Ebell; Kerrin Hansen; Luc Henry; Frank Becker; Stefan Hannus
G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.
Oncotarget | 2018
Florian Lüke; Raquel Blazquez; Rezan Fahrioglu Yamaci; Xin Lu; Benedikt Pregler; Stefan Hannus; Karin Menhart; Dirk Hellwig; Hans-Jürgen Wester; Saskia Kropf; Daniel Heudobler; Jirka Grosse; Jutta Moosbauer; Markus Hutterer; Peter Hau; Markus J. Riemenschneider; Michaela Bayerlová; Annalen Bleckmann; Bernhard Polzer; Tim Beißbarth; Christoph Klein; Tobias Pukrop
Brain and leptomeningeal metastasis (LMM) of non-small cell lung cancer is still associated with poor prognosis. Moreover, the current diagnostic standard for LMM often yields false negative results and the scientific progress in this field is still unsatisfying. We present a case of a 71-year old patient with an isolated LMM. While standard diagnostics could only diagnose a cancer of unknown primary, the use of [68Ga]-Pentixafor-PET/CT (CXCR4-PET/CT, a radiotracer targeting CXCR4) and a liquid biopsy of the cerebrospinal fluid revealed the primary NSCLC. The detection of L858R-EGFR, a common driver mutation in NSCLC, enabled us to treat the patient with Afatinib and monitor treatment using [68Ga]-Pentixafor PET/CT. To estimate the impact of CXCR4 signaling and its ligands in NSCLC brain metastasis we looked at their expression and correlation with EGFR mutations in a primary and brain metastasis data set and investigated the previously described binding of extracellular ubiquitin to CXCR4. In conclusion, we describe a novel approach to improve diagnostics towards LMM and underline the impact of the CXCL12/CXCR4 axis in brain metastasis in a subset of NSCLC patients. We cannot confirm a correlation of CXCR4 expression with EGFR mutations or the binding of extracellular ubiquitin as previously reported.
Oncotarget | 2017
Marsha Crochiere; Stefan Hannus; Kerrin Hansen; Frank Becker; Erkan Baloglu; Margaret S. Lee; Michael Kauffman; Sharon Shacham; Yosef Landesman
XPO1 (exportin 1) is the main nuclear export protein with over 200 different protein cargos. XPO1 is overexpressed in tumor cells and high levels are correlated with poor prognosis. Selective Inhibitor of Nuclear Export (SINE) compounds block nuclear export by inhibiting XPO1. The first SINE compound, selinexor, shows promising anti-cancer activity across hematological and solid tumors in Phase 2 and 3 clinical trials. The 2nd generation SINE compound KPT-8602 is being evaluated as an anti-cancer agent in a Phase 1 clinical trial. To predict patient response to treatment and confirm the selinexor recommended phase 2 dose (RP2D), an assay based on fluorescence cross correlation spectroscopy that measures XPO1 occupancy in cancer cells was developed. Studies comparing cytotoxicity and XPO1 occupancy in cell lines treated with selinexor or KPT-8602 indicated that XPO1 occupancy by both compounds could reach saturation regardless of drug sensitivity. However, higher levels of XPO1 protein correlated with lower sensitivity to SINE compound cytotoxicity. In vivo mouse studies showed XPO1 occupancy could be measured in tumors and was dose-dependent, with >90% target saturation at 10 mg/kg (∼50 mg flat dose in humans). Drug-target occupancy was measured in a dose-response time course and full occupancy occurred by 6 hours at all doses. The duration of occupancy was dose-dependent, where 10-15 mg/kg in mice (∼ 50-75 mg human flat dose) was necessary to maintain XPO1 occupancy up to 48 hours post-dose. These findings confirm the selinexor RP2D of 60 mg for achieving target occupancy and inhibition up to 48 hours.
Biospektrum | 2012
Stefan Hannus; Roland Brock
Fluorescence correlation spectroscopy (FCS) derives information on molecular interactions and concentrations from the analysis of timedependent fluctuations of a fluorescence signal, caused by diffusion of molecules through a confocal detection volume. Here, we present applications of FCS in drug discovery and molecular pharmacology, including the screening of drug-target interactions and peptide stability in crude cellular lysates, providing a physiological environment.