Publications by authors named "Andreas Stengl"

11 Publications

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HP1β carries an acidic linker domain and requires H3K9me3 for phase separation.

Nucleus 2021 Dec;12(1):44-57

Center for Molecular Biosystems (BioSysM), Faculty of Biology, Ludwig-Maximilians-Universität München, Munich, Germany.

Liquid-liquid phase separation (LLPS) mediated formation of membraneless organelles has been proposed to coordinate biological processes in space and time. Previously, the formation of phase-separated droplets was described as a unique property of HP1α. Here, we demonstrate that the positive net charge of the intrinsically disordered hinge region (IDR-H) of HP1 proteins is critical for phase separation and that the exchange of four acidic amino acids is sufficient to confer LLPS properties to HP1β. Surprisingly, the addition of mono-nucleosomes promoted H3K9me3-dependent LLPS of HP1β which could be specifically disrupted with methylated but not acetylated H3K9 peptides. HP1β mutants defective in H3K9me3 binding were less efficient in phase separationand failed to accumulate at heterochromatin . We propose that multivalent interactions of HP1β with H3K9me3-modified nucleosomes via its chromodomain and dimerization via its chromoshadow domain enable phase separation and contribute to the formation of heterochromatin compartments .
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http://dx.doi.org/10.1080/19491034.2021.1889858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939559PMC
December 2021

Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy.

Chembiochem 2021 Apr 17;22(7):1205-1209. Epub 2020 Dec 17.

Ludwig-Maximilians-Universität München, Department of Biology II, Human Biology and BioImaging, 82152, Planegg-Martinsried, Germany.

Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag® technology in combination with Cu-catalyzed azide-alkyne cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence-specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP-tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody-oligonucleotides for reversible immunofluorescence imaging.
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http://dx.doi.org/10.1002/cbic.202000727DOI Listing
April 2021

Systematic analysis of the binding behaviour of UHRF1 towards different methyl- and carboxylcytosine modification patterns at CpG dyads.

PLoS One 2020 21;15(2):e0229144. Epub 2020 Feb 21.

Center for Integrated Protein Science Munich at the TUM School of Life Sciences, Technische Universität München, Freising, Germany.

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein's NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229144PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034832PMC
May 2020

Ethynylphosphonamidates for the Rapid and Cysteine-Selective Generation of Efficacious Antibody-Drug Conjugates.

Angew Chem Int Ed Engl 2019 08 18;58(34):11631-11636. Epub 2019 Jul 18.

Chemical Biology Department, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125, Berlin, Germany.

Requirements for novel bioconjugation reactions for the synthesis of antibody-drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys-selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non-engineered monoclonal antibodies through a simple one-pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties. We demonstrate that ethynylphosphonamidate-linked ADCs have excellent properties for next-generation antibody therapeutics in terms of serum stability and in vivo antitumor activity.
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http://dx.doi.org/10.1002/anie.201904193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851832PMC
August 2019

TuPPL: Tub-tag mediated C-terminal protein-protein-ligation using complementary click-chemistry handles.

Org Biomol Chem 2019 05 1;17(20):4964-4969. Epub 2019 Apr 1.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, 82152 Planegg, Martinsried, Germany.

We introduce a chemoenzymatic strategy for straightforward in vitro generation of C-terminally linked fusion proteins. Tubulin tyrosine ligase is used for the incorporation of complementary click chemistry handles facilitating subsequent formation of functional bispecific antibody-fragments. This simple strategy may serve as central conjugation hub for a modular protein ligation platform.
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http://dx.doi.org/10.1039/c9ob00508kDOI Listing
May 2019

Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations.

Angew Chem Int Ed Engl 2019 08 29;58(34):11625-11630. Epub 2019 Apr 29.

Chemical Biology Department, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125, Berlin, Germany.

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide-modified building block reacts first with an ethynylphosphonite through a Staudinger-phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron-deficient triple bond subsequently undergoes a cysteine-selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine-selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.
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http://dx.doi.org/10.1002/anie.201814715DOI Listing
August 2019

FLEXamers: A Double Tag for Universal Generation of Versatile Peptide-MHC Multimers.

J Immunol 2019 04 13;202(7):2164-2171. Epub 2019 Feb 13.

Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, 81675 Munich, Germany;

Peptide-MHC (pMHC) multimers have become a valuable tool for immunological research, clinical immune monitoring, and immunotherapeutic applications. Biotinylated tetramers, reversible Streptamers, or dye-conjugated pMHC multimers are distinct pMHC reagents tailored for T cell identification, traceless T cell isolation, or TCR characterization, respectively. The specific applicability of each pMHC-based reagent is made possible either through conjugation of probes or reversible multimerization in separate production processes, which is laborious, time-consuming, and prone to variability between the different types of pMHC reagents. This prohibits broad implementation of different types of pMHC reagents as a standard toolbox in routine clinical immune monitoring and immunotherapy. In this article, we describe a novel method for fast and standardized generation of any pMHC multimer reagent from a single precursor ("FLEXamer"). FLEXamers unite reversible multimerization and versatile probe conjugation through a novel double tag (Strep-tag for reversibility and Tub-tag for versatile probe conjugation). We demonstrate that FLEXamers can substitute conventional pMHC reagents in all state-of-the-art applications, considerably accelerating and standardizing production without sacrificing functional performance. Although FLEXamers significantly aid the applicability of pMHC-based reagents in routine workflows, the double tag also provides a universal tool for the investigation of transient molecular interactions in general.
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http://dx.doi.org/10.4049/jimmunol.1801435DOI Listing
April 2019

Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation.

J Biol Chem 2017 09 7;292(37):15340-15351. Epub 2017 Aug 7.

From the Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany,

Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis. The E2-conjugating enzyme Ubc9 catalyzes the conjugation of SUMOs to ϵ-amino groups of lysine residues in target proteins. Attachment of SUMO moieties to internal lysines in Ubc9 itself can further lead to the formation of polymeric SUMO chains. Mono- and poly-SUMOylations of target proteins provide docking sites for distinct adapter and effector proteins important for regulating discrete SUMO-regulated pathways. However, molecular tools to dissect pathways depending on either mono- or poly-SUMOylation are largely missing. Using a protein-engineering approach, we generated high-affinity SUMO2 variants by phage display that bind the back side binding site of Ubc9 and function as SUMO-based Ubc9 inhibitors (SUBINs). Importantly, we found that distinct SUBINs primarily inhibit poly-SUMO chain formation, whereas mono-SUMOylation was not impaired. Proof-of-principle experiments demonstrated that in a cellular context, SUBINs largely prevent heat shock-triggered poly-SUMOylation. Moreover, SUBINs abrogated arsenic-induced degradation of promyelocytic leukemia protein. We propose that the availability of the new chain-selective SUMO inhibitors reported here will enable a thorough investigation of poly-SUMO-mediated cellular processes, such as DNA damage responses and cell cycle progression.
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http://dx.doi.org/10.1074/jbc.M117.794255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602394PMC
September 2017

A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies.

SLAS Discov 2017 03 13;22(3):309-315. Epub 2016 Dec 13.

1 Department of Biology II, LMU Munich, Planegg-Martinsried, Germany.

Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs.
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http://dx.doi.org/10.1177/1087057116677821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322830PMC
March 2017

Actin and PIP3 waves in giant cells reveal the inherent length scale of an excited state.

J Cell Sci 2014 Oct 8;127(Pt 20):4507-17. Epub 2014 Aug 8.

Max Planck Institute of Biochemistry, 82152 Martinsried, Germany

The membrane and actin cortex of a motile cell can autonomously differentiate into two states, one typical of the front, the other of the tail. On the substrate-attached surface of Dictyostelium discoideum cells, dynamic patterns of front-like and tail-like states are generated that are well suited to monitor transitions between these states. To image large-scale pattern dynamics independently of boundary effects, we produced giant cells by electric-pulse-induced cell fusion. In these cells, actin waves are coupled to the front and back of phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-rich bands that have a finite width. These composite waves propagate across the plasma membrane of the giant cells with undiminished velocity. After any disturbance, the bands of PIP3 return to their intrinsic width. Upon collision, the waves locally annihilate each other and change direction; at the cell border they are either extinguished or reflected. Accordingly, expanding areas of progressing PIP3 synthesis become unstable beyond a critical radius, their center switching from a front-like to a tail-like state. Our data suggest that PIP3 patterns in normal-sized cells are segments of the self-organizing patterns that evolve in giant cells.
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http://dx.doi.org/10.1242/jcs.156000DOI Listing
October 2014

Membrane and actin reorganization in electropulse-induced cell fusion.

J Cell Sci 2013 May 27;126(Pt 9):2069-78. Epub 2013 Feb 27.

Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

When cells of Dictyostelium discoideum are exposed to electric pulses they are induced to fuse, yielding motile polykaryotic cells. By combining electron microscopy and direct recording of fluorescent cells, we have studied the emergence of fusion pores in the membranes and the localization of actin to the cell cortex. In response to electric pulsing, the plasma membranes of two contiguous cells are turned into tangles of highly bent and interdigitated membranes. Live-imaging of cells double-labeled for membranes and filamentous actin revealed that actin is induced to polymerize in the fusion zone to temporarily bridge the gaps in the vesiculating membrane. The diffusion of green fluorescent protein (GFP) from one fusion partner to the other was scored using spinning disc confocal microscopy. Fusion pores that allowed intercellular exchange of GFP were formed after a delay, which lasted up to 24 seconds after exposure of the cells to the electric field. These data indicate that the membranes persist in a fusogenic state before pores of about 3 nm diameter are formed.
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http://dx.doi.org/10.1242/jcs.124073DOI Listing
May 2013