FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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Targeting G-protein-coupled receptors by Capture Compound Mass Spectrometry (CCMS) - a case study with sertindole
Blex(*), C., Michaelis(*), S., Schrey(*), A. K., Furkert, J., Eichhorst, J., Bartho(*), K., Quast(*), F. G., Marais(*), A., Hakelberg(*), M., Gruber(*), U., Niquet(*), S., Popp(*), O., Kroll(*), F., Sefkow(*), M., Schülein, R., Mathias(*), D.; Koster(*), H.
Chembiochem, 18:1639-1649

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner/Puchkov)

Abstract: Unbiased chemoproteomic profiling of small molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G-protein coupled receptors (GPCRs). These are hardly tractable by affinity pulldown from lysates. We report a Capture Compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK293 cells. The structure-activity relationship of sertindole for DRD2 binding is reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and CCMS. The activity pattern was rationalized by molecular modelling. The most active CC showed activities very similar to unmodifed sertindole. Well below 100 fmol of DRD2 in living cells used as experiment input were sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.

Molecular features of the L-type amino acid transporter 2 determine different import and export profiles for thyroid hormones and amino acids
Hinz, K. M., Neef, D., Rutz, C., Furkert, J., Köhrle(*), J., Schülein, R.; Krause, G.
Mol Cell Endocrinol, 443:163-174

Tags: Structural Bioinformatics and Protein Design (Krause, G.), Protein Trafficking (Schülein)

Abstract: The L-type amino acid transporter 2 (LAT2) imports amino acids (AA) and also certain thyroid hormones (TH), e.g. 3,3'-T2 and T3, but not rT3 and T4. We utilized LAT2 mutations (Y130A, N133S, F242W) that increase 3,3'-T2 import and focus here on import and export capacity for AA, T4, T3, BCH and derivatives thereof to delineate molecular features. Transport studies and analysis of competitive inhibition of import by radiolabelled TH and AA were performed in Xenopus laevis oocytes. Only Y130A, a pocket widening mutation, enabled import for T4 and increased it for T3. Mutant F242W showed increased 3,3'-T2 import but no import rates for other TH derivatives. No export was detected for any TH by LAT2-wild type (WT). Mutations Y130A and N133S enabled only the export of 3,3'-T2, while N133S also increased AA export. Thus, distinct molecular LAT2-features determine bidirectional AA transport but only an unidirectional 3,3'-T2 and T3 import.

In colon epithelia, Clostridium perfringens enterotoxin causes focal leaks by targeting claudins which are apically accessible due to tight junction derangement
Eichner(*), M., Augustin(*), C., Fromm(*), A., Piontek, A., Walther(*), W., Bücker(*), R., Fromm(*), M., Krause, G., Schulzke(*), J. D., Günzel(*), D.; Piontek(*), J.
The Journal of infectious diseases,

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (e.g. claudin-4) as receptors to form Ca2+-permeable pores in the membrane damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+-depletion, proinflammatory cytokine TNFalpha or dedifferentiation. Microscopy, Ca2+-monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis - if intestinal barrier dysfunction e.g. during inflammation facilitates claudin accessibility.

The differentiation and plasticity of Tc17 cells are regulated by CTLA-4-mediated effects on STATs
Arra(*), A., Lingel(*), H., Kuropka, B., Pick(*), J., Schnoeder(*), T., Fischer(*), T., Freund(*), C., Pierau(*), M.; Brunner-Weinzierl(*), M. C.
Oncoimmunology, 6:e1273300

Tags: Mass Spectrometry (Krause, E.)

Abstract: As the blockade of inhibitory surface-molecules such as CTLA-4 on T cells has led to recent advances in antitumor immune therapy, there is great interest in identifying novel mechanisms of action of CD8+ T cells to evoke effective cytotoxic antitumor responses. Using in vitro and in vivo models, we investigated the molecular pathways underlying the CTLA-4-mediated differentiation of IL-17-producing CD8+ T cells (Tc17 cells) that strongly impairs cytotoxicity. Our studies demonstrate that Tc17 cells lacking CTLA-4 signaling have limited production of STAT3-target gene products such as IL-17, IL-21, IL-23R and RORgammat. Upon re-stimulation with IL-12, these cells display fast downregulation of Tc17 hallmarks and acquire Tc1 characteristics such as IFNgamma and TNF-alpha co-expression, which is known to correlate with tumor control. Indeed, upon adoptive transfer, these cells were highly efficient in the antigen-specific rejection of established OVA-expressing B16 melanoma in vivo. Mechanistically, in primary and re-stimulated Tc17 cells, STAT3 binding to the IL-17 promoter was strongly augmented by CTLA-4, associated with less binding of STAT5 and reduced relative activation of STAT1 which is known to block STAT3 activity. Inhibiting CTLA-4-induced STAT3 activity reverses enhancement of signature Tc17 gene products, rendering Tc17 cells susceptible to conversion to Tc1-like cells with enhanced cytotoxic potential. Thus, CTLA-4 critically shapes the characteristics of Tc17 cells by regulating relative STAT3 activation, which provides new perspectives to enhance cytotoxicity of antitumor responses.

Intersectin associates with synapsin and regulates its nanoscale localization and function
Gerth(*), F., Jäpel, M., Pechstein, A., Kochlamazashvili, G., Lehmann, M., Puchkov, D., Onofri(*), F., Benfenati(*), F., Nikonenko(*), A. G., Maritzen, T., Freund(*), C.; Haucke, V.
Proc Natl Acad Sci U S A, 114:12057-12062

Tags: Molecular Pharmacology and Cell Biology (Haucke); Membrane Traffic and Cell Motility (Maritzen)

Abstract: Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery.

mTORC1 activity repression by late endosomal phosphatidylinositol 3,4-bisphosphate
Marat, A. L., Wallroth, A., Lo, W. T., Müller(*), R., Norata(*), G. D., Falasca(*), M., Schultz(*), C.; Haucke, V.
Science, 356:968-972

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Nutrient sensing by mechanistic target of rapamycin complex 1 (mTORC1) on lysosomes and late endosomes (LyLEs) regulates cell growth. Many factors stimulate mTORC1 activity, including the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] by class I phosphatidylinositol 3-kinases (PI3Ks) at the plasma membrane. We investigated mechanisms that repress mTORC1 under conditions of growth factor deprivation. We identified phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], synthesized by class II PI3K beta (PI3KC2beta) at LyLEs, as a negative regulator of mTORC1, whereas loss of PI3KC2beta hyperactivated mTORC1. Growth factor deprivation induced the association of PI3KC2beta with the Raptor subunit of mTORC1. Local PI(3,4)P2 synthesis triggered repression of mTORC1 activity through association of Raptor with inhibitory 14-3-3 proteins. These results unravel an unexpected function for local PI(3,4)P2 production in shutting off mTORC1.

Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning
Fricke, P., Chevelkov, V., Zinke, M., Giller(*), K., Becker(*), S.; Lange, A.
Nat Protoc, 12:764-782

Tags: Molecular Biophysics (Lange, A.)

Abstract: Solid-state NMR (ssNMR) is a technique that allows the study of protein structure and dynamics at atomic detail. In contrast to X-ray crystallography and cryo-electron microscopy, proteins can be studied under physiological conditions-for example, in a lipid bilayer and at room temperature (0-35 degrees C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years, 1H-detection of perdeuterated protein samples has been proposed as a method of alleviating the sensitivity issue. Such methods are, however, substantially more demanding to the spectroscopist, as compared with traditional 13C-detected approaches. As a guide, this protocol describes a procedure for the chemical shift assignment of the backbone atoms of proteins in the solid state by 1H-detected ssNMR. It requires a perdeuterated, uniformly 13C- and 15N-labeled protein sample with subsequent proton back-exchange to the labile sites. The sample needs to be spun at a minimum of 40 kHz in the NMR spectrometer. With a minimal set of five 3D NMR spectra, the protein backbone and some of the side-chain atoms can be completely assigned. These spectra correlate resonances within one amino acid residue and between neighboring residues; taken together, these correlations allow for complete chemical shift assignment via a 'backbone walk'. This results in a backbone chemical shift table, which is the basis for further analysis of the protein structure and/or dynamics by ssNMR. Depending on the spectral quality and complexity of the protein, data acquisition and analysis are possible within 2 months.

Bacteriophage Tail-Tube Assembly Studied by Proton-Detected 4D Solid-State NMR
Zinke, M., Fricke, P., Samson(*), C., Hwang, S., Wall(*), J. S., Lange, S., Zinn-Justin(*), S.; Lange, A.
Angew Chem Int Ed Engl,

Tags: Molecular Biophysics (Lange, A.)

Abstract: Obtaining unambiguous resonance assignments remains a major bottleneck in solid-state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three-dimensional (3D) spectra are used. Here, we present a proton-detected 4D solid-state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non-uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH, (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail-tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.

Chemical Approaches to Studying Labile Amino Acid Phosphorylation
Marmelstein, A. M., Moreno, J.; Fiedler, D.
Topics in current chemistry (Journal), 375:22

Tags: Chemical Biology I (Fiedler)

Abstract: Phosphorylation of serine, threonine, and tyrosine residues is the archetypal posttranslational modification of proteins. While phosphorylation of these residues has become standard textbook knowledge, phosphorylation of other amino acid side chains is underappreciated and minimally characterized by comparison. This disparity is rooted in the relative instability of these chemically distinct amino acid side chain moieties, namely phosphoramidates, acyl phosphates, thiophosphates, and phosphoanhydrides. In the case of the O-phosphorylated amino acids, synthetic constructs were critical to assessing their stability and developing tools for their study. As the chemical biology community has become more aware of these alternative phosphorylation sites, methodology has been developed for the synthesis of well-characterized standards and close mimics of these phosphorylated amino acids as well. In this article, we review the synthetic chemistry that is a prerequisite to progress in this field.

Unambiguous Identification of Serine and Threonine Pyrophosphorylation Using Neutral-Loss-Triggered Electron-Transfer/Higher-Energy Collision Dissociation
Penkert, M., Yates(*), L. M., Schümann, M., Perlman(*), D., Fiedler, D.; Krause, E.
Anal Chem, 89:3672-3680

Tags: Mass Spectrometry (Krause, E.), Chemical Biology I (Fiedler)

Abstract: Tandem mass spectrometry (MS/MS) has emerged as the core technology for identification of post-translational modifications (PTMs). Here, we report the mass spectrometry analysis of serine and threonine pyrophosphorylation, a protein modification that has eluded detection by conventional MS/MS methods. Analysis of a set of synthesized, site-specifically modified peptides by different fragmentation techniques shows that pyrophosphorylated peptides exhibit a characteristic neutral loss pattern of 98, 178, and 196 Da, which enables the distinction between isobaric pyro- and diphosphorylated peptides. In addition, electron-transfer dissociation combined with higher energy collision dissociation (EThcD) provides exceptional data-rich MS/MS spectra for direct and unambiguous pyrophosphosite assignment. Remarkably, sufficient fragmentation of doubly charged precursors could be achieved by electron-transfer dissociation (ETD) with increased supplemental activation, without losing the labile modification. By exploiting the specific fragmentation behavior of pyrophosphorylated peptides during collision-induced dissociation (CID), a data dependent neutral-loss-triggered EThcD acquisition method was developed. This strategy enables reliable pyrophosphopeptide identification in complex samples, without compromising speed and sensitivity.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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