FMP Publications

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

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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.

Functional Significance of the Signal Peptides of Corticotropin-Releasing Factor Receptors
Schülein, R., Gibert, A.; Rutz, C.
Current molecular pharmacology,

Tags: Protein Trafficking (Schülein)

Abstract: The corticotropin releasing factor (CRF) receptors belong to the large family of G protein-coupled receptors (GPCRs) and must be transported to the plasma membrane to function properly. The first step of the intracellular transport of GPCRs is their insertion into the membrane of the endoplasmic reticulum (ER). This process is mediated by the translocon complex of the ER membrane and the signal sequences of the receptors. Most GPCRs possess signal sequences which form part of the mature proteins, the so called signal anchor sequences (usually transmembrane domain 1). The CRF receptors possess instead signal sequences at their extreme N tails which were thought to be cleaved off following integration of the receptors into the ER membrane (signal peptides, SPs, also called cleaved signal sequences). Recent work, however, showed that not all subtypes of CRF receptors stick to this rule. Whereas the corticotropin-releasing factor receptor type 1 (CRF1R) and the corticotropin-releasing factor receptor type 2b (CRF2(b)R) possess conventional SPs which are indeed cleaved off following ER insertion, the SP of the cortictropin-releasing factor receptor type 2a (CRF2(a)R) remains uncleaved. It forms a unique N-terminal domain (pseudo signal peptide, PSP) which has surprising functions beyond the ER level. Its presence not only influences expression levels at the plasma membrane but also receptor homodimerisation and, as a consequence, G protein selectivity. In this mini-review, we summarize the progress in understanding the functions of SPs of CRF receptors. Recent data also allow deriving hypotheses for a physiological significance of these sequences.

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1
Chenge(*), J. T., Duyet(*), L. V., Swami(*), S., McLean(*), K. J., Kavanagh(*), M. E., Coyne(*), A. G., Rigby(*), S. E., Cheesman(*), M. R., Girvan(*), H. M., Levy(*), C. W., Rupp, B., von Kries, J. P., Abell(*), C., Leys(*), D.; Munro(*), A. W.
J Biol Chem, 292:1310-1329

Tags: Screening Unit (von Kries), Computational Chemistry and Protein Design (Kühne)

Abstract: The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands "moonlight" as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity.

Polar and charged extracellular residues conserved among barrier-forming claudins contribute to tight junction strand formation
Piontek, A., Rossa, J., Protze, J., Wolburg(*), H., Hempel(*), C., Günzel(*), D., Krause, G.; Piontek(*), J.
Annals of the New York Academy of Sciences,

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

Abstract: Claudins (Cldn) form the backbone of tight junction (TJ) strands and thereby regulate paracellular permeability for solutes and water. Polymeric strands are formed by homo- and heterophilic cis- and trans-interactions between claudin protomers. Crystal structures of some claudins have been resolved; however, the mechanism by which claudins assemble into TJ strands remains unclear. To elucidate strand architecture, TJ-like strands were reconstituted in HEK293 cells by claudin transfection. Determinants of prototypic, classic barrier-forming claudins (Cldn1, -3, and -5) involved in strand formation were analyzed by mutagenesis. The capability of claudin constructs to interact in trans and to form strands was investigated by cell contact-enrichment assays and freeze-fracture electron microscopy. Residues in extracellular loops 1 and 2 of the claudins affecting strand formation were identified. Using homology modeling and molecular docking, we tested working concepts for the arrangement of claudin protomers within TJ strands. We show that the charge of Lys65 in Cldn1 and Glu158 in Cldn3, but not of Arg30 or Asp145 in Cldn3, and the polarity of Gln56 and Gln62 in Cldn3 and of Gln57 in Cldn5 are necessary for TJ strand formation. These residues are all conserved among barrier-forming classic claudins. The results contribute to mechanistic understanding of claudin-based regulation of paracellular permeability.

Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin lymphoma
Du(*), J., Neuenschwander, M., Yu(*), Y., Dabritz(*), J. H., Neuendorff(*), N. R., Schleich(*), K., Bittner(*), A., Milanovic(*), M., Beuster(*), G., Radetzki, S., Specker, E., Reimann(*), M., Rosenbauer(*), F., Mathas(*), S., Lohneis(*), P., Hummel(*), M., Dörken(*), B., von Kries, J. P., Lee(*), S.; Schmitt(*), C. A.
Blood, 129:71-81

Tags: Screening Unit (von Kries)

Abstract: Classical Hodgkin lymphoma (cHL), although originating from B cells, is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell-committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoring the B-cell phenotype may not only correct a central malignant property, but it may also render cHL susceptible to clinically established antibody therapies targeting B-cell surface receptors or small compounds interfering with B-cell receptor signaling. We conducted a high-throughput pharmacological screening based on >28 000 compounds in cHL cell lines carrying a CD19 reporter to identify drugs that promote reexpression of the B-cell phenotype. Three chemicals were retrieved that robustly enhanced CD19 transcription. Subsequent chromatin immunoprecipitation-based analyses indicated that action of 2 of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the antileukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of cHL cell lines. When applied in combination with a screening-identified chemical, ATO evoked reexpression of the CD20 antigen, which could be further therapeutically exploited by enabling CD20 antibody-mediated apoptosis of cHL cells. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell non-Hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we report here a conceptually novel, redifferentiation-based treatment strategy for cHL.

Statin and rottlerin small-molecule inhibitors restrict colon cancer progression and metastasis via MACC1
Juneja(*), M., Kobelt(*), D., Walther(*), W., Voss(*), C., Smith(*), J., Specker, E., Neuenschwander, M., Gohlke(*), B. O., Dahlmann(*), M., Radetzki, S., Preissner(*), R., von Kries, J. P., Schlag(*), P. M.; Stein(*), U.
PLoS biology, 15:e2000784

Tags: Screening Unit (von Kries)

Abstract: MACC1 (Metastasis Associated in Colon Cancer 1) is a key driver and prognostic biomarker for cancer progression and metastasis in a large variety of solid tumor types, particularly colorectal cancer (CRC). However, no MACC1 inhibitors have been identified yet. Therefore, we aimed to target MACC1 expression using a luciferase reporter-based high-throughput screening with the ChemBioNet library of more than 30,000 compounds. The small molecules lovastatin and rottlerin emerged as the most potent MACC1 transcriptional inhibitors. They remarkably inhibited MACC1 promoter activity and expression, resulting in reduced cell motility. Lovastatin impaired the binding of the transcription factors c-Jun and Sp1 to the MACC1 promoter, thereby inhibiting MACC1 transcription. Most importantly, in CRC-xenografted mice, lovastatin and rottlerin restricted MACC1 expression and liver metastasis. This is-to the best of our knowledge-the first identification of inhibitors restricting cancer progression and metastasis via the novel target MACC1. This drug repositioning might be of therapeutic value for CRC patients.

Stable Positioning of Unc13 Restricts Synaptic Vesicle Fusion to Defined Release Sites to Promote Synchronous Neurotransmission
Reddy-Alla(*), S., Böhme, M. A., Reynolds(*), E., Beis(*), C., Grasskamp, A. T., Mampell(*), M. M., Maglione, M., Jusyte, M., Rey(*), U., Babikir(*), H., McCarthy, A. W., Quentin(*), C., Matkovic(*), T., Bergeron(*), D. D., Mushtaq, Z., Goettfert(*), F., Owald(*), D., Mielke(*), T., Hell(*), S. W., Sigrist(*), S. J.; Walter, A. M.

Tags: Molecular and Theoretical Neuroscience (Walter)

Abstract: Neural information processing depends on precisely timed, Ca2+-activated synaptic vesicle exocytosis from release sites within active zones (AZs), but molecular details are unknown. Here, we identify that the (M)Unc13-family member Unc13A generates release sites and show the physiological relevance of their restrictive AZ targeting. Super-resolution and intravital imaging of Drosophila neuromuscular junctions revealed that (unlike the other release factors Unc18 and Syntaxin-1A) Unc13A was stably and precisely positioned at AZs. Local Unc13A levels predicted single AZ activity. Different Unc13A portions selectively affected release site number, position, and functionality. An N-terminal fragment stably localized to AZs, displaced endogenous Unc13A, and reduced the number of release sites, while a C-terminal fragment generated excessive sites at atypical locations, resulting in reduced and delayed evoked transmission that displayed excessive facilitation. Thus, release site generation by the Unc13A C terminus and their specific AZ localization via the N terminus ensure efficient transmission and prevent ectopic, temporally imprecise release.

Tag-Free Semi-Synthesis of the Tau Protein
Reimann, O., Smet-Nocca(*), C.; Hackenberger, C. P.
Methods Mol Biol, 1523:215-235

Tags: Chemical Biology II (Hackenberger)

Abstract: Expressed protein ligation (EPL) is a valuable tool to study site-specific functionalities on proteins such as posttranslational modifications. The purification of such ligation products from EPL mixtures can be cumbersome due to a small size difference between the expressed protein portion and the desired ligated protein. Therefore, affinity tags are often required, which remain on the protein after purification. Herein, we present an efficient protocol to install a photocleavable biotin building block on synthetic C-terminal tau[390-441] and describe its use for purification of full-length semi-synthetic tau[1-441].

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells
Herce(*), H. D., Schumacher, D., Schneider, A. F. L., Ludwig(*), A. K., Mann, F. A., Fillies(*), M., Kasper, M. A., Reinke, S., Krause, E., Leonhardt(*), H., Cardoso(*), M. C.; Hackenberger, C. P. R.
Nature chemistry, 9:762-771

Tags: Chemical Biology II (Hackenberger), Mass Spectrometry (Krause, E)

Abstract: Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

NMR Hyperpolarization Techniques of Gases
Barskiy(*), D. A., Coffey(*), A. M., Nikolaou(*), P., Mikhaylov(*), D. M., Goodson(*), B. M., Branca(*), R. T., Lu(*), G. J., Shapiro(*), M. G., Telkki(*), V. V., Zhivonitko(*), V. V., Koptyug(*), I. V., Salnikov(*), O. G., Kovtunov(*), K. V., Bukhtiyarov(*), V. I., Rosen(*), M. S., Barlow(*), M. J., Safavi(*), S., Hall(*), I. P., Schroeder, L.; Chekmenev(*), E. Y.
Chemistry, 23:725-751

Tags: Molecular Imaging (Schröder)

Abstract: Nuclear spin polarization can be significantly increased through the process of hyperpolarization, leading to an increase in the sensitivity of nuclear magnetic resonance (NMR) experiments by 4-8 orders of magnitude. Hyperpolarized gases, unlike liquids and solids, can often be readily separated and purified from the compounds used to mediate the hyperpolarization processes. These pure hyperpolarized gases enabled many novel MRI applications including the visualization of void spaces, imaging of lung function, and remote detection. Additionally, hyperpolarized gases can be dissolved in liquids and can be used as sensitive molecular probes and reporters. This Minireview covers the fundamentals of the preparation of hyperpolarized gases and focuses on selected applications of interest to biomedicine and materials science.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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