FMP Publications

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

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Pknox1/Prep1 Regulates Mitochondrial Oxidative Phosphorylation Components in Skeletal Muscle
Kanzleiter(*), T., Rath(*), M., Penkov(*), D., Puchkov, D., Schulz(*), N., Blasi(*), F.; Schürmann(*), A.
Mol Cell Biol, 34:290-298

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: The homeodomain transcription factor Prep1 was previously shown to regulate insulin sensitivity. Our aim was to study the specific role of Prep1 for the regulation of energy metabolism in skeletal muscle. Muscle-specific ablation of Prep1 resulted in increased expression of respiratory chain subunits. This finding was consistent with an increase in mitochondrial enzyme activity without affecting mitochondrial volume fraction as assessed by electron microscopy. Metabolic phenotyping revealed no differences in daily energy expenditure or body composition. However, during treadmill exercise challenge, Prep1 ablation resulted in a higher maximal oxidative capacity and better endurance. Elevated PGC-1 alpha expression was identified as a cause for increased mitochondrial capacity in Prep1 ablated mice. Prep1 stabilizes p160 Mybbp1a, a known inhibitor of PGC-1 alpha activity. Thereby, p160 protein levels were significantly lower in the muscle of Prep1 ablated mice. By a chromatin immunoprecipitation-sequencing (ChIP-seq) approach, PREP1 binding sites in genes encoding mitochondrial components (e.g., Ndufs2) were identified that might be responsible for elevated proteins involved in oxidative phosphorylation (OXPHOS) in the muscle of Prep1 null mutants. These results suggest that Prep1 exhibits additional direct effects on regulation of mitochondrial proteins. We therefore conclude that Prep1 is a regulator of oxidative phosphorylation components via direct and indirect mechanisms.

Cell tracking with caged xenon: using cryptophanes as MRI reporters upon cellular internalization
Klippel, S., Döpfert, J., Jayapaul, J., Kunth, M., Rossella, F., Schnurr, M., Witte, C., Freund, C.; Schröder, L.
Angew Chem Int Ed Engl, 53:493-496

Tags: Molecular Imaging (Schröder)

Abstract: Caged xenon has great potential in overcoming sensitivity limitations for solution-state NMR detection of dilute molecules. However, no application of such a system as a magnetic resonance imaging (MRI) contrast agent has yet been performed with live cells. We demonstrate MRI localization of cells labeled with caged xenon in a packed-bed bioreactor working under perfusion with hyperpolarized-xenon-saturated medium. Xenon hosts enable NMR/MRI experiments with switchable contrast and selectivity for cell-associated versus unbound cages. We present MR images with 10(3) -fold sensitivity enhancement for cell-internalized, dual-mode (fluorescence/MRI) xenon hosts at low micromolar concentrations. Our results illustrate the capability of functionalized xenon to act as a highly sensitive cell tracer for MRI detection even without signal averaging. The method will bridge the challenging gap for translation to in vivo studies for the optimization of targeted biosensors and their multiplexing applications.

Multichannel MRI labeling of mammalian cells by switchable nanocarriers for hyperpolarized xenon
Klippel, S., Freund(*), C.; Schröder, L.
Nano Lett, 14:5721-5726

Tags: Molecular Imaging (Schröder)

Abstract: We demonstrate a concept for multichannel MRI cell-labeling using encapsulated laser-polarized xenon. Conceptually different Xe trapping properties of two nanocarriers, namely macrocyclic cages as individual hosts or compartmentalization into nanodroplets, ensure a large chemical shift separation for Xe bound in either of the carriers even after cellular internalization. Two differently labeled mammalian cell populations were imaged by frequency selective saturation transfer resulting in a switchable "two-color" xenon-MRI contrast at micro- to nanomolar Xe carrier concentrations.

Photoinactivation of glutamate receptors by genetically encoded unnatural amino acids
Klippenstein, V., Ghisi, V., Wietstruk, M.; Plested, A. J.
J Neurosci, 34:980-991

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Ionotropic glutamate receptors (iGluRs) are ubiquitous in the mammalian brain, and the AMPA-subtype is essential for fast, glutamate-activated postsynaptic currents. We incorporated photoactive crosslinkers into AMPA receptors using genetically encoded unnatural amino acid mutagenesis in a mammalian cell line. Receptors rescued by incorporation of unnatural amino acids, including p-benzoyl-l-phenylalanine (BzF, also known as Bpa), had largely similar properties to wild-type channels and were expressed at similar levels. BzF incorporation at subunit interfaces afforded photocrosslinking of subunits, as assessed by biochemical experiments. In electrophysiological recordings, BzF incorporation allowed selective and potent UV-driven photoinactivation of both homomeric (GluA2) and heteromeric (GluA2:GluA1) AMPA receptors. State dependence of trapping at two sites in the lower lobe of the ligand binding domain is consistent with deformation of these domains as well as intersubunit rearrangements during AMPA receptor desensitization.

Clathrin/AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses
Kononenko, N. L., Puchkov, D., Classen, G. A., Walter, A. M., Pechstein, A., Sawade, L., Kaempf, N., Trimbuch(*), T., Lorenz, D., Rosenmund(*), C., Maritzen, T.; Haucke, V.
Neuron, 82:981-988

Tags: Molecular Pharmacology and Cell Biology (Haucke), Membrane Traffic and Cell Motility (Maritzen), Cellular Imaging (Wiesner, Puchkov)

Abstract: Neurotransmission depends on presynaptic membrane retrieval and local reformation of synaptic vesicles (SVs) at nerve terminals. The mechanisms involved in these processes are highly controversial with evidence being presented for SV membranes being retrieved exclusively via clathrin-mediated endocytosis (CME) from the plasma membrane or via ultrafast endocytosis independent of clathrin. Here we show that clathrin and its major adaptor protein 2 (AP-2) in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate SVs but are not essential for membrane retrieval. Depletion of clathrin or conditional knockout of AP-2 result in defects in SV reformation and an accumulation of endosome-like vacuoles generated by clathrin-independent endocytosis (CIE) via dynamin 1/3 and endophilin. These results together with theoretical modeling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies.

Disturbed function of the blood-cerebrospinal fluid barrier aggravates neuro-inflammation
Kooij(*), G., Kopplin(*), K., Blasig, R., Stuiver(*), M., Koning(*), N., Goverse(*), G., van der Pol(*), S. M. A., Hof(*), B. V., Gollasch(*), M., Drexhage(*), J. A. R., Reijerkerk(*), A., Meij(*), I. C., Mebius(*), R., Willnow(*), T. E., Müller(*), D., Blasig, I. E.; de Vries(*), H. E.
Acta Neuropathol, 128:267-277

Tags: Molecular Cell Physiology (Blasig, I.E.)

Abstract: Multiple sclerosis (MS) is a chronic neuro-inflammatory disorder, which is marked by the invasion of the central nervous system by monocyte-derived macrophages and autoreactive T cells across the brain vasculature. Data from experimental animal models recently implied that the passage of leukocytes across the brain vasculature is preceded by their traversal across the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus. The correlation between the presence of leukocytes in the CSF of patients suffering from MS and the number of inflammatory lesions as detected by magnetic resonance imaging suggests that inflammation at the choroid plexus contributes to the disease, although in a yet unknown fashion. We here provide first insights into the involvement of the choroid plexus in the onset and severity of the disease and in particular address the role of the tight junction protein claudin-3 (CLDN3) in this process. Detailed analysis of human post-mortem brain tissue revealed a selective loss of CLDN3 at the choroid plexus in MS patients compared to control tissues. Importantly, mice that lack CLDN3 have an impaired BCSFB and experience a more rapid onset and exacerbated clinical signs of experimental autoimmune encephalomyelitis, which coincides with enhanced levels of infiltrated leukocytes in their CSF. Together, this study highlights a profound role for the choroid plexus in the pathogenesis of multiple sclerosis, and implies that CLDN3 may be regarded as a crucial and novel determinant of BCSFB integrity.

Efficient modification of alpha-synuclein serine 129 by protein kinase CK1 requires phosphorylation of tyrosine 125 as a priming event
Kosten, J., Binolfi, A., Stuiver, M., Verzini, S., Theillet, F. X., Bekei, B., van Rossum, M.; Selenko, P.
Acs Chem Neurosci, 5:1203-1208

Tags: In-Cell NMR (Selenko)

Abstract: S129-phosphorylated alpha-synuclein (alpha-syn) is abundantly found in Lewy-body inclusions of Parkinson's disease patients. Residues neighboring S129 include the alpha-syn tyrosine phosphorylation sites Y125, Y133, and Y136. Here, we use time-resolved NMR spectroscopy to delineate atomic resolution insights into the modification behaviors of different serine and tyrosine kinases targeting these sites and show that Y125 phosphorylation constitutes a necessary priming event for the efficient modification of S129 by CK1, both in reconstituted kinase reactions and mammalian cell lysates. These results suggest that alpha-syn Y125 phosphorylation augments S129 modification under physiological in vivo conditions.

Kinetics and efficiency of a methyl-carboxylated 5-Fluorouracil-bovine serum albumin adduct for targeted delivery
Koziol(*), M. J., Sievers(*), T. K., Smuda(*), K., Xiong(*), Y., Müller(*), A., Wojcik(*), F., Steffen(*), A., Dathe, M., Georgieva(*), R.; Bäumler(*), H.
Macromolecular bioscience, 14:428-439

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: 5-Fluorouracil (5-FU) is a clinically well-established anti-cancer drug effectively applied in chemotherapy, mainly for the treatment of breast and colorectal cancer. Substantial disadvantages are adverse effects, arising from serious damage of healthy tissues, and shortcoming pharmacokinetics due to its low molecular weight. A promising approach for improvement of such drugs is their coupling to suitable carriers. Here, a 5-FU adduct, 5-fluorouracil acetate (FUAc) is synthesized and covalently coupled to bovine serum albumin (BSA) as model carrier molecule. On average, 12 molecules FUAc are bound to one BSA. Circular dichriosm (CD)-spectra of BSA and FUAc-BSA are identical, suggesting no significant conformational differences. FUAc-BSA is tested on T-47D and MDA-MB-231 breast cancer cells. Proliferation inhibition of membrane albumin-binding protein (mABP)-expressing T-47D cells by FUAc-BSA is similar to that of 5-FU and only moderate for MDA-MB-231 cells that lack such expression. Therefore, a crucial role of mABP expression in effective cell growth inhibition by FUAc-BSA is assumed.

A novel twist in membrane dePHormation
Krauss, M.; Haucke, V.
Dev Cell, 31:3-4

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Bin-Amphiphysin-Rvs (BAR) domain-containing proteins form oligomeric assemblies that aid membrane remodeling. In this issue of Developmental Cell, Pang et al. (2014) show that the BAR domain of ACAP1, although architecturally similar to other BAR domains, cooperates with its neighboring pleckstrin homology domain to deform membranes and facilitate endosomal recycling.

Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer
Krüger(*), H. R., Schütz, I., Justies(*), A., Licha(*), K., Welker(*), P., Haucke, V.; Calderon(*), M.
J Control Release, 194:189-196

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Herein we present a FRET-based theranostic macromolecular prodrug (TMP) composed of (a) dendritic polyglycerol (PG) as polymeric nanocarrier, (b) doxorubicin (Dox) linked via a pH-sensitive hydrazone to (c) a tri-functional linker, and (d) an indodicarbocyanine dye (IDCC) attached in close proximity to Dox. The drug fluorescence is quenched via intramolecular FRET until the pH-sensitive hydrazone bond between the TMP and Dox is cleaved at acidic pH. By measuring its fluorescence, we characterized the TMP cleavage kinetics at different pH values in vitro. The intracellular release of Dox from the carrier was monitored in real time in intact cancer cells, giving more insight into the mode of action of a polymer drug conjugate.

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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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