FMP Publications

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

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References

2015

DOTAM derivatives as active cartilage-targeting drug carriers for the treatment of osteoarthritis
Hu(*), H. Y., Lim(*), N. H., Ding-Pfennigdorff(*), D., Saas(*), J., Wendt(*), K. U., Ritzeler(*), O., Nagase(*), H., Plettenburg(*), O., Schultz(*), C.; Nazare, M.
Bioconjug Chem, 26:383-388
(2015)

Tags: Medicinal Chemistry (Nazare)

Abstract: Targeted drug-delivery methods are crucial for effective treatment of degenerative joint diseases such as osteoarthritis (OA). Toward this goal, we developed a small multivalent structure as a model drug for the attenuation of cartilage degradation. The DOTAM (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid amide)-based model structure is equipped with the cathepsin D protease inhibitor pepstatin A, a fluorophore, and peptide moieties targeting collagen II. In vivo injection of these soluble probes into the knee joints of mice resulted in 7-day-long local retention, while the drug carrier equipped with a scrambled peptide sequence was washed away within 6-8 h. The model drug conjugate successfully reduced the cathepsin D protease activity as measured by release of GAG peptide. Therefore, these conjugates represent a promising first drug conjugate for the targeted treatment of degenerative joint diseases.

In vivo visualization of osteoarthritic hypertrophic lesions
Hu(*), H. Y., Lim(*), N. H., Juretschke(*), H. P., Ding-Pfennigdorff(*), D., Florian(*), P., Kohlmann(*), M., Kandira(*), A., von Kries(*), J. P., Saas(*), J., Rudolphi(*), K. A., Wendt(*), K. U., Nagase(*), H., Plettenburg(*), O., Nazare, M.; Schultz(*), C.
Chem Sci, 6:6256-6261
(2015)

Tags: Medicinal Chemistry (Nazare)

Abstract: Osteoarthritis (OA) is one of the most common diseases in the aging population. While disease progress in humans is monitored indirectly by X-ray or MRI, small animal OA lesions detection always requires surgical intervention and histology. Here we introduce bimodal MR/NIR probes based on cartilage-targeting 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid amide (DOTAM) that are directly administered to the joint cavity. We demonstrate applications in healthy and diseased rat joints by MRI in vivo. The same joints are inspected post-mortem by fluorescence microscopy, showing not only the precise location of the reagents but also revealing details such as focal cartilage damage and chondrophyte or osteophyte formation. This allows for determining the distinct pathological state of the disease and the regeneration capability of the animal model and will help to correctly assess the effect of potential disease modifying OA drugs (DMOADs) in the future.

Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier
Zhang(*), J., Ni(*), C., Yang(*), Z., Piontek, A., Chen(*), H., Wang(*), S., Fan(*), Y., Qin(*), Z.; Piontek(*), J.
Exp Dermatol, 24:605-610
(2015)

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

Abstract: Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.

Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability
Nischan, N., Herce(*), H. D., Natale(*), F., Bohlke(*), N., Budisa(*), N., Cardoso(*), M. C.; Hackenberger, C. P.
Angew Chem Int Ed Engl, 54:1950-1953
(2015)

Tags: Chemical Biology II (Hackenberger)

Abstract: The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

Stonin1 mediates endocytosis of the proteoglycan NG2 and regulates focal adhesion dynamics and cell motility
Feutlinske, F., Browarski, M., Ku(*), M. C., Trnka, P., Waiczies(*), S., Niendorf(*), T., Stallcup(*), W. B., Glass(*), R., Krause, E.; Maritzen, T.
Nat Commun, 6:8535
(2015)

Tags: Membrane Traffic and Cell Motility (Maritzen), Mass Spectrometry (Krause, E.)

Abstract: Cellular functions, ranging from focal adhesion (FA) dynamics and cell motility to tumour growth, are orchestrated by signals cells receive from outside via cell surface receptors. Signalling is fine-tuned by the exo-endocytic cycling of these receptors to control cellular responses such as FA dynamics, which determine cell motility. How precisely endocytosis regulates turnover of the various cell surface receptors remains unclear. Here we identify Stonin1, an endocytic adaptor of unknown function, as a regulator of FA dynamics and cell motility, and demonstrate that it facilitates the internalization of the oncogenic proteoglycan NG2, a co-receptor of integrins and platelet-derived growth factor receptor. Embryonic fibroblasts obtained from Stonin1-deficient mice display a marked surface accumulation of NG2, increased cellular signalling and defective FA disassembly as well as altered cellular motility. These data establish Stonin1 as a specific adaptor for the endocytosis of NG2 and as an important factor for FA dynamics and cell migration.

Anchoring dipalmitoyl phosphoethanolamine to nanoparticles boosts cellular uptake and fluorine-19 magnetic resonance signal
Waiczies(*), S., Lepore(*), S., Sydow, K., Drechsler(*), S., Ku(*), M. C., Martin(*), C., Lorenz, D., Schütz, I., Reimann(*), H. M., Purfurst(*), B., Dieringer(*), M. A., Waiczies(*), H., Dathe, M., Pohlmann(*), A.; Niendorf(*), T.
Sci Rep, 5:8427
(2015)

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe), Molecular Pharmacology and Cell Biology (Haucke), Cellular Imaging (Wiesner)

Abstract: Magnetic resonance (MR) methods to detect and quantify fluorine ((19)F) nuclei provide the opportunity to study the fate of cellular transplants in vivo. Cells are typically labeled with (19)F nanoparticles, introduced into living organisms and tracked by (19)F MR methods. Background-free imaging and quantification of cell numbers are amongst the strengths of (19)F MR-based cell tracking but challenges pertaining to signal sensitivity and cell detection exist. In this study we aimed to overcome these limitations by manipulating the aminophospholipid composition of (19)F nanoparticles in order to promote their uptake by dendritic cells (DCs). As critical components of biological membranes, phosphatidylethanolamines (PE) were studied. Both microscopy and MR spectroscopy methods revealed a striking (at least one order of magnitude) increase in cytoplasmic uptake of (19)F nanoparticles in DCs following enrichment with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). The impact of enriching (19)F nanoparticles with PE on DC migration was also investigated. By manipulating the nanoparticle composition and as a result the cellular uptake we provide here one way of boosting (19)F signal per cell in order to overcome some of the limitations related to (19)F MR signal sensitivity. The boost in signal is ultimately necessary to detect and track cells in vivo.

Different inhibition of Gbetagamma-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gbetagamma-dependent regulator of PI3Kgamma enzymatic activity
Shymanets(*), A., Vadas(*), O., Czupalla(*), C., LoPiccolo(*), J., Brenowitz(*), M., Ghigo(*), A., Hirsch(*), E., Krause, E., Wetzker(*), R., Williams(*), R. L., Harteneck(*), C.; Nürnberg(*), B.
Biochem J, 469:59-69
(2015)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Class IB phosphoinositide 3-kinases gamma (PI3Kgamma) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kgamma variants have one catalytic p110gamma subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110gamma and p101-p110gamma allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110gamma monoclonal antibody [mAb(A)p110gamma] to block PI3Kgamma enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110gamma interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110gamma, which was accompanied by conformational changes in the helical domain harbouring the Gbetagamma-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kgamma by the antibody. p87-p110gamma showed a significantly reduced Gbetagamma-mediated phospholipid recruitment as compared with p101-p110gamma. Concomitantly, in the presence of mAb(A)p110gamma, Gbetagamma did not bind to p87-p110gamma. These data correlated with the ability of the antibody to block Gbetagamma-stimulated lipid kinase activity of p87-p110gamma 30-fold more potently than p101-p110gamma. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gbetagamma-dependent regulation of p101 in PI3Kgamma activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kgamma variants downstream of GPCRs.

Evidence for a novel mechanism of antimicrobial action of a cyclic R-,W-rich hexapeptide
Scheinpflug, K., Krylova, O., Nikolenko, H., Thurm, C.; Dathe, M.
Plos One, 10:e0125056
(2015)

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

Abstract: The development of antimicrobial peptides as new class of antibiotic agents requires structural characterisation and understanding of their diverse mechanisms of action. As the cyclic hexapeptide cWFW (cyclo(RRRWFW)) does not exert its rapid cell killing activity by membrane permeabilisation, in this study we investigated alternative mechanisms of action, such as peptide translocation into the cytoplasm and peptide interaction with components of the phospholipid matrix of the bacterial membrane. Using fluorescence microscopy and an HPLC-based strategy to analyse peptide uptake into the cells we could confirm the cytoplasmic membrane as the major peptide target. However, unexpectedly we observed accumulation of cWFW at distinct sites of the membrane. Further characterisation of peptide-membrane interaction involved live cell imaging to visualise the distribution of the lipid cardiolipin (CL) and isothermal titration calorimetry to determine the binding affinity to model membranes with different bacterial lipid compositions. Our results demonstrate a distribution of the cyclic peptide similar to that of cardiolipin within the membrane and highly preferred affinity of cWFW for CL-rich phosphatidylethanolamine (POPE) matrices. These observations point to a novel mechanism of antimicrobial killing for the cyclic hexapeptide cWFW which is neither based on membrane permeabilisation nor translocation into the cytoplasm but rather on preferred partitioning into particular lipid domains. As the phospholipids POPE/CL play a key role in the dynamic organisation of bacterial membranes we discuss the consequences of this peptide-lipid-interaction and outline the impact on antimicrobial peptide research.

The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function
Albert(*), G. I., Schell(*), C., Kirschner(*), K. M., Schäfer(*), S., Naumann(*), R., Müller(*), A., Kretz(*), O., Kuropka, B., Girbig(*), M., Hübner(*), N., Krause, E., Scholz(*), H., Huber(*), T. B., Knobeloch(*), K. P.; Freund(*), C.
Journal of molecular cell biology, 7:402-414
(2015)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulators. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.

Disruption of adaptor protein 2mu (AP-2mu) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing
Jung(*), S., Maritzen, T., Wichmann(*), C., Jing(*), Z., Neef(*), A., Revelo(*), N. H., Al-Moyed(*), H., Meese(*), S., Wojcik(*), S. M., Panou(*), I., Bulut(*), H., Schu(*), P., Ficner(*), R., Reisinger(*), E., Rizzoli(*), S. O., Neef(*), J., Strenzke(*), N., Haucke, V.; Moser(*), T.
EMBO J, 34:2686-2702
(2015)

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

Abstract: Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2mu (AP-2mu) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2mu slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2mu-deficient IHCs, indicating a further role of AP-2mu in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.

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