FMP Publications

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

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References
In vivo properties of the disaggregase function of J-proteins and Hsc70 in Caenorhabditis elegans stress and aging
Kirstein, J., Arnsburg, K., Scior, A., Szlachcic(*), A., Guilbride(*), D. L., Morimoto(*), R. I., Bukau(*), B.; Nillegoda(*), N. B.
Aging cell,
(2017)

Tags: Proteostasis in Aging and Disease (Kirstein)

Abstract: Protein aggregation is enhanced upon exposure to various stress conditions and aging, which suggests that the quality control machinery regulating protein homeostasis could exhibit varied capacities in different stages of organismal lifespan. Recently, an efficient metazoan disaggregase activity was identified in vitro, which requires the Hsp70 chaperone and Hsp110 nucleotide exchange factor, together with single or cooperating J-protein co-chaperones of classes A and B. Here, we describe how the orthologous Hsp70s and J-protein of Caenorhabditis elegans work together to resolve protein aggregates both in vivo and in vitro to benefit organismal health. Using an RNAi knockdown approach, we show that class A and B J-proteins cooperate to form an interactive flexible network that relocalizes to protein aggregates upon heat shock and preferentially recruits constitutive Hsc70 to disaggregate heat-induced protein aggregates and polyQ aggregates that form in an age-dependent manner. Cooperation between class A and B J-proteins is also required for organismal health and promotes thermotolerance, maintenance of fecundity, and extended viability after heat stress. This disaggregase function of J-proteins and Hsc70 therefore constitutes a powerful regulatory network that is key to Hsc70-based protein quality control mechanisms in metazoa with a central role in the clearance of aggregates, stress recovery, and organismal fitness in aging.

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS
Leonhardt(*), J., Villela(*), D. C., Teichmann, A., Munter(*), L. M., Mayer(*), M. C., Mardahl(*), M., Kirsch(*), S., Namsolleck(*), P., Lucht(*), K., Benz(*), V., Alenina(*), N., Daniell(*), N., Horiuchi(*), M., Iwai(*), M., Multhaup(*), G., Schülein, R., Bader(*), M., Santos(*), R. A., Unger(*), T.; Steckelings(*), U. M.
Hypertension,
(2017)

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

Abstract: The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 +/- 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other.

Cross-over endocytosis of claudins is mediated by interactions via their extracellular loops
Gehne, N., Lamik, A., Lehmann, M., Haseloff, R. F., Andjelkovic(*), A. V.; Blasig, I. E.
Plos One, 12:e0182106
(2017)

Tags: Molecular Cell Physiology (Blasig, I.E.), Cellular Imaging (Wiesner, Puchkov)

Abstract: Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.

Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Muschiol(*), S., Erlendsson(*), S., Aschtgen(*), M. S., Oliveira(*), V., Schmieder, P., de Lichtenberg(*), C., Teilum(*), K., Boesen(*), T., Akbey(*), Ü.; Henriques-Normark(*), B.
J Biol Chem, 292:14134-14146
(2017)

Tags: Solution NMR (Schmieder)

Abstract: Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions, including attachment, twitching motility, biofilm formation, and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These "competence pili" are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three alpha-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second alpha-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting that the first helix is involved in forming the pilus structure core and that parts of helices two and three are primarily surface-exposed. Taken together, our results provide the first structure of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and corroborate the remarkable structural diversity among type IV pilin proteins.

Sleep & metabolism: The multitasking ability of lateral hypothalamic inhibitory circuitries
Herrera(*), C. G., Ponomarenko, A., Korotkova, T., Burdakov(*), D.; Adamantidis(*), A.
Front Neuroendocrinol, 44:27-34
(2017)

Tags: Behavioral Neurodynamics (Korotkova/Ponomarenko)

Abstract: The anatomical and functional mapping of lateral hypothalamic circuits has been limited by the numerous cell types and complex, yet unclear, connectivity. Recent advances in functional dissection of input-output neurons in the lateral hypothalamus have identified subset of inhibitory cells as crucial modulators of both sleep-wake states and metabolism. Here, we summarize these recent studies and discuss the multi-tasking functions of hypothalamic circuitries in integrating sleep and metabolism in the mammalian brain.

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells
Appelt-Menzel(*), A., Cubukova(*), A., Günther(*), K., Edenhofer(*), F., Piontek(*), J., Krause, G., Stüber(*), T., Walles(*), H., Neuhaus(*), W.; Metzger(*), M.
Stem cell reports, 8:894-906
(2017)

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

Abstract: In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Omega cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.

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
(2017)

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.

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
(2017)

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.

Trictide, a tricellulin-derived peptide to overcome cellular barriers
Cording, J., Arslan, B., Staat, C., Dithmer, S., Krug(*), S. M., Krüger(*), A., Berndt, P., Günther, R., Winkler, L., Blasig, I. E.; Haseloff, R. F.
Annals of the New York Academy of Sciences,
(2017)

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

Abstract: The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis-stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin-1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super-resolution stimulated-emission depletion microscopy revealed a tricellulin-free area at the tricellular region. Cis-interactions (as measured by fluorescence resonance energy transfer) of tricellulin-tricellulin (tTJs), tricellulin-claudin-1, tricellulin-marvelD3, and occludin-occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a beta-sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery.

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,
(2017)

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.

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