FMP Publications

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

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References
Occludin controls HIV transcription in brain pericytes via regulation of SIRT-1 activation
Castro(*), V., Bertrand(*), L., Luethen(*), M., Dabrowski, S., Lombardi(*), J., Morgan(*), L., Sharova(*), N., Stevenson(*), M., Blasig, I. E.; Toborek(*), M.
FASEB J, 30:1234-1246
(2016)

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

Abstract: HIV invades the brain early after infection; however, its interactions with the cells of the blood-brain barrier (BBB) remain poorly understood. Our goal was to evaluate the role of occludin, one of the tight junction proteins that regulate BBB functions in HIV infection of BBB pericytes. We provide evidence that occludin levels largely control the metabolic responses of human pericytes to HIV. Occludin in BBB pericytes decreased by 10% during the first 48 h after HIV infection, correlating with increased nuclear translocation of the gene repressor C-terminal-binding protein (CtBP)-1 and NFkappaB-p65 activation. These changes were associated with decreased expression and activation of the class III histone deacetylase sirtuin (SIRT)-1. Occludin levels recovered 96 h after infection, restoring SIRT-1 and reducing HIV transcription to 20% of its highest values. We characterized occludin biochemically as a novel NADH oxidase that controls the expression and activation of SIRT-1. The inverse correlation between occludin and HIV transcription was then replicated in human primary macrophages and differentiated monocytic U937 cells, in which occludin silencing resulted in 75 and 250% increased viral transcription, respectively. Our work shows that occludin has previously unsuspected metabolic properties and is a target of HIV infection, opening the possibility of designing novel pharmacological approaches to control HIV transcription.

Tubular Epithelial NF-kappaB Activity Regulates Ischemic AKI
Marko(*), L., Vigolo(*), E., Hinze(*), C., Park(*), J. K., Roel(*), G., Balogh(*), A., Choi(*), M., Wübken(*), A., Cording, J., Blasig, I. E., Luft(*), F. C., Scheidereit(*), C., Schmidt-Ott(*), K. M., Schmidt-Ullrich(*), R.; Müller(*), D. N.
Journal of the American Society of Nephrology : JASN, 27:2658-2669
(2016)

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

Abstract: NF-kappaB is a key regulator of innate and adaptive immunity and is implicated in the pathogenesis of AKI. The cell type-specific functions of NF-kappaB in the kidney are unknown; however, the pathway serves distinct functions in immune and tissue parenchymal cells. We analyzed tubular epithelial-specific NF-kappaB signaling in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. NF-kappaB reporter activity and nuclear localization of phosphorylated NF-kappaB subunit p65 analyses in mice revealed that IRI induced widespread NF-kappaB activation in renal tubular epithelia and in interstitial cells that peaked 2-3 days after injury. To genetically antagonize tubular epithelial NF-kappaB activity, we generated mice expressing the human NF-kappaB super-repressor IkappaBalphaDeltaN in renal proximal, distal, and collecting duct epithelial cells. Compared with control mice, these mice exhibited improved renal function, reduced tubular apoptosis, and attenuated neutrophil and macrophage infiltration after IRI-induced AKI. Furthermore, tubular NF-kappaB-dependent gene expression profiles revealed temporally distinct functional gene clusters for apoptosis, chemotaxis, and morphogenesis. Primary proximal tubular cells isolated from IkappaBalphaDeltaN-expressing mice and exposed to hypoxia-mimetic agent cobalt chloride exhibited less apoptosis and expressed lower levels of chemokines than cells from control mice did. Our results indicate that postischemic NF-kappaB activation in renal tubular epithelia aggravates tubular injury and exacerbates a maladaptive inflammatory response.

C-type natriuretic peptide and natriuretic peptide receptor B signalling inhibits cardiac sympathetic neurotransmission and autonomic function
Buttgereit(*), J., Shanks(*), J., Li(*), D., Hao(*), G., Athwal(*), A., Langenickel(*), T. H., Wright(*), H., da Costa Goncalves, A. C., Monti(*), J., Plehm(*), R., Popova(*), E., Qadri(*), F., Lapidus(*), I., Ryan(*), B., Ozcelik(*), C., Paterson(*), D. J., Bader(*), M.; Herring(*), N.
Cardiovasc Res, 112:637-644
(2016)

Tags: Anchored Signaling (Klussmann)

Abstract: AIMS: B-type natriuretic peptide (BNP)-natriuretic peptide receptor A (NPR-A) receptor signalling inhibits cardiac sympathetic neurotransmission, although C-type natriuretic peptide (CNP) is the predominant neuropeptide of the nervous system with expression in the heart and vasculature. We hypothesized that CNP acts similarly to BNP, and that transgenic rats (TGRs) with neuron-specific overexpression of a dominant negative NPR-B receptor would develop heightened sympathetic drive. METHODS AND RESULTS: Mean arterial pressure and heart rate (HR) were significantly (P < 0.05) elevated in freely moving TGRs (n = 9) compared with Sprague Dawley (SD) controls (n = 10). TGR had impaired left ventricular systolic function and spectral analysis of HR variability suggested a shift towards sympathoexcitation. Immunohistochemistry demonstrated co-staining of NPR-B with tyrosine hydroxylase in stellate ganglia neurons. In SD rats, CNP (250 nM, n = 8) significantly reduced the tachycardia during right stellate ganglion stimulation (1-7 Hz) in vitro whereas the response to bath-applied norepinephrine (NE, 1 muM, n = 6) remained intact. CNP (250 nM, n = 8) significantly reduced the release of 3H-NE in isolated atria and this was prevented by the NPR-B antagonist P19 (250 nM, n = 6). The neuronal Ca2+ current (n = 6) and intracellular Ca2+ transient (n = 9, using fura-2AM) were also reduced by CNP in isolated stellate neurons. Treatment of the TGR (n = 9) with the sympatholytic clonidine (125 microg/kg per day) significantly reduced mean arterial pressure and HR to levels observed in the SD (n = 9). CONCLUSION: C-type natriuretic peptide reduces cardiac sympathetic neurotransmission via a reduction in neuronal calcium signalling and NE release through the NPR-B receptor. Situations impairing CNP-NPR-B signalling lead to hypertension, tachycardia, and impaired left ventricular systolic function secondary to sympatho-excitation.

Substrate Hunting for the Myxobacterial CYP260A1 Revealed New 1alpha-Hydroxylated Products from C-19 Steroids
Khatri(*), Y., Ringle(*), M., Lisurek, M., von Kries, J. P., Zapp(*), J.; Bernhardt(*), R.
Chembiochem, 17:90-101
(2016)

Tags: Screening Unit (von Kries), Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: Cytochromes P450 catalyze a variety of synthetically useful reactions. However, it is difficult to determine their physiological or artificial functions when a plethora of orphan P450 systems are present in a genome. CYP260A1 from Sorangium cellulosum So ce56 is a new member among the 21 available P450s in the strain. To identify putative substrates for CYP260A1 we used high-throughput screening of a compound library (ca. 17,000 ligands). Structural analogues of the type I hits were searched for biotechnologically relevant compounds, and this led us to select C-19 steroids as potential substrates. We identified efficient surrogate redox partners for CYP260A1, and an Escherichia coli-based whole-cell biocatalyst system was developed to convert testosterone, androstenedione, and their derivatives methyltestosterone and 11-oxoandrostenedione. A detailed (1) H and (13) C NMR characterization of the product(s) from C-19 steroids revealed that CYP260A1 is the very first 1alpha-steroid hydroxylase.

Intracellular repair of oxidation-damaged alpha-synuclein fails to target C-terminal modification sites
Binolfi, A., Limatola, A., Verzini, S., Kosten, J., Theillet, F. X., Rose, H. M., Bekei, B., Stuiver, M., van Rossum, M.; Selenko, P.
Nat Commun, 7:10251
(2016)

Tags: In-Cell NMR (Selenko)

Abstract: Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (alpha-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched alpha-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal alpha-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of alpha-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered alpha-Syn in cells.

Structural disorder of monomeric alpha-synuclein persists in mammalian cells
Theillet, F. X., Binolfi, A., Bekei, B., Martorana(*), A., Rose, H. M., Stuiver, M., Verzini, S., Lorenz, D., van Rossum, M., Goldfarb(*), D.; Selenko, P.
Nature, 530:45-50
(2016)

Tags: In-Cell NMR (Selenko), Cellular Imaging (Wiesner)

Abstract: Intracellular aggregation of the human amyloid protein alpha-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of alpha-synuclein in different mammalian cell types. We show that the disordered nature of monomeric alpha-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, alpha-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-beta component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote alpha-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

Dynamics of the Ligand Binding Domain Layer during AMPA Receptor Activation
Baranovic, J., Chebli, M., Salazar, H., Carbone, A. L., Faelber(*), K., Lau(*), A. Y., Daumke(*), O.; Plested, A. J.
Biophys J, 110:896-911
(2016)

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: Ionotropic glutamate receptors are postsynaptic tetrameric ligand-gated channels whose activity mediates fast excitatory transmission. Glutamate binding to clamshell-shaped ligand binding domains (LBDs) triggers opening of the integral ion channel, but how the four LBDs orchestrate receptor activation is unknown. Here, we present a high-resolution x-ray crystal structure displaying two tetrameric LBD arrangements fully bound to glutamate. Using a series of engineered metal ion trapping mutants, we showed that the more compact of the two assemblies corresponds to an arrangement populated during activation of full-length receptors. State-dependent cross-linking of the mutants identified zinc bridges between the canonical active LBD dimers that formed when the tetramer was either fully or partially bound by glutamate. These bridges also stabilized the resting state, consistent with the recently published full-length apo structure. Our results provide insight into the activation mechanism of glutamate receptors and the complex conformational space that the LBD layer can sample.

How to build the fastest receptor on earth
Baranovic, J.; Plested, A. J. R.
Biol Chem, 397:195-205
(2016)

Tags: (Molecular Neuroscience and Biophysics (Plested)

Abstract: In 2014, a slew of structures of glutamate receptors were published, based on crystallography and electron microscopy. Here we review these insights, integrate them with existing knowledge about receptor function and try to understand how the structures relate to the key property of the AMPA receptor - its speed.

Single-Channel Recording of Glycine Receptors in Human Embryonic Kidney (HEK) Cells
Plested, A. J.; Baranovic, J.
Cold Spring Harb Protoc, 2016:pdb prot091652
(2016)

Tags: Molecular Neuroscience and Biophysics (Plested)

Abstract: This protocol describes how to record the single-channel activity of recombinant homomeric glycine receptors expressed in human embryonic kidney (HEK) cells. Cell-attached recordings readily reveal the large conductance (90 pS) and distinctive clusters of activations at high glycine concentration. This method for obtaining equilibrium recordings can be adapted to any ion channel receptor. The necessary extensions to outside-out patch for nonequilibrium recordings are also described, as are basic analyses of channel properties and activity.

Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies
Gupta(*), R., Lu(*), M., Hou(*), G., Caporini(*), M. A., Rosay(*), M., Maas(*), W., Struppe(*), J., Suiter(*), C., Ahn(*), J., Byeon(*), I. J., Franks, W. T., Orwick-Rydmark, M., Bertarello(*), A., Oschkinat, H., Lesage(*), A., Pintacuda(*), G., Gronenborn(*), A. M.; Polenova(*), T.
J Phys Chem B, 120:329-339
(2016)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

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