FMP Publications

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

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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

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.

Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca(2)(+) and GTPgammaS
Balletta(*), A., Lorenz, D., Rummel(*), A., Gerhard(*), R., Bigalke(*), H.; Wegner(*), F.
Toxicology, 328:48-56

Tags: Cellular Imaging (Wiesner)

Abstract: Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in human mast cell line-1 (HMC-1) cells, but not cell death or permeabilisation of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca(2+) and GTPgammaS concentrations are capable of inducing exocytosis as indicated by significant membrane capacitance (Cm) increases in HMC-1 cells. In this study, we investigated the direct effects of TcdB upon HMC-1 cell "stimulated" Cm increase, as well as on "constitutive" secretion of hexosaminidase and interleukin-16 (IL-16). Compared to untreated control cells, HMC-1 cells incubated with TcdB for 3-24h exhibited a significant reduction of the mean absolute and relative Cm increase in response to free-Ca(2+) and GTPgammaS suggesting an inhibition of secretory processes by TcdB. In conclusion, the HMC-1 cell line represents a suitable model for the study of direct effects of C. difficile toxins on human mast cell secretory activity.

Theta oscillations regulate the speed of locomotion via a hippocampus to lateral septum pathway
Bender, F., Gorbati, M., Cadavieco, M. C., Denisova, N., Gao, X., Holman, C., Korotkova, T.; Ponomarenko, A.
Nat Commun, 6:8521

Tags: Behavioral Neurodynamics (Korotkova/Ponomarenko)

Abstract: Hippocampal theta oscillations support encoding of an animal's position during spatial navigation, yet longstanding questions about their impact on locomotion remain unanswered. Combining optogenetic control of hippocampal theta oscillations with electrophysiological recordings in mice, we show that hippocampal theta oscillations regulate locomotion. In particular, we demonstrate that their regularity underlies more stable and slower running speeds during exploration. More regular theta oscillations are accompanied by more regular theta-rhythmic spiking output of pyramidal cells. Theta oscillations are coordinated between the hippocampus and its main subcortical output, the lateral septum (LS). Chemo- or optogenetic inhibition of this pathway reveals its necessity for the hippocampal regulation of running speed. Moreover, theta-rhythmic stimulation of LS projections to the lateral hypothalamus replicates the reduction of running speed induced by more regular hippocampal theta oscillations. These results suggest that changes in hippocampal theta synchronization are translated into rapid adjustment of running speed via the LS.

Gas-Phase Rearrangement in Lysine Phosphorylated Peptides During Electron-Transfer Dissociation Tandem Mass Spectrometry
Bertran-Vicente, J., Schümann, M., Hackenberger, C. P.; Krause, E.
Anal Chem, 87:6990-6994

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

Abstract: Tandem mass spectrometry (MS/MS) strategies coupled with collision-induced dissociation (CID) or radical-driven fragmentation techniques such as electron-capture dissociation (ECD) or electron-transfer dissociation (ETD) have been successfully used for comprehensive phosphoproteome analysis. However, the unambiguous characterization of the phosphorylation site remains a significant challenge due to phosphate-related neutral losses and gas-phase rearrangements, which have been observed during CID. In particular, for the analysis of labile N-phosphorylated peptides, ECD and ETD are emerging as a complementary method. In contrast to CID, the phosphorylation site of histidine, arginine, and lysine phosphorylated peptides can be characterized by ETD. Here, we present a study on the application of ETD for analysis of phospholysine (pLys) peptides. We show that, depending on the charge state of the precursor ion as well as the presence of basic amino acid side chains, phosphate transfer reactions during the ETD process can be observed leading to ambiguous fragment ion spectra. Basically, pLys is stable under ETD conditions allowing an unambiguous assignment of the site of phosphorylation, but some factors/parameters have to be considered to avoid gas-phase rearrangement which would lead to false positive results in phosphoproteomic studies.

Direct access to site-specifically phosphorylated-lysine peptides from a solid-support
Bertran-Vicente, J., Schümann, M., Schmieder, P., Krause, E.; Hackenberger, C. P. R.
Organic & Biomolecular Chemistry, 13:6839-6843

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

Abstract: Phosphorylation is a key process for changing the activity and function of proteins. The impact of phospho-serine (pSer), -threonine (pThr) and -tyrosine (pTyr) is certainly understood for some proteins. Recently, peptides and proteins containing N-phosphorylated amino acids such as phosphoarginine (pArg), phosphohistidine (pHis) and phospholysine (pLys) have gained interest because of their different chemical properties and stability profiles. Due to its high intrinsic lability, pLys is the least studied within this latter group. In order to gain insight into the biological role of pLys, chemical and analytical tools, which are compatible with the labile P(vO)-N bond, are highly sought-after. We recently reported an in-solution synthetic approach to incorporate pLys residues in a site-specific manner into peptides by taking advantage of the chemoselectivity of the Staudinger-phosphite reaction. While the in-solution approach allows us to circumvent the critical TFA cleavage, it still requires several transformations and purification steps to finally deliver pLys peptides. Here we report the synthesis of site-specific pLys peptides directly from a solid support by using a base labile resin. This straightforward and highly efficient approach facilitates the synthesis of various site-specific pLys-containing peptides and lays the groundwork for future studies about this elusive protein modification.

Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic profiling of protein lipidation during vertebrate development
Broncel(*), M., Serwa(*), R. A., Ciepla(*), P., Krause, E., Dallman(*), M. J., Magee(*), A. I.; Tate(*), E. W.
Angew Chem Int Ed Engl, 54:5948-5951

Tags: Mass Spectrometry (Krause, E.)

Abstract: Novel multifunctional reagents were applied in combination with a lipid probe for affinity enrichment of myristoylated proteins and direct detection of lipid-modified tryptic peptides by mass spectrometry. This method enables high-confidence identification of the myristoylated proteome on an unprecedented scale in cell culture, and allowed the first quantitative analysis of dynamic changes in protein lipidation during vertebrate embryonic development.

Myristoylation profiling in human cells and zebrafish
Broncel(*), M., Serwa(*), R. A., Ciepla(*), P., Krause, E., Dallman(*), M. J., Magee(*), A. I.; Tate(*), E. W.
Data Brief, 4:379-383

Tags: Mass Spectrometry (Krause, E.)

Abstract: Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaino et al., 2014 Nat. Biotechnol., 32, 223-6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development by Broncel et al., Angew. Chem. Int. Ed.

Light-Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-trans and 13-cis Retinal Isomers
Bruun(*), S., Stöppler, D., Keidel(*), A., Kuhlmann(*), U., Luck(*), M., Diehl, A., Geiger, M. A., Woodmansee(*), D., Trauner(*), D., Hegemann(*), P., Oschkinat, H., Hildebrandt(*), P.; Stehfes(*)t, K.
Biochemistry, 54:5389-5400

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named "light adaptation". The main objective of this spectroscopic study was to elucidate the molecular processes associated with light-dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all-trans configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all-trans,15-anti and 13-cis,15-syn configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13) horizontal lineC(14) and C(15) horizontal lineN bonds. Both isomers of this DAapp state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13) horizontal lineC(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DAapp) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-trans isomer than for the 13-cis isomer, possibly because of different chromophore-water interactions. Our data demonstrating two complementary photocycles of the DAapp isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light-dark adaptation, as suggested previously by electrical measurements.

Simian hemorrhagic fever virus cell entry is dependent on CD163 and uses a clathrin-mediated endocytosis-like pathway
Cai(*), Y., Postnikova(*), E. N., Bernbaum(*), J. G., Yu(*), S. Q., Mazur(*), S., Deiuliis(*), N. M., Radoshitzky(*), S. R., Lackemeyer(*), M. G., McCluskey(*), A., Robinson(*), P. J., Haucke, V., Wahl-Jensen(*), V., Bailey(*), A. L., Lauck(*), M., Friedrich(*), T. C., O'Connor(*), D. H., Goldberg(*), T. L., Jahrling(*), P. B.; Kuhn(*), J. H.
J Virol, 89:844-856

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: UNLABELLED: Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-beta-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, alpha-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component. IMPORTANCE: Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa virus. However, little is known about SHFV's ecology and molecular biology and the mechanism by which it causes disease. The results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low-pH-dependent, actin-independent endocytosis, likely with the help of a cellular surface protein.

Perspectives for sensitivity enhancement in proton-detected solid-state NMR of highly deuterated proteins by preserving water magnetization
Chevelkov, V., Xiang(*), S. Q., Giller(*), K., Becker(*), S., Lange, A.; Reif(*), B.
J. Biomol. NMR, 61:151-160

Tags: Molecular Biophysics (Lange, A.)

Abstract: In this work, we show how the water flip-back approach that is widely employed in solution-state NMR can be adapted to proton-detected MAS solid-state NMR of highly deuterated proteins. The scheme allows to enhance the sensitivity of the experiment by decreasing the recovery time of the proton longitudinal magnetization. The method relies on polarization transfer from non-saturated water to the protein during the inter-scan delay.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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13125 Berlin, Germany
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