FMP Publications

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

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Solid-state NMR Study of the YadA Membrane-Anchor Domain in the Bacterial Outer Membrane
Shahid, S. A., Nagaraj, M., Chauhan(*), N., Franks, T. W., Bardiaux(*), B., Habeck(*), M., Orwick-Rydmark(*), M., Linke(*), D.; van Rossum, B. J.
Angew Chem Int Ed Engl, 54:12602-12606

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using (13) C-(13) C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments.

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

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.

A Multiplexed NMR-Reporter Approach to Measure Cellular Kinase and Phosphatase Activities in Real-Time
Thongwichian, R., Kosten, J., Benary(*), U., Rose, H. M., Stuiver, M., Theillet, F. X., Dose, A., Koch(*), B., Yokoyama(*), H., Schwarzer, D., Wolf(*), J.; Selenko, P.
J. Am. Chem. Soc., 137:6468-6471

Tags: In-Cell NMR (Selenko), Protein Chemistry (Schwarzer)

Abstract: Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their individual activities in a semicontinuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions.

In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11
Varga(*), R. E., Khundadze(*), M., Damme(*), M., Nietzsche(*), S., Hoffmann(*), B., Stauber, T., Koch(*), N., Hennings(*), J. C., Franzka(*), P., Huebner(*), A. K., Kessels(*), M. M., Biskup(*), C., Jentsch, T. J., Qualmann(*), B., Braulke(*), T., Kurth(*), I., Beetz(*), C.; Hübner(*), C. A.
Plos Genet, 11:e1005454

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

Finding new drugs to enhance anion secretion in cystic fibrosis: Toward suitable systems for better drug screening. Report on the pre-conference meeting to the 12th ECFS Basic Science Conference, Albufeira, 25-28 March 2015
Verkman(*), A. S., Edelman(*), A., Amaral(*), M., Mall(*), M. A., Beekman(*), J. M., Meiners, T., Galietta(*), L. J.; Bear(*), C. E.
J Cyst Fibros, 14:700-705

Tags: Chemical Systems Biology (Frank)

A new variant in signal peptide of the human luteinizing hormone receptor (LHCGR) affects receptor biogenesis causing leydig cell hypoplasia
Vezzoli(*), V., Duminuco(*), P., Vottero(*), A., Kleinau(*), G., Schülein, R., Minari(*), R., Bassi(*), I., Bernasconi(*), S., Persani(*), L.; Bonomi(*), M.
Hum Mol Genet, 24:6003-6012

Tags: Protein Trafficking (Schülein)

Abstract: The human luteinizing hormone/chorionic gonadotropin receptor (LHCGR) plays a fundamental role in male and female reproduction. In males, loss-of-function mutations in LHCGR have been associated with distinct degrees of impairment in pre- and postnatal testosterone secretion resulting in a variable phenotypic spectrum, classified as Leydig cell hypoplasia (LCH) type 1 (complete LH resistance and disorder of sex differentiation) and type 2 (partial LH resistance with impaired masculinization and fertility). Here, we report the case of an adolescent who came to the pediatric endocrinologist at the age of 12 years old for micropenis and cryptorchidism. Testis biopsy showed profound LCH and absent germinal line elements (Sertoli-only syndrome). The sequence analysis of the LHCGR gene showed the presence of a compound heterozygosity, being one variation, c.1847C>A p.S616Y, already described in association to Hypergonadotropic Hypogonadism, and the other, c.29 C>T p.L10P, a new identified variant in the putative signal peptide (SP) of LHCGR. Functional and structural studies provide first evidence that LHCGR have a functional and cleavable SP required for receptor biogenesis. Moreover, we demonstrate the pathogenic role of the novel p.L10P allelic variant, which has to be considered a loss-of-function mutation significantly contributing, in compound heterozygosity with p.S616Y, to the LCH type 2 observed in our patient.

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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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