FMP Publications

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

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References
Synthesis and thermodynamic characterization of small cyclic antimicrobial arginine and tryptophan-rich peptides with selectivity for Gram-negative bacteria
Bagheri, M.
Methods Mol Biol, 618:87-109
(2010)

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

Abstract: One promising strategy to combat the proliferation of bacteria resistance toward current antibiotics is the development of peptide-based drug. Among these compounds is a group of small cyclic peptides rich in arginine (Arg) and tryptophan (Trp) residues with selective toxicity toward Gram-negative bacteria. The small size of these peptides with improved toxicity toward Gram-negative bacteria makes them an interesting candidate to understand the forces responsible for their selectivity and paves the way to develop new therapeutics with potent activity toward multi-resistant Gram-negative bacteria. To reach this goal, isothermal titration calorimetry (ITC) is a useful technique which may provide the complete set of thermodynamic parameters of the interaction of peptides with lipid bilayers mimicking the properties of bacterial membranes within a few hours. The purpose of this chapter is to describe the synthesis of this group of small synthetic antimicrobial peptides together with the application of ITC to study their interaction with lipid membranes.

New function for an old enzyme: NEP deficient mice develop late-onset obesity
Becker, M., Siems, W. E., Kluge(*), R., Gembardt(*), F., Schultheiss(*), H. P., Schirner(*), M.; Walther(*), T.
Plos One, 5
(2010)

Tags: Biochemical Neurobiology (Siems)

Abstract: BACKGROUND: According to the World Health Organization (WHO) there is a pandemic of obesity with approximately 300 million people being obese. Typically, human obesity has a polygenetic causation. Neutral endopeptidase (NEP), also known as neprilysin, is considered to be one of the key enzymes in the metabolism of many active peptide hormones. METHODOLOGY/PRINCIPAL FINDINGS: An incidental observation in NEP-deficient mice was a late-onset excessive gain in body weight exclusively from a ubiquitous accumulation of fat tissue. In accord with polygenetic human obesity, mice were characterized by deregulation of lipid metabolism, higher blood glucose levels, with impaired glucose tolerance. The key role of NEP in determining body mass was confirmed by the use of the NEP inhibitor candoxatril in wild-type mice that increased body weight due to increased food intake. This is a peripheral and not a central NEP action on the switch for appetite control, since candoxatril cannot cross the blood-brain barrier. Furthermore, we demonstrated that inhibition of NEP in mice with cachexia delayed rapid body weight loss. Thus, lack in NEP activity, genetically or pharmacologically, leads to a gain in body fat. CONCLUSIONS/SIGNIFICANCE: In the present study, we have identified NEP to be a crucial player in the development of obesity. NEP-deficient mice start to become obese under a normocaloric diet in an age of 6-7 months and thus are an ideal model for the typical human late-onset obesity. Therefore, the described obesity model is an ideal tool for research on development, molecular mechanisms, diagnosis, and therapy of the pandemic obesity.

A new phenotype of nongoitrous and nonautoimmune hyperthyroidism caused by a heterozygous thyrotropin receptor mutation in transmembrane helix 6
Winkler(*), F., Kleinau, G., Tarnow(*), P., Rediger(*), A., Grohmann(*), L., Gaetjens(*), I., Krause, G., L'Allemand(*), D., Grüters(*), A., Krude(*), H.; Biebermann(*), H.
The Journal of clinical endocrinology and metabolism, 95:3605-3610
(2010)

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

Abstract: CONTEXT: Activating mutations in the TSHR gene were found in patients suffering from nonautoimmune hyperthyroidism. In the past, it was assumed that thyroid hyperplasia is due to constitutive activation of the Gs/adenylyl cyclase signaling pathway; however, the physiological role of the Gq/11 pathway in this context remains unclear. OBJECTIVE: In this study, we investigated molecular details of the TSHR in a patient with nonautoimmune and nongoitrous hyperthyroidism. RESULTS: We detected a heterozygous mutation in exon 10 of the TSHR gene leading to an exchange of a cysteine residue for tryptophan at amino acid position 636 in transmembrane helix 6. Functional characterization of the mutant receptor revealed a slight reduction of the cell surface expression and TSH induced cAMP accumulation compared to the wild type. Additional observations included a constitutive activation of the Gs-mediated signaling pathway and a simultaneous nearly complete loss-of-function for the Gq/11 pathway after bovine TSH stimulation. Studies on TSHR models suggest significant changes of important amino acid interactions and the overall helix arrangement caused by mutation C636W. CONCLUSION: We report a patient in whom a TSHR mutation leads to nonautoimmune hyperthyroidism due to a mutation that constitutively activates the Gs signaling pathway but additionally completely inhibits the Gq/11 pathway. The absence of goiter in the patient suggests that the Gq/11 pathway is related to thyroid growth and that different signaling pathways are mediated and regulated by TSH. These functional data could be confirmed by reproducible findings of two siblings with a constitutive activation for both pathways.

Genetic defects, thyroid growth and malfunctions of the TSHR in pediatric patients
Biebermann(*), H., Winkler(*), F.; Kleinau, G.
Front Biosci-Landmrk, 15:913-933
(2010)

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

Abstract: Naturally occurring activating and inactivating mutations of the thyrotropin receptor (TSHR) were found as a molecular cause of diseases in patients suffering from non-autoimmune hyperthyroidism and syndromes of thyrotropin resistance, respectively. These mutations are mostly functionally characterized in vitro and therefore, they represent an excellent tool to study structure-function relationships of this G-protein-coupled receptor. In this review, we summarize published germline mutations of the TSHR with focus on 1) the phenotype of (pediatric) patients, 2) potential genotype/phenotype correlations, 3) structural implications for receptor activation and inactivation, 4) the impact on thyroid growth, and 5) finally on aspects of TSHR dimerization. In conclusion, this comprehensive analysis of medical and biological data opens an avenue to understand genetic defects and malfunctions of the TSHR in molecular detail and in their entirety. This knowledge is important to refine our insights in non-autoimmune diseases caused by defects of the TSHR gene and it might help to develop pharmacological means for compensation of uncontrolled thyroid growth.

Alpha-helical transmembrane peptides: a "divide and conquer" approach to membrane proteins
Bordag, N.; Keller, S.
Chem Phys Lipids, 163:1-26
(2010)

Tags: Biophysics of Membrane Proteins (Keller)

Abstract: Alpha-helical membrane proteins fulfill many vital roles in all living cells and constitute the majority of drug targets. However, their relevance is in no way paralleled by our current understanding of their structures and functions. This is because membrane proteins present a number of experimental obstacles that are difficult to surmount by classical methods developed for water-soluble proteins. Moreover, membrane proteins are not only challenging on their very own but, when embedded in a biological membrane, also reside in an outstandingly complex milieu. These difficulties have fostered a "divide and conquer" approach, in which a membrane protein is dissected into shorter and easier-to-handle transmembrane (TM) peptides. Under suitable conditions, such peptides fold independently and retain many of the properties displayed in the context of the full-length parent protein. This contribution reviews some of the most notable insights into alpha-helical membrane proteins gleaned from experiments on protein-derived TM peptides. We recapitulate some peculiar properties of lipid bilayers that render them such a complex and unique environment and discuss generic features pertaining to hydrophobic peptides derived from alpha-helical membrane proteins. The main part of the review is devoted to a critical discussion of particularly interesting examples of TM peptides studied in membrane-mimetic systems of increasing complexity: isotropic solvents, detergent micelles, lipid bilayers, and biological membranes. The unifying theme is to explore to what extent TM peptides in combination with different membrane-mimetic systems can aid in advancing our knowledge and comprehension of alpha-helical membrane proteins as well as in developing new pharmacological tools.

Protein folding in membranes
Fiedler, S., Broecker, J.; Keller, S.
Cellular and molecular life sciences : CMLS, 67:1779-1798
(2010)

Tags: Biophysics of Membrane Proteins (Keller)

Abstract: Separation of cells and organelles by bilayer membranes is a fundamental principle of life. Cellular membranes contain a baffling variety of proteins, which fulfil vital functions as receptors and signal transducers, channels and transporters, motors and anchors. The vast majority of membrane-bound proteins contain bundles of alpha-helical transmembrane domains. Understanding how these proteins adopt their native, biologically active structures in the complex milieu of a membrane is therefore a major challenge in today's life sciences. Here, we review recent progress in the folding, unfolding and refolding of alpha-helical membrane proteins and compare the molecular interactions that stabilise proteins in lipid bilayers. We also provide a critical discussion of a detergent denaturation assay that is increasingly used to determine membrane-protein stability but is not devoid of conceptual difficulties.

Membrane translocation assayed by fluorescence spectroscopy
Broecker, J.; Keller, S.
Methods Mol Biol, 606:271-289
(2010)

Tags: Biophysics of Membrane Proteins (Keller)

Abstract: Assessing the ability of biomolecules or drugs to overcome lipid membranes in a receptor-independent way is of great importance in both basic research and applications involving the use of liposomes. A combination of uptake, release, and dilution experiments performed by steady-state fluorescence spectroscopy provides a powerful, straightforward, and inexpensive way of monitoring membrane translocation of fluorescent compounds. This is particularly true for peptides and proteins carrying intrinsic tryptophan residues, which eliminates the need for attaching extrinsic labeling moieties to the compound of interest. The approach encompasses three different kinds of fluorescence titrations and some simple calculations that can be carried out in a spreadsheet program. A complete set of experiments and data analyses can typically be completed within two days.

Tricellulin forms homomeric and heteromeric tight junctional complexes
Westphal(*), J. K., Dörfel(*), M. J., Krug(*), S. M., Cording, J. D., Piontek, J., Blasig, I. E., Tauber(*), R., Fromm(*), M.; Huber(*), O.
Cellular and Molecular Life Sciences, 67:2057-2068
(2010)

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

Abstract: Sealing of the paracellular cleft by tight junctions is of central importance for epithelia and endothelia to function as efficient barriers between the extracellular space and the inner milieu. Occludin and claudins represent the major tight junction components involved in establishing this barrier function. A special situation emerges at sites where three cells join together. Tricellulin, a recently identified tetraspan protein concentrated at tricellular contacts, was reported to organize tricellular as well as bicellular tight junctions. Here we show that in MDCK cells, the tricellulin C-terminus is important for the basolateral translocation of tricellulin, whereas the N-terminal domain appears to be involved in directing tricellulin to tricellular contacts. In this respect, identification of homomeric tricellulin-tricellulin and of heteromeric tricellulin-occludin complexes extends a previously published model and suggests that tricellulin and occludin are transported together to the edges of elongating bicellular junctions and get separated when tricellular contacts are formed.

On the interaction of Clostridium perfringens enterotoxin with claudins
Veshnyakova, A., Protze, J., Rossa, J., Blasig, I. E., Krause, G.; Piontek, J.
Toxins (Basel), 2:1336-1356
(2010)

Tags: Molecular Cell Physiology (Blasig, I.E.), Structural Bioinformatics and Protein Design (Krause, G)

Abstract: Clostridium perfringens causes one of the most common foodborne illnesses, which is largely mediated by the Clostridium perfringens enterotoxin (CPE). The toxin consists of two functional domains. The N-terminal region mediates the cytotoxic effect through pore formation in the plasma membrane of the mammalian host cell. The C-terminal region (cCPE) binds to the second extracellular loop of a subset of claudins. Claudin-3 and claudin-4 have been shown to be receptors for CPE with very high affinity. The toxin binds with weak affinity to claudin-1 and -2 but contribution of these weak binding claudins to CPE-mediated disease is questionable. cCPE is not cytotoxic, however, it is a potent modulator of tight junctions. This review describes recent progress in the molecular characterization of the cCPE-claudin interaction using mutagenesis, in vitro binding assays and permeation studies. The results promote the development of recombinant cCPE-proteins and CPE-based peptidomimetics to modulate tight junctions for improved drug delivery or to treat tumors overexpressing claudins.

Participation of the second extracellular loop of claudin-5 in paracellular tightening against ions, small and large molecules
Piehl, C., Piontek, J., Cording, J., Wolburg(*), H.; Blasig, I. E.
Cellular and molecular life sciences : CMLS, 67:2131-2140
(2010)

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

Abstract: Tight junctions control paracellular permeability. Here, we analyzed the impact of residues in the second extracellular loop (ECL2) of mouse claudin-5 on paracellular permeability. Stable expression of claudin-5(wild type) in MDCK-II cells-but not that of mutants R145A, Y148A, Y158A or E159Q-increased transepithelial electrical resistance and decreased fluorescein permeation. Expression of claudin-5(Y148A), (Y158A) or (E159Q) enhanced permeability of FITC-dextran(10 kDa), which was unchanged in cells expressing claudin-5(wild type) or claudin-5(R145A). In contrast, targeting to tight junctions, strand morphology and tight junction assembly were unchanged. It is concluded that R145 is unessential for trans-interaction of claudin-5, but necessary for tightening against small solutes and ions. The highly conserved residues Y148, Y158 and E159 in ECL2 of claudin-5 contribute to homo- and/or heterophilic trans-interaction between classic claudins and thereby tighten the paracellular space against ions, small and large molecules. These results provide novel insights into the molecular function of tight junctions.

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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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