FMP Publications

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

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Renal ACE2 expression and activity is unaltered during established hypertension in adult SHRSP and TGR(mREN2)27
Kamilic(*), J., Hamming(*), I., Kreutz(*), R., Bolbrinker(*), J., Siems, W. E., Nassar(*), I., Sluimer(*), J. C., Walther(*), T., Navis(*), G. J.; van Goor(*), H.
Hypertens Res, 33:123-128

Tags: Biochemical Neurobiology (Siems)

Abstract: Differential renal expression of a homolog of the angiotensin-converting enzyme (ACE), that is, ACE2, has been implicated as a genetic basis of polygenetic hypertension in the stroke-prone spontaneously hypertensive rat model. However, data on the role of ACE2 in hypertension are still inconclusive. Therefore, we analyzed kidney ACE2 mRNA, ACE2 protein and ACE2 enzyme activities in the adult polygenetic stroke-prone spontaneously hypertensive rat (SHRSP) and the monogenetic TGR(mREN2)27 rat models, in comparison with their normotensive reference strains, Wistar-Kyoto (WKY) and Spraque-Dawley (SD) rats, respectively. Kidney ACE2 mRNA was studied using quantitative real-time reverse transcriptase-PCR (RT-PCR) in cortex and medulla, whereas protein expression was scored semiquantitatively in detail in different renal structures using immunohistochemistry. Furthermore, total renal tissue ACE2 activity was measured using a fluorimetric assay that was specified by the ACE2 inhibitor DX600. In SHRSP and homozygous TGR(mREN2)27 rats with established hypertension, kidney ACE2 mRNA, protein and tissue ACE2 activities were not different from their respective WKY and SD reference strain, respectively. In addition, when we looked at renal localization, we found ACE2 protein to be predominantly present in glomeruli and endothelium with weak staining in distal and negative staining in proximal tubuli. Thus, our data challenge previous work that implicates ACE2 as a candidate gene for hypertension in SHRSP by reporting a significant reduction of ACE2 in the kidneys of SHRSP. Taken together, renal ACE2 is not altered in the SHRSP and TGR(mREN2)27 genetic rat models with established hypertension. Hypertension Research (2010) 33, 123-128; doi: 10.1038/hr.2009.191; published online 20 November 2009

Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor
Kleinau, G., Haas, A. K., Neumann(*), S., Worth, C. L., Hoyer, I., Furkert, J., Rutz, C., Gershengorn(*), M. C., Schülein, R.; Krause, G.
Faseb j, 24:2347-2354

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

Abstract: The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept
Lisurek, M., Rupp, B., Wichard, J., Neuenschwander, M., von Kries, J. P., Frank(*), R., Rademann, J.; Kühne, R.
Mol Divers, 14:401-408

Tags: Screening Unit (von Kries), Medicinal Chemistry (Rademann), Computational Chemistry/Drug Design (Kühne)

Abstract: Success in small molecule screening relies heavily on the preselection of compounds. Here, we present a strategy for the enrichment of chemical libraries with potentially bioactive compounds integrating the collected knowledge of medicinal chemistry. Employing a genetic algorithm, substructures typically occurring in bioactive compounds were identified using the World Drug Index. Availability of compounds containing the selected substructures was analysed in vendor libraries, and the substructure-specific sublibraries were assembled. Compounds containing reactive, undesired functional groups were omitted. Using a diversity filter for both physico-chemical properties and the substructure composition, the compounds of all the sublibraries were ranked. Accordingly, a screening collection of 16,671 compounds was selected. Diversity and chemical space coverage of the collection indicate that it is highly diverse and well-placed in the chemical space spanned by bioactive compounds. Furthermore, secondary assay-validated hits presented in this study show the practical relevance of our library design strategy.

The late endosomal ClC-6 mediates proton/chloride countertransport in heterologous plasma membrane expression
Neagoe, I., Stauber, T., Fidzinski, P., Bergsdorf, E. Y.; Jentsch, T. J.
J Biol Chem, 285:21689-21697

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: Members of the CLC protein family of Cl(-) channels and transporters display the remarkable ability to function as either chloride channels or Cl(-)/H(+) antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl(-)/H(+) exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H(+) transport was abolished by mutations in either the "gating" or "proton" glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl(-) > I(-) conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the "anion-coordinating" tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO(3)(-) conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl(-)/H(+) antiporter.

Reciprocal regulation of aquaporin-2 abundance and degradation by protein kinase A and p38-MAP kinase
Nedvetsky, P. I., Tabor, V., Tamma(*), G., Beulshausen, S., Skroblin, P., Kirschner, A., Mutig(*), K., Boltzen, M., Petrucci, O., Vossenkamper, A., Wiesner, B., Bachmann(*), S., Rosenthal(*), W.; Klussmann, E.
Journal of the American Society of Nephrology : JASN, 21:1645-1656

Tags: Anchored Signalling (Klussmann), Cellular Imaging (Wiesner)

Abstract: Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK-dependent pathway.

Azides Derived from Colchicine and their Use in Library Synthesis: a Practical Entry to New Bioactive Derivatives of an Old Natural Drug
Nicolaus(*), N., Zapke, J., Riesterer(*), P., Neudörfl, J. M., Prokop(*), A., Oschkinat, H.; Schmalz(*), H. G.
Chemmedchem, 5:661-665

Tags: Protein Structure (Oschkinat)

No evidence for a role of CLCN2 variants in idiopathic generalized epilepsy
Niemeyer(*), M. I., Cid(*), L. P., Sepulveda(*), F. V., Blanz(*), J., Auberson(*), M.; Jentsch, T. J.
Nat Genet, 42:3-3

Tags: Physiology and Pathology of Ion Transport (Jentsch

Role of ClC-5 in Renal Endocytosis Is Unique among ClC Exchangers and Does Not Require PY-motif-dependent Ubiquitylation
Rickheit, G., Wartosch, L., Schaffer(*), S., Stobrawa(*), S. M., Novarino, G., Weinert, S.; Jentsch, T. J.
Journal of Biological Chemistry, 285:17595-17603

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: Inactivation of the mainly endosomal 2Cl(-)/H(+)-exchanger ClC-5 severely impairs endocytosis in renal proximal tubules and underlies the human kidney stone disorder Dent's disease. In heterologous expression systems, interaction of the E3 ubiquitin ligases WWP2 and Nedd4-2 with a "PY-motif" in the cytoplasmic C terminus of ClC-5 stimulates its internalization from the plasma membrane and may influence receptor-mediated endocytosis. We asked whether this interaction is relevant in vivo and generated mice in which the PY-motif was destroyed by a point mutation. Unlike ClC-5 knock-out mice, these knock-in mice displayed neither low molecular weight proteinuria nor hyperphosphaturia, and both receptor-mediated and fluid-phase endocytosis were normal. The abundances and localizations of the endocytic receptor megalin and of the Na(+)-coupled phosphate transporter NaPi-2a (Npt2) were not changed, either. To explore whether the discrepancy in results from heterologous expression studies might be due to heteromerization of ClC-5 with ClC-3 or ClC-4 in vivo, we studied knock-in mice additionally deleted for those related transporters. Disruption of neither ClC-3 nor ClC-4 led to proteinuria or impaired proximal tubular endocytosis by itself, nor in combination with the PY-mutant of ClC-5. Endocytosis of cells lacking ClC-5 was not impaired further when ClC-3 or ClC-4 was additionally deleted. We conclude that ClC-5 is unique among CLC proteins in being crucial for proximal tubular endocytosis and that PY-motif-dependent ubiquitylation of ClC-5 is dispensable for this role.

Endosomal chloride-proton exchange rather than chloride conductance is crucial for renal endocytosis
Novarino, G., Weinert, S., Rickheit, G.; Jentsch, T. J.
Science, 328:1398-1401

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: Loss of the endosomal anion transport protein ClC-5 impairs renal endocytosis and underlies human Dent's disease. ClC-5 is thought to promote endocytosis by facilitating endosomal acidification through the neutralization of proton pump currents. However, ClC-5 is a 2 chloride (Cl-)/proton (H+) exchanger rather than a Cl- channel. We generated mice that carry the uncoupling E211A (unc) mutation that converts ClC-5 into a pure Cl- conductor. Adenosine triphosphate (ATP)-dependent acidification of renal endosomes was reduced in mice in which ClC-5 was knocked out, but normal in Clcn5(unc) mice. However, their proximal tubular endocytosis was also impaired. Thus, endosomal chloride concentration, which is raised by ClC-5 in exchange for protons accumulated by the H+-ATPase, may play a role in endocytosis.

Amyloid beta 42 peptide (Abeta42)-lowering compounds directly bind to Abeta and interfere with amyloid precursor protein (APP) transmembrane dimerization
Richter(*), L., Munter(*), L. M., Ness(*), J., Hildebrand(*), P. W., Dasari, M., Unterreitmeier(*), S., Bulic(*), B., Beyermann, M., Gust(*), R., Reif, B., Weggen(*), S., Langosch(*), D.; Multhaup(*), G.
Proc Natl Acad Sci U S A, 107:14597-14602

Tags: Solid-State NMR Spectroscopy (Reif), Peptide Synthesis (Beyermann)

Abstract: Following ectodomain shedding by beta-secretase, successive proteolytic cleavages within the transmembrane sequence (TMS) of the amyloid precursor protein (APP) catalyzed by gamma-secretase result in the release of amyloid-beta (Abeta) peptides of variable length. Abeta peptides with 42 amino acids appear to be the key pathogenic species in Alzheimer's disease, as they are believed to initiate neuronal degeneration. Sulindac sulfide, which is known as a potent gamma-secretase modulator (GSM), selectively reduces Abeta42 production in favor of shorter Abeta species, such as Abeta38. By studying APP-TMS dimerization we previously showed that an attenuated interaction similarly decreased Abeta42 levels and concomitantly increased Abeta38 levels. However, the precise molecular mechanism by which GSMs modulate Abeta production is still unclear. In this study, using a reporter gene-based dimerization assay, we found that APP-TMS dimers are destabilized by sulindac sulfide and related Abeta42-lowering compounds in a concentration-dependent manner. By surface plasmon resonance analysis and NMR spectroscopy, we show that sulindac sulfide and novel sulindac-derived compounds directly bind to the Abeta sequence. Strikingly, the attenuated APP-TMS interaction by GSMs correlated strongly with Abeta42-lowering activity and binding strength to the Abeta sequence. Molecular docking analyses suggest that certain GSMs bind to the GxxxG dimerization motif in the APP-TMS. We conclude that these GSMs decrease Abeta42 levels by modulating APP-TMS interactions. This effect specifically emphasizes the importance of the dimeric APP-TMS as a promising drug target in Alzheimer's disease.

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