@article{ShaoMaPairaetal.2018, author = {Shao, Yi-Ming and Ma, Xiaohua and Paira, Priyankar and Tan, Aaron and Herr, Deron Raymond and Lim, Kah Leong and Ng, Chee Hoe and Venkatesan, Gopalakrishnan and Klotz, Karl-Norbert and Federico, Stephanie and Spalluto, Giampiero and Cheong, Siew Lee and Chen, Yu Zong and Pastorin, Giorgia}, title = {Discovery of indolylpiperazinylpyrimidines with dual-target profiles at adenosine A2A and dopamine D2 receptors for Parkinson's disease treatment}, series = {PLoS ONE}, volume = {13}, journal = {PLoS ONE}, doi = {10.1371/journal.pone.0188212}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-237766}, year = {2018}, abstract = {Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra of the human brain, leading to depletion of dopamine production. Dopamine replacement therapy remains the mainstay for attenuation of PD symptoms. Nonetheless, the potential benefit of current pharmacotherapies is mostly limited by adverse side effects, such as drug-induced dyskinesia, motor fluctuations and psychosis. Non-dopaminergic receptors, such as human A2A adenosine receptors, have emerged as important therapeutic targets in potentiating therapeutic effects and reducing the unwanted side effects. In this study, new chemical entities targeting both human A2A adenosine receptor and dopamine D2 receptor were designed and evaluated. Two computational methods, namely support vector machine (SVM) models and Tanimoto similarity-based clustering analysis, were integrated for the identification of compounds containing indole-piperazine-pyrimidine (IPP) scaffold. Subsequent synthesis and testing resulted in compounds 5 and 6, which acted as human A2A adenosine receptor binders in the radioligand competition assay (Ki = 8.7-11.2 μM) as well as human dopamine D2 receptor binders in the artificial cell membrane assay (EC50 = 22.5-40.2 μM). Moreover, compound 5 showed improvement in movement and mitigation of the loss of dopaminergic neurons in Drosophila models of PD. Furthermore, in vitro toxicity studies on compounds 5 and 6 did not reveal any mutagenicity (up to 100 μM), hepatotoxicity (up to 30 μM) or cardiotoxicity (up to 30 μM).}, language = {en} } @article{NabeebaccusVermaZoccaratoetal.2021, author = {Nabeebaccus, Adam A and Verma, Sharwari and Zoccarato, Anna and Emanuelli, Giulia and Santos, Celio XC. and Streckfuss-B{\"o}meke, Katrin and Shah, Ajay M.}, title = {Cardiomyocyte protein O-GlcNAcylation is regulated by GFAT1 not GFAT2}, series = {Biochemical and Biophysical Research Communications}, volume = {583}, journal = {Biochemical and Biophysical Research Communications}, doi = {10.1016/j.bbrc.2021.10.056}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-371510}, pages = {121-127}, year = {2021}, abstract = {In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart.}, language = {en} } @article{AngayFriedrichPinneckeretal.2018, author = {Angay, Oguzhan and Friedrich, Mike and Pinnecker, J{\"u}rgen and Hintzsche, Henning and Stopper, Helga and Hempel, Klaus and Heinze, Katrin G.}, title = {Image-based modeling and scoring of Howell-Jolly Bodies in human erythrocytes}, series = {Cytometry Part A}, volume = {93}, journal = {Cytometry Part A}, doi = {10.1002/cyto.a.23123}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221140}, pages = {305-313}, year = {2018}, abstract = {The spleen selectively removes cells with intracellular inclusions, for example, detached nuclear fragments in circulating erythrocytes, called Howell-Jolly Bodies (HJBs). With absent or deficient splenic function HJBs appear in the peripheral blood and can be used as a simple and non-invasive risk-indicator for fulminant potentially life-threatening infection after spleenectomy. However, it is still under debate whether counting of the rare HJBs is a reliable measure of splenic function. Investigating HJBs in premature erythrocytes from patients during radioiodine therapy gives about 10 thousand times higher HJB counts than in blood smears. However, we show that there is still the risk of false-positive results by unspecific nuclear remnants in the prepared samples that do not originate from HJBs, but from cell debris residing above or below the cell. Therefore, we present a method to improve accuracy of image-based tests that can be performed even in non-specialized medical institutions. We show how to selectively label HJB-like clusters in human blood samples and how to only count those that are undoubtedly inside the cell. We found a "critical distance" dcrit referring to a relative HJB-Cell distance that true HJBs do not exceed. To rule out false-positive counts we present a simple inside-outside-rule based on dcrit—a robust threshold that can be easily assessed by combining conventional 2D imaging and straight-forward image analysis. Besides data based on fluorescence imaging, simulations of randomly distributed HJB-like objects on realistically modelled cell objects demonstrate the risk and impact of biased counting in conventional analysis. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.}, language = {en} } @article{Gohla2019, author = {Gohla, Antje}, title = {Do metabolic HAD phosphatases moonlight as protein phosphatases?}, series = {BBA - Molecular Cell Research}, volume = {1866}, journal = {BBA - Molecular Cell Research}, doi = {10.1016/j.bbamcr.2018.07.007}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-233168}, pages = {153-166}, year = {2019}, abstract = {Mammalian haloacid dehalogenase (HAD)-type phosphatases have evolved to dephosphorylate a wide range of small metabolites, but can also target macromolecules such as serine/threonine, tyrosine-, and histidine-phosphorylated proteins. To accomplish these tasks, HAD phosphatases are equipped with cap domains that control access to the active site and provide substrate specificity determinants. A number of capped HAD phosphatases impact protein phosphorylation, although structural data are consistent with small metabolite substrates rather than protein substrates. This review discusses the structures, functions and disease implications of the three closely related, capped HAD phosphatases pyridoxal phosphatase (PDXP or chronophin), phosphoglycolate phosphatase (PGP, also termed AUM or glycerol phosphatase) and phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP or HDHD2B). Evidence in support of small metabolite and protein phosphatase activity is discussed in the context of the diversity of their biological functions.}, language = {en} } @article{JeanclosAlbersenRamosetal.2019, author = {Jeanclos, Elisabeth and Albersen, Monique and Ramos, R{\´u}ben J. J. and Raab, Annette and Wilhelm, Christian and Hommers, Leif and Lesch, Klaus-Peter and Verhoeven-Duif, Nanda M. and Gohla, Antje}, title = {Improved cognition, mild anxiety-like behavior and decreased motor performance in pyridoxal phosphatase-deficient mice}, series = {BBA - Molecular Basis of Disease}, volume = {1865}, journal = {BBA - Molecular Basis of Disease}, doi = {10.1016/j.bbadis.2018.08.018}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323396}, pages = {193-205}, year = {2019}, abstract = {Pyridoxal 5′-phosphate (PLP) is an essential cofactor in the catalysis of ~140 different enzymatic reactions. A pharmacological elevation of cellular PLP concentrations is of interest in neuropsychiatric diseases, but whole-body consequences of higher intracellular PLP levels are unknown. To address this question, we have generated mice allowing a conditional ablation of the PLP phosphatase PDXP. Ubiquitous PDXP deletion increased PLP levels in brain, skeletal muscle and red blood cells up to 3-fold compared to control mice, demonstrating that PDXP acts as a major regulator of cellular PLP concentrations in vivo. Neurotransmitter analysis revealed that the concentrations of dopamine, serotonin, epinephrine and glutamate were unchanged in the brains of PDXP knockout mice. However, the levels of γ-aminobutyric acid (GABA) increased by ~20\%, demonstrating that elevated PLP levels can drive additional GABA production. Behavioral phenotyping of PDXP knockout mice revealed improved spatial learning and memory, and a mild anxiety-like behavior. Consistent with elevated GABA levels in the brain, PDXP loss in neural cells decreased performance in motor tests, whereas PDXP-deficiency in skeletal muscle increased grip strength. Our findings suggest that PDXP is involved in the fine-tuning of GABA biosynthesis. Pharmacological inhibition of PDXP might correct the excitatory/inhibitory imbalance in some neuropsychiatric diseases.}, language = {en} } @article{MeralProvasiPradaGraciaetal.2018, author = {Meral, Derya and Provasi, Davide and Prada-Gracia, Diego and M{\"o}ller, Jan and Marino, Kristen and Lohse, Martin J. and Filizola, Marta}, title = {Molecular details of dimerization kinetics reveal negligible populations of transient µ-opioid receptor homodimers at physiological concentrations}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, doi = {10.1038/s41598-018-26070-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223995}, year = {2018}, abstract = {Various experimental and computational techniques have been employed over the past decade to provide structural and thermodynamic insights into G Protein-Coupled Receptor (GPCR) dimerization. Here, we use multiple microsecond-long, coarse-grained, biased and unbiased molecular dynamics simulations (a total of ~4 milliseconds) combined with multi-ensemble Markov state models to elucidate the kinetics of homodimerization of a prototypic GPCR, the µ-opioid receptor (MOR), embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol lipid bilayer. Analysis of these computations identifies kinetically distinct macrostates comprising several different short-lived dimeric configurations of either inactive or activated MOR. Calculated kinetic rates and fractions of dimers at different MOR concentrations suggest a negligible population of MOR homodimers at physiological concentrations, which is supported by acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments. This study provides a rigorous, quantitative explanation for some conflicting experimental data on GPCR oligomerization.}, language = {en} } @article{JoosSaadatmandSchnabeletal.2018, author = {Joos, J. P. and Saadatmand, A. R. and Schnabel, C. and Viktorinov{\´a}, I. and Brand, T. and Kramer, M. and Nattel, S. and Dobrev, D. and Tomancak, P. and Backs, J. and Kleinbongard, P. and Heusch, G. and Lorenz, K. and Koch, E. and Weber, S. and El-Armouche, A.}, title = {Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, doi = {10.1038/s41598-018-21372-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323637}, year = {2018}, abstract = {Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, we studied a Drosophila strain with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. We also found changes in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, we analyzed posttranslational modification of H3S28 in an experimental heart failure model and observed increased H3S28 phosphorylation levels in HF hearts. Our data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure.}, language = {en} } @article{HommersRichterYangetal.2018, author = {Hommers, L. G. and Richter, J. and Yang, Y. and Raab, A. and Baumann, C. and Lang, K. and Schiele, M. A. and Weber, H. and Wittmann, A. and Wolf, C. and Alpers, G. W. and Arolt, V. and Domschke, K. and Fehm, L. and Fydrich, T. and Gerlach, A. and Gloster, A. T. and Hamm, A. O. and Helbig-Lang, S. and Kircher, T. and Lang, T. and Pan{\´e}-Farr{\´e}, C. A. and Pauli, P. and Pfleiderer, B. and Reif, A. and Romanos, M. and Straube, B. and Str{\"o}hle, A. and Wittchen, H.-U. and Frantz, S. and Ertl, G. and Lohse, M. J. and Lueken, U. and Deckert, J.}, title = {A functional genetic variation of SLC6A2 repressor hsa-miR-579-3p upregulates sympathetic noradrenergic processes of fear and anxiety}, series = {Translational Psychiatry}, volume = {8}, journal = {Translational Psychiatry}, doi = {10.1038/s41398-018-0278-4}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-322497}, year = {2018}, abstract = {Increased sympathetic noradrenergic signaling is crucially involved in fear and anxiety as defensive states. MicroRNAs regulate dynamic gene expression during synaptic plasticity and genetic variation of microRNAs modulating noradrenaline transporter gene (SLC6A2) expression may thus lead to altered central and peripheral processing of fear and anxiety. In silico prediction of microRNA regulation of SLC6A2 was confirmed by luciferase reporter assays and identified hsa-miR-579-3p as a regulating microRNA. The minor (T)-allele of rs2910931 (MAFcases = 0.431, MAFcontrols = 0.368) upstream of MIR579 was associated with panic disorder in patients (pallelic = 0.004, ncases = 506, ncontrols = 506) and with higher trait anxiety in healthy individuals (pASI = 0.029, pACQ = 0.047, n = 3112). Compared to the major (A)-allele, increased promoter activity was observed in luciferase reporter assays in vitro suggesting more effective MIR579 expression and SLC6A2 repression in vivo (p = 0.041). Healthy individuals carrying at least one (T)-allele showed a brain activation pattern suggesting increased defensive responding and sympathetic noradrenergic activation in midbrain and limbic areas during the extinction of conditioned fear. Panic disorder patients carrying two (T)-alleles showed elevated heart rates in an anxiety-provoking behavioral avoidance test (F(2, 270) = 5.47, p = 0.005). Fine-tuning of noradrenaline homeostasis by a MIR579 genetic variation modulated central and peripheral sympathetic noradrenergic activation during fear processing and anxiety. This study opens new perspectives on the role of microRNAs in the etiopathogenesis of anxiety disorders, particularly their cardiovascular symptoms and comorbidities.}, language = {en} } @article{HarnošCanizalJuraseketal.2019, author = {Harnoš, Jakub and Ca{\~n}izal, Maria Consuelo Alonso and Jur{\´a}sek, Miroslav and Kumar, Jitender and Holler, Cornelia and Schambony, Alexandra and Han{\´a}kov{\´a}, Kateřina and Bernat{\´i}k, Ondřej and Zdr{\´a}hal, Zbyn{\^e}k and G{\"o}m{\"o}ryov{\´a}, Krist{\´i}na and Gybeľ, Tom{\´a}š and Radaszkiewicz, Tomasz Witold and Kravec, Marek and Trant{\´i}rek, Luk{\´a}š and Ryneš, Jan and Dave, Zankruti and Fern{\´a}ndez-Llamazares, Ana Iris and V{\´a}cha, Robert and Tripsianes, Konstantinos and Hoffmann, Carsten and Bryja, V{\´i}tězslav}, title = {Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-09651-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227837}, year = {2019}, abstract = {Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.}, language = {en} } @article{HartmannKnierimMaureretal.2023, author = {Hartmann, Nico and Knierim, Maria and Maurer, Wiebke and Dybkova, Nataliya and Hasenfuß, Gerd and Sossalla, Samuel and Streckfuss-B{\"o}meke, Katrin}, title = {Molecular and functional relevance of Na\(_V\)1.8-induced atrial arrhythmogenic triggers in a human SCN10A knock-out stem cell model}, series = {International Journal of Molecular Sciences}, volume = {24}, journal = {International Journal of Molecular Sciences}, number = {12}, issn = {1422-0067}, doi = {10.3390/ijms241210189}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-362708}, year = {2023}, abstract = {In heart failure and atrial fibrillation, a persistent Na\(^+\) current (I\(_{NaL}\)) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that Na\(_V\)1.8 contributes to arrhythmogenesis by inducing a I\(_{NaL}\). Genome-wide association studies indicate that mutations in the SCN10A gene (Na\(_V\)1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these Na\(_V\)1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the I\(_{NaL}\) and action potential duration. Ca\(^{2+}\) measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca\(^{2+}\) leak. The I\(_{NaL}\) was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of Na\(_V\)1.8. No effects on atrial APD\(_{90}\) were detected in any groups. Both SCN10A KO and specific blockers of Na\(_V\)1.8 led to decreased Ca\(^{2+}\) spark frequency and a significant reduction of arrhythmogenic Ca\(^{2+}\) waves. Our experiments demonstrate that Na\(_V\)1.8 contributes to I\(_{NaL}\) formation in human atrial CMs and that Na\(_V\)1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore Na\(_V\)1.8 could be a new target for antiarrhythmic strategies.}, language = {en} }