TY - JOUR A1 - Lorenz, Kristina A1 - Rosner, Marsha Rich T1 - Harnessing RKIP to combat heart disease and cancer JF - Cancers N2 - Cancer and heart disease are leading causes of morbidity and mortality worldwide. These diseases have common risk factors, common molecular signaling pathways that are central to their pathogenesis, and even some disease phenotypes that are interdependent. Thus, a detailed understanding of common regulators is critical for the development of new and synergistic therapeutic strategies. The Raf kinase inhibitory protein (RKIP) is a regulator of the cellular kinome that functions to maintain cellular robustness and prevent the progression of diseases including heart disease and cancer. Two of the key signaling pathways controlled by RKIP are the β-adrenergic receptor (βAR) signaling to protein kinase A (PKA), particularly in the heart, and the MAP kinase cascade Raf/MEK/ERK1/2 that regulates multiple diseases. The goal of this review is to discuss how we can leverage RKIP to suppress cancer without incurring deleterious effects on the heart. Specifically, we discuss: (1) How RKIP functions to either suppress or activate βAR (PKA) and ERK1/2 signaling; (2) How we can prevent cancer-promoting kinase signaling while at the same time avoiding cardiotoxicity. KW - RKIP KW - ERK1/2 KW - PKA KW - βAR KW - heart failure KW - cancer Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262185 SN - 2072-6694 VL - 14 IS - 4 ER - TY - JOUR A1 - Jarzina, Sebastian A1 - Di Fiore, Stefano A1 - Ellinger, Bernhard A1 - Reiser, Pia A1 - Frank, Sabrina A1 - Glaser, Markus A1 - Wu, Jiaqing A1 - Taverne, Femke J. A1 - Kramer, Nynke I. A1 - Mally, Angela T1 - Application of the adverse outcome pathway concept to in vitro nephrotoxicity assessment: kidney injury due to receptor-mediated endocytosis and lysosomal overload as a case study JF - Frontiers in Toxicology N2 - Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling. KW - adverse outcome pathway (AOP) KW - nephrotoxicity KW - QIVIVE KW - risk assessment KW - key event relationship KW - In vitro toxicity testing Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284796 SN - 2673-3080 VL - 4 ER - TY - JOUR A1 - Mally, Angela A1 - Jarzina, Sebastian T1 - Mapping adverse outcome pathways for kidney injury as a basis for the development of mechanism-based animal-sparing approaches to assessment of nephrotoxicity JF - Frontiers in Toxicology N2 - In line with recent OECD activities on the use of AOPs in developing Integrated Approaches to Testing and Assessment (IATAs), it is expected that systematic mapping of AOPs leading to systemic toxicity may provide a mechanistic framework for the development and implementation of mechanism-based in vitro endpoints. These may form part of an integrated testing strategy to reduce the need for repeated dose toxicity studies. Focusing on kidney and in particular the proximal tubule epithelium as a key target site of chemical-induced injury, the overall aim of this work is to contribute to building a network of AOPs leading to nephrotoxicity. Current mechanistic understanding of kidney injury initiated by 1) inhibition of mitochondrial DNA polymerase γ (mtDNA Polγ), 2) receptor mediated endocytosis and lysosomal overload, and 3) covalent protein binding, which all present fairly well established, common mechanisms by which certain chemicals or drugs may cause nephrotoxicity, is presented and systematically captured in a formal description of AOPs in line with the OECD AOP development programme and in accordance with the harmonized terminology provided by the Collaborative Adverse Outcome Pathway Wiki. The relative level of confidence in the established AOPs is assessed based on evolved Bradford-Hill weight of evidence considerations of biological plausibility, essentiality and empirical support (temporal and dose-response concordance). KW - adverse outcome pathway KW - nephrotoxicity KW - protein alkylation KW - lysosomal disruption KW - mitochondrial DNA polymerase γ Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284405 SN - 2673-3080 VL - 4 ER - TY - JOUR A1 - Maurer, Wiebke A1 - Hartmann, Nico A1 - Argyriou, Loukas A1 - Sossalla, Samuel A1 - Streckfuss-Bömeke, Katrin T1 - Generation of homozygous Na\(_{v}\)1.8 knock-out iPSC lines by CRISPR Cas9 genome editing to investigate a potential new antiarrhythmic strategy JF - Stem Cell Research N2 - The sodium channel Na\(_{v}\)1.8, encoded by SCN10A, is reported to contribute to arrhythmogenesis by inducing the late I\(_{Na}\) and thereby enhanced persistent Na\(^{+}\) current. However, its exact electrophysiological role in cardiomyocytes remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) with a homozygous SCN10A knock-out from a healthy iPSC line by CRISPR Cas9 genome editing. The edited iPSCs maintained full pluripotency, genomic integrity, and spontaneous in vitro differentiation capacity. The iPSCs are able to differentiate into iPSC-cardiomyocytes, hence making it possible to investigate the role of Na\(_{v}\)1.8 in the heart. KW - arrhythmogenesis KW - cardiomyocytes KW - induced pluripotent stem cells Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300936 VL - 60 ER - TY - JOUR A1 - Jeanclos, Elisabeth A1 - Schlötzer, Jan A1 - Hadamek, Kerstin A1 - Yuan-Chen, Natalia A1 - Alwahsh, Mohammad A1 - Hollmann, Robert A1 - Fratz, Stefanie A1 - Yesilyurt-Gerhards, Dilan A1 - Frankenbach, Tina A1 - Engelmann, Daria A1 - Keller, Angelika A1 - Kaestner, Alexandra A1 - Schmitz, Werner A1 - Neuenschwander, Martin A1 - Hergenröder, Roland A1 - Sotriffer, Christoph A1 - von Kries, Jens Peter A1 - Schindelin, Hermann A1 - Gohla, Antje T1 - Glycolytic flux control by drugging phosphoglycolate phosphatase JF - Nature Communications N2 - Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates. KW - phosphoglycolate phosphatase KW - glycolytic flux control KW - intrinsic metabolism Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300928 VL - 13 IS - 1 ER - TY - JOUR A1 - Klenk, Christoph A1 - Hommers, Leif A1 - Lohse, Martin J. T1 - Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor JF - Frontiers in Endocrinology N2 - Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G\(_s\) and increased activation of G\(_q\) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling. KW - GPCRs KW - parathyroid hormone 1 receptor KW - matrix metalloproteinase KW - ectodomain cleavage KW - biased signaling Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262055 SN - 1664-2392 VL - 13 ER - TY - THES A1 - İşbilir, Ali T1 - Localization and Trafficking of CXCR4 and CXCR7 T1 - Lokalisation und Verteilung von CXCR4 und CXCR7 N2 - G protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins, and are the master components that translate extracellular stimulus into intracellular signaling, which in turn modulates key physiological and pathophysiological processes. Research within the last three decades suggests that many GPCRs can form complexes with each other via mechanisms that are yet unexplored. Despite a number of functional evidence in favor of GPCR dimers and oligomers, the existence of such complexes remains controversial, as different methods suggest diverse quaternary organizations for individual receptors. Among various methods, high resolution fluorescence microscopy and imagebased fluorescence spectroscopy are state-of-the-art tools to quantify membrane protein oligomerization with high precision. This thesis work describes the use of single molecule fluorescence microscopy and implementation of two confocal microscopy based fluorescence fluctuation spectroscopy based methods for characterizing the quaternary organization of two class A GPCRs that are important clinical targets: the C-X-C type chemokine receptor 4 (CXCR4) and 7 (CXCR7), or recently named as the atypical chemokine receptor 3 (ACKR3). The first part of the results describe that CXCR4 protomers are mainly organized as monomeric entities that can form transient dimers at very low expression levels allowing single molecule resolution. The second part describes the establishment and use of spatial and temporal brightness methods that are based on fluorescence fluctuation spectroscopy. Results from this part suggests that ACKR3 forms clusters and surface localized monomers, while CXCR4 forms increasing amount of dimers as a function of receptor density in cells. Moreover, CXCR4 dimerization can be modulated by its ligands as well as receptor conformations in distinct manners. Further results suggest that antagonists of CXCR4 display distinct binding modes, and the binding mode influences the oligomerization and the basal activity of the receptor: While the ligands that bind to a “minor” subpocket suppress both dimerization and constitutive activity, ligands that bind to a distinct, “major” subpocket only act as neutral antagonists on the receptor, and do not modulate neither the quaternary organization nor the basal signaling of CXCR4. Together, these results link CXCR4 dimerization to its density and to its activity, which may represent a new strategy to target CXCR4. N2 - G protein-gekoppelte Rezeptoren (GPCRs) bilden die größte Klasse der Membranproteine und sind entscheidend an der Übersetzung extrazellulärer Reize in intrazelluläre Signale beteiligt, welche wiederum unzählige physiologische und pathophysiologische Prozesse regulieren. Die Forschungsergebnisse der letzten drei Jahrzehnte deutet darauf hin, dass viele GPCRs mittels noch weitgehend unbekannter Mechanismen miteinander Komplexe bilden können. Trotz vielfältiger Beobachtungen, die für die funktionelle Relevanz von GPCR-Dimeren und -Oligomeren sprechen, ist deren Existenz dennoch weiterhin umstritten, vor allem da verschiedene Methoden auf unterschiedliche quaternäre Anordnungen derselben Rezeptoren hinweisen. Von den derzeit verfügbaren Methoden zur genauen Untersuchung der GPCR Dimerisierung/-Oligomerisierung, stellen die hochauflösende Fluoreszenzmikroskopie sowie die bildbasierte Fluoreszenzspektroskopie die Techniken der Wahl dar. Die hier vorliegende Arbeit beschreibt die Anwendung der Einzelmolekül Fluoreszenzmikroskopie sowie zweier konfokalmikroskopischer Methoden zur Messung der Fluoreszenzfluktuation, mit deren Hilfe die quaternäre Anordnung zweier klinisch hochattraktiver Klasse A GPCRs untersucht wurde: der C-X-C Typ Chemokinrezeptoren 4 (CXCR4) und 7 (CXCR7), letzterer auch bekannt als atypischer Chemokinrezeptor 3 (ACKR3). Der erste Teil der Ergebnisse legt anhand Untersuchungen an einzelnen Molekülen dar, dass CXCR4 überwiegend in Form monomerer Einheiten auftritt, die bei sehr geringen Expressionsleveln kurzlebige Dimere bilden können. Der zweite Teil beschreibt die Etablierung und Anwendung räumlicher und zeitlicher Brillanzmethoden, die auf der spektroskopischen Untersuchung der Fluoreszenzfluktuation beruhen. Die Ergebnisse dieses Abschnitts deuten darauf hin, dass ACKR3 sowohl in Form beständiger Rezeptor-Cluster, und monomere Einheit an der Oberfläche lebender Zellen auftritt. CXCR4 ist bei zunehmender Rezeptordichte hingegen vermehrt in Form von Dimeren zu finden. Zudem kann die Dimerisierung von CXCR4 von dessen Liganden, als auch von der drei dimensionalen Anordnung der Rezeptorteilstrukturen (Rezeptorkonformation)auf unterschiedliche Weise reguliert werden. Die weiteren Ergebnisse legen nahe, dass Antagonisten auf unterschiedliche Weise an CXCR4 binden können und dass der jeweilige Bindungsmodus entscheidend für den Einfluss des Liganden auf Oligomerisierung und basale Aktivität von CXCR4 ist: Während Liganden, die an eine kleinere Untertasche des Rezeptors binden, sowohl die Dimerisierung als auch die Basalaktivität unterdrücken, fungieren Verbindungen, die an eine andere, größere Untertasche binden, lediglich als neutrale Antagonisten und zeigen keinerlei Einfluss auf die quaternäre Anordnung und basale Aktivität von CXCR4. Zusammenfassend verknüpfen diese Ergebnisse CXCR4-Dimerisierung mit der Rezeptordichte in Zellen und seiner Aktivität, was die Grundlage für neue Strategien zur phamakologischen Modulation von CXCR4 darstellen könnte. KW - G-Protein gekoppelter Rezeptor KW - GPCR KW - Receptor KW - Chemokine KW - oligomerization KW - CXCR4 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249378 ER - TY - JOUR A1 - Obidiegwu, Jude E. A1 - Lyons, Jessica B. A1 - Chilaka, Cynthia A. T1 - The Dioscorea genus (yam) — an appraisal of nutritional and therapeutic potentials JF - Foods N2 - The quest for a food secure and safe world has led to continuous effort toward improvements of global food and health systems. While the developed countries seem to have these systems stabilized, some parts of the world still face enormous challenges. Yam (Dioscorea species) is an orphan crop, widely distributed globally; and has contributed enormously to food security especially in sub-Saharan Africa because of its role in providing nutritional benefits and income. Additionally, yam has non-nutritional components called bioactive compounds, which offer numerous health benefits ranging from prevention to treatment of degenerative diseases. Pharmaceutical application of diosgenin and dioscorin, among other compounds isolated from yam, has shown more prospects recently. Despite the benefits embedded in yam, reports on the nutritional and therapeutic potentials of yam have been fragmented and the diversity within the genus has led to much confusion. An overview of the nutritional and health importance of yam will harness the crop to meet its potential towards combating hunger and malnutrition, while improving global health. This review makes a conscious attempt to provide an overview regarding the nutritional, bioactive compositions and therapeutic potentials of yam diversity. Insights on how to increase its utilization for a greater impact are elucidated. KW - yam KW - Dioscorea KW - nutritional composition KW - bioactive compounds KW - therapeutic potential Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213102 SN - 2304-8158 VL - 9 IS - 9 ER - TY - JOUR A1 - Vazquez-Rodriguez, Saleta A1 - Vilar, Santiago A1 - Kachler, Sonja A1 - Klotz, Karl-Norbert A1 - Uriarte, Eugenio A1 - Borges, Fernanda A1 - Matos, Maria João T1 - Adenosine receptor ligands: coumarin−chalcone hybrids as modulating agents on the activity of hARs JF - Molecules N2 - Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin–chalcone hybrids were synthesized (compounds 1–8) and screened in radioligand binding (hA\(_1\), hA\(_{2A}\) and hA\(_3\)) and adenylyl cyclase (hA\(_{2B}\)) assays in order to evaluate their affinity for the four human AR subtypes (hARs). Coumarin–chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for hA\(_1\) or hA\(_3\) subtypes. In general, hydroxy-substituted hybrids showed some affinity for the hA\(_1\), while the methoxy counterparts were selective for the hA\(_3\). The most potent hA\(_1\) ligand was compound 7 (K\(_i\) = 17.7 µM), whereas compound 4 was the most potent ligand for hA\(_3\) (K\(_i\) = 2.49 µM). In addition, docking studies with hA\(_1\) and hA\(_3\) homology models were established to analyze the structure–function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity. KW - coumarin KW - chalcone KW - neurodegenerative diseases KW - adenosine receptors KW - binding affinity KW - docking Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-213165 SN - 1420-3049 VL - 25 IS - 18 ER - TY - JOUR A1 - Weigand, Isabel A1 - Ronchi, Cristina L. A1 - Vanselow, Jens T. A1 - Bathon, Kerstin A1 - Lenz, Kerstin A1 - Herterich, Sabine A1 - Schlosser, Andreas A1 - Kroiss, Matthias A1 - Fassnacht, Martin A1 - Calebiro, Davide A1 - Sbiera, Silviu T1 - PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser\(^{114}\) phosphorylation JF - Science Advances N2 - Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing’s syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser\(^{114}\) phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing’s syndrome. KW - mutation triggers KW - phosphorylation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270445 VL - 7 IS - 8 ER -