@article{NwoghaAbtewRaveendranetal.2023, author = {Nwogha, Jeremiah S. and Abtew, Wosene G. and Raveendran, Muthurajan and Oselebe, Happiness O. and Obidiegwu, Jude E. and Chilaka, Cynthia A. and Amirtham, Damodarasamy D.}, title = {Role of non-structural sugar metabolism in regulating tuber dormancy in white yam (Dioscorea rotundata)}, series = {Agriculture}, volume = {13}, journal = {Agriculture}, number = {2}, issn = {2077-0472}, doi = {10.3390/agriculture13020343}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304486}, year = {2023}, abstract = {Changes in sugar composition occur continuously in plant tissues at different developmental stages. Tuber dormancy induction, stability, and breaking are very critical developmental transitions in yam crop production. Prolonged tuber dormancy after physiological maturity has constituted a great challenge in yam genetic improvement and productivity. In the present study, biochemical profiling of non-structural sugar in yam tubers during dormancy was performed to determine the role of non-structural sugar in yam tuber dormancy regulation. Two genotypes of the white yam species, one local genotype (Obiaoturugo) and one improved genotype (TDr1100873), were used for this study. Tubers were sampled at 42, 56, 87, 101, 115, and 143 days after physiological maturity (DAPM). Obiaoturugo exhibited a short dormant phenotype and sprouted at 101-DAPM, whereas TDr1100873 exhibited a long dormant phenotype and sprouted at 143-DAPM. Significant metabolic changes were observed in non-structural sugar parameters, dry matter, and moisture content in Obiaoturugo from 56-DAPM, whereas in TDr1100873, significant metabolic changes were observed from 101-DAPM. It was observed that the onset of these metabolic changes occurred at a point when the tubers of both genotypes exhibited a dry matter content of 60\%, indicating that a dry matter content of 60\% might be a critical threshold for white yam tuber sprouting. Non-reducing sugars increased by 9-10-fold during sprouting in both genotypes, which indicates their key role in tuber dormancy regulation in white yam. This result implicates that some key sugar metabolites can be targeted for dormancy manipulation of the yam crop.}, language = {en} } @phdthesis{Zink2023, author = {Zink, Christoph}, title = {Biochemische und strukturbiologische Charakterisierung der Inhibition der Pyridoxal 5´-Phosphat Phosphatase durch 7,8-Dihydroxyflavon}, doi = {10.25972/OPUS-25151}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251511}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Die Pyridoxal-5'-Phosphat Phosphatase (PDXP), auch bekannt als Chronophin (CIN), ist eine HAD-Phosphatase, die beim Menschen ubiquit{\"a}r exprimiert wird und eine entscheidende Rolle im zellul{\"a}ren Vitamin-B6-Metabolismus einnimmt. PDXP ist in der Lage Pyridoxal-5'-Phosphat (PLP), die co-enzymatisch aktive Form von Vitamin B6, zu dephosphorylieren. In-vivo Studien mit M{\"a}usen zeigten, dass die Abwesenheit von PDXP mit verbesserten kognitiven Leistungen und einem verringerten Wachstum von Hirntumoren assoziiert ist. Dies begr{\"u}ndet die gezielte Suche nach einem pharmakologischen Inhibitor f{\"u}r PDXP. Ein Hochdurchsatz-Screen legte nahe, dass 7,8-Dihydroxyflavon (7,8-DHF) hierf{\"u}r ein potenzieller Kandidat ist. Zahlreiche Studien beschreiben bereits vielf{\"a}ltige positive neurologische Effekte nach in-vivo Administration von 7,8-DHF, allerdings bleibt der genaue Wirkmechanismus umstritten und wird bis dato nicht mit PDXP in Zusammenhang gebracht. Ziel dieser Arbeit ist es, die Inhibition von PDXP durch 7,8-DHF n{\"a}her zu charakterisieren und damit einen Beitrag zur Beantwortung der Frage zu leisten, ob PDXP an den 7,8-DHF-induzierten Effekten beteiligt ist. Hierzu wurde der Effekt von 7,8-DHF auf die enzymatische Aktivit{\"a}t von rekombinant hergestelltem, gereinigtem PDXP in in-vitro Phosphatase-Assays charakterisiert. Um die Selektivit{\"a}t von 7,8-DHF gegen{\"u}ber PDXP zu untersuchen, wurden f{\"u}nf weitere HAD-Phosphatasen getestet. Unter den analysierten Phosphatasen zeigte einzig die dem PDXP nah verwandte Phosphoglykolat Phosphatase (PGP) eine geringer ausgepr{\"a}gte Sensitivit{\"a}t gegen 7,8-DHF. Ein Vergleich von 7,8-DHF mit sechs strukturell verwandten, hydroxylierten Flavonen zeigte, dass 7,8-DHF unter den getesteten Substanzen die h{\"o}chste Potenz und Effektivit{\"a}t aufwies. Außerdem wurde eine Co-Kristallisation von PDXP mit 7,8-DHF durchgef{\"u}hrt, deren Struktur bis zu einer Aufl{\"o}sung von 2,0 {\AA} verfeinert werden konnte. Die in der Kristallstruktur identifizierte Bindungsstelle von 7,8-DHF an PDXP wurde mittels verschiedener, neu generierter PDXP-Mutanten enzymkinetisch best{\"a}tigt. Zusammenfassend zeigen die hier beschriebenen Ergebnisse, dass 7,8-DHF ein direkter, selektiver und vorwiegend kompetitiver Inhibitor der PDXP-Aktivit{\"a}t ist, mit einer IC50 im submikromolaren Bereich. Die Ergebnisse dieser in-vitro Untersuchungen motivieren zu weiterer Forschung bez{\"u}glich der 7,8-DHF-vermittelten Inhibition der PDXP-Aktivit{\"a}t in Zellen, um die Frage beantworten zu k{\"o}nnen, ob PDXP auch in-vivo ein relevantes Target f{\"u}r 7,8-DHF darstellt.}, subject = {Pyridoxalphosphat}, language = {de} } @phdthesis{Eppli2023, author = {Eppli, Nenad}, title = {Untersuchung des Einflusses der ERK1/2-Autophosphorylierung an Threonin 188 auf Mausherzen mittels transgener M{\"a}use mit ubiquit{\"a}rer {\"U}berexpression von ERK2\(^{T188D}\)}, doi = {10.25972/OPUS-21655}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216558}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Die ERK2Thr188-Autophosphoylierung stellt einen regulatorischen Signalweg dar, der infolge einer hypertrophen Stimulation die kardiale Hypertrophie beg{\"u}nstigt. Eine Hemmung dieser Phosphorylierung in Kardiomyozyten verhindert die Ausbildung der kardialen Hypertrophie ohne Beeinflussung der kardioprotektiven Funktionen von ERK1/2. Demgegen{\"u}ber f{\"u}hrt die dauerhafte Simulation zu einem gain-of-function-Ph{\"a}notypen mit ausgepr{\"a}gter Hypertophie, Fibrose und einer reduzierten Herzfunktion. In dieser Arbeit wurde die dauerhafte Simulation ERK2Thr188-Phosphorylierung (T188D) in einem Mausmodell mit ubiquit{\"a}rer Expression dieser Mutation untersucht. Dabei konnte gezeigt werden, dass sich nach Stimulation durch TAC in diesen Tieren ein etwas st{\"a}rkerer hypertropher Ph{\"a}notyp mit vergr{\"o}ßerten Kardiomyozyten, gesteigerter interstitieller Fibrosierung und reduzierter Herzfunktion ausbildet als in M{\"a}usen mit kardiomyozyten-spezifischer {\"U}berexpression diese Mutante. In Fibroblasten- und VSMC-Zelllinien wurde eine gesteigerte Proliferation der T188D-{\"u}berexprimierenden Zellen im Vergleich zu Kontrollen festgestellt. Somit scheint die ERK2Thr188-Phosphorylierung auch in kardialen Nicht-Myozyten einen maladaptiven Einfluss auf das Herz auszu{\"u}ben.}, subject = {Herzhypertrophie}, language = {de} } @article{SedaghatHamedaniRebsKayvanpouretal.2022, author = {Sedaghat-Hamedani, Farbod and Rebs, Sabine and Kayvanpour, Elham and Zhu, Chenchen and Amr, Ali and M{\"u}ller, Marion and Haas, Jan and Wu, Jingyan and Steinmetz, Lars M. and Ehlermann, Philipp and Streckfuss-B{\"o}meke, Katrin and Frey, Norbert and Meder, Benjamin}, title = {Genotype complements the phenotype: identification of the pathogenicity of an LMNA splice variant by nanopore long-read sequencing in a large DCM family}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {20}, issn = {1422-0067}, doi = {10.3390/ijms232012230}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290415}, year = {2022}, abstract = {Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20-40\% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.}, language = {en} } @article{SchanbacherHermannsLorenzetal.2023, author = {Schanbacher, Constanze and Hermanns, Heike M. and Lorenz, Kristina and Wajant, Harald and Lang, Isabell}, title = {Complement 1q/tumor necrosis factor-related proteins (CTRPs): structure, receptors and signaling}, series = {Biomedicines}, volume = {11}, journal = {Biomedicines}, number = {2}, issn = {2227-9059}, doi = {10.3390/biomedicines11020559}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-304136}, year = {2023}, abstract = {Adiponectin and the other 15 members of the complement 1q (C1q)/tumor necrosis factor (TNF)-related protein (CTRP) family are secreted proteins composed of an N-terminal variable domain followed by a stalk region and a characteristic C-terminal trimerizing globular C1q (gC1q) domain originally identified in the subunits of the complement protein C1q. We performed a basic PubMed literature search for articles mentioning the various CTRPs or their receptors in the abstract or title. In this narrative review, we briefly summarize the biology of CTRPs and focus then on the structure, receptors and major signaling pathways of CTRPs. Analyses of CTRP knockout mice and CTRP transgenic mice gave overwhelming evidence for the relevance of the anti-inflammatory and insulin-sensitizing effects of CTRPs in autoimmune diseases, obesity, atherosclerosis and cardiac dysfunction. CTRPs form homo- and heterotypic trimers and oligomers which can have different activities. The receptors of some CTRPs are unknown and some receptors are redundantly targeted by several CTRPs. The way in which CTRPs activate their receptors to trigger downstream signaling pathways is largely unknown. CTRPs and their receptors are considered as promising therapeutic targets but their translational usage is still hampered by the limited knowledge of CTRP redundancy and CTRP signal transduction.}, language = {en} } @article{GmachBathePetersTeluguetal.2022, author = {Gmach, Philipp and Bathe-Peters, Marc and Telugu, Narasimha and Miller, Duncan C. and Annibale, Paolo}, title = {Fluorescence spectroscopy of low-level endogenous β-adrenergic receptor expression at the plasma membrane of differentiating human iPSC-derived cardiomyocytes}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {18}, issn = {1422-0067}, doi = {10.3390/ijms231810405}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288277}, year = {2022}, abstract = {The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the β\(_1\)- and β\(_2\)-adrenergic receptors (β\(_{1/2}\)-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of β-ARs in adult CMs.}, language = {en} } @article{DjelićBorozanDimitrijevićSrećkovićetal.2022, author = {Djelić, Ninoslav and Borozan, Sunčica and Dimitrijević-Srećković, Vesna and Pajović, Nevena and Mirilović, Milorad and Stopper, Helga and Stanimirović, Zoran}, title = {Oxidative stress and DNA damage in peripheral blood mononuclear cells from normal, obese, prediabetic and diabetic persons exposed to thyroid hormone in vitro}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {16}, issn = {1422-0067}, doi = {10.3390/ijms23169072}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285988}, year = {2022}, abstract = {Diabetes, a chronic group of medical disorders characterized byhyperglycemia, has become a global pandemic. Some hormones may influence the course and outcome of diabetes, especially if they potentiate the formation of reactive oxygen species (ROS). There is a close relationship between thyroid disorders and diabetes. The main objective of this investigation was to find out whether peripheral blood mononuclear cells (PBMCs) are more prone to DNA damage by triiodothyronine (T\(_3\)) (0.1, 1 and 10 μM) at various stages of progression through diabetes (obese, prediabetics, and type 2 diabetes mellitus—T2DM persons). In addition, some biochemical parameters of oxidative stress (catalase-CAT, thiobarbituric acid reactive substances—TBARS) and lactate dehydrogenase (LDH) were evaluated. PBMCs from prediabetic and diabetic patients exhibited increased sensitivity for T\(_3\) regarding elevated level of DNA damage, inhibition of catalase, and increase of TBARS and LDH. PBMCs from obese patients reacted in the same manner, except for DNA damage. The results of this study should contribute to a better understanding of the role of thyroid hormones in the progression of T2DM.}, language = {en} } @phdthesis{Nemec2023, author = {Nemec, Katarina}, title = {Modulation of parathyroid hormone 1 receptor (PTH1R) signaling by receptor activity-modifying proteins (RAMPs)}, doi = {10.25972/OPUS-28858}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288588}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {The receptor activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that interact with several G protein-coupled receptors (GPCRs), the largest and pharmacologically most important family of cell surface receptors. RAMPs can regulate GPCR function in terms of ligand-binding, G-protein coupling, downstream signaling, trafficking, and recycling. The integrity of their interactions translates to many physiological functions or pathological conditions. Regardless of numerous reports on its essential importance for cell biology and pivotal role in (patho-)physiology, the molecular mechanism of how RAMPs modulate GPCR activation remained largely elusive. This work presents new insights that add to the common understanding of the allosteric regulation of receptor activation and will help interpret how accessory proteins - RAMPs - modulate activation dynamics and how this affects the fundamental aspects of cellular signaling. Using a prototypical class B GPCR, the parathyroid hormone 1 receptor (PTH1R) in the form of advanced genetically encoded optical biosensors, I examined RAMP's impact on the PTH1R activation and signaling in intact cells. A panel of single-cell FRET and confocal microscopy experiments as well canonical and non-canonical functional assays were performed to get a holistic picture of the signaling initiation and transduction of that clinically and therapeutically relevant GPCR. Finally, structural modeling was performed to add molecular mechanistic details to that novel art of modulation. I describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity and kinetics of cAMP accumulation. Additionally, RAMP2 increases PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R and modulates cytosolic ERK1/2 phosphorylation. Structural homology modeling shows that structural motifs governing GPCR-RAMP interaction originate in allosteric hotspots and rationalize functional modulation. Moreover, to interpret the broader role of RAMP's modulation in GPCRs pharmacology, different fluorescent tools to investigate RAMP's spatial organization were developed, and novel conformational biosensors for class B GPCRs were engineered. Lastly, a high throughput assay is proposed and prototyped to expand the repertoire of RAMPs or other membrane protein interactors. These data uncover the critical role of RAMPs in GPCR activation and signaling and set up a novel platform for studying GPCR modulation. Furthermore, these insights may provide a new venue for precise modulation of GPCR function and advanced drug design.}, subject = {G-Protein gekoppelter Rezeptor}, language = {en} } @article{SchanbacherBieberReindersetal.2022, author = {Schanbacher, Constanze and Bieber, Michael and Reinders, Yvonne and Cherpokova, Deya and Teichert, Christina and Nieswandt, Bernhard and Sickmann, Albert and Kleinschnitz, Christoph and Langhauser, Friederike and Lorenz, Kristina}, title = {ERK1/2 activity is critical for the outcome of ischemic stroke}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {2}, issn = {1422-0067}, doi = {10.3390/ijms23020706}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-283991}, year = {2022}, abstract = {Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2\(^{wt}\)) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood-brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIP\(^{wt}\)) and its phosphorylation-deficient mutant RKIP\(^{S153A}\), known inhibitors of the ERK1/2 signaling cascade. RKIP\(^{wt}\) and RKIP\(^{S153A}\) attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.}, language = {en} } @article{BauerMallyLiedtke2021, author = {Bauer, Benedikt and Mally, Angela and Liedtke, Daniel}, title = {Zebrafish embryos and larvae as alternative animal models for toxicity testing}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {24}, issn = {1422-0067}, doi = {10.3390/ijms222413417}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284225}, year = {2021}, abstract = {Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.}, language = {en} }