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Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
The melanocortin 4 receptor (MC4R) is a key player in hypothalamic weight regulation and energy expenditure as part of the leptin–melanocortin pathway. Mutations in this G protein coupled receptor (GPCR) are the most common cause for monogenetic obesity, which appears to be mediated by changes in the anorectic action of MC4R via G\(_S\)-dependent cyclic adenosine-monophosphate (cAMP) signaling as well as other signaling pathways. To study potential bias in the effects of MC4R mutations between the different signaling pathways, we investigated three major MC4R mutations: a G\(_S\) loss-of-function (S127L) and a G\(_S\) gain-of-function mutant (H158R), as well as the most common European single nucleotide polymorphism (V103I). We tested signaling of all four major G protein families plus extracellular regulated kinase (ERK) phosphorylation and β-arrestin2 recruitment, using the two endogenous agonists, α- and β-melanocyte stimulating hormone (MSH), along with a synthetic peptide agonist (NDP-α-MSH). The S127L mutation led to a full loss-of-function in all investigated pathways, whereas V103I and H158R were clearly biased towards the G\(_{q/11}\) pathway when challenged with the endogenous ligands. These results show that MC4R mutations can cause vastly different changes in the various MC4R signaling pathways and highlight the importance of a comprehensive characterization of receptor mutations.
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.
Adenosine receptor ligands: coumarin−chalcone hybrids as modulating agents on the activity of hARs
(2020)
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.
Pyridoxal 5′‐phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ‐aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca\(^{2+}\)‐ and integrin‐binding protein 1 (CIB1) as a PDXP interactor by yeast two‐hybrid screening and find a calmodulin (CaM)‐binding motif that overlaps with the PDXP‐CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca\(^{2+}\) concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP‐CIB1 complex may functionally differ from the PDXP‐Ca\(^{2+}\)‐CaM complex.
Long-term fate of etoposide-induced micronuclei and micronucleated cells in Hela-H2B-GFP cells
(2020)
Micronuclei are small nuclear cellular structures containing whole chromosomes or chromosomal fragments. While there is a lot of information available about the origin and formation of micronuclei, less is known about the fate of micronuclei and micronucleated cells. Possible fates include extrusion, degradation, reincorporation and persistence. Live cell imaging was performed to quantitatively analyse the fates of micronuclei and micronucleated cells occurring in vitro. Imaging was conducted for up to 96 h in HeLa-H2B-GFP cells treated with 0.5, 1 and 2 µg/ml etoposide. While a minority of micronuclei was reincorporated into the main nucleus during mitosis, the majority of micronuclei persisted without any alterations. Degradation and extrusion were observed rarely or never. The presence of micronuclei affected the proliferation of the daughter cells and also had an influence on cell death rates. Mitotic errors were found to be clearly increased in micronucleus-containing cells. The results show that micronuclei and micronucleated cells can, although delayed in cell cycle, sustain for multiple divisions.
Aims Acute myocardial infarction (MI) is the major cause of chronic heart failure. The activity of blood coagulation factor XIII (FXIIIa) plays an important role in rodents as a healing factor after MI, whereas its role in healing and remodelling processes in humans remains unclear. We prospectively evaluated the relevance of FXIIIa after acute MI as a potential early prognostic marker for adequate healing.
Methods and results This monocentric prospective cohort study investigated cardiac remodelling in patients with ST-elevation MI and followed them up for 1 year. Serum FXIIIa was serially assessed during the first 9 days after MI and after 2, 6, and 12 months. Cardiac magnetic resonance imaging was performed within 4 days after MI (Scan 1), after 7 to 9 days (Scan 2), and after 12 months (Scan 3). The FXIII valine-to-leucine (V34L) single-nucleotide polymorphism rs5985 was genotyped. One hundred forty-six patients were investigated (mean age 58 ± 11 years, 13% women). Median FXIIIa was 118 % (quartiles, 102–132%) and dropped to a trough on the second day after MI: 109%(98–109%; P < 0.001). FXIIIa recovered slowly over time, reaching the baseline level after 2 to 6 months and surpassed baseline levels only after 12 months: 124 % (110–142%). The development of FXIIIa after MI was independent of the genotype. FXIIIa on Day 2 was strongly and inversely associated with the relative size of MI in Scan 1 (Spearman’s ρ = –0.31; P = 0.01) and Scan 3 (ρ = –0.39; P < 0.01) and positively associated with left ventricular ejection fraction: ρ = 0.32 (P < 0.01) and ρ = 0.24 (P = 0.04), respectively.
Conclusions FXIII activity after MI is highly dynamic, exhibiting a significant decline in the early healing period, with reconstitution 6 months later. Depressed FXIIIa early after MI predicted a greater size of MI and lower left ventricular ejection fraction after 1 year. The clinical relevance of these findings awaits to be tested in a randomized trial.
Aims
Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β1-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β\(_1\)-adrenoceptor (β1ECII) that is targeted by stimulating β\(_1\)-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope.
Methods and results
Non-conserved amino acids within the β\(_1\)EC\(_{II}\) loop (compared with the amino acids constituting the ECII loop of the β\(_2\)-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine–serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β1ECII ± the above replacements, and (ii) by (auto)antibody stimulation of human β\(_1\)-adrenoceptors bearing corresponding point mutations. With the use of stimulating β\(_1\)-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β\(_1\)EC\(_{II}\) loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK\(^{211–214}\) motif and (ii) the intra-loop disulfide bond C\(^{209}\)↔C\(^{215}\). Of note, aberrant intra-loop disulfide bond C\(^{209}\)↔C\(^{216}\) almost fully disrupted the functional auto-epitope in cyclopeptides.
Conclusions
The conformational auto-epitope targeted by cardio-pathogenic β\(_1\)-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β\(_1\)EC\(_{II}\) loop bearing the NDPK\(^{211–214}\) motif and the C\(^{209}\)↔C\(^{215}\) bridge while lacking cysteine C216. Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β\(_1\)-autoantibodypositive CHF.
Neurodegenerative diseases show an increase in prevalence and incidence, with the most prominent example being Alzheimer's disease. DNA damage has been suggested to play a role in the pathogenesis, but the exact mechanisms remain elusive. We enrolled 425 participants with and without neurodegenerative diseases and analyzed DNA damage in the form of micronuclei in buccal mucosa samples. In addition, other parameters such as binucleated cells, karyolytic cells, and karyorrhectic cells were quantified. No relevant differences in DNA damage and cytotoxicity markers were observed in patients compared to healthy participants. Furthermore, other parameters such as lifestyle factors and diseases were also investigated. Overall, this study could not identify a direct link between changes in buccal cells and neurogenerative diseases, but highlights the influence of lifestyle factors and diseases on the human buccal cytome.
Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions.
Infants and young children (IYC) remain the most vulnerable population group to environmental hazards worldwide, especially in economically developing regions such as sub-Saharan Africa (SSA). As a result, several governmental and non-governmental institutions including health, environmental and food safety networks and researchers have been proactive toward protecting this group. Mycotoxins, toxic secondary fungal metabolites, contribute largely to the health risks of this young population. In SSA, the scenario is worsened by socioeconomic status, poor agricultural and storage practices, and low level of awareness, as well as the non-establishment and lack of enforcement of regulatory limits in the region. Studies have revealed mycotoxin occurrence in breast milk and other weaning foods. Of concern is the early exposure of infants to mycotoxins through transplacental transfer and breast milk as a consequence of maternal exposure, which may result in adverse health effects. The current paper presents an overview of mycotoxin occurrence in foods intended for IYC in SSA. It discusses the imperative evidence of mycotoxin exposure of this population group in SSA, taking into account consumption data and the occurrence of mycotoxins in food, as well as biomonitoring approaches. Additionally, it discusses the health implications associated with IYC exposure to mycotoxins in SSA.
The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β‐arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β‐arrestin‐dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors.
In this thesis, different Förster resonance energy transfer (FRET)‐based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild‐type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET‐based receptor sensors in the field of chemokine receptors. Additional FRET‐based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand‐induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12‐induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands.
Die Phosphatase PDXP (auch bekannt als Chronophin) gehört zur Familie der HAD Phosphatasen, einer ubiquitär exprimierten Enzymklasse mit wichtigen physiologischen Funktionen. PDXP zeigt Phosphatase-Aktivität gegenüber seinem Substrat Pyridoxal 5´-Phosphat (PLP), der aktivierten Form von Vitamin B6. PDXP-defiziente Mäuse (Knockout-Mäuse) weisen im Vergleich zu Wildtypen verdoppelte PLP-Konzentrationen in Erythrozyten sowie im Gesamthirn auf. Vermutlich kommt PDXP daher eine wichtige Funktion in Erythrozyten und im Hirn zu. Ziel dieser Arbeit war es, erste Einblicke in diese Funktion(en) von PDXP zu erlangen.
Hierzu wurden HPLC-basierte Analysen der erythrozytären PLP-Konzentrationen in Wildtyp- sowie PDXP-defizienten Mäusen durchgeführt. Dabei ließen sich die rund doppelt so hohen erythrozytären PLP-Level in den KO-Mäusen bestätigen. Zudem ist es gelungen, eine Methode zur Messung der endogenen Phosphatase-Aktivität von PDXP in Erythrozytenlysaten zu etablieren. So konnte im Wildtyp anhand der Verringerung der PLP-Konzentrationen pro Zeiteinheit eine erythrozytäre PDXP-Aktivität nachgewiesen werden. Dazu waren die Inkubation mit Pyridoxin, sowie die Anwendung eines Inhibitors der PDXK notwendig. Eine bis dato vermutete Funktion der PDXP, zur Mobilisation von erythrozytärem PLP während Fastenzeiten, konnte ausgeschlossen werden. So zeigte der Vergleich der erythrozytären PLP-Konzentrationen aus gefasteten mit normal gefütterten Tieren in beiden Genotypen exakt dieselbe prozentuale PLP-Verringerung. Während Nahrungszufuhr ließ sich jedoch eine Funktion der Phosphatase PDXP als „Converter“ von Pyridoxin zu Pyridoxal erkennen. Ausgehend von PN konnte im Wildtyp (über die Zwischenprodukte PNP und PLP) eine PDXP-abhängige Dephosphorylierung von PLP zu PL erfolgen. So wies der Wildtyp eine rund vierfach höhere PL-Produktion auf, verglichen mit der PDXP-defizienten Maus. Die Phosphatase PDXP erwies sich als essenziell für die erythrozytäre Konversion von Pyridoxin zu Pyridoxal. Dadurch erreicht der Organismus eine metabolische Flexibilität, die ihn bis zu einem gewissen Grad unabhängig von der Nahrungsauswahl macht. Zudem können Zellen oder Organe, denen durch das Fehlen der PNPO, die Konversion zu PLP nicht möglich ist, mit PL versorgt werden.
Aus der hohen Reaktivität von PLP mit umliegenden Nucleophilen ergibt sich eine gewisse Problematik für die Zelle im Umgang mit freiem PLP. So liegt der Großteil des erythrozytären PLPs gebunden an Proteine (vor allem Hämoglobin) vor. Anhand von Filtern (MWCO, 3000) ließ sich zwischen der hier definiert als „freien“ und der „gebundenen“ Form von PLP differenzieren. So konnten erste Erkenntnisse zur Rolle von PDXP als Determinator freier PLP-Konzentrationen in Erythrozyten und insbesondere im Hippocampus erlangt werden. Im Hippocampus ergaben sich insgesamt deutlich höhere Konzentrationen an freiem PLP als in den Erythrozyten und es bestand zudem ein Unterschied zwischen den Genotypen. So wiesen die KO-Mäuse ~1/3 höhere freie PLP-Konzentrationen im Vergleich zu den Wildtypen auf. Schließlich konnte ein Effekt des Tieralters auf den PLP-Metabolismus festgestellt werden. Sowohl in den Erythrozyten als auch im Hippocampus ergaben sich alterskorrelierte Änderungen ihrer PLP-Konzentrationen. Zudem zeigten Western Blot Analysen altersbedingte Unterschiede ihrer Vitamin B6-Enzymexpressionen. So wiesen ältere Wildtypen im Hippocampus eine fünffach erhöhte PDXP-Expression verglichen mit jüngeren Tieren auf. In den Erythrozytenlysaten hingegen zeigten ältere Tiere beider Genotypen eine rund vierfach geringere PNPO-Expression gegenüber jüngeren Tieren. Die mit dem Alter eintretende physiologische Verringerung der erythrozytären PNPO-Expression würde somit für den Organismus einen Verlust seiner metabolischen Flexibilität bedeuten, die mit der Konversion von PN zu PL einhergeht.
In den letzten Jahrzehnten ist die Akzeptanz stetig größer geworden, dass oxidativer Stress eine bedeutende Rolle bei der Entstehung von chronischen Erkrankungen, malignen Neoplasien sowie der Beschleunigung des Alterungsprozesses spielt. Als eine der häufigsten chronischen Erkrankungen ist Hypertonie oft mit einem fehlregulierten Renin-Angiotensin-Aldosteron-System assoziiert, welches chronisch oxidativen Stress verursacht. Bluthochdruck ist ein Risikofaktor für neurologische Erkrankungen wie der vaskulären Demenz (VaD) und viele neurologischen Störungen, einschließlich der VaD, haben eine ROS-assoziierte beziehungsweise inflammatorische Komponente in ihrer Entstehung.
Unsere Arbeitsgruppe konnte bereits eine AT-II-induzierte Genotoxizität in Nieren- und Myokardzellen bzw. -Gewebe nachweisen. Ziel dieser Dissertation war es, einen möglichen Zusammenhang zwischen AT-II und Neurodegeneration zu untersuchen, welche durch eine neuronale Genotoxizität von AT-II ausgelöst wird.
Zunächst zeigten wir in zwei neuronalen Zelllinien, dass AT-II eine Dosis-abhängige Genomschädigung verursacht. Nachfolgende Experimente konnten diese Toxizität auf NOX-produziertes Superoxid zurückführen, das nach Bindung von AT-II an den AT1R generiert wird. Zudem konnte ein AT-II-induzierter Verbrauch des wichtigsten intrazellulären Antioxidans – Glutathion - nachgewiesen werden.
In vivo konnten wir zeigen, dass AT1aR-Knockout-Mäuse nach AT-II-Behandlung signifikant mehr Genomschäden im Subfornikalorgan (SFO) aufwiesen als Wildtypmäuse. Das SFO hat als eine der wenigen Strukturen im Gehirn eine unterbrochene Blut-Hirn-Schranke, was es für zirkulierendes AT-II zugänglich und besonders empfindlich macht. Diese Genomschäden wurden in der neueren Literatur auch in Nieren- und Herzgewebe beschrieben und belegen eine zusätzliche, AT1aR- und damit Blutdruck-unabhängige Genotoxizität von AT-II.
Zusammenfassend zeigt diese Arbeit, dass erhöhte AT-II-Konzentrationen in Nervenzellen Genomschäden durch NOX-produziertes Superoxid verursachen. Die Hoffnung ist, dass diese Ergebnisse dabei helfen, eines Tages die vollständige Entstehung der VaD zu entschlüsseln.
Das Ziel der vorliegenden Arbeit war die Mikrokernbildung in Mundschleimhautzellen von 35 Patienten mit Kopf-Hals-Tumoren während einer sechswöchigen Radio-/Radiochemotherapie und sechs Wochen danach darzustellen. Die Ergebnisse der vorliegenden Arbeit zeigten, dass Patienten mit Kopf-Hals-Tumoren im Vergleich zu gesunden Probanden erhöhte Mikrokernraten aufwiesen. Ebenfalls konnte gezeigt werden, dass es zu einer vermehrten Bildung von Mikrokernen während einer sechswöchigen Radio-/Radiochemotherapie kam. Nach Therapiebeendigung sanken die Werte nach drei bis sechs Wochen und lagen unter dem Ausgangswert, in dem Bereich von spontan entstehenden Mikrokernen. In Bezug auf die Tumorgröße konnte nur in der zweiten Woche ein signifikanter Unterschied in der Mikrokernrate zwischen T1- und T4-Stadium beobachtet werden. Es konnte keine Korrelation zwischen einer zusätzlich verabreichten Chemotherapie, Grading des Tumors, Alter sowie Geschlecht der Patienten und einem Anstieg der Mikrokernrate festgestellt werden.
Durch Phosphorylierung inaktiviert GRK2 kardiale ß-Adrenorezeptoren und vermindert dadurch die Kontraktilität von neonatalen Rattenkardiomyozyten. Als natürlicher Inhibitor der GRK2 beeinflusst RKIP die Signalweiterleitung bei Stimulation von ß-Adrenorezeptoren. In dieser Arbeit konnte gezeigt werden, dass eine Überexpression von RKIP die Kontraktilität von neonatalen Rattenkardiomyozyten erhöht. Es wurde die Anzahl auftretender Spontankontraktionen vor und nach Stimulation mit Isoproterenol erfasst sowie eine zeitliche Analyse der Calciumfreisetzung und –aufnahme nach elektrischer Stimulation durchgeführt. Im unstimulierten Zustand zeigten neonatale Rattenkardiomyozyten, die Wildtyp-RKIP überexprimierten, verglichen mit der Kontrollgruppe, keine signifikanten Unterschiede hinsichtlich auftretender Spontankontraktionen. Nach Stimulation mit Isoproterenol zeigten neonatale Rattenkardiomyozyten, in denen Wildtyp-RKIP überexprimiert wurde, eine signifikant höhere Anzahl auftretender Spontankontraktionen. Eine Analyse der Calciumtransienten zeigte bei RKIP-Wildtyp überexprimierenden neonatalen Rattenkardiomyozyten eine erhöhte Calciumfreisetzung während der Systole sowie eine beschleunigte Calciumwiederaufnahme während der Diastole. Zudem wurden die RKIP-Mutanten RKIPS51V und RKIPS52V im Hinblick auf ihre Kontraktilität untersucht. Neonatale Rattenkardiomyozyten welche RKIPS51V und RKIPS52V überexprimierten zeigten weder im Hinblick auf auftretende Spontankontraktionen noch bei der Analyse der Calciumtransienten signifikante Unterschiede zur Kontrollgruppe. Da auch eine Phosphorylierung an Aminosäureposition 51 bzw. Aminosäureposition 52 ohne direkte Auswirkung auf die Kontraktilität möglich ist, wurde in einem in vitro Kinase Assay analysiert, ob neben der bekannten Phosphorylierungsstelle S153 eine weitere Phosphorylierung von RKIP durch PKA oder PKC erfolgt. Bei Einsatz der an Aminosäureposition 153 phosphorylierungsdefizienten RKIP Mutante RKIPS153A konnte keine Phosphorylierung beobachtet werden. In der vorliegenden Arbeit konnte neben einer Phosphorylierung an S153 keine weitere Phosphorylierung von RKIP durch PKC oder PKA beobachtet werden.
Übergewicht, das als Volkskrankheit ein wachsendes globales Problem darstellt, ist mit mehreren folgenreichen Komorbiditäten behaftet und die Assoziation der Erkrankung mit nachweisbarer Schädigung des Erbguts durch oxidativen Stress ist mittlerweile unangefochten. In der vorliegenden Studie wurden periphere Lymphozyten stark bis morbid adipöser Patienten mit Hilfe des Mikrokern-Assays untersucht und es konnte – begleitend zu der zu erwartenden BMI-Abnahme – eine signifikante Reduktion des Genomschadens durch Magenbypass bzw. Sleeve-Gastrektomie 12 Monate postoperativ detektiert werden. Daneben demonstrierte die Analyse zusätzlich erhobener Patientendaten, die u. a. Nüchternglucose, HbA1c, Blutdruck und Herzfrequenz sowie ein Blutbild der Patienten (inklusive CRP als Entzündungsmarker, Transaminasen, gGT sowie Lipidprofil) umfasste, eine deutliche Besserung vieler Parameter bis auf teilweise wieder physiologische Normwerte. All diese Ergebnisse stützen die These der metabolischen Wirksamkeit sowohl des Roux-en-Y-Magenbypass als auch der Sleeve- Gastrektomie. Ihre Bedeutung liegt nicht zuletzt in der angenommenen Reduktion des Krebserkrankungsrisikos, da jeweils ein Zusammenhang mit der Adipositas an sich, dem Diabetes und durch oxidativen Stress verursachter DNA-Schädigung besteht. Mit Blick auf eine gegenwärtig in der Forschung diskutierte potentielle therapeutische Anwendung von Antioxidantien zur Reduktion von Erbgutschädigungen, die durch oxidativen Stress zustande kommen, wurden die Substanzen Tricetinidin, Curcumin und Resveratrol im Rahmen des Mikrokerntests an HL60- und NRK-Zellen untersucht, in denen mit der Mischung aus Insulin und Angiotensin II eine gentoxische Wirkung erzielt worden war.
μ‐Opioid receptors (μ‐ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level.
Recently, it was shown that MDA-MB-231 breast cancer cells express very high levels of the A2BAR as the sole adenosine receptor subtype, and stimulation of the A2BAR in MDA-MB-231 cells triggers an unusual inhibitory signal on ERK1/2 phosphorylation. The ERK1/2 pathway is reported to be associated with the control of growth, proliferation and differentiation of cells and as such might serve as a promising target for tumor treatment. The present study investigated signaling mechanisms involved in linking A2BAR to ERK1/2 phosphorylation in MDA-MB-231 cells. The A2BAR mediated reduction of ERK1/2 phosphorylation and of proliferation of MDA-MB-231 cell is in good agreement with previous results from (Dubey et al., 2005). These observations provide support to the hypothesis that activation of A2BAR could attenuate the growth of some types of cancer cell and argue against a stimulation of proliferation resulting from the activation of A2BAR as discussed by (Fernandez-Gallardo et al., 2016). AC activation by forskolin has recently been shown to enhance the activity of the chemotherapeutic agent doxorubicin in TNBC cells via a mechanism dependent on the PKA-mediated inhibition of ERK1/2 phosphorylation. Furthermore, forskolin also increased the sensitivity of MDA-MB-231 and MDA-MB-468 triple negative breast cancer cells to 5-fluorouracil and taxol (Illiano et al., 2018), and sustains the evidence of anticancer activity mediated by cAMP/PKA-mediated ERK1/2 inhibition. Similar to these studies, a reduced amount of pERK1/2 was also observed after stimulation of AC with FSK, application of cAMP-AM or inhibition of PDE-4. The inhibition of ERK1/2 phosphorylation was mimicked by UTP and abolished with the PLC inhibitor U73122 or by chelating intracellular Ca2+ with BAPTA-AM. These results point to an important role for both cAMP and Ca2+ signaling in the pathway leading to a decrease in ERK1/2 phosphorylation. This study encourages the idea that A2BAR could be used as target in cancer therapy. But A2BAR did not only stimulate signaling cascades associated with cell survival and proliferation reduction, but also key phases relevant in angiogenesis like Ca2+ mobilization (Kohn et al., 1995). Whereas the potency toward AC and Ca2+ are similar for the diverse agonists, the potency to promote ERK1/2 reduction is much higher. Interestingly, the proliferation of MDA-MB-231 cells is inhibited by low nanomolar agonist concentration which is inactive in Ca2+ mobilization. This means that it is certainly possible to reduce the proliferation without promoting angiogenesis. LUF6210 is particularly interesting when considering that it preferentially stimulates a reduction in ERK1/2 phosphorylation over Ca2+ and therefore may not promote angiogenesis. LUF6210 is therapeutically appealing as adjuvant in treatment of cancer. Given that stimulation of AC can activate a reduction of ERK1/2 phosphorylation and proliferation in cancer cells, agonist bias toward Gs-AC-PKA-mediated ERK1/2 inhibition represent a potential therapy of various malignancies. The fact that the reduction of ERK1/2 phosphorylation followed by reduced proliferation observed in MDA-MB-231 cells were mediated by the activation of the A2BAR illustrates the importance of this receptor subtype in cancer. A2BARs must be considered as a key factor in cancer treatment and deserve attention for the development of new therapeutic strategies.
Bei vielen Erkrankungen wie z.B. Herzhypertrophie, Diabetes und Entzündungen spielt die Raf-MEK-ERK-Signalkaskade eine wichtige Rolle. ERK1/2 ist in vielen zellulären Prozessen, u.a. Proliferation, Differenzierung, Wachstum, Hypertrophie und Apoptose involviert. Auch in der Tumorentstehung besitzt dieser MAPK-Signalweg eine signifikante Funktion, da er bei ca. 50% aller Krebsarten deutlich aktiviert ist. Ziel dieser Arbeit war es, die Rolle einer neu entdeckten Phosphorylierungsstelle an Threonin188 an ERK2 bei der Entstehung und der möglichen Therapie von Tumoren zu erarbeiten. Dafür wurde ein Myc-ERK2309-357-Peptid verwendet, das 2013 in der Arbeitsgruppe Lorenz entwickelt wurde. Myc-ERK2309-357 zeigte in bisher unveröffentlichten Versuchen, dass es direkt an ERK2 bindet, eine Dimerisierung von ERK1/2 hemmen und eine vermehrte Lokalisation von ERK2 im Zellkern verhindern kann. Im Rahmen dieses Projekts konnten wir belegen, dass mit Hilfe des Myc-ERK2309-357-Peptids die Tumorzellproliferation von verschiedenen Krebszelllinien (Caco-2, SCC68, PC/1-1 und PC/13-1) um 60-80% vermindert werden konnte. Des Weiteren konnten wir zeigen, dass Myc-ERK2309-357 keinen Einfluss auf die Phosphorylierung von ERK1/2 am TEY-Motiv besitzt. Die Aktivierung von ERK1/2 durch die Kinasen MEK1/2 wird somit nicht beeinflusst und die zytosolischen ERK-Funktionen, wie z.B. der anti-apoptotische Effekt, würden somit bestehen bleiben. Außerdem fanden wir heraus, dass Myc-ERK2309-357 im Vergleich zu den MEK-Inhibitoren U0126 und PD98059 und verglichen mit dem EGF-Rezeptor-Antikörper Cetuximab die Proliferation signifikant besser hemmt.
Investigation of dynamic processes of prototypical class A GPCRs by single-molecule microscopy
(2020)
In this work, two projects were pursued.
In the first project, I investigated two different subtypes of opioid receptors, which play a key role as target for analgesia. A set of subtype specific fluorescent ligands for μ opioid receptor (MOR) and δ opioid receptor (DOR) was characterised and used to gain insights into the diffusion behaviour of those receptors. It was shown that the novel ligands hold photophysical and pharmacological properties making them suitable for single-molecule microscopy. Applying them to wild-type receptors expressed in living cells revealed that both sub-types possess a heterogeneous diffusion behaviour. Further- more, the fluorescent ligands for the MOR were used to investigate homodomerisation, a highly debated topic. The results reveal that only ≈ 5 % of the receptors are present as homodimers, and thus the majority is monomeric. G-protein coupled receptors (GPCRs) play a major role as drug targets. Accordingly, understanding the activation process is very important. For a long time GPCRs have been believed to be either active or inactive. In recent years several studies have shown, that the reality is more complex, involving more substates. [1, 2, 3, 4] In this work the α 2A AR was chosen to investigate the activation process on a single-molecule level, thus being able to distinguish also rare or short-lived events that are hidden in ensemble mea- surements. With this aim, the receptor was labelled intracellular with two fluorophores using supported membranes. Thus it was possible to acquire movies showing qualita- tively smFRET events. Unfortunately, the functionality of the used construct could not be demonstrated. To recover the functionality the CLIP-tag in the third intracellular loop was replaced successfully with an amber codon. This stop codon was used to insert an unnatural amino acid. Five different mutants were created and tested and the most promising candidate could be identified. First ensemble FRET measurements indicated that the functionality might be recovered but further improvements would be needed. Overall, I could show that single-molecule microscopy is a versatile tool to investigate the behaviour of typical class A GPCRs. I was able to show that MOR are mostly monomeric under physiological expression levels. Furthermore, I could establish intra- cellular labelling with supported membranes and acquire qualitative smFRET events.
G protein coupled receptor kinases (GRK) phosphorylate and thereby desensitize G protein coupled receptors (GPCR) including β-adrenergic receptors (βAR), which are critical regulators of cardiac function. We identified the Raf kinase inhibitor protein (RKIP) as an endogenous inhibitor of GRK2 that leads to increased cardiac contractility via βAR activation. RKIP binds to the N-terminus (aa1-185) of GRK2, which is important for the GRK2/receptor interaction. Thereby it interferes with the GRK2/receptor interaction without interference with cytosolic GRK2 target activation. In this project, the RKIP/GRK interface was investigated to develop strategies that simulate the effects of RKIP on βAR.
RKIP binding to different isoforms of GRK expressed in the heart was analyzed by protein interaction assays using full-length and N-termini of GRK2, GRK3 and GRK5: 1-53, 54-185 and 1-185. Co-immunoprecipitation (Co-IPs) and pull-down assays revealed that RKIP binds to the peptides of GRK2 and GRK3 but not to the ones of GRK5, which suggests the existence of several binding sites of RKIP within the N-termini of GRK2 and GRK3. To analyze whether the peptides of GRK2 and GRK3 are able to simulate the RKIP mediated interference of the GRK2/receptor interaction, we analyzed the β2-AR phosphorylation in the absence and presence of the peptides. Interestingly, N-termini (aa1-185) of GRK2 and GRK3 reduced β2AR phosphorylation to a comparable extent as RKIP. In line with reduced receptor phosphorylation, the peptides also reduced isoproterenol-stimulated receptor internalization as shown by [3H] CGP-12177 radioligand binding assay and fluorescence microscopy compared to control cells. Subsequently, these peptides increased downstream signaling of β2AR, i.e. the phosphorylation of the PKA substrate phosducin. In an attempt to elucidate the mechanism behind the observed effects, Co-IPs were performed in order to investigate whether the peptides bind directly to the β2-AR and block its phosphorylation by GRK2. Indeed, GRK2 1-185 and GRK3 1-185 could bind the receptor, suggesting that this way GRK2 is prevented from inhibiting the receptor. To investigate the physiological effect of GRK2 1-185, GRK3 1-185 and GRK5 1-185, their effect on neonatal mouse cardiomyocyte contractility and hypertrophy was analyzed. After long-term isoproterenol stimulation, in the presence of GRK2 1 185 and GRK3 1-185 the cross-sectional area of the cardiomyocytes showed no significant increase in comparison to the unstimulated control cells. In addition, upon isoproterenol stimulation, GRK2 1-185 and GRK3 1-185 increased the beat rate in cardiomyocytes, mimicking RKIP while the base impedance, an indicator of viability, remained stable.
The N-termini (1-185) of GRK2 and GRK3 simulated RKIP’s function and had a significant influence on β2AR phosphorylation, on its downstream signaling and internalization, could bind β2-AR, increased beat rate and did not significantly induce hypertrophy, suggesting that they may serve as a model for the generation of new and more specific targeting strategies for GRK mediated receptor regulation.
Die Raf-MEK-ERK1/2-Kaskade spielt eine wichtige Rolle in der Vermittlung von kardialer Hypertrophie und Zellüberleben. Durch unsere Arbeitsgruppe konnte im Vorfeld gezeigt werden, dass die Dimerisierung von ERK2 eine Voraussetzung für dessen Autophosphorylierung an Thr188 darstellt, welche wiederum für die Übermittlung der hypertrophen Effekten von ERK1/2 erforderlich ist. Im Rahmen dieser Arbeit wurde daraus abgeleitet die Fragestellung untersucht, ob mit Verhinderung der ERK2-Dimerisierung eine nützliche Strategie zur Inhibition von Hypertrophie vorliegt und welchen Einfluss diese auf das Zellüberleben hat.
Die Auswirkungen der Dimerisierungsdefizienz von ERK2 wurden in neonatalen Kardiomyozyten der Ratte und in transgenen Mäusen mithilfe einer ERK2-Mutante untersucht, der einige Aminosäuren in der ERK-ERK-Interaktionsfläche fehlen und daher keine Dimere bilden kann (ERK2Δ174-177). Eine Überexpression von ERK2Δ174-177 in neonatalen Kardiomyozyten verringerte signifikant die Antwort auf hypertrophe Stimuli (Phenylephrin, Endothelin 1). Im Anschluss daran wurden die Effekte der Dimerisierungsdefizienz von ERK2 in vivo an transgenen Mäusen mit kardialer Überexpression von ERK2Δ174-177 erforscht. Diese Mäuse zeigten unter basalen Bedingungen keine Unterschiede gegenüber Wildtyp-Mäusen hinsichtlich Kardiomyozytengröße, Ventrikelwanddicke und kardialer Funktion. Unter chronischer Druckbelastung mittels TAC ließ sich hingegen ein signifikant vermindertes Ausmaß an Hypertrophie im Vergleich zu Wildtyp quantifizieren. Da der ERK1/2-Signalweg auch am Überleben von Kardiomyozyten beteiligt ist, wurde die Apoptose an histologischen Schnitten von Mausherzen analysiert. Interessanterweise fand sich bei Herzen, die das dimerisierungsdefiziente ERK2-Protein überexprimierten, eine mit Wildtyp vergleichbare Anzahl TUNEL-positiver Zellen. Ein ähnliches Ergebnis konnte bei der Messung des Fibrosegrades an Sirius-Rot gefärbten histologischen Schnitten beobachtet werden. Zuletzt wurden die Folgen der ERK2-Dimerisierungsdefizienz auf physiologische Hypertrophie mit einem Laufrad-Versuchsaufbau evaluiert. Transgene ERK2Δ174-177- und Wildtyp-Mäuse zeigten unter diesem physiologischen Stimulus keine Unterschiede im Hinblick auf die Zunahme an kardialer Hypertrophie.
Da die Dimerisierungsdefizienz von ERK2 zu einer reduzierten pathologischen Hypertrophie, ohne negative Auswirkungen auf ERK1/2-vermittelte anti-apoptotische Effekte noch auf kardiale Funktion oder physiologische Hypertrophieprozesse führt, stellt die Hemmung der ERK-Dimerisierung ein attraktives Ziel zur Therapie pathologischer Hypertrophie sowie potentiell auch anderer auf den ERK1/2-Signalweg basierenden Krankheiten dar.