ABSTRACT
Oxytocin is a 9-amino acid peptide hormone. Since its discovery in 1954, it has most commonly been studied in relation to its role in stimulating parturition and lactation. However, it is now known that oxytocin has a widely diverse set of functions throughout the body including neuromodulation, bone growth, and inflammation. Previous research has suggested that divalent metal ions may be required for oxytocin activity, but the exact metal species and specific pathways have yet to be fully elucidated. In this work, we focus on characterizing copper and zinc bound forms of oxytocin and related analogs through far-UV circular dichroism. We report that Cu(ii) and Zn(ii) bind uniquely to oxytocin and all analogs investigated. Furthermore, we investigate how these metal bound forms may affect downstream signaling of MAPK activation upon receptor binding. We find that both Cu(ii) and Zn(ii) bound oxytocin attenuates the activation of the MAPK pathway upon receptor binding relative to oxytocin alone. Interestingly, we observed that Zn(ii) bound forms of linear oxytocin facilitate increased MAPK signaling. This study lays the foundation for future work on elucidating the metal effects on oxytocin's diverse bioactivity.
ABSTRACT
The development of bioluminescence-based tools has seen steady growth in the field of chemical biology over the past few decades ranging in uses from reporter genes to assay development and targeted imaging. More recently, coelenterazine-utilizing luciferases such as Gaussia, Renilla, and the engineered nano-luciferases have been utilized due to their intense luminescence relative to firefly luciferin/luciferase. The emerging importance of these systems warrants investigations into the components that affect their light production. Previous work has reported that one marine luciferase, Gaussia, is potently inhibited by copper salt. The mechanism for inhibition was not elucidated but was hypothesized to occur via binding to the enzyme. In this study, we provide the first report of a group of nonhomologous marine luciferases also exhibiting marked decreases in light emission in the presence of copper (II). We investigate the mechanism of action behind this inhibition and demonstrate that the observed copper inhibition does not stem from a luciferase interaction but rather the chemical oxidation of imidazopyrazinone luciferins generating inert, dehydrated luciferins.
Subject(s)
Copper , Luciferases, Firefly , Copper/pharmacology , Luciferases/genetics , Oxidation-Reduction , Luciferases, Firefly/metabolism , Firefly Luciferin , Luminescent Measurements/methods , LuminescenceABSTRACT
[This corrects the article DOI: 10.1039/D1SC07177G.].
ABSTRACT
Copper is an essential redox-active metal that plays integral roles in biology ranging from enzymatic catalysis to mitochondrial respiration. However, if not adequately regulated, this redox activity has the potential to cause oxidative stress through the production of reactive oxygen species. Indeed, the dysregulation of copper has been associated with a variety of disease states including diabetes, neurodegenerative disorders, and multiple cancers. While increasing tools are being developed for illuminating labile intracellular copper pools and the trafficking pathways in which they are involved, significantly less attention has been given to the analogous extracellular labile pool. To address this gap, we have developed a bioluminescence-based imaging probe, picolinic ester caged-diphenylterazine (pic-DTZ) for monitoring labile, extracellular copper using a coelenterazine-like imidazopyrazinone and the genetically-engineered, marine-based luciferase, nanoluciferase. Unlike the more commonly-used firefly luciferase, nanoluciferase does not require ATP, allowing its application to the extracellular milieu. pic-DTZ demonstrates high metal and oxidation state selectivity for Cu(ii) in aqueous buffer as well as selectivity for labile pools over coordinatively inaccessible protein-bound Cu(ii). We demonstrate the potential of pic-DTZ as a diagnostic tool in human serum and plasma for copper-associated diseases. Additionally, we apply pic-DTZ to lend insight into the extracellular copper dynamic in anticancer treatments.
ABSTRACT
This work reports a new ATP-independent bioluminescent probe (bor-DTZ) for detecting hydrogen peroxide that is compatible with the Nanoluciferase enzyme. The probe is designed with an arylboronate ester protecting group appended to a diphenylterazine core via a self-immolative phenolate linker. Reaction with hydrogen peroxide reveals diphenylterazine, which can then react with Nanoluciferase to produce a detectable bioluminescent signal. Bor-DTZ shows a dose-dependent response to hydrogen peroxide and selectivity over other biologically relevant reactive oxygen species and can be applied to detect either intra- or extracellular species. We further demonstrate the ability of this platform to monitor fluxes in extracellular hydrogen peroxide in a breast cancer cell line in response to the anticancer treatment, cisplatin.