Mercator Project: Redox engineering tools and application of biosensors in vivo
Mercator Fellow: V.V. Belousov
Within the Mercator project we are aiming at developing new in vivo and in vitro tools for unravelling the importance of redox biology for cardiomyocyte physiological and pathophysiological functions. This includes establishing mouse models expressing engineered tools for studying the consequences of subcellular localized production of H2O2 for cardiomyocyte function as well as new sensitive biosensors, which allow localized and specific detection of H2O2.
Redox engineered cardiomyocyte-specific mice
A central principle of synthetic biology is engineered trans-species or even trans-kingdom transfer of molecular blocks from the "donor" organism to the "acceptor" one to bring to the acceptor organism some new properties. We aim to apply synthetic biology principles to design new redox engineering tools for the application in the cardiovascular system. Many prokaryotic and fungal species metabolize substrates normally not present in higher eukaryotes. One of such substrate classes is D-amino acids (D-aa) that are present in vertebrates in very small amounts. Bacteria, archaea and fungi can use D-aa in biochemical reactions catalysed by stereospecific oxidases, dehydrogenases, aminotransferases etc.. Amino acids in mammalian cells are almost exclusively L-type and the enzymes utilizing amino acids are highly stereospecific. Therefore, the enzymes utilizing D-aa, if placed in mammalian cells, remain inactive in the absence of D-aa substrate, but upon addition of D-aa substrate these enzymes would catalyze specific reactions. The main important point is that other secondary substrates and cofactors of D-aa utilizing enzymes are shared between species. This applies to the D-aa oxidase (DAO). DAO catalyzes the deamination of D-aa using oxygen as a secondary substrate and produces H2O2 (Pollegioni et al.,J. Mol. Biol., 324, 2002, 535-546; Lee and Chu, Lett. Appl. Microbiol., 23, 1996, 283–286). We successfully utilized this enzyme for controlled production of H2O2 in mammalian cells. The activity of the expressed enzyme in cells is controlled using external additions of D-alanine (D-ala) as the substrate for DAO. D-ala is a specific substrate for DAO, and in the absence of it, no H2O2 is produced. Since HydrogenPeroxide (HyPer) is a suitable probe to detect H2O2 levels, it was coupled to the DAO enzyme. The DAO/HyPer fusion protein allows the determination of the dynamics of H2O2 production and the sensitivity to externally added D-ala in the living cells. This answers the question of how much H2O2 is produced by DAO in mammalian cells. Even with a millimolar excess of added D-ala, the concentration of H2O2 produced by DAO in cells does normally not exceed 50 - 200 nM (Matlashov et al., Antioxid. Redox Signal., 20, 2014, 1039-1044).
In order to better understand cardiac myocytes physiologically under various levels of H2O2 and various locations of its production we aim to generate transgenic mouse models expressing DAO-HyPer2-NLS, DAO-HyPer2-NES and DAO-HyPer2-mito located to the nucleus, the cytoplasm, and the mitochondrial matrix respectively, in a cardiomyocytes restricted fashion using the αMHC promoter. All three compartments have a distinct redox regulation. To unravel the physiological importance of this compartmentalization it is crucial to be able to generate H2O2 locally. Our preliminary results using cell culture demonstrate that patterns of H2O2 migration clearly differ in case of intranuclear versus cytoplasmic H2O2 production by DAO. For the planned mouse models HyPer2 fused to DAO will be used as i) a marker of proper intracellular localization of DAO and ii) as a readout of H2O2 generation. HyPer2, but not HyPer or HyPer3, will be used because of higher brightness in vivo. Administering mice, Langendorff-perfused hearts or isolated cardio-myocytes with D-Ala we will be able to change oxidative load in cardiac myocytes in vivo. Mice will be phenotyped regarding heart function after induction of localized H2O2 production. The mouse models will be additionally used to study the physiological role of compartmentalized production of H2O2 for Ca2+ cycling. H2O2 application is known to increase intracellular Ca2+ release from SR through cardiac RyR2, the major Ca2+ source in cardiac myocytes. Furthermore, abnormally increased SR Ca2+ leak is a well-established and central pathogenetic mechanism of intracellular Ca2+ overload in different forms of heart disease. Recently, cysteine based redox modifications of RyR2 channels were observed in chronic forms of heart disease and shown to correlate with increased SR Ca2+ leak. However, the subcellular domains that produced increased redox equivalents, in particular H2O2, which lead to RyR2 channel modification are not known. Furthermore, RyR2 dependent Ca2+ leak has been proposed to cause mitochondrial Ca2+ overload as a mechanism of increased mitochondrial H2O2 production, which can reciprocally increase RyR2 channel dysfunction. Hence, targeting of DAO-HyPer2 biosensors to the nucleus, cytosol versus mitochondria will be instrumental to characterize the sources of RyR2 channel redox modifications and to develop interventions, which may inhibit the detrimental reciprocal interactions therapeutically.
New generation of redox biosensors
Recently we generated a new family of H2O2 probes of enhanced brightness, pH stability and sensitivity (unpublished results). Blue, green and red probes can be used for multicolor imaging of H2O2 simultaneously in various compartments. Out of three different green sensors one has enhanced dynamic range and the other has 10x higher sensitivity compared to previous HyPer ge-nerations. All the green sensors are brighter compared to EGFP. The red fluorescent probe is absolutely pH-stable in a physiological pH range. All the probes exist in intensiometric and ra-tiometric versions. We aim to generate adenoviral vectors expressing nuclear, cytosolic and mitochondrial versions of the new probes to test them in cardiomyocytes and cardiac fibroblasts. This source will be used as pipeline for feeding new biosensor probes into the IRTG 1816 projects in Göttingen and London. Doctoral researchers will directly benefit from these new technical deve-lopments and will be trained to use the new generation of probes. Probes that will show the best performance in situ will be used in future for mouse transgenesis similar to DAO-HyPer2 constructs.
IRTG 1816 Mercator Fellow from Moscow State University, Russia
Research interests: Biochemistry, redox engineering