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Claudette M. St. Croix - Research
1. The general focus of our research activities is the identification of post-translational protein modifications induced by nitric oxide (NO) related species in pulmonary endothelium, and determination of the physiological consequences of these events in vivo. Current studies concentrate on the zinc-binding protein metallothionein, which is an important target for trans-S-nitrosation, with resulting effects on intracellular zinc homeostasis, gene expression, and cellular sensitivity to toxic stressors. Specifically, we use advanced fluorescent microscopy techniques and direct gene transfer of green fluorescent protein (GFP)-modified metallothionein to study the effects of NO on metallothionein structure and intracellular zinc concentrations in living cells. We also use a variety of molecular and cell biology techniques, including Northern and Western blotting, electrophoretic mobility shift assays and live cell imaging of GFP-modified transcription factors to study the effects of NO and changes in intracellular zinc on the expression of protective genes like metallothionein.
Figure 1. We use fluorescence resonance energy transfer (FRET) techniques to monitor the interactions of nitric oxide (NO) related species with GFP-modified target proteins including the cysteine-rich heavy metal binding protein, metallothionein (MT), in live cells in real time. FRET is a physicochemical phenomenon whereby two fluorophores (i.e., donor and acceptor pair) that have appropriate spectral properties and are closely apposed (10-40 ?) transfer photon energy in a non-radiative fashion. The technique is suitable for live cells and capable of detecting changes in the conformational state of green fluorescent protein (GFP)-modified proteins. In this image, lung endothelial cells were infected with an adenoviral vector encoding the FRET-MT reporter molecule. FRET was detected in real time, using full spectral confocal imaging. The NO donors, S-nitroso-L-cysteine ethyl ester (SNCEE) caused an increase in the peak emission intensity of the donor (cyan, ~485 nm) and a decrease in that of the acceptor (yellow, ~525 nm) suggestive of nitrosothiol-mediated conformational changes in MT that are consistent with the NO-mediated release of metals from thiolate clusters of the protein shown in Figure 2.
Figure 2. Live pulmonary artery endothelial cells loaded with the zinc-specific fluorophore, Zinquin under control conditions (A), after exposure to nitric oxide donors (B), and after exposure to exogenous Zn+ (C). NO donors increased Zinquin fluorescence by 15-20%.
Figure 3. We have recently been successful in imaging Zinquin in an intact, isolated and perfused lung using multiphoton laser scanning microscopy with Dr. Simon Watkins at the Center for Biological Imaging. Figure 3 shows that baseline Zinquin fluorescence (A) was significantly increased 10 min following the addition of exogenous zinc to the perfusate (B) and attenuated by the zinc chelator, TPEN (C).