Background and purpose
Nitric oxide (NO) activates soluble guanylyl cyclase (sGC) for cGMP production, but in disease, sGC becomes insensitive towards NO activation. What changes occur to sGC during its inactivation in cells is not clear. Experimental approach: We utilized HEK293 cells expressing sGC proteins to study the changes that occur regarding its haem content, heterodimer status and sGCβ protein partners when the cells were given the oxidant ODQ or the NO donor NOC12 to inactivate sGC. Haem content of sGCβ was monitored in live cells through use of a fluorescent-labelled sGCβ construct, whereas sGC heterodimer status and protein interactions were studied by Western blot analysis. Experiments with purified proteins were also performed. Key
Purpose
Nitric oxide (NO) activates soluble guanylyl cyclase (sGC) for cGMP production, but in disease, sGC becomes insensitive towards NO activation. What changes occur to sGC during its inactivation in cells is not clear. Experimental approach: We utilized HEK293 cells expressing sGC proteins to study the changes that occur regarding its haem content, heterodimer status and sGCβ protein partners when the cells were given the oxidant ODQ or the NO donor NOC12 to inactivate sGC. Haem content of sGCβ was monitored in live cells through use of a fluorescent-labelled sGCβ construct, whereas sGC heterodimer status and protein interactions were studied by Western blot analysis. Experiments with purified proteins were also performed. Key
Results
ODQ- or NOC12-driven inactivation of sGC in HEK293 cells was associated with haem oxidation (by ODQ), S-nitrosation of the sGCβ subunit (by NOC12), sGC heterodimer breakup and association of the freed sGCβ subunit with cell chaperone Hsp90. These changes occurred without detectable loss of haem from the sGCβ reporter construct. Treating a purified ferrous haem-containing sGCβ with ODQ or NOC12 caused it to bind with Hsp90 without showing any haem loss.