Inactivation of soluble guanylyl cyclase in living cells proceeds without loss of haem and involves heterodimer dissociation as a common step

Abstract

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 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. Conclusion and Implications Oxidative (ODQ) or nitrosative (NOC12) inactivation of cell sGC involves sGC heterodimer dissociation and rearrangement of the sGCβ protein partners without any haem loss from sGCβ. Clarifying what changes do and do not occur to sGC during its inactivation in cells may direct strategies to preserve or recover NO‐dependent cGMP signalling in health and disease. LINKED ARTICLES This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc

NO rapidly mobilizes cellular heme to trigger assembly of its own receptor

“…BAY41, the sGC activator BAY58, and the A23187 calcium ionophore were purchased from Sigma-Aldrich. All other reagents and materials were obtained from sources reported elsewhere ( 20 , 23 ).…”

“…These fall into two groups: those that only activate the heme-containing, NO-responsive, mature sGC, as exemplified by BAY 41-2272 (BAY41) ( 21 ), and those that only activate the heme-free, NO-unresponsive sGC, as exemplified by BAY 58-2667 (BAY58) ( 22 ). Studies with these compounds and related biochemical data have indicated that cells and tissues typically contain a significant level of immature heme-free (apo) sGC even in normal healthy conditions ( 17 , 23 ), with estimates indicating apo-sGCβ represents from 40 to 80% of the total sGC. Why cells and tissues maintain such high levels of heme-free NO-insensitive sGC is puzzling, and how they might convert their apo-sGCβ to an active sGC is currently unclear, but answering these questions would improve our understanding of NO–sGC–cGMP signaling in biology.…”

“…This provides a way to measure the sGC heme level that has advantages over other methods that are less direct and have inherently poor time resolution (i.e., measuring sGC catalytic activity, heterodimer level, or level of [ 14 C]heme). We have recently utilized FlAsH-labeled TC-sGCβ to monitor real-time changes to its heme content in living cells, and in doing so have discovered that GAPDH is involved in sGC heme delivery ( 20 ), uncovered molecular details of the hsp90-driven sGC heme insertion rection ( 17 , 19 ), and defined how sGC inactivation impacts its heme content in living cells ( 23 ). In our current study, we utilized FlAsH–TC-sGCβ to study how physiologic levels of NO might impact its heme content and maturation.…”

Decoding signaling mechanisms: unraveling the targets of guanylate cyclase agonists in cardiovascular and digestive diseases

“…In brief, sGC contains a heme moiety, which is either ferrous (reduced sGC) or ferric (oxidized sGC). In these forms, the sGC stimulator can only target the reduced, heme-containing sGC, whereas the sGC activator binds to the oxidized or heme-free sGC, resulting in increased production of cGMPs ( Dai and Stuehr, 2022 ). Although sGC activators stimulate heme-containing enzymes independently of NO, NO enhances their activity; even when oxidative stress occurs, cGMP is released by sGC activators ( Dai and Stuehr, 2022 ).…”

Recent developments in cGMP research: From mechanisms to medicines and back

“…Clarifying what changes occur to NO‐GC during its inactivation in living cells may direct strategies to preserve or recover NO‐dependent cGMP signalling in disease. In this issue, Dai and Stuehr (2022) studied NO‐GC inactivation triggered by the oxidant ODQ or by the NO donor NOC12 in HEK293 cells expressing a fluorescent NO‐GC reporter construct allowing tracking of its haem content. They found that either inactivation regime led to dissociation of the NO‐GC α/β‐heterodimer and binding of the β subunit to heat shock protein 90.…”