Hemeproteins are essential for life, and heme insertion is an essential step in their maturation and function. Although the mechanisms by which mammals insert heme during hemeprotein maturation are mostly unknown, studies from our group uncovered a specific involvement of the chaperon hsp90 in heme insertion into four key hemeproteins, inducible nitric oxide synthase (iNOS), soluble guanylyl cyclase (sGC), hemoglobin (Hb) and myoglobin (Mb). Our studies indicate that a strong sGC-hsp90 interaction can be a measure of heme-free sGC in cells and that this interaction is mutually exclusive with respect to sGC-subunit heterodimerization. Together, these findings have potential applications in the clinical diagnosis of diseased conditions where sGC is dysfunctional. We discovered that sGC becomes dysfunctional in inflammatory asthma under elevated nitric oxide (NO), which impedes the NO-based bronchodilation, but can be overcome by sGC activators which can induce bronchodilation despite this loss. Such sGC dysfunction in asthma is associated with a strong molecular signature of sGC dysfunction which comprises of a weak sGC-?1?1 heterodimer, a strong sGC?1-hsp90 interaction and a high S-nitrosylation (SNO) on sGC-?1. Our current and past studies have revealed that NO levels are critical in biology and can act both ways to make or break sGC. While high NO levels as in asthma can induce sGC dysfunction by breaking the sGC-?1?1 heterodimer, low NO levels can trigger heme-insertion in sGC-?1, increasing and stabilizing the sGC heterodimer. Moreover in human asthmatic ASMCs (airway smooth muscle cells), our studies suggest that sGC is unresponsive to NO due to it being heme deficient, but can be activated by sGC activators. Based on these exciting new findings we propose (i) molecular and cellular- level studies to define mechanisms to understand how sGC becomes dysfunctional under high NO as in asthma. This includes mechanisms to determine whether a denitrosylase such as thioredoxin-1 (Trx-1) or Hb present in the apical epithelium (as A549 cells express Hb in our new find) can have a protective role for underlying airway smooth muscle sGC. (ii) Establish, whether a defective sGC heme exists in asthmatic HASMCs (human airway smooth muscle cells) or in mouse models of asthma (OVA, CFA/HDME) causing defective bronchodilation, explore the basis of this heme-deficient sGC and firmly establish the molecular signatures of sGC dysfunction in human asthma, such that this can be applied in future as a dysfunction indicator of sGC in blood platelets of live asthma patients. (iii) Finally coupling the effect of low NO levels in inducing sGC heterodimerization, and overexpressing enzymes which are downregulated (Hsp90, Trx-1, Catalase) in asthmatic HASMCs, we propose to restore sGC dysfunction in such HASMCs that display a predomiant heme-free sGC phenotype. Together our project will advance the current knowledge of how hsp90, NO and inflammation can regulate sGC maturation, and will provide new information on sGC maturation in healthy and asthmatic airways.
Our current findings provide new insight on how the sGC signaling enzyme becomes dysfunctional toward nitric oxide (NO) in inflammatory asthma. Our findings reveal a clearly defined molecular signature of sGC dysfunction in asthma that impedes the NO-based bronchodilation, which can be overcome by sGC activators by inducing bronchodilation and such sGC activators could be new bronchodilators for asthma. Our proposal stands to reveal cellular mechanisms by which sGC becomes dysfunctional in asthma, establish the hallmarks of sGC dysfunction in human asthma, will explore pathways to restore sGC dysfunction and better stabilize the lung sGC heterodimer in airway smooth muscle cells (ASMCs) from asthma.
