Hemeproteins are essential for life and heme insertion is an essential step in their maturation. 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 three key hemeproteins, inducible nitric oxide synthase (iNOS), soluble guanylyl cyclase (sGC) and hemoglobin (Hb). 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 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 dysfunctional due to it being heme deficient, but can be activated by sGC activators. Based on these exciting new findings we propose (i) to determine the molecular basis of sGC dysfunction in asthma, and the cellular mechanisms that impair or protect sGC. This includes mechanisms to determine whether a denitrosylase such as thioredoxin-1 (Trx-1) or NO scavenger Hb expressed in the apical epithelium can have a protective role for underlying airway smooth muscle sGC. (ii) Establish the molecular hallmarks of sGC dysfunction in two mouse asthma models (OVA albumin and house dust mite model [HDME]) and in human severe asthmatic HASMCs & lung tissue samples. (iii) Determine the genetic, epigenetic, and biochemical mechanisms causing the defective sGC. (iv) Explore means to restore sGC function in severe asthmatic HASMC, including therapeutic NO exposure and overexpressing beneficial proteins (Hsp90, Trx-1, Catalase) whose expression may be lowered in asthmatic HASMCs. Together our project will advance the current knowledge of how chaperones, redox enzymes, NO, and inflammation regulate sGC in healthy and asthmatic airways, and suggest ways to restore its function.
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 as biomarkers 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.
