Heat shock protein 90 regulates soluble guanylyl cyclase maturation by a dual mechanism

Abstract

Edited by Ruma Banerjee The enzyme soluble guanylyl cyclase (sGC) is a heterodimer composed of an ␣ subunit and a heme-containing ␤ subunit. It participates in signaling by generating cGMP in response to nitric oxide (NO). Heme insertion into the ␤1 subunit of sGC (sGC␤) is critical for function, and heat shock protein 90 (HSP90) associates with heme-free sGC␤ (apo-sGC␤) to drive its heme insertion. Here, we tested the accuracy and relevance of a modeled apo-sGC␤-HSP90 complex by constructing sGC␤ variants predicted to have an impaired interaction with HSP90. Using site-directed mutagenesis, purified recombinant proteins, mammalian cell expression, and fluorescence approaches, we found that (i) three regions in apo-sGC␤ predicted by the model mediate direct complex formation with HSP90 both in vitro and in mammalian cells; (ii) such HSP90 complex formation directly correlates with the extent of heme insertion into apo-sGC␤ and with cyclase activity; and (iii) apo-sGC␤ mutants possessing an HSP90-binding defect instead bind to sGC␣ in cells and form inactive, heme-free sGC heterodimers. Our findings uncover the molecular features of the cellular apo-sGC␤-HSP90 complex and reveal its dual importance in enabling heme insertion while preventing inactive heterodimer formation during sGC maturation.

Maturation, inactivation, and recovery mechanisms of soluble guanylyl cyclase

“…In mammalian cells heme insertion into apo-sGCβ also depends on Hsp90. Heme insertion requires that Hsp90 has intact ATP-ase activity ( 52 ) and relies on direct interaction between Hsp90 and apo-sGCβ ( 76 ) ( Fig. 2 ).…”

New Insights Into Heat Shock Protein 90 in the Pathogenesis of Pulmonary Arterial Hypertension

“…Therefore, we mainly present a brief overview to provide their basic results for understanding the Hsp90 impact on sGC under physiological and pathological conditions. Hsp90 regulates sGC, involving the association of Hsp90 MD with two regions of sGC, by a dual mechanism ( Papapetropoulos et al, 2005 ; Dai et al, 2019 ). On the one hand, Hsp90 drives heme insertion into apo-sGC β1 after forming a complex.…”

“…Once heme insertion is complete, Hsp90 dissociates from sGC β1, which partners with sGC α1 to form a mature and functional sGC heterodimer. On the other hand, apo-sGC β1 associates with Hsp90 much more strongly than sGC α1, which prevents the formation of heme-free and non-functional sGC heterodimer ( Dai et al, 2019 ). It is well established that oxidative stress, which plays a major role in the development of pulmonary vascular remodeling and a consequent increase of pulmonary pressure, can cause the oxidation of the sGC heme resulting in desensitization to NO signaling ( Shah et al, 2018 ).…”

“…NO stimulates the insertion of heme in apo-sGC-β1, the dissociation of Hsp90 from the complex, and the association of sGC-α1 to form the active enzyme ( Ghosh et al, 2014 ). It seems that impaired NO and the upregulation of Hsp90 in PAH break the insertion of heme into sGC-β1 and the equilibrium between apo-sGC-β1 and holo-sGC-β1 ( Ghosh et al, 2014 ; Dai et al, 2019 ). However, how this equilibrium is managed remains to be elucidated.…”

“…In contrast, sGC activators increase enzymatic activity of the oxidized heme or heme-deficient apo-sGC without relying on NO. The pharmacologic sGC activators were found to mimic all the changes in sGC β1 protein associations and consequent increase in the sGC heterodimer formation ( Dai et al, 2019 ). Thus, inhibition of Hsp90 may further enhance the efficacy of sGC activators to treat PAH by increasing the levels of heme-free sGC ( Figure 4 ).…”