Soluble guanylyl cyclase requires heat shock protein 90 for heme insertion during maturation of the NO-active enzyme

Abstract

Heme insertion is key during maturation of soluble guanylyl cyclase (sGC) because it enables sGC to recognize NO and transduce its multiple biological effects. Although sGC is often associated with the 90-kDa heat shock protein (hsp90) in cells, the implications are unclear. The present study reveals that hsp90 is required to drive heme insertion into sGC and complete its maturation. We used a mammalian cell culture approach and followed heme insertion into transiently and endogenously expressed heme-free sGC. We used pharmacological hsp90 inhibitors, an ATP-ase inactive hsp90 mutant, and heme-dependent or heme-independent sGC activators as tools to decipher the role of hsp90. Our findings suggest that hsp90 complexes with apo-sGC, drives heme insertion through its inherent ATPase activity, and then dissociates from the mature, heme-replete sGC. Together, this improves our understanding of sGC maturation and reveals a unique means to control sGC activity in cells, and it has important implications for hsp90 inhibitor-based cancer therapy.

Nitric Oxide and Heat Shock Protein 90 Activate Soluble Guanylate Cyclase by Driving Rapid Change in Its Subunit Interactions and Heme Content

“…In all cases, the cells were treated with cycloheximide (10 g/ml) for 30 min before sGC activation. To inhibit heme biosynthesis and deplete stores of intracellular heme, 400 M SA was added to the cells 48 h prior to transfection or to activation of sGC (14). In such cases, the heme depletion was followed either by transient transfection or sGC activation.…”

“…Western Blots and Immunoprecipitations-Standard protocols were followed as mentioned previously (14). For immunoprecipitations, 500 g of the total cell supernatant was precleared with 20 l of protein G-Sepharose beads (Amersham Biosciences) for 1 h at 4°C, beads were pelleted, and the supernatants incubated overnight at 4°C with 3 g of anti-V5 or anti-sGC-␤1 antibody.…”

“…We recently found that hsp90 drives heme insertion into sGC during its maturation in cells (14). Hsp90 is bound primarily to the heme-free sGC-␤1 subunit in cells, drives heme insertion into the apo-sGC-␤1 in an ATP-dependent process, and then dissociates afterward.…”

Regulation of heme biosynthesis and transport in metazoa

“…Heme insertion into sGC was found to be mediated by HSP90, which preferentially interacts with apo-sGC [77]. HSP90-mediated heme incorporation converts apo-sGC into a mature active form that is responsive to NO [77]. The insertion of heme into sGC is positively regulated by NO [89].…”

“…The product of iNOS, nitric oxide (NO), also inhibited heme insertion into iNOS through S-nitrosylation of GAPDH [76]. Heme insertion into sGC was found to be mediated by HSP90, which preferentially interacts with apo-sGC [77]. HSP90-mediated heme incorporation converts apo-sGC into a mature active form that is responsive to NO [77].…”