Characterization of a Novel Type of Endogenous Activator of Soluble Guanylyl Cyclase

Abstract

Nitric oxide (NO) remains the only firmly established endogenous modulator of soluble guanylyl cyclase (sGC) activity, but physiological, structural, and biochemical evidence now suggests that in vivo regulation of sGC involves direct interaction with other factors. We searched for such endogenous modulators in human umbilical vein endothelial cells and COS-7 cells. The cytosolic fraction of both cell types stimulated the activity of semipurified sGC severalfold in the absence or presence of a saturating concentration of NO. The cytosolic factor was sensitive to proteinase K and destroyed by boiling, suggesting that it contains a protein component. Size exclusion chromatography revealed peaks of activity between 40 and 70 kDa. The sGC-activating effect was further purified by ion exchange chromatography. In the presence of the benzylindazole YC-1 or NO, the partially purified factor synergistically activated sGC, suggesting that this factor had a mode of activation different from that of YC-1 or NO. Four candidate activators were identified from the final purification step by matrix-assisted laser desorption ionization mass spectrometry analysis. Using an sGC affinity matrix, one of them, the molecular chaperone Hsp70, was shown to directly interact with sGC. This interaction was further confirmed by co-immunoprecipitation in lung tissues and by co-localization in smooth muscle cells. sGC and Hsp70 co-localized at the plasma membrane, supporting the idea that sGC can be translocated to the membrane. Hsp70 co-purifies with the sGC-activating effect, and immunodepletion of Hsp70 from COS-7 cytosol coincided with a marked attenuation of the sGC-activating effect, yet the effect was not rescued by the addition of pure Hsp70. Thus, Hsp70 is a novel sGC-interacting protein that is responsible for the sGC-activating effect, probably in association with other factors or after covalent modification.

Interaction between the 90-kDa Heat Shock Protein and Soluble Guanylyl Cyclase: Physiological Significance and Mapping of the Domains Mediating Binding

“…In the case of sGC, CCT interacts with ␤ 1 -inhibiting NO-stimulated sGC activity (Hanafy et al, 2004), whereas interaction of sGC with hsp70 increases the cGMP-forming ability of the cyclase (Balashova et al, 2005). Subcellular localization of sGC is also regulated by protein-protein interactions.…”

Acrylamide Analog as a Novel Nitric Oxide-Independent Soluble Guanylyl Cyclase Activator

“…On the other hand, the heme-independent activator BAY58-2667 activates sGC even after it has been oxidized by ODQ or rendered heme-deficient (22,39,40), and its effect is additive rather than synergistic activation with NO. Recently, Hsp70 was found to be a sGC-interacting protein that is responsible for the activating effect in association with other NO-independent activators (41). A-778935 increased cGMP level dose-dependently in smooth muscle cells (Fig.…”

Inhibition of Proinflammatory Tumor Necrosis Factor-α-Induced Inducible Nitric-Oxide Synthase by Xanthine-Based 7-[2-[4-(2-Chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-Nitrobenzene)piperazinyl]ethyl]-1, 3-dimethylxanthine (KMUP-3) in Rat Trachea: The Involvement of Soluble Guanylate Cyclase and Protein Kinase G

“…Two xanthine-or imidazole-based derivatives, KMUP-1 and KMUP-3, have been found to increase the expression of cGMP and PKG expression (Wu et al, 2004;Lin et al, 2006), which might make them useful in the treatment of pulmonary airway dysfunctions. In comparison, the more previously discovered indazole derivatives YC-1 and BAY-41-2272 have been shown to activate sGC by a binding manner, leading to increases of cGMP (Ko et al, 1994;Chang et al, 2004;Balashova et al, 2005). The chemical relationship between KMUP-1/KMUP-3 and YC-1/BAY-41-2272 were thus noted for their sGC activation, PKG expression, and cGMP-enhancing activities.…”