CCTη, a Novel Soluble Guanylyl Cyclase-interacting Protein

Abstract

Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC results in the production of cGMP from GTP. In this paper, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit and the ␣ 1 ␤ 1 isoform of sGC. CCT was found to interact with the ␤ 1 subunit of sGC via a yeast-two-hybrid screen. This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain. The interaction between these two proteins was further supported by a co-localization of the proteins within rat brain. Using the yeast two-hybrid system, CCT was found to bind to the N-terminal portion of sGC. In vitro assays with purified CCT and Sf9 lysate expressing sGC resulted in a 30 -50% inhibition of diethylamine diazeniumdiolate-NO-stimulated sGC activity. The same assays were then performed using BAY41-2272, an NOindependent allosteric sGC activator, and CCT had no effect on this activity. Furthermore, CCT had no effect on basal or sodium nitroprusside-stimulated ␣␤ Cys-105 sGC, a constitutively active mutant that only lacks the heme group. The N-terminal 94 amino acids of CCT seem to be critical for the mediation of this inhibition. Lastly, a 45% inhibition of sGC activity by CCT was seen in vivo in BE2 cells stably transfected with CCT and treated with sodium nitroprusside. These data suggest that CCT binds to sGC and, in cooperation with some other factor, inhibits its activity by modifying the binding of NO to the heme group or the subsequent conformational changes.Nitric oxide (NO), 1 a simple diatomic gas, plays anything but a simple role in signal transduction. NO is produced by the enzyme nitric-oxide synthase. The main target of NO, mediating most of its downstream effects, is a cytosolic heterodimer of ␣ and ␤ subunits known as soluble guanylyl cyclase (sGC). Upon NO binding, the activated sGC catalyzes the formation of cGMP from GTP (1-3). Cyclic GMP is then involved in the activation of a variety of effectors such as cyclic nucleotidegated channels, protein kinases, and phosphodiesterases (4).There has been evidence suggesting that there are in vivo mechanisms other than nitric oxide that regulate sGC activity. Bellamy et al. (5) show that deactivation of sGC on the removal of NO occurred 25-fold faster in intact cerebellar cells than the fastest estimate for purified sGC. These data suggest that there is probably another protein or group of proteins that regulate sGC activity, because it occurs in the order of seconds.In addition to the indirect evidence of protein regulation of sGC provided by Bellamy et al. (5), PSD95 (post-synaptic density protein 95) (6) and heat shock protein 90 (HSP90) (7) have both been shown to interact with isoforms of soluble guanylyl cyclase. Both PSD95 and HSP90 were shown to localize different isoforms of sGC to the membrane. Additionally, HSP90 was shown to enhance the response of sGC to NO. In both of these cases, the interaction ...

α1-A680T Variant in GUCY1A3 as a Candidate Conferring Protection From Pulmonary Hypertension Among Kyrgyz Highlanders

“…In addition, heat shock protein 90 and CCTη are also thought to be interacting with sGC in vivo. 28,29 A recent study demonstrated that loss of function mutations in α 1 -sGC and CCTη are associated with myocardial infarction. 30 We hypothesize that the physiological effects of the α 1 -A680T sGC variant are mediated by the combined effect of a subtle increase in NO sensitivity as well as changes in protein-protein interactions or localization in cells.…”

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.…”

Molecular cloning and characterization of α1-soluble guanylyl cyclase gene promoter in rat pituitary cells

“…The human isoforms isolated from an adult brain library, termed a3 and b3, represent the human homologues of rat and bovine a1 and b1 (Zabel et al 1998). The a1 and a2 subunits are interchangeable in terms of sGC activity when coexpressed with b1, but the alb1 heterodimer is most common in mammalian tissues (Hanafy et al 2004). The dimer activity is regulated directly at protein level.…”

“…The enzyme is also subjected to phosphorylation by protein kinase, src-like kinases, and protein kinases C and G (Pyriochou & Papapetropoulos 2005). Proteinprotein interactions also contribute to the direct control of sGC activity, since documented by interaction between the chaperonin containing t-complex polypeptide subunit h and b1-sGC, which leads to inhibition of the enzyme activity (Hanafy et al 2004).…”