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  • Renal disease is characterized by aberrant fibrotic remodeli

    2021-10-12

    Renal disease is characterized by aberrant fibrotic remodeling of renal tissue, elevated apoptosis in kidney tissue, and eventual organ failure. In addition, patients with renal disease are more likely to suffer from systemic hypertension due to oxidative damage (reviewed in Ref. [18]). Treatment of rats with progressive renal fibrosis using GC-1 stimulator BAY 41-2272 elevated cGMP levels and reduced kidney fibrosis and systemic blood pressure [19]. Rat models of renal disease induced by a high-salt diet were given cinaciguat. After 21 weeks, increased cGMP levels were measured and renal function was improved over the placebo group [20]. NO supplementation has also been used to improve renal function. After 1 week of supplemented dietary arginine, the substrate for NO synthesis, increased cGMP levels were measured in urine, improved renal function and reduced renal fibrosis and apoptosis [21].
    GC-1 domain architecture GC-1 is a 150 kDa heterodimer and its α1β1 isoform is found ubiquitously. While another isoform of the enzyme does exist (GC-2; α2β1) [22], this review will focus on the predominant α1β1 isoform. Each α/β polypeptide contains four domains from N- to C-terminus connected by short linkers: NO-sensor domain, PAS domain, coiled-coil domain, and catalytic domain (Fig. 2). The N-terminal NO-sensor domain is predicted to adopt a structure similar to bacterial heme nitric oxide-oxygen binding (HNOX) proteins (Fig. 2). These proteins are typically standalone proteins and use a heme cofactor to sense gaseous ligands and activate response proteins to elicit the desired effect, usually through gene regulation [23]. While αHNOX and βHNOX domains are predicted to share a similar HNOX-like fold, only the βHNOX domain of GC-1 carries a heme prosthetic group that binds NO; the role of αHNOX remains to be determined, however recent work suggests it regulates GC-1 activity by lowering the affinity of βHNOX for CO and NO [24]. βHNOX uses a His ligand to bind the heme cofactor in its reduced Fe2+ redox state. NO binding to the distal heme side severs the His-iron bond, which is thought to play a crucial role in NO-induced stimulation of GC-1 [25,26]. In addition, several groups have found that excess NO is required to fully activate GC-1 [27,28]. The proximal side of the heme [28,29] or Cys residues [30,31] have been suggested as potential PPACK Dihydrochloride synthesis for the extra NO. However, recent spectroscopic data in Shewanella oneidensis HNOX shows that the distal site is preferred, thus questioning the biological significance of proximal heme-NO binding [32]. The subsequent domain belongs to the Per-Arnt-Sim (PAS) family of proteins, typically used to transduce signals to effector domains through subtle conformational shifts. While some PAS domains use cofactors for signal transduction, the PAS domain in GC-1 has none. Crystal structures of a bacterial homolog [33] and of the GC-1 αPAS domain [34] have been solved and show the predicted fold consisting of six β-sheets surrounded by several short α helices (Fig. 2). The GC-1 PAS domain has been suggested to play a key role in GC-1 dimerization [33]. More recently, the PAS domain was shown to interact with heat-shock protein 90 and mediate heme insertion in the βHNOX domain [35]. Crystallographic studies showed that the coiled-coil (CC) domain folds as long α-helices (Fig. 2) in an antiparallel orientation [36]. However cross-linking studies later demonstrated that the heterodimeric CC assembles in a parallel orientation [37]. The CC domain has been shown to play a key role in GC-1 heterodimerization [38], and to act as a scaffold for other GC-1 domains [37,39]. The C-terminal catalytic domain (αβGCcat) contains the substrate-binding pocket located at the interface of the α and β subunits, which both contribute key residues for GTP binding (Fig. 2). Several structures of apo inactive cyclase domains from bacteria, algae, fungus, and human have been solved, but a structure of the holo active form remains elusive [[40], [41], [42], [43], [44]]. Several groups have reported a high propensity for ββGCcat homodimers to form both in solution and during crystallization attempts [42,43]. Despite these structural characterizations, several key questions remain about the αβGCcat domain: what is the mechanism by which αβGCcat transitions from the inactive apo conformation to the catalytically-active conformation upon NO binding? What are the residues involved in the transition from inactive to active αβGCcat? How is the NO-activating signal transduced from the N-terminal NO-sensor domain to the C-terminal catalytic domain? Answering these questions will aid in the design of novel therapeutics that target GC-1 and promote NO-sensitization and cGMP generation.