C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

Abstract

It is established that suicide inactivation of neuronal nitricoxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind L-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N G -nitro-L-arginine, but not its inactive D-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.Nitric-oxide synthases (NOS) are cytochrome P450-like hemoprotein enzymes that catalyze the conversion of L-arginine to nitric oxide and citrulline by a process that requires NADPH and molecular oxygen (1). There are three major mammalian isoforms as follows: neuronal NOS (nNOS), 2 endothelial NOS, and inducible NOS. NOS is bidomain in structure with an oxygenase domain, which contains the binding site for the heme, L-arginine, and tetrahydrobiopterin, and a reductase domain, which contains the binding sites for FMN, FAD, and NADPH (2). NOS is a highly regulated enzyme requiring homodimerization and bound calmodulin for efficient electron transfer from the flavins to the heme moiety to enable synthesis of NO. Another mechanism of regulation is the ubiquitination and proteasomal degradation of NOS (3). Of particular pharmacological interest is the finding that certain drugs cause the suicide inactivation, covalent alteration, ubiquitination, and proteasomal degradation of nNOS (3-8). This phenomenon is not unique to nNOS as it is well documented that the suicide inactivation of other P450 cytochromes leads to covalent alteration, enhanced ubiquitination, and proteasomal turnover of the enzymes (9). The C terminus of Hsc70-interacting protein (CHIP) has been shown to be an E3 ligase that ubiquitinates cytochromes P450 3A4 and 2E1 as well as nNOS (10 -12). The...

Ubiquitination of Neuronal Nitric-oxide Synthase in the Calmodulin-binding Site Triggers Proteasomal Degradation of the Protein

“…Consistent with this notion, N G -nitro-L-arginine decreases the amount of Hsp70 and CHIP bound to nNOS, whereas the N G -nitro-D-arginine has no effect (18). Accordingly, N G -nitro-L-arginine decreases ubiquitination of nNOS in a variety of in vitro and cell systems, decreases the turnover of the protein in cells, and increases the amount of nNOS protein in vivo (11,14,16,18,39). Defining the sites of ubiquitination should ultimately provide a framework for understanding how the chaperones and CHIP recognize and bind to nNOS to ubiquitinate lysine residue 739 as a signal for degradation of nNOS to maintain protein quality control.…”

“…1B, open squares) remains the same, suggesting that the majority of the nNOS protein is accounted for in each lane. It is important to note that unlike our previous studies where only a few percent of the total nNOS was ubiquitinated (6,11,16), the current system gives a much higher yield (ϳ50%) of ubiquitinated nNOS. As shown in Fig.…”

“…We have previously shown that nNOS is proteasomally degraded in an ubiquitin-dependent manner in this system (15). The fraction II-dependent proteasomal degradation is also responsive to the nature of the nNOS heme cleft and dependent on ATP, CHIP, and Hsp70 (11). As shown in Fig.…”

“…We have established that the substrate-binding cleft of nNOS controls the ubiquitination and degradation of the enzyme by Hsp70⅐CHIP (11,14). A diagnostic test of this mechanism is the decreased nNOS ubiquitination caused by L-isomer, but not the D-isomer, of N G -nitro-arginine (NNA) that can be demonstrated in in vitro studies with cell extracts, in intact cells, as well as in rats (11,14,17).…”

“…CHIP has been shown to be in native complexes with all three NOS isoforms as follows: neuronal NOS (nNOS), endothelial NOS, and inducible NOS (5)(6)(7)(8). Although CHIP is not the only ubiquitin ligase for NOS, as Parkin was found to ubiquitinate nNOS (9) and SPSB2 was found to direct the ubiquitination of inducible NOS independent of CHIP (10), the CHIP-dependent pathway appears to be the major one involved in the recognition and ubiquitination of altered dysfunctional forms of nNOS (11,12). The discovery that dysfunctional forms of nNOS were selectively culled came from in vitro and in vivo studies on the suicide inactivation of nNOS (13)(14)(15)(16).…”