The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitinprotein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 M), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOSubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70. Nitric-oxide synthases (NOSs)1 are cytochrome P450-like hemoprotein enzymes that catalyze the conversion of L-arginine to citrulline and nitric oxide by a process that requires NADPH and molecular oxygen. NOS is bidomain in structure with an oxygenase domain, which contains the cysteine residue that is coordinated to the prosthetic heme as well as the tetrahydrobiopterin binding site, and a reductase domain, which contains the binding sites for FMN, FAD, and NADPH. The NOS is a highly regulated enzyme requiring homodimerization and association with Ca 2ϩ -calmodulin for activity. Another mechanism for regulation involves the selective ubiquitin-dependent proteasomal degradation of dysfunctional NOS (1). This is evident, for example, when metabolism-based or suicide inactivators cause the covalent alteration and inactivation of neuronal NOS (nNOS) and trigger enhanced proteasomal degradation of the enzyme (2). The nature of the factor(s) that selectively recognize dysfunctional nNOS is unknown; however, inhibition of hsp90 leads to enhanced proteasomal degradation of nNOS, implicating the hsp90-based chaperone machinery as a potential regulator of nNOS protein levels (3).Hundreds, perhaps thousands, of cellular proteins are chaperoned by the hsp90/hsp70-based chaperone machinery (4), and it has been proposed that these chaperones play a key role in protein triage decisions that maintain quality control of cellular proteins. CHIP is a U-box-containin...

NO production by neuronal nitric oxide synthase (nNOS) requires calmodulin and is enhanced by the chaperone Hsp90, which cycles dynamically with the enzyme. The proteasomal degradation of nNOS is enhanced by suicide inactivation and by treatment with Hsp90 inhibitors, the latter suggesting that dynamic cycling with Hsp90 stabilizes nNOS. Here, we use a purified ubiquitinating system containing CHIP (carboxyl terminus of Hsp70-interacting protein) as the E3 ligase to show that Hsp90 inhibits CHIP-dependent nNOS ubiquitination. Like the established Hsp90 enhancement of NO synthesis, Hsp90 inhibition of nNOS ubiquitination is Ca 2+ /calmodulin-dependent, suggesting that the same interaction of Hsp90 with the enzyme is responsible for both enhancement of nNOS activity and inhibition of ubiquitination. It is established that CHIP binds to Hsp90 as well as to Hsp70, but we show here the two chaperones have opposing actions on nNOS ubiquitination, with Hsp70 stimulating and Hsp90 inhibiting. We have used two mechanism-based inactivators, guanabenz and N G -amino-L-arginine, to alter the heme/substrate binding cleft and promote nNOS ubiquitination that can be inhibited by Hsp90. We envision that as nNOS undergoes toxic damage, the heme/substrate binding cleft opens exposing hydrophobic residues as the initial step in unfolding. As long as Hsp90 can form even transient complexes with the opening cleft, ubiquitination by Hsp70-dependent ubiquitin E3 ligases, like CHIP, is inhibited. When unfolding of the cleft progresses to a state that cannot cycle with Hsp90, Hsp70-dependent ubiquitination is unopposed. In this way, the Hsp70/Hsp90 machinery makes the quality control decision for stabilization versus degradation of nNOS.Both the function and turnover of a wide variety of signaling proteins, such as steroid receptors and protein kinases, are regulated by Hsp90 1 (reviewed in Ref. 1). These Hsp90 'client' proteins are assembled into complexes with the chaperone by a multichaperone machinery in which Hsp90 and Hsp70 function together as essential components (1). Formation of heterocomplexes with Hsp90 stabilizes client proteins, and treatment with an Hsp90 inhibitor such as geldanamycin uniformly triggers their degradation (2). Degradation of the Hsp90-regulated signaling proteins occurs via the ubiquitin-proteasome pathway, which in this case is initiated by Hsp70-dependent E3 ubiquitin ligases, such as CHIP (3) and parkin (4).

Background: NOS enzymes are large, dimeric complexes essential in mammalian physiology. Results: EM structural analysis and three-dimensional models reveal nNOS reductase-oxygenase arrangements and a CaMdependent rotation of the FMN domain. Conclusion: Coordinated conformational changes act to reposition the FMN domain for electron transfer. Significance: This work captures structural states of the NOS holoenzyme that drive the NO synthesis cycle.

Iron and copper are redox active metals essential for life. In the budding yeast Saccharomyces cerevisiae, expression of iron and copper genes involved in metal acquisition and utilization is tightly regulated at the transcriptional level. In addition iron and copper metabolism are inextricably linked because of the dependence on copper as a co-factor for iron uptake or mobilization. To further identify genes that function in iron and copper homeostasis, we screened for novel yeast mutants defective for iron limiting growth and thereby identified the CTI6 gene. Cti6 is a PHD finger-containing protein that has been shown to participate in the interaction of the Ssn6-Tup1 co-repressor with the Gcn5-containing SAGA chromatin-remodeling complex. In this report we show that CTI6 mRNA levels are increased under iron-limiting conditions, and that cti6 mutants display a growth defect under conditions of iron deprivation. Furthermore, we demonstrate that Cti6 is a nuclear protein that functionally associates with the Rpd3-Sin3 histone deacetylase complex involved in transcriptional repression. Cti6 demonstrates Rpd3-dependent transcriptional repression, and cti6 mutants exhibit an enhanced silencing of telomeric, rDNA and HMR loci, similar to mutants in genes encoding other Rpd3-Sin3-associated proteins. Microarray experiments with cti6 mutants grown under iron-limiting conditions show a down-regulation of telomeric genes and an upregulation of Aft1 and Tup1 target genes involved in iron and oxygen regulation. Taken together, these data suggest a specific role for Cti6 in the regulation of gene expression under conditions of iron limitation.

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ABSTRACT:Smoking causes a dysfunction in endothelial nitric-oxide synthase (eNOS), which is ameliorated, in part, by administration of tetrahydrobiopterin (BH 4 ). The exact mechanism by which the nitric oxide deficit occurs is unknown. We have previously shown that aqueous extracts of chemicals in cigarettes (CE) cause the suicide inactivation of neuronal NO synthase (nNOS) by interacting at the substrate-binding site. In the current study, we have found that CE directly inactivates eNOS by a process that is not affected by the natural substrate L-arginine and is distinct from the mechanism of inactivation of nNOS. We discovered that CE causes a time-, concentration-, and NADPH-dependent inactivation of eNOS in an in vitro system containing the purified enzyme, indicating a metabolic component to the inactivation. The CE-treated eNOS but not nNOS was nearly fully reactivated upon incubation with excess BH 4 , suggesting that BH 4 depletion is a potential mechanism of inactivation. Moreover, in the presence of CE, eNOS catalyzed the oxidation of BH 4 to dihydrobiopterin and biopterin by a process attenuated by high concentrations of superoxide dismutase but not catalase. We speculate that a redox active component in CE, perhaps a quinone compound, causes oxidative uncoupling of eNOS to form superoxide, which in turn oxidizes BH 4 . The discovery of a direct inactivation of eNOS by a compound(s) present in tobacco provides a basis not only for further study of the mechanisms responsible for the biological effects of tobacco but also a search for a potentially novel inactivator of eNOS.Cigarette smoking is dose dependently associated with impairment of endothelial-dependent dilation in humans (Adams et al., 1997). The role of NO deficit in the genesis of vascular disease has been reviewed (Cooke and Dzau, 1997b); moreover, it is known that acute as well as chronic smoking decreases exhaled NO in humans (Kharitonov et al., 1995). A variety of mechanisms have been postulated to explain this NO deficit (Cooke and Dzau, 1997a), including the enhanced formation of superoxide, which would rapidly react with NO and thereby decrease its bioactivity (Kelm et al., 1997). More recently, Heitzer et al. (2000) reported that administration of tetrahydrobiopterin (BH 4 ) to chronic smokers improves endothelium-dependent vasodilation as determined by forearm blood-flow measurements. Administration of tetrahydroneopterin, which has the same antioxidant properties as BH 4 but is not a cofactor for NO synthase, did not improve vascular function and further supports the notion of a specific dysfunction in NOS rather than a general enhancement of oxidative stress (Heitzer et al., 2000). In addition, the vasodilator response due to sodium nitroprusside is not affected in these smokers irrespective of BH 4 administration, indicating that the responsiveness to NO is not changed. Thus, taken together, these observations strongly implicate a dysfunctional NO synthase in smokers (Heitzer et al., 2000). This is consistent with an earlier r...

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...