Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.

Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved.

Dynamic cycling with a unique Hsp90/Hsp70-dependent chaperone machinery and GAPDH is needed for heme insertion and activation of neuronal NO synthase.

Heat shock protein 90 (Hsp90) is known to mediate heme insertion and activation of heme-deficient neuronal nitric oxide (NO) synthase (apo-nNOS) in cells by a highly dynamic interaction that has been extremely difficult to study mechanistically with the use of subcellular systems. In that the heme content of many critical hemeproteins is regulated by Hsp90 and the heme chaperone GAPDH, the development of an in vitro system for the study of this chaperone-mediated heme regulation would be extremely useful. Here, we show that use of an antibody-immobilized apo-nNOS led not only to successful assembly of chaperone complexes but the ability to show a clear dependence on Hsp90 and GAPDH for heme-mediated activation of apo-nNOS.

Mapping protein-protein interactions in homodimeric CYP102A1 by crosslinking and mass spectrometry.

Covalent crosslinking and mass spectrometry techniques hold great potential in the study of multiprotein complexes, but a major challenge is the inability to differentiate intra- and inter- protein crosslinks in homomeric complexes. In the current study we use CYP102A1, a well-characterized homodimeric P450, to examine a subtractive method that utilizes limited crosslinking with disuccinimidyl dibutyric urea (DSBU) and isolation of the monomer, in addition to the crosslinked dimer, to identify inter-monomer crosslinks. The utility of this approach was examined with the use of MS-cleavable crosslinker DSBU and recently published cryo-EM based structures of the CYP102A1 homodimer.

Hsp70:CHIP Ubiquitinates Dysfunctional but Not Native Neuronal NO Synthase.

Heat shock protein (Hsp) 70 modulators are being developed to enhance the removal of toxic proteins in a variety of protein misfolding diseases. In the course of our studies on neuronal nitric oxide synthase (nNOS), a client of the Hsp90 and Hsp70 chaperone system, we have established that inactivation of nNOS by heme or tetrahydrobiopterin (BH) alteration and loss triggers ubiquitination by the Hsp70-associated E3 ligase c-terminus of Hsp70-interacting protein (CHIP) and subsequent degradation in cells. Although in cells Hsp90 and Hsp70 work together to maintain protein quality control, in this study, we specifically developed an assay to assess the selectivity of the Hsp70:CHIP complex for inactivated nNOS.

Chaperone Activity and Dimerization Properties of Hsp90 and Hsp90 in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery.

Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90 and Hsp90, and their relative chaperone activities and composition in these client protein•Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90 and Hsp90 in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)•Hsp90 heterocomplexes to generate high-affinity steroid-binding activity.

Improved method for assembly of hemeprotein neuronal NO-synthase heterodimers.

The assembly of mutated and wild type monomers into functional heterodimeric hemeproteins has provided important mechanistic insights. As in the case of NO synthase (NOS), the existing methods to make such heterodimeric NOSs are inefficient and labor intensive with typical yields of about 5%. We have found that expression of neuronal NOS heterodimers in insect cells, where we take advantage of an exogenous heme-triggered chaperone-assisted assembly process, provides an approximately 43% yield in heterodimeric NOS.

Architecture of the nitric-oxide synthase holoenzyme reveals large conformational changes and a calmodulin-driven release of the FMN domain.

Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca(2+)-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis.

Heme-dependent activation of neuronal nitric oxide synthase by cytosol is due to an Hsp70-dependent, thioredoxin-mediated thiol-disulfide interchange in the heme/substrate binding cleft.

We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90.

Plant-Derived Small Molecule Inhibitors of Neuronal NO-Synthase: Potential Effects on Protein Degradation.

Cigarette smoking is known to cause a decrease in NO production in man resulting in a variety of pathological effects, including vascular dysfunction. Aqueous extracts of cigarette and cigarette smoke contain chemical inhibitors to NO-synthases, a heme-containing cytochrome P450 enzymes. More recently, it was shown that freshly harvested leaves from the tobacco plant (Nicotiana tabacum, Solanaceae) also contain chemical inhibitors to neuronal NO-synthase (nNOS).

Naturally occurring neuronal NO-synthase inactivators found in Nicotiana tabacum (Solanaceae) and other plants.

NO-synthase (NOS) is a heme-containing enzyme that catalyzes the oxidation of L-arginine to nitric oxide, an important cellular signaling molecule. Recently, it was found that aqueous extracts of tobacco cigarettes cause the inactivation of the neuronal isoform of NOS (nNOS) and that this may explain some of the toxicological effects of smoking. Although the exact identity of the chemical inactivator(s) is not known, we wondered if extracts prepared from other plants, including those closely related to tobacco, Nicotiana tabacum (Solanaceae), would similarly inactivate nNOS.

Tetrahydrobiopterin protects against guanabenz-mediated inhibition of neuronal nitric-oxide synthase in vitro and in vivo.

It is established that guanabenz inhibits neuronal nitric-oxide (NO) synthase (nNOS) and causes the enhanced proteasomal degradation of nNOS in vivo. Although the time- and NADPH-dependent inhibition of nNOS has been reported in studies where guanabenz was incubated with crude cytosolic preparations of nNOS, the exact mechanism for inhibition is not known. Moreover, even less is known about how the inhibition of nNOS triggers its proteasomal degradation.

Metabolism of aminoguanidine, diaminoguanidine, and NG-amino-L-arginine by neuronal NO-synthase and covalent alteration of the heme prosthetic group.

It is established that aminoguanidine (AG), diaminoguanidine (DAG), and NG-amino-l-arginine (NAA) are metabolism-based inactivators of the three major isoforms of nitric oxide synthase (NOS). In the case of neuronal NOS (nNOS), heme alteration is known to be a major cause of inactivation, although the exact mechanism by which this occurs is not well-understood. We show here by the use of LC/MS/MS techniques that AG, DAG, and NAA are metabolized by nNOS to products with corresponding mass ions at m/z of 45.

Tetrahydrobiopterin depletion and ubiquitylation of neuronal nitric oxide synthase.

Tetrahydrobiopterin is a necessary cofactor for the synthesis of nitric oxide by the hemeprotein enzyme, NO-synthase (NOS). It is widely thought that inadequate levels of tetrahydrobiopterin lead to tissue injury and organ dysfunction due, in part, to formation of superoxide from pterin-deficient NOS. In the course of studies on the ubiquitylation of neuronal NOS (nNOS), we have found that certain substrate analogs, such as N(G)-nitro-L-arginine, stabilize the dimeric form of nNOS and protect the enzyme from ubiquitylation.

Cti6 is an Rpd3-Sin3 histone deacetylase-associated protein required for growth under iron-limiting conditions in Saccharomyces cerevisiae.

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.

Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake.

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.