This review briefly summarizes what was known about NOS enzymology at the time of the Nobel Prize award in 1998 and then discusses from the author's perspective some of the advances in NOS enzymology ...over the subsequent 20 years, focused on five aspects: the maturation process of NOS enzymes and its regulation; the mechanism of NO synthesis; the redox roles played by the 6R‐tetrahydrobiopterin cofactor; the role of protein conformational behaviour in enabling NOS electron transfer and its regulation by NOS structural elements and calmodulin, and the catalytic cycling pathways of NOS enzymes and their influence on NOS activity.
Linked Articles
This article is part of a themed section on Nitric Oxide 20 Years from the 1998 Nobel Prize. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.2/issuetoc
Background and Purpose
Nitric oxide (NO) activates soluble guanylyl cyclase (sGC) for cGMP production, but in disease, sGC becomes insensitive towards NO activation. What changes occur to sGC during ...its inactivation in cells is not clear.
Experimental Approach
We utilized HEK293 cells expressing sGC proteins to study the changes that occur regarding its haem content, heterodimer status and sGCβ protein partners when the cells were given the oxidant ODQ or the NO donor NOC12 to inactivate sGC. Haem content of sGCβ was monitored in live cells through use of a fluorescent‐labelled sGCβ construct, whereas sGC heterodimer status and protein interactions were studied by Western blot analysis. Experiments with purified proteins were also performed.
Key Results
ODQ‐ or NOC12‐driven inactivation of sGC in HEK293 cells was associated with haem oxidation (by ODQ), S‐nitrosation of the sGCβ subunit (by NOC12), sGC heterodimer breakup and association of the freed sGCβ subunit with cell chaperone Hsp90. These changes occurred without detectable loss of haem from the sGCβ reporter construct. Treating a purified ferrous haem‐containing sGCβ with ODQ or NOC12 caused it to bind with Hsp90 without showing any haem loss.
Conclusion and Implications
Oxidative (ODQ) or nitrosative (NOC12) inactivation of cell sGC involves sGC heterodimer dissociation and rearrangement of the sGCβ protein partners without any haem loss from sGCβ. Clarifying what changes do and do not occur to sGC during its inactivation in cells may direct strategies to preserve or recover NO‐dependent cGMP signalling in health and disease.
LINKED ARTICLES
This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc
Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygenase (TDO) catalyze the conversion of L-tryptophan to N-formyl-kynurenine and thus play primary roles in metabolism, inflammation, and ...tumor immune surveillance. Because their activities depend on their heme contents, which vary in biological settings and go up or down in a dynamic manner, we studied how their heme levels may be impacted by nitric oxide (NO) in mammalian cells. We utilized cells expressing TDO or IDO1 either naturally or via transfection and determined their activities, heme contents, and expression levels as a function of NO exposure. We found NO has a bimodal effect: a narrow range of low NO exposure promoted cells to allocate heme into the heme-free TDO and IDO1 populations and consequently boosted their heme contents and activities 4- to 6-fold, while beyond this range the NO exposure transitioned to have a negative impact on their heme contents and activities. NO did not alter dioxygenase protein expression levels, and its bimodal impact was observed when NO was released by a chemical donor or was generated naturally by immune-stimulated macrophage cells. NO-driven heme allocations to IDO1 and TDO required participation of a GAPDH–heme complex and for IDO1 required chaperone Hsp90 activity. Thus, cells can up- or downregulate their IDO1 and TDO activities through a bimodal control of heme allocation by NO. This mechanism has important biomedical implications and helps explain why the IDO1 and TDO activities in animals go up and down in response to immune stimulation.
Soluble guanylate cyclase (sGC) is a heme-containing heterodimeric enzyme that generates many molecules of cGMP in response to its ligand nitric oxide (NO); sGC thereby acts as an amplifier in ...NO-driven biological signaling cascades. Because sGC helps regulate the cardiovascular, neuronal, and gastrointestinal systems through its cGMP production, boosting sGC activity and preventing or reversing sGC inactivation are important therapeutic and pharmacologic goals. Work over the last two decades is uncovering the processes by which sGC matures to become functional, how sGC is inactivated, and how sGC is rescued from damage. A diverse group of small molecules and proteins have been implicated in these processes, including NO itself, reactive oxygen species, cellular heme, cell chaperone Hsp90, and various redox enzymes as well as pharmacologic sGC agonists. This review highlights their participation and provides an update on the processes that enable sGC maturation, drive its inactivation, or assist in its recovery in various settings within the cell, in hopes of reaching a better understanding of how sGC function is regulated in health and disease.
Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygense (TDO) are heme-containing dioxygenases that catalyze the conversion of tryptophan to N-formyl-kynurenine and thus enable generation ...of l-kynurenine and related metabolites that govern the immune response and broadly impact human biology. Given that TDO and IDO1 activities are directly proportional to their heme contents, it is important to understand their heme delivery and insertion processes. Early studies established that TDO and IDO1 heme levels are sub-saturating in vivo and subject to change but did not identify the cellular mechanisms that provide their heme or enable dynamic changes in their heme contents. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into IDO1 and TDO expressed in both normal and heme-deficient HEK293T cells and into IDO1 naturally expressed in HeLa cells in response to IFN-γ, and also investigated the interactions of TDO and IDO1 with GAPDH and Hsp90 in cells and among their purified forms. We found that GAPDH delivered both mitochondrially-generated and exogenous heme to apo-IDO1 and apo-TDO in cells, potentially through a direct interaction with either enzyme. In contrast, we found Hsp90 interacted with apo-IDO1 but not with apo-TDO, and was only needed to drive heme insertion into apo-IDO1. By uncovering the cellular processes that allocate heme to IDO1 and TDO, our study provides new insight on how their activities and l-kynurenine production may be controlled in health and disease.
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•Tryptophan metabolism by catalytic activities of TDO and IDO1 is directly proportional to their heme contents.•TDO and IDO1 naturally exist with variable heme levels that can change dynamically in response to inflammation.•Heme allocation to TDO and IDO1 depends on expression level and heme binding to GAPDH, and IDO1 also requires chaperone hsp90.•Identifying the proteins involved in TDO and IDO1 heme acquisition provides a new way to regulate their activities.
Nitric oxide (NO) signaling regulates various physiological processes in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. Although NOS-like ...activities that are sensitive to mammalian NOS inhibitors have been detected in plant extracts, few bona fide plant NOS enzymes have been identified. We searched the data set produced by the 1000 Plants (1KP) international consortium for the presence of transcripts encoding NOS-like proteins in over 1000 species of land plants and algae. We also searched for genes encoding NOS-like enzymes in 24 publicly available algal genomes. We identified no typical NOS sequences in 1087 sequenced transcriptomes of land plants. In contrast, we identified NOS-like sequences in 15 of the 265 algal species analyzed. Even if the presence of NOS enzymes assembled from multipolypeptides in plants cannot be conclusively discarded, the emerging data suggest that, instead of generating NO with evolutionarily conserved NOS enzymes, land plants have evolved finely regulated nitrate assimilation and reduction processes to synthesize NO through a mechanism different than that in animals.
Nitric oxide (NO)-unresponsive forms of soluble guanylyl cyclase (sGC) exist naturally and in disease can disable NO-sGC-cGMP signaling. Agonists like BAY58-2667 (BAY58) target these sGC forms, but ...their mechanisms of action in living cells are unclear. We studied rat lung fibroblast-6 cells and human airway smooth muscle cells that naturally express sGC and HEK293 cells that we transfected to express sGC and variants. Cells were cultured to build up different forms of sGC, and we used fluorescence and FRET-based measures to monitor BAY58-driven cGMP production and any protein partner exchange or heme loss events that may occur for each sGC species. We found that: (i) BAY58 activated cGMP production by the apo-sGC
-Hsp90 species after a 5-8 minute delay that was associated with apo-sGC
exchanging its Hsp90 partner with an sGC
subunit. (ii) In cells containing an artificially constructed heme-free sGC heterodimer, BAY58 initiated an immediate and three times faster cGMP production. However, this behavior was not observed in cells expressing native sGC under any condition. (iii) BAY58 activated cGMP production by ferric heme sGC only after a 30-minute delay, coincident with it initiating a delayed, slow ferric heme loss from sGC
We conclude that the kinetics favor BAY58 activation of the apo-sGC
-Hsp90 species over the ferric heme sGC species in living cells. The protein partner exchange events driven by BAY58 account for the initial delay in cGMP production and also limit the speed of subsequent cGMP production in the cells. Our findings clarify how agonists like BAY58 may activate sGC in health and disease. SIGNIFICANCE STATEMENT: A class of agonists can activate cyclic guanosine monophosphate (cGMP) synthesis by forms of soluble guanylyl cyclase (sGC) that do not respond to NO and accumulate in disease, but the mechanisms of action are unclear. This study clarifies what forms of sGC exist in living cells, which of these can be activated by the agonists, and the mechanisms and kinetics by which each form is activated. This information may help to hasten deployment of these agonists for pharmaceutical intervention and clinical therapy.
A natural heme deficiency that exists in cells outside of the circulation broadly compromises the heme contents and functions of heme proteins in cells and tissues. Recently, we found that the ...signaling molecule, nitric oxide (NO), can trigger or repress the deployment of intracellular heme in a concentration‐dependent hormetic manner. This uncovers a new role for NO and sets the stage for it to shape numerous biological processes by controlling heme deployment and consequent heme protein functions in biology.
We hypothesize that tissues exist naturally in a heme‐deficient condition that limits the heme content and functions of heme proteins. The nitric oxide (NO) that is made in our tissues can control cell heme availability in a bimodal way, and so regulate heme protein functions in health and disease.
Nitric oxide (NO) signaling in biology relies on its activating cyclic guanosine monophosphate (cGMP) production by the NO receptor soluble guanylyl cyclase (sGC). sGC must obtain heme and form a ...heterodimer to become functional, but paradoxically often exists as an immature heme-free form in cells and tissues. Based on our previous finding that NO can drive sGC maturation, we investigated its basis by utilizing a fluorescent sGC construct whose heme level can be monitored in living cells. We found that NO generated at physiologic levels quickly triggered cells to mobilize heme to immature sGC. This occurred when NO was generated within cells or by neighboring cells, began within seconds of NO exposure, and led cells to construct sGC heterodimers and thus increase their active sGC level by several-fold. The NO-triggered heme deployment involved cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-heme complexes and required the chaperone hsp90, and the newly formed sGC heterodimers remained functional long after NO generation had ceased. We conclude that NO at physiologic levels triggers assembly of its own receptor by causing a rapid deployment of cellular heme. Redirecting cellular heme in response to NO is a way for cells and tissues to modulate their cGMP signaling and to more generally tune their hemeprotein activities wherever NO biosynthesis takes place.
Asthma is characterized by airway remodeling and hyperreactivity. Our earlier studies determined that the nitric oxide (NO)–soluble guanylyl cyclase (sGC)–cGMP pathway plays a significant role in ...human lung bronchodilation. However, this bronchodilation is dysfunctional in asthma due to high NO levels, which cause sGC to become heme‐free and desensitized to its natural activator, NO. In order to determine how asthma impacts the various lung segments/lobes, we mapped the inflammatory regions of lungs to determine whether such regions coincided with molecular signatures of sGC dysfunction. We demonstrate using murine models of asthma (OVA and CFA/HDM) that the inflamed segments of these murine lungs can be tracked by upregulated expression of HO1 and these regions in turn overlap with regions of heme‐free sGC as evidenced by a decreased sGC‐α1β1 heterodimer and an increased response to heme‐independent sGC activator, BAY 60‐2770, relative to naïve uninflamed regions. We also find that NO generated from iNOS upregulation in the inflamed segments has a higher impact on developing heme‐free sGC as increasing iNOS activity correlates linearly with elevated heme‐independent sGC activation. This excess NO works by affecting the epithelial lung hemoglobin (Hb) to become heme‐free in asthma, thereby causing the Hb to lose its NO scavenging function and exposing the underlying smooth muscle sGC to excess NO, which in turn becomes heme‐free. Recognition of these specific lung segments enhances our understanding of the inflamed lungs in asthma with the ultimate aim to evaluate potential therapies and suggest that regional and not global inflammation impacts lung function in asthma.
Model establishing the molecular underpinnings in mouse asthma between point of high inflammation and sGC dysfunction. Severe inflammation points in mouse asthma (OVA and CFA/HDM) lungs are characterized by high NO generated from iNOS induction, and these can be tracked by following upregulated expression of HO1 in the different lung segments. High NO generated in the airway epithelium makes lung hemoglobin (Hb) heme‐free, which is unable to scavenge the excess NO, thereby losing its protection for the smooth muscle sGC.
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