Cytochrome P450 enzymes (CYPs) play diverse roles in human health and disease, and although their activities depend on their heme contents, the cellular mechanisms governing CYP heme levels are unclear. Because CYP activities are influenced by biological nitric oxide (NO), we investigated how a range of NO exposures would impact the heme levels and activities of CYP2D6 and 3A4 expressed in Chinese hamster ovary cells and in the human liver cell line HepG2. Following expression, both CYPs were present as a 60:40 mix of heme-free and heme-bound forms. A low range of NO concentrations (approximately 1-10 nM) generated in cultures by a chemical NO donor or by added activated macrophages caused cells to allocate heme into their heme-free CYP3A4 and 2D6 populations such that the levels of heme-replete and active CYPs increased by twofold to threefold. NO concentrations above this range (approximately 25-100 nM) gradually lost the positive effect and at the higher level caused heme loss from the CYPs and corresponding losses in activity. The positive or negative effects of NO began within the first 2 h of exposure and were completed within 6 h. The NO-driven increase in CYP heme content relied on a GAPDH-heme complex forming and chaperone heat shock protein 90 activity in the cells. Thus, NO can upregulate or downregulate cellular CYP3A4 and 2D6 activities by exerting a concentration-dependent change in their heme contents. These findings may help explain how NO generation in disease or inflammation can change CYP activities and impact drug pharmacokinetics and the generation of immune-active metabolites.

Heme is an iron-containing cofactor generated in mitochondria that must leave this organelle to reach protein targets in other cell compartments. Because mitochondrial heme binding by cytosolic GAPDH enables its distribution in cells, we sought to uncover how heme reaches GAPDH. Experiments utilizing two human cell lines and a GAPDH reporter protein whose heme binding can be followed by fluorescence reveal that the mitochondrial protein FLVCR1b provides heme to GAPDH in concert with a rise and fall in their association. An absence of FLVCR1b diminishes GAPDH association with mitochondria and prevents GAPDH and cell hemeproteins from receiving heme. GAPDH heme procurement also requires the TANGO2 protein, which interacts with FLVCR1b to presumably support heme export. In isolated mitochondria, GAPDH associates with FLVCR1b to trigger heme release and delivery to client hemeproteins. Identifying FLVCR1b as the source of mitochondrial heme for GAPDH reveals a path by which this essential cofactor can reach multiple protein targets within eukaryotic cells.

Intracellular trafficking of mitochondrial heme to create functional heme proteins presents a fundamental challenge in animal cells. This article provides some background on heme allocation, discusses some of the concepts, and then reviews research from the last two decades that has uncovered unexpected and important roles for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), heat shock protein 90 (Hsp90), and nitric oxide (NO) in enabling and regulating cell heme allocations to hemeproteins that mature and function outside of the mitochondria. A model for how hemeprotein heme contents and functions in cells can be regulated through the coordinate participation of GAPDH, Hsp90, and NO is presented.

In eukaryotes, the last steps of heme biosynthesis occur in mitochondria and so heme must be transported to reach many heme-dependent proteins that mature and function outside this organelle. Although the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has emerged as a key intracellular heme chaperone, how it performs heme deliveries to its numerous clients is poorly understood. It is unknown if handoffs of the GAPDH-bound heme require that it make direct contact with its clients or instead involve GAPDH passing its heme to middlemen proteins to execute the final heme transfers. To address this question, we studied GAPDH heme transfer to the client protein indoleamine 2,3-dioxygenase 1 (IDO1), whose enzyme activity is heme-dependent and regulates mammalian immune responses and cancer progression. A chemical crosslinking-mass spectrometry approach identified two Lys residues that formed an inter-protein crosslink across a previously uncharacterized GAPDH-IDO1 interface. This guided our building a model of the GAPDH-IDO1 complex so we could interrogate by point mutagenesis the role of the GAPDH-IDO1 contact in enabling delivery of GAPDH heme to IDO1. We characterized behaviors of the GAPDH and IDO1 variants in their purified form and when they were expressed in the HEK293T human cell line. This revealed GAPDH heme transfer to IDO1 in cells requires that they make a direct contact which relies on a specific Lys-Asp charge pairing interaction forming across the complex interface. These findings illuminate a key step in the maturation of functional IDO1 and improve our understanding of how GAPDH may perform its heme trafficking function in mammals.

In order to determine the underpinnings of a dysfunctional NO-sGC signal pathway which occurs in pulmonary arterial hypertension (PAH), we investigated pulmonary arterial smooth muscle cells (PASMCs) derived from PAH patients. We found low expression of sGC, a poor sGC alpha 1(31 heterodimer and this correlated with low expression of its facilitator chaperon, hsp90. Treating PASMCs overnight (16 h) with low micromolar doses of a slow release NO donor DETANONOate, reinstated the sGC alpha 1(31 heterodimer and restored its NO-heme dependent activity. Transwell co-culture of HEK cells stably expressing eNOS with PAH PASMCs also restored the sGC heterodimer and its heme-dependent activity with sGC stimulator, BAY 41-2272. To determine whether the dysfunctionality in the NO-sGC pathway stems from a dysfunctional eNOS producing negligible NO, we did transwell co-cultures of pulmonary arterial endothelial cells (PAECs) with PASMCs. Our results indicated that PAECs from both control and PAH samples when activated for eNOS restored both sGC heterodimer and its heme-dependent sGC activity in the corresponding PASMCs, suggesting that PAECs from PAH can also generate NO. In line with these results expression of eNOS, its support chaperon hsp90, its specific kinase Akt, p-Akt or post-translational modifications (PTMs) like OGlcNAc or phospho-tyrosine were unchanged in PAH relative to controls. Additionally there was uniform expression of Hb alpha/(3 and Mb in PASMCs or PAECs in PAH or controls and these globins can effectively scavenge the eNOS generated NO, as there was evidence of strong eNOS-Hb/Mb interactions. Our studies suggest that factors such as globin NO scavenging along with vascular remodeling in PAH can cause hampered vasodilation which in the face of poor NO levels as occurs in PAH are additional impediments for effective vasodilation. However importantly our studies suggests that future therapies can use low doses of NO along with sGC stimulators as a potential drug for PAH subjects.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45–65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. 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 CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation process and how this may help to regulate their functions in health and disease.

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-alpha 1 beta 1 heterodimer and an increased response to heme-independent sGC activator, BAY 60-2770, relative to naive 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.image

To function in cGMP generation the sGCb subunit must incorporate heme and form a heterodimer with the partner a-subunit. Cellular studies have shown that GAPDH associates with and supplies heme to the apo-sGCb subunit, which is in complex with the cell chaperone Hsp90. The Hsp90 then drives heme insertion into the apo-sGCb subunit in an ATP-driven process, which in turn enables sGCb to interact with the a-subunit and form a functional sGC heterodimer. NO at physiologic levels enhances cellular heme delivery into apo-sGCb and the consequent formation of the heterodimer, by a process that also depends on GAPDH and Hsp90.To gain mechanistic understanding, we developed an in vitro reconstitution system that utilizes purified proteins and allows us to follow in real time heme transfer from a pre-formed GAPDH-heme complex into apo-GCb by monitoring fluorescence changes in their respective FlAsH tags. Results from the defined system show that the heme transfer mechanism greatly depends on direct protein-protein interactions of apo-sGCb with GAPDH & with Hsp90 and requires ATP hydrolysis by Hsp90. We also found that added NO drives a faster and greater heme transfer into the apo-sGCb subunit, and its ability to do so depends on NO binding to the heme in the GAPDH. Our findings clarify the processes by which sGC matures inside cells and provide a platform for molecular-level investigations.

Hemoglobin (Hb) present in the lung epithelium is of unknown significance. However Hb being an nitric oxide (NO) scavenger can bind to NO and reduce its deleterious effects. Hence we postulated an NO scavenging role for this lung Hb. Doing transwell co-culture with bronchial epithelial cells, A549/16-HBE (apical) and human airway smooth muscle cells (HASMCs as basal), we found that Hb can protect the smooth muscle soluble guanylyl cyclase (sGC) from excess NO. Inducing the apical A549/16-HBE cells with cytokines to trigger iNOS expression and NO generation caused a time dependent increase in SNO-sGC and this was accompanied with a concomitant drop in sGC-α1β1 heterodimerization. Silencing Hbαβ in the apical cells further increased the SNO on sGC with a faster drop in the sGC heterodimer and these effects were additive along with further silencing of thioredoxin 1 (Trx1). Since heme of Hb is critical for NO scavenging we determined the Hb heme in a mouse model of allergic asthma (OVA) and found that Hb in the inflammed OVA lungs was low in heme or heme-free relative to those of naïve lungs. Further we established a direct correlation between the status of the sGC heterodimer and the Hb heme from lung samples of human asthma, iPAH, COPD and cystic fibrosis. These findings present a new mechanism of protection of lung sGC by the epithelial Hb, and suggests that this protection maybe lost in asthma or COPD where lung Hb is unable to scavenge the NO due to it being heme-deprived.

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 models of mouse asthma (OVA, CFA/HDM) that the inflammed segments of the mouse asthma 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 in 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 enhance our understanding of the inflammed lungs in asthma with the ultimate aim to evaluate potential therapies and suggests that regional and not global inflammation impacts lung function in asthma.

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.