Biology subjects

Stuehr, D. J.

Publications and source records attributed to Stuehr, D. J..

7 recordsLinked to original sources

bioRxiv2026

Soluble guanylyl cyclase subunits act as Hsp90 co-chaperones to ensure the expression and functional maturation of hemeproteins in mammalian cells

The cofactor Fe-protoporphyrin IX cofactor (heme) performs many functions in biology. Animal cells must stabilize their newly generated heme-free (apo)-hemeproteins and deliver mitochondrial heme to them so they can mature to functional form. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) typically accomplishes the heme deliveries, and for many apo-hemeproteins, heat shock protein 90 (Hsp90) drives their heme insertions. We previously observed hemeproteins express poorly in a cell line (COS-7) that does not express soluble guanylyl cyclase (sGC), a heme-binding enzyme that typically functions through its cGMP generation. To understand sGC involvement, we expressed four hemeproteins, Hemoglobin beta (Hb{beta}), Myoglobin (Mb), Indoleamine 2,3-dioxygenase 1 (IDO1), and Tryptophan 2,3-dioxygenase (TDO) in a cell line expressing sGC (HEK293) or in two cell lines (COS-7, DU145) that do not. We assessed hemeprotein expression levels, their abilities to acquire heme, and when relevant if these facets could be rescued by co-expressing individual sGC subunits, including variants with defects in either sGC heme binding, Hsp90 association, heterodimerization, or cGMP production. We found that co-expression of either sGC subunit was essential for three of the four apo-hemeproteins to accumulate in the COS7 and DU145 cells and acquire heme. This did not involve heme binding, heterodimer formation, or cGMP generation by the sGC subunits, and instead depended on a subunits ability to recruit Hsp90 and GAPDH to the apo-hemeproteins via their own Hsp90 binding. Recruiting Hsp90 and GAPDH to apo-hemeprotein clients to ensure they can accumulate and mature to functional form broadens our understanding of sGC and Hsp90 functions in biology.

cell biology↗

bioRxiv2026

NO modulates human airway smooth muscle function by altering glucose-6-phosphate dehydrogenase effects on sGC function in asthma

Since NO can modulate mesenchymal cell function, we posit that NO can modulate gene expression associated with excitation-contraction coupling. Our study shows that treating asthma-derived HASMCs with a low dose of NO plus sGC stimulator BAY-41, in most cases sensitized smooth muscle sGC towards activation via an elevated sGC heterodimer and in some cases also improved sGC{beta}1, catalase, Cyb5r3 or Trx1 expression (n=24 non-asthma and n=25 asthma). Interestingly we found that majority of asthma HASMCs showed a marked downregulation of G6PD expression inducing a low GSH/GSSG ratio in asthma, and these findings were replicated in murine lungs of allergic asthma (OVA and CFA/HDM). Studies with HEK/COS-7 cells showed G6PD synergizing with hsp90 in enabling sGC heme-maturation. G6PD overexpression in HASMCs enhanced the sGC heterodimerization while silencing of endogenous G6PD abrogated it. Complementation of these cellular results with whole animal models of G6PD deficiency or overexpression provided verification to our findings. Mouse lung tissue from the humanized variant of G6PD deficiency, V68M (G6PD A-deficiency) showed significant downregulation in the sGC heterodimer, with a concomitant reduction in its NO heme-dependent activity, thereby showing that G6PD deficiency lowers sGC heme. Conversely, G6PD overexpressing mouse lung tissue displayed an elevated sGC heterodimer and also showed a robust G6PD-sGC{beta}1 interaction, suggesting G6PD to be involved in the heme-maturation of sGC{beta}1. While G6PD maintains the cell redox by generating NADPH, its new role in regulating sGC maturation links sGC dysfunction in asthma to G6PD deficiency and may potentially uncover new targets for asthma treatment.

cell biology↗

bioRxiv2025

Myoglobin leaching into the serum of IDA mice is driven by the high activation of sGC under anemic conditions which induces myoglobin expression

Our study reveals that status of the sGC heterodimer or its subsequent activation aligns with active erythropoiesis, and this heterodimer also correlates with the expression of myoglobin (Mb) or HO1. In this study we found that Mb expression which is driven by iron restriction and high sGC activation in iron deficiency anemia (IDA) leaches out more into the serum relative to non-anemic WTs. Tissues from IDA mice of both models developed either by nutritional iron deprivation or by ablation of ferroportin (Fpn) gene or from iron refractory iron deficiency anemia (IRIDA) mice found that Mb expression follows a variable pattern in different tissues but always correlates to the status of the sGC heterodimer or its subsequent activation. Here higher Mb expression happening in anemic (IDA, Fe<5 ppm or IDA, Fpn) or non-anemic WT mice is both due to iron restriction and an elevated sGC heterodimer that corroborated with greater sGC activation. More importantly we find significant leaching of Mb into the serum of these anemic (IDA) mice from both models and our spectral data suggests that this Mb is heme-free. This Mb leaching in anemia is a cumulative impact of Mb secreting out from various tissues including lungs, spleen, skeletal or cardiac muscles where Mb is expressed and not just in the skeletal muscles where Mb expression is low. Based on these findings we construct a working model of anemia, where high activation of sGC under anemic conditions (Fpn ablation or restricted Fe diet) induces Apo-Mb or heme-free Mb expression which can then leach out into the serum. Our findings of Mb leaching are novel and can find further application as a diagnostic strategy in anemia.

biochemistry↗

bioRxiv2024

Heme allocation in eukaryotic cells relies on mitochondrial heme export through FLVCR1b to cytosolic GAPDH

Heme is an iron-containing cofactor essential for life. In eukaryotes heme is generated in the mitochondria and must leave this organelle to reach protein targets in other cell compartments. Mitochondrial heme binding by cytosolic GAPDH was recently found essential for heme distribution in eukaryotic cells. Here, we sought to uncover how mitochondrial heme reaches GAPDH. Experiments involving a human cell line and a novel GAPDH reporter construct whose heme binding in live cells can be followed by fluorescence revealed that the mitochondrial transmembrane protein FLVCR1b exclusively transfers mitochondrial heme to GAPDH through a direct protein-protein interaction that rises and falls as heme transfers. In the absence of FLVCR1b, neither GAPDH nor downstream hemeproteins received any mitochondrial heme. Cell expression of TANGO2 was also required, and we found it interacts with FLVCR1b to likely support its heme exporting function. Finally, we show that purified GAPDH interacts with FLVCR1b in isolated mitochondria and triggers heme transfer to GAPDH and its downstream delivery to two client proteins. Identifying FLVCR1b as the sole heme source for GAPDH completes the path by which heme is exported from mitochondria, transported, and delivered into protein targets within eukaryotic cells.

cell biology↗

bioRxiv2024

Visualizing Mitochondrial Heme Flow through GAPDH to Targets in Living Cells and its Regulation by NO

Iron protoporphyrin IX (heme) is an essential cofactor that is chaperoned in mammalian cells by GAPDH in a process regulated by NO. To gain further understanding we generated a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after being expressed and labeled with fluorescent FlAsH reagent could indicate heme binding by fluorescence quenching. When purified or expressed in HEK293T mammalian cells, FlAsH-labeled TC-hGAPDH displayed physical, catalytic, and heme binding properties like native GAPDH and its heme binding (2 mol per tetramer) quenched its fluorescence by 45-65%. In live HEK293T cells we could visualize TC-hGAPDH binding mitochondrially-generated heme and releasing it to the hemeprotein target IDO1 by monitoring cell fluorescence in real time. In cells with active mitochondrial heme synthesis, a low-level NO exposure increased heme allocation into IDO1 while keeping steady the level of heme-bound TC-hGAPDH. When mitochondrial heme synthesis was blocked at the time of NO exposure, low NO caused cells to reallocate existing heme from TC-hGAPDH to IDO1 by a mechanism requiring IDO1 be present and able to bind heme. Higher NO exposure had an opposite effect and caused cells to reallocate existing heme from IDO1 to TC-hGAPDH. Thus, with TC-hGAPDH we could follow mitochondrial heme as it travelled onto and through GAPDH to a downstream target (IDO1) in living cells, and to learn that NO acted at or downstream from the GAPDH heme complex to promote a heme reallocation in either direction depending on the level of NO exposure.

cell biology↗

bioRxiv2023

Regions of Inflammation in mouse asthma correspond to regions of heme-free soluble guanylyl cyclase and can be tracked by marked expression of heme-oxygenase-1

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-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 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.

immunology↗

bioRxiv2022

Indoleamine Dioxygenase and Tryptophan Dioxygenase Activities are Regulated through Control of Cell Heme Allocation by Nitric Oxide

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 range from 30- 60% heme-saturated 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 very low NO exposure promoted cells to allocate heme into TDO and IDO1 and boosted their activities several 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 down-regulate 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.

cell biology↗