Asthma is defined by airway inflammation and hyperresponsiveness, contributes to morbidity mortality worldwide. Although bronchodilation a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC its natural stimulant nitric oxide (NO), or pharmacologic agonists BAY 41-2272 60-2770, triggered in normal human lung...

Soluble guanylyl cyclase (sGC) is a key component of NO–cGMP signaling in mammals. Although heme must bind the sGC β1 subunit (sGCβ) for to function, how delivered sGCβ remains unknown. Given that GAPDH displays properties chaperone inducible NO synthase, here we investigated whether delivery apo-sGCβ involves GAPDH. We utilized an reporter construct, tetra-Cys sGCβ, whose insertion can be followed by fluorescence quenching live cells, assessed lowering cell expression impacts delivery, and...

Significance Nitric oxide (NO) performs many biological functions, but how it operates at the molecular and cellular levels is not fully understood. We discovered that cell NO generation physiologic triggers a rapid redeployment of intracellular heme, an iron-containing cofactor, we show this drives assembly natural receptor protein, soluble guanylyl cyclase, which needed for to perform its signaling functions. Our study uncovers way can shape processes cells may use control their...

Heme insertion is key during maturation of soluble guanylyl cyclase (sGC) because it enables sGC to recognize NO and transduce its multiple biological effects. Although often associated with the 90-kDa heat shock protein (hsp90) in cells, implications are unclear. The present study reveals that hsp90 required drive heme into complete maturation. We used a mammalian cell culture approach followed transiently endogenously expressed heme-free sGC. pharmacological inhibitors, an ATP-ase inactive...

Maturation of NOS enzymes requires that they incorporate heme to become active, but how this cellular process occurs is unclear. We investigated a role for chaperone heat shock protein 90 (hsp90) in enabling insertion into the cytokine-inducible mouse NOS. used macrophage cell line RAW 264.7 and human embryonic kidney HEK293T cells studied native during iNOS expression exogenous preformed apo-iNOS. Pulldown experiments showed hsp90-iNOS complex was present cells, extent their association...

Maturation of adult (α2β2) and fetal hemoglobin (α2γ2) tetramers requires that heme be incorporated into each globin. While alpha (Hb-α) relies on a specific erythroid chaperone (alpha Hb-stabilizing protein, AHSP), the other chaperones may help mature partner globins (Hb-γ or Hb-β) in cells, enable nonerythroid cells to express Hb, are unknown. We investigated role for heat-shock protein 90 (hsp90) Hb maturation precursor naturally Hb-α with either Hb-γ (K562 HiDEP-1 cells) Hb-β (HUDEP-2)...

Islet transplantation is an alternative to pancreas cure type 1 diabetes, but both require chronic immunosuppression, which often accompanied by deleterious side effects. The purpose of this study was explore prolongation islet allograft survival cotransplantation with myeloid-derived suppressor cells (MDSCs) without requirement immunosuppression and determine the role inducible nitric oxide synthase (iNOS) produced MDSCs in immune regulation.Bone marrow were isolated from wild-type (WT) or...

GAPDH, a heme chaperone, has been previously implicated in the incorporation of into iNOS and soluble guanylyl cyclase (sGC). Since sGC is critical for myoglobin (Mb) heme-maturation, we investigated role GAPDH maturation this globin, as well hemoglobins α, β, γ. Utilizing cell culture systems, found that overexpression wild-type increased, whereas mutants H53A K227A decreased, content Mb Hbα Hbβ. Overexpression fully recovered heme-maturation inhibition observed with mutants. Partial rescue...

Nitric oxide (NO) is a signal molecule and plays critical role in the regulation of vascular tone, displays anti-platelet anti-inflammatory properties. While our earlier current studies found that low NO doses trigger rapid heme insertion into immature heme-free soluble guanylyl cyclase β subunit (apo-sGCβ), resulting mature sGC-αβ heterodimer, more recent evidence suggests can also heme-maturation hemoglobin myoglobin. This phenomena was not only limited to sGC globins, but occur all three...

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 scavenging role for this Hb. Doing transwell co-culture with bronchial epithelial cells, A549/16-HBE (apical) human airway smooth muscle cells (HASMCs as basal), found that protect soluble guanylyl cyclase (sGC) from excess NO. Inducing apical cytokines trigger iNOS expression generation caused a time...

Abstract A natural heme deficiency that exists in cells outside of the circulation broadly compromises contents and functions proteins tissues. Recently, we found signaling molecule, nitric oxide (NO), can trigger or repress deployment intracellular a concentration‐dependent hormetic manner. This uncovers new role for NO sets stage it to shape numerous biological processes by controlling consequent protein biology.

Overproduction of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) has been etiologically linked to several inflammatory, immunological, and neurodegenerative diseases. As dimerization NOS is required for its activity, inhibitors, including pyrimidine imidazoles, are being evaluated therapeutic inhibition iNOS. However, the precise mechanism their action still unclear. Here, we examined iNOS a imidazole core compound derivative (PID), having low cellular toxicity high affinity...

Summary Objective Drug toxicity is a hurdle to drug development and clinical translation of basic research. Antiepileptic drugs such as carbamazepine ( CBZ ) selective serotonin reuptake inhibitors sertraline SRT are commonly co‐prescribed patients with epilepsy comorbid depression. Because may interfere cytochrome P450 CYP enzyme activity s have been implicated in the conversion reactive cytotoxic metabolites, we investigated vitro models determine whether affects neurotoxic potential...

Heat shock protein 90 (hsp90) drives heme insertion into the β1 subunit of soluble guanylate cyclase (sGC) β1, which enables it to associate with a partner sGCα1 and mature nitric oxide (NO)-responsive active form. We utilized fluorescence polarization measurements hydrogen-deuterium exchange mass spectrometry define molecular interactions between specific human isoforms hsp90β apo-sGCβ1. its isolated M domain, but not N C domains, bind low micromolar affinity heme-free, truncated version...

The soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate signaling pathway evokes vascular smooth muscle relaxation; whether this mediates airway relaxation remains controversial. We posit that sGC activators are equi-effective as β-agonists in reversing contractile agonist-induced shortening. To provide clarity, we tested the efficacy of stimulator and activator drugs, BAY 41-2272 60-2270, respectively, bronchoconstriction human small airways using precision-cut lung slices...

A subset of asthmatics develop a severe form the disease whose etiology involves airway inflammation along with inherent drivers that remain ill-defined. To address this, we studied human smooth muscle cells (HASMC), relaxation drives bronchodilation and dysfunction contributes to obstruction hypersensitivity in asthma. Because HASMC can be driven by NO-soluble guanylyl cyclase (sGC)-cGMP signaling pathway, questioned if from asthma donors might possess defects their sGC or redox enzymes...

Significance: Soluble guanylate cyclase (sGC) is an intracellular enzyme that plays a primary role in sensing nitric oxide (NO) and transducing its multiple signaling effects mammals. Recent Advances: The chaperone heat shock protein 90 (hsp90) associates with proteins cells, including sGC, where it helps to drive heme insertion into the sGC-β1 subunit. This allows associate partner sGC-α1 subunit mature NO-responsive active form. Critical Issues: In this article, we review evidence date...

Myoglobin (Mb) maturation involves heme incorporation as a final step. We investigated role for heat shock protein (hsp) 90 in Mb C2C12 skeletal muscle myoblasts and cell lines. found the following: 1) Hsp90 directly interacts preferentially with heme-free both purified form cells. 2) drives insertion into apoprotein-Mb an ATP-dependent process. 3) During differentiation of myotubes, apo-Mb-hsp90 complex associates 5 cochaperons, Hsp70, activator hsp90 ATPase 1 (Aha1), alanyl-tRNA synthetase...

Eukaryotic initiation factor 2- (eIF2-) associated glycoprotein p67 blocks eIF2α phosphorylation by kinases, and its N-terminal 1−97 amino acid segment can induce efficient translation. To investigate whether glycosylation at the serine/threonine clusters this region is important in protein synthesis, we selected 27TSST30 60SGTS63 for further analysis. By site-directed mutagenesis, were substituted with 27AAGA30 60AGAA63 residues full-length p67, their EGFP fusions constitutively expressed...