ABSTRACT
Objectives: Continuous and excessive secretion of pro-inflammatory and anti-inflammatory chemicals and cytokines may further deteriorate inflammation. Anti-inflammatory drugs play an imperative role in inhibiting the evolution of inflammatory diseases. As per the Unani doctrine, a holistic treatment approach is used to treat illnesses. Therefore, drugs having different actions are used to achieve the synergic effect. Three drugs (Cinnamomum zeylanicum, Alpinia galanga, and Withania somnifera), which are frequently used in Unani medicine for joint disorders were selected to evaluate the anti-inflammatory activity of the extract derived from them. Methods: We used RAW 264.7 macrophage cells to see the expression of inflammatory markers IL-1ß, IL-6, and TNF-α. Cytotoxic activity was assessed with MTT assay and Nitric Oxide (NO) was evaluated using Griess reagent. Further, anti-inflammatory activity was evaluated in Wistar Albino rats using carrageenan-induced paw oedema and immunohistochemistry assays for Cyclooxygenase-2 (COX-2). All the data were analyzed using ANOVA and Dunnett t test for multiple comparisons. Results: This extract did not show any cytotoxic effect and the gene expression was significantly reduced for IL-1ß, IL-6, and TNF-α in a dose-dependent manner. Further, NO production was also significantly reduced in the test groups. Immunohistochemistry revealed that the test groups had less inflammation as compared to the control group. Conclusion: It may be inferred that the ethanolic extract of the three herbs has strong anti-inflammatory activity in the tested inflammatory models and the extract is safe as it did not show any cytotoxic effects in both in vitro and in vivo conditions.
Subject(s)
Alpinia , Anti-Inflammatory Agents , Cinnamomum zeylanicum , Plant Extracts , Rats, Wistar , Withania , Animals , Withania/chemistry , Plant Extracts/pharmacology , Anti-Inflammatory Agents/pharmacology , Rats , Alpinia/chemistry , Mice , Cinnamomum zeylanicum/chemistry , RAW 264.7 Cells , Male , Edema/drug therapy , CarrageenanABSTRACT
With the continuous development of new sensor features and tracking algorithms for object tracking, researchers have opportunities to experiment using different combinations. However, there is no standard or agreed method for selecting an appropriate architecture for autonomous vehicle (AV) crash reconstruction using multi-sensor-based sensor fusion. This study proposes a novel simulation method for tracking performance evaluation (SMTPE) to solve this problem. The SMTPE helps select the best tracking architecture for AV crash reconstruction. This study reveals that a radar-camera-based centralized tracking architecture of multi-sensor fusion performed the best among three different architectures tested with varying sensor setups, sampling rates, and vehicle crash scenarios. We provide a brief guideline for the best practices in selecting appropriate sensor fusion and tracking architecture arrangements, which can be helpful for future vehicle crash reconstruction and other AV improvement research.
ABSTRACT
Arsenic (As) is a global carcinogenic contaminant, and is one of the significant environmental constraints that limits the development and yield of crop plants. It is always tagged along with rice as rice takes up As and tends to accumulate it in grains. This amassment makes a way for As to get into the food chain that leads to unforeseen human health risks. Being viewed as parallel with toxicity, As in rice is an important global risk that calls for an urgent solution. WRKY Transcription Factors (TFs) seems to be promising in this area. The classical and substantial progress in the molecular mechanism of WRKY TFs, strengthened the understanding of innovative solutions for dealing with As in rice. Here, we review the potential of WRKY TFs under As stressed rice as a genetic solution and also provide insights into As and rice. Further, we develop an understanding of WRKY TF gene family and its regulation in rice. To date, studies on the role of WRKY TFs under As stressed rice are lacking. This area needs to be explored more so that this gene family can be utilized as an effective genetic tool that can break the As cycle to develop low or As free rice cultivar.
Subject(s)
Arsenic , Oryza , Humans , Oryza/genetics , Oryza/metabolism , Transcription Factors/metabolism , Arsenic/toxicity , Plants/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Stress, Physiological/genetics , Gene Expression Regulation, Plant/genetics , PhylogenyABSTRACT
Mechanisms that regulate nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions and is activated by calmodulin (CaM) binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two NOS electron transfer domains in an FRET dye-labeled endothelial NOS reductase domain (eNOSr) and to understand how CaM affects the dynamics to regulate catalysis by shaping the spatial and temporal conformational behaviors of eNOSr. In addition, we developed and applied a new imaging approach capable of recording three-dimensional FRET efficiency versus time images to characterize the impact on dynamic conformal states of the eNOSr enzyme by the binding of CaM, which identifies clearly that CaM binding generates an extra new open state of eNOSr, resolving more detailed NOS conformational states and their fluctuation dynamics. We identified a new output state that has an extra open conformation that is only populated in the CaM-bound eNOSr. This may reveal the critical role of CaM in triggering NOS activity as it gives conformational flexibility for eNOSr to assume the electron transfer output FMN-heme state. Our results provide a dynamic link to recently reported EM static structure analyses and demonstrate a capable approach in probing and simultaneously analyzing all of the conformational states, their fluctuations, and the fluctuation dynamics for understanding the mechanism of NOS electron transfer, involving electron transfer among FAD, FMN, and heme domains, during nitric oxide synthesis.
Subject(s)
Calmodulin , Nitric Oxide Synthase Type III , Calmodulin/metabolism , Electron Transport , Heme/metabolism , Nitric Oxide , Nitric Oxide Synthase , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide Synthase Type III/metabolismABSTRACT
Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr being an abundant post-translational modification on calmodulin, the mechanistic role of this modification in altering calmodulin function and eNOS activation has not been investigated. Here, using genetic code expansion to site-specifically nitrate calmodulin at its two tyrosine residues, we assessed the effects of these alterations on calcium binding by calmodulin and on binding and activation of eNOS. We found that nitroTyr-calmodulin retains affinity for eNOS under resting physiological calcium concentrations. Results from in vitro eNOS assays with calmodulin nitrated at Tyr-99 revealed that this nitration reduces nitric-oxide production and increases eNOS decoupling compared with WT calmodulin. In contrast, calmodulin nitrated at Tyr-138 produced more nitric oxide and did so more efficiently than WT calmodulin. These results indicate that the nitroTyr post-translational modification, like tyrosine phosphorylation, can impact calmodulin sensitivity for calcium and reveal Tyr site-specific gain or loss of functions for calmodulin-induced eNOS activation.
Subject(s)
Calcium/metabolism , Calmodulin/metabolism , Nitric Oxide Synthase Type III/metabolism , Tyrosine/metabolism , Animals , Cattle , Cell Extracts , Fluorescence , HEK293 Cells , Humans , Interferometry , Nitrosation , Protein BindingABSTRACT
The enzyme soluble guanylyl cyclase (sGC) is a heterodimer composed of an α subunit and a heme-containing ß subunit. It participates in signaling by generating cGMP in response to nitric oxide (NO). Heme insertion into the ß1 subunit of sGC (sGCß) is critical for function, and heat shock protein 90 (HSP90) associates with heme-free sGCß (apo-sGCß) to drive its heme insertion. Here, we tested the accuracy and relevance of a modeled apo-sGCß-HSP90 complex by constructing sGCß variants predicted to have an impaired interaction with HSP90. Using site-directed mutagenesis, purified recombinant proteins, mammalian cell expression, and fluorescence approaches, we found that (i) three regions in apo-sGCß predicted by the model mediate direct complex formation with HSP90 both in vitro and in mammalian cells; (ii) such HSP90 complex formation directly correlates with the extent of heme insertion into apo-sGCß and with cyclase activity; and (iii) apo-sGCß mutants possessing an HSP90-binding defect instead bind to sGCα in cells and form inactive, heme-free sGC heterodimers. Our findings uncover the molecular features of the cellular apo-sGCß-HSP90 complex and reveal its dual importance in enabling heme insertion while preventing inactive heterodimer formation during sGC maturation.
Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Soluble Guanylyl Cyclase/metabolism , Animals , COS Cells , Cattle , Cells, Cultured , Chlorocebus aethiops , HumansABSTRACT
NO synthase (NOS) enzymes perform interdomain electron transfer reactions during catalysis that may rely on complementary charge interactions at domain-domain interfaces. Guided by our previous results and a computer-generated domain-docking model, we assessed the importance of cross-domain charge interactions in the FMN-to-heme electron transfer in neuronal NOS (nNOS). We reversed the charge of three residues (Glu-762, Glu-816, and Glu-819) that form an electronegative triad on the FMN domain and then individually reversed the charges of three electropositive residues (Lys-423, Lys-620, and Lys-660) on the oxygenase domain (NOSoxy), to potentially restore a cross-domain charge interaction with the triad, but in reversed polarity. Charge reversal of the triad completely eliminated heme reduction and NO synthesis in nNOS. These functions were partly restored by the charge reversal at oxygenase residue Lys-423, but not at Lys-620 or Lys-660. Full recovery of heme reduction was probably muted by an accompanying change in FMN midpoint potential that made electron transfer to the heme thermodynamically unfavorable. Our results provide direct evidence that cross-domain charge pairing is required for the FMN-to-heme electron transfer in nNOS. The unique ability of charge reversal at position 423 to rescue function indicates that it participates in an essential cross-domain charge interaction with the FMN domain triad. This supports our domain-docking model and suggests that it may depict a productive electron transfer complex formed during nNOS catalysis.
Subject(s)
Electrons , Heme/metabolism , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide/metabolism , Animals , Catalysis , Cytochromes c/metabolism , Electron Transport , Flavin Mononucleotide/metabolism , Kinetics , Models, Molecular , Mutation , Nitric Oxide Synthase Type I/chemistry , Nitric Oxide Synthase Type I/genetics , Oxidation-Reduction , Protein Domains , RatsABSTRACT
Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within hemeproteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH heme binding, antagonized heme delivery to both cytosolic and nuclear hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile heme to rise dramatically. We conclude that by virtue of its heme-binding property, GAPDH binds and chaperones labile heme to create a heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring heme homeostasis in health and disease.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Heme/metabolism , Molecular Chaperones/metabolism , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Heme/chemistry , Humans , Mice , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Sequence Data , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Protein Binding , Sequence AlignmentABSTRACT
The signaling molecule nitric oxide (NO) is synthesized in animals by structurally related NO synthases (NOSs), which contain NADPH/FAD- and FMN-binding domains. During catalysis, NADPH-derived electrons transfer into FAD and then distribute into the FMN domain for further transfer to internal or external heme groups. Conformational freedom of the FMN domain is thought to be essential for the electron transfer (ET) reactions in NOSs. To directly examine this concept, we utilized a "Cys-lite" neuronal NOS flavoprotein domain and substituted Cys for two residues (Glu-816 and Arg-1229) forming a salt bridge between the NADPH/FAD and FMN domains in the conformationally closed structure to allow cross-domain disulfide bond formation or cross-linking by bismaleimides of various lengths. The disulfide bond cross-link caused a ≥95% loss of cytochrome c reductase activity that was reversible with DTT treatment, whereas graded cross-link lengthening gradually increased activity, thus defining the conformational constraints in the catalytic process. We used spectroscopic and stopped-flow techniques to further investigate how the changes in FMN domain conformational freedom impact the following: (i) the NADPH interaction; (ii) kinetics of electron loading (flavin reduction); (iii) stabilization of open versus closed conformational forms in two different flavin redox states; (iv) reactivity of the reduced FMN domain toward cytochrome c; (v) response to calmodulin binding; and (vi) the rates of interflavin ET and the FMN domain conformational dynamics. Together, our findings help explain how the spatial and temporal behaviors of the FMN domain impact catalysis by the NOS flavoprotein domain and how these behaviors are governed to enable electron flow through the enzyme.
Subject(s)
Flavoproteins/chemistry , Nitric Oxide Synthase Type I/metabolism , Animals , Calmodulin/chemistry , Catalysis , Cross-Linking Reagents/chemistry , Cysteine/chemistry , Cytochromes c/chemistry , Disulfides/chemistry , Electrons , Flavins/chemistry , Hydrogen-Ion Concentration , Kinetics , Maleimides/chemistry , Mutation , NADP/chemistry , Nitric Oxide/chemistry , Oxidation-Reduction , Protein Domains , Rats , Spectrophotometry, Ultraviolet , Sulfhydryl Compounds/chemistryABSTRACT
Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.
Subject(s)
Calmodulin/metabolism , Fluorescence Resonance Energy Transfer/methods , Nitric Oxide Synthase Type I/chemistry , Nitric Oxide Synthase Type I/metabolism , Animals , Carbocyanines/metabolism , Cattle , Protein Structure, Secondary , Protein Structure, Tertiary , Spectrophotometry, Ultraviolet , Time FactorsABSTRACT
The activity of endothelial NO synthase (eNOS) is triggered by calmodulin (CaM) binding and is often further regulated by phosphorylation at several positions in the enzyme. Phosphorylation at Ser1179 occurs in response to diverse physiologic stimuli and increases the NO synthesis and cytochrome c reductase activities of eNOS, thereby enhancing its participation in biological signal cascades. Despite its importance, the mechanism by which Ser1179 phosphorylation increases eNOS activity is not understood. To address this, we used stopped-flow spectroscopy and computer modeling approaches to determine how the phosphomimetic mutation (S1179D) may impact electron flux through eNOS and the conformational behaviors of its reductase domain, both in the absence and presence of bound CaM. We found that S1179D substitution in CaM-free eNOS had multiple effects; it increased the rate of flavin reduction, altered the conformational equilibrium of the reductase domain, and increased the rate of its conformational transitions. We found these changes were equivalent in degree to those caused by CaM binding to wild-type eNOS, and the S1179D substitution together with CaM binding caused even greater changes in these parameters. The modeling indicated that the changes caused by the S1179D substitution, despite being restricted to the reductase domain, are sufficient to explain the stimulation of both the cytochrome c reductase and NO synthase activities of eNOS. This helps clarify how Ser1179 phosphorylation regulates eNOS and provides a foundation to compare its regulation by other phosphorylation events.
Subject(s)
Nitric Oxide Synthase Type III/chemistry , Nitric Oxide Synthase Type III/metabolism , Amino Acid Motifs , Amino Acid Substitution , Animals , Cattle , Cytochromes c/metabolism , Flavins/chemistry , Flavins/metabolism , Kinetics , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/genetics , Phosphorylation , Protein ConformationABSTRACT
Heat shock protein 90 (hsp90) drives heme insertion into the ß1 subunit of soluble guanylate cyclase (sGC) ß1, which enables it to associate with a partner sGCα1 subunit and mature into a nitric oxide (NO)-responsive active form. We utilized fluorescence polarization measurements and hydrogen-deuterium exchange mass spectrometry to define molecular interactions between the specific human isoforms hsp90ß and apo-sGCß1. hsp90ß and its isolated M domain, but not its isolated N and C domains, bind with low micromolar affinity to a heme-free, truncated version of sGCß1 (sGCß1(1-359)-H105F). Surprisingly, hsp90ß and its M domain bound to the Per-Arnt-Sim (PAS) domain of apo-sGC-ß1(1-359), which lies adjacent to its heme-binding (H-NOX) domain. The interaction specifically involved solvent-exposed regions in the hsp90ß M domain that are largely distinct from sites utilized by other hsp90 clients. The interaction strongly protected two regions of the sGCß1 PAS domain and caused local structural relaxation in other regions, including a PAS dimerization interface and a segment in the H-NOX domain. Our results suggest a means by which the hsp90ß interaction could prevent apo-sGCß1 from associating with its partner sGCα1 subunit while enabling structural changes to assist heme insertion into the H-NOX domain. This mechanism would parallel that in other clients like the aryl hydrocarbon receptor and HIF1α, which also interact with hsp90 through their PAS domains to control protein partner and small ligand binding interactions.
Subject(s)
Guanylate Cyclase/chemistry , Guanylate Cyclase/metabolism , HSP90 Heat-Shock Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Amino Acid Sequence , Animals , Cattle , Deuterium Exchange Measurement , HSP90 Heat-Shock Proteins/chemistry , Heme/metabolism , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Soluble Guanylyl CyclaseABSTRACT
The impairment of vasodilator nitric oxide (NO) production is well accepted as a typical marker of endothelial dysfunction in vascular diseases, including in the pathophysiology of pulmonary arterial hypertension (PAH), but the molecular mechanisms accounting for loss of NO production are unknown. We hypothesized that low NO production by pulmonary arterial endothelial cells in PAH is due to inactivation of NO synthase (eNOS) by aberrant phosphorylation of the protein. To test the hypothesis, we evaluated eNOS levels, dimerization, and phosphorylation in the vascular endothelial cells and lungs of patients with PAH compared with controls. In mechanistic studies, eNOS activity in endothelial cells in PAH lungs was found to be inhibited due to phosphorylation at T495. Evidence pointed to greater phosphorylation/activation of protein kinase C (PKC) α and its greater association with eNOS as the source of greater phosphorylation at T495. The presence of greater amounts of pT495-eNOS in plexiform lesions in lungs of patients with PAH confirmed the pathobiological mechanism in vivo. Transfection of the activating mutation of eNOS (T495A/S1177D) restored NO production in PAH cells. Pharmacological blockade of PKC activity by ß-blocker also restored NO formation by PAH cells, identifying one mechanism by which ß-blockers may benefit PAH and cardiovascular diseases through recovery of endothelial functions.
Subject(s)
Endothelial Cells/enzymology , Hypertension, Pulmonary/enzymology , Nitric Oxide Synthase Type III/metabolism , Protein Processing, Post-Translational , Adult , Cells, Cultured , Female , Humans , Hypertension, Pulmonary/pathology , Lung/enzymology , Lung/pathology , Male , Middle Aged , Nitric Oxide/biosynthesis , Phosphorylation , Protein Kinase C/metabolismABSTRACT
Nitric oxide synthases (NOSs) are haem-thiolate enzymes that catalyse the conversion of L-arginine (L-Arg) into NO and citrulline. Inducible NOS (iNOS) is responsible for delivery of NO in response to stressors during inflammation. The catalytic performance of iNOS is proposed to rely mainly on the haem midpoint potential and the ability of the substrate L-Arg to provide a hydrogen bond for oxygen activation (O-O scission). We present a study of native iNOS compared with iNOS-mesohaem, and investigate the formation of a low-spin ferric haem-aquo or -hydroxo species (P) in iNOS mutant W188H substituted with mesohaem. iNOS-mesohaem and W188H-mesohaem were stable and dimeric, and presented substrate-binding affinities comparable to those of their native counterparts. Single turnover reactions catalysed by iNOSoxy with L-Arg (first reaction step) or N-hydroxy-L-arginine (second reaction step) showed that mesohaem substitution triggered higher rates of Fe(II)O2 conversion and altered other key kinetic parameters. We elucidated the first crystal structure of a NOS substituted with mesohaem and found essentially identical features compared with the structure of iNOS carrying native haem. This facilitated the dissection of structural and electronic effects. Mesohaem substitution substantially reduced the build-up of species P in W188H iNOS during catalysis, thus increasing its proficiency towards NO synthesis. The marked structural similarities of iNOSoxy containing native haem or mesohaem indicate that the kinetic behaviour observed in mesohaem-substituted iNOS is most heavily influenced by electronic effects rather than structural alterations.
Subject(s)
Arginine/chemistry , Heme/chemistry , Mesoporphyrins/chemistry , Models, Molecular , Nitric Oxide Synthase Type II/chemistry , Nitric Oxide/metabolism , Up-Regulation , Amino Acid Substitution , Animals , Arginine/metabolism , Biocatalysis , Dimerization , Enzyme Stability , Heme/metabolism , Hydrogen Bonding , Hydroxylation , Kinetics , Mesoporphyrins/metabolism , Mice , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
The NOS (nitric oxide synthase; EC 1.14.13.39) enzymes contain a C-terminal flavoprotein domain [NOSred (reductase domain of NOS)] that binds FAD and FMN, and an N-terminal oxygenase domain that binds haem. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain [FNR (ferredoxin NADP-reductase)] and the oxygenase domain. Previously we have shown that three residues on the FMN domain (Glu762, Glu816 and Glu819) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred (neuronal NOSred). In the present study, we show that charge neutralization or reversal at each of these residues alters the setpoint [Keq(A)] of the NOSred conformational equilibrium to favour the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have higher conformational transition rates (1.5-4-fold) and rates of interflavin electron transfer (1.5-2-fold) relative to wild-type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations.
Subject(s)
Flavin Mononucleotide/metabolism , Nitric Oxide Synthase Type I/chemistry , Nitric Oxide Synthase Type I/metabolism , Protein Interaction Domains and Motifs/physiology , Binding Sites/genetics , Electron Transport , Humans , Kinetics , Models, Biological , Models, Molecular , Molecular Docking Simulation , Movement/physiology , Mutagenesis, Site-Directed , NADPH-Ferrihemoprotein Reductase/chemistry , NADPH-Ferrihemoprotein Reductase/metabolism , Nitric Oxide Synthase Type I/genetics , Protein Binding/genetics , Protein Conformation , Protein Folding , Protein Interaction Domains and Motifs/genetics , Static ElectricityABSTRACT
PIM-1 kinase belongs to the Ser/Thr kinases family, an attractive therapeutic target for prostate cancer. Here, we screened about 100 natural substances to find potential PIM-1 inhibitors. Two natural compounds, Naringenin and Quercetin, were finally selected based on their PIM-1 inhibitory potential and binding affinities. The docking score of Naringenin and Quercetin with PIM-1 is -8.4 and - 8.1 kcal/mol, respectively. Fluorescence binding studies revealed a strong affinity (Ka values, 3.1 × 104 M-1 and 4.6 × 107 M-1 for Naringenin and Quercetin, respectively) with excellent IC50 values for Naringenin and Quercetin (28.6 µM and 34.9 µM, respectively). Both compounds inhibited the growth of prostate cancer cells (LNCaP) in a dose-dependent manner, with the IC50 value of Naringenin at 17.5 µM and Quercetin at 8.88 µM. To obtain deeper insights into the PIM-1 inhibitory effect of Naringenin and Quercetin, we performed extensive molecular dynamics simulation studies, which provided insights into the binding mechanisms of PIM-1 inhibitors. Finally, Naringenin and Quercetin were suggested to serve as potent PIM-1 inhibitors, offering targeted treatments of prostate cancer. In addition, our findings may help to design novel Naringenin and Quercetin derivatives that could be effective in therapeutic targeting of prostate cancer.
Subject(s)
Flavanones , Molecular Docking Simulation , Molecular Dynamics Simulation , Prostatic Neoplasms , Proto-Oncogene Proteins c-pim-1 , Quercetin , Flavanones/pharmacology , Flavanones/chemistry , Quercetin/pharmacology , Quercetin/chemistry , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Proto-Oncogene Proteins c-pim-1/metabolism , Humans , Male , Cell Line, Tumor , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Cell Proliferation/drug effects , Protein BindingABSTRACT
Head and neck squamous cell carcinoma (HNSC) is a diverse group of tumors arising from oral cavity, oropharynx, larynx, and hypopharynx squamous epithelium, posing significant morbidity. Aquaporins (AQPs) are membrane proteins forming water channels, some associated with carcinomas. Chromobox (CBX) family is known to modulate physiological and oncological processes. In our study, we analyzed AQPs and CBXs having significant expression followed by their prognostic and mutational assessment. Next, we performed enrichment and tumor infiltration analysis followed by HPA validation. Lastly, we established a 3-node miRNA-TF-mRNA regulatory network and performed protein-protein docking of the highest-degree subnetwork motif between TF and mRNA. Significant upregulation of CBX3/2 and downregulation of AQP3/5/7 correlated with poor overall survival (OS) in HNSC patients. The most significant pathway, GO-BP, GO-MF, and GO-CC terms associated with AQP3 and CBX3 were passive transport by aquaporins, response to vitamin, glycerol channel activity, and condensed chromosome, centromeric region. AQP3 negatively correlated with [Formula: see text] T cells, positively with [Formula: see text] T cells and B cells, and negatively with tumor purity, whereas CBX3 positively correlated with [Formula: see text] T cells, negatively with [Formula: see text] T cells and B cells, and positively with tumor purity. Three-node miRNA-TF-mRNA regulatory network revealed a highest-degree subnetwork motif comprising one TF (SMAD3), one miRNA (miR-423-5p), and one mRNA (AQP3). Protein-protein interaction studies suggested a direct interaction between AQP3 and Smad3 proteins. We concluded that AQP3 and CBX3 hold potential as treatment strategies and individual prognostic biomarkers, while further protein-protein interaction studies of AQP3 could offer insights into its interactions with Smad3 proteins.
ABSTRACT
In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.
Subject(s)
Calmodulin/chemistry , Nitric Oxide Synthase Type I/chemistry , Nitric Oxide/biosynthesis , Animals , Calmodulin/metabolism , Cytochromes c/chemistry , Cytochromes c/metabolism , Electron Transport/physiology , Flavin Mononucleotide/chemistry , Flavin Mononucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Kinetics , NADP/chemistry , NADP/metabolism , Nitric Oxide/chemistry , Nitric Oxide Synthase Type I/metabolism , Oxidation-Reduction , Protein Structure, Tertiary , RatsABSTRACT
The proper functioning of any protein depends on its three dimensional conformation which is achieved by the accurate folding mechanism. Keeping away from the exposed stress conditions leads to cooperative unfolding and sometimes partial folding, forming the structures like protofibrils, fibrils, aggregates, oligomers, etc. leading to several neurodegenerative diseases like Parkinson's disease, Alzheimer's, Cystic fibrosis, Huntington, Marfan syndrome, and also cancers in some cases, too. Hydration of proteins is necessary, which may be achieved by the presence of organic solutes called osmolytes within the cell. Osmolytes belong to different classes in different organisms and play their role by preferential exclusion of osmolytes and preferential hydration of water molecules and achieves the osmotic balance in the cell otherwise it may cause problems like cellular infection, cell shrinkage leading to apoptosis and cell swelling which is also the major injury to the cell. Osmolyte interacts with protein, nucleic acids, intrinsically disordered proteins by non-covalent forces. Stabilizing osmolytes increases the Gibbs free energy of the unfolded protein and decreases that of folded protein and vice versa with denaturants (urea and guanidinium hydrochloride). The efficacy of each osmolyte with the protein is determined by the calculation of m value which reflects its efficiency with protein. Hence osmolytes can be therapeutically considered and used in drugs.
Subject(s)
Intrinsically Disordered Proteins , Water/chemistry , Solutions , Molecular Conformation , Protein Folding , ThermodynamicsABSTRACT
Breast cancer (BC) is the second-most common type and among the leading causes of worldwide cancer-related deaths. There is marked person-to-person variability in susceptibility to, and phenotypic expression and prognosis of BC, a predicament that calls for personalized medicine and individually tailored therapeutics. In this study, we report new observations on prognostic hub genes and key pathways involved in BC. We used the data set GSE109169, comprising 25 pairs of BC and adjacent normal tissues. Using a high-throughput transcriptomic approach, we selected data on 293 differentially expressed genes to establish a weighted gene coexpression network. We identified three age-linked modules where the light-gray module strongly correlated with BC. Based on the gene significance and module membership features, peptidase inhibitor 15 (PI15) and KRT5 were identified as our hub genes from the light-gray module. These genes were further verified at transcriptional and translational levels across 25 pairs of BC and adjacent normal tissues. Their promoter methylation profiles were assessed based on various clinical parameters. In addition, these hub genes were used for Kaplan-Meier survival analysis, and their correlation with tumor-infiltrating immune cells was investigated. We found that PI15 and KRT5 may be potential biomarkers and potential drug targets. These findings call for future research in a larger sample size, which could inform diagnosis and clinical management of BC, thus paving the way toward personalized medicine.