Manifold active-state conformations in GPCRs: Agonist-activated constitutively active mutant AT1 receptor preferentially couples to Gq compared to the wild-type AT1 receptor

AbstractThe angiotensin II type I (AT1) receptor mediates regulation of blood pressure and water-electrolyte balance by Ang II. Substitution of Gly for Asn111 of the AT1 receptor constitutively activates the receptor leading to Gq-coupled IP3 production independent of Ang II binding. The Ang II-activated conformation of the AT1N111G receptor was proposed to be similar to that of the wild-type AT1 receptor, although, various aspects of the Ang II-induced conformation of this constitutively active mutant receptor have not been systematically studied. Here, we provide evidence that the conformation of the active state of the wild-type and the constitutively active AT1 receptors are different. Upon Ang II binding an activated conformation of the wild-type AT1 receptor activates G protein and recruits β-arrestin. In contrast, the agonist-bound AT1N111G mutant receptor preferentially couples to Gq and is inadequate in β-arrestin recruitment

New prognostic scoring system for mortality in idiopathic pulmonary fibrosis by modifying the gender, age, and physiology model with desaturation during the six-minute walk test

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease (ILD) with variable and heterogeneous clinical course. The GAP (gender, age, and physiology) model had been used to predict mortality in patients with IPF, but does not contain exercise capacity. Therefore, our aim in this study was to develop new prognostic scoring system in the Korea IPF Cohort (KICO) registry.Materials and methodsThis is a retrospective study of Korean patients with IPF in KICO registry from June 2016 to August 2021. We developed new scoring system (the GAP6) based on the GAP model adding nadir saturation of percutaneous oxygen (SpO2) during six-minute walk test (6MWT) in the KICO registry and compared the efficacy of the GAP and the GAP6 model.ResultsAmong 2,412 patients in KICO registry, 966 patients were enrolled. The GAP6 model showed significant prognostic value for mortality between each stage [HR Stage II vs. Stage I = 2.89 (95% CI = 2.38–3.51), HR Stage III vs. Stage II = 2.68 (95% CI = 1.60–4.51)]. In comparison the model performance with area under curve (AUC) using receiver operating characteristic (ROC) curve analysis, the GAP6 model showed a significant improvement for predicting mortality than the GAP model (AUC the GAP vs. the GAP6, 0.646 vs. 0.671, p < 0.0019). Also, the C-index values slightly improved from 0.674 to 0.691 for mortality.ConclusionThe GAP6 model adding nadir SpO2 during 6WMT for an indicator of functional capacity improves prediction ability with C-index and AUC. Additional multinational study is needed to confirm these finding and validate the applicability and accuracy of this risk assessment system

Different Effects of Salt Bridges near the Active Site of Cold-Adapted <i>Proteus mirabilis</i> Lipase on Thermal and Organic Solvent Stabilities

Organic solvent-tolerant (OST) enzymes have been discovered in psychrophiles. Cold-adapted OST enzymes exhibit increased conformational flexibility in polar organic solvents resulting from their intrinsically flexible structures. Proteus mirabilis lipase (PML), a cold-adapted OST lipase, was used to assess the contribution of salt bridges near the active site involving two arginine residues (R237 and R241) on the helix η1 and an aspartate residue (D248) on the connecting loop to the thermal and organic solvent stabilities of PML. Alanine substitutions for the ion pairs (R237A, R241A, D248A, and R237A/D248A) increased the conformational flexibility of PML mutants compared to that of the wild-type PML in an aqueous buffer. The PML mutants became more susceptible to denaturation after increasing the dimethyl sulfoxide or methanol concentration than after a temperature increase. Methanol was more detrimental to the structural stability of PML compared to dimethyl sulfoxide. These results suggest that direct interactions of dimethyl sulfoxide and methanol with the residues near the active site can have a destructive effect on the structure of PML compared with the global effect of heat on the protein structure. This study provides insight into the conformational changes within an OST enzyme with different effects on its thermal and organic solvent stabilities

Different Effects of Salt Bridges near the Active Site of Cold-Adapted Proteus mirabilis Lipase on Thermal and Organic Solvent Stabilities

Organic solvent-tolerant (OST) enzymes have been discovered in psychrophiles. Cold-adapted OST enzymes exhibit increased conformational flexibility in polar organic solvents resulting from their intrinsically flexible structures. Proteus mirabilis lipase (PML), a cold-adapted OST lipase, was used to assess the contribution of salt bridges near the active site involving two arginine residues (R237 and R241) on the helix &eta;1 and an aspartate residue (D248) on the connecting loop to the thermal and organic solvent stabilities of PML. Alanine substitutions for the ion pairs (R237A, R241A, D248A, and R237A/D248A) increased the conformational flexibility of PML mutants compared to that of the wild-type PML in an aqueous buffer. The PML mutants became more susceptible to denaturation after increasing the dimethyl sulfoxide or methanol concentration than after a temperature increase. Methanol was more detrimental to the structural stability of PML compared to dimethyl sulfoxide. These results suggest that direct interactions of dimethyl sulfoxide and methanol with the residues near the active site can have a destructive effect on the structure of PML compared with the global effect of heat on the protein structure. This study provides insight into the conformational changes within an OST enzyme with different effects on its thermal and organic solvent stabilities

Cloning, Expression, and Characterization of a Psychrophilic Glucose 6-Phosphate Dehydrogenase from Sphingomonas sp. PAMC 26621

Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5&ndash;8.5 and 30 &deg;C for NADP+ and 20 &deg;C for NAD+. SpG6PD1 utilized both NADP+ and NAD+, with the preferential utilization of NADP+. A high Km value for glucose 6-phosphate and low activation enthalpy (&Delta;H&Dagger;) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4&ndash;40 &deg;C, its activity and tertiary structure were better preserved between 4&ndash;20 &deg;C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat