The mammalian odorant receptors (ORs) form a chemical-detecting interface between the atmosphere and the nervous system. This large gene family is composed of hundreds of membrane proteins predicted ...to form as many unique small molecule binding niches within their G-protein coupled receptor (GPCR) framework, but very little is known about the molecular recognition strategies they use to bind and discriminate between small molecule odorants. Using rationally designed synthetic analogs of a typical aliphatic aldehyde, we report evidence that among the ORs showing specificity for the aldehyde functional group, a significant percentage detect the aldehyde through its ability to react with water to form a 1,1-geminal (gem)-diol. Evidence is presented indicating that the rat OR-I7, an often-studied and modeled OR known to require the aldehyde function of octanal for activation, is likely one of the gem-diol activated receptors. A homology model based on an activated GPCR X-ray structure provides a structural hypothesis for activation of OR-I7 by the gem-diol of octanal.
Protein kinases are key signaling enzymes which are dysregulated in many health disorders and therefore represent major targets of extensive drug discovery efforts. Their regulation in the cell is ...exerted via various mechanisms, including control of the 3D conformation of their catalytic domains. We developed a procedure, BlockMaster, for partitioning protein structures into semirigid blocks and flexible regions based on residue−residue correlations calculated from normal modes. BlockMaster provided correct partitioning into domains and subdomains of several test set proteins for which documented expert annotation of subdomains exists. When applied to representative structures of protein kinases, BlockMaster identified semirigid blocks within the traditional N-terminal and C-terminal lobes of the kinase domain. In general, the block regions had elevated helical content and reduced, but significant, coil content compared to the nonblock (flexible) regions. The specificity-determining regions, previously used to derive inhibitory peptides, were found to be more flexible in the tyrosine kinases than in serine/threonine kinases. Two blocks were identified which spanned both lobes. The first, which we termed the “pivot” block, included the αC−β4 loop in the N-terminal lobe and part of the activation loop in the C-terminal lobe and appeared in both the active and inactive conformations of the kinases. The second, which we termed the “loop” block, differed between the active and inactive conformations. In the structures of active kinases, this block included part of the activation loop in the C-terminal lobe and the αC helix in the N-terminal lobe, representing a known interaction that stabilizes the active conformation. In the inactive structures, this block included G loop residues instead of the αC residues. This novel inactive “loop” block may stabilize the inactive conformation and thus downregulate kinase activity.
Highlights • Structural modeling analysis of tilapia GTHs, GTHRs and their complexes is presented. • In vivo immunoneutralization of FSH abolished basal and GnRH-stimulated 11KT and LH. • In vivo ...immunoneutralization of LH reduced 11KT and slightly reduced FSH levels. • Results suggest a feedback mechanism between FSH and LH release in tilapia.
The 25 human bitter taste receptors (hT2Rs) recognize thousands of structurally and chemically diverse bitter substances. The binding modes of human bitter taste receptors hT2R10 and hT2R46, which ...are responsible for strychnine recognition, were previously established using site-directed mutagenesis, functional assays, and molecular modeling. Here we construct a phylogenetic tree and reconstruct ancestral sequences of the T2R10 and T2R46 clades. We next analyze the binding sites in view of experimental data to predict their ability to recognize strychnine. This analysis suggests that the common ancestor of hT2R10 and hT2R46 is unlikely to bind strychnine in the same mode as either of its two descendants. Estimation of relative divergence times shows that hT2R10 evolved earlier than hT2R46. Strychnine recognition was likely acquired first by the earliest common ancestor of the T2R10 clade before the separation of primates from other mammals, and was highly conserved within the clade. It was probably independently acquired by the common ancestor of T2R43-47 before the homo-ape speciation, lost in most T2Rs within this clade, but enhanced in the hT2R46 after humans diverged from the rest of primates. Our findings suggest hypothetical strychnine T2R receptors in several species, and serve as an experimental guide for further study. Improved understanding of how bitter taste receptors acquire the ability to be activated by particular ligands is valuable for the development of sensors for bitterness and for potential toxicity.
Smell loss is important for coronavirus disease-2019 (COVID-19) screening and diagnosis. Particular attention should be paid to individuals with pre-COVID-19 chronic hyposmia or anosmia. We report a ...case of reversible taste impairment in a COVID-19 patient with chronically impaired sense of smell. This case emphasizes the importance of COVID-19-related taste assessment.
T2R bitter taste receptors play a crucial role in sinonasal innate immunity by upregulating mucociliary clearance and nitric oxide (NO) production in response to bitter gram-negative quorum-sensing ...molecules in the airway surface liquid. Previous studies showed that phytochemical flavonoid metabolites, known as anthocyanidins, taste bitter and have antibacterial effects. Our objectives were to examine the effects of anthocyanidins on NO production by human sinonasal epithelial cells and ciliary beat frequency, and their impact on common sinonasal pathogens Pseudomonas aeruginosa and Staphylococcus aureus.
Ciliary beat frequency and NO production were measured by using digital imaging of differentiated air-liquid interface cultures prepared from primary human cells isolated from residual surgical material. Plate-based assays were used to determine the effects of anthocyanidins on bacterial swimming and swarming motility. Biofilm formation and planktonic growth were also assessed.
Anthocyanidin compounds triggered epithelial cells to produce NO but not through T2R receptors. However, anthocyanidins did not impact ciliary beat frequency. Furthermore, they did not reduce biofilm formation or planktonic growth of P. aeruginosa. In S. aureus, they did not reduce planktonic growth, and only one compound had minimal antibiofilm effects. The anthocyanidin delphinidin and anthocyanin keracyanin were found to promote bacterial swimming, whereas anthocyanidin cyanidin and flavonoid myricetin did not. No compounds that were tested inhibited bacterial swarming.
Results of this study indicated that, although anthocyanidins may elicited an innate immune NO response from human cells, they do not cause an increase in ciliary beating and they may also cause a pathogenicity-enhancing effect in P. aeruginosa. Additional studies are necessary to understand how this would affect the use of anthocyanidins as therapeutics. This study emphasized the usefulness of in vitro screening of candidate compounds against multiple parameters of both epithelial and bacterial physiologies to prioritize candidates for in vivo therapeutic testing.
Protein kinase RNA-activated (PKR) plays an important role in a broad range of intracellular regulatory mechanisms and in the pathophysiology of many human diseases, including microbial and viral ...infections, cancer, diabetes and neurodegenerative disorders. Recently, several potent PKR inhibitors have been synthesized. However, the enzyme’s multifunctional character and a multitude of PKR downstream targets have prevented the successful transformation of such inhibitors into effective drugs. Thus, the need for additional PKR inhibitors remains. With the help of computer-aided drug-discovery tools, we designed and synthesized potential PKR inhibitors. Indeed, two compounds were found to inhibit recombinant PKR in pharmacologically relevant concentrations. One compound, 6-amino-3-methyl-2-oxo-
N
-phenyl-2,3-dihydro-1H-benzodimidazole-1-carboxamide, also showed anti-apoptotic properties. The novel molecules diversify the existing pool of PKR inhibitors and provide a basis for the future development of compounds based on PKR signal transduction mechanism.
Scope
Brown adipose tissue (BAT) is the main regulator of thermogenesis by increasing energy expenditure through the uncoupling of oxidative metabolism from ATP synthesis. There is a growing body of ...evidence for BAT being the key responsible organ in combating obesity and its related disorders. Herein we propose the fungal protein ostreolysin (Oly), which has been previously shown to bind to cholesterol‐enriched raft‐like membrane domains (lipid rafts) of mammalian cells, as a suitable candidate for interaction with brown preadipocytes. The aim of the present study was therefore to characterize the mechanism by which a recombinant version of ostreolysin (rOly) induces brown adipocyte differentiation.
Methods and results
Primary isolated brown preadipocytes or HIB‐1B brown preadipocyte cells were treated with rOly and the effects on morphology, lipid accumulation, respiration rate, and associated gene and protein expression were measured. rOly upregulated mRNA and protein levels of factors related to brown adipocyte differentiation, induced lipid droplet formation, and increased cellular respiration rate due to expression of uncoupling protein 1. rOly also upregulated β‐tubulin expression, and therefore microtubules might be involved in its mechanism of action.
Conclusion
rOly promotes brown adipocyte differentiation, suggesting a new mechanism for rOly's contribution to the battle against obesity.
The fungal protein ostreolysin (Oly), which has been previously shown to bind to cholesterol‐enriched raft‐like membrane domains (lipid rafts) of mammalian cells, is a suitable candidate for interaction with brown preadipocytes. We used a recombinant version of ostreolysin (rOly), and we demonstrate that rOly affects morphology, lipid accumulation, respiration rate due to enhanced expression of uncoupling protein 1, and concomitantly differentiation associated gene and protein expression, in summary induces brown adipocyte differentiation.
Analysis of 501 melanoma exomes revealed RGS7, which encodes a GTPase-accelerating protein (GAP), to be a tumor-suppressor gene. RGS7 was mutated in 11% of melanomas and was found to harbor three ...recurrent mutations (p.R44C, p.E383K and p.R416Q). Structural modeling of the most common recurrent mutation of the three (p.R44C) predicted that it destabilizes the protein due to the loss of an H-bond and salt bridge network between the mutated position and the serine and aspartic acid residues at positions 58 as 61, respectively. We experimentally confirmed this prediction showing that the p.R44C mutant protein is indeed destabilized. We further show RGS7 p.R44C has weaker catalytic activity for its substrate Gα
, thus providing a dual mechanism for its loss of function. Both of these effects are expected to contribute to loss of function of RGS7 resulting in increased anchorage-independent growth, migration and invasion of melanoma cells. By mutating position 56 in the R44C mutant from valine to cysteine, thereby enabling the formation of a disulfide bridge between the two mutated positions, we slightly increased the catalytic activity and reinstated protein stability, leading to the rescue of RGS7's function as a tumor suppressor. Our findings identify RGS7 as a novel melanoma driver and point to the clinical relevance of using strategies to stabilize the protein and, thereby, restore its function.
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