RESUMO
New chiral carbon dots (CDs), L-PCDs, for discriminating tryptophan (Trp) enantiomers were prepared in this work. Firstly, original CDs were synthesized through a hydrothermal method using pyridine-2,6-dicarboxylic acid and o-phenylenediamine as raw materials. Then, the surface of original CDs was modified with L-phenylalanine to create chiral fluorescent carbon L-PCDs. In the presence of D-Trp, the fluorescence intensity of L-PCDs decreased significantly while it remained unchanged in the presence of L-Trp. The chiral sensing system used in this study has a rapid response time of 3 minutes and can identify enantiomers with an enantioselectivity (ID/IL) of up to 3.3. For D-Trp, a good linear relationship can be obtained in the range of 0.3-4.2 mM with a limit of detection of 0.06 mM. This sensor allows for both quantitative detection of D-Trp and determination of enantiomeric percentage in the racemate. The chiral recognition mechanism is attributed to the different interaction between D-/L-Trp and L-PCDs.
RESUMO
The histamine H4 receptor (H4R) plays key role in immune cell function and is a highly valued target for treating allergic and inflammatory diseases. However, structural information of H4R remains elusive. Here, we report four cryo-EM structures of H4R/Gi complexes, with either histamine or synthetic agonists clobenpropit, VUF6884 and clozapine bound. Combined with mutagenesis, ligand binding and functional assays, the structural data reveal a distinct ligand binding mode where D943.32 and a π-π network determine the orientation of the positively charged group of ligands, while E1825.46, located at the opposite end of the ligand binding pocket, plays a key role in regulating receptor activity. The structural insight into H4R ligand binding allows us to identify mutants at E1825.46 for which the agonist clobenpropit acts as an inverse agonist and to correctly predict inverse agonism of a closely related analog with nanomolar potency. Together with the findings regarding receptor activation and Gi engagement, we establish a framework for understanding H4R signaling and provide a rational basis for designing novel antihistamines targeting H4R.
Assuntos
Agonismo Inverso de Drogas , Histamina , Imidazóis , Tioureia/análogos & derivados , Histamina/metabolismo , Receptores Histamínicos H4 , Receptores Acoplados a Proteínas G/metabolismo , Ligantes , Receptores Histamínicos/metabolismo , Antagonistas dos Receptores Histamínicos/farmacologiaRESUMO
Although GPR3 plays pivotal roles in both the nervous system and metabolic processes, such as cold-induced thermogenesis, its endogenous ligand remains elusive. Here, by combining structural approach (including cryo-electron microscopy), mass spectrometry analysis, and functional studies, we identify oleic acid (OA) as an endogenous ligand of GPR3. Our study reveals a hydrophobic tunnel within GPR3 that connects the extracellular side of the receptor to the middle of plasma membrane, enabling fatty acids to readily engage the receptor. Functional studies demonstrate that OA triggers downstream Gs signaling, whereas lysophospholipids fail to activate the receptor. Moreover, our research reveals that cold stimulation induces the secretion of OA in mice, subsequently activating Gs/cAMP/PKA signaling in brown adipose tissue. Notably, brown adipose tissues from Gpr3 knockout mice do not respond to OA during cold stimulation, reinforcing the significance of GPR3 in this process. Finally, we propose a "born to be activated and cold to enhance" model for GPR3 activation. Our study provides a starting framework for the understanding of GPR3 signaling in cold-stimulated thermogenesis.
Assuntos
Tecido Adiposo Marrom , Ácido Oleico , Receptores Acoplados a Proteínas G , Animais , Camundongos , Membrana Celular , Microscopia Crioeletrônica , Ligantes , Camundongos Knockout , Ácido Oleico/metabolismo , Ácido Oleico/farmacologia , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Adhesion G-protein-coupled receptors (aGPCRs) play key roles in a diversity of physiologies. A hallmark of aGPCR activation is the removal of the inhibitory GAIN domain and the dipping of the cleaved stalk peptide into the ligand-binding pocket of receptors; however, the detailed mechanism remains obscure. Here, we present cryoelectron microscopy (cryo-EM) structures of ADGRL3 in complex with Gq, Gs, Gi, and G12. The structures reveal unique ligand-engaging mode, distinctive activation conformation, and key mechanisms of aGPCR activation. The structures also reveal the uncharted structural information of GPCR/G12 coupling. A comparison of Gq, Gs, Gi, and G12 engagements with ADGRL3 reveals the key determinant of G-protein coupling on the far end of αH5 of Gα. A detailed analysis of the engagements allows us to design mutations that specifically enhance one pathway over others. Taken together, our study lays the groundwork for understanding aGPCR activation and G-protein-coupling selectivity.
Assuntos
Proteínas de Ligação ao GTP , Receptores Acoplados a Proteínas G , Ligantes , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Ligação ao GTP/metabolismoRESUMO
Plant litter decomposition is mainly affected by litter properties and environmental factors, but the influence of terrain on litter decomposition is not well understood. We studied the effects of terrain on litter decomposition over a period of 12 months at six locations in a typical steppe ecoregion and measured the concomitant release of carbon (C), nitrogen (N), and phosphorus (P). The study site has two aspects, shaded and sunny, each aspect having three slopes: 15°, 30°, and 45°. The same mixed litter was used at each location to exclude the influence of litter quality variation. Results showed that soil temperature and moisture, solar radiation, and plant species diversity varied by terrain, which in turn, affected the k-value (standardized total effects, 0.78, 0.12, 0.92, 0.23, respectively) and the release of C (0.72, -0.25, 0.83, 0.24, respectively), N (0.89, -0.45, 0.76, 0.40, respectively) and P (0.88, 0.77, 0.58, 0.57, respectively). K-value and C release decreased with increasing slope on shaded aspect, while increased with increasing slope on sunny aspect. The release of N and P decreased with increasing slope on the shaded aspect. K-value and C, N, and P release were significantly higher on shaded than that on sunny aspect at 15° and 30°, while at 45°, it was higher on sunny than on shaded aspect. The litter mass loss was slower on shaded 45° and sunny 15°. So moderate grazing or mowing could be used to reduce litter accumulation and accelerate litter decomposition on these terrains. Structural equation modeling indicated that soil temperature and solar radiation had the greatest influence on k-value and C, N, and P release, and these two factors were directly related to soil moisture and plant species diversity. Overall, our results emphasize the need to consider terrain for litter decomposition in typical steppe ecoregions.