Identification of a carbohydrate recognition motif of purinergic receptors.

As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly modulate important drug targets, such as G-protein coupled receptors (GPCRs). Here, we employed P2Y purinoceptor 14 (P2Y14), a drug target for inflammation and immune responses, to uncover the sugar nucleotide activation of GPCRs. Integrating molecular dynamics simulation with functional study, we identified the uridine diphosphate (UDP)-sugar-binding site on P2Y14, and revealed that a UDP-glucose might activate the receptor by bridging the transmembrane (TM) helices 2 and 7. Between TM2 and TM7 of P2Y14, a conserved salt bridging chain (K2.60-D2.64-K7.35-E7.36 [KDKE]) was identified to distinguish different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine. We identified the KDKE chain as a conserved functional motif of sugar binding for both P2Y14 and P2Y purinoceptor 12 (P2Y12), and then designed three sugar nucleotides as agonists of P2Y12. These results not only expand our understanding for activation of purinergic receptors but also provide insights for the carbohydrate drug development for GPCRs.

Sugars and other types of carbohydrates are biomolecules which play a range of key roles in the body. In particular, they are important messengers that help to coordinate immune responses. For example, a carbohydrate known as UDP-Glucose (a kind of UDP-sugar) can activate P2Y14, a receptor studded through the surface of many cells; this event then triggers a cascade of molecular events associated with asthma, kidney injury and lung inflammation. Yet it remains unclear how exactly UDP-Glucose recognizes P2Y14 ­ and, more broadly, how carbohydrates interact with purinergic receptors, the class of proteins that P2Y14 belongs to. To examine this question, Zhao et al. combined functional experiments in the laboratory with molecular dynamics simulations, a computational approach. This work revealed that UDP-Glucose may activate P2Y14 by bridging its segments anchored within the cell membrane. A component of P2Y14, known as the KDKE chain, was found to have an important role in distinguishing between highly similar types of UDP-sugars. This allowed Zhao et al. to design three sugar molecules which could activate another purinergic receptor that also contained a KDKE chain. Purinergic receptors are promising therapeutic targets. A finer understanding of how they recognise the molecules that activate them is therefore important to be able to identify and design new drug compounds.

A nomogram for distinguishing benign and malignant parotid gland tumors using clinical data and preoperative blood markers: development and validation.

PURPOSE: This study aimed to construct and validate a nomogram that incorporated clinical data and preoperative blood markers to differentiate BPGTs from MPGTs more efficiently and at low cost. METHODS: We retrospectively analyzed patients who underwent parotidectomy and histopathological diagnosis at the First Affiliated Hospital of Guangxi Medical University from January 2013 to June 2022. Subjects were randomly divided into training and validation sets with a 7:3 ratio. In the training set, the least absolute shrinkage and selection operator (LASSO) regression analysis was performed to select the most relevant features from 19 variables and built a nomogram using logistic regression. We evaluated the model's performance using receiver-operating characteristic (ROC) curves, calibration curves, clinical decision curve analysis (DCA), and clinical impact curve analysis (CICA). RESULTS: The final sample consisted of 644 patients, of whom 108 (16.77%) had MPGTs. The nomogram included four features: current smoking status, pain/tenderness, peripheral facial paralysis, and lymphocyte-to-monocyte ratio (LMR). The optimal cut-off value for the nomogram was 0.17. The areas under the ROC curves (AUCs) of the nomogram were 0.748 (95% confidence interval [CI] = 0.689-0.807) and 0.754 (95% CI = 0.636-0.872) in the training and validation sets, respectively. The nomogram also showed good calibration, high accuracy, moderate sensitivity, and acceptable specificity in both sets. The DCA and CICA demonstrated that the nomogram had significant net benefits for a wide range of threshold probabilities (0.06-0.88 for the training set; 0.06-0.57 and 0.73-0.95 for the validation set). CONCLUSION: The nomogram based on clinical characteristics and preoperative blood markers was a reliable tool for discriminating BPGTs from MPGTs preoperatively.

Systematic Enzymatic Synthesis of dTDP-Activated Sugar Nucleotides.

Deoxythymidine diphosphate (dTDP)-activated sugar nucleotides are the most diverse sugar nucleotides in nature. They serve as the glycosylation donors of glycosyltransferases to produce various carbohydrate structures in living organisms. However, most of the dTDP-sugars are difficult to obtain due to synthetic difficulties. The limited availability of dTDP-sugars has hindered progress in investigating the biosynthesis of carbohydrates and exploring new glycosyltransferases in nature. In this study, based on the de novo and salvage biosynthetic pathways, a variety of dTDP-activated sugar nucleotides were successfully prepared in high yields and on a large scale from readily available starting materials. The produced sugar nucleotides could provide effective tools for fundamental research in glycoscience.

A Sensitive and Reversible Labeling Strategy Enables Global Mapping of the Core-Fucosylated Glycoproteome on Cell Surfaces.

Core fucosylation, the attachment of α1,6-fucose to the innermost N-acetylglucosamine (GlcNAc) residue of N-glycans, has a strong relationship with tumor growth, invasion, metastasis, prognosis, and immune evasion by regulating many membrane proteins. However, details about the functional mechanism are still largely unknown due to the lack of an effective analytical method to identify cell-surface core-fucosylated glycoproteins, and especially glycosylation sites. Here, we developed a sensitive and reversible labeling strategy for probing core fucosylation, by which core-fucosylated glycoproteins that located on cell-surface were selectively tagged by a biotinylated probe with high sensitivity. The labeled probe can be further broken enzymatically after the capture by affinity resin. The on-bead traceless cleavage allowed the global mapping of core-fucosylated glycoproteins and glycosylation sites by mass spectrometry (MS). The profile of core-fucosylated glycoproteome provides an in-depth understanding of the biological functions of core fucosylation.

Facile Synthesis of Sugar Nucleotides from Common Sugars by the Cascade Conversion Strategy.

Sugar nucleotides are essential glycosylation donors in the carbohydrate metabolism. Naturally, most sugar nucleotides are derived from a limited number of common sugar nucleotides by de novo biosynthetic pathways, undergoing single or multiple reactions such as dehydration, epimerization, isomerization, oxidation, reduction, amination, and acetylation reactions. However, it is widely believed that such complex bioconversions are not practical for synthetic use due to the high preparation cost and great difficulties in product isolation. Therefore, most of the discovered sugar nucleotides are not readily available. Here, based on de novo biosynthesis mainly, 13 difficult-to-access sugar nucleotides were successfully prepared from two common sugars D-Man and sucrose in high yields, at a multigram scale, and without the need for tedious purification manipulations. This work demonstrated that de novo biosynthesis, although undergoing complex reactions, is also practical and cost-effective for synthetic use by employing a cascade conversion strategy.

Cofactor-Driven Cascade Reactions Enable the Efficient Preparation of Sugar Nucleotides.

Glycosylation is catalyzed by glycosyltransferases using sugar nucleotides or occasionally lipid-linked phosphosugars as donors. However, only very few common sugar nucleotides that occur in humans can be obtained readily, while the majority of sugar nucleotides that exist in bacteria, plants, archaea, or viruses cannot be synthesized in sufficient quantities by either enzymatic or chemical synthesis. The limited availability of such rare sugar nucleotides is one of the major obstacles that has greatly hampered progress in glycoscience. Herein we describe a general cofactor-driven cascade conversion strategy for the efficient synthesis of sugar nucleotides. The described strategy allows the large-scale preparation of rare sugar nucleotides from common sugars in high yields and without the need for tedious purification processes.

Age-related Activation of Cyclic GMP-AMP synthase-Stimulator of Interferon Genes Signaling in the Auditory System is Associated with Presbycusis in C57BL/6J Male Mice.

Presbycusis, or age-related hearing loss (ARHL), is primarily associated with sensory or transduction nerve cell degeneration in the peripheral and/or central auditory systems. During aging, the auditory system shows mitochondrial dysfunction and increased inflammatory responses. Mitochondrial dysfunction promotes leakage of mitochondrial DNA (mtDNA) into the cytosol, which activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to induce type I interferon and inflammatory responses. However, whether this pathway is involved in the occurrence and development of ARHL is unknown. This study aimed to determine whether there are age-related changes in the levels of cytosolic mtDNA and cGAS-STING pathway activation in the auditory pathway and to explore their relationship with ARHL. The results showed that cGAS-positive immunoreactive cells were observed in the cochlea, inferior colliculus, and auditory cortex. Levels of cytosolic mtDNA, cGAS, STING, phosphorylated interferon regulatory factor 3, and cytokines were significantly increased in the cochlea, inferior colliculus, and auditory cortex of 6-, 9-, and 12-month-old mice compared with 3-month-old mice. These findings suggested that cytosolic mtDNA may play an important role in the pathogenesis of ARHL by activating cGAS-STING-mediated type I interferon and inflammatory responses.

Salicylate Induced GABAAR Internalization by Dopamine D1-Like Receptors Involving Protein Kinase C (PKC) in Spiral Ganglion Neurons.

BACKGROUND Sodium salicylate (SS) induces excitotoxicity of spiral ganglion neurons (SGNs) by inhibiting the response of γ-aminobutyric acid type A receptors (GABAARs). Our previous studies have shown that SS can increase the internalization of GABAARs on SGNs, which involves dopamine D1-like receptors (D1Rs) and related signaling pathways. In this study, we aimed to explore the role of D1Rs and their downstream molecule protein kinase C (PKC) in the process of SS inhibiting GABAARs. MATERIAL AND METHODS The expression of D1Rs and GABARγ2 on rat cochlear SGNs cultured in vitro was tested by immunofluorescence. Then, the SGNs were exposed to SS, D1R agonist (SKF38393), D1R antagonist (SCH23390), clathrin/dynamin-mediated endocytosis inhibitor (dynasore), and PKC inhibitor (Bisindolylmaleimide I). Western blotting and whole-cell patch clamp technique were used to assess the changes of surface and total protein of GABARγ2 and GABA-activated currents. RESULTS Immunofluorescence showed that D1 receptors (DRD1) were expressed on SGNs. Data from western blotting showed that SS promoted the internalization of cell surface GABAARs, and activating D1Rs had the same result. Inhibiting D1Rs and PKC decreased the internalization of GABAARs. Meanwhile, the phosphorylation level of GABAARγ2 S327 affected by PKC was positively correlated with the degree of internalization of GABAARs. Moreover, whole-cell patch clamp recording showed that inhibition of D1Rs or co-inhibition of D1Rs and PKC attenuated the inhibitory effect of SS on GABA-activated currents. CONCLUSIONS D1Rs mediate the GABAAR internalization induced by SS via a PKC-dependent manner and participate in the excitotoxic process of SGNs.

Salicylate-Induced Ototoxicity of Spiral Ganglion Neurons: Ca2+/CaMKII-Mediated Interaction Between NMDA Receptor and GABAA Receptor.

Sodium salicylate (SS) is one of the nonsteroidal anti-inflammatory drugs and widely used in clinical practice. Therefore, we aimed to investigate the potential ototoxicity mechanism of sodium salicylate: the influence of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaMKII) in interaction between NMDA receptors (NMDAR) and GABAA receptors (GABAAR) in rat cochlear spiral ganglion neurons (SGNs). After treatment with SS, NMDA, and an NMDAR inhibitor (APV), the changes of GABAAR ß3 (GABR ß3) mRNA, surface and total protein, and GABAAR currents in SGNs were assessed by quantitative PCR, Western blot, and whole-cell patch clamp. Mechanistically, SS and/or NMDA increased the GABR ß3 mRNA expression, while decreased GABR ß3 surface protein levels and GABAAR-mediated currents. Moreover, application of SS and/or NMDA showed promotion in phosphorylation levels at S383 of GABR ß3. Collectively, Ca2+ chelator (BAPTA) or Ca2+/CaMKII inhibitor (KN-93) reversed the effects of SS and/or NMDA on GABAAR. Therefore, we hypothesize that the interaction between NMDAR and GABAAR is involved in the SGNs damage induced by SS. In addition, the underlying molecular mechanism is related to Ca2+/CaMKII-mediated signaling pathway, which suggests that the interaction between calcium signal-regulated receptors mediates SS ototoxicity.