Lysosome Targeting Chimaeras for Glut1-Facilitated Targeted Protein Degradation.

Targeted protein degradation technology holds great potential in biomedicine, particularly in treating tumors and other protein-related diseases. Research on intracellular protein degradation using molecular glues and PROTAC technology is leading, while research on the degradation of membrane proteins and extracellular proteins through the lysosomal pathway is still in the preclinical stage. The scarcity of useful targets is an immense limitation to technological advancement, making it essential to explore novel, potentially effective approaches for targeted lysosomal degradation. Here, we employed the glucose transporter Glut1 as an innovative lysosome-targeting receptor and devised the Glut1-Facilitated Lysosomal Degradation (GFLD) strategy. We synthesized potential Glut1 ligands via reversible addition-fragmentation chain transfer (RAFT) polymerization and acquired antibody-glycooligomer conjugates through bioorthogonal reactions as lysosome-targeting protein degradation molecules, utilized in the management of PD-L1 high-expressing triple-negative breast cancer. The glucose transporter Glut1 as a lysosome-targeting receptor exhibits potential for the advancement of a broader array of medications in the future.

Structural insights into ligand recognition, selectivity, and activation of bombesin receptor subtype-3.

Bombesin receptor subtype-3 (BRS3) is an important orphan G protein-coupled receptor that regulates energy homeostasis and insulin secretion. As a member of the bombesin receptor (BnR) family, the lack of known endogenous ligands and high-resolution structure has hindered the understanding of BRS3 signaling and function. We present two cryogenic electron microscopy (cryo-EM) structures of BRS3 in complex with the heterotrimeric Gq protein in its active states: one bound to the pan-BnR agonist BA1 and the other bound to the synthetic BRS3-specific agonist MK-5046. These structures reveal the architecture of the orthosteric ligand pocket underpinning molecular recognition and provide insights into the structural basis for BRS3's selectivity and low affinity for bombesin peptides. Examination of conserved micro-switches suggests a shared activation mechanism among BnRs. Our findings shed light on BRS3's ligand selectivity and signaling mechanisms, paving the way for exploring its therapeutic potential for diabetes, obesity, and related metabolic disorders.

Stock Assessment of the Commercial Small Pelagic Fishes in the Beibu Gulf, the South China Sea, 2006-2020.

Long-term variations in population structure, growth, mortality, exploitation rate, and recruitment pattern of two major commercial small pelagic fishes (CSPFs) (Decapterus maruadsi and Trachurus japonicus) are reported based on bottom trawl survey data collected during 2006-2020 in the Beibu Gulf, South China Sea. All individuals collected during each sampling quarter over a period of 15 years were subjected to laboratory-based analysis. In this study, the stock of D. maruadsi and T. japonicus inhabiting the Beibu Gulf was assessed using length-based methods (bootstrapped electronic length frequency analysis (ELEFAN)) to complete stock assessment in different fishery management periods (the division of fisheries management periods was based on China's input and output in the South China Sea offshore fisheries over 15 years, specifically divided into period I (2006-2010), period II (2011-2015), and period III (2016-2020)). The results showed that the mean body length, dominant body size, and estimated asymptotic length of two CSPFs decreased, whereas their growth coefficient decreased, indicating miniaturization and slower growth, respectively. Estimated exploitation rates and catching body length for two CSPFs indicated that both stocks in the Beibu Gulf were overexploited in period I and moderately exploited after 2011. These stocks were taking a good turn in status in period III, with the exploitation rate much lower than the initial period and reversing the downward trend in catching body length. Furthermore, the variations in the spawning season of the two CSPF stocks and their barely satisfactory expected yield indicated the complexity of the current fishery management in the Beibu Gulf. These results suggest that management measures to reduce fishing pressure may have a positive influence on the biological characteristics of those CSPFs in the Beibu Gulf; however, the stock structure already affected by overfishing will be a huge challenge for the conservation and restoration of fisheries resources in the future. Given that the current stocks of D. maruadsi and T. japonicus in the Beibu Gulf still have low first-capture body length (Lc) and high fishing mortality (F) (compared to F0.1), we identify a need to refine population structure by controlling fishing efforts and increasing catchable size, and more consideration should be given to the local fishery resource status in fisheries management.

Cryo-EM structures of adenosine receptor A3AR bound to selective agonists.

The adenosine A3 receptor (A3AR), a key member of the G protein-coupled receptor family, is a promising therapeutic target for inflammatory and cancerous conditions. The selective A3AR agonists, CF101 and CF102, are clinically significant, yet their recognition mechanisms remained elusive. Here we report the cryogenic electron microscopy structures of the full-length human A3AR bound to CF101 and CF102 with heterotrimeric Gi protein in complex at 3.3-3.2 Å resolution. These agonists reside in the orthosteric pocket, forming conserved interactions via their adenine moieties, while their 3-iodobenzyl groups exhibit distinct orientations. Functional assays reveal the critical role of extracellular loop 3 in A3AR's ligand selectivity and receptor activation. Key mutations, including His3.37, Ser5.42, and Ser6.52, in a unique sub-pocket of A3AR, significantly impact receptor activation. Comparative analysis with the inactive A2AAR structure highlights a conserved receptor activation mechanism. Our findings provide comprehensive insights into the molecular recognition and signaling of A3AR, paving the way for designing subtype-selective adenosine receptor ligands.

Molecular recognition and activation of the prostacyclin receptor by anti-pulmonary arterial hypertension drugs.

The prostacyclin (PGI2) receptor (IP) is a Gs-coupled receptor associated with blood pressure regulation, allergy, and inflammatory response. It is a main therapeutic target for pulmonary arterial hypertension (PAH) and several other diseases. Here we report cryo-electron microscopy (cryo-EM) structures of the human IP-Gs complex bound with two anti-PAH drugs, treprostinil and MRE-269 (active form of selexipag), at global resolutions of 2.56 and 2.41 angstrom, respectively. These structures revealed distinct features governing IP ligand binding, receptor activation, and G protein coupling. Moreover, comparison of the activated IP structures uncovered the mechanism and key residues that determine the superior selectivity of MRE-269 over treprostinil. Combined with molecular docking and functional studies, our structures provide insight into agonist selectivity, ligand recognition, receptor activation, and G protein coupling. Our results provide a structural template for further improving IP-targeting drugs to reduce off-target activation of prostanoid receptors and adverse effects.

Structural basis of psychedelic LSD recognition at dopamine D1 receptor.

Understanding the kinetics of LSD in receptors and subsequent induced signaling is crucial for comprehending both the psychoactive and therapeutic effects of LSD. Despite extensive research on LSD's interactions with serotonin 2A and 2B receptors, its behavior on other targets, including dopamine receptors, has remained elusive. Here, we present cryo-EM structures of LSD/PF6142-bound dopamine D1 receptor (DRD1)-legobody complexes, accompanied by a ß-arrestin-mimicking nanobody, NBA3, shedding light on the determinants of G protein coupling versus ß-arrestin coupling. Structural analysis unveils a distinctive binding mode of LSD in DRD1, particularly with the ergoline moiety oriented toward TM4. Kinetic investigations uncover an exceptionally rapid dissociation rate of LSD in DRD1, attributed to the flexibility of extracellular loop 2 (ECL2). Moreover, G protein can stabilize ECL2 conformation, leading to a significant slowdown in ligand's dissociation rate. These findings establish a solid foundation for further exploration of G protein-coupled receptor (GPCR) dynamics and their relevance to signal transduction.

miR-218-5p promotes hepatic lipogenesis through targeting Elovl5 in non-alcoholic fatty liver disease.

Investigating and identifying pathogenic molecules of non-alcoholic fatty liver disease (NAFLD) has become imperative, which would serve as effective targets in the future. We established high-fat diet (HFD)-induced NAFLD model in mice and palmitic acid (PA)-induced model in mouse AML12 cells. The level of miR-218-5p was examined by qRT-PCR, and Elovl5 was identified as the potential target gene of miR-218-5p. The binding relationship between miR-218-5p and Elovl5 was validated by double luciferase reporter gene assay, and inhibition/overexpression of miR-218-5p in vitro. The functional mechanisms of miR-218-5p/Elovl5 in regulating lipogenesis in NAFLD were investigated in vivo and in vitro through gain- and loss-of-function studies. MiR-218-5p was significantly increased, and Elovl5 was decreased in model group. According to the double luciferase reporter and gene interference experiments in AML12 cells, Elovl5 was a target gene of miR-218-5p and its expression was regulated by miR-218-5p. The SREBP1-mediated lipogenesis signaling pathway regulated by Elovl5 was upregulated in model group. Moreover, silencing of miR-218-5p significantly upregulated Elovl5 expression, and suppressed SREBP1 signaling pathway in PA-induced AML-12 cells. Correspondingly, the cell injury, elevated TC, TG contents and lipid droplet accumulation were ameliorated. Furthermore, the effect of miR-218-5p on lipogenesis in vitro and in vivo was obstructed by si-Elovl5, implicating that miR-218-5p promotes lipogenesis by targeting ELOVL5 in NAFLD. miR-218-5p could promote fatty acid synthesis by targeting Elovl5, thereby accelerating the development of NAFLD, which is one of the key pathogenic mechanisms of NAFLD and provides a new molecular target for the management of NAFLD.

Structural basis for recognition of 26RFa by the pyroglutamylated RFamide peptide receptor.

The neuropeptide 26RFa, a member of the RF-amide peptide family, activates the pyroglutamylated RF-amide peptide receptor (QRFPR), a class A GPCR. The 26RFa/QRFPR system plays critical roles in energy homeostasis, making QRFPR an attractive drug target for treating obesity, diabetes, and eating disorders. However, the lack of structural information has hindered our understanding of the peptide recognition and regulatory mechanism of QRFPR, impeding drug design efforts. In this study, we determined the cryo-EM structure of the Gq-coupled QRFPR bound to 26RFa. The structure reveals a unique assembly mode of the extracellular region of the receptor and the N-terminus of the peptide, and elucidates the recognition mechanism of the C-terminal heptapeptide of 26RFa by the transmembrane binding pocket of QRFPR. The study also clarifies the similarities and distinctions in the binding pattern of the RF-amide moiety in five RF-amide peptides and the RY-amide segment in neuropeptide Y. These findings deepen our understanding of the RF-amide peptide recognition, aiding in the rational design of drugs targeting QRFPR and other RF-amide peptide receptors.

Molecular basis for the activation of PAF receptor by PAF.

Platelet-activating factor (PAF) is a potent phospholipid mediator crucial in multiple inflammatory and immune responses through binding and activating the PAF receptor (PAFR). However, drug development targeting the PAFR has been limited, partly due to an incomplete understanding of its activation mechanism. Here, we present a 2.9-Å structure of the PAF-bound PAFR-Gi complex. Structural and mutagenesis analyses unveil a specific binding mode of PAF, with the choline head forming cation-π interactions within PAFR hydrophobic pocket, while the alkyl tail penetrates deeply into an aromatic cleft between TM4 and TM5. Binding of PAF modulates conformational changes in key motifs of PAFR, triggering the outward movement of TM6, TM7, and helix 8 for G protein coupling. Molecular dynamics simulation suggests a membrane-side pathway for PAF entry into PAFR via the TM4-TM5 cavity. By providing molecular insights into PAFR signaling, this work contributes a foundation for developing therapeutic interventions targeting PAF signal axis.

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells

Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells

Berberine, a constituent of some traditional Chinese medicinal plants, has been reported to have cytotoxicity effects on different human cancer cell lines. There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8. In this paper, the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay, fluorescence microscopy and flow cytometry analysis. Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose- and time-dependent manner. Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining. The concentrations of lactate dehydrogenase and both acid and alkaline phosphatases were significantly increased in cell supernatants after berberine treatment, suggesting cell death. Furthermore, flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner. To further investigate the apoptotic molecular mechanism, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting methods were used. The up-regulated mRNA and/or protein expressions of Fas, FasL, TNF-a, caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine. Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis. We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin (PHB), and decreased vimentin expression. These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.(AU)