Structural insights into human organic cation transporter 1 transport and inhibition.

The human organic cation transporter 1 (hOCT1), also known as SLC22A1, is integral to hepatic uptake of structurally diversified endogenous and exogenous organic cations, influencing both metabolism and drug pharmacokinetics. hOCT1 has been implicated in the therapeutic dynamics of many drugs, making interactions with hOCT1 a key consideration in novel drug development and drug-drug interactions. Notably, metformin, the frontline medication for type 2 diabetes, is a prominent hOCT1 substrate. Conversely, hOCT1 can be inhibited by agents such as spironolactone, a steroid analog inhibitor of the aldosterone receptor, necessitating a deep understanding of hOCT1-drug interactions in the development of new pharmacological treatments. Despite extensive study, specifics of hOCT1 transport and inhibition mechanisms remain elusive at the molecular level. Here, we present cryo-electron microscopy structures of the hOCT1-metformin complex in three distinct conformational states - outward open, outward occluded, and inward occluded as well as substrate-free hOCT1 in both partially and fully open states. We also present hOCT1 in complex with spironolactone in both outward and inward facing conformations. These structures provide atomic-level insights into the dynamic metformin transfer process via hOCT1 and the mechanism by which spironolactone inhibits it. Additionally, we identify a 'YER' motif critical for the conformational flexibility of hOCT1 and likely other SLC22 family transporters. Our findings significantly advance the understanding of hOCT1 molecular function and offer a foundational framework for the design of new therapeutic agents targeting this transporter.

Structures of human prostaglandin F2α receptor reveal the mechanism of ligand and G protein selectivity.

Prostaglandins and their receptors regulate various physiological processes. Carboprost, an analog of prostaglandin F2α and an agonist for the prostaglandin F2-alpha receptor (FP receptor), is clinically used to treat postpartum hemorrhage (PPH). However, off-target activation of closely related receptors such as the prostaglandin E receptor subtype EP3 (EP3 receptor) by carboprost results in side effects and limits the clinical application. Meanwhile, the FP receptor selective agonist latanoprost is not suitable to treat PPH due to its poor solubility and fast clearance. Here, we present two cryo-EM structures of the FP receptor bound to carboprost and latanoprost-FA (the free acid form of latanoprost) at 2.7 Å and 3.2 Å resolution, respectively. The structures reveal the molecular mechanism of FP receptor selectivity for both endogenous prostaglandins and clinical drugs, as well as the molecular mechanism of G protein coupling preference by the prostaglandin receptors. The structural information may guide the development of better prostaglandin drugs.

Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing.

Genomes can be edited by homologous recombination stimulated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide 9]-induced DNA double-strand breaks. However, this approach is inefficient for inserting or deleting long fragments in mammalian cells. Here, we describe a simple genome-editing method, termed transcription-coupled Cas9-mediated editing (TEd), that can achieve higher efficiencies than canonical Cas9-mediated editing (CEd) in deleting genomic fragments, inserting/replacing large DNA fragments and introducing point mutations into mammalian cell lines. We also found that the transcription on DNA templates is crucial for the promotion of homology-directed repair, and that tethering transcripts from TEd donors to targeted sites further improves editing efficiency. The superior efficiency of TEd for the insertion and deletion of long DNA fragments expands the applications of CRISPR for editing mammalian genomes.

Late embryogenesis abundant group3 protein (DrLEA3) is involved in antioxidation in the extremophilic bacterium Deinococcus radiodurans.

Deinococcus radiodurans is able to survive under extreme conditions, including high doses of ionizing radiation, desiccation and oxidative stress. In addition to enhanced DNA repair capabilities, an effective antioxidation system plays an important role in its robustness. Previous studies have linked the radiation resistance of D. radiodurans to its prolonged desiccation tolerance phenotype, which both cause DNA damage. In the current study, we investigated the roles of dr_1172 in D. radiodurans, the gene encoding a typical group 3 LEA protein (DrLEA3) conserved within Deinococcus species. In addition to the increased transcriptional level under oxidative stress, the inactivation of dr_1172-sensitized cells to H2O2 treatments and the reduced cellular antioxidation activities suggested that dr_1172 is involved in the cellular defense against oxidative stress. Moreover, DrLEA3 was enriched at the cell membrane and bound to various types of metal ions. Cells devoid of DrLEA3 showed a decreased intracellular Mn/Fe concentration ratio, indicating that DrLEA3 also plays a role in maintaining metal ion homeostasis in vivo.

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in the Extreme Resistance of Deinococcus radiodurans.

Increasing evidence shows that the succinylation of lysine residues mainly regulates enzymes involved in the carbon metabolism pathway, in both prokaryotic and eukaryotic cells. Deinococcus radiodurans is one of the most radioresistant organisms on earth and is famous for its robust resistance. A major goal in the current study of protein succinylation is to explore its function in D. radiodurans. High-resolution LC-MS/MS is used for qualitative proteomics to perform a global succinylation analysis of D. radiodurans and 492 succinylation sites in 270 proteins are identified. These proteins are involved in a variety of biological processes and pathways. It is found that the enzymes involved in nucleic acid binding/processing are enriched in D. radiodurans compared with their previously reported levels in other bacteria. The mutagenesis studies confirm that succinylation regulates the enzymatic activities of species-specific proteins PprI and DdrB, which belong to the radiation-desiccation response regulon. Together, these results provide insight into the role of lysine succinylation in the extreme resistance of D. radiodurans.

Understanding the Relationship Between Grit and Foreign Language Performance Among Middle School Students: The Roles of Foreign Language Enjoyment and Classroom Environment.

OBJECTIVE: This study aims to examine the effect of grit on foreign language performance (FLP) among middle school students. A mediated moderation model was constructed to assess the mediating role of foreign language enjoyment (FLE) and the moderating role of classroom environment (CE) in the relationship between grit and FLP. METHODS: The study adopted the Grit Scale-Short Version, the Chinese Version of the FLE Scale, and the English CE Inventory to investigate 832 middle school students, and recorded the students' FLP in their final exam after 1 month. Correlation and regression analyses were used to evaluate the relationships between grit, FLE, CE, and FLP. RESULTS: The results indicated that grit positively affected FLP. In addition, FLE mediated the relationship between grit and FLP, and CE moderated the relationship between grit and FLE, and between grit and FLP. CONCLUSION: Grit not only directly promotes the FLP of middle school students but also indirectly improves FLP by promoting FLE. In addition, the impact of grit on FLE and FLP increases in a positive CE.

Synergistic anticancer effect of Grb2 and ITGA1 on cancer cells highly expressing Grb2 through suppressing ERK phosphorylation.

BACKGROUND: Growth factor receptor bound protein 2 (Grb2) is known to be an adaptor protein that provides a critical link between cell surface growth factor receptors and the MAPK signaling. It was shown that high expression of Grb2 enhances cancer cells proliferation, invasion and malignant transformation. OBJECTIVE: In this study, we aimed to systemically understand the function of Grb2 in cancer. METHODS: The expression of Grb2 in different cancer cell lines was examined from a publicly available database and we chose two cancer cell lines highly expressing Grb2 to investigate the role of Grb2. To systemically understand the function of Grb2 in cancer cells, proteomic profiles also were analyzed. RESULT: The results suggested that downregulation of Grb2 reduced cell proliferation in Hela cells and Jurkat cells. In addition, knockdown of Grb2 reduced the expression of ITGA1 and inhibited the phosphorylation of ERK. Intriguingly, simultaneous inhibition of Grb2 and ITGA1 resulted in a greater inhibition of phosphorylated ERK than either inhibition of Grb2 or ITGA1, and thus triggered marked apoptosis in Hela cells and Jurkat cells. These results suggest a synergistic anticancer effect of Grb2 and ITGA1 mediated by the ERK pathway in cancer cells highly expressing Grb2. In conclusion, we provided evidence that inhibition of Grb2 and ITGA1 might be an attractive target for therapeutic intervention against the cancer growth of cancers with high Grb2 expression.

The relationship between DNA repair genes (XPA, XPF, XPG) polymorphism and the risk of preeclampsia in Chinese Han Women.

Our study aimed to investigate the association between polymorphism of three core genes belonging to NER pathway and preeclampsia (PE) risk in Chinese Han Women. The DNA used in our experiment was extracted from 1004 cases and 1274 normal pregnant women. All single nucleotide polymorphisms (SNPs) were analyzed with TaqMan allelic discrimination real-time PCR. Our findings showed the genotype and allele frequency of XPA rs1800975, XPF rs1799801, XPG rs17655 in case group has no significant differences compared with control group (all P > 0.05). However, we found the genotype of rs1799801 in the control group was significantly different from severe PE group in cases (P < 0.05). Moreover, the genotype frequency of rs1800975 and rs1799801in the early-onset PE were significantly different from control group (all P < 0.05). These results indicated that the polymorphism of the three SNPs had no significant correlation with risk of PE as a whole. However, we found XPA rs1800975 played a role in the development of early-onset PE, and XPF rs1799801 was associated with severe PE and early-onset PE. So we may need to continue to increase the sample size to clarify their relationship.