Archives of biochemistry and biophysics最新文献

Sickle cell disease (SCD) is an inherited disorder characterized by abnormal hemoglobin molecules that cause the sickling of red blood cells (RBCs) which occlude blood vessels, increase hemolysis, and leads to chronic inflammation. Accumulating evidence has strongly linked SCD and other inflammatory conditions to the persistent activation of neutrophils which generate the antimicrobial enzyme myeloperoxidase (MPO) that produces hypohalous acids such as hypochlorous acid (HOCl) and hypothiocyanous acid. The MPO-HOCl system can induce oxidative damage of erythrocytes and vasculature through the accumulation of toxic free iron and the deficiency of nitric oxide (NO) and zinc (Zn), which are common features observed in SCD. MPO is also a crucial regulator in neutrophil extracellular trap (NET) formation that significantly contributes to SCD pathogenesis and vaso-occlusive crises. The objective of this review is to discuss the potential role of MPO-HOCl in SCD pathophysiology and summarize the current state of knowledge regarding the role of NETs, oxidative stress, and NO in SCD. Here, we highlight the novel pathway of MPO-HOCl in SCD pathology that drives NET formation, alters hemoglobin oxidation states, accelerates hemolysis, generates free iron and protein aggregation through hemoglobin heme destruction, and increases vascular constriction through endothelin-1 upregulation and nitric oxide depletion.

Selenoproteins are critical regulators of redox homeostasis, protein folding, and metabolism, and their dysregulation has been implicated in cancer biology. Among them, selenoprotein F (SELENOF) has been reported to be tumor suppressive, whereas the RNA-binding protein EIF4A3, a component of the exon junction complex, has been implicated in post-transcriptional repression of selenoproteins. The regulatory and clinical significance of this interaction in colorectal adenocarcinoma (COAD) remains unclear. We performed an integrative analysis of transcriptomic data from The Cancer Genome Atlas (TCGA), proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC), and patient tissue microarrays. Western blotting, qRT-PCR, and immunofluorescence staining were used to examine SELENOF, GPX1, and EIF4A3 expression in colon cancer cell lines and tumor tissues. Correlation, regression, and survival analyses were conducted, and pathway enrichment was assessed using gene set enrichment analysis (GSEA) of RNA and proteome correlation profiles. Motif discovery and translational efficiency analyses were performed to identify 3'-UTR features associated with EIF4A3 repressive activity. SELENOF and EIF4A3 showed inverse, stage-dependent protein expression patterns in COAD in the CPTAC cohort. Survival analyses demonstrated that SELENOF alone was not prognostic but acquired significance in EIF4A3-high tumors, where low SELENOF was associated with poor outcomes. Motif analyses identified enriched 3'UTR elements in SELENOF, suggesting that EIF4A3 represses translation through non-SECIS motifs positioned near canonical SECIS elements. Our findings explore a novel EIF4A3-SELENOF regulatory axis in colorectal cancer. SELENOF acquires conditional prognostic significance only in the context of elevated EIF4A3, highlighting the importance of molecular interaction specificity in biomarker discovery.

Myocardial ischemia-reperfusion (I/R) injury remains a major clinical challenge that undermines the benefits of reperfusion therapy and contributes to adverse cardiac remodeling. Despite their regenerative potential, the therapeutic efficacy of mesenchymal stem cells (MSCs) is limited by poor engraftment and low survival under ischemic conditions. To address these limitations, we developed a combinatorial strategy utilizing low-density lipoprotein receptor-related protein 6 (LRP6)-targeted chimeric antigen receptor-engineered MSCs (CAR-MSCs) to enhance site-specific homing to injured myocardium, coupled with overexpression of microsomal glutathione S-transferase 1 (MGST1) to strengthen cellular antioxidant defense. We evaluated this approach in both cellular and animal models of I/R injury. In vitro, under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions, treatment with MSCs, CAR-MSCs, or MGST1 overexpression alone each attenuated OGD/R-induced injury. The combination of unmodified MSCs with MGST1 overexpression (MSCs+MGST1) further improved cell viability and reduced apoptosis compared to MSCs alone. Notably, the combination of CAR-MSCs and MGST1 overexpression (CAR-MSCs+MGST1) exhibited the most pronounced protective effects, significantly outperforming all other groups, including MSCs+MGST1, in enhancing cell viability, reducing apoptosis and intracellular reactive oxygen species (ROS) levels, modulating oxidative stress markers (MDA, SOD, CAT), and promoting the secretion of reparative growth factors (VEGF, IGF-1, HGF). In a rat I/R model, combined treatment significantly reduced infarct size, ameliorated histological damage, decreased collagen deposition and apoptosis, and consistently modulated serum oxidative and regenerative biomarkers. Mechanistically, the combined intervention activated the Nrf2/Keap1 signaling pathway, upregulating downstream effectors NQO1 and HO-1. The cardioprotective effects were partially abolished by Nrf2 inhibition. In summary, LRP6-targeted CAR-MSCs coupled with MGST1 overexpression deliver synergistic protection against I/R injury by activating the Nrf2/Keap1 antioxidant program, offering a clinically translatable strategy to enhance precision MSC therapy and mitigate reperfusion-driven cardiac damage.

C-X-C motif chemokine ligand 17 (CXCL17) has recently been identified as an agonist of the poorly characterized G protein-coupled receptor 25 (GPR25). Although GPR25 orthologs are widely distributed across vertebrates, non-mammalian CXCL17 orthologs have only been identified in some fish species in our recent studies. In this study, we systematically searched public databases for amphibian CXCL17 orthologs based on conserved C-terminal motif, gene synteny, and genomic architecture. Using this approach, we identified up to eighteen possible CXCL17 orthologs from diverse amphibian species. These amphibian CXCL17s exhibit no significant overall sequence similarity to known mammalian or fish CXCL17s, and most of them display distinctive features, including four cysteine residues in their mature peptide and an additional residue following the conserved C-terminal Xaa-Pro-Yaa motif. A representative ortholog from the tropical clawed frog (Xenopus tropicalis) was recombinantly expressed and functionally characterized using cell-based assays, inducing ligand‒receptor binding, β-arrestin recruitment, and chemotactic cell migration. The recombinant amphibian CXCL17 directly bound to and efficiently activated its cognate GPR25 receptor and induced chemotactic migration of the transfected human embryonic kidney (HEK) 293T cells, but deletion of four C-terminal residues largely abolished its activity, indicating that all CXCL17 orthologs employ a conserved mechanism for receptor binding and activation. These findings provide new insights into the phylogenetic distribution and sequence diversity of CXCL17 orthologs across vertebrate lineages. In future, more studies are needed to clarify in vivo functions of the CXCL17‒GPR25 system in amphibians.

ATP-dependent kinases play central roles in metabolism and cellular regulation, making their accurate quantification essential for biochemical and kinetic studies. Classical coupled assays used to measure ATP-dependent kinase activity suffer from technical limitations, including instability of substrates of the auxiliary enzymes, dependence on monovalent ions, and the need to detect cofactor disappearance. Here, we describe the characterization and standardization of an alternative coupled assay for the quantitative and continuous measurement of ATP-dependent kinase activity, based on an ADP-dependent glucokinase from Thermococcus litoralis (TlGK) coupled to glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (LmG6PDH). The assay relies on the auxiliary enzymes TlGK and LmG6PDH, both of which were produced recombinantly and kinetically characterized at pH 7.0 and 25 °C. In this system, ADP generated by the kinase reaction is used to produce glucose-6-phosphate, which is subsequently oxidized to form NADH, allowing direct spectrophotometric monitoring at 340 nm. The effect of the Mg-ATP complex on assay performance was systematically analyzed, identifying the kinetic mechanism of inhibition, which is relevant for the experimental design of the coupled assay. Using a kinetic model for sequentially coupled reactions, we developed a general mathematical framework to estimate the minimum amounts of auxiliary enzymes required to ensure steady-state conditions and maintain the analytical validity of the assay. The method was experimentally validated using the ATP-dependent kinase ThiD, demonstrating that the measured initial velocity is independent of auxiliary enzyme concentrations and directly proportional to the kinase activity. Together, these results establish a kinetic framework and provide a versatile alternative assay for the quantitative measurement of ATP-dependent kinase activity.

Background: Epithelial ovarian cancer (EOC) has high tumor drug resistance, recurrence rate, and risk of metastasis.

Objectives: To elucidate the role of the long non-coding RNA SLC2A1-AS1 in EOC.

Methods: A cohort of 140 EOC patients was enrolled. SLC2A1-AS1 and related transcripts were quantified by RT-qPCR. A panel of OC cell lines was then employed, and the oncogenic functions of SLC2A1-AS1 were systematically interrogated through CCK-8 proliferation, Transwell migration/invasion, and western blot analysis. Dual-luciferase reporter and RIP assays were performed to confirm the targeting interactions.

Results: SLC2A1-AS1 expression was significantly increased in EOC tissues. High expression of SLC2A1-AS1 significantly increased the recurrence rate and mortality. Multifactor Cox suggested that high expression of SLC2A1-AS1 was an independent risk factor for overall survival (OS) and recurrence-free survival (RFS). In addition, SLC2A1-AS1 targeted miR-508-5p, which was generally down-regulated in EOC and was significantly negatively correlated with SLC2A1-AS1. Furthermore, miR-508-5p directly targeted FOXO1, a gene markedly up-regulated in EOC and inversely correlated with miR-508-5p levels. Depletion of SLC2A1-AS1 liberated miR-508-5p, which in turn binds FOXO1 mRNA, thereby suppressing proliferation, migration, and invasion of SKOV3 and OVCAR3 cells. This tumor-suppressive program could be largely weakened by miR-508-5p-inhibitor, underscoring the SLC2A1-AS1/miR-508-5p/FOXO1 axis as a critical driver of EOC aggressiveness. Consistent results were obtained in additional EOC cell lines A2780 and CAOV3, confirming the generalizability of the SLC2A1-AS1/miR-508-5p/FOXO1 axis across different cellular backgrounds.

Conclusion: SLC2A1-AS1 may drive the progression and recurrence of EOC through the miR-508-5p/FOXO1 axis.