Biochemistry and Cell Biology最新文献

Tumor necrosis factor receptor-associated factor 7 (TRAF7), a member of the tumor necrosis factor receptor superfamily, is known as an E3 ubiquitin ligase and has been shown to contribute to the progression of various cancers. However, the function of TRAF7 in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, our findings demonstrate a marked downregulation of TRAF7 protein expression in ESCC cell lines compared to non-neoplastic esophageal epithelial cells. Overexpression of TRAF7 inhibited cell proliferation and migration of ESCC cells, as well as promoted cell apoptosis and blocked cell cycle at the G2/M phase. In this study, we observed that TRAF7 interacted with the SOX12 protein and promoted ubiquitin-proteasome-mediated degradation of SOX12 via K48-linked ubiquitination in ESCC cells. Rescue experiments further confirmed that TRAF7's inhibitory effects on tumor cell proliferation and migration in ESCC cells partly depended on SOX12. In summary, our research reveals that TRAF7 functions as a tumor suppressor partially by promoting K48-linked ubiquitination-mediated degradation of the SOX12 protein.

Gateway cloning is an easy, efficient, accurate, and versatile cloning strategy. During Expression clone validation, we sometimes see an additional band co-migrating with the pDONR (Entry) backbone. We show that this "mystery" band is not an artifact of aberrant recombination but instead originates from a cotransformation event, where more than one different plasmid is transformed into a single Escherichia coli cell simultaneously and in the absence of antibiotic selection. We find that the unselected pDONR Entry plasmid is cotransformed into E. coli with the desired Expression vector in 9%-29% of colonies and is maintained without antibiotic selection, despite plasmid incompatibility. We propose an easy strategy to screen for and eliminate cotransformants. Our results challenge accepted beliefs of bacterial plasmid transformation, selection, and maintenance and comprise the first documented occurrence of cotransformation in Gateway cloning.

Methyl CpG binding protein 2 (MeCP2) is a chromatin-associated protein that remains enigmatic despite more than 30 years of research, primarily due to the ever-growing list of its molecular functions, and, consequently, its related pathologies. Loss of function MECP2 mutations cause the neurodevelopmental disorder Rett syndrome (RTT); in addition, dysregulation of MeCP2 expression and/ or function are involved in numerous other pathologies, but the mechanisms of MeCP2 regulation are unclear. Advancing technologies and burgeoning mechanistic theories assist our understanding of the complexity of MeCP2 but may inadvertently cloud it if not rigorously tested. Here, rather than focus on RTT, we examine relatively underexplored aspects of MeCP2, such as its dosage homeostasis at the gene and protein levels, its controversial participation in phase separation, and its overlooked role in depression and oxidative stress. All these factors may be essential to understanding the full scope of MeCP2 function in healthy and diseased states, but are relatively infrequently studied and require further criticism. The aim of this review is to discuss the esoteric facets of MeCP2 at the molecular and pathological levels and to consider to what extent they may be necessary for general MeCP2 function.

Prostate cancer (PCa) is a complex disease with diverse molecular alterations. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that exhibits pleiotropic roles in PCa, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent ligand for AhR. While targeting ferroptosis is an innovative PCa therapeutic strategy, the impact of AhR on this process remains unclear. This study aimed to investigate the influence of AhR on lipid peroxidation and ferroptosis. Results showed that TCDD activated AhR, as evidenced by increased CYP1A1 expression, leading to reduced cell viability. TCDD caused mitochondria shrinkage, decreased the GSH/GSSG ratio, and elevated the MDA levels and lipid peroxidation. Interestingly, AhR knockdown reversed these effects, similar to the action of ferroptosis inhibitors. Mechanistically, TCDD suppressed nuclear receptor subfamily 4 group A member 1 (NR4A1) expression, in part due to AhR activation. This suppression subsequently led to a reduction in the expression of the NR4A1 downstream target stearoyl-CoA desaturase 1 (SCD1). NR4A1 overexpression counteracted the effects of TCDD. In vivo, TCDD activated AhR, downregulated NR4A1 and SCD1 expression, induced mitochondria shrinkage, and increased the MDA and 4-hydroxynonenal (4-HNE) levels. In summary, TCDD promotes ferroptosis in androgen-dependent PCa via inhibiting the NR4A1/SCD1 axis, in part dependent on AhR activation.

Bovine lactoferrin (bLf) confers significant functional benefits for human health, but low concentrations in milk and high cost of commercial production limit availability and thus product application. Precision fermentation offers a solution to increase availability of biosimilar recombinant bLf (rbLf) thereby opening new opportunities for this high-value ingredient. To comply with regulatory requirements, we aimed to establish that rbLf from Komagataella phaffii is substantially similar to native bLf in structure and key functions. Intact mass analysis showed a molecular weight of 84 kDa for rbLf, comparable to 82-83 kDa of bLf. LC-MS N-linked glycan profiling revealed predominantly high-mannose-based glycans on rbLf, similar to ∼50% of bLf glycans. The isoelectric point and core amino acid sequence of rbLf and bLf are identical. rbLf retains the functional ability to bind and release iron, bind to intestinal Lf receptors, increase epithelial cell growth (>120% of control, P < 0.0001), reduce enteropathogenic Escherichia coli growth (>50% reduction, P < 0.0001), bind lipopolysaccharide (LPS) (+4-fold, P < 0.001), and antagonize LPS-induced toll-like receptor 4 activity (>40% reduction, P < 0.0001). These results demonstrate similarity of rbLf in structure and function to native bLf, supporting the effective application for expanded market opportunities for infant and adult health.

Fusarium graminearum FgSte2 and FgSte3 are G-protein-coupled receptors (GPCRs) shown to play roles in hyphal chemotropism and fungal plant pathogenesis in response to activity arising from host-secreted peroxidases. Here, we follow up on the observation that chemotropism is dependent on both FgSte2 and FgSte3 being present; testing the possibility that this might be due to formation of an FgSte2-FgSte3 heterodimer. Bioluminescence resonance energy transfer (BRET) analyses were conducted in Saccharomyces cerevisiae, where the addition of horse radish peroxidase (HRP) was found to increase the transfer of energy from the inducibly expressed FgSte3-Nano luciferase donor, to the constitutively expressed FgSte2-yellow fluorescent protein (YFP) acceptor, compared to controls. A partial response was also detected when an HRP-derived ligand-containing extract was enriched from F. graminearum spores and applied instead of HRP. In contrast, substitution with pheromones or an unrelated bovine GPCR, rhodopsin-YFP used as acceptor, eliminated all BRET responses. Interaction results were validated by affinity pulldown and receptor expression was validated by confocal immunofluorescence microscopy. Taken together these findings demonstrate the formation of HRP and HRP-derived ligand stimulated heterodimers between FgSte2 and FgSte3. Outcomes are discussed from the context of the roles of ligands and reactive oxygen species in GPCR dimerization.

Astronauts experience the reactivation of latent viruses in spaceflight, an indicator of reduced immunity. It is unclear how the immune system responds to pathogens in a microgravity environment. A longitudinal profile of leukocytes' transcriptome changes from participants to an Earth model of microgravity and from astronauts sojourning aboard the International Space Station revealed a reduced expression of immune-related genes while in microgravity. In the current study, we identified transcriptomic changes specific to the transition to and from bed/space, as well as the adaptation, and the recovery from microgravity/space exposure. The expression of immune-related gene shifted in opposite direction at phase transition compared to within the bed rest and reambulation phases. Differential expression of cytokine genes supported a reduced immune-response during the head down tilt bed rest phase and return to baseline levels at reambulation. Immunoglobulin gene expression increased after participants left the facility. The enrichment analysis of the differentially expressed genes identified the gene ontology terms virus/viral and genes previously involved in the modulation of the response to latent reactivation, including IFNL1, TNFSF14, IL10, and ISG15. Leukocytes' transcriptomic analysis revealed dynamic changes of immune-related gene expression timed with phases of spaceflight. The current analysis combined with previous evidence of herpesvirus reactivation during space mission represent a valuable model for the study of viral latency in vivo.

Aluminium (Al) toxicity has attracted considerable interest due to its bioavailability, environmental persistence, and adverse health effects. The present study investigates the effect of Al on rat erythrocytes under in vivo conditions. Rats were administered 0 (control), 25 (Al 1), 35 (Al 2), 45 (Al 3), and 55 (Al 4) mg/kg b.wt. of AlCl₃ orally for 30 days. Hemolysates were prepared from different experimental groups and assayed for various biochemical parameters. AlCl₃ administration significantly increased protein oxidation and lipid peroxidation, while decreasing total sulfhydryl and glutathione levels in rat erythrocytes. Methemoglobin level was increased and methemoglobin reductase activity was decreased upon AlCl₃ treatment. Prolonged AlCl₃ exposure inhibited the activities of antioxidant enzymes, and lowered the cells' antioxidant power. It also caused an increase in H₂O₂ and NO levels showing generation of oxidative and nitrosative stress. AlCl₃ intoxication adversely affected the membrane-bound and metabolic enzyme activities. Alterations in all the biochemical parameters were found in an AlCl₃ dose-dependent manner. Scanning electron microscopy showed gross morphological changes in erythrocytes from discocytes to acanthocytes and echinocytes, further supporting the damaging effect of aluminium. The aluminium-induced oxidative stress seems to be the key mechanism of damage to the cellular components that could lead to red cell senescence.