Journal of biochemistry最新文献

Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication, transporting diverse molecular cargoes such as proteins, lipids and nucleic acids. Despite their growing importance in both basic biology and clinical applications, the remarkable heterogeneity of EVs remains a major obstacle to their functional characterization. In a recent study, Maeda and colleagues developed a highly sensitive and quantitative method for monitoring EV release using high-affinity binary technology (HiBiT)-tagged marker proteins, combined with a novel chromatographic approach that fractionates EVs based on surface charge properties. This strategy enabled the identification of distinct EV subpopulations harbouring specific protein markers and differing in their biogenesis and intracellular origin. By integrating CRISPR-mediated tagging, live-cell luminescence assays and ion-exchange chromatography, the study establishes surface charge as a new physicochemical parameter for EV classification. These findings offer a powerful framework for dissecting EV heterogeneity and lay the foundation for the development of more precise EV-based diagnostic strategies.

Recent advances in mass spectrometry-based proteomics have enabled increasingly precise characterization of protein modifications in clinical specimens. Among these, glycosylation is one of the most structurally complex and biologically informative post-translational modifications, reflecting cellular differentiation and disease states. Ohashi et al. (J. Biochem. 2024; 175: 561-572) performed a site-specific N-glycosylation analysis of LAMP1 in breast cancer tissue samples, demonstrating the feasibility of targeted glycoproteomics in patient-derived specimens and revealing tumor-associated glycoform heterogeneity. Their study exemplifies how focusing on a single glycoprotein target can provide detailed insight into disease-specific glycan remodeling within the tumor microenvironment. In this commentary, I discuss the significance of such targeted approaches in the broader context of clinical glycoproteomics and highlight their potential contribution to cancer biomarker discovery and precision medicine. Continued integration of glycoproteomic data with genomic and clinical information is expected to further advance our understanding of tumor biology and therapeutic response.

Lactoferrin is a multifunctional protein mainly involved in the immune defense of organisms against various pathogens. It has been reported that intestinal inflammation was reduced by lactoferrin administration. However, the precise mechanism underlying lactoferrin's involvement in intestinal inflammation is not yet fully understood. In this study, we purified a mouse intestinal lactoferrin-binding protein with a molecular mass of ~ 400 kDa that was expressed in the small intestine and colon. Sequence analysis revealed that the intestinal lactoferrin-binding protein represented an ortholog of rat immunoglobulin G fragment crystallizable gamma-binding protein (IgGFcγBP). N-linked glycans of lactoferrin were not necessary for binding to IgGFcγBP. After reduction, IgGFcγBP was separated into 120, 70, 65, 60, and 55 kDa proteins, but these did not bind to lactoferrin. The expression of IgGFcγBP was lost in a mouse model of dextran sodium sulfate induced colitis and restored during the convalescence period of colitis, suggesting a role in mucosal protection and immune regulation. Furthermore, we discuss potential links between IgGFcγBP and mucin-associated microbiota which may contribute to lactoferrin's immunomodulatory effects. These findings provide new insights into the interaction between lactoferrin, mucosal immunity, and gut microbiota.

Long non-coding RNAs (lncRNAs) regulate a wide array of cellular processes through interactions with RNA-binding proteins (RBPs). Taurine Upregulated Gene 1 (TUG1) is an lncRNA that is overexpressed in many types of cancer and has been implicated in resolving R-loops, thereby maintaining genomic integrity. However, the full spectrum of its protein interactions and stress-responsive dynamics remains unclear. Here, we employed CRISPR-assisted RNA-protein interaction detection (CARPID) combined with mass spectrometry to comprehensively identify the interacting proteins of TUG1 in HEK293T cells. Using three distinct single-guide RNAs (sgRNAs) targeting different regions of TUG1, we consistently identified 17 TUG1-interacting proteins under basal conditions. Upon camptothecin (CPT) treatment, which induces R-loop formation, the number of associated proteins increased to 25. Under these stress conditions, the protein sets identified by each sgRNA showed greater overlap, suggesting a more conserved pattern of TUG1-protein interactions in response to R-loop accumulation. Many of these proteins are known R-loop-associated factors, including DEAD/DEAH-box RNA helicases, poly(ADP-ribose) polymerase 1 (PARP1) and heterogeneous nuclear ribonucleoproteins (HNRNPs), indicating that TUG1 engages R-loop regulatory machinery to maintain genome integrity. Our study provides new insights into lncRNA-mediated R-loop regulation and its role in genome maintenance.

To progress the RNA-binding small molecule drug discovery, the specific interaction between RNAs having a single bulge and a fluoroquinolone derivative, KG022, was analysed by NMR spectroscopy. In our previous work, it was found that KG022 is located between the two base pairs at the 3' and 5' side of the bulged residue. KG022 prefers G or C as the bulged residue and, in the present study, the reason for this preference was analysed by using RNAs with modified nucleoside residues as the bulged residue. It was found that the amino groups of bulged guanine and cytidine bases interact with the oxygen atoms of the backbone phosphate groups, and the oxygen and nitrogen atoms of bulged guanine and cytidine bases interact with the piperazine group of KG022. Thus, this work presents an example of the mechanism of the specific recognition of a small molecule by RNAs.

S-adenosylmethionine (SAM) is the major cellular methyl donor and regulates gene expression through epigenetic and other methylation-related processes. While SAM biosynthesis influences a variety of biological phenomena including ageing and disease, its cell type-specific regulation and functional implications remain poorly understood. In this study, we report that the Drosophila germline exhibits a uniquely repressive SAM biosynthesis status during gametogenesis, as indicated by low expression of SAM synthetase (Sam-S), a key enzyme for SAM production. Experimentally enhancing SAM biosynthesis in the germline led to increased expression of retrotransposons, with HeT-A, a telomere-specific element, showing the most pronounced response. We also observed increased promoter activity of HeT-A under high SAM conditions, along with accumulation of N6-methyladenine (6 mA), the major form of DNA methylation in the Drosophila genome. Although a direct causal link between 6 mA levels and transcription was not broadly observed across other retrotransposons or genes, these results raise the possibility that SAM levels modulate HeT-A expression at least in part through DNA methylation. Our findings highlight a previously underexplored metabolic feature of the Drosophila germline and suggest that SAM availability contributes to the regulation of retrotransposon activity in a lineage-specific manner.

Despite being a carcinogen, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits metastatic melanoma growth by downregulating the signal transducer and activator of transcription 3. However, the molecular mechanisms remain unclear. The aim of this study was to identify tyrosine phosphatases that are involved in TPA-induced inhibition of cell proliferation in metastatic melanoma cells. We screened protein tyrosine phosphatases (PTPs) required for TPA-mediated inhibition of cell proliferation. We identified two PTPs, SH2 domain-containing PTP2 (SH-PTP2/PTPN11) and T-cell PTP (TC-PTP/PTPN2) that play key roles in TPA-mediated inhibition of metastatic melanoma cell growth. Transient expression of SH-PTP2 and TC-PTP induced G0/G1 cell cycle arrest in a phosphatase-dependent manner. Furthermore, SH-PTP2 was translocated to the cell membrane upon TPA treatment, resulting in a decrease in Janus kinase 2 activity. TC-PTP is localized in the nucleus together with the adapter protein ubiquitin-like protein 4A; TC-PTP was translocated to the nuclear periphery upon TPA stimulation. These two signaling pathways, involving SH-PTP2 and TC-PTP, are distinct from those observed in normal melanocytes and benign melanoma cells. These pathways represent previously unknown responses to TPA specific to metastatic melanoma cells. Overall, these findings may contribute to the development of new anticancer agents.

Ectodomain shedding (shedding) is a processing mechanism that cleaves the juxtamembrane region of membrane proteins and solubilizes almost the entire extracellular domain. Shedding irreversibly regulates the localization and function of membrane proteins; however, its physiological role is not fully understood. Previously, we showed that the shedding susceptibility of multiple membrane proteins is altered by skipping or inclusion of skipping exon(s) that encode their juxtamembrane region. In this study, we screened the skipping exon encoding the juxtamembrane region of membrane proteins and found that the shedding susceptibility of UNC5B, a Netrin-1 receptor, is altered by skipping or inclusion of the skipping exon encoding its juxtamembrane region. These results raise the possibility that the biological phenomena involving UNC5B, including neural circuit formation, angiogenesis and cancer development, are regulated by shedding in a splice isoform-dependent manner.

Previous studies have reported several O-linked N-acetylglucosamine (O-GlcNAc) modifications of core histones H2A, H2B, H3 and H4. In parallel, the characteristics and functions of O-GlcNAcylated histones are also shown, and they are involved in various cellular processes, such as development and tumorigenesis, indicating that the exploration of new histone O-GlcNAcylation contributes significantly to the elucidation of molecular mechanisms occurring in cells. Here, we report that O-GlcNAcylation occurs at threonine 71 of histone H4 (H4T71Gc) by developing a monoclonal antibody that recognizes the O-GlcNAcylated H4T71 peptide. Threonine 71 of histone H4 is highly conserved from metazoans to mammals, and H4T71Gc can be detected. Chromatin immunoprecipitation-seq and biochemical analysis revealed that H4T71Gc was localized to the region where histone H3 modified by trimethylation of lysine 9 (H3K9me3) was enriched in a genome-wide manner. H3K9me3 is known to function in chromatin condensation, suggesting that H4T71Gc plays a role in both the progression and maintenance of condensed chromatin in several species.

Cholesterol is a crucial lipid that lowers the phase transition temperature of phospholipid membranes and enhances their stability. Artificial cells with diverse functionalities have been developed by encapsulating transcription-translation reactions within liposomes, with the expectation that cholesterol would similarly contribute to the stabilization of membrane compartments in these artificial cells. In this study, we examined whether cholesterol influences the efficiency of reactions within liposomes. Our results demonstrated that the efficiency of transcription-translation reactions decreases in liposomes containing 40 mol% cholesterol, a level comparable to that of the outer leaflet of the human cell membrane. Furthermore, this decrease in reaction efficiency was found to be independent of liposome size or the efficiency of molecule encapsulation. This study highlights the critical role of cholesterol content in the design of artificial cells and drug delivery systems via liposome fusion, emphasizing the need for careful optimization.