Journal of biochemistry最新文献

The T1r family of receptors is essential for the detection of sweet and umami tastants, which are categorized as class C G protein-coupled receptors (GPCRs). Although these receptors share structural characteristics with other class C GPCRs, such as metabotropic glutamate receptors, they are uniquely characterized by a significantly shorter C-terminal intracellular domain, consisting of approximately 30-40 amino acid residues. Yoshida et al. recently demonstrated that the C-terminal region of mouse T1rs directly binds to calmodulin in a Ca2+-dependent manner. This interaction highlights a previously unrecognized aspect of the intracellular signalling mechanism of T1rs and indicates that the C-terminal region contributes to taste signal regulation, particularly through Ca2+-dependent feedback mechanisms.

Proteasomal degradation of ubiquitinated proteins involves various accessory factors, including p97 and shuttle factors, but their requirements and relationship with substrate structural properties are not fully understood, especially in human cells. Here, we demonstrate that substrate structure dictates the dependency on p97 and RAD23A/B for proteasomal degradation in human cells, using two ubiquitin-fusion model substrates, Ub-GFP (well-folded) and Ub-GFP-tail (with an unstructured tail). Both substrates exhibited similar ubiquitin chain composition, primarily mediated by the UBR4-KCMF1 E3 ligase. Interactome analyses revealed that Ub-GFP preferentially interacts with p97 and RAD23B, while Ub-GFP-tail binds more strongly with the proteasome. The degradation of Ub-GFP depends on p97 and RAD23A/B, whereas that of Ub-GFP-tail bypasses these accessory factors. RAD23A/B knockdown resulted in a reduction in the apparent lengths of ubiquitin chains on both substrates, yet it only affected Ub-GFP degradation, suggesting that even a lower level of ubiquitination is sufficient to support proteasomal degradation of substrates with an unstructured tail. Overall, our findings highlight substrate structure as a key determinant of accessory factor requirement, offering valuable insights for the development of targeted protein degradation.

(Pro)renin receptor [(P)RR], encoded by Atp6ap2, is a transmembrane protein found in many organs. It functions in lysosomes as part of the vacuolar-ATPase complex, facilitating autophagy and degradation. Mutations in ATP6AP2 are linked to neurological conditions, including X-linked parkinsonism with spasticity. However, our understanding of the role of (P)RR in whole brain development remains incomplete. Here, we generated mice with neural stem cell (NSC)-specific (P)RR deficiency (CKO). CKO mice exhibited significant brain atrophy during mid-gestation, leading to perinatal lethality. Foetal CKO brains showed lateral ventricular enlargement with malformation of neocortex and ganglionic eminence from mid-gestation. CKO brains showed massive apoptosis in multiple regions along with microglial activation at E15. On the contrary, CKO NSCs showed normal self-renewal ability, suggesting that (P)RR is critical for survival of differentiated cells. In line with this, the mechanistic study using RNA-seq of primary NSCs revealed downregulation of genes related to neurodevelopment and myelination. We also found p62 and LC3-II protein accumulation, hallmarks of deregulated autophagic pathways, in CKO foetal brains and NSCs. These findings demonstrate that (P)RR is crucial for guiding NSC differentiation and ensuring the coordinated construction of brain architecture during development.

REV1 is a eukaryotic error-prone DNA polymerase belonging to the Y-family, with a central role in translesion DNA synthesis (TLS) to continue DNA replication even in the presence of DNA damage in the template strand. TLS is stimulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), a toroidal-shaped protein functioning as a scaffold for DNA polymerases and repair enzymes. Mammals possess four types of Y-family DNA polymerases: Pol η, Pol κ, Pol ι and REV1. Among those, Pol η, Pol κ and Pol ι interact with PCNA through PCNA-binding motifs, low-affinity variants of PCNA-interacting protein box (PIP-box). To date, several studies have reported that REV1 interacts with PCNA, but identified PCNA-binding regions are inconsistent; therefore, a structural basis for interaction between REV1 and PCNA also remains unclear. Here, we identified a signature sequence conserved within vertebrates REV1 responsible for PCNA-binding. Furthermore, we unveiled a mechanism underlying the physical interaction between the PCNA-binding motif of human REV1 and PCNA by X-ray crystallography, thus revealing that REV1 binds to PCNA through a PIP-box variant located in the C-terminal side of the little finger domain. Our study provides a convincing answer for a long-standing controversy regarding the physical interaction between REV1 and PCNA.

Phospholipase C (PLC) plays crucial roles in phospholipid metabolism by generating second messengers regulating diverse cellular processes. PLCs are classified into phosphatidylinositol-specific PLCs (PI-PLCs) and nonspecific PLCs. Among them, PI-PLCs are highly conserved across mammals, plants and bacteria, with distinct structural organizations and regulatory mechanisms. Mammalian PI-PLCs participate in intracellular signalling and modulate various pathways, such as the G protein-coupled receptor- and receptor tyrosine kinase-mediated cascades, whereas plant PI-PLCs regulate the growth and stress responses via phosphatidic acid and inositol phosphate signalling. Bacterial PI-PLCs contribute to virulence by targeting the host cell membranes and glycosylphosphatidylinositol-anchored proteins. Despite structural variations, PI-PLCs share conserved catalytic mechanisms integral to cellular physiology across different organisms. This review article highlights the structural diversity and functional significance of PI-PLCs in mammals, plants and bacteria.

Human serum albumin (HSA) has three major binding sites for drugs: Site I, Site II, and FA1 site. Dansyl amino acids (Dans-AAs) have long been used as convenient markers to judge whether a low molecular weight molecule of interest (ligand) binds to Sites I or II. However, crystal structures of HSA-Dans-AA complexes have revealed that Dans-AAs with strict site specificity are also bound to non-marker sites. To characterize the multiple binding of Dans-AAs in detail, the average number of the bound ligands per HSA molecule were obtained in a free ligand concentration of 1-400 μM for dansylate (DA) and 17 Dans-AAs using microbore frontal gel filtration chromatography. Analysis of the binding curves indicated that there are three specific binding sites for Dans-AAs. Four Dans-AAs with hydrophobic sidechain bind to all the sites with identical affinity, whereas DA and four Dans-AAs bind equally to two of them. Nine Dans-AAs bind to one of the three sites with the maximum occupancy ranging from 72 to 94%. The UV-vis absorption spectrum of HSA-ligand complex was obtained for DA and 10 Dans-AAs, revealing that the dansyl moiety is in hydrophobic environment and conformational changes in the binding site residues are induced.

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.

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.