Journal of biochemistry最新文献

The risk of breast cancer (BC) recurrence is high in postmenopausal women, though the underlying molecular mechanisms are not yet fully understood. We developed a long-term estrogen-deprived (LTED) cell line from MCF-7 cells, which we used as an in vitro model for aromatase inhibitor (AI)-resistant estrogen receptor α (ERα)-positive postmenopausal BC. We also describe the involvement of fatty acid 2-hydroxylase (FA2H) in the modulation of LTED cell migration. Small interfering RNA specific to FA2H (siFA2H) could reduce cell migration, whereas the introduction of plasmid expressing FA2H, but not its inactive mutant, resulted in enhanced migration. Moreover, proliferation of the LTED cells was not affected by modulation of FA2H expression. Fulvestrant (FUL), a selective estrogen receptor degrader used to treat AI-resistant ERα-positive postmenopausal BC, was found to induce degradation of ERα together with a decrease in ER-mediated transcription; however, FA2H protein expression and migration remained unchanged. Overall, the findings of this study suggest that FA2H is one of the drivers of LTED cell migration, and that LTED cells resistant to FUL therapy may be involved in malignancy and metastatic mechanisms.

The nuclear envelope (NE) is a double-membrane structure critical for genome maintenance and cellular function, composed of the inner and outer nuclear membranes. In fission yeast, the inner nuclear membrane (INM) proteins Lem2 and Bqt4 are essential for maintaining NE integrity. The study published by Hiraoka group (Hirano et al. 2023) explores the interactions between Lem2 and Bqt4 with lipid synthesis enzymes, addressing their roles in NE maintenance. The authors identified Lem2- and Bqt4-binding proteins using immunoprecipitation and mass spectrometry, revealing that Lem2 interacts with lipid synthesis enzymes, while Bqt4 binds to an enzyme that involves in glucosylceramide synthesis. These findings suggest that Lem2 and Bqt4 independently contribute to NE structure and its integrity through distinct lipid metabolic pathways, highlighting their complementary roles in nuclear membrane homeostasis. This study represents a significant step forward in the field of NE biology to unravel the complexities of nuclear membrane dynamics.

A recent study reported that isomerization of aspartyl residues (Asp) occurs in various tissues and proteins in vivo. For a comprehensive analysis of post-translational modifications, the MS-based proteomic approach is a straightforward method; however, the isomerization of Asp does not alter its molecular weight. Therefore, a unique method is required to analyze Asp isomers using mass spectrometry. Herein, we present a novel strategy, isoAsp-Quest, which is a database search-oriented isoAsp identification method. isoAsp is specifically converted to 18O-labeled Lα-Asp by the enzymatic reaction of protein L-isoaspartyl-O-methyltransferase (PIMT) in 18O water with a mass shift of 2 Da, which, in principle, enables us to distinguish Asp isomers. However, in practice, a labeled Lα-Asp signal overlaps with that of endogenous Lα-Asp, making detection challenging. Therefore, degradation of the endogenous Lα-Asp peptide by AspN and subsequent removal of AspN were performed prior to the PIMT reaction. This strategy was applied to bovine lens α-crystallin. Consequently, several Asp isomerization sites, consistent with human αA-crystallin, were identified in bovine αA-crystallin, indicating that this strategy is also effective for biological proteins. Therefore, isoAsp-Quest enables the analysis of Lβ-Asp in a straightforward and rapid workflow, which may be useful for the quality control of protein products and biomarker discovery.

The incidence of malignant mesothelioma (MM), a disease linked to refractory asbestos exposure, continues to increase globally, and remains largely resistant to various treatments. Our previous studies have identified a strong correlation between connective tissue growth factor (CTGF) protein expression and MM malignancy, underscoring the importance of understanding CTGF regulation in MM cells. In this study, we demonstrate for the first time that stimulation with platelet-derived growth factor receptor (PDGFR) ligand, PDGF-BB, increases CTGF protein expression levels without affecting CTGF mRNA levels. Inhibition of PDGFR resulted in a reduction of CTGF protein expression, indicating that PDGFR activation is essential in regulating CTGF protein expression in MM cells. PDGF-BB also activated the protein kinase B (AKT) pathway, and inhibition of AKT phosphorylation abolished the PDGFR-induced CTGF protein expression, suggesting that PDGFR acts upstream of CTGF via the AKT pathway. This reinforces the role of CTGF protein as a key regulator of MM malignancy. Additionally, PDGFR activation led to the phosphorylation of mTOR and 4E-BP1, critical regulators of protein synthesis downstream of AKT, suggesting that PDGFR controls CTGF protein expression through the regulation of CTGF mRNA translation.

L-arginine oxidase (AROD, EC 1.4.3.25) is an oxidoreductase that catalyzes the deamination of L-arginine, with flavin adenine dinucleotide (FAD) as a cofactor. Recently identified AROD from Pseudomonas sp. TPU 7192 (PT-AROD) demonstrates high selectivity for L-arginine. This enzyme is useful for accurate assays of L-arginine in biological samples. The structural characteristics of the FAD-dependent AROD, however, remain unknown. Here, we report the structure of PT-AROD at a resolution of 2.3 Å by cryo-electron microscopy. PT-AROD adopts an octameric structure with D4 symmetry, which is consistent with its molecular weight in solution, estimated by mass photometry. Comparative analysis of this structure with that determined using X-ray crystallography reveals open and closed forms of the lid-like loop at the entrance to the substrate pocket. Furthermore, mutation of Glu493, located at the substrate binding site, diminishes substrate selectivity, suggesting that this residue contributes significantly to the high selectivity of PT-AROD.

Balancing energy production and storage is a fundamental process critical for cellular homeostasis in most eukaryotes that relies on the intimate interplay between mitochondria and lipid droplets. In the oleaginous yeast Lipomyces starkeyi under nitrogen starvation, lipid droplet forms a single giant spherical structure that is easily visible under a light microscope. Currently, how mitochondria behave in L. starkeyi cells undergoing giant lipid droplet formation remains unknown. Here we show that mitochondria transition from fragments to elongated tubules and sheet-like structures that are in close proximity to a giant lipid droplet in nitrogen-depleted L. starkeyi cells. Under the same conditions, mitochondrial degradation and autophagy are strongly suppressed, suggesting that these catabolic events are not required for giant lipid droplet formation. Conversely, carbon-depleted cells suppress mitochondrial elongation and lipid droplet expansion, whereas they promote mitochondrial degradation and autophagy. We propose a potential link of mitochondrial proximity and autophagic suppression to giant lipid droplet formation.

Tendons and ligaments, crucial components of the musculoskeletal system, connect muscles to bones. In the realm of sports, tendons and ligaments are vulnerable tissues, with injuries such as Achilles tendon rupture and anterior cruciate ligament tears directly impacting an athlete's career. Furthermore, repetitive trauma and tissue degeneration can lead to conditions like secondary osteoarthritis, ultimately affecting the overall quality of life. Recent research highlights the pivotal role of mechanical stress in maintaining homeostasis within tendons and ligaments. This review delves into the latest insights on the structure of tendons and ligaments and the plasticity of tendon tissue in response to mechanical loads.

Erythrocytes are important vascular components that play vital roles in maintaining vascular homeostasis, in addition to carrying oxygen. Previously, we reported that the changes in the internal milieu (e.g. hyperglycemia or hypercholesterolemia) increase erythrocyte adhesion to various extracellular matrix components, potentially through altering glycosaminoglycans (GAGs). In this study, we have investigated the expression of syndecan (Sdc) family members that could be involved in mediating cytoadherence under conditions of dyslipidemia and hyperglycemia. Among the Sdc family members analysed, we found significant overexpression of Sdc-3 in erythrocyte membranes harvested from high-fat-fed control and diabetic animals. Animal studies revealed a positive correlation between Sdc-3 expression, blood sugar levels and erythrocyte adhesion. In the human study, diabetic cohorts with body mass index >24.9 showed significantly increased expression of Sdc-3. Interestingly, blocking the Sdc-3 moiety with an anti-Sdc-3 antibody revealed that the core protein might not be directly involved in erythrocyte adhesion to fibronectin despite the GAGs bringing about adhesion. Lastly, Nano liquid chromatography-mass spectrometry/MS verified the presence of Sdc-3 in erythrocyte membranes. In conclusion, the high-fat diet and diabetes modulated Sdc-3 expression in the erythrocyte membrane, which may alter its adhesive properties and promote vascular complications.

In most organs, resources such as nutrients, oxygen and physiologically active substances are unevenly supplied within the tissue spaces. Consequently, different tissue functions are exhibited in each space. This spatial heterogeneity of tissue environments arises depending on the spatial arrangement of nutrient vessels and functional vessels, leading to continuous changes in the metabolic states and functions of various cell types from regions proximal to these vessels to distant regions. This phenomenon is referred to as 'zonation'. Traditional analytical methods have made it difficult to investigate this zonation in detail. However, recent advancements in intravital imaging, spatial transcriptomics and single-cell transcriptomics technologies have facilitated the discovery of 'zones' in various organs and elucidated their physiological roles. Here, we outline the spatial differences in the immune system within each zone of organs. This information provides a deeper understanding of organs' immune systems.

Prostate cancer (PCa) has become a worldwide health burden among men. Previous studies have suggested that cellular retinoic acid binding protein 2 (CRABP2) significantly affects the regulation of cell proliferation, motility and apoptosis in multiple cancers; however, the effect of CRABP2 on PCa is poorly reported. CRABP2 expression in different PCa cell lines and its effect on different cellular functions varied. While CRABP2 promotes cell migration and invasion, it appears to inhibit cell proliferation specifically in PC-3 cells. However, the proliferation of DU145 and 22RV1 cells did not appear to be significantly affected by CRABP2. Additionally, CRABP2 had no influence on the cell cycle distribution of PCa cells. The RNA-seq assay showed that overexpressing CRABP2 upregulated laminin subunit beta-3 (LAMB3) mRNA expression, and the enrichment analyses revealed that the differentially expressed genes were enriched in the phosphoinositide 3-kinase (PI3K)/activated protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signalling pathways. The following western blot experiments also confirmed the upregulated LAMB3 protein level and the activation of the PI3K/AKT and MAPK signalling pathways. Moreover, overexpressing CRABP2 significantly inhibited tumour growth in vivo. In conclusion, CRABP2 facilitates cell migration and invasion by activating PI3K/AKT and MAPK signalling pathways through upregulating LAMB3 in PCa.