Cellular and Molecular Life Sciences最新文献

Imbalances in gut microbiota and their metabolites have been implicated in osteoporotic disorders. Trimethylamine-n-oxide (TMAO), a metabolite of L-carnitine produced by gut microorganisms and flavin-containing monooxygenase-3, is known to accelerate tissue metabolism and remodeling; however, its role in bone loss remained unexplored. This study investigates the relationship between gut microbiota dysbiosis, TMAO production, and osteoporosis development. We further demonstrate that the loss of beneficial gut microbiota is associated with the development of murine osteoporosis and alterations in the serum metabolome, particularly affecting L-carnitine metabolism. TMAO emerges as a functional metabolite detrimental to bone homeostasis. Notably, transplantation of mouse gut microbiota counteracts obesity- or estrogen deficiency-induced TMAO overproduction and mitigates key features of osteoporosis. Mechanistically, excessive TMAO intake augments bone mass loss by inhibiting bone mineral acquisition and osteogenic differentiation. TMAO activates the PERK and ATF4-dependent disruption of endoplasmic reticulum autophagy and suppresses the folding of ATF5, hindering mitochondrial unfolding protein response (UPR^mt) in osteoblasts. Importantly, UPR^mt activation by nicotinamide riboside mitigates TMAO-induced inhibition of mineralized matrix biosynthesis by preserving mitochondrial oxidative phosphorylation and mitophagy. Collectively, our findings revealed that gut microbiota dysbiosis leads to TMAO overproduction, impairing ER homeostasis and UPR^mt, thereby aggravating osteoblast dysfunction and development of osteoporosis. Our study elucidates the catabolic role of gut microflora-derived TMAO in bone integrity and highlights the therapeutic potential of healthy donor gut microbiota transplantation to alter the progression of osteoporosis.

Cell metabolism is crucial for orchestrating the differentiation and function of regulatory T cells (Tregs). However, the underlying mechanism that coordinates cell metabolism to regulate Treg activity is not completely understood. As a pivotal molecule in lipid metabolism, the role of SHIP-1 in Tregs remains unknown. In this study, we found SHIP-1 Treg KO mice (SHIP-1 specifically deleted in regulatory T cells) had severe autoimmunity with increased Tregs in the thymus and disrupted peripheral T cell homeostasis. Mechanistically, CD4^Cre Ship-1^flox/flox mice were found to have increased Treg precursors and SHIP-1 KO Tregs had reduced migration and stability, which caused decreased Tregs in the spleen. Additionally, the suppressive function of Tregs from SHIP-1 KO mice was diminished, along with their promotion of anti-tumor immunity. Interestingly, the PI3K-mTORC1, but not mTORC2, signaling axis was enhanced in SHIP-1 KO Tregs. In vivo treatment of SHIP-1 Treg KO mice with rapamycin rescued the abnormal Treg percentages and peripheral T cell homeostasis, as well as Treg suppressive function. Furthermore, the treatment of wild-type mice with SHIP-1 inhibitor enhanced anti-tumor activity. Our study highlights the SHIP-1-PI3K-mTORC1 axis that regulates Treg differentiation and function, and it is a potential target for cancer treatment.

Asthma is a chronic inflammatory respiratory disease characterized by recurrent breathing difficulties caused by airway obstruction and hypersensitivity. Although there is diversity in their specific mechanisms, microRNAs (miRNAs) have a significant impact on the development of asthma. Currently, the contribution of miR-130b-3p to asthma remains elusive. The goal of this study was to examine whether miR-130b-3p attenuates house dust mite (HDM)-induced asthma through High-mobility group box protein 1 (HMGB1)/Toll-like receptor 4 (TLR4)/mitochondrial fission protein (DRP1) signaling pathway. We elucidate that miR-130b-3p can bind to the HMGB1 3'UTR, attenuating HMGB1 mRNA and protein levels, and nucleo-cytoplasmic translocation of HMGB1. We observed that miR-130b-3p agomir or HMGB1 CKO attenuated HDM-induced airway inflammation and hyperresponsiveness, and decreased Th2-type cytokines in bronchoalveolar lavage fluid (BALF) and mediastinal lymph nodes. Further, HMGB1 CKO contributes to alleviating Th2 inflammation in AT-II cells (CD45.2^-/CD31^-/Epcam^-+/proSP-C⁺/MHC-II⁺) from lung single cell suspensions of asthmatic mice by flow cytometry. Our findings identified miR-130b-3p as a potent regulator in asthma that exerts its anti-inflammatory effects by targeting HMGB1 and the subsequent HMGB1/TLR4/DRP1axis, presenting a prospective novel therapeutic avenue for asthma management.

ENPP1/CD203a is a membrane-bound ectonucleotidase capable of hydrolyzing ATP, cGAMP and other substrates. Its enzymatic activity plays an important role in the balance of extracellular adenine nucleotides and the modulation of purinergic signaling, in soft tissue calcification, and in the regulation of the cGAS/STING pathway. However, a detailed analysis of ENPP1 surface expression on human immune cells has not been performed. Here, we selected VHH domains from human ENPP1-immunized alpacas to generate heavy-chain antibodies targeting ENPP1, and analyzed cell surface expression on all circulating immune cell subsets using flow cytometry. We find high expression of ENPP1 in CD141^high conventional dendritic cells (cDC1), while ENPP1 was not detectable on other dendritic cells and monocytes. In the lymphocytic compartment, only CD56^bright natural killer cells and mucosal-associated invariant T cells (MAIT) express ENPP1. In contrast, all other T cell subpopulations, CD56^dim natural killer cells and B lymphocytes do not or only minimally express ENPP1. In summary, we describe highly cell type-specific expression of ENPP1 in the immune system using a newly generated heavy-chain antibody. This reagent will help to decipher the function of ENPP1 in the regulation of the immune response, allow a quick identification of ENPP1-deficiency and of ENPP1-positive tumors, and constitutes the basis for targeted anti-tumor intervention.

Methylation of cytosine in CpG dinucleotides is an epigenetic modification carried out by DNA-methyltransferases (DNMTs) that contributes to chromatin condensation and structure and, thus, to gene transcription regulation and chromosome stability. DNMT1 maintains the DNA methylation pattern of the genome at each cell cycle by copying it to the newly synthesized DNA strand during the S-phase. DNMT1 pharmacological inhibition as well as genetic knockout and knockdown, leads to passive DNA methylation loss. However, these strategies have been associated with different cell fates, even in the same cell background, suggesting that they can question the interpretation of the obtained results. Using a cell system in which endogenous DNMT1 is fused with an inducible degron and can be rapidly degraded, we found that in non-tumoral RPE-1 cells, DNMT1 loss progressively induced cell proliferation slowing-down and cell cycle arrest at the G1/S transition. The latter is due to p21 activation, which is partly mediated by p53 and leads to a global reduction in DNA methylation. DNMT1 restoration rescues cell proliferation, indicating that its deregulation is sensed as tunable cellular stress.

Flower, a highly conserved protein, crucial for endocytosis and cellular fitness, has been implicated in cytotoxic T lymphocyte (CTL) killing efficiency through its role in cytotoxic granule (CG) endocytosis at the immune synapse (IS). This study explores the molecular cues that govern Flower-mediated CG endocytosis by analyzing uptake of Synaptobrevin2, a protein specific to CG in mouse CTL. Using immunogold electron microscopy and total internal fluorescence microscopy, we found that Flower translocates in a stimulus-dependent manner from small vesicles to the IS, thereby ensuring specificity in CG membrane protein recycling. Using confocal live-cell imaging, we assessed the ability of a range of naturally occurring mouse, human and Drosophila isoforms to rescue defective endocytosis in Flower KO CTLs. This analysis demonstrated that the N-terminal portion of the protein, encompassing amino acids 1-106 in mice, is the minimal domain necessary for Synaptobrevin2 endocytosis. Additionally, we identified two pivotal sites through site-specific mutation: a putative AP2-binding site, and a tyrosine at position 104 in mouse Flower. These findings provide insights into Flower's specific functional domain essential for CG endocytosis, which is a key process in mediating T cell serial killing required for the effective fight against cancer.

Post-translational modifications (PTMs) are implicated in many biological processes including receptor activation, signal transduction, transcriptional regulation and protein turnover. Lysine's side chain is particularly notable, as it can undergo methylation, acetylation, SUMOylation and ubiquitination. Methylation affects not only lysine but also arginine residues, both of which are implicated in epigenetic regulation. Beyond histone-tails as substrates, dynamic methylation of transcription factors has been described. The focus of this review is on these non-histone substrates providing a detailed discussion of what is currently known about methylation of hypoxia-inducible factor (HIF), P53, nuclear receptors (NRs) and RELA. The role of methylation in regulating protein stability and function by acting as docking sites for methyl-reader proteins and via their crosstalk with other PTMs is explored.

Background: Endometrial cancer (EC) represents a serious health concern among women globally. Excessive activation of the protooncogene c-Myc (c-Myc) is associated with the proliferation and stemness of EC cells. Phosphocholine (PC), which is synthesized by choline kinase alpha (CHKA) catalysis, is upregulated in EC tumor tissues. The present study aimed to investigate the effect of PC accumulation on EC cells and clarify the relationship between PC accumulation and c-Myc activity in EC.

Methods: The c-Myc and CHKA expression in EC tumor tissues were examined using immunohistochemistry. Cell Counting Kit-8 assay, colony formation assay, flow cytometry, western blotting, BrdU staining, and tumorsphere formation assay were used to assess the effect of PC accumulation on EC cells. The mechanism by which PC accumulation inhibits c-Myc was evaluated using RNA-sequencing. Patient-derived organoid (PDO) models were utilised to explore the preclinical efficacy of PC against EC cells.

Results: PC accumulation suppressed EC cell proliferation and stemness by inhibiting the activation of the mammalian target of rapamycin (mTOR)-c-Myc signaling. PC accumulation promoted excessive reactive oxygen species production, which reduced the expression of GTPase HRAS. This, in turn, inhibited the mTOR-c-Myc axis and induced EC cell apoptosis. Finally, PC impeded proliferation and downregulated the expression of the mTOR-MYC signaling in EC PDO models.

Conclusions: PC accumulation impairs the proliferation ability and stem cell characteristics of EC cells by inhibiting the activated mTOR-c-Myc axis, potentially offering a promising strategy to enhance the efficacy of EC clinical therapy through the promotion of PC accumulation in tumor cells.

Loss-of-function variants in ATP6V0A2, encoding the trans Golgi V-ATPase subunit V0a2, cause wrinkly skin syndrome (WSS), a connective tissue disorder with glycosylation defects and aberrant cortical neuron migration. We used knock-out (Atp6v0a2^-/-) and knock-in (Atp6v0a2^RQ/RQ) mice harboring the R755Q missense mutation selectively abolishing V0a2-mediated proton transport to investigate the WSS pathomechanism. Homozygous mutants from both strains displayed a reduction of growth, dermis thickness, and elastic fiber formation compatible with WSS. A hitherto unrecognized male infertility due to globozoospermia was evident in both mouse lines with impaired Golgi-derived acrosome formation and abolished mucin-type O-glycosylation in spermatids. Atp6v0a2^-/- mutants showed enhanced fucosylation and glycosaminoglycan modification, but reduced levels of glycanated decorin and sialylation in skin and/or fibroblasts, which were absent or milder in Atp6v0a2^RQ/RQ. Atp6v0a2^RQ/RQ mutants displayed more abnormal migration of cortical neurons, correlating with seizures and a reduced O-mannosylation of α-dystroglycan. While anterograde transport within the secretory pathway was similarly delayed in both mutants the brefeldin A-induced retrograde fusion of Golgi membranes with the endoplasmic reticulum was less impaired in Atp6v0a2^RQ/RQ. Measurement of the pH in the trans Golgi compartment revealed a shift from 5.80 in wildtype to 6.52 in Atp6v0a2^-/- and 6.25 in Atp6v0a2^RQ/RQ. Our findings suggest that altered O-glycosylation is more relevant for the WSS pathomechanism than N-glycosylation and leads to a secondary dystroglycanopathy. Most phenotypic and cellular properties correlate with the different degrees of trans Golgi pH elevation in both mutants underlining the fundamental relevance of pH regulation in the secretory pathway.

Human induced pluripotent stem cells (hiPSCs) generate multiple clones with inherent heterogeneity, leading to variations in their differentiation capacity. Previous studies have primarily addressed line-to-line variations in differentiation capacity, leaving a gap in the comprehensive understanding of clonal heterogeneity. Here, we aimed to profile the heterogeneity of hiPSC clones and identify predictive biomarkers for cardiomyocyte (CM) differentiation capacity by integrating transcriptomic, epigenomic, endogenous retroelement, and protein kinase phosphorylation profiles. We generated multiple clones from a single donor and validated that these clones exhibited comparable levels of pluripotency markers. The clones were classified into two groups based on their differentiation efficiency to CMs-productive clone (PC) and non-productive clone (NPC). We performed RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq). NPC was enriched in vasculogenesis and cell adhesion, accompanied by elevated levels of phosphorylated ERK1/2. Conversely, PC exhibited enrichment in embryonic organ development and transcription factor activation, accompanied by increased chromatin accessibility near transcription start site (TSS) regions. Integrative analysis of RNA-seq and ATAC-seq revealed 14 candidate genes correlated with cardiac differentiation potential. Notably, TEK and SDR42E1 were upregulated in NPC. Our integrative profiles enhance the understanding of clonal heterogeneity and highlight two novel biomarkers associated with CM differentiation. This insight may facilitate the identification of suboptimal hiPSC clones, thereby mitigating adverse outcomes in clinical applications.