Alpha-mannosidosis (AMD) is a well-known lysosomal storage disorder caused by the loss of α-mannosidase activity due to the mutation of the MAN2B1 gene. Defective α-mannosidase cannot completely degrade the sugar chains of upstream input glycoproteins, leading to the accumulation of oligosaccharides with α-mannosidic linkages in lysosomes, finally causing AMD. However, till now, the upstream input mediating AMD remains elusive, which hinders the development of alternative therapeutic treatments. To address this question, we establish the first Drosophila model of AMD and, through genetic screen, identify three novel upstream factors named Jer1, Ga2, and LpR1. We demonstrate that knocking down either of them can rescue the lethal phenotype of AMD flies and they mediate upstream input of AMD through a Jer1-Ga2-LpR1 axis. Mechanistically, Jer1 recruits the E3 ligase Ga2, which mediates the ubiquitination of the glycoprotein LpR1 for subsequent lysosomal degradation. Therefore, knockdown of Jer1 or Ga2 downregulates LpR1 ubiquitination and prevents it from degradation in lysosomes, reducing the burden on lysosomes and alleviating the symptoms of AMD. Importantly, our study further demonstrates that IPP, UBE3C, and VLDLR, the mammalian counterparts of Jer1, Ga2, and LpR1, respectively, are functionally conserved during evolution, suggesting that they can be used as potential therapeutic targets for the treatment of AMD.
Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, monitored by the spindle assembly checkpoint machinery, which prevents precocious chromosome segregation and reduces the risk of aneuploidy. MAD2B, a homologue of MAD2, is critical for mitotic quality control and DNA damage repair. However, it remains elusive how MAD2B guides genome surveillance during the cell cycle. Here, we show that MAD2B safeguards genome integrity through interactions with CIP2A in a context-dependent manner. To delineate the molecular mechanisms underlying MAD2B-dependent signaling in mitosis, we carried out affinity purification of FLAG-MAD2B followed by mass spectrometry to identify MAD2B-associated proteins. Our biochemical characterization uncovered a previously uncharacterized interaction between MAD2B and CIP2A, mediated by the N-terminus of CIP2A. Importantly, MAD2B depletion resulted in DNA damage response and replication stress phenotypes, leading to aberrant mitotic DNA synthesis primarily in HeLa cells. Notably, loss of MAD2B disrupted CIP2A recruitment to γH2AX-marked DNA lesions and attenuated DNA damage repair. Together, these results establish MAD2B as a context-sensitive regulator of genome stability that links replication stress surveillance to mitotic chromosome repair via the MAD2B-CIP2A signaling axis.
In end-stage liver disease, impaired hepatocyte proliferation prevents regeneration. Biliary epithelial cell transdifferentiation thus becomes a critical alternative for liver repair, yet its efficiency remains low in mammals. Elucidating the initiation mechanisms of this process may unlock novel therapeutic targets for end-stage liver disease treatment. By leveraging the zebrafish severe liver injury model and its transparency, as well as the availability of small-molecule research, we identified that topoisomerase 1 (Top1) is essential for the initiation of biliary-derived liver regeneration. Top1 upregulation occurred early after liver injury. Moreover, genetic knockout (top1 mutants) or pharmacological inhibition (topotecan) impeded liver regeneration by suppressing cholangiocyte dedifferentiation, promoting cell apoptosis, and blocking the redifferentiation of bipotential progenitor cells into hepatocytes and cholangiocytes. Mechanistically, Top1 activates DNA methyltransferase 1 (Dnmt1) in cholangiocytes, thereby depressing p53 during dedifferentiation, which subsequently maintains the activity of mTOR signalling in cholangiocytes to trigger regeneration initiation. These findings suggest that the Top1-Dnmt1-p53 axis orchestrates the initiation of biliary-driven liver regeneration.
Monoamine neurotransmitters dopamine and 5-hydroxytryptamine (serotonin, 5-HT), traditionally implicated in the central nervous system, are increasingly recognized as contributors to hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms remain unclear. Here, we demonstrate that the E3 ubiquitin ligase F-box-only protein 6 (FBXO6) is highly expressed in HCC patients. FBXO6 promotes accumulation of 5-HT and dopamine in HCC by mediating K48-linked ubiquitination and degradation of monoamine oxidase A (MAOA). Elevated monoamine levels activate their cognate receptors, triggering the PI3K/AKT/mTOR signaling axis. Simultaneously, these neurotransmitters induce transglutaminase 2 (TGM2)-dependent histone monoaminylation (H3Q5ser and H3Q5dop), forming a synergistic oncogenic circuit that drives HCC development. Clinical specimens reveal a strong inverse correlation between FBXO6 and MAOA protein levels, validating this regulatory axis. Preclinical studies demonstrate that combined treatment with the mTOR inhibitor everolimus and the TGM2 inhibitor GK921 exerts synergistic antitumor effects against FBXO6-driven HCC, providing a rational therapeutic strategy for patients with FBXO6 overexpression.
Lysophosphatidic acid (LPA) is known to be crucial for oocyte developmental competence, but the underlying molecular mechanisms remain unclear. In this study, we investigated the mechanistic relationship between follicular fluid LPA levels and oocyte quality determinants using a cohort of assisted reproductive technology patients, correlating LPA abundance with follicle sensitivity indices. Transcriptome sequencing and metabolic flux analyses compared low versus high LPA groups. Functional validation employed gabapentin-mediated inhibition of branched-chain amino acid (BCAA) catabolism in oocytes, assessing meiotic maturation, mitochondrial function, and oxidative stress. We found that reduced LPA levels were associated with significant suppression of BCAA catabolic pathways, leading to accumulation of valine and leucine. Gabapentin inhibition of BCAA oxidation recapitulated the low-LPA phenotype, inducing severe meiotic defects characterized by aberrant spindle assembly, chromosomal misalignment, and pronounced DNA damage. These oocytes also exhibited mitochondrial dysfunction with disrupted organellar distribution and severely compromised membrane polarization, along with elevated reactive oxygen species. Our findings indicate that impaired BCAA metabolism under low-LPA conditions compromises oocyte quality through mitochondrial dysfunction and oxidative stress, suggesting LPA as a potential modulator of metabolic pathways during oocyte maturation and also suggesting novel therapeutic targets for improving assisted reproduction outcomes.
Cell state transitions are essential for diverse cell fate decision processes. Such transitions are mostly irreversible in natural environments, but they can be reversed under certain conditions. This raises a question on the largely unknown regulatory mechanisms underlying the asymmetric cell state transitions in forward and backward directions. Here, we present scDECIPHER (single-cell dynamic explorer of complex interactions and pathway hierarchies), an integrative framework designed to address this question using single-cell resolution transcriptome data measured over cell state transitions. By applying scDECIPHER to human and mouse cancer samples, we uncover the hidden molecular regulatory mechanisms to overcome drug resistance in cancer, suggesting the potential to control cell state transitions towards desired directions.
Heart failure is characterized by profound structural and metabolic remodeling. Members of the solute carrier family 25 (SLC25), as inner mitochondrial membrane proteins, play critical roles in mitochondrial dysfunction during heart failure; however, the role of cardiac-enriched SLC25 family members remains incompletely understood. In this study, we identify solute carrier family 25 member 34 (SLC25A34) as a previously unrecognized mitochondrial regulator of cardiac energy metabolism in heart failure. SLC25A34 is highly enriched in the heart and predominantly expressed in adult cardiomyocytes. Its expression progressively increases during postnatal cardiac development, is closely associated with fatty acid-based energy utilization, and is inducible by palmitate. In contrast, SLC25A34 expression is markedly reduced in failing human hearts and multiple mouse models of heart failure. Mechanistically, oxidative stress during heart failure suppresses SLC25A34 expression in cardiomyocytes. Genetic deletion of Slc25a34 exacerbates cardiac dysfunction and adverse remodeling following transverse aortic constriction, without significantly affecting cardiac hypertrophy. Integrated biochemical, transcriptomic, and functional analyses demonstrate that loss of SLC25A34 suppresses the AMPK-CPT1B axis, thereby impairing fatty acid oxidation, leading to reduced ATP production, lipid droplet accumulation, and disrupted redox homeostasis. Conversely, adeno-associated virus-mediated cardiac-specific overexpression of Slc25a34 significantly mitigates heart failure progression. Collectively, these findings establish SLC25A34 as a key mitochondrial regulator linking fatty acid metabolism, energy sensing, and redox homeostasis and suggest that targeting SLC25A34 may represent a novel therapeutic strategy for heart failure.
Decidualization of human endometrial stromal cells (hESCs) during the secretory phase is critical for implantation and pregnancy. In this study, we assessed 25 established in vitro decidualization markers using in vivo single-cell transcriptomic data and found that the expression levels of most of them, including PRL and IGFBP1, were comparable between the proliferative- and secretory-phase hESCs or between the decidualized and non-decidualized hESCs. We hypothesized that the lack of endometrial glands in vitro causes the discrepancy. However, the role of the endometrial glands in decidualization remains unclear. Using a co-culture model, we demonstrated that the secretome of human endometrial organoids attenuates decidualization by reducing canonical marker upregulation and hESC mesenchymal-epithelial transition. Transcriptomic analysis revealed the downregulation of key decidualization regulators CEBPA and EDN1. Consistent with the in vitro findings, an estradiol-stimulated mouse model with increased glandular formation showed reduced decidualization. Collectively, our results identify a novel homeostatic function of endometrial gland secretome in decidualization regulation.
Src-homology-2-containing protein tyrosine phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a master regulator of cell growth, survival, and differentiation. Normally, SHP2 is kept in a self-inhibited state, in which its N-terminal SH2 domain blocks the catalytic site. SHP2 activation is directly tied to the disruption of the self-inhibition. While mutations that disrupt this self-inhibited state can cause developmental disorders and cancer, most SHP2 variants remain functionally uncharacterized. Using computational saturation mutagenesis, we systematically analyzed > 9000 single-amino acid substitutions in the SH2 and PTP domains. Our analysis of binding energy changes identified key residues-especially A72 and G503-whose mutation destabilizes the autoinhibited conformation. Functional assays further demonstrated that mutations within the SH2 and PTP domains significantly enhance enzymatic activity, downstream signaling, and cellular proliferation. By integrating clinical data, we found that pathogenic variants preferentially adopt the destabilizing conformations, directly linking structural changes to disease. This work provides a comprehensive map of SHP2 mutation effects, highlights conformational opening as a key driver of pathogenicity, and establishes a predictive framework for interpreting the functional impact of uncharacterized variants in multi-domain proteins.
Double-negative T (DNT) cells (TCRαβ+CD4-CD8-NK1.1-/CD56-) exhibit strong tumor-killing capabilities. Our single-cell transcriptome analysis has revealed high Fcer1g expression in DNT cells, but its role in tumor immunity remains unclear. In this study, we demonstrated that IgG1 stimulation significantly upregulated IgG Fc receptors and cytotoxic molecules in DNT cells, enhancing their cytotoxicity against MC38 tumor cells in vitro. FcεRIγ-deficient DNT cells failed to respond effectively to IgG1 stimulation. Inhibiting the downstream spleen tyrosine kinase (Syk) of FcεRIγ reduced cytotoxicity of DNT cells and phosphorylation levels of molecules such as AKT and NF-κB. In a subcutaneous tumor model, combined treatment with DNT cells and tumor-specific antibodies more effectively inhibited tumor growth compared to DNT cells alone, while FcεRIγ-deficient DNT cells combined with antibodies showed no significant difference in efficacy compared to DNT cells alone, suggesting that DNT cells enhance tumor cell killing via FcεRIγ-mediated antibody-dependent cellular cytotoxicity (ADCC). These results indicate that DNT cells mediate antitumor ADCC effects through high FcεRIγ expression. Binding of IgG1 to FcεRIγ activates the FcεRIγ/Syk/AKT/NF-κB pathway, consequently enhancing tumor cell killing. Thus, DNT cells may play a significant role in cancer immunity, providing a basis for novel immune cell and antibody combination therapies.
The parathyroid gland, a pivotal organ regulating calcium and phosphorus homeostasis, harbors two primary cell types: chief cells and the enigmatic oxyphil cells. While scarce in healthy individuals, oxyphil cells undergo pronounced proliferation in uremic secondary hyperparathyroidism (SHPT), and their abundance is strongly associated with resistance to first-line therapies like calcitriol and calcimimetics. This correlation underscores a critical clinical challenge, yet the origin, functional role, and mechanisms driving oxyphil cell proliferation have remained poorly understood. Integrated multi-omics studies have decisively illuminated the underlying mechanisms, revealing uremic milieu-driven transdifferentiation from chief cells to oxyphil cells and the pivotal role of mitochondrial biogenesis activation in this process. This paradigm shift redefines oxyphil cells from passive entities to metabolically hyperactive, autonomous units capable of heightening parathyroid hormone synthesis and secretion. The core mechanism of therapy resistance is explained by the profound downregulation of key regulatory receptors, rendering them insensitive to conventional drugs. This review synthesizes current knowledge and, more importantly, highlights how integrated multi-omics approaches are illuminating the pathobiology of oxyphil cells, providing groundbreaking insights into their function, origin, and proliferation mechanisms. We conclude that these advances are pivotal for developing novel therapeutic strategies to overcome treatment resistance in uremic SHPT.
The endoplasmic reticulum lipid raft proteins (Erlins) belong to the stomatin-prohibitin-flotillin-HflC/K (SPFH) family and form highly oligomeric platforms that mediate the degradation of activated inositol 1,4,5-trisphosphate receptors by facilitating their interaction with the E3 ligase RNF170. However, the molecular mechanisms underlying this process remain unclear. Here, we successfully reconstituted the Erlin1-Erlin2 complex and its complex with RNF170 by overexpressing these components in HEK293F cells. We also isolated the Erlin2 oligomer by solely expressing Erlin2 in the cells. Using cryo-electron microscopy, we determined the structures of the Erlin1-Erlin2 complex, the Erlin1-Erlin2-RNF170 complex, and the Erlin2 oligomer at resolutions of 3.29 Å, 3.05 Å, and 2.12 Å, respectively. Both the Erlin1-Erlin2 complex and the Erlin2 oligomer exhibit similar cage-like architectures, with the Erlin1-Erlin2 complex containing 13 pairs of Erlin1 and Erlin2 subunits, whereas the Erlin2 oligomer comprises 26 Erlin2. Although RNF170 was clearly identified during protein purification, it was invisible in the final 3D reconstruction, suggesting a high degree of flexibility between RNF170 and the Erlin complex. Multiple water molecules were identified in the Erlin2 oligomer, underscoring their critical roles in facilitating the high degree of oligomerization of the Erlin2 complex. Taken together, our structural investigation elucidates the molecular basis for the assembly of the Erlin complex and provides a framework for further investigation.
BRG1/BRM-associated factor (BAF)-family chromatin remodelers regulate transcription and genome organization by repositioning nucleosomes. The human non-canonical BAF (ncBAF) complex is uniquely defined by the presence of BRD9 and GLTSCR1/GLTSCR1L, as well as the absence of the ARID1/2 scaffold and the canonical nucleosome-binding module found in cBAF and PBAF. How ncBAF assembles and engages nucleosomes remains elusive. Here, we present a cryo-EM structure of ncBAF bound to a nucleosome, integrated with biochemical assays and crosslinking mass spectrometry analyses. The ncBAF complex adopts a three-module architecture comprising an ATPase motor module, a repositioned actin-related protein (ARP) module, and a highly flexible Base module. The ncBAF-specific subunits BRD9 and GLTSCR1L are largely dispensable for complex assembly and nucleosome remodeling. Instead, BCL7A directly engages the H2A-H2B acidic patch and the H2A N-terminal tail, providing a structural substitute for SMARCB1 in stimulating remodeling activity. These findings reveal how ncBAF compensates for the loss of the canonical nucleosome-binding module through modular reorganization, providing a structural framework for understanding ncBAF-mediated chromatin regulation and its roles in development and disease.
Defective sperm motility is frequently associated with male infertility. The immotile short-tail sperm (ISTS) defect is a rare condition in which most sperm cells have abnormally short tails, impairing their motility and leading to infertility. Despite its implications, the genetic factors related to the ISTS defect remain largely unclear. In this study, we identify coiled-coil domain-containing 112 (CCDC112) as a critical factor for sperm tail formation. CCDC112 can associate with microtubules and interact with multiple centrosomal proteins, including PCM1, TEX9, and taxilin. Disruption of CCDC112 expression profoundly affects the cellular distribution of PCM1. Interestingly, the knockout of Ccdc112 in mice impacts spermiogenesis, resulting in the ISTS defect and ultimately causing male infertility. Furthermore, quantitative proteomic analysis highlights the essential role of CCDC112 in regulating axonemal microtubule inner proteins. We thus characterize CCDC112 as a microtubule-associated protein and substantiate its genetic link to ISTS-related male infertility.
The NLRP3 inflammasome plays a pivotal role in mediating pro-inflammatory cytokine release and inducing pyroptosis. Its aberrant activation is implicated in various inflammatory diseases, including gout, a condition characterized by monosodium urate crystal deposition in the ankle joint. Here, we identify β-alanine, an endogenous amino acid, as a novel NLRP3 inflammasome inhibitor with promising therapeutic potential for gout. Mechanistic investigations reveal that β-alanine binds to NLRP3, sequestering it within the trans-Golgi network. This interaction disrupts NLRP3 inflammasome assembly, thereby inhibiting the secretion of interleukin-1β (IL-1β) and IL-18. Moreover, in vivo experiments demonstrate that β-alanine administration significantly alleviates monosodium urate crystal-induced inflammation and joint swelling in mice without evident toxicity. Collectively, our findings not only uncover a novel endogenous regulatory mechanism for NLRP3-driven inflammation but also position β-alanine as a potential therapeutic candidate for gout.
The intracellular abundance of NAD+, a vital metabolic cofactor, critically influences muscle stem cell (MuSC) function. However, the spatial regulation of NAD+ and its impact on MuSC function remain unclear. In this study, we demonstrated that the loss of miR-183 and miR-96 leads to inefficient skeletal muscle regeneration upon injury and triggers premature differentiation of MuSC-derived primary myoblasts. The underlying mechanism involves miRNA-mediated regulation through targeting SLC25A51, a mitochondrial transporter for NAD+ that elevates mitochondrial NAD+ while reducing cytoplasmic NAD+ levels. Our results suggest that the reduction in cytoplasmic NAD+ diminishes SIRT1-mediated deacetylation, increasing H4K16ac at the promoters of myogenic genes to promote differentiation. Concurrently, the mitochondrial NAD+ accumulation stimulates the tricarboxylic acid cycle, leading to elevated levels of ATP and citrate. These metabolites allosterically activate the ACLY pathway, which in turn increases acetyl-CoA production, thereby supplying acetyl groups for H4K16ac. Furthermore, SIRT3 knockdown impaired myogenic differentiation and attenuated the increased levels of both ATP and acetyl-CoA in miR-183/96-deficient cells, suggesting that the elevated mitochondrial NAD+ also enhances differentiation via SIRT3-mediated regulation of mitochondrial metabolism and acetyl-CoA production. Our work establishes miR-183 and miR-96 as critical regulators of epigenetic-metabolic networks that influence MuSC differentiation through subcellular partitioning of NAD+, ensuring proper regeneration timing.
Cyclin B3 (CCNB3) plays a critical regulatory role in mammalian meiosis. Studies in mice have demonstrated that CCNB3 interacts with CDK1 to modulate the activity of MPF, thereby driving meiotic progression. However, the functional mechanisms of CCNB3 in porcine oocytes remain unclear. In this study, we reveal for the first time that knockdown of CCNB3 in porcine oocytes induces meiotic arrest at metaphase I, accompanied by impaired degradation of cyclin B1 and securin. Further investigation identifies that the antisense long non-coding RNA CCNB3-AS forms a double-stranded RNA (dsRNA) structure with the CCNB3 mRNA, significantly enhancing its stability by resisting PAT1 homolog 1 (PATL1)-mediated degradation. Mechanistically, CCNB3-AS interacts with the scaffold protein Vimentin (VIM). Structural analysis reveals that VIM binds to the PAT1 domain of PATL1 and is capable of influencing the ability of CNOT7, the core subunit of the CCR4-NOT complex, to bind to PATL1, ultimately maintaining stable CCNB3 mRNA expression. Our study elucidates the molecular mechanism by which the CCNB3-AS/CCNB3 dsRNA duplex cooperates with VIM and PATL1 to collectively regulate meiosis in porcine oocytes. Furthermore, we reveal the non-canonical role of VIM in mRNA degradation, providing new theoretical support for understanding the mechanisms underlying porcine oocyte meiosis.
