Neurotransmitter release requires the precise localization and assembly of the SNARE machinery at presynaptic release sites. Although liquid-liquid phase separation of active zone scaffolds is known to organize these sites, the mechanism for the specific enrichment of the SNARE machinery has remained unclear. In this study, we establish that within RIM1/RIM-BP2 biomolecular condensates, Munc13-1 functions as an organizational hub that spatially sorts and concentrates cognate SNAREs, orchestrating their stepwise assembly. In particular, this condensate environment empowers Munc13-1 to cluster Syb2 into nanodomains on the vesicle membrane, dramatically increasing local SNARE density and ensuring efficient complex formation. Consequently, the introduction of RIM1/RIM-BP2 into PC12 cells enhances spatiotemporally precise dense-core vesicle exocytosis in a Munc13-dependent manner. Our findings suggest a model in which phase separation creates a privileged platform that enables Munc13-1 to direct SNARE complex assembly, thereby ensuring the speed and precision of synaptic vesicle exocytosis.

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease with limited treatment options and poorly understood molecular underpinnings. Dysregulated TGF-β/SMAD signaling is a key driver of fibrotic remodeling, promoting persistent myofibroblast activation and excessive extracellular matrix deposition. Here we identify the Farnesoid X receptor (FXR), a bile acid activated nuclear receptor, as a previously unrecognized suppressor of pulmonary fibrosis. FXR expression is significantly reduced in lung tissues from patients with PF and in myofibroblasts derived from BLM-induced mouse models, correlating inversely with fibrosis severity. Genetic ablation of FXR exacerbates BLM-induced pulmonary fibrosis by promoting fibroblast hyperactivation and dysregulation of the TGF-β/SMAD signaling pathway. Mechanistically, we identify PPP1CB as a previously unrecognized FXR-interacting protein in primary myofibroblasts derived from IPF patients. We further show that FXR both increases chromatin accessibility at the PPP1CB locus and assembles a functional complex with PPP1CB, which in turn promotes SMAD2/3 dephosphorylation and suppresses their nuclear translocation. Notably, the clinical-stage FXR agonist TERN101 exhibits potent therapeutic efficacy in a BLM-induced mouse pulmonary fibrosis model. These findings establish FXR as a critical antifibrotic regulator in lung tissue and suggest that pharmacological activation of FXR may offer a promising therapeutic strategy for IPF.

Abstract The synaptic delivery of the AMPA-type glutamate receptors (AMPARs) is crucial for longterm potentiation (LTP) of excitatory synapses, yet the mechanisms underlying neuronal activity-dependent AMPAR exocytosis at the plasma membrane (PM) remain unclear. We previously demonstrated that the PM synthesis of the phosphoinositide PI4P is enhanced upon LTP induction and that PM PI4P, not PI(4,5)P 2 , is required for activity-induced AMPAR exocytic trafficking. Here, we show that AMPARs are exocytosed at PI4P-enriched dendritic PM microdomains in potentiated hippocampal neurons. The Q-SNARE SNAP47 binds PI4P via its pleckstrin homology (PH)-like domain. This interaction recruits SNAP47 to the PM, promoting the exocytic fusion of AMPAR vesicles through the SNAP47–Syntaxin-3–VAMP2 SNARE complex. In the hippocampus, the SNAP47–PI4P interaction is necessary for both LTP and long-term memory. Our findings reveal a mechanistic role for PI4P in mediating activity-dependent, SNARE-driven fusion of AMPAR exocytic vesicles with the PM.

The prevalence of prediabetes among adults in the U.S. is three times higher than that of diabetes, highlighting a greater disease burden. Both diabetes and prediabetes have been demonstrated to be associated with an increased risk of cardiovascular disease (CVD). However, research has primarily focused on diabetes, with limited attention to CVD risk prediction in prediabetes. Emerging 13 metabolic health-related indicators have been proposed to optimize the predictive effect on CVD risk in patients with prediabetes. This study aimed to compare the predictive efficacy of these biomarkers and further develop a nomogram to improve predictive performance of the CVD risk in patients with prediabetes. All eligible participants in the National Health and Nutrition Examination Survey (NHANES) 1999–2020 were enrolled in this study and randomly assigned to the development and validation cohorts in a ratio of 7:3. In the development cohort, the efficacy of 13 indicators used to predict the CVD risk was assessed by receiver operative characteristic (ROC) curves. Independent risk predictors identified by multivariate logistic regression were used to construct a nomogram, and internal and external validation were further implemented. The ROC curve demonstrated that the triglyceride-glucose (TyG) index was an effective predictor of CVD risk [area under the curve (AUC) = 0.694] and exhibited the best predictive performance among the 13 metabolic health-related indices. Based on independent risk factors identified by multivariate logistic regression, the CVD risk nomogram [including age, gender, hypertension, TyG, stress hyperglycemia ratio (SHR), and neutrophil-to-lymphocyte ratio (NLR)] was successfully constructed and validated with good performance (AUCs/C-indexes > 0.70 for all). This study developed a reliable nomogram for predicting CVD risk in patients with prediabetes. The model demonstrated robust performance and offered a simple yet individualized approach for predicting the CVD risk in patients with prediabetes.

Synaptotagmins (Syts) are the primary Ca2+-sensors for synaptic vesicle exocytosis, while most mammalian Syts are non-Ca2+-affinitive and play critical roles in neurotransmission and synaptic plasticity with unclear mechanisms. Here, we show that high-alkaline non-Ca2+-binding Syt11 exhibits higher affinity for acidic phospholipids and Ca2+-inhibited liposome-binding, thereby competing with the Ca2+-binding Syt1. Physiological levels of Ca2+ eliminate this competition by promoting Ca2+-dependent membrane insertion of Syt1 while suppressing Syt11's binding through electrostatic shielding of the membrane surface. Site-directed mutagenesis reveals a dual-regional lipid-binding mode (a lysine-rich motif for Ca2+-independent binding and Ca2+-binding loops for Ca2+-facilitation) for Syt1, and a redundant multi-point lipid-binding interface for Syt11. Consistent with the Ca2+-dependent competition, Syt11 inhibits both the early stages of exocytosis and endocytosis in neurons, while the maximal rate of exocytosis remains intact. This Ca2+-sensitivity of Syt11 proposes Syt1-Syt11 inter-switching in membrane-occupancy as a critical step precisely controlling exocytosis and endocytosis during synaptic transmission.

The balance between synaptic excitation and inhibition (E/I) is essential for coordinating motor behavior, yet the differential roles of exocytosis regulators in this balance are less understood. In this study, we investigated the roles of 2 conserved exocytosis regulators, complexin/CPX-1 and CAPS/UNC-31, in excitatory versus inhibitory synapses at Caenorhabditis elegans neuromuscular junctions. cpx-1 null mutants exhibited a marked increase in spontaneous release specifically at excitatory synapses, alongside an unequal reduction in excitatory and inhibitory evoked release. A clamping-specific knockin mutant, cpx-1(Δ12), which preserved evoked release, also showed a biased enhancement in excitatory spontaneous release. Conversely, the unc-31 null mutation, while maintaining normal spontaneous release, displayed a more pronounced reduction in evoked release at excitatory synapses. Notably, we found that CPX-1's clamping function is dependent on UNC-31 and is sensitive to external Ca2+. Pull-down experiments confirmed that CAPS/UNC-31 does not directly interact with complexin, implying an indirect regulatory mechanism. Moreover, complexin regulates activity-dependent synaptic plasticity, which is also UNC-31 dependent. The unexpected role of CAPS/UNC-31 in the absence of CPX-1 clamping function may underpin the synaptic E/I balance and coordinated behavioral outputs in different species.

Munc13 family proteins are crucial for the secretion of neurotransmitters and hormones necessary for cell communication. They share a conserved C-terminal region that includes C 2 and the MUN domains, which facilitate membrane interactions and the assembly of soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes. Neuronal isoforms of Munc13 possess a variable N-terminal region that is essential for neurotransmitter release and short-term plasticity, although the precise functions of this region remain not fully understood. Here, we identified a negatively charged sequence within the N terminus of Munc13-1, termed polyE, which is specific to Munc13-1 among all Munc13 isoforms and potentially derived from a common ancestor of homeotherms. We found that polyE binds significantly to the MUN domain through charge–charge interactions, inhibiting MUN activity in promoting SNARE complex assembly. Disrupting the polyE–MUN interaction by introducing pseudophosphorylated mutations in the MUN domain alleviates this inhibition, thereby enhancing neurotransmitter release. Strikingly, Ca 2+ ions exhibit significant binding to polyE. We found that 40 μM of Ca 2+ adequately competes with the polyE–MUN interaction to reduce polyE inhibition. This concentration is comparable to presynaptic local [Ca 2+ ] i triggered by a single action potential. Taken together, these results indicate an autoinhibition conformation of Munc13-1 mediated by the polyE–MUN interaction. In addition, the relief of this autoinhibition conformation of Munc13-1 by presynaptic Ca 2+ influx and/or posttranslational modifications in the MUN domain may underlie Munc13-1 function in neurotransmitter release and short-term plasticity.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin mediates neuronal exocytosis and self-assembles into large clusters in the plasma membrane. The formation and function of these clusters, and whether they promote or inhibit synaptic-vesicle fusion, remain unclear. Here using optogenetic control of syntaxin clustering in vitro and in vivo, as a light-inducible gain-of-function assay, we show that light-enhanced clustering reduces both spontaneous and triggered vesicle fusion, and this impairs mouse hunting behavior. Cluster formation is induced by liquid-liquid phase separation (LLPS) of the SNARE domain of syntaxin. For the regulatory mechanism, Munc18, which is known to alter syntaxin conformation, acts to reduce LLPS for cluster formation, thereby promoting active syntaxin. These results suggest that exocytosis regulation involves LLPS-induced syntaxin clusters that serve as a syntaxin reservoir from which Munc18 captures syntaxin monomers to form a syntaxin-Munc18 complex, setting the stage for efficient fusion.

Munc13 plays a crucial role in short-term synaptic plasticity by regulating synaptic vesicle (SV) exocytosis and neurotransmitter release at the presynaptic terminals. However, the intricate mechanisms governing these processes have remained elusive due to the presence of multiple functional domains within Munc13, each playing distinct roles in neurotransmitter release. Here we report a coordinated mechanism in the C. elegans Munc13 homolog UNC-13 that controls the functional switch of UNC-13 during synaptic transmission. Mutations disrupting the interactions of C1 and C2B with diacylglycerol (DAG) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) on the plasma membrane induced the gain-of-function state of UNC-13L, the long UNC-13 isoform, resulting in enhanced SV release. Concurrent mutations in both domains counteracted this enhancement, highlighting the functional interdependence of C1 and C2B. Intriguingly, the individual C1 and C2B domains exhibited significantly stronger facilitation of SV release compared to the presence of both domains, supporting a mutual inhibition of C1 and C2B under basal conditions. Moreover, the N-terminal C2A and X domains exhibited opposite regulation on the functional switch of UNC-13L. Furthermore, we identified the polybasic motif in the C2B domain that facilitates SV release. Finally, we found that disruption of C1 and C2B membrane interaction in UNC-13S, the short isoform, leads to functional switch between gain-of-function and loss-of-function. Collectively, our findings provide a novel mechanism for SV exocytosis wherein UNC-13 undergoes functional switches through the coordination of its major domains, thereby regulating synaptic transmission and short-term synaptic plasticity.

Munc13 plays a crucial role in short-term synaptic plasticity by regulating synaptic vesicle (SV) exocytosis and neurotransmitter release at the presynaptic terminals. However, the intricate mechanisms governing these processes have remained elusive due to the presence of multiple functional domains within Munc13, each playing distinct roles in neurotransmitter release. Here, we report a coordinated mechanism in the Caenorhabditis elegans Munc13 homolog UNC-13 that controls the functional switch of UNC-13 during synaptic transmission. Mutations disrupting the interactions of C1 and C2B with diacylglycerol (DAG) and phosphatidylinositol 4,5-bisphosphate (PIP2) on the plasma membrane induced the gain-of-function state of UNC-13L, the long UNC-13 isoform, resulting in enhanced SV release. Concurrent mutations in both domains counteracted this enhancement, highlighting the functional interdependence of C1 and C2B. Intriguingly, the individual C1 and C2B domains exhibited significantly stronger facilitation of SV release compared to the presence of both domains, supporting a mutual inhibition of C1 and C2B under basal conditions. Moreover, the N-terminal C2A and X domains exhibited opposite regulation on the functional switch of UNC-13L. Furthermore, we identified the polybasic motif in the C2B domain that facilitates SV release. Finally, we found that disruption of C1 and C2B membrane interaction in UNC-13S, the short isoform, leads to functional switch between gain-of-function and loss-of-function. Collectively, our findings provide a novel mechanism for SV exocytosis wherein UNC-13 undergoes functional switches through the coordination of its major domains, thereby regulating synaptic transmission and short-term synaptic plasticity.

Autophagy, a conserved catabolic process implicated in a diverse array of human diseases, requires efficient fusion between autophagosomes and lysosomes to function effectively. Recently, SNAP47 has been identified as a key component of the dual-purpose SNARE complex mediating autophagosome-lysosome fusion in both bulk and selective autophagy. However, the spatiotemporal regulatory mechanisms of this SNARE complex remain unknown. In this study, we found that SNAP47 undergoes acetylation followed by deacetylation during bulk autophagy and mitophagy. The acetylation status of SNAP47 is regulated by the acetyltransferase CBP and the deacetylase HDAC2. Notably, the spatiotemporal regulatory dynamics of SNAP47 acetylation differ between bulk autophagy and mitophagy due to distinct regulation on the activity of acetyltransferase and deacetylase. Acetylated SNAP47 inhibits autophagosome-lysosome fusion by indirectly impeding SNARE complex assembly. Mechanistically, deacetylated SNAP47 recruits HOPS components to autophagic vacuoles independently of STX17 and STX17-SNAP47 interaction, while acetylated SNAP47 inhibits this recruitment, consequently leading to the failure of SNARE complex assembly. Taken together, our study uncovers a SNAP47 acetylation-dependent regulatory mechanism governing autophagosome-lysosome fusion by modulating the recruitment of HOPS to autophagic vacuoles without involving STX17, SNAP47-STX17 interaction and ternary SNARE complex formation.

Tumor-derived extracellular vesicles primarily carry PD-L1 via exosomes, which interact with PD-1 receptors on T cells, impacting immune responses in the tumor microenvironment and beyond, leading to a more extensive immunosuppressive landscape. However, the mechanisms governing exosomal PD-L1 sorting and secretion remain elusive. In this study, we identified Munc13-4 as a crucial regulator of exosomal PD-L1 sorting and secretion. Deletion of Munc13-4 in breast tumors enhances T cell-mediated anti-tumor immunity, suppresses tumor growth, and improves the efficacy of immune checkpoint inhibitors. Our results illustrate how Munc13-4 collaborates with HRS, Rab27, and SNAREs to facilitate PD-L1 sorting and secretion via exosomes. The cryo-EM structure of the Munc13-4–Rab27a complex provide new insights into its role in exosome secretion. Importantly, we discovered that Munc13-4 has a novel role in sorting PD-L1 onto exosomes, which relies on the formation of a ternary complex with PD-L1 and HRS. In addition, IFNγ stimulation modifies Munc13-4 and HRS, establishing a dynamic regulatory mechanism that enables tumor cells to adapt to immune pressure by modulating PD-L1 sorting. Using a specially designed peptide to disrupt the Munc13-4–PD-L1 interaction and impede PD-L1 sorting significantly enhances anti-tumor immunity and slows tumor growth in vivo. These results highlight the potential of targeting the Munc13-4–PD-L1 axis to suppress tumor immune evasion.

Autophagy, an essential process in eukaryotic cells, entails the sequestration and degradation of cytosolic components and organelles following fusion with the lysosome or vacuole. Autophagy-related protein 18 (Atg18), a key autophagy-related protein, binds phosphatidylinositol-3-phosphate (PI3P) to localize to autophagosomal membranes, where it recruits Atg2 to mediate lipid transfer during autophagosome biogenesis. Although the roles of Atg18 in autophagy are well established, whether this protein exerts additional regulatory functions in this process remains to be elucidated. Here, we report the weak interactions between Atg18 and Atg8 or Atg16 mediated by the Atg8-interacting motif (AIM) within Atg18. Disruption of the AIM in Atg18 leads to reduced autophagosome formation and diminished autophagic activity. Moreover, we demonstrate that Atg18 is involved in the recruitment of Atg8 to the autophagosome and facilitates the C-terminal cleavage of Atg8 by Atg4. Furthermore, the Atg18-Atg8 complex can be dissociated by Atg3, enabling free Atg18 to subsequently recruit Atg16 to the autophagosome, preparing for Atg8 lipidation. Thus, our findings unveil previously unknown roles for Atg18 in downstream factor recruitment and Atg4 cleavage during autophagosome formation via its AIM.

OBJECTIVE:Nuclear import/export of HIV-1 is regulated by the nuclear pore complexes (NPCs), but the impact of many individual nucleoporins on viral infection is unclear. Here, we investigated the role of a transmembrane nucleoporin Nup210 in HIV-1 infection. DESIGN/METHODS:TZM-bl cells with Nup210-knockdown or overexpression were infected with either wildtype HIV-1 NL4-3 or VSV-G pseudotyped NL4-3-KFS. The efficiency of viral infection was assessed by measuring luciferase activity. The DNA levels of reverse transcription, nuclear entry, and proviral DNA integration were determined by qPCR. The levels of unspliced, singly spliced, and fully spliced mRNA were determined by RT-qPCR. The levels of viral key proteins were determined by western blotting. The viral DNA, mRNA, and protein assays were also performed in Raltegravir-treated Nup210-knockdown or overexpression cells. RESULTS:Generally, Nup210-knockdown promoted HIV-1 infection, whereas Nup210-overexpression had no significant effect on entire infection. Several findings were obtained in further investigations. First, Nup210-knockdown increased the accumulation of integrated proviral DNA, while the levels of reverse transcription (RT) products and 2-LTR circles remained unaffected by either Nup210-knockdown or overexpression. Second, Nup210 regulated viral mRNA alternative splicing, particularly, Nup210-knockdown resulted in the highest increase in singly spliced Vpr mRNA, whereas Nup210-overexpression led to the biggest rise in unspliced Gag mRNA. Third, Vpr expression was elevated by Nup210-knockdown, suggesting that Vpr may act as a viral antagonist of Nup210. Additionally, Raltegravir, together with Nup210, inhibit viral infection by interfering proviral DNA integration, subsequent transcription and translation. CONCLUSION:The endogenous Nup210 is sufficient to suppress HIV-1 infection by downregulating late steps of viral nuclear entry.

Tumor-derived exosomes carry programmed death-ligand 1 (PD-L1), which binds programmed cell death protein 1 (PD-1) on T cells, suppressing immune responses locally and systemically. However, the mechanisms governing exosomal PD-L1 sorting and secretion remain elusive. Here, we identify Munc13-4 as a crucial regulator of this process. Deletion of Munc13-4 in breast tumors enhances T cell-mediated anti-tumor immunity, suppresses tumor growth, and improves the efficacy of immune checkpoint inhibitors. Mechanistically, Munc13-4 collaborates with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), Rab27, and SNAREs to facilitate PD-L1 sorting and secretion via exosomes. Cryogenic electron microscopy (cryo-EM) analysis of the Munc13-4-Rab27a complex provide structural insights into exosome secretion. Importantly, PD-L1 sorting relies on a ternary complex composed of Munc13-4, PD-L1 and HRS, which is regulated by interferon gamma (IFNγ) signaling. A designed peptide that disrupts Munc13-4-PD-L1 interaction impedes PD-L1 sorting, enhances antitumor immunity, and suppresses tumor growth, highlighting the therapeutic potential of targeting this pathway.

Despite medical interventions, the regenerative capacity of the peripheral nervous system is limited. Dorsal root ganglion (DRG) neurons possess the capacity to detect mechanical signals from their microenvironment, but the impact and mechanism by which these signals regulate axon regrowth and even regeneration in DRG neurons remain unclear. In this study, DRG neurons from newborn rats are cultured on substrates with varying degrees of stiffness in vitro to investigate the role of mechanical signals in axon regrowth. The findings reveal that substrate stiffness plays a crucial role in regulating axon regrowth, with an optimal stiffness required for this process. In addition, the data demonstrate that Piezo1, a mechanosensitive cation channel, detects substrate stiffness at the growth cone and regulates axon regrowth through activating downstream Ca2+-CaMKII-FAK-actin cascade signaling pathway. Interestingly, knocking down Piezo1 in adult rat DRG neurons leads to enhanced axon regeneration and accelerated recovery of sensory function after sciatic nerve injury. Overall, these findings contribute to the understanding of the role of mechanical signals in axon regeneration and highlight microenvironmental stiffness as a promising therapeutic target for repairing nerve injuries.

Physical exercise has beneficial effect on anxiety disorders, but the underlying molecular mechanism remains largely unknown. Here, it is demonstrated that physical exercise can downregulate the S-nitrosylation of gephyrin (SNO-gephyrin) in the basolateral amygdala (BLA) to exert anxiolytic effects. It is found that the level of SNO-gephyrin is significantly increased in the BLA of high-anxiety rats and a downregulation of SNO-gephyrin at cysteines 212 and 284 produced anxiolytic effect. Mechanistically, inhibition of SNO-gephyrin by either Cys212 or Cys284 mutations increased the surface expression of GABAAR γ2 and the subsequent GABAergic neurotransmission, exerting anxiolytic effect in male rats. On the other side, overexpression of neuronal nitric oxide synthase in the BLA abolished the anxiolytic-like effects of physical exercise. This study reveals a key role of downregulating SNO-gephyrin in the anxiolytic effects of physical exercise, providing a new explanation for protein post-translational modifications in the brain after exercise.

At the synapse, presynaptic neurotransmitter release is tightly controlled by release machinery, involving the soluble N -ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins and Munc13. The Ca 2+ sensor Doc2 cooperates with Munc13 to regulate neurotransmitter release, but the underlying mechanisms remain unclear. In our study, we have characterized the binding mode between Doc2 and Munc13 and found that Doc2 originally occludes Munc13 to inhibit SNARE complex assembly. Moreover, our investigation unveiled that EphB2, a presynaptic adhesion molecule (SAM) with inherent tyrosine kinase functionality, exhibits the capacity to phosphorylate Doc2. This phosphorylation attenuates Doc2 block on Munc13 to promote SNARE complex assembly, which functionally induces spontaneous release and synaptic augmentation. Consistently, application of a Doc2 peptide that interrupts Doc2-Munc13 interplay impairs excitatory synaptic transmission and leads to dysfunction in spatial learning and memory. These data provide evidence that SAMs modulate neurotransmitter release by controlling SNARE complex assembly.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAER) family proteins are the engines of most intra-cellular and exocytotic membrane fusion pathways (Jahn and Scheller 2006). Over the past two decades, in-vitro liposome fusion has been proven to be a powerful tool to reconstruct physiological SNARE-mediated membrane fusion processes (Liu et al. 2017). The reconstitution of the membrane fusion process not only provides direct evidence of the capability of the cognate SNARE complex in driving membrane fusion but also allows researchers to study the functional mechanisms of regulatory proteins in related pathways (Wickner and Rizo 2017). Heretofore, a variety of delicate methods for in-vitro SNARE-mediated liposome fusion have been established (Bao et al. 2018; Diao et al. 2012; Duzgunes 2003; Gong et al. 2015; Heo et al. 2021; Kiessling et al. 2015; Kreye et al. 2008; Kyoung et al. 2013; Liu et al. 2017; Scott et al. 2003). Although technological advances have made reconstitution more physiologically relevant, increasingly elaborate experimental procedures, instruments, and data processing algorithms nevertheless hinder the non-experts from setting up basic SNARE-mediated liposome fusion assays. Here, we describe a low-cost, timesaving, and easy-to-handle protocol to set up a foundational in-vitro SNARE-mediated liposome fusion assay based on our previous publications (Liu et al. 2023; Wang and Ma 2022). The protocol can be readily adapted to assess various types of SNARE-mediated membrane fusion and the actions of fusion regulators by using appropriate alternative additives (e.g., proteins, macromolecules, chemicals, etc.). The total time required for one round of the assay is typically two days and could be extremely compressed into one day.