This study aims to elucidate the protective effects of melatonin against Bisphenol A (BPA) and its metabolite 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP)-induced apoptosis in Leydig cells, focusing on its ability to alleviate oxidative stress and endoplasmic reticulum stress. Results showed that melatonin significantly alleviated testicular damage caused by BPA, increased testicular index in mice, improved the function of seminiferous tubules, and enhanced the survival rate and density of epididymal sperm. Furthermore, melatonin treatment significantly reduced the number and proportion of TUNEL-positive Leydig cells, decreased cleaved caspase-3 expression and the cleaved caspase-3/total caspase-3 ratio, increased testosterone secretion from Leydig cells, and elevated the expression of steroidogenic enzymes cytochrome P450 17α-hydroxylase/20-lyase and 17β-hydroxysteroid dehydrogenase 3. Melatonin also significantly lowered the elevated ROS levels in Leydig cells induced by MBP and enhanced the expression of antioxidant enzymes superoxide dismutase 1/2 and catalase. Additionally, melatonin effectively countered endoplasmic reticulum stress in Leydig cells induced by BPA and MBP, significantly decreasing levels of GRP94 and GRP78, while inhibiting activation of the PERK/EIF2α/ATF4/CHOP signaling pathway. Notably, melatonin significantly alleviated endoplasmic reticulum stress and apoptosis in TM3 cells, and this effect was associated with enhanced SIRT1 nuclear localization and deacetylase activity, as demonstrated by reduced acetylated-p53 levels. Collectively, melatonin effectively protects Leydig cells from BPA and MBP-induced damage by alleviating oxidative stress and endoplasmic reticulum stress through inhibition of the PERK signaling pathway, an effect that may involve SIRT1 activation. It may serve as a therapeutic agent to safeguard male reproductive health against environmental pollutants.
Membrane proteins contain hydrophobic sequences that must be chaperoned during protein folding and transport to the lipid bilayer. Failures in this essential process result in the formation of potentially toxic aggregates. Our mechanistic understanding of how chaperones interact with hydrophobic sequences is limited due to the technical challenges of measuring interactions between aggregation-prone proteins in a detergent-free environment. As hydrophobic substrates are prone to non-specific interactions, traditional binding assays that utilize one substrate and one chaperone per assay struggle to demonstrate that an interaction is specific and physiological. To address these technical challenges, we developed the barcoded binding assay, which allows for the measurement of chaperone binding selectivity to a complex pool of substrates in a detergent-free environment. In this assay, a pool of 35S-labeled barcoded substrates is produced by IVT in the presence of the chaperone of interest, and chaperone selectivity is quantified by immunoprecipitation, SDS PAGE, and autoradiography. We previously used this assay to identify novel substrate binding preferences for the Ubiquilin family of chaperones. Here, we provide an overview of the assay, demonstrate the benefits of the multiplexed barcoded binding assay over a single substrate approach, and provide a detailed protocol for how this assay can be adapted to work with a broad array of chaperones.
Background Knee osteoarthritis (KOA) is a degenerative joint disease characterized by progressive cartilage degradation, in which chondrocyte apoptosis plays a pivotal role. Acupuncture is widely used for KOA management, but its underlying molecular mechanisms remain poorly understood. Endoplasmic reticulum (ER) stress-induced apoptosis via the c-Jun N-terminal kinase (JNK) pathway is a critical driver of chondrocyte death in KOA. This study aimed to investigate whether acupuncture exerts its chondroprotective effects by modulating the ER stress-JNK signaling axis. Methods A rat model of KOA was established by joint immobilization. Fifty rats were randomly assigned to five groups: Normal, Model, Model+Acupuncture (Acu), Model+ Acu+Anisomycin(JNK activator), and Model+SP600125(JNK inhibitor). The therapeutic efficacy of a 4-week acupuncture regimen was evaluated through behavioral scores, radiographic imaging, and histological analysis. Unbiased RNA-sequencing was performed to identify key signaling pathways. The mechanism was further validated through TUNEL staining, Transmission electron microscopy (TEM), Western blot, RTqPCR, immunofluorescence, and ELISA. Results Unbiased transcriptomic analysis identified the ER stress and MAPK/JNK signaling pathways as key targets of acupuncture. Acupuncture significantly improved joint function, alleviated cartilage degradation, and preserved proteoglycan content in KOA rats. Mechanistically, acupuncture suppressed the expression of the ER stress marker IRE1α, inhibited the phosphorylation of JNK, and consequently downregulated the pro-apoptotic Bax/Bcl-2 ratio and Caspase-3 expression. This led to a marked decrease in chondrocyte apoptosis and restoration of ER homeostasis, as confirmed by TUNEL and TEM. Crucially, the anti-apoptotic effects of acupuncture were significantly counteracted by the JNK activator Anisomycin. In addition, acupuncture remodels the systemic inflammatory network by suppressing the production of proinflammatory cytokines IL-1β and TNF-α, which triggers a compensatory upregulation of the anti-inflammatory mediator IL-10. Conclusion Our findings demonstrate for the first time that acupuncture ameliorates knee osteoarthritis by inhibiting chondrocyte apoptosis via suppression of the ER stress-JNK signaling pathway. This study provides a novel and robust mechanistic rationale for the clinical application of acupuncture and highlights the ER stress-JNK axis as a promising therapeutic target for KOA.
In our previous report, PERK/NRF2/CX43/StAR/progesterone pathway activation in ovarian granulosa cells was shown to mediate cold-induced female reproductive disorders. However, how PERK is activated by low temperature remained unclear. In the present study, we found that the circadian protein E4BP4 was significantly upregulated in ovarian granulosa cells following exposure to cold or isoproterenol (ISO), a non-selective β-adrenergic receptor agonist that pharmacologically activates β-adrenergic signaling, a key component of the cold stress response. Mechanistically, E4BP4 interacted with PERK and was required for PERK activation and subsequent NRF2/CX43/StAR signaling, leading to increased progesterone secretion. Interestingly, NRF2 also acted as a transcriptional activator of E4BP4 under ISO treatment, and blocking PERK or NRF2 expression attenuated ISO-induced E4BP4 accumulation, suggesting a positive feedback loop involving PERK/NRF2/E4BP4. Collectively, these findings identify E4BP4 as a cold-responsive circadian protein that interacts with PERK and may contribute to cold-induced reproductive disorders via a bidirectional E4BP4-PERK feedback loop.
Background Epichaperomes are stable, stress-induced supramolecular assemblies formed through extensive integration of molecular chaperones, co-chaperones, signaling proteins, and client proteins. Although increasing evidence indicates that epichaperomes act as organizational hubs that coordinate proteostasis and signaling networks in cancer, neurodegenerative disorders, and chronic inflammatory diseases, their potential physical roles in cellular organization remain largely unexplored. Results Here, the Epichaperome Matrix Theory, a systems-level theoretical framework that conceptualizes the epichaperome as a dynamic, nonequilibrium biomolecular matrix possessing emergent transport-regulatory properties is proposed. In this model, epichaperome assemblies generate heterogeneous electrostatic landscapes through the collective distribution of charged amino acid residues, phosphorylation-dependent charge accumulation, ATP-driven conformational dynamics, and high-order network connectivity. By integrating principles from Poisson-Boltzmann electrostatics, Nernst-Planck transport theory, active matter physics, percolation theory, graph theory, biomolecular condensate thermodynamics, and porous hydrogel transport models, a mathematical description in which epichaperomes function as adaptive organizational scaffolds capable of influencing molecular flux, signaling efficiency, and spatial coordination within cells is developed. This framework is further extended through the Transcellular Epichaperome Continuum Hypothesis, proposing that intracellular epichaperomes may be functionally coupled to plasma membrane-associated and extracellular epichaperome assemblies, forming a multiscale organizational network spanning individual cells, tissues, and organ systems. In this extended model, membrane-bound epichaperomes act as coupling interfaces between intracellular and extracellular compartments, while secreted chaperones, extracellular vesicles, and extracellular protein assemblies contribute to intercellular connectivity. Mathematical analysis predicts the emergence of percolating transport networks, electrostatic coupling domains, synchronized conformational dynamics, and stress-responsive communication pathways when epichaperome connectivity exceeds critical thresholds. Conclusions The proposed framework suggests that epichaperomes may represent more than stress-associated protein interaction networks and could function as dynamic organizational matrices integrating molecular organization, signaling, and adaptive responses across multiple biological scales. Although the theory remains speculative and currently lacks direct experimental validation, it generates testable predictions regarding membrane-associated epichaperomes, extracellular epichaperome assemblies, electrostatic organization, and intercellular transport behaviors. By providing a unified theoretical foundation linking stress biology, chaperone networks, systems biology, and biophysics, this work expands the conceptual landscape of epichaperome research and identifies new directions for investigating the role of higher-order chaperome organization in health and disease.
