Heat shock proteins (HSPs) are essential molecular chaperones that maintain protein homeostasis and protect cells under stress. Their extracellular functions have emerged as pivotal in cancer biology. Extracellularly released HSPs are selectively packaged through stress-responsive pathways and exhibit context-dependent effects within the tumor microenvironment. They can modulate immune cell functions, act as danger signals, and promote immune activation or suppression. Clinically, extracellular vesicle (EV)-HSPs demonstrate potential as diagnostic and prognostic biomarkers. It has been shown that they can outperform their soluble counterparts due to enhanced stability and tumor specificity. This mini-review aims to highlight findings related to the roles of EV-HSPs and their potential applications.
The phenylalanine hydroxylase (PAH) pathway is an enzymatic pathway focused primarily on the hydroxylation of the amino acid L-phenylalanine (Phe) to L-tyrosine (Tyr). However, the functioning of this pathway can also have significant impacts on other downstream metabolites. In addition, both common and rare genetic variants have been demonstrated to significantly impair PAH pathway function. However, to our knowledge, the comprehensive PAH pathway including influences on related downstream metabolic pathways has not been mapped in an integrative manner. As such, we conducted a narrative review of the literature to describe the comprehensive PAH pathway, including influences on downstream related metabolic pathways, while mapping a visual depiction of these integrative processes. In addition to the hydroxylation of Phe to Tyr, the comprehensive pathway narrative describes Phe metabolism and transport across the blood brain barrier including competition with other large neutral amino acids, Tyr catabolism, and others. Discussion of the importance of these metabolic processes in the context of PAH genetic variation are also provided. With an increased focus on conducting health research from a systems biology perspective, this work is critical to improving our understanding of the downstream effects of amino acid metabolism and their possible impacts on health outcomes.
High-density lipoprotein (HDL) functionality has emerged as a key determinant of residual cardiovascular risk despite advances in LDL-cholesterol lowering therapies. Small HDL particles, generated through ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux, are particularly effective in promoting cholesterol removal from macrophage foam cells and are associated with reduced atherosclerotic risk. Probucol, a lipid-lowering drug, inhibits ABCA1-dependent HDL biogenesis, whereas its major metabolite, 3,3',5,5'-tetra-tert-butyldiphenoquinone (DQ), preserves the generation of small HDL particles. However, the molecular basis for these distinct effects has remained unclear. To investigate the structural mechanisms underlying ABCA1-mediated lipid export, we performed molecular docking analyses using the human ABCA1 structure (PDB: 5XJY) and the GOLD in silico docking platform. Cholesterol and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) docking poses were successfully identified in DQ-bound ABCA1. In contrast, no ligand-binding poses were detected in probucol-bound ABCA1. These findings suggest that DQ preserves the lipid-recognition and transport properties of ABCA1, whereas probucol disrupts ligand accommodation within the transporter. This study provides the first atomic-level insight into the interactions among ABCA1, cholesterol, probucol, and DQ, offering a structural explanation for their distinct effects on HDL biogenesis and identifying a potential framework for developing novel HDL-targeted therapeutics.
Hypertrophic scars (HS) represent pathological outcomes of traumatic or surgical injuries with dysregulated wound healing. We investigated the therapeutic effects and underlying mechanism of autologous chyle fat (ALCF) in HS formation. A thermal injury-induced burn wound model was established in mice. Histopathological alterations, fibrotic deposition, the levels of α-smooth muscle actin (α-SMA), collagen I and collagen III were quantified. To elucidate the mechanism, tumor necrosis factor-stimulated gene-6 (TSG-6) was silenced, and TSG-6, binding immunoglobulin protein (Bip), C/EBP homologous protein (CHOP), inositol-requiring enzyme 1α (IRE1α), p-IRE1α, c-Jun N-terminal kinase (JNK), and p-JNK were assessed to evaluate endoplasmic reticulum stress (ERS) and downstream signaling. The JNK signaling pathway was pharmacologically activated to validate the IRE1α/JNK axis. Clinically, ALCF injection yielded favorable outcomes. ALCF markedly alleviated fibrosis in burn wound scar tissues and reduced Bip and CHOP, indicating ERS attenuation. ALCF upregulated TSG-6 expression in healing wound tissues, whereas TSG-6 knockdown partially abolished the antifibrotic effects of ALCF. ALCF suppressed IRE1α phosphorylation and JNK activation. Reactivation of the IRE1α/JNK pathway reversed the inhibitory effects of ALCF on ERS and promoted fibrotic deposition. Collectively, ALCF attenuates burn wound fibrosis through TSG-6-mediated suppression of ERS, associated with reduced IRE1α phosphorylation and JNK signaling inhibition.
The alpha subunit of mitochondrial trifunctional protein (αTFP) catalyzes the acylation of monolysocardiolipin (MLCL) to cardiolipin (CL). We determined whether the beta subunit of mitochondrial trifunctional protein (βTFP) impacted the ability of αTFP to promote CL resynthesis from MLCL. Purified recombinant αTFP, but not βTFP, exhibited acylation of MLCL to CL with [1-14C]linoleoyl-coenzyme A. Incubation of both αTFP and βTFP together did not alter the ability of αTFP to catalyze the acylation of MLCL to CL with [1-14C]linoleoyl-coenzyme A. HeLa cells were transfected with recombinant αTFP, βTFP, or both, then incubated with [1-14C]linoleate, and radioactivity incorporated into CL was determined. Expression of αTFP increased [1-14C]linoleate incorporation into CL 2-fold (p < 0.05), whereas expression of βTFP did not affect [1-14C]linoleate incorporation into CL. Expression of both αTFP and βTFP together increased [1-14C]linoleate incorporation into CL to an identical level to that achieved with αTFP alone. Barth syndrome (BTHS) patient lymphoblasts, which exhibit elevated MLCL and reduced CL, were incubated with βTFP inhibitory RNA, and CL mass was determined. Incubation of BTHS lymphoblasts with βTFP inhibitory RNA increased CL mass 1.5-2-fold (p < 0.05). We hypothesize that the βTFP may regulate CL resynthesis in BTHS lymphoblasts.
The direct binding of cAMP to the hyperpolarization-activated cyclic nucleotide-gated channel form 4 (HCN4) protein contributes to autonomic modulation of heart rate. Here, we determine affinity for binding of cyclic nucleotides directly to the cytosolic C-terminus of the HCN4 channel of three disease-linked mutations that are found in the C-linker of the C-terminus, which attaches the more distal binding domain to the pore. Two of these mutations, one in the A'-helix (K530N) and another in the B'-helix (D553N), reduced binding affinity but did not alter the negatively cooperative pattern of binding found in the wild type channel. Finally, a third mutation in the A'-helix (R524Q) did not alter either the pattern or affinity of binding to the cyclic nucleotides despite a dramatic, and previously reported, increase in cAMP potency in the full channel. Our data may be explained by location of each mutation and its interactions with domains external to this region. The data support negatively cooperative binding of cAMP and cGMP to HCN4 and help to identify distinct structural contributions to ligand binding and gating, both of which determine potency.
We aimed to explore the effect and the mechanism of PPP2R2B on cell proliferation, migration, and ferroptosis in breast cancer cells. By bioinformatic analysis and data mining, PPP2R2B was associated with the prognosis and ferroptosis of breast cancer. Breast cancer cells were transfected with PPP2R2B overexpression or PPP2R2B knockdown plasmids, and cell proliferation, apoptosis, migration, and invasion were examined. Moreover, transfected breast cancer cells were treated with ferroptosis inducer, and the relative iron level, Fe2+ level, MDA level, lipid ROS level, MitoSOX intensity, and fluorescence intensity were detected. Protein expressions of GPX4, ACSL4, and SLC7A11 were evaluated by Western blot. The effect of PPP2R2B on JAK2-STAT3 signaling was studied. Finally, the tumor xenograft model in nude mice was constructed to study the PPP2R2B overexpression on tumor growth in vivo. PPP2R2B was down-regulated in the breast cancer tissues and predicted poor prognosis. PPP2R2B inhibited cell proliferation and induced cell apoptosis. PPP2R2B suppressed cell migration and invasion of MCF-7 and MDA-MB-231 cells. PPP2R2B promoted erastin-induced ferroptosis in breast cancer cells and regulated the expression of GPX4, ACSL4, and SLC7A11. In addition, PPP2R2B inhibited the phosphorylation of JAK2 and STAT3. PPP2R2B also inhibited tumor growth in vivo. PPP2R2B targets the JAK2-STAT3 signaling pathway to regulate cell proliferation, apoptosis, migration, invasion, and ferroptosis in breast cancer cells.
CREB binding protein (CBP) and its homolog E1A binding protein p300 (p300) are crucial lysine acetyltransferases that have been shown to play key roles in various human malignancies. However, the precise functions and regulatory mechanisms of CBP/p300 in hepatocellular carcinoma (HCC) remain to be explored. Herein, we report that the expression levels of CBP and p300 are upregulated in HCC compared to normal tissues, and high CBP expression is associated with poor overall survival based on TCGA data. Combined knockdown of CBP and p300 or treatment with the CBP/p300 histone acetyltransferases (HAT) inhibitor A-485 reduces cell viability and promotes cell death in HCC cell lines. Additionally, inhibition of CBP/p300 activates autophagy, which mediates cell death in HCC cells. Mechanistically, the inhibition of CBP/p300 HAT activity attenuates H3K27ac at Ephrin type-A receptor 2 (EPHA2) super-enhancer and suppresses the expression of EPHA2. EPHA2 overexpression can partially rescue CBP/p300 HAT inhibition-mediated autophagy and loss of cell viability. These findings reveal a novel epigenetic mechanism of CBP/p300 in HCC and identify the CBP/p300 inhibitor A-485 as a promising therapeutic candidate for HCC.
Somatic cell-based reproductive technologies (SCRTs) offer a promising approach for preserving the genetic diversity of threatened species. SCRTs require the nuclear reprogramming of a donor somatic cell, which has low success rates (1%-5%) and can vary significantly between individuals and cell lines derived from the same individual. Somatic and pluripotent cells differ in several key characteristics, and donor cells presenting more pluripotent-like traits have been found to result in improved reprogramming outcomes. Here, we characterized 18 standardized fibroblast cell lines from six Angus bulls for several of these characteristics at the inter- and intra-individual levels: bioenergetic status, targeted metabolite abundance, global DNA methylation levels, and presence of key histone modifications. Differences in the metaboloepigenetic profiles of these cell lines were observed at both inter- and intra-individual levels. By integrating these data, we positioned cell lines along a somatic-to-pluripotent-like continuum and identified a reduced set of features that captured the dominant separation between profiles. Although functional reprogramming outcomes were not assessed here, these results provide a data-driven framework to prioritise donor cell lines for prospective induced pluripotent stem cell/somatic cell nuclear transfer validation and to guide the development of practical biomarker panels for SCRT workflows.
S-palmitoylation is a reversible post-translational modification that adds palmitic acid onto cysteine residues of proteins through the formation of a thioester bond. The reaction is catalyzed by protein acyl transferases and reversed by acyl protein thioesterases, also known as S-depalmitoylases. Here, we optimized acyl resin-assisted capture (acyl-RAC) for identifying S-palmitoylated proteins in the model eukaryote Dictyostelium discoideum. Using this optimized protocol and Western blotting, we revealed S-palmitoylated proteins in D. discoideum including calcium-dependent cell adhesion protein A, calreticulin, and glucose-regulated protein 78. Overall, this work establishes acyl-RAC as a tool for studying S-palmitoylation in D. discoideum and reveals a subset of S-palmitoylated proteins in this model organism that can be further studied.
Caspases are well recognized and studied as the central executioners of apoptosis, while recent studies have unraveled their diverse roles beyond cell apoptotic function. One emerging area of interest is nonapoptotic caspase activity as a potential regulator in lipid metabolism. This review explores the differential nonapoptotic role of caspases in lipid metabolic process, encompassing the influence of caspases on lipid reprogramming, lipid synthesis, degradation, transport, and signaling. We discuss the mechanisms underlying these interactions, their relevance to physiological processes, and their implications for various diseases. This reveals a hidden layer of regulation where the machinery of cell death is repurposed to control the fundamental processes of fat handling in living animal. Dysregulation of this system forms a vicious cycle that propagates cellular dysfunction, directly contributing to the pathogenesis of major human diseases in aging, including redox stress, nonalcoholic steatohepatitis, type 2 diabetes, neurodegenerative disorders, and cancer. Consequently, the caspase-metabolism axis emerges as a compelling therapeutic frontier. However, the clinical translation of caspase modulators is challenged by the context-dependent duality of caspase functions. Future therapeutic strategies must therefore advance beyond pan-inhibition toward the precise, context-specific modulation of discrete caspase-mediated metabolic events to halt disease progression effectively.
Microglia, the primary immune cells of the brain parenchyma, play critical roles in neurodevelopment and homeostasis. The human microglia clone 3 (HMC3) cell line has long been a staple in vitro tool for exploring microglia dynamics. However, recent evidence suggests that HMC3 cells behave less like natural microglia and more like pericytes and astrocytes. Until a large-scale shift in replacing HMC3 cells with alternative in vitro models occurs, it is important to standardize characterization techniques used to study HMC3 polarization dynamics to enhance repeatability of findings. Here, we characterized the morphological, cellular, and molecular responses of HMC3 cells in response to 10 and 50 ng/mL interferon-gamma (IFN-γ) treatment. We created an HMC3-specific morphology atlas, conducted manual and automated morphology assessments, quantified live and dead cell counts, and assessed mitochondrial output and the accumulation of reactive oxygen species. We also quantified transcript and protein abundance of candidate markers of chemokine secretion, cytokine signaling, endosomal processes, and cytoprotective responses. We presented detailed methodology on cell culturing, treatments, target selection, internal reference controls, primer design, validation, testing, and quantification to enhance the reproducibility of the data. Our study will increase the rigor of target selection and shed light on the unique polarization dynamics of HMC3 cells.
This study reports the p-toluenesulfonic acid-catalyzed synthesis of three novel 3,4-dihydropyrimidin-2(1H)-one derivatives (4a = PMe, 4b = PPh, 4c = PNO2) based on a 1,1'-biphenyl, and the evaluation of their cytotoxic, genotoxic, antioxidant, and apoptosis-related effects in vitro and in vivo. Structural characterization was performed using FT-IR, 1H NMR, APT-13C NMR, and LC/MS-MS. Cytotoxicity was assessed on A549 human lung cancer and MRC-5 normal lung fibroblast cells using the WST-8 assay, while genotoxicity was evaluated using the Drosophila wing SMART assay. qRT-PCR was conducted to analyze gene expression related to cellular stress responses, antioxidant defense, and apoptosis in both cell lines and Drosophila melanogaster. All compounds exhibited dose-dependent cytotoxicity toward A549 cells with low toxicity in MRC-5 cells, indicating selective anticancer activity. Compound 4b showed the strongest cytotoxic effect (highest potency), whereas compound 4c exhibited the highest selectivity index (SI = 1.22), indicating a more favorable therapeutic window. Compound 4a induced a pro-apoptotic shift in the BAX/BCL-2 ratio, and all compounds enhanced antioxidant gene expression, particularly 4a. No significant genotoxic effects were detected. These findings suggest that the synthesized dihydropyrimidinone derivatives possess selective anticancer potential and favorable genotoxic safety profile, with mechanistic diversity supporting their relevance for targeted cancer therapy.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), continues to be the second leading cause of infectious disease-related mortality globally, surpassed only by COVID-19. The Wnt/β-catenin signaling pathway is involved in regulating TB pathogenesis, with AXIN1-a key negative regulator of this pathway-emerging as a potential target for modulating immune responses. Our study explored the function of AXIN1 in TB by examining its expression in peripheral blood mononuclear cells (PBMCs) from TB patients and its effects on macrophage and CD4+T cell functions. We found that AXIN1 expression was significantly downregulated the PBMCs of TB patients and in macrophages infected with Bacillus Calmette-Guérin (BCG). In Tcf/Lef-Gfp transgenic mice intratracheally infected with Mycobacterium bovis BCG, treatment with XAV939 to upregulate AXIN1 expression inhibited Wnt/β-catenin signaling, reduced the proportions of CD4+T cells and macrophages, and decreased the percentage of MHC-II-positive macrophages. Conversely, downregulating AXIN1 in macrophages enhanced CD4+T cell activation and MHC-II-dependent antigen presentation following BCG infection. The accumulated evidence indicates that AXIN1 plays a critical role in modulating macrophage phagocytosis and CD4+T cell reactions because of M. tb infection. Understanding the mechanisms underlying the regulation of the innate-to-adaptive immune transition by AXIN1 could provide new insights into TB pathogenesis and inform the development of novel therapeutic and vaccine strategies.
Bone marrow mesenchymal stem cells (BMSCs) play crucial roles in bone tissue regeneration and repair due to their self-renewal ability and multidifferentiation potential. Additionally, miRNAs play crucial roles in controlling osteogenic differentiation. The purpose of this study was to investigate the impact of miR-20b-5p on the osteogenic differentiation of BMSCs under inflammatory conditions. Human BMSCs were treated with 1 µg/mL lipopolysaccharide (LPS) for 24 h to establish an in vitro inflammatory model simulating inflammation in bone injury. The levels of miR-20b-5p, proteins, and inflammatory cytokines were detected via RT-qPCR, Western blotting, and ELISA, and osteogenic differentiation of the BMSCs was evaluated via ALP and alizarin red staining. Consistent with previous findings, LPS downregulated osteogenic markers (RUNX2, OCN, OPN) and upregulated inflammatory cytokines (TNF-α, IL-6, and IL-1β) in BMSCs. Furthermore, the levels of miR-20b-5p were decreased, and the levels of endoplasmic reticulum stress-related proteins (p-EIF2S1, ATF4, CHOP, and GRP78) were increased in LPS-induced BMSCs. However, overexpression of miR-20b-5p weakened the effect of LPS and promoted osteogenic differentiation. Mechanistically, miR-20b-5p overexpression alleviated ER stress caused by LPS-induced inflammation by decreasing EIF2S1 levels, thereby promoting the osteogenic differentiation of BMSCs. Our research indicates that increasing miR-20b-5p expression could be an innovative approach to promote osteogenic differentiation of BMSCs.
The latest research on epigenetics in health and disease was reported at the 10th Canadian Epigenetics, Environment and Health Research Consortium (CEEHRC) annual meeting at Blue Mountain Resort in Ontario in October 2024. Canadian and international researchers from a wide range of disciplines and career stages convened to engage in interdisciplinary discussions on the latest advances in epigenomics, promoting collaboration and the exchange of knowledge in this rapidly evolving field. The meeting emphasized a comprehensive understanding of epigenetic mechanisms—such as DNA methylation, histone modification, and chromatin accessibility—that regulate key biological processes, from embryonic development to disease progression and treatment resistance. The CEEHRC Annual Meeting offered valuable insights from fundamental biology and basic science, helping to elucidate the epigenetic foundations of development and laying the groundwork for translational research aimed at combating epigenetically driven diseases.
Transcription factor SOX2 is essential for a number of biological processes, including mammalian nervous system development and adult brain stem cell maintenance. Expression of this gene requires precise control by a complex network of regulatory elements, which still remains poorly understood. Years of research by different groups have generated a large amount of data on multiple SOX2 enhancers, with varying levels of evidence for their activity across species and tissues. However, the volume of information in the field and inconsistent nomenclature, with the same enhancer referred to by different study-specific names, make understanding progress in SOX2 enhancer regulation challenging. In this review, we brought together current knowledge on predicted and experimentally validated SOX2 enhancers, highlighting links between conserved elements studied in different species. We also propose a unified enhancer naming system based on the distance from the SOX2 transcription start site in the genome of interest, aiming to improve consistency and make communication in the field more straightforward.
Abnormal lipid accumulation following myocardial infarction (MI) serves as a critical pathological factor contributing to cardiomyocyte injury. The nuclear receptor corepressor 1 (NCOR1) is famous as a key regulator in atherosclerosis, fatty liver, and other metabolic diseases, and recent evidence suggested that NCOR1 exerted a protective action in damaged heart cells. In this study, in a murine MI model induced by left anterior descending coronary artery ligation, we observed a significant downregulation of NCOR1 in myocardial tissues. NCOR1 was also downregulated in oxygen–glucose deprivation (OGD)-treated H9C2 cells, in which NCOR1 overexpression improved lipid metabolic dysregulation and peroxidation. Mechanistically, NCOR1 interacted with peroxisome proliferator activated receptor gamma (PPARγ) protein, which transcriptionally activated the expression of the mitophagy marker gene PINK1. Either knockdown of PPARγ or PINK1 was able to reverse the improvement of NCOR1 overexpression on OGD-induced dysregulation of mitophagy, lipid peroxidation, and cardiomyocyte damage. Finally, we demonstrated that NCOR overexpression (mediated by lentiviral vector) reduced infarct size, attenuated myocardial damage, and significantly improved cardiac function in MI mice. These findings not only identify NCOR1 as a novel protector in hypoxic-ischemic myocardium but also delineate the “NCOR1-PPARγ-PINK1” axis as a novel mechanism for improving mitochondrial function and lipid peroxidation, offering a promising therapeutic target for MI treatment.
