The Warburg effect-induced lactate production in T-cell acute lymphoblastic leukemia (T-ALL) cell proliferation is well established, however, the role of lactate-mediated protein lactylation in this process remains poorly understood. In this study, we demonstrated that inhibiting lactate levels through the lactate dehydrogenase A (LDHA) inhibitor significantly reduced both cell proliferation and protein lactylation levels in T-ALL cells. Lactate deficiency led to a marked decrease in protein lactylation, accompanied by impaired cell proliferation. Meanwhile, lactate deficiency also induced the S‑phase cell cycle arrest and reduced DNA synthesis, which collectively impaired cell proliferation. Mechanistically, we observed that the decreased expression of lactylation in histone H3 at lysine 18 (H3K18lac) reduced the enrichment of this mark on the promoter region of neurotrophic receptor tyrosine kinase 3 (NTRK3), which supported the cell proliferation of T-ALL cells. Moreover, the overexpression of NTRK3 rescued the lactate deficiency-induced proliferation inhibition of T-ALL cells. Furthermore, exogenous lactate supplementation dramatically restored the decreased cell proliferation in T-ALL cells and restored H3K18lac levels. Our findings reveal a novel role of lactate-mediated protein lactylation in regulating T-ALL cell proliferation.
To investigate the role and mechanism of METTL14-mediated regulation of cuproptosis in myocardial ischemia-reperfusion injury (MI/RI). An MI/RI rat model was established. H9C2 cells were cultured and subjected to hypoxia/reoxygenation (H/R) modeling. Cells were transfected with siRNAs targeting METTL14 and ATP7A. Cell viability was detected by CCK-8 assay. Intracellular Cu2+ levels were measured using a biochemical kit. The interaction between METTL14 and ATP7A mRNA was verified by RNA immunoprecipitation (RIP) assay. Adenovirus containing si-METTL14 was constructed and injected into rats, followed by the induction of the MI/RI model. Cardiac function was evaluated by measuring ejection fraction (EF) and fractional shortening (FS) using echocardiography. Rat myocardial tissues were collected for Hematoxylin and Eosin (HE) staining. The mRNA expression of m6A regulatory enzymes was detected by quantitative PCR (qPCR). Cu2+ levels in myocardial tissue were measured using a biochemical assay kit. The expression of cuproptosis-related proteins was assessed by Western blot. MI/RI rats showed significantly decreased FS and EF values, with necrosis and hemorrhage, increased Cu2+ content, elevated mRNA levels of FTO, METTL3, ALKBH5, and METTL14, decreased ATP7A protein expression, and increased protein expression of FDX1, LIAS, and DLAT in the myocardial tissue. Intramyocardial injection of adenovirus containing si-METTL14 reversed the MI/RI-induced changes. In vitro, decreased cell viability, increased Cu2+ content, decreased ATP7A protein expression, and increased FDX1, LIAS, and DLAT protein expression were observed in H/R stimulated H9C2 cells, which were remarkably rescued by si-METTL14. Moreover, co-transfection with both si-METTL14 and si-ATP7A reversed effects induced by si-METTL14 alone. METTL14 promotes cuproptosis-associated changes and exacerbates MI/RI.
Non-enzymatic glycosylation of proteins leads to the formation of advanced glycation end products (AGEs), implicated in oxidative stress and the progression of several chronic diseases. Among the reactive carbonyl compounds, methylglyoxal (MGO) is highly potent in inducing glycoxidative damage by modifying proteins. Herein, we investigated the protective effect of plumbagin, a naphthoquinone derived from Plumbago zeylanica, to counteract MGO-induced glycation of catalase, employing integrated biophysical and computational approaches. AGE-specific fluorescence at 335 and 370 nm, ThT dye fluorescence, and microscopic visualizations confirmed that plumbagin markedly reduces AGEs formation and aggregation. Fluorescence quenching experiments revealed a stable catalase-plumbagin complex formation having binding affinity (Ka): 0.205 × 106 M-1 and a stoichiometry of one binding site (n ≈ 1.1), suggesting a single, specific binding pocket. Isothermal titration calorimetry (ITC) confirmed an exothermic, enthalpy-driven interaction. Molecular dynamics simulations (MDS) analysis displayed a more compact and ordered conformation of complex, as evidenced by reduced Rg, SASA, and RMSF values. H-bonds stabilized the complex without altering the catalytic residues (His74, Asn147, Tyr357). PCA and FEL showed complex remained confined to a single deep energy minimum, indicating enhanced thermodynamic stability. These findings highlight plumbagin as a potent multifunctional compound capable of mitigating MGO-induced glycation.
Lung cancer remains a major global health challenge, and the oncogenic function of KDM1B (Lysine-specific Demethylase 1B) is still poorly characterized. This study employed integrated bioinformatics and experimental approaches to investigate KDM1B's function in lung cancer. Pan-cancer analysis using databases such as TIMER revealed notably elevated KDM1B mRNA expression in LUAD datasets, suggesting its potential as a diagnostic biomarker. A strong association was also found between increased KDM1B levels and immune cell infiltration in LUAD datasets. Protein interaction networks constructed using STRING and Cytoscape revealed close associations between KDM1B and key regulatory genes in NSCLC. KEGG enrichment analysis linked KDM1B to the mTOR signaling, which is critical for cell proliferation and survival. RT-PCR and western blotting for experimental validation showed KDM1B expression was significantly increased in A549 and NCI-H460 lung cancer cells. The deletion of KDM1B inhibits cell growth, induces G0/G1 phase cell cycle arrest, and promotes apoptosis in A54 cells. Moreover, cell proliferation was significantly inhibited by the KDM1B inhibitor, tranylcypromine, and induced G0/G1 phase cell cycle arrest, increased apoptosis, ROS, and glycolytic activity in A549 cells. Collectively, these findings highlight KDM1B as a valuable therapeutic target in lung adenocarcinoma and emphasize its key role in lung cancer development.
ALKBH5, as an m6A demethylase, plays a crucial regulatory role in various kidney diseases such as acute kidney injury, chronic kidney disease, renal fibrosis and renal cell carcinoma. However, its function often exhibits contradictory effects: In renal fibrosis, ALKBH5 has been reported to both promote and suppress fibrogenesis, whereas in renal cell carcinoma most studies support an oncogenic role, but some clinical observations link ALKBH5 downregulation to poor prognosis. These contradictions may stem from differences in cell types, disease stages, and microenvironments. This manuscript systematically reviews the complex function of ALKBH5 in kidney diseases and its underlying mechanisms, explores its potential as a therapeutic target, evaluates its therapeutic potential, and highlights the need for cell- and pathology-specific strategies to enable precise intervention. Future research should focus on its cell and pathological background specificity to achieve precise intervention.
Immune cell activation and differentiation are tightly coupled to metabolic reprogramming, with glucose availability and intracellular glycolytic flux serving as central determinants of immune cell fate and function. While increased glucose uptake and aerobic glycolysis support rapid proliferation and effector programs, persistent glucose abundance or dysregulated glycolytic signaling can contribute to immune dysfunction, chronic inflammation, and impaired host defense. Conversely, controlled limitation of glucose availability-through altered systemic supply, tissue microenvironmental competition, or targeted modulation of glycolysis-can rebalance immune metabolism toward oxidative phosphorylation, fatty acid oxidation, and mitochondrial fitness. This review examines how glucose availability and glycolytic flux operate as metabolic checkpoints that integrate with nutrient-sensing pathways, including mTORC1, AMPK, and HIF-1α, to shape immune activation, effector differentiation, and memory formation. We discuss evidence across disease contexts, including hyperglycemia-associated immune dysfunction, viral infection, autoimmunity, and cancer, and summarize emerging strategies to therapeutically modulate glucose metabolism using dietary interventions and pharmacologic tools. By distinguishing dietary carbohydrate intake from systemic glucose availability and cell-intrinsic glycolytic control, this review provides a coherent framework for understanding when glucose modulation can enhance immunity and when it risks immune suppression or metabolic exhaustion.
Organ fibrosis represents a terminal pathological consequence of chronic tissue injury and contributes substantially to global morbidity and mortality, yet effective therapeutic options remain limited. Characterized by the persistent activation of fibroblasts into myofibroblasts, fibrosis results in excessive extracellular matrix (ECM) deposition and progressive disruption of normal organ architecture, ultimately leading to functional failure. Increasing evidence places zinc finger proteins (ZNFs) as key mediators within the regulatory hierarchy of fibrotic diseases. As the largest and most diverse family of transcriptional regulators in the human genome, ZNFs utilize specialized zinc-coordinating motifs to regulate gene expression, protein stability, and intracellular signaling pathways. Beyond their classical roles in DNA binding and transcriptional control, ZNFs are now recognized as key orchestrators of fibrotic programs through regulation of ubiquitin-mediated protein turnover, cytoskeletal dynamics, and core pro-fibrotic signaling pathways, including transforming growth factor-β/Smad, mitogen-activated protein kinase, and phosphoinositide 3-kinase/protein kinase B cascades. Dysregulation of specific ZNFs has emerged as a critical driver of myofibroblast differentiation, ECM synthesis, and pathological tissue remodeling across multiple organ systems, including the liver, lung, kidney, skin, and heart. This review provides a comprehensive synthesis of the molecular roles of ZNFs in organ-specific fibrosis, integrating their structural classification with their mechanistic influence on key signaling networks. Furthermore, we discuss the clinical potential of ZNFs as biomarkers of fibrotic progression and evaluate emerging therapeutic strategies aimed at targeting ZNF-mediated pathways. Finally, we outline key knowledge gaps and future research directions. By consolidating recent advances, this review highlights ZNFs as promising molecular targets for the next generation of anti-fibrotic interventions.
Protein tags are widely used for purification, solubilization, detection, and imaging, yet they can substantially alter protein self-assembly. This interference is particularly significant for intrinsically disordered proteins and low-complexity domains, whose aggregation and phase separation are mediated by weak multivalent interactions that are easily disrupted by exogenous elements. In this review, we examine how affinity tags, solubility tags, fluorescent proteins, and chemical labels influence aggregation, amyloid formation, and liquid-liquid phase separation (LLPS). We first classify recurring perturbation mechanisms into six primary categories: solubility enhancement, artificial multivalency, electrostatic interactions, local effects, metal coordination, and positional dependence. Crucially, these non-exclusive mechanisms often operate simultaneously within a single construct. We then review representative case studies across pathogenic amyloids, RNA-binding proteins, viral inclusions, functional amyloids, yeast prions, and membrane proteins. These examples demonstrate that tags alter assembly kinetics, phase boundaries, material properties, fibril morphology, oligomeric states, and observed phenotypes, rather than merely serving as neutral tools for detection. In some systems, tags suppress intrinsic assembly; in others, they promote non-native condensation or stabilize alternative aggregate states. Finally, we discuss practical experimental strategies to distinguish intrinsic protein behavior from construct-dependent effects, emphasizing matched comparisons, orthogonal validation, and the careful interpretation of measurements based on tag cleavage or fluorescence. Collectively, the evidence indicates that protein tags should be treated as experimental variables that shape assembly states rather than as inert technical additions.
SARS-CoV-2 infection is driven by extensive interactions between viral proteins and host cellular factors, yet the structural properties of host proteins within these interaction networks remain incompletely understood. Intrinsically disordered proteins and regions are key contributors to protein-protein interaction networks due to their conformational flexibility and associated with it multifunctionality, binding promiscuity, and regulatory versatility. In this study, we performed a systematic, proteome-wide analysis of intrinsic disorder in human proteins interacting with SARS-CoV-2 by integrating five experimentally validated interaction datasets comprising 2055 unique host proteins. Using disorder prediction, structural confidence assessment, functional and domain annotation, protein-protein interaction network analysis, phase-separation propensity estimation, and independent validation with the D2P2 platform on a selected set of proteins, we characterized the structural organization of the SARS-CoV-2 human interactome. Our results reveal a balanced distribution of ordered and disordered host proteins, distinct functional and domain signatures across disorder classes, consistent inverse relationships between disorder and structural confidence, and increased network connectivity and phase-separation propensity among highly disordered interactors. These findings indicate that SARS-CoV-2 exploits structural diversity within the host proteome rather than preferentially targeting a single disorder class and highlight intrinsic disorder as a key contributor to interaction plasticity and network organization at the systems level.
Inflammatory response induced cell apoptosis plays a crucial role in the pathological process of secondary injury in spinal cord injury (SCI), and targeted reduction of secondary inflammatory response can effectively promote neuronal recovery after SCI. TRIM32, an E3 ubiquitin ligase, has been shown to modulate inflammation by influencing ubiquitination modifications. In this study, we sought to investigate the function of TRIM32 in the progression of SCI. LPS treated PC12 cells and SD rats were utilized to establish the SCI model. HE staining and BBB score was conducted to analyze the SCI development of rats. The inflammatory factor contents were detected using ELISA kits. Cell growth was analyzed by CCK-8 and flow cytometry assays. Western blot was performed to detect ubiquitination levels and protein levels. In addition, CO-IP assay was carried out to analyze the relationship between TRIM32 and TLR4. TRIM32's ubiquitination modification of TLR4 is achieved through K48 linkage. TRIM32 was down-regulated in the LPS treated PC12 cells and SCI rats. Overexpression of TRIM32 decreased the IL-1β, IL-6, and TNF-α contents in vivo and in vitro. Additionally, TRIM32 overexpression increased the ubiquitination levels of TLR4, which further decreased the protein stability and expression of TLR4. TRIM32's ubiquitination modification of TLR4 is achieved through K48 linkage. Furthermore, the overexpression of TLR4 counteracted the influence of TRIM32 on cell viability, the rate of apoptosis, and the levels of IL-1β, IL-6, and TNF-α in PC12 cells subjected to LPS treatment. In rats with SCI, the upregulation of TRIM32 alleviated damage to spinal cord tissues and enhanced the BBB scoring. This study demonstrated that TRIM32 overexpression inhibited the inflammation in SCI progression through inducing the ubiquitination degradation of TLR4. TRIM32 might be an intriguing host therapeutic target for the treatment of SCI.
ATP synthase (ATPase) is a crucial molecular motor in Mycobacterium tuberculosis (Mtb), essential for energy production and oxygen-dependent pathogenesis. The enzyme consists of two distinct rotors: a membrane-embedded Fₒ unit and a cytosolic catalytic F1 unit, along with a stator, a central stalk, and a heterodimeric peripheral stalk (PS). As the F0 region hosts critical drug-binding pockets, it has gained significant interest. This study focuses on local structural dynamics at the leading site in the presence of bedaquiline (BDQ). All-atom molecular dynamics simulations were performed using GROMACS in a heterogeneous bilayer composed of phosphoinositol, phosphoethanolamine, phosphoglycerol, and cardiolipin (PI: PE: PG: CL) in a 32:42:4:50 ratio. The results revealed key interactions of BDQ with cL59, cF65, cE61, cA62, cI55, cI66, cG58, aI215, and aF219 at the a/c interface, consistent with energetically favored binding conformation. Quantitative lipid contact analysis revealed higher CL interactions with BDQ at leading site together with interfacial water molecules, whereas protein-lipid contacts based on only lipid headgroup (P-atoms) analysis remained independent of lipid abundance in the system. RMSD and RMSF revealed BDQ-induced fluctuations in the outer helix of subunit-c, while subunit-a remained comparatively more stable during the simulation. Distance analysis further indicated that the ligand remains confined within the binding region despite local flexibility. We further identified putative non-collinear proton channels, which showed no significant global perturbation upon BDQ binding. Residues aG195, aN105, aD220, aN190, aQ227, cE61 (inlet side), cE61 (outlet side), aE176, aE175, aA178, aK179, aS182, aY238, aQ110, aF192, aL122 form the two half channels in Mtb. The pooled water-count analysis for channels showed similar hydration levels in all simulated systems. We hypothesize that selective targeting of the leading pocket by newer drugs, in the presence of CL lipids, can modulate the proton inlet channel. The heterogeneous bilayer supports the structural and functional integrity of the membrane and ATPase complex. A CL-enriched membrane environment may provide a useful framework for investigating membrane-associated effects of BDQ and its analogs. Chain-wise analysis showed synchronous movement of the PS subunits and twisting of the δ-binding region, which may be perturbed in the presence of the F1 unit. The dynamics also revealed subunit-bδ involvement with the subunit-a at the leading pocket, a less studied PS and stator function. Identifying residue-specific interactions between PS could help to reveal its mechanical function. Together, these results provide complementary dynamic insights into BDQ at the leading pocket in a physiologically mimicked membrane environment and potentially support its relevance as a target site for the development of anti-TB compounds targeting ATPase.
Agrin is an important factor in maintaining skeletal architecture. However, the role of osteoblast-expressed agrin in bone tissue remains unexplored. Thus, we hypothesized that agrin-deficient osteoblasts negatively affect bone tissue homeostasis by disrupting osteoblastic differentiation of mesenchymal stem cells (MSCs). To investigate this hypothesis, mice with deletion of agrin in Runx2-expressing osteoblasts were generated using Runx2-Cre mice. Microtomographic analyses revealed that agrin ablation in osteoblasts resulted in deleterious effects on the cortical bone of the femurs, leading to a tendency towards reduced bone stiffness, as indicated by the three-point bending test. This negative impact of agrin-deficient osteoblasts on bone tissue may be partially attributed to the reduced osteoblastic differentiation of MSCs derived from these mice, as evidenced by lower expression of osteoblastic marker genes and alkaline phosphatase activity. This disruption in osteoblastic differentiation is directly linked to agrin-deficient osteoblasts, as demonstrated by diminished gene expression of agrin and its receptors in MSCs collected from these animals. Our findings highlight the role of osteoblast-secreted agrin in maintaining long bone structure, mainly by promoting MSC differentiation into osteoblasts. The findings indicate that agrin could be an effective therapeutic target for treating bone loss-related conditions, such as osteoporosis and aging.
Leptin, a hormone historically recognized for regulating appetite and energy homeostasis, is increasingly appreciated as a central mediator of immune function, metabolic integration, and tissue-specific signaling. Despite decades of research, leptin resistance limits the efficacy of conventional therapies, such as hormone replacement, in treating obesity and related disorders. Emerging evidence demonstrates that leptin interacts bidirectionally with the gut microbiome, influencing systemic metabolism and immune responses. Advances in multi-omics profiling, synthetic biology, and tissue-targeted therapeutics provide unprecedented opportunities to overcome these barriers. Here, we present a perspective emphasizing integrative strategies that combine precision medicine, immune modulation, engineered leptin analogs, and microbiome-targeted interventions. Leveraging these innovations could redefine leptin-based therapies, enabling system-level restoration of metabolic and immune homeostasis. Understanding leptin as a pleiotropic, multi-system hormone positions it at the forefront of the next endocrine paradigm.
Diabetes is a major risk factor for osteoporosis, which negatively impacts bone health, but the mechanisms underlying the effects of hyperglycemia on bone marrow mesenchymal/stromal cells (BMSC) are not fully understood. This study investigated how high glucose levels influence BMSC differentiation, proliferation, viability, and metabolism. The results demonstrated that high glucose inhibits osteogenesis in human BMSC, as evidenced by reduced alkaline phosphatase activity, impaired calcium deposition, and downregulation of key osteogenic genes (RUNX2, ALP). Conversely, high glucose conditions promoted adipogenesis, characterized by increased percentage of cells with lipid droplets, and upregulation of adipogenic genes (PPARγ2, CEBPα, AdipoQ), suggesting a shift towards fat cell differentiation. Furthermore, BMSC cultured in high glucose showed decreased proliferation, elevated DNA damage, increased oxidative stress, enhanced apoptosis and senescence, particularly in later passages, highlighting the negative impact of hyperglycemia on BMSC viability. Metabolomic profiling of osteogenic and adipogenic differentiation in normal and high glucose conditions revealed key metabolic shifts, with nicotinamide adenine dinucleotide (NAD+) and l-glutamate/α-ketoglutarate (α-KG) identified as critical metabolites driving these processes. Supplementation with NAD+ and α-KG in high glucose conditions significantly enhanced ALP activity. These findings suggest that high glucose promotes adipogenesis at the expense of osteogenesis, exacerbating cellular damage and accelerating aging in BMSC. The identification of NAD+ and α-KG as key regulators in this process provides new insights into the metabolic mechanisms behind impaired bone health in diabetes and highlights potential therapeutic avenues to counteract these detrimental effects to better manage diabetes-related bone diseases.
It has been suggested that orexin-A (OXA) exerts neuroprotective and anti-inflammatory effects in the nervous system, while there is limited understanding of the role of OXA in cortical astrocytes under inflammation. This study was designed to investigate whether OXA could inhibit astrocyte migration induced by lipopolysaccharide (LPS) treatment and whether this action of OXA is mediated by activation of orexin 1 receptor (OX1R) in cultured mouse cortical astrocytes. OXA and OX1R were expressed in glial fibrillary acidic protein (GFAP)-positive cultured mouse astrocytes, and their expression was significantly increased by lipopolysaccharide (LPS) treatment. In addition, treatment of LPS induced significant increases in not only astrocyte migration but also phosphorylation of K + -Cl- cotransporter 2 (KCC2), ERK, and p38 MAPK, and these increases were inhibited by OXA treatment. This inhibitory effect of OXA was restored by treatment of the OX1R antagonist, SB334867. Furthermore, OXA treatment increased GABA immunoreactivity in LPS-treated cultured astrocytes and restored the expression of the GABA transporters GAT1 and GAT3 to levels comparable to those of the control group. This effect was abolished by SB334867 treatment. Collectively, these results suggest that OXA inhibits LPS-induced abnormal astrocyte migration via direct activation of orexin 1 receptor and that this inhibitory effect may be related to the modulation of intracellular GABA levels and GAT expression as well as dephosphorylation of KCC2, ERK, and p38 MAPK.
Polo-like kinase 3 (PLK3) plays major roles in cell cycle regulation, DNA repair, and cellular responses to hypoxia. Our prior studies demonstrated that PLK3 negatively regulates the hypoxic response by directly phosphorylating and destabilizing HIF-1α and by destabilizing the E3 ubiquitin ligase SIAH2. We also find that PLK3 stabilizes PTEN by direct phosphorylation. Plk3 knockout mice exhibit increased spontaneous tumorigenesis in multiple organs, particularly the lung, at an advanced age. Tumors from these mice tend to be highly vascularized, consistent with the function of PLK3 in the hypoxic response. However, another study only observed increased tumorigenesis in female Plk3 knockout mice. The present study further explored the role of PLK3 in lung tumorigenesis. We find that PLK3 can phosphorylate SIAH2 in vitro, confirming our hypothesis that PLK3 regulates SIAH2 by direct phosphorylation. We detected a negative correlation between the levels of PLK3 and SIAH2 and a positive correlation between HIF-1α and SIAH2 in both human lung adenocarcinoma and squamous cell carcinoma. We observed an increase in lung tumorigenesis in Plk3 knockout mice in the A/J strain background. Our RNA-Seq analysis revealed significantly increased expression of genes involved in oncogenic pathways and the immune response in lung tumors from Plk3 knockout mice. Finally, we find that induced systemic SIAH2 expression promotes CD8 T cell infiltration into subcutaneous tumors in a syngeneic mouse model. Our work further supports the tumor suppressive role of PLK3 in lung cancer and discovered a novel involvement of PLK3 in the regulation of the immune microenvironment of lung tumors.
Lyn kinase, a member of the Src family of kinases, is a critical regulator of immune cell signaling, regulating key processes including activation, proliferation, survival, and apoptosis. Dysregulated Lyn kinase activity causes haematological malignancies, autoimmune disorders, solid tumors, neurodegenerative, cardiovascular, and metabolic disorders. Here, we aimed to provide a comprehensive account of Lyn kinase biology, highlighting its structural features, regulatory roles in immune and cellular signaling, and pathological implications across diseases, including cancers, neurodegenerative disorders, autoimmune diseases, and metabolic disorders. We further examined emerging therapeutic strategies, including small-molecule inhibitors, monoclonal antibodies, and natural compounds, and highlighted the therapeutic potential of Lyn kinase as a promising drug target. Overexpression of Lyn contributes to tumor growth, metastasis, and resistance to treatment in leukaemia, prostate, breast, lung, and pancreatic cancers. Beyond oncology, emerging evidence links Lyn kinase to neuroinflammation, synaptic dysfunction, and metabolic regulation, further underscoring its broad disease relevance. Lyn kinase is considered a viable therapeutic target, with ongoing research focusing on small-molecule inhibitors, monoclonal antibodies, and natural compounds like flavonoids and polyphenols that have exhibited promising preclinical and clinical outcomes. We summarized current insights into Lyn kinase biology, highlighting its pathological significance and discussing therapeutic opportunities arising from the modulation of this enzyme.
Lysosomal function can be affected by components in cell culture. This in turn may influence cellular metabolism and, consequently, research and diagnostics outcomes. One such component is the commonly used pH buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). HEPES specifically impacts the trafficking of the lysosomal enzyme glucocerebrosidase, which is deficient in Gaucher disease (GD). Understanding how HEPES affects cellular models of GD is essential, since glucocerebrosidase is central to diagnostic testing and the investigation of GD pathophysiology. Therefore, we examined the broader effects of HEPES on cultured fibroblasts from individuals with GD and healthy controls. We cultured dermal fibroblasts of eight adults with GD and seven healthy age- and sex-matched controls. The cells were cultured in two culture media, Ham's F10 and DMEM, both with and without HEPES. We assessed glucocerebrosidase enzyme activity and sphingolipid concentrations using a quantitative UPLC-MS/MS method. Additionally, we conducted multi-omics analyses, consisting of lipidomics, metabolomics and proteomics, to explore the broader impact of HEPES in cell culture on fibroblasts. Glucocerebrosidase activity in cell lysates increased after HEPES exposure in both GD and control fibroblasts, to an extent that may influence diagnostic outcomes. In GD fibroblasts, substrate accumulation was absent and not altered by HEPES exposure. GD fibroblasts exhibited a multi-omics profile largely overlapping with healthy controls and lacking the typical pathological features associated with GD in other cell types, such as mitochondrial dysfunction, dysregulated autophagy, disruption of intracellular calcium homeostasis, ER stress and chronic oxidative stress. In addition, the multi-omics profile was altered by HEPES, however in a non-specific manner. In conclusion, HEPES influences fibroblasts in culture, both from healthy controls and from patients with GD. Furthermore, GD fibroblasts lack a specific disease-related profile. This renders cultured fibroblasts unsuitable for studying pathophysiological processes in GD. Culturing GD fibroblasts with HEPES may compromise the reliability of diagnostics.
Mitophagy, a selective autophagic process, is critical for maintaining mitochondrial quality and cellular homeostasis. It plays a dual role, facilitating cell survival by removing damaged mitochondria or contributing to programmed cell death in certain conditions. Dysregulation of mitophagy is implicated in various diseases, including neurodegenerative disorders, metabolic syndromes, cardiovascular diseases, and cancers. This review examines the key regulatory mechanisms of mitophagy, focusing on pathways such as the PINK1-Parkin, BNIP3/NIX, and FUNDC1 pathways, alongside emerging modulators. Notably, mitophagy is frequently associated with various cell death pathways, such as apoptosis, necroptosis, ferroptosis, and pyroptosis. Primarily, mitophagy functions as a protective mechanism rather than a direct trigger of cell death. It may be connected to cell death when its capacity is overwhelmed rather than actively promoting the process. For instance, impaired mitophagy exacerbates neurodegeneration in Parkinson's and Alzheimer's diseases, while its activation protects against ischemic injury in cardiovascular diseases. In cancer, mitophagy is paradoxical, as it either inhibits tumor growth or promotes survival under stress. Therapeutic interventions targeting mitophagy, including small-molecule modulators, show promise in preclinical studies; however, they require further clinical validation. Advancements in imaging techniques, single-cell omics, and high-throughput screenings are anticipated to deepen our understanding of mitophagy dynamics and therapeutic potential. This review highlights mitophagy as a pivotal target for treating diseases associated with mitochondrial dysfunction, providing insights into innovative therapeutic strategies.
Myocardial ischemia/reperfusion injury (MIRI) commonly arises during medical procedures for coronary artery disease (CAD), a global health issue. Inhibiting autophagy-dependent ferroptosis has emerged as an effective strategy for MIRI treatment, yet its precise mechanisms warrant further exploration. A murine model of myocardial ischemia/reperfusion (I/R) was employed, and cardiac myocytes were subjected to hypoxia/reoxygenation (H/R). Myocardial tissue alterations were assessed using Evans blue/TTC staining, HE staining, and TUNEL assays. An automated biochemical analyzer was used to quantify serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels. Myocardial cell viability was evaluated using Cell Counting Kit-8 (CCK-8) assays. The interaction of the ATG7 promoter with SPI1 was explored through ChIP experiments. The expression levels of autophagy markers (Beclin-1, LC3The expr, ATG7, and SPI1 were assessed via immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. Various indicators, including LDH, ROS, MDA, Fe2 + , GSH, GPx4, and FTH1, were measured to characterize the ferroptosis process. In MIRI model mice, autophagy-dependent ferroptosis clearly occurred, and ATG7 expression was elevated. ATG7 knockdown effectively alleviated MIRI and inhibited autophagy-induced ferroptosis. SPI1 was identified as a key regulator in this process. SPI1 bound to the ATG7 promoter region, enhancing ATG7 transcription during myocardial I/R and thereby modulating both ferroptosis and autophagy. SPI1 knockdown inhibited ferroptosis and alleviated MIRI by suppressing autophagy. The results of our study revealed that SPI1 promoted ATG7 transcription, exacerbating ferroptosis in MIRI. These findings suggest that therapeutic strategies targeting ferroptosis and autophagy may mitigate cardiovascular diseases in MIRI.
