搜索结果：Journal of cellular biochemistry

Biosynthesis of lipids and fatty acids (FAs) is essential for the normal functioning of cellular processes, and lipid availability determines the progression of multiple malignant tumor types. To date, the roles of individual steps in lipid biosynthesis during tumor growth and their interaction with intracellular signaling pathways are not well understood. Our study demonstrates that upregulation of de novo FA and lipid synthesis is a conserved characteristic of malignant tumors. In vivo tumor cell-specific silencing of components of the neutral lipid biosynthetic apparatus revealed that loss of several enzymes involved in FA and diacylglycerol synthesis inhibited tumor growth. Specifically, acetyl-CoA carboxylase (ACC), which catalyzes the first step of FA synthesis, drives late-stage tumor growth. FA synthesis perturbation led to inactivation of TORC1 (mechanistic Target of Rapamycin Complex 1)-accompanied by activation of the catabolic process autophagy. Moreover, TORC1 activity cannot be fully restored by hyperactivation of upstream Insulin/PI3K signaling or inhibition of AMP-activated kinase (AMPK) in ACC-deficient tumor cells, but supplementation with ectopic oleic acid can partially increase TORC1 activity and tumor progression. In addition to their metabolic value, the role of FAs in promoting TORC1 gives us new insight into cancer cell dependence on de novo FA synthesis.

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.

Existing studies have revealed that RNA modification regulators and cellular senescence can affect the Alzheimer's disease (AD) process. This study investigated the synergistic mechanism in the brains of AD. Based on brain tissue proteomics of patients with AD, we screened out the subtypes of patients that are coordinately regulated by cellular senescence-related proteins and RNA modification regulators. Transcriptome datasets were used to validate and evaluate 20 hub proteins identified using 100 integrated machine learning algorithms. Finally, protein and metabolic data were employed to explore the characteristics of metabolic subtypes and pathways in AD progression. The diagnostic model had good diagnostic performance, as revealed by the average area under the receiver operating characteristic curve (AUC) = 0.885 of the internal datasets and the average AUC = 0.89 of transcriptome datasets. Risk score can be used to assess disease progression and the corresponding changes in metabolic characteristics. Finally, metabolic analysis indicates significant abnormalities in amino acid and lipid metabolism during the progression of AD. We revealed the potential role of RNA modification regulators and cellular senescence-related proteins in AD pathogenesis and related diagnostic markers through proteomic analysis and machine learning-based methods.

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.

Jamuar syndrome (Developmental and Epileptic Encephalopathy 84, OMIM# 618792) is a rare autosomal recessive congenital disorder of glycosylation (CDG), caused by variations in the gene encoding UDP-glucose dehydrogenase (UGDH). Although a number of UGDH variants have been functionally characterized, there is an incomplete catalogue of variants and their impacts on development. Here, we present functional data characterizing new missense variants from three unrelated individuals who were D379N homozygous, Y356D homozygous, and compound heterozygous A436G/R442W, respectively. UGDH activity was low to undetectable in patient-derived fibroblasts bearing either UGDH D379N or UGDH A436G/R442W, relative to WT fibroblasts, despite robust UGDH expression in both. Measurement of nucleotide sugar levels revealed a significant decrease in the UGDH product, UDP-glucuronate, and consequent reductions in hyaluronan production, Notch1 levels, and rate of O-and N-linked glycan synthesis, consistent with loss of UGDH activity. These features support the designation of UGDH D379N and UGDH A436G as causative variants in Jamuar Syndrome. We expressed and purified UGDH D379N, A436G, R442W, R443H, and Y356D variants to examine underlying molecular mechanisms. Kinetic properties and structural stability assays selectively revealed significant changes in conformational dynamics that manifested strong effects on endogenous inhibitor binding and product inhibition. The results suggest that alterations to the C-terminal domain impact activity of UGDH in cells by impairing its cofactor exchange rate and diminishing quaternary association. These effects would be maximized at developmental milestones in which hypoxia drives morphological change, since NADH accumulation would then decrease glycosaminoglycan production, with profound developmental consequences.

The mechanistic target of rapamycin complex 2 (mTORC2), a key regulator of cellular metabolism, growth, and survival, remains poorly characterized in the context of dopaminergic neurotoxicity. In this study, we investigated the role of mTORC2 signaling in the survival of SH-SY5Y neuroblastoma cells exposed to the Parkinsonian neurotoxins 1-methyl-4-phenylpyridinium (MPP⁺) and 6-hydroxydopamine (6-OHDA), and examined its interplay with oxidative stress and major stress-responsive signaling pathways. Both neurotoxins induced oxidative stress and mitochondrial damage, accompanied by PINK1 accumulation and culminating in caspase-3 activation, PARP1 cleavage, and apoptotic cell death. These effects were associated with reactive oxygen species (ROS)-dependent phosphorylation of the mTORC2 components Rictor and SIN1, as well as the downstream mTORC2 target Akt (Ser473), indicating activation of mTORC2 signaling in response to neurotoxic insult. RNA interference-mediated depletion of the mTORC2 subunits Rictor, SIN1, or mLST8 reduced Akt phosphorylation and potentiated 6-OHDA-induced cytotoxicity by exacerbating oxidative stress, mitochondrial damage, PINK1 accumulation, and the apoptotic cleavage of caspase-3 and PARP1. In contrast, only Rictor depletion, but not SIN1 or mLST8 knockdown, increased the susceptibility of SH-SY5Y cells to MPP⁺-induced toxicity. Genetic inactivation of mTORC2 reduced basal phosphorylation of the cellular energy sensor AMP-activated protein kinase (AMPK), but did not alter neurotoxin-induced phosphorylation of AMPK or the mitogen-activated protein kinases ERK and JNK. Together, these findings demonstrate a protective role of mTORC2 components against 6-OHDA-induced, and to a lesser extent, MPP+-induced, mitochondrial damage and apoptotic cell death.

Rapamycin (Rapa) is a potent inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) with possible applications in multiple diseases; however, it and its analogues exhibit low solubility, variable bioavailability, and dose-limiting side effects. To engineer a long-release carrier, we employ Rapa's cognate receptor (FKBP12) to modulate its solubility, rate of release, and cellular uptake. To target its internalization into cancer cells under stress with an unfolded protein response (UPR), we use an L-peptide that binds cell-surface glucose-regulated protein 78 (GRP78). Herein, the L-peptide was fused to five FKBP domains linked by an elastin-like polypeptide (ELP) selected to form a biomolecular condensate depot at body temperature. This novel GRP78-targeted carrier (L-5FV) was characterized by UV-vis spectrophotometry, dynamic light scattering (DLS), surface plasmon resonance (SPR), and dialysis under sink conditions to assess its thermosensitivity, particle assembly, binding kinetics to both Rapa and GRP78, and drug release, respectively. Functional delivery of cellular internalization and mTORC1 inhibition were confirmed using fluorescence microscopy and Western blot in dose- and time-dependent manners in a breast cancer cell line, BT-474. Both targeted and untargeted formulations are phase-separated at physiological temperatures and exhibit nanomolar affinity for FKBP12 and Rapa. Notably, L-5FV demonstrated a more significant cellular association and inhibition of p-rpS6, a mechanistic target of mTORC1 activity. This report provides insight into how to construct long-release, molecularly targeted drug carriers with applications in UPR-active cancers.

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.

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.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a widespread modification in metazoans. Cumulative evidence has revealed the altered A-to-I editing profiles in cancers, but the underlying mechanism remains unclear. Here, we discover the well-known histone lysine methyltransferase enhancer of zeste homologue 2 (EZH2) as an unexplored ADAR interactor and editing regulator in prostate cancer (PCa). Through competing with interleukin enhancer binding factor 2 (ILF2) for ADAR1 binding, EZH2 reshapes the substrate selectivity of ADAR1 and thus exhibits a bidirectional role in editing regulation. Moreover, EZH2 depletion induces the translational repression of transportin-1 (TRN1), which further results in the accumulation of cytoplasmic ADAR1p110 isoform to protect many oncogenic transcripts from degradation. Consistently, depletion of ADAR1 dramatically enhances the sensitivity of cancer cells and tumors to EZH2 selective degraders. Collectively, our study sheds new light on a link between two layers of epigenetic regulations at histone modification and RNA editing levels, demonstrates a previously uncharacterized role of EZH2 in RNA editing and mRNA stability independently of its lysine methyltransferase activity, and reveals the significance of EZH2-ADAR1 cascade in governing RNA editing and mRNA stability, which may provide additional perspectives for the advancement of EZH2-targeting cancer therapies.

Mechanistic target of rapamycin complex I (mTORC1) is a key regulator of cell growth and metabolism, and its activity increases with aging. Hyperactivation of mTORC1 is associated with the pathology of sarcopenia and mitochondrial dysfunction. Exercise training has been shown to improve muscle quality and function in people with sarcopenia. However, it is unknown if hyperactive mTORC1 will alter exercise training-induced adaptations. In this study, we examined the effect of endurance training on muscle function and metabolism in a mouse model of hyperactive mTORC1 [DEP domain-containing protein 5 muscle-specific knockout (DEPDC5 mKO)]. After 8 wk of exercise training, DEPDC5 mKO mice had increased mitochondrial activity and tibialis anterior (TA) muscle mass, despite no change in physical function. Furthermore, DEPDC5 mKO mice had a trend for reduction in the phosphorylation of the mTORC1 downstream target, ribosomal protein S6, which may have contributed to the lack of functional adaptations. In addition, there was a reduction in triglycerides (TGs) and phosphatidylcholines (PCs) in DEPDC5 mKO mice, suggesting an increase in lipid fuel use and alterations in lipid membrane composition due to an increase in mitochondrial activity. We conclude that hyperactive mTORC1 in muscle may attenuate functional adaptations to endurance exercise training, despite increasing mitochondrial respiration and alterations in lipid metabolism.NEW & NOTEWORTHY Endurance exercise training in mice with hyperactive muscle mechanistic target of rapamycin complex I (mTORC1) was associated with increase in mitochondrial activity and TA muscle mass despite lack of changes in physical function. These findings could be attributed to altered autophagy-related signaling and a reduction in the phosphorylation of ribosomal protein S6, downstream target of mTORC1, after exercise training in DEPDC5 mKO mice. Reduction in phosphatidylcholines (PCs) and triglycerides (TGs) may suggest an increase in lipid fuel use and alterations in lipid membrane composition due to an increase in mitochondrial activity.

Chronic Kidney Disease of unknown etiology (CKDu) is a major public health concern in several agricultural regions of Sri Lanka. In areas with a high prevalence of CKDu, elevated concentrations of fluoride and water hardness have been consistently reported in domestic wells, along with emerging evidence of cyanotoxin contamination. This study investigated the potential renal toxicity associated with environmentally relevant concentrations of fluoride, water hardness, and microcystin-LR (MC-LR) using a zebrafish (Danio rerio) model. Twenty groundwater samples were collected from the CKDu-endemic Padaviya region in the North Central Province. Mean fluoride and total hardness concentrations were 1.12&#x2009;&#xb1;&#x2009;0.64&#xa0;mg&#xa0;L-1 and 424.2&#x2009;&#xb1;&#x2009;126.1&#xa0;mg&#xa0;L-1, respectively, while MC-LR was detected in 45% of the samples (mean: 0.96&#xa0;&#xb5;g&#xa0;L-1). Zebrafish embryos (2&#xa0;h post fertilisation) were exposed to the concentrations reflecting maximum and minimum field observation values. Single exposures to fluoride, hardness, and MC-LR resulted in moderate mortality and developmental abnormalities, whereas combined exposure produced significantly higher mortality, 50.0&#x2009;&#xb1;&#x2009;1.15% (p&#x2009;<&#x2009;0.05), and increased incidence of oedema, spinal curvature, and growth retardation in larvae. Histopathological examinations of larvae exposed to the combined treatment revealed noticeable pronephric alterations, including tubular vacuolation, cellular swelling, and inflammation indicative of renal damage. Chronic exposure to zebrafish resulted in significant upregulation in Kidney Injury Molecule (KIM-1) gene expression, representing renal tubular injury. These findings suggest that the combined chronic exposure to multiple well water contaminants may induce nephrotoxic effects at concentrations relevant to CKDu-endemic surroundings. Although the results do not establish direct causation, they highlight the importance of considering mixture toxicity in environmental risk assessment and drinking-water management in CKDu-affected regions to address the health issue.

The chimeric protein p210 BCR-ABL is a major causative factor of chronic myeloid leukemia (CML). Previously, we found that p210 BCR-ABL translocates from the cytosol to the mitochondria upon mitochondrial damage via the interaction of its pleckstrin homology domain (p210-PH) with cardiolipin (CL), a mitochondria-specific phospholipid. However, the precise pathological functions of this event are unknown. Here, using multivalent peptide library screens, we identified a tetravalent peptide, WDD-R4-tet, which binds to the CL-binding region of p210-PH and inhibits the translocation of p210 BCR-ABL to the mitochondria. Notably, WDD-R4-tet induced the apoptosis of CML cells by specifically suppressing the expression of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2), ubiquitin ligases with anti-apoptotic functions, leading to the activation of caspases. Other compounds that inhibited cIAP1/2 also efficiently inhibited the proliferation of CML cells. Thus, WDD-R4-tet might be a novel therapeutic agent for CML, which functions by inhibiting novel cell-survival signaling pathways generated on the mitochondrial outer membrane of CML cells.

Calcific aortic valve disease (CAVD) is a multifactorial condition characterized by progressive leaflet calcification with a potential role for bacterial colonization in its pathogenesis. This study investigates clinical, microbiological and molecular features of calcified versus non-calcified aortic regurgitation (AR) valves. This is a prospective, observational study, whose primary objective was to compare the occurrence of bacterial detection between CAVD and AR. The secondary objectives included the evaluation of bone-related calcification markers in valves from CAVD and AR patients. We analysed 31 CAVD and 8 AR valves, yielding 111 leaflets (84 calcified, 27 non-calcified). Light microscopy of CAVD leaflets revealed near-complete disruption of the three-layered valve architecture, with calcified masses extending through the leaflets, sparse cellularity and focal micro-angiogenesis; no bacteria were detected by GRAM, PAS or TEM. Enrichment culture detected low-virulence bacteria in 5.95% of CAVD and 4.16% of AR leaflets; 16S rRNA PCR was positive in 22.5% of CAVD and 12.5% of AR cases, with Staphylococcus and Streptococcus spp. predominating. Calcium content was significantly higher in CAVD leaflets (p&#x2009;=&#x2009;.001) and correlated with dyslipidemia (p&#x2009;=&#x2009;.02). Osterix expression was higher in valves with positive microbiological findings (p&#x2009;<&#x2009;.0001), while ALP was increased in CAVD and bicuspid valves regardless of microbial status. Valve interstitial cells from CAVD exhibited spontaneous in&#xa0;vitro calcification, unlike controls. The early osteogenic marker osterix was found to be upregulated in patients whose valves tested positive for microbial DNA, suggesting a potential role for bacteria in driving cellular differentiation towards an osteoblastic phenotype in CAVD.

Regulated in development and DNA damage response-1 (REDD1/DDIT4) is induced in response to environmental stress to restrain the mechanistic target of rapamycin complex 1 (mTORC1) signaling as an adaptive strategy to restore cellular homeostasis. Interestingly, REDD1/DDIT4 expression is upregulated in several tumor types including colorectal cancer, suggesting it may have a role in tumourigenesis. Here, we report that activating transcription factor 4 (ATF4)-dependent REDD1/DDIT4 expression is required for survival of colon tumor cells undergoing endoplasmic reticulum (ER) stress through the modulation of TRAILR2/DR5 gene expression. Our findings further demonstrate that resistance to ER stress-induced apoptosis in multicellular tumor spheroids (MCTS) is associated with constitutive expression of REDD1/DDIT4 and diminished mTORC1 activity. CRISPR/Cas9-mediated deletion of REDD1/DDIT4 markedly increases TRAILR2/DR5 expression and enhances apoptosis in spheroids exposed to ER stress. Interestingly, RNA sequencing analysis reveals that the loss of the transcriptional regulator EVI-1/MECOM in cells deficient in REDD1/DDIT4 amplifies the ER stress-induced upregulation of TRAILR2/DR5, leading to enhanced apoptosis. In summary, our findings underscore the crucial role of REDD1/DDIT4 in regulating TRAILR2/DR5-induced caspase-8 activation and apoptosis under chronic ER stress, by inhibiting mTORC1 activity and promoting EVI-1/MECOM-mediated suppression of TRAILR2/DR5 gene expression.

The metabolic enzyme lactate dehydrogenase C4 (LDHC4) is aberrantly expressed in cancers and linked to poor prognosis. However, its role in lung adenocarcinoma (LUAD) and the molecular mechanisms beyond glycolysis remain unclear. This study investigates whether LDHC4 promotes LUAD by modulating protein lactylation, a lactate-derived post-translational modification, focusing on the tumor suppressor retinoblastoma protein (RB1). LDHC4 expression and its correlation with clinicopathological features and survival were analyzed using public databases (UALCAN, Kaplan-Meier Plotter, LOGpc) and validated in a cohort of 90 paired LUAD tissues via immunohistochemistry. The functional impact of LDHC4 on proliferation, migration, and invasion was assessed in A549 and PC-9 cells using gain- and loss-of-function models. The global lactylation profile was analyzed using DIA-based lactylation proteomics on the Astral platform. The interaction between RB1 and E2F1 (E2F transcription factor 1) was examined through molecular dynamics simulations, co-immunoprecipitation (Co-IP), and immunofluorescence. The functional consequences of site-specific RB1 lactylation at lysine 900 (RB1-K900lac) were determined using RB1-K900R mutant constructs and cell cycle analysis. LDHC4 was significantly overexpressed in LUAD tissues, correlating with poor patient survival, and was an independent prognostic risk factor. In vitro, LDHC4 promoted LUAD cell proliferation, migration, and invasion, and its tumor-promoting role was corroborated in an LUAD xenograft model, in which derived tumors exhibited increased volume and weight compared with mock-transfected controls. Mechanistically, LDHC4 overexpression elevated global protein lactylation levels and specifically increased lactylation of RB1. Bioinformatics and molecular dynamics simulations identified K900 as a key conserved residue for RB1-E2F1 binding; its lactylation destabilized the complex by increasing structural fluctuation and weakening intermolecular interactions. Cellular experiments confirmed that the lactylation-resistant RB1-K900R mutant bound E2F1 more strongly than wild-type RB1. Functionally, cells expressing RB1-K900R exhibited suppressed malignant phenotypes and G1/S cell cycle arrest, accompanied by downregulation of CDKs/cyclins and upregulation of P21. This study uncovers a novel LDHC4-driven oncogenic axis in LUAD. LDHC4 facilitates RB1 lactylation at the K900 residue, which disrupts the RB1-E2F1 tumor-suppressive complex, leading to cell cycle dysregulation and tumor progression. These findings may position the "LDHC4-RB1 lactylation" axis as a promising therapeutic target for LUAD.

Three-dimensional (3D) in vitro models of depression are valuable platforms for investigating disease pathophysiology, elucidating the mechanisms of antidepressant action, assessing drug efficacy, and facilitating the development of novel therapeutics. In our previous study, 3D cortical spheroids exposed to the synthetic glucocorticoid dexamethasone exhibited marked impairments in neuroplasticity, representing a key pathological feature of depression. The present study aimed to evaluate the applicability of this model under escitalopram treatment and to elucidate the molecular mechanisms involved. Primary rat cortical cell-derived 3D spheroids were exposed to dexamethasone (100 &#xb5;M) and subsequently treated with escitalopram at concentrations of 0.1, 1, and 10 &#xb5;M. Western blot analysis was performed to measure brain-derived neurotrophic factor (BDNF), mTORC1&#x2013;related signaling proteins, and synaptic proteins including PSD-95 and GluA1. Neurite outgrowth was visualized via immunofluorescence and quantified using Sholl analysis. Dexamethasone significantly reduced BDNF expression, neurite complexity, and phosphorylation of mTORC1, 4E-BP1, and p70S6K, along with decreasing synaptic protein levels. Escitalopram dose&#x2013;dependently reversed these deficits, with the most pronounced effects observed at 10 &#xb5;M. These findings demonstrate that escitalopram is associated with enhanced neuroplasticity and increased activation of mTORC1 signaling in dexamethasone-treated cortical spheroids, validating this system as a 3D in vitro for mechanistic investigation of stress-related neuroplasticity impairment.

The p47ING1a isoform of the ING1 tumor suppressor regulates cellular senescence through Rb-dependent pathways via its plant homeodomain (PHD) zinc-finger, which recognizes the H3K4me3 histone mark. However, the mutational landscape of p47ING1a and the functional consequences of PHD-domain nonsynonymous single-nucleotide polymorphisms (nsSNPs) remain poorly characterized. This study aimed to identify and structurally evaluate the most deleterious nsSNPs in p47ING1a and clarify their potential role in disrupting ING1 tumor-suppressor activity. A total of 347 missense nsSNPs were retrieved from the NCBI dbSNP database and screened using 12 sequence-based computational tools. Variants consistently predicted as deleterious were further evaluated by I-Mutant stability analysis and ConSurf evolutionary conservation profiling. Three-dimensional structural modeling was performed using AlphaFold3, refined through GalaxyRefine, and validated by ERRAT, PROCHECK, and TM-align. Mutation-induced structural and binding effects were assessed using Missense3D, mCSM, and BeAtMuSiC. Post-translational modification sites were predicted via NetPhos 3.1, GPS 3.0, BDM-PUB, and NetOGlyc 4.0. Protein-protein interaction networks were constructed using STRING and Gene MANIA. Pan-cancer expression was analyzed through UALCAN and the Human Protein Atlas. Twelve computational tools converged on six high-priority variants, namely, C358S, C374G, W378G, F379V, S382L, and R400P. All localized exclusively within the PHD zinc-finger domain, residues 353-402. All six mutations were consistently predicted to destabilize the p47ING1a protein across multiple stability analyses. Six nsSNPs in the PHD domain of p47ING1a are predicted to disrupt protein stability, H3K4me3 binding, and Sin3A/HDAC complex interactions, thereby impairing ING1 tumor-suppressor function. These findings provide a computational basis for prioritizing variants for experimental validation through site-directed mutagenesis, chromatin-binding assays, and structure-guided therapeutic targeting of the PHD-H3K4me3 interface.

Malformations of cortical development (MCD) are major causes of refractory epilepsies, particularly in children. Cannabidiol (CBD) has demonstrated efficacy in treatment of refractory pediatric epilepsy syndromes. However, preclinical studies addressing its developmental stage-dependent effects, particularly in experimental models of MCD, remain limited. We evaluated the effects of CBD on induced hyperexcitability in cortical brain slices from Wistar rats with and without MCD at distinct developmental stages and examined whether alterations in endocannabinoid system (ECS) components are associated with CBD responsiveness. MCD was induced by bilateral cortical freeze lesion at postnatal day (P0-1) to generate microgyria in the somatosensory cortex. Local field potentials were recorded from cortical slices of juvenile (P21-30) and adolescent (P35-60) Sham and MCD rats. CBD was applied under three different timing paradigms to assess its effects on epileptiform activity induced by modified artificial cerebrospinal fluid containing 4-aminopiridine (4-AP) and 0&#xa0;Mg2+ (mACSF). Gene expression of ECS components was quantified in cortical tissue by RT-qPCR at both developmental stages. CBD co-applied with mACSF reduced short (>2-10&#xa0;s) ictal events in slices from Sham and decreased prolonged (>100&#xa0;s) ictal events in slices mainly from MCD animals at both ages. CBD did not attenuate pre-established hyperexcitability. However, pre-exposure to CBD delayed ictal onset, reduced overall ictal events frequency, particularly in juvenile Sham animals, and abolished long-lasting ictal events in slices from adolescent animals. Cortical samples from juvenile MCD animals exhibited increased gene expression of NAPE-PLD, MGLL, CB1R and CB2R, whereas DAGL was reduced in adolescence. CBD exerted age- and context-dependent modulatory effects on cortical hyperexcitability, with stronger preventive than therapeutic actions. Developmental stage, cortical organization and alterations in ECS components may influence CBD responsiveness. These findings highlight the importance of maturational, cortical network and molecular context when evaluating cannabinoid-based strategies for MCD-related refractory epilepsies.