Journal of Cellular Biochemistry

Lung adenocarcinomas frequently harbour actionable oncogenic mutations that are vulnerable to treatment with targeted therapies. While responses to targeted therapies are often initially dramatic, relapse is almost inevitable and prevents durable responses in advanced-stage patients. Relapse is, in part, caused by drug tolerant persister cells (DTPs) which are able to survive treatment by entering a reversible, dormant state. Although long non-coding RNAs (lncRNAs) regulate processes thought to allow DTPs to survive and become stably resistant, the potential roles of lncRNAs in DTPs are largely unknown. In this study, we sought to investigate the expression of lncRNAs in in vitro DTP models of lung adenocarcinoma. We found that the lncRNAs Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) were enriched in DTPs and that knocking down MALAT1 enhanced the effect of targeted therapies in both EGFR- and KRAS-mutant DTP models. To better understand pathways that MALAT1 might regulate in DTPs, bulk RNA-sequencing was performed and several pathways that may contribute to the actions of MALAT1 in DTPs were identified. Overall, our work describes a role for the lncRNA MALAT1 in DTPs in NSCLC and suggests that MALAT1 may be a novel target for the prevention of drug tolerance and subsequent resistance to targeted therapy in NSCLC.

Parathyroid hormone-related peptide (PTHrP), encoded by PTHLH, has been implicated in tumor progression through its involvement in epithelial-mesenchymal transition (EMT), angiogenesis, and tumor cell migration. Previous experimental studies suggest that PTHrP may promote these processes in colorectal cancer (CRC), partly through the modulation of factors such as secreted protein acidic and rich in cysteine (SPARC) and vascular endothelial growth factor (VEGFA). These events play a key role in the acquisition of an aggressive phenotype in our experimental models. In this study, we performed an integrative in silico analysis of multiple transcriptomic datasets to investigate the potential role of PTHLH in CRC. Differential expression analysis identified a set of consistently dysregulated genes across independent datasets. Functional enrichment and network analyses revealed that PTHLH expression is associated with biological processes related to extracellular matrix remodeling, EMT, and angiogenesis. Correlation analyses showed a positive association between PTHLH and SPARC expression, while network-based approaches suggested a potential functional connection with VEGFA. To assess the clinical relevance of these findings, survival analysis was performed using publicly available datasets. High expression levels of PTHLH, SPARC, and VEGFA were significantly associated with reduced overall survival in patients. Notably, a combined gene signature based on these three factors demonstrated a stronger prognostic effect than individual genes, indicating enhanced predictive value. These findings suggest that PTHrP is associated with molecular pathways involved in tumor progression and, together with SPARC and VEGF, may contribute to a coordinated regulatory axis with prognostic relevance in CRC, warranting further experimental validation.

Salla disease is caused by a genetic mutation in sialin, a lysosomal membrane transporter, which exports sialic acid from lysosomes. Substrate translocation occurs via a rocker-switch mechanism that alternately exposes the substrate-binding site to the lysosomal lumen and the cytosol. The pathogenic mutation R39C found in most Salla disease patients decreases the lysosomal localisation and the transport activity. In this study, we used computational and mutagenesis approaches to elucidate the molecular effects of the R39C mutation. Using three-dimensional models of human sialin in the lumen-open (LO) and cytosol-open (CO) states combined with the mutagenesis of selected residues, we identify a critical "triplet" motif comprising R39, E194, and E262, which is associated with an ionic lock formed between K197 and D350 in the LO conformation. Molecular dynamics simulations suggest that the electrostatic triplet negatively modulates the ionic lock, and are consistent with a strengthened ionic lock in R39C sialin, potentially favouring the LO state. To assess the global effects of the R39C mutation, we computed dynamic cross-correlation matrices and identified correlation patterns consistent with an allosteric coupling between the ionic lock K197/D350 and the region surrounding the sialic acid binding site in wild-type sialin, whereas in the LO state of R39C sialin, this communication preferentially bypasses this region. Therefore, the R39C mutation may impede the LO to CO conformational transition required for sialic acid transport, providing a plausible mechanistic framework for the decreased transport activity, and possibly the decreased lysosomal localisation, observed in Salla disease. HighlightsO_LIThe R39 residue participates in an interaction triplet, which negatively regulates an ionic lock stabilising the lumen-open conformation C_LIO_LIThe R39C mutation is associated with a stronger ionic lock in the simulations, and may favour the lumen-open state C_LIO_LICorrelation network analysis suggests an allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LIO_LIThe R39C mutation alters the inferred allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/719580v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1bf7144org.highwire.dtl.DTLVardef@1a53ab8org.highwire.dtl.DTLVardef@b2249forg.highwire.dtl.DTLVardef@1827244_HPS_FORMAT_FIGEXP M_FIG C_FIG

The Na+/K+-ATPase (NKA) regulates ion balance in the kidney and influences cellular processes like proliferation and apoptosis through its signal transduction. The endogenous ligand 20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to inflammation and fibrosis in chronic kidney disease (CKD) and inhibits NKA activity in renal tubules. However, the molecular mechanism of this interaction remains unclear. In this study, we used in-silico approach to investigate the potential interaction between 20-HETE and NKA. Various ligands, including known NKA ligands such as cardiotonic steroids (CTS), 20-HETE, and negative controls, were docked using rigid and Induced Fit Docking to predict the affinity of the ligands toward NKA. Binding free energy calculations with the Prime Molecular mechanics with generalized Born and surface area (Prime MM/GBSA) tools were used to confirm the involvement of key amino acids in ligand-receptor interactions. The docking analyses revealed that 20-HETE exhibited a binding affinity comparable to negative control, with some differences between rigid and induced fit docking. Binding free energy data highlighted key amino acids in the 20-HETE and NKA interaction. Interaction fingerprint and mutations such as Ala330Gly and Val329Ala significantly reduced binding free energy, while Thr804Ala showed a notable decrease, underscoring the potential importance of these amino acids in ligand stabilization. These findings provide computational evidence supporting potential direct interaction between 20-HETE and NKA and identify candidate residues for future experimental validation.

Purine moieties are essential for many functions within the eukaryotic cell, including energy, signaling and nucleic acid synthesis. While purine starvation is known to induce stress resistance in eukaryotic model organism budding yeast Saccharomyces cerevisiae, it remains unclear whether the physiological response is related to disruption of synthesis pathway in particular position or it is uniform across all genetic deficiencies within the de novo adenine biosynthesis pathway. It is also not known how purine starved cells perceive purine shortage - weather they share the same signaling elements with nitrogen starvation or not. MethodsWe characterised physiology of strains with deletions in adenine biosynthesis pathway when cultivated in full or purine deficient and compared to cell physiological parameters when cultivated in nitrogen deficient media. We tested stress tolerance, carbon flux, cell cycle arrest and did transcription profiling (RNA-seq). ResultsOur findings demonstrate that purine starvation-induced stress resistance is significantly modulated by the specific step at which the pathway is interrupted. Transcriptional analysis revealed that purine starvation in many aspects phenocopies nitrogen starvation, particularly - in both starvations strong downregulation of ribosome related genes occurs. In the same time several metabolic features which differ from N- and ade- starvations: pentose phosphate pathway is specifically upregulated within ade4{Delta}-ade2{Delta} and downregulated in N-cells. Notably, the expression of stress-responsive genes such as HSP12, HSP26, and GRE1 varied between mutants, suggesting that the accumulation of pathway intermediates (e.g., AIR in ade2{Delta}) or the absence of downstream precursors (AICAR) alters the perception of starvation especially in the case of ade16{Delta}ade17{Delta} strain. ConclusionsMetabolic and stress-tolerance phenotypes of purine auxotrophs are not merely a result of purine depletion but seems that the response is signalled via the same pathways, like TOR1. The results suggest that strains having mutations within various positions of the purine pathway "perceive" purine limitation a bit differently - especially when we compare the end of the pathway with the other mutants. Different phenotypic outcomes of the occasional purine depletion might give preferences for organisms which have mutations in the beginning rather at the end of the pathway. Besides, our findings might have implications in the design of synthetic pathways and the use of auxotrophic markers in yeast research.

MRPL47 (Mitochondrial Ribosomal Protein Large Subunit 47) gene in chromosome 3q26 encodes a protein that is part of the large subunit of the mitochondrial ribosome. We observed that MRPL47 is frequently amplified and overexpressed in ovarian cancer samples. Importantly, increased expression of MRPL47 mRNA is associated with high levels of MRPL47 protein in ovarian cancer patients. High expression of MRPL47 is also associated with poor overall and recurrence free survival of ovarian cancer patients. Notably, MRPL47 improved metabolic fitness by enhancing cellular respiration, and glycolysis in cancer cells. Gene set enrichment analysis and target specific knockdown assays revealed that MYC transcription factor regulates MRPL47 expression. Furthermore, MRPL47 was identified very high in the plasma samples of ovarian cancer patients compared to those of healthy volunteers. MRPL47 was also associated with cisplatin resistance, whereas its expression predicted sensitivity to cisplatin therapy. Taken together, we demonstrated that MRPL47 can be used as a diagnostic biomarker for ovarian cancer and other cancers with 3q26 chromosomal amplification.

Efficient transport of membrane proteins through the endosomal network is essential for brain development and function, with perturbation implicated in disease. Deficiencies in Retromer, a key regulator of endosomal transport, have been linked to aging-related neurodegenerative disorders including Alzheimers and Parkinsons disease. To better define the neuroprotective role of Retromer, we have applied cell surface restricted proteomics to identify those integral membrane proteins whose recycling to the plasma membrane is mediated by Retromer and associated cargo adaptors, sorting nexin 3 (SNX3), its paralogue sorting nexin 12 (SNX12), and sorting nexin 27 (SNX27) (data available via ProteomeXchange: PXD078277). By comparing primary rat cortical neurons and astrocytes we have identified several cargoes that require either SNX3/SNX12- or SNX27-Retromer complexes for endosomal recycling, including proteins involved in synapse organisation, synaptic signalling and Alzheimers disease pathology. We highlight that perturbed Retromer function leads to endosomal enlargement, and we establish a key role of SNX27-Retromer in modulating transport of glutamate across both neuronal and astrocytic membranes via recycling of glutamate transporters EAAT3 (SLC1A1) and EAAT1 (SLC1A3) respectively. Our study provides further mechanistic insight into the consequences of Retromer deficiency for neuronal and astrocytic function, offering new avenues of research in the treatment of neurodegenerative disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/724903v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@98277forg.highwire.dtl.DTLVardef@1490534org.highwire.dtl.DTLVardef@f4a9feorg.highwire.dtl.DTLVardef@c48402_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Suppression of Retromer and the sorting nexins (SNX27, SNX3/SNX12) leads to a significant change in the surface proteome of rat cortical neurons and astrocytes. Focusing on the glutamate transporters, SLC1A1 and SLC1A3, we have validated that SNX27-Retromer is required for their trafficking, with SNX27-Retromer suppression in astrocytes leading to a loss of glutamate uptake. C_FIG

SignificanceQuantifying lipid droplet (LD) remodeling in 3D hepatic organoids is often limited to endpoint staining or phototoxic live fluorescence imaging, thereby obscuring droplet-level kinetics. AimWe aimed to develop a label-free method to track LD dynamics in living hepatic organoids under different fatty-acid loads. ApproachTime-lapse 3D refractive-index tomograms were acquired using holotomography and analyzed with a depth-adaptive, multi-threshold segmentation pipeline to quantify LD number, volume, sphericity, and refractive-index-derived concentration and dry mass at single-droplet resolution. ResultsOleic acid and linoleic acid induced LD accumulation while preserving organoid integrity, whereas palmitic acid triggered rapid structural collapse. Despite increases in total LD burden under both oleic acid and linoleic acid, droplet-level dynamics diverged: oleic acid produced volume-dominated accumulation via enlargement of fewer LDs and increased size heterogeneity, whereas linoleic acid produced number-dominated accumulation via sustained increases in LD number, yielding a more uniform population of small droplets. ConclusionsLabel-free holotomography with depth-adaptive analysis enables non-invasive, longitudinal, and multi-scale quantification of LD dynamics in intact organoids and reveals fatty-acid- dependent temporal modes of lipid storage. Statement of DiscoveryWe developed a label-free, longitudinal 3D holotomography framework with depth-adaptive lipid droplet segmentation that quantifies single-droplet dynamics in living mouse hepatic organoids. Using this platform, we found that oleic acid and linoleic acid induce LD accumulation via distinct strategies--oleic acid via droplet enlargement and linoleic acid via sustained increases in droplet number--while palmitic acid rapidly compromises organoid integrity.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide. Current treatments offer limited efficacy and no definitive cure, underscoring the urgent need for more selective and effective therapeutic strategies. This study investigated the synthetic lethality caused by co-targeting two metabolic genes, ATP citrate lyase (ACLY) and oxoglutarate dehydrogenase (OGDH), in HCC cells. Using valproic acid (VPA) and bempedoic acid (BA) as pharmacological inhibitors of OGDH and ACLY, respectively, we observed a strong synergistic effect in inhibiting the proliferation of HCC cell lines (Hep3B and Huh7), compared to using these drugs individually. Importantly, this combination treatment exhibited little increased cytotoxicity in the non-cancerous liver cell line THLE-2, indicating a degree of selectivity. Our findings are consistent with previous reports implicating USP13 as a metabolic regulator of ACLY and OGDH in various cancers, suggesting that the inhibition of USP13 may prevent HCC cell proliferation primarily through its downstream effects on ACLY and OGDH. By directly co-targeting ACLY and OGDH, our approach may offer a more precise and safer alternative to USP13 inhibition. Additionally, while both VPA and BA have been individually associated with beneficial effects in liver disease, their combined application in the context of HCC has not been previously investigated. Limitations include the reliance on cell line models, highlighting the need for validation in more physiologically relevant systems such as human organoids and animal models. Overall, this study provides a compelling rationale for further investigation into ACLY and OGDH as a synthetic lethal pair and the therapeutic potential of the VPA-BA combination treatment in HCC.

BackgroundIdiopathic epilepsy (IE) is the most common chronic nervous system disorder of dogs, and its cause is poorly understood. Emerging evidence suggests that microbiome alterations can occur with IE via the microbiota-gut-brain axis. Therefore, we analyzed the fecal microbiomes of 98 dogs (49 IE, 49 control) in a pairwise case-control observational study using 16S rRNA gene sequencing. ResultsAlthough the microbial community was mostly similar between groups, IE was associated with a modest but significant shift in Weighted-Unifrac distance (P = 0.042). We used six differential abundance (DA) methods to identify differentially abundant amplicon sequencing variants (ASVs) between IE and control groups. Notably, one Collinsella ASV was found to be significantly more abundant in IE dogs by all six methods. The gut microbial compositions varied drastically across households (accounting for about 69% of the total variation), but did not have significant differences between sex, age, or breed. Phenobarbital administration in IE dogs had a significant effect on seizure control, and was not associated with changes in the microbiome. ConclusionOur findings suggest a relationship between gut microbiomes and IE. However, the specific mechanism needs to be further investigated.

OXA-232, an OXA-48 like carbapenemase stands amongst newly identified beta-lactamases that causes of the extensive of beta-lactam resistance. While active-site residues are well characterised, the contributions of conserved non-active-site residues in exerting enzymatic activity remain unexplored, limiting our understanding about the roles of these residues in the overall OXA-232 function. To address these gaps, the conserved residues S118, V120, L158, and D159 of OXA-232 positioned adjacent to the active-site motifs and within the omega-like loop were substituted with alanine. Substitutions of S118A and D159A rendered the expressing cells susceptible to penicillins, cephalosporins, and carbapenems, whereas the cells harbouring OXA-232V120A and OXA-232L158A proteins exhibited substrate-selective susceptibility changes. Kinetic analysis with purified proteins revealed the reduction in catalytic efficiency of all the mutants compared to wild-type protein. Though the L158A and D159A mutated proteins become deacylation-deficient, the mutations S118A and V120A exhibited selective acylation defects without trapping intermediates. It is evident from circular dichroism spectroscopy and molecular dynamics simulations that OXA-232S118A, OXA-232V120A, and OXA-232L158A nearly retained their secondary structures and compactness, except for OXA-232D159A, which presumably triggered a misfolding leading to destabilisation of the omega-loop. Interestingly, bicarbonate supplementation partially rescued the lost activities in soluble mutants, underscoring the carbamylation dependence. Taken together, these findings establish S118 and D159 as essential for core catalysis and structural integrity, with V120 and L158 modulating substrate-specific turnover and orientation. The current study reappraised the mechanistic insights of OXA-48-like carbapenemases, providing significant resources in rationally designing future therapeutics to combat carbapenem resistance.

PurposePancreatic ductal adenocarcinoma (PDAC) is characterized by a nutrient-deprived and hypoxic tumor microenvironment (TME) that imposes severe metabolic stress on cancer cells. Under these conditions, tumor cells frequently activate the integrated stress response (ISR) to adapt to TME and develop resistance to therapies. However, how TME components support tumor adaptation to mitochondrial metabolic stress remains incompletely understood. Here, we aimed to identify key metabolite involved in ISR adaptation under oxidative phosphorylation (OXPHOS) inhibition and to elucidate the metabolic symbiosis between cancer-associated fibroblasts (CAFs) and PDAC cells. MethodsWe integrated transcriptomic and metabolomic analyses with functional assays. ISR activation was evaluated by assessing the phosphorylation of eIF2 (p-eIF2) following treatment with carboxyamidotriazole orotate (CTO), an Complex I inhibitor. Metabolomic profiling was used to identify metabolites involved in ISR activation alleviation. Mouse models were used to assess therapeutic responses following depletion of the identified metabolite under CTO treatment. Genetic perturbation of Slc38a4 was performed to assess its functional role in tumor cell adaptation to metabolic stress. ResultsWe identified asparagine (ASN) as a critical metabolite supplied by CAFs to PDAC cells under OXPHOS inhibition. A minimum level of ASN is required for PDAC cells to execute ISR downstream adaptation. ASN depletion significantly enhanced the anti-tumor efficacy of OXPHOS inhibition both in vitro and in vivo. SLC38A4 emerged as a potential mediator of this interaction. SLC38A4 expression was associated with c-Myc, and its loss increased the sensitivity of PDAC cells to CTO-induced metabolic stress. ConclusionOur findings reveal a CAF-tumor metabolic crosstalk in which stromal-derived ASN supports PDAC cell adaptation to mitochondrial metabolic stress. Adaptive outcome of ISR signaling depends on the availability of key metabolic substrates such as ASN. When extracellular ASN supply is limited, the ATF4-dependent adaptive program collapses, converting ISR from a pro-survival response into a therapeutic vulnerability. SLC38A4 may function as a key mediator of this metabolic coupling and represents a potential target for enhancing the efficacy of OXPHOS inhibition in PDAC.

Colorectal cancer (CRC) poses a severe global health threat with high incidence, mortality, and poor 5-year survival rates for advanced cases despite existing treatments. This study aims to explore the role of STRIP2 in CRC progression and its underlying mechanisms. Impact of STRIP2 on CRC in vitro was investigated via CRC cell proliferation, migration, invasion, and apoptosis. The in vivo impact was investigated via nude mice models. The role of STRIP2 in CRC was investigated via transcriptomic analysis, Western blot, Co-immunoprecipitation assays and ferroptosis validations. STRIP2 is overexpressed in CRC, driving malignant phenotypes in vitro and in vivo. Mechanically, STRIP2 stabilizes the IL17 downstream effector LCN2 by blocking its K48-linked ubiquitination and degradation, enhances anti-ferroptosis of CRC cells. Oe-STRIP2 suppresses ferroptosis, boosting proliferation and reducing oxidative stress; while si-STRIP2 induces the opposite effect. This study suggests STRIP2-mediated stabilization of LCN2 and enhances CRC cells ferroptosis resistance, thus promoting CRC cell survival and mediates malignant progression in CRC, which provides a novel link between STRIP2 and ferroptosis regulation in CRC. HighlightO_LISTRIP2 is overexpressed in CRC tissues and cells C_LIO_LISTRIP2 blocks LCN2 Ubiquitination and stabilizes LCN2 C_LIO_LISTRIP2 suppresses CRC ferroptosis C_LIO_LISTRIP2 drives CRC malignant phenotypes both in vitro & in vivo C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/725308v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1baf7baorg.highwire.dtl.DTLVardef@1de15d9org.highwire.dtl.DTLVardef@16c8078org.highwire.dtl.DTLVardef@667840_HPS_FORMAT_FIGEXP M_FIG C_FIG

The NAD+ dependent deacetylase sirtuin-1 (SIRT1) is known to elicit cellular defenses against aging, cancer, and other aberrant pathologies. Previous studies have identified an intrinsically disordered region of SIRT1 comprised of N-terminal residues 1-52, herein referred to as motif A, which activates SIRT1 activity, likely through intramolecular interactions. Additionally, phosphorylation of N-terminal residues Ser27 and Ser47 has been shown to be important for regulating SIRT1 activity and stability. The lack of in vitro characterization of these effects hampers our further understanding of the role of motif A in SIRT1 regulation. In this study, we elucidate the role phosphorylation plays in motif As structure as well as its regulatory effects on SIRT1 activity against Ac-p65. We find that phosphomimetic mutation at Ser27 significantly increases the activation effect of motif A towards SIRT1. This result is supported by molecular dynamics simulations of the phosphomimetics, which reveal stabilization of different transient structures for motif A depending on whether Ser27 and Ser47 have been modified. A key finding suggested by this study is that phosphorylation of S27 appears to activate SIRT1 by causing motif A, which is intrinsically disordered in the WT, to fold into an ordered structure. This conclusion is based on both the experimental findings and simulation results. These findings contribute to our understanding of SIRT1 regulation, specifically the role played by phosphorylation within the N-terminal disordered region.

Genetic deletion or pharmacological blockade of P2X7 receptors (Rs) counteract status epilepticus (SE) in animal models of epilepsy. It is, however, unclear whether P2X7Rs are localized at astrocytes or neurons, and the reason for astrocytic atrophy arising in consequence of SE is also ambiguous. We conducted a combined morphological/electrophysiological study in order to investigate these issues. It has been shown that kainic acid (KA)-induced SE in mice led to the atrophy of hippocampal astrocytes and at the same time to the decrease of ezrin immunoreactivity and its co-expression with mCherry, whose synthesis has been initiated by the injection of a virus complex. mCherry expression in astrocytes enabled us to study changes in cell somata and processes brought about by KA-injection. Ezrin is a plasmalemmal-cytoskeleton linker; its grade of expression indicates changes in the existence/function of small peripheral astrocytic processes. Pretreatment of mice with the blood-brain barrier-permeable P2X7R antagonist JNJ-47965567 prevented the SE-induced damage of astrocytes. KA caused a potentiation of dibenzoyl-ATP (Bz-ATP) currents in astrocytes but not neurons of the hippocampus. This effect was also abolished by pre-treatment of mice with JNJ-47965567 before applying KA, although no similar changes occurred in hippocampal CA1 neurons. The measurement of spontaneous postsynaptic currents (sPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) indicated a presynaptic facilitation of neurotransmitter release by Bz-ATP. In conclusion, we suggest that astrocytic P2X7Rs are the primary target of ATP release from damaged CNS cells in the hippocampus which simultaneously causes damage to astrocytic somata and processes.

The PI3K-AKT-MTOR signalling axis is pivotal in regulating cell survival, proliferation, and growth. TSC2 (tuberous sclerosis complex subunit 2) is a well-established negative regulator of this pathway, which primarily acts by suppressing the MTORC1 activity. While the cytoplasmic role of TSC2 is well characterized, emerging evidence suggests its additional nuclear functions. Previous work from our laboratory identified TSC2 as a transcriptional repressor of the EREG (Epiregulin) gene. Building on this foundation, the present study investigates the transcriptional role of TSC2 in miRNA (microRNA) gene regulation. A genome-wide miRNA microarray profiling of TSC2-depleted cells from an oral squamous cell carcinoma (OSCC) cell line, SCC131, identified 19 upregulated and 24 downregulated miRNAs. Of them, miR-514b-3p emerged as one of the most significantly upregulated miRNAs. TSC2 knockdown resulted in robust miR-514b-3p upregulation, whereas TSC2 overexpression suppressed its expression. Moreover, TSC2 negatively regulates MIR514B promoter activity in an NLS-dependent manner. The chromatin immunoprecipitation analysis showed direct binding between TSC2 and MIR514B promoter, establishing miR-514b-3p as a transcriptional target of TSC2. We further identified TSPAN9 (Tetraspanin 9) as a direct downstream target of miR-514b-3p. The dual-luciferase reporter assay and Western blot analysis confirmed direct interaction between miR-514b-3p and TSPAN9 3UTR. Furthermore, TSC2 positively regulates TSPAN9 levels by repressing miR-514b-3p, thereby establishing a novel TSC2-miR-514b-3p-TSPAN9 regulatory axis. Additionally, we uncovered crosstalk between TSC2-miR-514b-3p-TSPAN9 axis and the canonical PI3K-AKT-MTOR signalling, where miR-514b-3p positively, and TSPAN9 negatively regulates the PI3K-AKT-MTOR pathway. Interestingly, AKT functions as an upstream regulator of this axis by modulating TSC2 nuclear localization. Collectively, this study provides new insights into the non-canonical, nucleus-dependent transcriptional functions of TSC2, thus expanding its role beyond cytoplasmic signalling regulation and underscoring its significance in the cellular signalling networks.

Parkinsons disease (PD) is the second most common neurodegenerative disease, resulting from accumulation of -synuclein (-syn) in midbrain dopaminergic neurons and progressive neuronal loss. The most relevant species of -syn, oligomers, may exert neurotoxicity in a variety of mechanisms. Accumulation of misfolded -syn in the endoplasmic reticulum (ER) lumen induces ER stress conditions that leads to activation of the Unfolded Protein Response (UPR) and its main sensor PKR-like ER kinase (PERK). PERK is critical for cell fate determination - under prolonged ER stress, it may direct cell towards pro-apoptotic pathways. Targeting of -syn aggregation or UPR by genetic and pharmacological approaches proved effective in preclinical models of PD by previous research. Thus, in the present study, we aimed to determine the potential effect of combination of small-molecule inhibitors of -syn aggregation and ER stress-mediated PERK signaling (namely anle138b and AMG44) in a novel, 3D in vitro model of PD. We demonstrate that combination of both anti-aggregation and ER stress-targeting approaches amplifies neuroprotection against PD in organoid model in terms of increased neuronal metabolic activity, decreased -syn phosphorylation and aggregation, reduced dopaminergic cell death, and restoration of proteostasis.

Modification by ubiquitination governs the half-lives of thousands of proteins that are fated for elimination by either the proteasome or autophagy pathways, depending on the intricate architectures of ubiquitin modification. This system mediates quality control for individual proteins, protein complexes, and organelles, as well as myriad purely regulatory functions. Here we provide a comprehensive survey of the ubiquitin-proteasome system (UPS), the scope of which is at present poorly defined. The UPS, with the inclusion of pathways involving ubiquitin-like modifiers, comprises in our estimate over 1400 distinct proteins in humans, a vast set of activities whose collective impact on the biology of the cell is pervasive. The UPS is an integral component of the proteostasis network (PN), the remainder of which we have also surveyed in recent studies. With the addition of molecular chaperones, proteins from autophagy-lysosome pathway, and related activities, the PN includes in total over 3100 components by our estimates. Comprehensive and systematic definition of these pathways should support a range of ongoing investigations in the areas of genomics, proteomics, biochemistry, cell biology, and disease research.

Background & AimsGastric epithelial cells maintain homeostasis through dynamic self-renewal mechanisms involving stem and progenitor cells; however, identifying them has been challenging. This study aims to identify stem cells of healthy gastric epithelium and cell type-specific regulators defining gastric epithelial homeostasis via single-nucleus multiome analysis. MethodsTen unique gastric samples were collected from 8-12 week old wildtype mice. Isolated nuclei were subjected to simultaneous profiling of gene expression and chromatin accessibility. After quality control, 31,598 cells were analyzed with Seurat and Signac using weighted-nearest neighbors analysis for joint RNA and ATAC clustering. Furthermore, SCENIC+, MultiVelo, EpiCHAOS and Cell plasticity score were used to uncover gene regulatory networks, cell state dynamics and lineage trajectories. ResultsOur analyses were validated by the identification of known regulators of stem-cell differentiation into mature cell types. More importantly, it revealed previously uncharacterized regulatory networks comprising novel transcription factor combinations that define cell identities, including Ppara, Pparg, Arid5b and Sox5 as candidate regulators of parietal, foveolar, chief and neck cells, respectively. Further, our data support the identity of isthmus cells as stem-like cells of healthy gastric epithelium, as evidenced by epigenetic plasticity that simultaneously contains open chromatin states of all differentiated cell types in the absence of transcriptional reprogramming. ConclusionConsistent with Waddingtons epigenetic landscape hypothesis, gastric epithelial homeostasis is controlled by orchestrated epigenetic and transcriptional programs. Contrary to the prevailing hypothesis, stem cells can be defined not by a separate epigenetic state but by epigenetic superposition of differentiated cell states. Future work is needed to define the universality of these results.

WD40 domains share a widespread {beta}-propeller fold, and often act as versatile scaffold proteins. Despite their central role in organizing dynamic cellular complexes, the molecular and structural mechanisms of many WD40 proteins remain poorly understood. Among them, DCAF7, an ubiquitously expressed and essential gene in human, also encodes a highly conserved WD40 protein in eukaryotic organisms. It is known to interact with multiple and functionnally diverse partners to coordinates cellular activity of several protein kinases as well as transcriptional regulators, thereby modulating key cellular processes such as cell growth, differentiation, and transcriptional regulation. However, the precise mode of action of DCAF7 is unknown and its important divergence in sequence from better characterize WD40 prevent information transfer by similarity. Structural interactomic can reveal how protein-protein interactions (PPIs) occur within an organism and are essential for understanding biological functions and developing new therapeutic strategies. Using SLiMAn2, AlphaFold2/3 and PSSMsearch, we identified a conserved -helical short linear motif (SLiM) in several well known DCAF7 partners that binds to the top surface of its {beta}-propeller. This motif was subsequently used to generate a regular expression, to identify potential new direct binders across the DCAF7 meta-interactome and the human proteome. Domain-domain interactions were also predicted for some other partners. Finally, modeling of oligomeric complexes with such new hits reveals the structural basis of DCAF7 scaffolding, with links to neurodevelopmental disorders such as autism.