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.

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.

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

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.

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

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.

BackgroundHepatic stellate cells (HSC) are Vitamin A storing non-parenchymal cells of the liver. During injury and inflammation, HSCs are the major contributors of excessive extracellular matrix (ECM) leading to Liver Fibrosis (LF). Emerging evidence suggests a fibrosis-independent role of these cells as key regulators of liver homeostasis and liver regeneration, emphasising on the dual role of HSCs in liver. HSCs are known to secrete several growth factors through which they largely execute their functions. However, the role of secretome (exosomes) from early activated or undifferentiated HSCs in a fibrotic milieu nor its composition are completely understood. MethodsLX-2 cells were cultured in low to no serum conditions and their isolated exosomes were transplanted into fibrotic severe combined immune deficient (SCID) mice livers, followed by post-transplantation analysis of the liver tissue and compared to the untreated controls. Total proteomic profiling of cell and exosomal cargo was performed using mass spectrometry and the data analysed and compared with the total HSC cell proteome. ResultsSignificant reduction in collagen in the transplanted mice livers compared to untreated fibrotic controls was observed with both the cells and exosomes transplantation. Comparative analysis revealed distinct enrichment of proteins and signaling pathways associated with extracellular matrix regulation, cellular communication, and metabolism in exosomes. Notably, these pathways are prominently represented in the exosomal fraction, suggesting a selective packaging of functional mediators. ConclusionThis study suggests the potential role of HSCs in regulating the complex liver homeostasis via exosomal network of proteins that contribute significantly to liver repair by ECM remodelling and growth factor-mediated signalling to regulate metabolism, fibrosis and liver regeneration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/721862v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@99bbf4org.highwire.dtl.DTLVardef@1029dd0org.highwire.dtl.DTLVardef@c6f578org.highwire.dtl.DTLVardef@1dba81_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chemotherapeutic treatment of breast cancer with Doxorubicin (DOX) can induce tumor and stromal cell senescence leading to therapy-resistance. Senescence-associated secretory phenotype (SASP) promotes secretion of pro-inflammatory and tumorigenic factors causing systemic inflammation. Combined, this can result in immune suppression, tumor growth and secondary spread of cancer. Targeting and removing senescent and cancerous cells using a combination of chemotherapeutic and senolytic drugs may reduce systemic inflammation, improve therapeutic efficacy, and prevent metastasis. Exposure of triple-negative breast cancer (MDA-MB-231), hormone-responsive (MCF-7) and HER2+ (MDA-MB-453) cells, and primary spine osteoblasts to DOX showed significant induction of p21-positive senescent cells. DOX and senolytics (RG-7112, o-Vanillin) treatment of co-culture spheroids showed a significant additive effect in reducing tumor sphere viability and growth, indicating reduced metastatic potential. This was correlated with reduced SASP in triple-negative and hormone responsive lines and decreased levels of senescent cells in all subtypes and primary stromal cells, while proliferation was decreased, and apoptosis increased across all breast cancer subtypes. Future chemotherapeutic treatment in breast cancer models may be optimized by adding senolytic drugs to more effectively clear senescent tumor and stromal cells, reducing risk for relapse and metastatic potential, while allowing for tissue regeneration in the bone metastatic environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/724653v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@c4cb8forg.highwire.dtl.DTLVardef@105219org.highwire.dtl.DTLVardef@17e0517org.highwire.dtl.DTLVardef@802bd2_HPS_FORMAT_FIGEXP M_FIG C_FIG Senolytics selectively eliminate senescent cancer and stromal cells and enhance Doxorubicin efficacy in a 3D bone-like tumor microenvironment model.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

The nuclear receptor superfamily is comprised of ligand-regulated transcription factors that contain an intrinsically disordered domain at the amino-terminal end, known as the N-terminal domain (NTD). While this poorly conserved domain is known to possess ligand-independent activation function (AF-1), few NTD functions are conserved between nuclear receptors (NRs). Identified roles in other receptors include androgen receptor (AR), estrogen receptor (ER) and mineralocorticoid receptor (MR). Here, we aim to define the function of the NTD of the farnesoid X receptor (FXR), a crucial regulator of lipid and bile acid metabolism. We show that the NTD engages in interdomain contact with other FXR domains. We also observe that the NTD interacts directly with coregulator proteins. Using mutagenesis, mammalian two-hybrid assays and molecular dynamics simulations, we identify and validate a novel SXXLF motif in the NTD which mediates interactions with both coregulators and the ligand binding domain. Mutation of the motif induces large changes in conformational and allosteric coupling in FXR. Our study identifies a new nuclear receptor-interacting motif that modulates the transcriptional activity of FXR. Graphical AbstractFXR-NTD regulates transcriptional activity through interdomain communication with the LBD and is also involved in co-activator recruitment. The SENLF motif is the first defined functional element within the FXR-NTD and mediates both NTD-LBD interaction and selective co-activator engagements to drive NTD-mediated transcriptional activity. O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/724725v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5a37aorg.highwire.dtl.DTLVardef@2fa9e1org.highwire.dtl.DTLVardef@13a19daorg.highwire.dtl.DTLVardef@1775ed2_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundRising antimicrobial resistance rates, require new therapeutic approaches such as antimicrobial peptides (AMPs), which are part of the innate immune defense, as alternatives to antibiotics. In this study, we aim to unravel the antibacterial activity of human histone H1.2 peptide against Pseudomonas aeruginosa and its potential immune modulatory role. MethodsWe used a hemofiltrate peptide database for antimicrobial peptide prediction to identify novel human AMPs. Thirteen sequences of histone H1 were identified as putative AMPs, synthesized, and tested against bacterial ESKAPE pathogens in a radial diffusion assay. SYTOX green assay, electrophoretic mobility shift assay, and differential proteomics assays were conducted to determine the mode of action of H1.2 peptide fragment. A crystal violet assay was performed to evaluate the inhibition of biofilm formation. The cytotoxicity of the peptide was tested in LDH and Alamar assays. Finally, to visualize the contributions of H1.2 in NETs formation, scanning electron microscopy was performed. ResultsThe H1.2 peptide inhibited the growth of P. aeruginosa in a dose and pH-dependent manner without cytotoxicity towards mammalian THP-1 cells. It acts on intracellular targets to inhibit the growth of P. aeruginosa. STRING analysis from the differential proteomics assay showed that H1.2 targets the downregulation of proteins involved in the biogenesis of outer membrane proteins, including the folding and trafficking of outer membrane proteins across the cytoplasmic membrane. Scanning electron microscopy images showed that H1.2 forms NET-like structures capable of trapping and immobilizing P. aeruginosa. ConclusionThe characterized antimicrobial activity of H1.2 points to a role for human histone H1 fragments in innate immunity and may represent a promising approach for the development of novel antibacterial therapies. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/724237v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1778ddborg.highwire.dtl.DTLVardef@26430org.highwire.dtl.DTLVardef@ffbfa2org.highwire.dtl.DTLVardef@7e38ae_HPS_FORMAT_FIGEXP M_FIG C_FIG Sec transport and BAM complex system including chaperone proteins and quality control proteases are inhibited by H1.2 in Pseudomonas aeruginosa.Outer membrane proteins (OMPs) are synthesized in the cytoplasm and transported across the inner membrane via the Sec translocase, assisted by SecA/SecB or ribosomes. In the periplasm, they are escorted by chaperones such as SurA to the BAM complex for insertion into the outer membrane. Here, we show that H1.2, an antimicrobial peptide, targets membrane biogenesis in P. aeruginosa through downregulating Sec translocase (SecA/SecB and SecYEG), SurA, and BAM complex. Therefore, leading to improper transfer, folding and insertion of OMPs into the outer membrane. Normally, misfolded proteins are degraded by the protease MucD to prevent toxic aggregation in the bacteria. However, with H1.2 inhibiting MucD the proteotoxic stress is exacerbated, ultimately compromising bacterial homeostasis and viability. Figure created using BioRender.com.

Cells employ a bevy of transcriptional and post-translational stress responses to tolerate the burden of misfolded proteins induced by stress. In particular, the heat shock response facilitates the upregulation of molecular chaperones and protein remodeling factors that mediate proteostasis in response to accumulated misfolded proteins in the nucleus and cytosol. However, in response to stress neurons struggle to induce a canonical heat shock response, highlighting our poor understanding of how neurons maintain proteostasis. Specifically, the ability of post-mitotic respiring cells to regulate the heat shock response in comparison to their rapidly dividing, predominantly glycolytic counterparts has been under-studied. In this study, we employ yeast models that are easily manipulated to generate energy via glycolysis or mitochondrial respiration by changing the carbon source in the media. Using this model, we demonstrate that Hsf1 activity, the heat shock response and proteostasis are impaired in respiring cells. Interestingly, our data show that reduced Hsf1 activity regulates viability of respiring cells, with respiring cells poorly tolerating constitutively activated Hsf1. Finally, we describe alternative post-translational programming of the molecular chaperones Hsp70 and Hsp104 that plausibly enables respiring cells to mediate proteostasis despite a dampened heat shock response. Our findings offer new insights into possible proteostatic strategies employed by cells in different metabolic conditions.

Conflicting roles have been proposed for the E. coli protein RatA. Initially described as a ribosome targeting toxin, a later report pronounced it the bacterial homologue to the inner mitochondrial membrane protein Coq10. Coq10 proteins are conserved from prokaryotes to human and implicated to serve a lipid chaperone role in the biosynthesis of ubiquinone, a crucial electron carrier during aerobic respiration. We recently identified that the contradictory results published for RatA can be attributed to a mis-annotation of the gene in the reference genome. Here, we further elucidate the molecular function of RatA. We clarify that RatA is not a toxin but serves as a lipid shuttle for ubiquinone from its cytosolic biosynthesis complex to the inner membrane. Furthermore, we show that the loss of RatA results in an impaired, but not abolished electron transport chain and demonstrate broad metabolic adaptations of the cells as a consequence. Therefore, we propose to rename RatA to UbiM to reflect its function and to be in accordance with the naming convention of other ubiquinone biosynthesis proteins.

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype lacking well-defined molecular targets, leaving chemotherapy as the primary treatment despite drug resistance, systemic toxicity, and high recurrence rates. Therefore, the development of effective and less toxic therapeutic agents is essential. This study investigated the anti-cancer potential of gloriosine, a bioactive alkaloid with antiproliferative activity and low toxicity toward normal breast cells. MethodsPotential targets of gloriosine were predicted using SwissTargetPrediction, TargetNet, and PharmMapper, and overlapping genes related to TNBC and glutamine metabolism were selected. Protein-protein interaction networks, Gene Ontology, and KEGG pathway enrichment analyses were performed. Molecular docking evaluated binding affinity, followed by in vitro validation using cell viability, colony formation, and wound healing assays. ROS levels were measured by DCFDA and GSH assays, and ferroptosis was assessed by Western blot and FerroOrange staining in MDA{square}MB{square}231 cells. ResultsA total of 100 potential targets were identified, with 60 overlapping with TNBC and glutamine metabolism-related genes. Key targets included SRC, EGFR, mTOR, and HSP90AA1. Enrichment analyses indicated involvement in cancer progression, metabolic regulation, and resistance pathways, including central carbon metabolism, EGFR inhibitor resistance, and ErbB signaling. Gloriosine showed strong binding affinity toward hub targets. Experimental studies confirmed concentration-dependent inhibition of cell proliferation and migration. Mechanistically, gloriosine suppressed glutamine metabolism via GLS1 downregulation and induced ferroptosis, evidenced by increased ROS, glutathione depletion, GPX4 downregulation, and elevated intracellular iron levels. ConclusionsGloriosine exerts significant anti-cancer effects in TNBC through multi-target modulation and induction of ferroptosis, highlighting its potential as a promising therapeutic candidate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/725321v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@ce0ebcorg.highwire.dtl.DTLVardef@29603borg.highwire.dtl.DTLVardef@6d0025org.highwire.dtl.DTLVardef@249700_HPS_FORMAT_FIGEXP M_FIG C_FIG Flow chart of the network pharmacological and in vitro study of gloriosine

BackgroundIn RAS-mutant tumors, ERK phosphorylates the mitochondrial fission GTPase DRP1 to promote mitochondrial fission. DRP1 activity is tumor-promoting in pancreatic and other RAS-driven cancers, but its role in therapeutic resistance is unknown. MethodsWe developed a panel of patient-derived pancreatic cancer cell lines resistant to the MEK inhibitor trametinib. We used immunofluorescence imaging, in vitro growth assays and orthotopic xenografts to determine the role of DRP1 in trametinib resistance. ResultsWe find that trametinib-resistant cells exhibit increased expression and phosphorylation of DRP1 compared to sensitive counterparts. Quantitative analysis of mitochondrial structure reveals that mitochondria in resistant cells are morphologically distinct and relatively smaller than sensitive cells treated with trametinib. Genetic and pharmacological inhibition of both c-Myc and CDK6 are sufficient to block DRP1 phosphorylation in resistant cells, suggesting that activation of a c-Myc-CDK6 signaling axis drives reactivation of mitochondrial fission in the absence of MAPK signaling. Importantly, deletion of DRP1 leads to either growth inhibition or re-sensitization to trametinib in resistant lines. ConclusionThese findings suggest DRP1 contributes to drug resistance, and that inhibition of mitochondrial fission might be a promising therapeutic strategy to combat resistance to MAPK and RAS inhibitors.

Centrosome dysfunction is linked to developmental disorders affecting brain and body size, including microcephaly and primordial dwarfism. However, the cellular mechanisms underlying these rare conditions remain poorly understood. In this study, we investigate a rare variant of the centrosome-associated protein Pericentrin, which was discovered in a single family with Majewski/microcephalic osteodysplastic primordial dwarfism type II (MOPD II). Unlike the majority of pathogenic PCNT variants that cause severe protein truncation, the p.Lys3154del variant ({Delta}K3154) involves a single amino acid deletion in the proteins only conserved functional domain, providing a unique opportunity to explore PCNT function in MOPD II. To model PCNT{Delta}K3154, we examined the effects of Drosophila Pericentrin-like protein (PLP) carrying an orthologous deletion (Plp{Delta}R). Our results show that plp{Delta}R animals exhibit smaller tissues that recapitulate MOPD II phenotypes. Behavioral assays revealed defects in climbing and mechanosensation, suggesting impaired sensory cilia function. We also found that Plp{Delta}R cells exhibit accelerated mitosis, increased apoptosis, and reduced pericentriolar material recruitment. In silico structural modeling, yeast two-hybrid, and co-immunoprecipitation experiments show that Plp{Delta}R produces a protein that disrupts PLP dimerization and PLP interaction with Asterless, another centrosome protein. Overall, modeling the human MOPD II patient variant PCNT{Delta}K3154 in Drosophila reveals how a single amino acid deletion affects biological processes from the molecular level to the organismal level. Our work offers new insights into the defective cellular mechanisms underlying MOPD II in patients with the PCNT{Delta}K3154 variant, potentially linking the etiology of the disease in these individuals to the loss of a single protein-protein interaction.

The biological conservation between fungi and mammals due to a common ancestor has made development of selective antifungal drugs a difficult challenge. Further complicating this situation is the selection of antifungal drug-resistant organisms during drug treatment. The pathogenic yeast Nakaseomyces glabratus (called here Candida glabrata) presents an especially challenging organism due to its tendency to frequently lose susceptibility to the major antifungal drug class the azoles. Additionally, C. glabrata develops resistance to echinocandin drugs, a second, more recently described antifungal agent at 10 times the rate of other organisms. Previous work has established that the sterol responsive transcriptional regulator Upc2A is a key determinant of azole susceptibility in C. glabrata and plays a role in echinocandin resistance. We used a biochemical approach to identify proteins that co-purified with Upc2A and identified the Ypk2 AGC kinase as an interacting protein. Strains lacking YPK2 exhibited increased susceptibility to fluconazole and the echinocandin caspofungin. A ypk2{Delta} strain failed to normally induce transcription of several ERG genes but exhibited normal induction of the CDR1 ATP-binding cassette transporter gene. Isogenic ypk2{Delta} strains were also highly susceptible to the three major classes of antifungal drugs, indicating that this kinase behaves as a multidrug susceptibility factor. RNA-seq analyses indicated that the transcriptional response to exposure is different for each drug and each response is differentially altered upon loss of Ypk2. Our data indicate that Ypk2 plays an important role in coordinating gene expression that impacts susceptibility to all major antifungal drug classes.

The activation of thermogenesis in brown adipose tissue (BAT) represents a pivotal target for ameliorating disorders of glucose and lipid metabolism. This study sought to elucidate the regulatory effects of quercetin on thermogenesis and glucose-lipid metabolism within brown adipocytes, alongside its underlying molecular mechanisms. The findings demonstrated that quercetin markedly upregulated the expression of uncoupling protein 1 (UCP1), a critical thermogenic protein in brown adipocytes, thereby enhancing cellular thermogenic capacity and effectively mitigating glucose and lipid metabolism disorders. Subsequent mechanistic investigations confirmed that quercetin activated the COX2-PGE2-EP4-UCP1 signaling axis by augmenting the stability of cyclooxygenase 2 (COX2) protein, thus mediating its thermogenic-promoting and metabolism-improving effects. This study identifies quercetin as a potential therapeutic agent for the improvement of glucose and lipid metabolism disorders, uncovers a novel molecular mechanism through which quercetin regulates brown adipocyte thermogenesis, and provides a theoretical and experimental foundation for the application of quercetin in the prevention and treatment of obesity and related metabolic diseases.

NKCC2, localized to the apical membrane of thick ascending limb epithelial cells, is essential for renal salt handling and systemic electrolyte homeostasis. NKCC2 undergoes extensive ubiquitylation, with the E3 protein ligase Nedd4-2 implicated as a key regulator. However, progress has been limited by challenges expressing NKCC2 in mammalian cell lines, hindering mechanistic studies of NKCC2 ubiquitylation. Therefore, the aims of this study were to develop a mammalian cell model enabling mechanistic investigations of NKCC2 ubiquitylation, including the role of Nedd4-2 and the functional consequences of site-specific modification. A tetracycline-inducible MDCKI cell line was generated expressing human NKCC2 and used to assess Nedd4-2-dependent and site-specific ubiquitylation of NKCC2 using biochemical, imaging, and functional assays. The MDCKI cell line demonstrated stable, inducible expression of full-length human NKCC2. In this cell line, mutating the ubiquitylation site at K871 increased membrane abundance and uptake activity, without altering internalization rates. Nedd4-2 co-immunoprecipitated with NKCC2, and Nedd4-2 deletion increased total, but not membrane NKCC2 abundance. In summary, ubiquitylation on NKCC2 at K871 plays a key role in controlling NKCC2 membrane localization and thus function. Although Nedd4-2 can modulate NKCC2 abundance, it is not involved in NKCC2 trafficking. We conclude that the generated cell line provides a robust model for mechanistic studies of NKCC2 and will aid studies examining posttranslational regulation of NKCC2.

Background and aimsTherapeutic outcomes for advanced hepatocellular carcinoma remain inadequate, despite recent advances using immunotherapy. Long-term effectiveness of systemic therapies, including second-line multi-tyrosine kinase inhibitor sorafenib, is limited by resistance mechanisms and adverse effects. Upregulated deubiquitinase UCH-L1 is frequently correlated with poor prognosis in cancers. Here, we investigated the therapeutic potential of combining pharmacological UCH-L1-inhibition with sorafenib in HCC. MethodsUCH-L1 expression was analysed in TCGA-LIHC data and patient-derived HCC tissues. Sorafenib and LDN57444 effects were evaluated in vitro in cytotoxicity and invasion assays. Gene and protein expression were examined by RT-qPCR, Western blotting and immunohistochemistry. In vivo efficacy of drug synergy was assessed in an orthotopic xenograft mouse HCC model. ResultsIn silico data-analysis revealed significantly higher UCH-L1 levels in patient HCC tumours versus non-tumour, associated with reduced overall survival. Low-dose sorafenib upregulated UCH-L1 in HCC cell line Hep3B. Paradoxically, this also promoted invasiveness and sustained MEK1/2-ERK1/2-pathway activation. Combining low-dose sorafenib with LDN57444 produced strong synergistic cytotoxicity in vitro, reverted MAPK-activation and suppressed invasion. Consistently, at low sorafenib dose co-treatment with LDN57444 completely inhibited tumour growth of Hep3B xenografts and enhanced sorafenib efficacy. ConclusionLDN57444 sensitises HCC cells to low-dose sorafenib by reverting drug-induced pro-oncogenic signalling and thereby strongly synergises with sorafenib to enhance anti-tumour efficacy in a HCC mouse model. This presents UCH-L1 as a player in treatment-induced adaptive response and supports further exploring UCH-L1-targeting in combination with sorafenib as therapeutic avenue for advanced HCC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/725527v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@176dc91org.highwire.dtl.DTLVardef@8acae8org.highwire.dtl.DTLVardef@f71bborg.highwire.dtl.DTLVardef@1f3c5aa_HPS_FORMAT_FIGEXP M_FIG C_FIG Lay summaryThis study explores a new treatment approach for hepatocellular carcinoma (HCC) by combining two drugs: LDN57444, which blocks the enzyme UCH-L1, and sorafenib, a FDA-approved multi-tyrosine kinase inhibitor. We evaluated the effect of this drug combination in vitro using a HCC cell line and in an mouse HCC-model. The drug combination displayed strong, synergy in lowering HCC cell viability, and greatly reduced invasiveness and in vivo tumour growth. LDN57444 sensitised HCC cells to low doses of sorafenib by preventing UCH-L1-mediated activation of pro-oncogenic signalling. These findings highlight the potential of this new drug combination for treating advanced HCC thereby potentially reducing side-effects and countering drug resistance. Impact and implicationsOur preclinical research introduces a novel combination strategy against advanced HCC that holds potential to improve existing therapies, particularly the second-line multi-tyrosine kinase inhibitor sorafenib. The proposed combination of sorafenib with an inhibitor of the deubiquitinase UCH-L1 not only enhances sorafenib efficacy but present promise to also counter resistance mechanisms. Moreover, because effective responses are achieved at lower drug doses, this may in addition reduce therapy-associated adverse effects further increasing potential impact. While sorafenib is FDA-approved, the UCH-L1 inhibitor LDN57444 needs further (clinical) development to bring our promising findings to full translational potential for HCC patients and physicians.