Search Results (110)

Background: Systemic mastocytosis (SM) is a clonal mast cell (MC) neoplasm driven by somatically acquired activating mutations in the KIT receptor tyrosine kinase (CD117), resulting in pathological accumulation of morphologically atypical MCs in extracutaneous organs. The KIT D816V substitution is detectable in over 95% of cases by high-sensitivity next-generation sequencing (NGS) or allele-specific PCR. This gain-of-function variant confers ligand-independent receptor autophosphorylation, leading to constitutive activation of downstream signaling cascades that promote MC progenitor survival, clonal expansion, and resistance to apoptosis. Co-occurring somatic mutations in TET2, SRSF2, ASXL1, CBL, and RUNX1, increasingly identified in the context of clonal hematopoiesis of indeterminate potential, are associated with more aggressive phenotypes and independently confer adverse prognostic impact. Results: The 2022 WHO classification delineates indolent forms from advanced-phase SM, aggressive SM, SM with an associated hematologic neoplasm (SM-AHN), and MC leukemia, which produce progressive end-organ damage through both neoplastic tissue infiltration and uncontrolled mediator release. Formal diagnosis requires integration of histological criteria (multifocal bone marrow MC aggregates of ≥15 cells), immunophenotypic aberrancies (CD25, CD2, and/or CD30 coexpression on MCs by flow cytometry or immunohistochemistry), biochemical markers (baseline serum tryptase ≥ 20 ng/mL), and molecular confirmation of KIT D816V or equivalent pathogenic KIT mutation. The development of type I KIT inhibitors with selectivity for the D816V-mutant conformation has fundamentally restructured the therapeutic field of advanced SM. Conclusions: This review provides a thorough synthesis of SM pathobiology, WHO-defined diagnostic and classification criteria, validated prognostic tools, and the developing landscape of KIT-directed and combination therapies, with direct translational relevance for specialist practitioners managing this heterogeneous myeloid neoplasm. Full article

Escherichia coli, a facultative anaerobic Gram-negative member of the Enterobacteriaceae, is an increasingly important opportunistic pathogen driven in part by rising resistance to clinically important antibiotics. Regulation of multidrug efflux systems by two-component signal transduction pathways, particularly the BaeSR system, plays a central role in this process. However, the functional residues governing signal transduction through the sensor kinase BaeS remain incompletely defined. In this study, we integrated domain prediction, homology-guided site-directed mutagenesis, in vitro protein purification, autophosphorylation assays, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR)-based transcriptional analysis of selected BaeSR-regulated genes to delineate key residues required for BaeS function. Sequence analysis identified His250 as a candidate autophosphorylation site and Asn364 as a conserved residue within the catalytic domain. Biochemical characterization of purified wild-type BaeS and an H250A mutant demonstrated that His250 is indispensable for autophosphorylation. Consistently, RT-qPCR analysis showed that BaeS activation markedly induced the transcription of BaeSR-regulated efflux-associated genes, whereas genetic deletion of baeS or selective disruption of kinase activity by the N364A mutation abolished this response. Together, these findings establish His250 as a key residue for BaeS autophosphorylation and identify Asn364 as essential for inducible BaeSR signaling and activation of resistance-associated target genes, thereby establishing an experimental framework for elucidating BaeSR-mediated efflux regulation and informing future studies of resistance regulatory networks and potential intervention strategies centered on key signaling nodes. Full article

Background: Acquired resistance to the third-generation EGFR tyrosine kinase inhibitor osimertinib, often mediated by EGFR triple mutations, poses a major clinical challenge in non-small cell lung cancer (NSCLC) treatment. Among these, some rare mutations, such as L858R/T790M/L792H and L858R/T790M/G796R, create steric hindrance that directly interferes with osimertinib binding, yet effective targeted therapeutic strategies for these specific mutations remain lacking. Cudratricusxanthone A (CTXA), a natural xanthone derivative isolated from Cudrania tricuspidata Bur., has demonstrated various pharmacological activities, but its effects against EGFR triple-mutant NSCLC have not been systematically investigated. Methods: Stable Ba/F3 and NIH/3T3 cell lines expressing EGFR L858R/T790M/L792H or L858R/T790M/G796R triple mutations were generated via electroporation. The antiproliferative effects of CTXA were evaluated by MTT/MTS assays, colony formation, and wound healing assays. Cell cycle distribution and apoptosis were analyzed by flow cytometry. Protein expression of EGFR signaling pathway components (p-EGFR, p-ERK, p-AKT, p-STAT3) and cell cycle regulators (Cyclin D1, CDK4) were examined by Western blotting. Molecular docking and 200 ns molecular dynamics simulations were performed to investigate the stability and binding modes of CTXA to the mutant EGFR kinase domains. Results: The successfully established triple-mutant cell lines exhibited high EGFR expression, IL-3-independent growth, and significant resistance to osimertinib. CTXA inhibited the proliferation of all triple-mutant cell lines in a time- and concentration-dependent manner, with 48 h IC₅₀ values ranging from 0.362 to 2.488 μM. Mechanistically, CTXA suppressed EGFR autophosphorylation and downregulated downstream p-ERK, p-AKT, and p-STAT3. CTXA induced G1 phase cell cycle arrest by downregulating Cyclin D1 and CDK4, significantly promoted apoptosis, and inhibited cell migration. Molecular docking revealed that while osimertinib binding was blocked by steric hindrance from His-792 or Arg-796, CTXA adapted to the mutated ATP-binding pockets through multiple hydrogen bonds and extensive hydrophobic interactions. Molecular dynamics simulations confirmed the stable binding of CTXA to both mutant EGFR proteins over the 200 ns simulations. Conclusions: This study demonstrates for the first time that the natural compound CTXA possesses antitumor efficacy against EGFR L858R/T790M/L792H and L858R/T790M/G796R mutants by regulating EGFR-ERK/AKT/STAT3 signaling. Our findings position CTXA as a promising lead compound for tackling this challenging form of acquired resistance and highlight the value of natural products in multi-target antitumor drug discovery. Full article

Sucrose non-fermenting 1-related kinase (SNRK) is an understudied serine/threonine kinase of the CAMKL family, known for its role in metabolic regulation and cell signaling. Despite its emerging relevance in various biological processes and diseases, the phosphoregulatory landscape of human SNRK (valid substrates or role of its phosphosites) remains unexplored and demands robust, large-scale, data-oriented approaches to predict the potential substrates. A comprehensive analysis of global human phosphoproteomics datasets was performed to systematically identify class I phosphosites on SNRK, along with their predicted upstream kinases, potential downstream substrates, and coregulated phosphoproteins. Our analysis resulted in the identification of 33 dark SNRK phosphosites, of which 19 were differentially regulated across an array of experimental conditions. Among them, S518 and S569, outside their kinase domain, were the most frequently regulated and co-occurred phosphosites under diverse conditions. Notably, S569 is predicted as a candidate autophosphorylation site of SNRK. In these contexts, coregulation analysis of proteins and their phosphorylation sites suggested associations of phospho-SNRK in cell cycle progression, chromatin organization, and DNA replication. Uncovering candidate upstream kinases and potential substrates for prioritized validation, this study provides the first comprehensive phosphoproteomic map of SNRK, serving as a foundation for future investigations into its signaling network associations and therapeutic approaches. Full article

Marine bacteria are increasingly explored as alternative microbial platforms for the production of high-value biopharmaceuticals. In this study, we investigate the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125), an unconventional host capable of yielding soluble and biologically active human cyclin-dependent kinase-like 5 (hCDKL5). This serine/threonine kinase plays a crucial role in neuronal development, and its deficiency causes CDKL5 Deficiency Disorder, a severe and currently untreatable neurodevelopmental disease. Recombinant production of hCDKL5 is a prerequisite for the development of enzyme replacement therapy; however, current manufacturing processes remain insufficient for industrial translation, particularly in terms of product quality and functional consistency. To address these limitations, we developed dedicated analytical strategies: protein accumulation was quantified using a customised sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed to selectively detect full-length hCDKL5, while protein functionality was assessed by mass spectrometry-based quantification of autophosphorylation, a critical determinant of kinase activation. These complementary tools were applied to characterise hCDKL5 production under different growth conditions. Overall, this work establishes an integrated analytical framework aligned with a Quality by Design approach, enabling the simultaneous assessment of yield, structural integrity, and functional activation, and providing a robust basis for rational process optimisation towards scalable hCDKL5 manufacturing. Full article

Neurogranin (Ng) is a postsynaptic calmodulin-binding protein highly enriched in forebrain neurons and widely implicated in synaptic plasticity. However, whether Ng contributes more broadly to neuronal network maturation and cellular homeostasis remains unclear. Here, we examined the consequences of silencing or restoring Ng to adult physiological levels in primary hippocampal neurons. Ng expression promoted dendritic expansion, increased synaptic number, and shifted the axon initial segment toward the soma, consistent with structural adaptations to enhanced connectivity. Calcium (Ca²⁺) imaging revealed a marked increase in spontaneous neuronal activity and network synchronization, which was confirmed by electrophysiological recordings showing enhanced burst firing and spike synchrony. At the molecular level, Ng altered Ca²⁺/calmodulin (CaM) signaling by increasing total CaM levels, reducing Ca²⁺/CaM-dependent protein kinase II (CaMKII) abundance while increasing its relative autophosphorylation, and downscaling specific ionotropic glutamate receptors. Despite elevated network activity, Ng expression enhanced neuronal metabolic competence and viability, reduced cellular stress signaling and induced modest caspase-3 activation without engagement of apoptotic pathways. Together, these results indicate that Ng promotes neuronal maturation and coordinated network activity while engaging compensatory mechanisms that preserve excitatory balance and neuronal resilience. Our findings identify Ng as a molecular integrator linking Ca²⁺/CaM signaling with the structural and functional maturation of neuronal networks. Full article

Cells rely heavily on DNA repair networks to survive genomic damage. For repairing double-strand breaks, Non-Homologous End Joining (NHEJ) remains the primary pathway, which is largely controlled by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Researchers have long studied how phosphorylation drives this kinase. However, recent data point to an important additional layer of control. Drawing on evidence accumulated over the past two decades, we propose a “Spatiotemporal Logic Circuit” model for DNA-PKcs regulation. In this model, SUMO-associated interactions may help stabilize synaptic assembly, HUWE1-mediated neddylation may facilitate kinase activation at Lys4007, and K48-linked ubiquitination—potentially involving RNF144A—may contribute to the turnover of persistent repair complexes. Importantly, we frame these UBL-mediated events within the broader autophosphorylation-driven conformational cycle of DNA-PKcs, which remains central to NHEJ progression. Additionally, we highlight the structural interface where activation and degradation signals may converge and the extraction barrier posed by the massive DNA-PKcs scaffold. From a translational perspective, we argue that the exceptional size of DNA-PKcs (~470 kDa) and its topological entrapment on DNA render it an unusually challenging PROTAC target—one that may require p97/VCP-assisted extraction before proteolysis can proceed. We also highlight the underappreciated risk that E3 ligase loss-of-function, already documented in BET-PROTAC resistance, may similarly undermine DNA-PKcs degrader strategies. Full article

Drought stress severely constrains plant growth and productivity. To mitigate water loss, plants primarily regulate stomatal aperture through the Abscisic acid (ABA) signaling pathway, where the Sucrose Nonfermenting 1-Related Protein Kinase 2 (SnRK2) family kinase Open Stomata 1 (OST1) acts as a central positive regulator. However, the upstream regulators that fine-tune OST1 activity remain incompletely characterized. Aliphatic Suberin Feruloyl Transferase (ASFT), a BAHD acyltransferase essential for suberin aromatic monomer biosynthesis, was previously uncharacterized regarding its function in leaves. Here, we report that ASFT negatively regulates drought tolerance in Arabidopsis thaliana by directly interacting with OST1 and inhibiting its autophosphorylation, thereby restricting stomatal aperture. Consistent with this, the asft mutant exhibited decreased water loss and enhanced survival under drought, whereas ASFT-overexpressing lines showed opposite phenotypes. BiFC, Co-IP and in vitro kinase assays confirmed that ASFT directly interacts with OST1 and suppresses its autophosphorylation, while dehydration-induced OST1 phosphorylation was elevated in the asft mutant. Genetic evidence confirmed that ASFT functions upstream of OST1. This study reveals a moonlighting role for this suberin biosynthetic enzyme in ABA signaling and provides a potential target for breeding drought-resistant crops. Full article

Histidine kinases are an integral component of bacterial two-component systems (TCSs), playing a pivotal role in signal transduction pathways, resulting in both resistance and virulence. However, their inherent membrane-bound nature often results in poor solubility, making them difficult to isolate and rendering them incompatible with most in vitro biochemical techniques. Consequently, much of the research on two-component systems has centered on response regulators, limiting both drug discovery efforts and our broader understanding of key signal transduction mechanisms. To address these challenges, we sought to straightforwardly generate cytosolic truncation mutants of histidine kinases that retain their autophosphorylation and phosphotransfer capabilities. Previously, we successfully developed a cytosolic truncation mutant of PmrB (PmrBc) that maintained these critical functions, demonstrating its suitability as a viable surrogate for in vitro investigations, including inhibitor compound screening. Building upon this foundation, we have refined our methods and here demonstrate these improvements by producing functional histidine kinase truncation mutants from the following diverse bacterial species: Escherichia coli; PhoQ, BasS and Klebsiella pneumoniae; and PmrB and PhoQ. Full article

Mutations in leucine-rich repeat kinase 2 (LRRK2) are among the most common genetic causes of Parkinson’s disease (PD), yet substantial heterogeneity exists among pathogenic variants. How mutations in distinct functional domains of LRRK2 differentially perturb cellular homeostasis remains incompletely understood. Here, we compared two pathogenic LRRK2 mutations—G2019S in the kinase domain and I1371V in the GTPase domain—across multiple cellular models, including SH-SY5Y and U87 cells, and healthy human iPSC-derived floor plate cells. We demonstrate that the I1371V mutation induces markedly more severe cellular dysfunction than G2019S. I1371V-expressing cells exhibited elevated LRRK2 autophosphorylation at S1292 and robust hyperphosphorylation of Rab8A and Rab10, indicating enhanced downstream signaling. These alterations impaired sterol trafficking, leading to selective depletion of membrane cholesterol without changes in total cellular cholesterol. Consequently, I1371V cells displayed increased membrane fluidity, disrupted microdomain organization, altered membrane topology, reduced caveolin-1 expression, and impaired dopamine transporter surface expression and dopamine uptake. Lipidomic profiling further revealed a broad disruption of lipid homeostasis, including reductions in cholesteryl esters, sterols, sphingolipids, and glycerophospholipids, whereas G2019S cells showed comparatively modest changes. Pharmacological intervention revealed mutation-specific responses, with the non-selective LRRK2 modulator GW5074 outperforming the kinase-selective inhibitor MLi-2 in restoring Rab8A phosphorylation, membrane integrity, and dopaminergic function. Collectively, these findings identify membrane lipid dysregulation as a central cell biological mechanism in LRRK2-associated PD and underscore the importance of variant-specific therapeutic strategies. Full article

Focal Adhesion Kinase (FAK) is a key regulator of tumor cell migration and survival, and its persistent overexpression in aggressive cancers has motivated ongoing efforts to identify novel small-molecule inhibitors. Despite this interest, progress in discovering new potent scaffolds has been limited. In this work, we applied a multistep computational workflow followed by experimental testing to refine hit selection and reduce the false positives typically associated with docking. DrugBank and several commercial libraries were screened using Exponential Consensus Ranking (ECR) docking, and molecular dynamics simulations were used to assess pose stability and interaction persistence. A subset of predicted binders was then tested in MG-63 (bone cancer) and MDA-MB-231 (breast cancer) cells using cell viability and wound-healing assays, followed by direct autophosphorylation assays with recombinant FAK. Several repurposed compounds, including clofazimine and tafamidis, produced clear dose-dependent effects on cell migration, although their inhibitory activity in biochemical assays remained weak (${\mathrm{IC}}_{50}$ values above 100 $\mu$M), far from the potency of the reference inhibitor TAE226. Retrospective analysis of the computational workflow showed that standard MM-GBSA calculations did not correlate with these experimental outcomes. However, incorporating explicit water molecules through the NWAT-MMGBSA approach improved agreement with the biochemical data and helped to rationalize the limited affinity observed experimentally. Taken together, the results underline the relevance of explicit solvation in modeling the FAK active site and suggest that refined solvent-aware protocols may provide more reliable guidance for future screening efforts. Full article

Drought is the single largest abiotic threat to soybean yield, yet the lectin genes that mediate drought perception and signaling in this crop have never been systematically mapped. We reanalyzed the public RNA-seq dataset GSE237798 (Williams 82 leaves, 7-day water withdrawal) with an updated fastp–HISAT2–featureCounts–DESeq2 pipeline and a curated catalog of 359 soybean lectin loci. Of the 127 lectin transcripts showing any drought-dependent shift, only 15 were stringently differentially expressed with substantial fold changes: 7 were upregulated and 8 downregulated. These genes span four families, GNA, legume, LysM and Nictaba-related lectins, and are heavily biased toward lectin receptor-like kinases (11 of 15), pinpointing the plasma membrane as the main control node. Gene Ontology enrichment highlights protein autophosphorylation and signal-transduction terms, and the inspection of AlphaFold models together with established lectin knowledge indicates that G- and L-type lectin domains have largely lost canonical carbohydrate-binding residues, whereas LysM and Nictaba proteins retain conserved folds compatible with ligand binding. The data expose a focused, modular lectin program rather than the broad activation often assumed: most soybean lectins stay silent under drought conditions, and only a defined subset toggles their expression, albeit mildly. Full article

Abnormal expressions and genetic mutations of EGFR are broadly involved in the progression of many human solid tumors, which has led to the development of small molecule inhibitors (TKIs). However, patients’ tumors usually develop resistance to targeted therapeutic TKIs after a period of treatment, mostly due to secondary mutations in EGFR. To date, three major and prevalent point mutations in EGFR, including L858R, T790M, and C797S, impact the use of TKIs in non-small cell lung cancer patients. Although at least four generations of TKIs have been designed and developed by targeting these mutations, how each mono, dual, or triple variant responds to clinical TKIs remains largely undeciphered. To fill this gap, we constructed a series of EGFR mutants and assessed their responses to clinical TKIs in vitro. The first-generation TKI, erlotinib, completely blocked the autophosphorylation of WT, L858R, C797S, and C797S/L858R, but only partially, if at all, in EGFR containing the T790M mutation alone or in combination. The third generation, osimertinib, completely abolished the autophosphorylation of WT, T790M, L858R, and T790M/L858R. It also significantly inhibited C797S and C790S/L858R, but had no effect on T790M/C797S or T790M/C797S/L858R. EAI045, as the fourth-generation TKI, almost completely inhibited WT and all mutants in complete growth media, but EGF-mediated phosphorylation of WT, C797S, and C797S/L858R were only partially inhibited in quiescence media, while the other mutants were fully inhibited. Furthermore, the abolishment of the enhanced tolerance to Dox in cells transiently expressing T790M/L858R and T790M/C797S/L858R by EAI045 suggests that their enhanced autophosphorylation is involved in their resistant ability. These findings provide some insights into how patients carrying typical mutations should be correctly and efficiently treated and why patients present side effects (because of non-specific inhibitory effects on cells without EGFR mutations). Full article

Leucine-rich repeat receptor-like kinases (LRR-RLKs) have evolved to perceive environmental changes. Among LRR-RLKs, PEPR1 perceives the pep1 peptide and triggers defense signal transduction in Arabidopsis thaliana. In the present study, we focused on PEPR1 and PEPR2, which are the receptors of pep1, to understand the role of tyrosine phosphorylation. PEPR1-CD (cytoplasmic domain) recombinant protein exhibited strong tyrosine autophosphorylation, including threonine autophosphorylation. We subjected all tyrosine residues in PEPR1-CD to site-directed mutagenesis. The recombinant proteins were purified along with PEPR1-CD, and Western blotting was performed using a tyrosine-specific antibody. Among the 13 tyrosine residues in PEPR1-CD, the PEPR1(Y995F)-CD recombinant protein showed significantly reduced tyrosine autophosphorylation intensity compared to PEPR1-CD and other tyrosine mutants, despite little change in threonine autophosphorylation. To confirm the autophosphorylation site, we generated a phospho-specific peptide Ab, pY995. As a result, Tyr-995 of PEPR1-CD was a major tyrosine autophosphorylation site in vitro. To understand the function of tyrosine phosphorylation in vivo, we generated transgenic plants, expressing PEPR1-Flag, PEPR1(Y995F)-Flag, and PEPR1(Y995D)-Flag in a pepr1/2 double mutant background. Interestingly, the root growths of PEPR1(Y995F)-Flag and PEPR1(Y995D)-Flag were not inhibited by pep1 peptide treatment, compared to Col-0 and PEPR1-Flag (pepr1/2) transgenic plants. Also, we analyzed downstream components, which included PROPEP1, MPK3, WRKY33, and RBOHD gene expressions in four different genotypes (Col-0, PEPR1-Flag, PEPR1(Y995F)-Flag, and PEPR1(Y995D)-Flag) of plants in the presence of the pep1 peptide. Interestingly, the expressions of PROPEP1, MPK3, WRKY33, and RBOHD were not regulated by pep1 peptide treatment in PEPR1(Y995F)-Flag and PEPR1(Y995D)-Flag transgenic plants, in contrast to Col-0 and PEPR1-Flag. These results suggest that specific tyrosine residues play an important role in vivo in the plant receptor function. Full article

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that develop in the gastrointestinal tract; KIT mutations are present in both canine and human GISTs. In this study, genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) sections of 55 canine GIST cases, and mutation searches were performed for exons 8, 9, and 11. The results revealed novel mutations, A434T and F436S, in exon 8. In contrast to the A434T mutation without functional changes, the F436S mutant retained its dimerization ability, but lost its phosphorylation function and attenuated downstream Akt signaling, which is reflected in wound healing and migration activities. A comparison of the subcellular localization of WT KIT and the F436S mutant revealed no differences. In silico simulations indicated that the F436S mutation alters the structure of the near-membrane region and that its effects may extend to the transmembrane and intracellular domains compared to the WT. F436S is a point mutation that affects the entire molecule because co-mutation with the F436S mutation and the known autophosphorylation mutation reduces the autophosphorylation abilities. Full article