Src Kinase Research Articles

CD11b deficiency attenuates the ischemia/reperfusion-induced AKI-to-CKD process by regulatingmacrophage polarization.

Severe acute kidney injury (AKI) is a risk factor for the future development of chronic kidney disease (CKD), and macrophages are an essential cell group implicated in injury, repair, and fibrosis during this progress. However, the underlying mechanism of how macrophages participate in the development of the disease is largely unclear. CD11b (Integrin αM) is highly expressed in monocytes/macrophages and has been verified to mediate broad functions. We found that CD11b-knockout mice were protected from the ischemia/reperfusion-induced AKI-to-CKD process. Additionally, renal macrophages from the kidneys of CD11b-deficient mice presented an M2-like, anti-inflammatory phenotype. We demonstrated both invitro and invivo that enhanced alternative activation in CD11b-knockout macrophages is regulated by the interleukin (IL)-4/signal transducer and activator of transcription (STAT) 6 axis, which is mediated by the inactivation of the Src-family kinase (SFK) member Lyn. Importantly, the therapeutical administration of CD11b-neutralizing antibody can effectively prevent AKI-to-CKD progress. Thus, our study verifies that CD11b plays a critical role in limiting the alternative activation of macrophages and may be a potential therapeutic target during the AKI-to-CKD process.

DRIM modulates Src activation and regulates angiogenic functions in vascular endothelial cells.

Downregulated in Metastasis Protein (DRIM) was discovered in malignant epithelial cells and was thought to be mainly a nucleus protein affecting cancer cells. Recent single-cell sequencing analysis suggests that DRIM is abundantly expressed in vascular endothelial cells. There has been no knowledge of the role of DRIM in the endothelium. In the present study, using protein fraction method and cell imaging, we identified that the DRIM protein was abundantly present in both nucleus and the cytoskeletal fractions of human vascular endothelial cells. Knockdown of DRIM in the endothelial cells significantly affected growth, migration, and angiogenic tubule formation. Proteomics analyses revealed that Src was an important direct target protein of DRIM, a finding further confirmed by protein interaction assay. Silencing DRIM activated the tyrosine 419 site phosphorylation of Src kinase in endothelial cells, thereby affecting the downstream proteins of Src including p-FAK and p-STAT3, and exerting biological effects. To conclude, our results provide evidence of DRIM being a nuclear and cytoskeletal-associated protein, having a novel key role of the protein in vascular endothelial cells.

Extrajunctional CLDN10 cooperates with LAT1 and accelerates clear cell renal cell carcinoma progression

Background & aimsIn addition to their adhesive properties, cell adhesion molecules such as claudins (CLDNs) exhibit signaling ability to organize diverse cellular events. Although the CLDN-adhesion signaling stimulates or inhibits cancer progression, the underlying mechanism remains poorly established. Here, we verified whether and how CLDN10 promotes intracellular signals and malignant phenotypes in clear cell renal cell carcinoma (ccRCC).MethodsWe developed a novel monoclonal antibody that specifically recognizes CLDN10. By immunohistochemistry using this antibody, the clinicopathological significance of aberrant CLDN10 expression in 165 ccRCC patients was determined. We next generated the ccRCC cells (786-O, ACHN, and OS-RC-2) expressing CLDN10, and compared their phenotypes with those of control cells. Immunoprecipitation-mass spectrometry was used to identify a CLDN10-interacting protein, followed by evaluation of its association with CLDN10 and loss-of-functions in ccRCC cells.ResultsHigh CLDN10 expression predicted poor outcome in ccRCC patients and represented an independent prognostic marker for cancer-specific survival. Cell surface CLDN10 promoted cell viability, proliferation, and migration of ccRCC cells, as well as their tumor growth. CLDN10 also activated mTOR signaling and expression of downstream targets, including MYC target genes. Notably, we found that CLDN10 forms a complex with an amino acid transporter, LAT1, and that CLDN10–LAT1 signaling facilitates malignant phenotypes in ccRCC cells. Structural prediction and immunoprecipitation analysis results strongly suggest an interaction between CLDN10-TM1 (transmembrane domain 1) and LAT1-TM4.ConclusionsWe conclude that CLDN10–LAT1 signaling drives ccRCC progression. Taken together with our previous findings on CLDN–Src-family kinases signaling, CLDNs propagate distinct intracellular signals depending on their association with different binding partners.

Fyn, an important molecule in the brain, is a potential therapeutic target for brain tumours.

Under normal physiological conditions, Fyn, a nonreceptor tyrosine kinase, is involved in signal transduction pathways in the nervous system and in the formation and activation of T lymphocytes. Fyn is a member of the Src family of kinases (SFKs) and plays a role in cell morphogenic transformation, motility, proliferation, and death, which in turn influences the development and progression of various cancer types. SFKs are overexpressed or hyperactive in tumours, and they are engaged in several signalling pathways that lead to tumour development. Inhibition of Fyn can enhance patient outcomes and prolong survival. Thus, Fyn is a desirable therapeutic target in a variety of tumour types. To lay the groundwork for further investigation and targeted therapy in tumours, in this article, we review the most recent findings on the function of Fyn in tumours, with an emphasis on its role in gliomas. Understanding the function of Fyn during tumourigenesis and development and in resistance to anticancer therapeutic agents can aid in the development and application of innovative medicines that specifically target this kinase, thus improving the management of cancers.

Systematic pharmacology-based strategy to investigate the mechanism of beta-sitosterol for the treatment of rheumarthritis.

Objective: β-Sitosterol, which is derived from Vladimiriae Radix (VR), is used for the treatment of rheumatoid arthritis (RA), but the pharmacological mechanisms through which β-sitosterol affects RA have not been fully elucidated. Methods: Through the Traditional Chinese Medicine Systems Pharmacology and Analysis (TCMSP), PubChem, SwissTargetPrediction, GeneCards, DisGeNET, and OMIM databases, "β-sitosterol-RA"-related genes were obtained, and a target protein interaction network (protein-protein interaction [PPI]) was constructed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out for the intersecting genes. Discovery Studio 2019 software was used to perform molecular docking on MMP9, CASP3, HSP90AA1, SRC, EGFR, and ALB genes. β-Sitosterol was co-cultured with MH7A cells in three experimental groups: control group (DMSO), positive drug group (methotrexate, 80μmol/L), and drug intervention group (10, 20, 40, 80, and 160μmol/L β-sitosterol). The CCK8 method was used to investigate the inhibitory effect of β-sitosterol on the proliferation of MH7A cells. RT-PCR was used to analyze the mRNA expression of the abovementioned core targets. Results: A total of 41 genes associated with β-sitosterol and RA were obtained, mainly involving the FoxO signaling pathway and PI3K/AKT signaling pathway. The molecular docking results suggested that β-sitosterol could bind effectively to six core targets. The experimental results showed that β-sitosterol could significantly inhibit the excessive proliferation of MH7A cells (p< 0.05). The RT-PCR results showed that the expression of MMP9, HSP90AA1, SRC, EGFR, and ALB core genes in the control group was significantly upregulated, while the CASP3 gene was downregulated. Compared to the control group, the mRNA expression of MMP9, HSP90AA1, SRC, EGFR, and ALB decreased (p< 0.01), while the apoptosis-related gene CASP3 increased in both the drug intervention (80μmol/L β-sitosterol) and positive drug groups (80μmol/L methotrexate). Conclusion: Hence, β-sitosterol could contribute to the inhibition of RA by modulating cell proliferation and regulating the aforementioned six core proteins, potentially through the regulation of the FoxO and PI3K/AKT signaling pathways.

N-methyl-D-aspartate receptor activation is downstream coupled to pannexin 1 opening by Src kinase in dorsal horn neurons: an essential link for mechanical hyperalgesia in nerve-injured rats.

A well-recognized molecular entity involved in pain-related neuroplasticity is the N-methyl-D-aspartate receptor (NMDAR), which is crucial for developing chronic pain. Likewise, the pannexin 1 (Panx1) channel has been described as necessary for initiating and maintaining neuropathic pain, driving nociceptive signals dependent on spinal NMDAR through several possible mechanisms. Through behavioral, pharmacological, and molecular approaches, our study in male rats has revealed several key findings: (1) neurons located in spinal cord laminae I and II express functional Panx1 channels in both neuropathic and sham rats. These channels can open (indicated by YOPRO-1 uptake) through the stimulation of NMDARs with intrathecal NMDA; (2) intrathecal NMDA leads to increased expression of pSrc and pPanx1 in dorsal horn neurons. This elevation exacerbates existing mechanical hyperalgesia in nerve-injured rats; (3) inhibition of Src with intrathecal PP2 or blockade of Panx1 with intrathecal 10Panx effectively mitigates NMDA-induced effects and reduces the spontaneous mechanical hyperalgesia of nerve-injured rats. Notably, while 10Panx successfully alleviates hyperalgesia, it does not alter pSrc expression; and (4) NMDA-stimulated YOPRO-1 uptake in neurons of laminae I-II of spinal cord slices were prevented by the NMDAR antagonist D-AP5, the Src inhibitor PP2 (but not PP3), as well as with the 10Panx and carbenoxolone. Therefore, NMDAR activation in dorsal horn neurons triggers an NMDAR-Src-Panx1 signaling pathway, where Panx1 acts as an enhancing effector in neuropathic pain. This implies that disrupting the NMDAR-Panx1 communication (eg, through Src inhibitors and/or Panx1 blockers) may offer a valuable strategy for managing some forms of chronic pain.

Cryo-EM Detection of AMPylated Histidine Implies Covalent Catalysis in AMPylation Mediated by a Bacterial Effector.

AMPylation is a post-translational modification (PTM) whereby adenosine monophosphate (AMP) from adenosine triphosphate (ATP) is transferred onto protein hydroxyl groups of serine, threonine, or tyrosine. Recently, an actin-dependent AMPylase namely LnaB from the bacterial pathogen Legionella pneumophila was found to AMPylate phosphate groups of phosphoribosylated ubiquitin and Src family kinases. LnaB represents an evolutionarily distinct family of AMPylases with conserved active site Ser-His-Glu residues. Here, we capture the structure of the LnaB-actin complex in a putative intermediate state via single-particle cryogenic electron microscopy (cryo-EM) and find that the catalytic histidine of LnaB is covalently attached to AMP through a phosphoramidate linkage at the Nδ1 atom. This observation provides direct structural evidence of histidine AMPylation as a PTM and implies the possibility of covalent catalysis in LnaB-mediated AMPylation, a mechanism distinct from known AMPylases. Subsequent biochemical studies confirm the observed AMP binding site and provide additional insights into the catalytic properties of LnaB. Together, our work highlights the power of cryo-EM in capturing labile PTMs and transient species during enzymatic reactions, while opening new avenues of mechanistic investigation into the LnaB family.

The inhibition of rutin on Src kinase blocks high glucose-induced EGFR/ERK transactivation in diabetic nephropathy by integrative approach of network pharmacology and experimental verification

BackgroundAlthough clinical strategies for diabetic nephropathy (DN) therapy include stringent blood pressure control through blockade of the renin-angiotensin system and management of hyperglycemia, the condition is still observed to progress relentlessly. PurposeTo elucidate the protective effects of rutin on podocytes in db/db mice with integrative approach of network pharmacology and experimental verification. MethodsThe study employs network pharmacology to identify common targets between rutin and DN, constructs a potential protein-protein interaction (PPI) network, and conducts Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Molecular docking is utilized to evaluate the interaction between rutin and protein targets. Additionally, experimental validation is performed using db/db mice and human podocyte cell models. ResultsRutin has been found to have a significant renoprotective effect, reducing blood glucose, proteinuria, and improving renal function in db/db mice. Rutin's inhibition of Src kinase reduces the phosphorylation levels of EGFR and ERK, which may mitigate podocyte injury. Additionally, rutin exhibits antioxidant properties, capable of lowering the levels of reactive oxygen species (ROS) in kidney tissue and increasing the activity of antioxidant enzymes like superoxide dismutase (SOD). These effects help protect podocytes from oxidative stress, further supporting the potential application of rutin in the treatment of DN. ConclusionsThe inhibition of rutin on Src kinase blocks high glucose-induced EGFR/ERK transactivation and protects podocyte injury in DN, indicating it might serve as a promising therapeutic agent for podocyte-targeted therapies.

Structure of Full-Length Src Kinase and Its Key Phosphorylated States: Molecular Dynamics Study.

Src kinase is one of the key regulators of cellular metabolism and is dysregulated in numerous diseases, including cancer, neurodegenerative diseases, and particularly Alzheimer's disease. Despite its therapeutic importance, its full-length structure has never been obtained before, as it contains an intrinsically disordered regulatory region, SH4UD. The SH4UD region is crucial for Src activation, functional dimerization, and regulation by other kinases. In this study, we used the replica exchange molecular dynamics approach with a hybrid temperature and Hamiltonian tempering to obtain the conformational ensemble of full-length Src kinase in its non-phosphorylated state and in the presence of its two key regulatory phosphorylations: pY419 and pY530. The representative structures and simulation trajectories of non-phosphorylated pY419 and pY530 Src are available in open access. We demonstrate that pY419 phosphorylation, which is associated with Src activation, enhances its motility, whereas inhibited pY530 Src preserves relatively compact conformation. This study also provides insights into how SH4UD contributes to Src substrate binding, dimerization, and autophosphorylation, highlighting the putative role of 14-RRR-16 in this process.

20-Hydroxyeicosatetraenoic Acid Regulates the Src/EGFR/NF-κB Signaling Pathway Via GPR75 to Activate Microglia and Promote TBI in the Immature Brain.

20-Hydroxyeicosatetraenoic acid (20-HETE) is associated with secondary damage in traumatic brain injury (TBI) of the immature brain. Microglial activation is pivotal in this process. However, the underlying mechanism of action remains unknown. While 20-HETE interacts with G protein-coupled receptor 75 (GPR75) in some pathological processes, their interaction in brain tissue remains uncertain. This study aimed to investigate whether 20-HETE can activate microglia by binding to GPR75 in TBI of the immature brain. Drug affinity responsive molecular target stability (DARTS) assays, cycloheximide (CHX) chase assays, and auto-dock assays were employed to analyze the interaction between 20-HETE and GPR75. The expression levels of cytochrome P450 4A (CYP4A) and GPR75 in activated microglia in an immature brain TBI model were observed by western blot and multiple immunofluorescence staining. The effects of different levels of 20-HETE expression and lentivirus-mediated GPR75 gene silencing on 20-HETE-induced inflammatory factor release from BV-2 cells were observed by enzyme-linked immunoassay (ELISA). The phosphorylation levels of the downstream Src kinase, epidermal growth factor receptor (EGFR), and nuclear factor (NF)-κB were assessed using western blot. Cell viability and apoptosis were detected by CCK-8 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. 20-HETE bound to the GPR75 protein and inhibited its degradation. GPR75 gene silencing reversed the 20-HETE-induced inflammatory activation of BV-2 cells, effectively inhibiting the activation of the Src/EGFR/NF-κB pathway and the effects of 20-HETE on cell viability and the apoptosis rate. In contrast, overexpression of GPR75 had the opposite effect. In addition, after immature brain TBI, the 20-HETE and GPR75 expression levels were upregulated in microglia, with significant activation of the Src/EGFR/NF-κB pathway. Inhibition of 20-HETE synthesis with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) produced the opposite effect. 20-HETE regulates the Src/EGFR/NF-κB signaling pathway via GPR75 to activate microglia, promoting immature brain TBI. These findings offer a novel target for promoting the brain injury effect of 20-HETE.

Antimicrobial Peptide Pro10-1D Exhibits Anti-Allergic Activity: A Promising Therapeutic Candidate.

Although antimicrobial peptides (AMPs) exhibit a range of biological functions, reports on AMPs with therapeutic effects in allergic disorders are limited. In this study, we investigated the anti-allergic effects of Pro10-1D, a 10-meric AMP derived from insect defensin protaetiamycine. Our findings demonstrate that Pro10-1D effectively inhibits antigen-induced degranulation of mast cells (MCs) with IC50 values of approximately 11.6 μM for RBL-2H3 cells and 2.7 μM for bone marrow-derived MCs. Furthermore, Pro10-1D suppressed the secretion of cytokines with IC50 values of approximately 2.8 μM for IL-4 and approximately 8.6 μM for TNF-α. Mechanistically, Pro10-1D inhibited the Syk-LAT-PLCγ1 signaling pathway in MCs and decreased the activation of mitogen-activated protein kinases (MAPKs). Pro10-1D demonstrated a dose-dependent reduction in IgE-mediated passive cutaneous anaphylaxis in mice with an ED50 value of approximately 7.6 mg/kg. Further investigation revealed that Pro10-1D significantly reduced the activity of key kinases Fyn and Lyn, which are critical in the initial phase of the FcεRI-mediated signaling pathway, with IC50 values of approximately 22.6 μM for Fyn and approximately 1.5 μM for Lyn. Collectively, these findings suggest that Pro10-1D represents a novel therapeutic candidate for the treatment of IgE-mediated allergic disorders by targeting the Lyn/Fyn Src family kinases in MCs.

Abstract 4139195: Tipping Point Analysis Identifies C-terminal src kinase-Collagen 22 Regulation by Inflammation in the Pathogenesis of Early-Stage Pulmonary Arterial Hypertension

Inflammatory injury to pulmonary artery endothelial cells (PAECs) is a key pathogenetic event that drives fibrotic vascular remodeling in pulmonary arterial hypertension (PAH), a progressive disease that lacks early stage-specific therapeutic targets. In an inflammatory rodent model of PAH, we established early-stage disease based on abnormalities in right ventricular (RV) afterload, pulmonary vascular resistance, and RV cardiomyocyte phenotype that were evident without substantial elevation in pulmonary artery pressure. We performed RNA-Seq on PAEC mRNA isolated ex vivo from control, early-stage, and advanced-stage PAH rats and deployed a ‘tipping point’ methodology to identify key pulmonary endothelial protein-protein interactions (PPIs) that regulate the transition from control to early-stage PAH in silico . Building on prior data on the intersection between glucose and proline signaling in fibrosis, we interrogated our early-stage PAH network for convergent PPIs involving metabolism and fibrosis endotypes. C-terminal src kinase (Csk), a conserved inhibitor of Src, was predicted to regulate PAEC fibrosis and was increased in early-stage PAH. In human PAECs, inflammatory stimuli affected markers of Src activation as well as collagen 22 (Col22A1). We observed that pulmonary arterial Col22A1 expression increased progressively across all PAH stages in vivo and that arterial Col22A1 expression is upregulated in human PAH. Further, although inflammation upregulated total Csk mRNA and protein expression in HPAECs, it decreased phosphorylation of Src at tyrosine 527 (pTyr527Src), a Csk-dependent brake on Src kinase activity. Targeting Src through small molecule inhibition or adenovirus overexpression of Csk to increase pTyr527Src, in turn, attenuated inflammation-induced Col22A1 in vitro . Taken together, these data suggest that acquired inhibition of Csk is a novel molecular mechanism by which inflammation induces pulmonary endothelial dysfunction, Src activation, and fibrosis. In early-stage PAH, disrupted Csk inhibition of Src may induce transcriptional upregulation of Csk as a negative feedback response to Src activation. These data suggest that the Csk-Src-Col22A1 axis is a novel target for advancing early-stage therapeutics in patients with PAH subtypes characterized by inflammatory vascular injury.

CSIG-34. TYROSINE PHOSPHORYLATION OF NON-CANONICAL NF-ΚB P52 PROMOTES GLIOMA GROWTH AND CHEMORESISTANCE IN GBM

Abstract p52 is a nuclear factor-κB (NF-κB) transcription factor that comprises the non-canonical NF-κB pathway. The impact and mechanism of non-canonical NF-κB signaling remains understudied in glioblastoma (GBM). Subunit post-translational modification is a known mechanism of NF-κB activation, however phosphorylation of p52 has never been demonstrated. Elevated tyrosine kinase activity is a known-feature of GBM and portends worse survival. Further, p52 has been shown to upregulate factors that likely promote invasion of GBM, thereby reducing survival. Activation of p52 may serve as a link between increased kinase activity and poor survival in GBM. p52-knockout cell lines were generated using the CRISPR-Cas9 system and assessed for growth and survival differences in vitro and in syngeneic murine glioma models. Public glioma datasets and an institutional cohort were surveyed to corroborate p52-dependent survival differences in GBM. To determine whether p52 is post-translationally modified, we performed tandem mass spectrometry on p52 in GBM cells. Interacting factors were also analyzed and tested for site-specific modification of p52 via co-IP studies. We then generated PTM-null, knock-in glioma cell lines using CRISPR-CAS9 editing to demonstrate the functional significance of p52 PTMs, which were assessed using assays of relative cell growth, stemness, chemosensitivity and survival. Finally, we performed unbiased analysis of the p52 PTM-dependent transcriptome via RNA-seq. p52 loss significantly reduced glioma growth rate in vitro and improves murine survival. Retrospective analysis of GBM in patients suggests that p52 levels are negatively prognostic. Mass spectrometry identified multiple p52 PTMs with tyrosine phosphorylation sites as top hits. Identified phospho-tyrosine residues specifically interact with SRC-family kinases. PTM site editing significantly reduces glioma cell growth and stemness while enhancing chemosensitivity, reflecting broad transcriptome level changes. In sum, p52 activity enhances GBM growth and chemoresistance, which is partially modulated by tyrosine-specific phosphorylation. p52 warrants investigation as a therapeutic target in GBM, possibly in combination with kinase inhibitors and standard alkylating chemotherapy.

Multi-bioinformatics revealed potential biomarkers and repurposed drugs for gastric adenocarcinoma-related gastric intestinal metaplasia

Biomarkers associated with the progression from gastric intestinal metaplasia (GIM) to gastric adenocarcinoma (GA), i.e., GA-related GIM, could provide valuable insights into identifying patients with increased risk for GA. The aim of this study was to utilize multi-bioinformatics to reveal potential biomarkers for the GA-related GIM and predict potential drug repurposing for GA prevention in patients. The multi-bioinformatics included gene expression matrix (GEM) by microarray gene expression (MGE), ScType (a fully automated and ultra-fast cell-type identification based solely on a given scRNA-seq data), Ingenuity Pathway Analysis, PageRank centrality, GO and MSigDB enrichments, Cytoscape, Human Protein Atlas and molecular docking analysis in combination with immunohistochemistry. To identify GA-related GIM, paired surgical biopsies were collected from 16 GIM-GA patients who underwent gastrectomy, yielding 64 samples (4 biopsies per stomach x 16 patients) for MGE. Co-analysis was performed by including scRNAseq and immunohistochemistry datasets of endoscopic biopsies of 37 patients. The results of the present study showed potential biomarkers for GA-related GIM, including GEM of individual patients, individual genes (such as RBP2 and CD44), signaling pathways, network of molecules, and network of signaling pathways with key topological nodes. Accordingly, potential treatment targets with repurposed drugs were identified including epidermal growth factor receptor, proto-oncogene tyrosine-protein kinase Src, paxillin, transcription factor Jun, breast cancer type 1 susceptibility protein, cellular tumor antigen p53, mouse double minute 2, and CD44.

Allosteric changes in the conformational landscape of Src kinase upon substrate binding

Precise regulation of protein kinase activity is crucial in cell functions, and its loss is implicated in various diseases. The kinase activity is regulated by interconverting active and inactive states in the conformational landscape. However, how protein kinases switch conformations in response to different signals such as the binding at distinct sites remains incompletely understood. Here, we predict the binding mode for the peptide substrate to Src tyrosine kinase using enhanced conformational sampling simulations (totaling 24 μs) and then investigate changes in the conformational landscape upon substrate binding by conducting unbiased molecular dynamics simulations (totaling 50 μs) initiated from the apo and substrate-bound forms. Unexpectedly, the peptide substrate binding significantly facilitates the transitions from active to inactive conformations in which the αC helix is directed outward, the regulatory spine is broken, and the ATP-binding domain is perturbed. We also explore an underlying residue-contact network responsible for the allosteric conformational changes. Our results are in accord with the recent experiments reporting the negative cooperativity between the peptide substrate and ATP binding to tyrosine kinases and will contribute to advancing our understanding of the regulation mechanisms for kinase activity.

A non-catalytic role of IPMK is required for PLCγ1 activation in T cell receptor signaling by stabilizing the PLCγ1-Sam68 complex

BackgroundPhospholipase C gamma 1 (PLCγ1) is an important mediator of the T cell receptor (TCR) and growth factor signaling. PLCγ1 is activated by Src family kinases (SFKs) and produces inositol 1,4,5-triphosphate (InsP3) from phosphatidylinositol 4,5-bisphosphate (PIP2). Inositol polyphosphate multikinase (IPMK) is a pleiotropic enzyme with broad substrate specificity and non-catalytic activities that mediate various functional protein-protein interactions. Therefore, IPMK plays critical functions in key biological events such as cell growth. However, the contribution of IPMK to the activation of PLCγ1 in TCR signaling remains mostly unelucidated. The current study aimed to elucidate the functions of IPMK in TCR signaling and to uncover the mode of IPMK-mediated signaling action in PLCγ1 activation.MethodsConcanavalin A (ConA)-induced acute hepatitis model was established in CD4+ T cell-specific IPMK knockout mice (IPMKΔCD4). Histological analysis was performed to assess hepatic injury. Primary cultures of naïve CD4+ T cells were used to uncover the role of mechanisms of IPMK in vitro. Western blot analysis, quantitative real-time PCR, and flow cytometry were performed to analyze the TCR-stimulation-induced PLCγ1 activation and the downstream signaling pathway in naïve CD4+ T cells. Yeast two-hybrid screening and co-immunoprecipitation were conducted to identify the IPMK-binding proteins and protein complexes.ResultsIPMKΔCD4 mice showed alleviated ConA-induced acute hepatitis. CD4+ helper T cells in these mice showed reduced PLCγ1 Y783 phosphorylation, which subsequently dampens calcium signaling and IL-2 production. IPMK was found to contribute to PLCγ1 activation via the direct binding of IPMK to Src-associated substrate during mitosis of 68 kDa (Sam68). Mechanistically, IPMK stabilizes the interaction between Sam68 and to PLCγ1, thereby promoting PLCγ1 phosphorylation. Interfering this IPMK-Sam68 binding interaction with IPMK dominant-negative peptides impaired PLCγ1 phosphorylation.ConclusionsOur results demonstrate that IPMK non-catalytically promotes PLCγ1 phosphorylation by stabilizing the PLCγ1–Sam68 complex. Targeting IPMK in CD4+ T cells may be a promising strategy for managing immune diseases caused by excessive stimulation of TCR.

Zelenirstat Inhibits N-Myristoyltransferases to Disrupt Src Family Kinase Signalling and Oxidative Phosphorylation Killing Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a hematological malignancy with limited treatment options and a high likelihood of recurrence after chemotherapy. We studied N-myristoylation, the myristate modification of proteins linked to survival signaling and metabolism, as a potential therapeutic target for AML. N-myristoylation is catalyzed by two N-myristoyltransferases (NMTs), NMT1 and NMT2, with varying expressions in AML cell lines and patient samples. We identified NMT2 expression as a marker for AML patient survival, and low NMT2 expression was associated with poor outcomes. We used the first-in-class pan-NMT inhibitor, zelenirstat, to investigate the role of N-myristoylation in AML. Zelenirstat effectively inhibits myristoylation in AML cell lines and patient samples, leading to degradation of Src family kinases (SFKs), induction of endoplasmic reticulum (ER) stress, apoptosis, and cell death. Zelenirstat was well tolerated in vivo and reduced the leukemic burden in an ectopic AML cell line and in multiple orthotopic AML patient-derived xenograft models. The leukemia stem cell (LSC)-enriched fractions of the hierarchical OCI-AML22 model were highly sensitive to myristoylation inhibition. Zelenirstat also impairs mitochondrial complex I and oxidative phosphorylation, which are critical for LSC survival. These findings suggest that targeting N-myristoylation with zelenirstat represents a novel therapeutic approach for AML, with promise in patients with currently poor outcomes.

Csk controls leukocyte extravasation via local regulation of Src family kinases and cortactin signaling.

C-terminal Src kinase (Csk) targets Src family kinases (SFKs) and thereby inactivates them. We have previously shown that Csk binds to phosphorylated tyrosine 685 of VE-cadherin, an adhesion molecule of major importance for the regulation of endothelial junctions. This tyrosine residue is an SFK target, and its mutation (VE-cadherin-Y685F) inhibits the induction of vascular permeability in various inflammation models. Nevertheless, surprisingly, it increases leukocyte extravasation. Here, we investigated whether endothelial Csk is involved in these effects. We found that the deficiency of Csk in endothelial cells augments SFK activation and the phosphorylation of VE-cadherin-Y685 but had no net effect on vascular leak formation. In contrast, the lack of endothelial Csk enhanced leukocyte adhesion and transmigration in vitro and in vivo. Furthermore, the silencing of Csk increased tyrosine phosphorylation of the SFK substrate cortactin. Importantly, the effects of Csk silencing on the increase in SFK activation, cortactin phosphorylation, and neutrophil diapedesis were all dependent on Y685 of VE-cadherin. Deletion of cortactin, in turn, erased the supporting effect of Csk silencing on leukocyte transmigration. We have previously shown that leukocyte transmigration is regulated by endothelial cortactin in an ICAM-1-dependent manner. In line with this, blocking of ICAM-1 erased the supporting effect of Csk silencing on leukocyte transmigration. Collectively, our results establish a negative feedback loop that depends on the phosphorylation of VE-cadherin-Y685, which recruits Csk, which in turn dampens the activation of SFK and cortactin and thereby the clustering of ICAM-1 and the extravasation of neutrophils.

Exploring the potential mechanisms of polysaccharides against gastric ulcer: Network pharmacology analysis and molecular docking validation

AbstractGastric ulcer is a common peptic ulcer that affects human health and life quality seriously. As anti‐gastric ulcer drugs usually cause side‐effects, polysaccharides may be the potential alternatives because of better effectiveness and less toxicity. Although the anti‐gastric ulcer activities of polysaccharides have been widely reported, the mechanisms have not yet been well‐disclosed. In this study, network pharmacology analysis was performed to explore the potential mechanisms of polysaccharides against gastric ulcer, and the results were validated by molecular docking. Results indicated that β‐glucan, arabinogalactan, xylan, and arabinan were the key structures, and ABL1, AKT1, androgen receptor, epidermal growth factor receptor, v‐Ha‐ras Harvey rat sarcoma viral oncogene homolog, HSP90AA1, mitogen‐activated protein kinase 8 (MAPK8), MAPK14, NOS2, PIK3R1, RAC1, ras homolog gene family member A, and proto‐oncogene tyrosine‐protein kinase Src were the core targets for polysaccharides in treating gastric ulcer. Polysaccharides have influences on 1958 GO items and 199 KEGG pathways, and their anti‐gastric ulcer activities are related to MAPK, Ras, PI3K‐Akt, vascular endothelial growth factor, prolactin, FoxO and Rap1 signaling pathways, etc. Molecular docking validation showed that the results of network pharmacology analysis were credible, and interactions between polysaccharide structures and core targets were observed. This study contributes to understanding the mechanisms of polysaccharides in treating gastric ulcer and provides references for future activity screening and mechanism research in anti‐gastric ulcer.

SKI-606, a Src inhibitor, ameliorates benzene-induced hematotoxicity via blocking ROS/Src kinase-mediated p38 and Akt signaling pathways

Exposure to benzene causes acute myelosuppression and other hematologic disorders. However, the detailed mechanism by which benzene exerts its severe hematotoxicity and potential treatments still require further deciphering and exploration. Herein, we found that hydroquinone (HQ), a main benzene metabolite, significantly increased intracellular reactive oxygen species (ROS) formation and subsequently caused damage to DNA, leading to impaired colony formation capacity and induction of apoptosis in human hematopoietic stem/progenitor cells (HSPCs) in vitro. The effects were mediated by activation of Src kinase, which subsequently activated the p38 signaling pathway while inhibiting the Akt signaling pathway. The mechanism was further verified by pre-treatment with a Src kinase inhibitor SKI-606, which effectively reversed the dampened self-renewal capacity and increased apoptosis of HSPCs induced by HQ in vitro. Furthermore, administration of SKI-606 partially reversed benzene-induced hematotoxicity and prolonged the survival time in benzene-poisoned mice. Taken together, these findings highlight that HQ-induced hematotoxicity in HSPCs is attributed to the Src kinase-mediated activation of p38 signaling pathway and repression of Akt signaling pathway. Notably, SKI-606 as a tyrosine kinase inhibitor may be a promising and potential agent for alleviating benzene-induced hematotoxicity.