Co-immunoprecipitation Assays Research Articles

Wheat Tae-MIR1118 Constitutes a Functional Module With Calmodulin TaCaM2-1 and MYB Member TaMYB44 to Modulate Plant Low-N Stress Response.

Distinct target genes are modulated by microRNA members and affect various biological processes associated with abiotic stress responses in plants. In this study, we characterized a functional module comprising miRNA/target and a downstream MYB transcription factor partner, Tae-MIR1118/TaCaM2/TaMYB44, in Triticum aestivum to mediate the plant low-nitrogen (N) stress response. Dual luciferase (LUC) assay and expression analysis indicated that TaCaM2 is regulated by Tae-MIR1118 through a posttranscriptional cleavage mechanism. Reporter LUC activity in N. benthamiana leaves co-transformed with effector CaMV35S::Tae-MIR1118 and reporter TaCaM2::LUC was significantly reduced, and the transcripts of Tae-MIR1118 and TaCaM2 in tissues exhibited converse expression patterns under varying N levels. Specifically, the transcripts of Tae-MIR1118 decreased, whereas those of TaCaM2 increased under low-N stress in a temporal-dependent manner. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays indicated that TaCaM2 interacted with the MYB transcription factor TaMYB44. Transgene analysis revealed the negative roles of Tae-MIR1118 and the positive functions of TaCaM2 and TaMYB44 in regulating plants for low-N stress adaptation by modulating glutamine synthetase activity, N uptake capacity, and root morphology. Yeast one-hybrid, transcriptional activation, and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-PCR) assays indicated that TaMYB44 could bind to the promoters of genes TaGS2.2, TaNRT2.1, and TaPIN4 and induce transcription of these stress-defensive genes. Knockdown of these three genes reduced GS activity, N accumulation, and root growth traits in plants subjected to N starvation. The yield in the wheat variety panel was highly correlated with the transcripts of Tae-MIR1118, TaCaM2, and TaMYB44 in plants cultured under N-deprived field conditions. A major haplotype of Tae-MIR1118, TaMIR1118-Hap1, enhanced the low-N stress tolerance of plants. Our findings indicate that the Tae-MIR1118/TaCaM2/TaMYB44 pathway primarily affects the low-N response of plants by modulating associated physiological processes.

PRIM2 promotes proliferation and metastasis of pancreatic ductal adenocarcinoma through interactions with FAM111B.

Pancreatic ductal adenocarcinomas (PDAC) are huge threat to human for the extreme malignancy. PRIM2 was reported as tumor marker, while the functions and regulatory mechanisms in PDAC are still unclear. The study aimed to investigate the function of PRIM2 in PDAC. Expression was detected using immunohistochemistry (IHC), Western blot, and real-time quantitative PCR (RT-qPCR) methods. Cell assays and xenograft model confirmed the phenotypes. Co-Immunoprecipitation (Co-IP) and protein stability assays were used for protein interactions. Inhibiting PRIM2 resulted in decreased proliferation and migration both in vitro and in vivo. PRIM2 upregulated FAM111B at increased RNA levels and protein stability. PRIM2/FAM111B axis promoted proliferation and migration by modulating the PI3K/AKT and epithelial-mesenchymal transition (EMT) markers. The axis has the potential to be targeted for PDAC treatment.

USP11 promotes lipogenesis and tumorigenesis by regulating SREBF1 stability in hepatocellular carcinoma

BackgroundThe relationship between hepatocellular carcinoma (HCC) metastasis and cancer metabolism reprogramming is becoming increasingly evident. Ubiquitin-specific protease 11 (USP11), a member of the deubiquitinating enzyme family, has been linked to various cancer-related processes. While USP11 is known to promote HCC metastasis and proliferation, the precise mechanisms, especially those related to cancer metabolism, remain unclear.MethodsThrough mass spectrometry, co-immunoprecipitation, immunofluorescence, and ubiquitination assays, we identified USP11 as the key deubiquitinase for SREBF1.Lipogenesis was evaluated using Oil Red O and Nile Red staining, along with the detection of triglycerides and cholesterol. To assess HCC cell proliferation, migration, and invasion in vitro, Transwell assays, EDU, colony formation, and CCK-8 were conducted. Xenograft models in nude mice were developed to verify the role of the USP11/SREBF1 axis in lipogenesis and tumor growth in vivo.ResultsUSP11 directly interacts with SREBF1, and its silencing leads to the disruption of SREBF1 stabilization through K48-linked deubiquitination and degradation. Importantly, the truncated mutant USP11 (503–938 aa) interacts with the truncated mutant SREBF1 (569–1147aa), with K1151 playing a crucial role in this interaction. Higher levels of USP11 enhance lipogenesis, proliferation, and metastasis in HCC cells. Importantly, the knockdown of SREBF1 weakened the effects of USP11 in enhancing lipogenesis and tumorigenesis. Futhermore, the elevated expression of USP11 and SREBF1 in HCC tissue serves as an indicator of poor prognosis in HCC patients.ConclusionsIn summary, our study reveals that USP11 promotes HCC proliferation and metastasis through SREBF1-induced lipogenesis. These findings provide a foundation for novel therapies targeting lipid metabolism in HCC.

The wheat CC-NBS-LRR protein TaRGA3 confers resistance to stripe rust by suppressing ascorbate peroxidase 6 activity.

Nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune receptors that activate innate immune responses upon sensing pathogen attack. However, the molecular mechanisms by which NLR proteins initiate downstream signal transduction pathways to counteract pathogen invasion remain poorly understood. In this study, we identified the wheat (Triticum aestivum) NLR protein Resistance Gene Analogs3 (TaRGA3), which was significantly upregulated during Puccinia striiformis f. sp. tritici (Pst) infection. TaRGA3 and its coiled-coil (CC) domain, localized to the cytoplasm and nucleus, can induce cell death in Nicotiana benthamiana. Virus-induced gene silencing and overexpression suggested that TaRGA3 contributed to wheat resistance to stripe rust by facilitating reactive oxygen species (ROS) accumulation. Yeast 2-hybrid, luciferase complementation imaging, and co-immunoprecipitation assays revealed that TaRGA3 interacted with wheat protein Ascorbate Peroxidase 6 (TaAPX6). Further analysis showed that TaAPX6 specifically targeted the CC domain of TaRGA3. The TaRGA3-TaAPX6 interplay led to reduced enzyme activity of TaAPX6. Notably, TaAPX6 negatively regulated wheat resistance to Pst by removing excessive ROS accompanying Pst-induced hypersensitive responses. Our findings reveal that TaRGA3 responding to Pst infection confers enhanced wheat resistance to stripe rust, possibly by suppressing TaAPX6-modulated ROS scavenging, and demonstrate that TaRGA3 can be used to engineer stripe rust resistance in wheat.

Abstract B007: Identification of Asporin as a HER3 ligand exposes a therapeutic vulnerability in metastatic prostate cancer

Abstract Background: Rates of prostate cancer incidence and advanced-stage diagnoses are increasing. Despite curative-intent therapies, nearly all patients with advanced disease progress to metastatic castration-resistant prostate cancer (mCRPC), a lethal form of the disease. There is an urgent unmet need to understand the mechanisms driving prostate cancer progression and identify alternative therapies for patients with mCRPC. It is well-established that cancer-associated fibroblasts (CAF) in the tumor microenvironment secrete factors that enable cancer cells to establish metastases. Asporin (ASPN) is an extracellular protein secreted by a subset of CAF and is associated with worse oncologic outcomes, yet the mechanisms remain poorly understood. In this study, we sought to identify the molecular interactions between the CAF-secreted ASPN and metastatic prostate cancer cells. Methods: ASPN-induced HER2/HER3 activation and downstream signaling was identified by RNA-sequencing and then confirmed by immunoblotting in multiple human metastatic prostate cancer cell lines. Binding interactions were assessed computationally by AlphaFOld2 with Rosetta refinement and substantiated by co-immunoprecipitation assays. CRISPR-cas9 targeting of HER2 and HER3 and therapeutic inhibition of HER2 with Tucatinib were used to determine the role of HER2/HER3 in ASPN-induced signaling and migration. The efficacy of Tucatinib and antibody-drug conjugates (ADC) targeting HER2 (Trastuzumab-deruxtecan) and HER3 (Patritumab-deruxtecan) were assessed in multiple metastatic prostate cancer cell lines in vitro. Trastuzumab-deruxtecan was assessed in PC3 xenografts in vivo. Prostate cancer metastases from 33 patients were assessed by IHC for HER2 and HER3 and by RNAscope for ASPN. Results: We report that ASPN is a novel ligand of HER3 and induces HER3 heterodimerization with its preferred dimerization partner, HER2. ASPN activates established ErbB-associated pathways including Phosphoinositide 3-kinase (PI3K), Mitogen-activated protein kinase (MAPK), and calcium signaling, in multiple metastatic prostate cancer cell lines to promote cell migration. Genetic and molecular inhibition of HER2/HER3 mitigates ASPN-induced signaling and cell migration, suggesting these receptors are required for paracrine activation by ASPN. Importantly, small molecule and ADC therapies targeting HER2/HER3 significantly diminished prostate cancer cell growth in vitro, with enzalutamide-resistant cells showing increased sensitivity. Trastuzumab-deruxtecan nearly resolved tumors in an in vivo xenograft model. Lastly, ASPN+ CAF in the TME of HER2/HER3-expressing metastatic prostate cancer is frequently observed in patient samples, supporting the clinical relevance of these findings. Conclusions: Collectively, these findings indicate ASPN functions as a HER3 ligand to induce cellular migration, and inhibition with anti-HER2/HER3 therapies highlights potential clinical utility for patients with HER2-expressing metastatic prostate cancer. Citation Format: Amanda B. Hesterberg, Hong Yuen Wong, Jorgen Jackson, Monika Antunovic, Brenda L. Rios, Evan Watkins, Riley E. Bergman, Brad A. Davidson, Sarah E. Ginther, Diana Graves, Elliott F. Nahmias, Jared A. Googel, Lillian B. Martin, Violeta Sanchez, Paula Gonzalez Ericsson, Quanhu Sheng, Benjamin P. Brown, Jens Meiler, Ben H. Park, Kerry R. Schaffer, Jennifer B. Gordetsky, Paula J. Hurley. Identification of Asporin as a HER3 ligand exposes a therapeutic vulnerability in metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B007.

DLGAP3 suppresses malignant behaviors of glioma cells via inhibiting RGS12-mediated MAPK/ERK signaling

BackgroundGlioma is the most common malignant tumor of the central nervous system, and is characterized by high recurrence, poor prognosis and especially complex pathogenesis. The synaptic plasticity-related protein DLGAP3 is mainly involved in the assembly and function of postsynaptic density complex. It’s widely known that DLGAP3 participating in the occurrence of various neuropsychiatric diseases, but its role in glioma tumorigenesis remains largely unclear. MethodsWe ectopically expressed and knocked down DLGAP3 in glioma cells to perform a series of functional studies in vitro. Meanwhile, western blot analysis, co-immunoprecipitation, enrichment analysis and dual-luciferase reporter system assays were performed to explore the mechanism of DLGAP3 suppressing glioma tumorigenesis and progression. ResultsWe found that DLGAP3 was low expressed in gliomas, and decreased DLGAP3 expression was strongly correlated with poor survival of glioma patients. Ectopic expression of DLGAP3 in glioma cell lines dramatically inhibited cell proliferation, invasion and migration. In addition, our data also showed that DLGAP3 can tightly connected with RGS12, and DLGAP3 overexpression significantly increased the expression of RGS12 and inhibited the phosphorylation levels of MEK and ERK. Furthermore, the RGS12 inhibited transcription and translation of BRAF, which further decreased the activity of MAPK/ERK signaling pathway. This suggests that DLGAP3 may act as a tumor suppressor in gliomas and inhibits glioma tumorigenesis by regulating RGS12 and the downstream MAPK/ERK signals axis. ConclusionOur data indicates that DLGAP3 is a potential tumor suppressor and valuable prognostic biomarker in gliomas.

Role of the CTCF/p300 axis in osteochondrogenic-like differentiation of polyploid giant cancer cells with daughter cells

BackgroundPolyploid giant cancer cells (PGCCs) have properties of cancer stem cells (CSCs). PGCCs with daughter cells (PDCs) undergo epithelial–mesenchymal transition and show enhanced cellular plasticity. This study aimed to elucidate the mechanisms underlying the osteo/chondrogenic-like differentiation of PDCs, which may be exploited therapeutically by transdifferentiation into post-mitotic and functional cells.MethodsCobalt chloride was used to induce PGCC formation in MDA-MB-231 and HEY cells, and PDCs were cultured in osteo/chondrogenic differentiation media. Alcian blue staining was used to confirm osteo/chondrogenic differentiation, and the cell cycle was detected using flow cytometry. The expression of osteo/chondrogenic differentiation-related proteins was compared, and a co-immunoprecipitation assay was used to demonstrate the interactions between proteins. Bioinformatic analysis was used to explore the regulatory mechanism of osteo/chondrogenic differentiation, and a dual-luciferase reporter assay was performed to validate the interaction between transcriptional factors and target genes. Animal xenograft models were used to confirm the osteo/chondrogenic differentiation of PDCs.ResultsWhen cultured in osteo/chondrogenic medium, the stemness of PDCs decreased, and the expression of osteo/chondrogenic-related markers increased. This osteo/chondrogenic-like process was regulated by the transforming growth factor-β pathway in a time-dependent manner. A concurrent increase in the expression of histone acetyltransferase p300 and the transcription factor CCCTC-binding factor (CTCF) was observed. Co-immunoprecipitation assays revealed that p300 acetylated the osteo/chondrogenic marker RUNT-related transcription factor 2 (RUNX2). Analysis of chromatin immunoprecipitation sequencing datasets revealed that both CTCF and histone H3 lysine 27 acetylation (H3K27ac) were enriched in the promoter region of E1A-associated protein p300 (P300). The four predicted binding sites for CTCF and P300 were validated using dual-luciferase reporter assays. We examined the interaction between CTCF and H3K27ac and found that these two proteins had a combined effect on the transactivation of P300.ConclusionCTCF, in synergy with H3K27ac, amplified the expression of P300, facilitating acetyl group transfer to RUNX2. This acetylation stabilized RUNX2 and promoted osteo/chondrogenic differentiation, thereby reducing the incidence of PDC malignancies.

Deciphering sepsis: An observational bioinformatic analysis of gene expression in granulocytes from GEO dataset GSE123731.

Sepsis triggers severe inflammatory responses leading to organ dysfunction and demands early diagnostic and therapeutic intervention. This study identifies differentially expressed genes (DEGs) in sepsis patients using the Gene Expression Omnibus database to find potential diagnostic and therapeutic markers. We analyzed the dataset GSE123731 via GEO2R to detect DEGs, constructed protein-protein interaction networks, and performed transcription factor analyses using Cytoscape. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted using R and FunRich software. Key genes were validated by Quantitative Reverse Transcription Polymerase Chain and co-immunoprecipitation assays in granulocytes from sepsis patients. We identified 59 DEGs significantly involved in neutrophil degranulation and immune system activation. Cytokine signaling pathways were highlighted in Kyoto Encyclopedia of Genes and Genomes analysis. Co-immunoprecipitation assays confirmed interactions involving matrix metallopeptidase 8, matrix metallopeptidase 9, and arginase 1, supporting their roles as biomarkers. The identified DEGs and validated interactions reveal crucial molecular mechanisms in sepsis, offering new avenues for diagnostic and therapeutic strategies, potentially enhancing patient outcomes.

KGF secreted from HSCs activates PAK4/BMI1, promotes HCC stemness through PI3K/AKT pathway.

In our present study, we investigated the interaction between HSCs and HCC, also explored the molecular mechanism. Clinical samples were collected from HCC and adjacent tissue with different degree of liver fibrosis. HCC cells were co-cultured with LX-2 cell by Transwell system or cultured with conditioned medium (CM), which was collected from LX-2. The tumor spheroid growth and colony formation analyses were performed to evaluate the cell stemness. Flow cytometry analysis was conducted on cell apoptosis after 5-Fu treatment. Co-immunoprecipitation assay confirmed the interaction between BMI1 and PAK4. Our results showed that BMI1 was highly expressed in HCC and was correlated with HCC liver fibrosis. Both co-cultured with LX-2 and cultured with CM promoted HCC stemness, also increased KGF level and BMI1 expression. KGF treatment had a similar effect with co-culture with LX-2 on HCC. BMI1 overexpression promoted HCC stemness and activated PI3K/AKT pathway, which was reversed by PI3K inhibition. PAK4 was activated by KGF, then phosphorylated S315 site and promoted protein stability of BMI1, therefore enhanced HCC stemness. BMI1 also had a promote effect on liver fibrosis. In summary, we found that KGF secreted by HSCs activated PAK4, which phosphorylated S315 and promoted protein stability of BMI1, and further promoted liver fibrosis and HCC stemness through the PI3K/AKT signaling pathway. Our present study deeply studied the interaction and mechanism between HSCs and HCC, which might provide a new insight for HCC therapy.

Modulation of SRC by SNTB1 activates the Hippo-YAP pathway during colon adenocarcinoma metastasis

BackgroundColon adenocarcinoma (COAD) ranks as the third most prevalent and lethal cancer in 2020, with metastasis being the primary cause of cancer-related mortality. A comprehensive understanding of the mechanism underlying distant metastasis is imperative for enhancing the prognosis and quality of life of patients with COAD.MethodsThis study employed gene set enrichment analysis (GSEA) on RNA-sequencing data from 408 patients with COAD in The Cancer Genome Atlas (TCGA) database. GSEA analysis was applied to find the significant hallmark gene set, and genes in the significant hallmark gene set was performed by univariate Cox regression to select the key gene. Then, multivariate Cox regression model was constructed. And various databases were utilized to validate and find the significant oncoprotein and hallmark gene set of the key gene. In addition, the expression of key regulators in para-carcinoma tissue, colon cancer and distant metastases samples were detected by real-time PCR, Immunohistochemistry and Western blot. Additionally, the biological functions were examined by in vitro and in vivo experiments. The scRNA-seq and CellphoneDB were performed to explore cell characters in COAD and the potential mechanism of metastasis.ResultsThe regulatory network analysis revealed SRC/YAP1 as the most significant oncoprotein and signature gene set associated with SNTB1. Moreover, significant SNTB1 overexpression in COAD metastatic tissues was observed compared to para-carcinoma and primary COAD tissues. Co-immunoprecipitation assays demonstrated the formation of a complex between SNTB1 and SRC proteins. Furthermore, the overexpression of SNTB1 enhanced the proliferation, migration and invasion capacities of COAD cell lines. Caudal vein injection of COAD cells overexpressing SNTB1 in nude mice resulted in increased tumour growth and metastasis to the lung, liver and bone. Finally, single cell RNA-seq revealed alterations in the cellular subtypes of COAD, and CellphoneDB indicated that the interaction between cancer cells exhibiting high SNTB1 expression and enterocytes promoted EMT through cellular communications involving TGF-β, accelerating metastasis in COAD.ConclusionThis study postulates that SNTB1 interacts with SRC to activate the Hippo-YAP pathway, thereby promoting COAD metastasis. Furthermore, cellular communication with enterocytes promotes EMT, facilitating metastasis. These findings propose novel therapeutic targets for preventing or treating metastatic COAD.

Thorase deficiency causes both Aβ accumulation and tau hyperphosphorylation in mouse brain.

The pathogenesis of two major pathogenic characters-amyloid beta (Aβ) accumulation and hyperphosphorylated tau protein-in the brains of patients with Alzheimer's disease (AD) remains unclear. Western blot and immunofluorescence staining were performed to detect the proteins in the brains of Thorase conditional knockout/transgenic mice and their littermates. A co-immunoprecipitation assay was applied to examine the Thorase-interacting proteins. Genetic deletion of Thorase resulted in tau hyperphosphorylation and promoted Aβ accumulation in the mouse brain. Conversely, Thorase overexpression alleviated the pathogenesis of AD. Thorase regulated the phosphorylation of tau by targeting specific kinases and theprotein phosphatase 2B (PP2B). Thorase deficiency also impaired microglial phagocytosis and induced neuroinflammation by the activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3)inflammasomes in microglia. Thorase may be a potential druggable target for developing therapeutic approaches to treat AD and other neurodegenerative diseases. Thorase deletion leads to elevated amyloid beta (Aβ) deposition and hyperphosphorylated tau accumulation in the brain. Thorase regulates the phosphorylation of tau protein via PP2B. Thorase deficiency impairs microglial phagocytosis and promotesNLRP3-mediated neuroinflammation. Overexpression of Thorase alleviates Aβ deposition and tau phosphorylation in the AD mouse model.

TRIM46 accelerates H1N1 influenza virus-induced ferroptosis and inflammatory response by regulating SLC7A11 ubiquitination.

Influenza A (H1N1) virus is an acute respiratory infection responsible for enormous morbidity and mortality worldwide. The tripartite motif-containing protein 46 (TRIM46) has an antiviral function that inhibits various viral infections. This study is designed to explore the role and mechanism of TRIM46 in the progress of H1N1 infection. Herein, we infected A549 or 16HBE cells with the H1N1 virus at different times to assess TRIM46 and solute carrier family 7 member 11 (SLC7A11) expression. TRIM46 and Influenza A nucleoprotein mRNA levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). TRIM46, solute carrier family 7 member 11 (SLC7A11), and Nucleoprotein protein levels were detected using protein level were detected by western blot assay. Cell virulence was determined using Virulence assay (TCID50) assay. Cell viability was determined using Cell Counting Kit-8 (CCK-8) assay. Reactive oxygen species (ROS), intracellular iron content, Malondialdehyde (MDA), and Glutathione (GSH) levels were determined using special assay kits. The stability of SLC7A11 was assessed by Cycloheximide (CHX) assay. Interaction between TRIM46 and SLC7A11 was verified using Co-immunoprecipitation (CoIP) assay. The biological role of TRIM46 was assessed in H1N1 virus-challenged lung injury mice in vivo. TRIM46 level was significantly increased during H1N1 virus infection, and SLC7A11 expression was decreased. TRIM46 downregulation could suppress H1N1 virus replication and relieve H1N1 infection-induced ferroptosis and inflammation in A549 or 16HBE cells. Mechanistically, TRIM46 could promote SLC7A11 ubiquitination and decrease its stability. TRIM46 knockdown repressed H1N1 virus-induced lung injury in vivo. TRIM46 could contribute to influenza A H1N1 virus infection by promoting SLC7A11 ubiquitination in A549 cells, which indicates that targeting TRIM46 may improve the prognosis of patients.

Abstract 4134837: Checkpoint Kinase 1 Attenuates Myocardial Ischemia-Reperfusion Injury Through Maintaining SIRT1-Dependent Mitochondrial Homeostasis

Background: Mitochondrial dysfunction is linked to myocardial ischemia-reperfusion (I/R) injury. Checkpoint kinase 1 (CHK1) could facilitate cardiomyocyte proliferation and cardiac repair post myocardial infarction, however, its role on mitochondrial function in I/R injury remains unknown. Research Questions: The purpose of this study is to explore the potential effects and mechanisms of CHK1 on mitochondrial homeostasis during myocardial I/R injury. Methods: To investigate the role of CHK1 on mitochondrial function following I/R injury, cardiomyocyte-specific knockout/knock-in mouse models were generated. Mass spectrometry-proteomics analysis and protein co-immunoprecipitation assays were conducted to dissect the molecular mechanism of CHK1. The interaction between CHK1 and SIRT1 was explored using truncated plasmids. Results: CHK1 was downregulated in myocardium post I/R and neonatal mouse cardiomyocytes (NMCMs) post oxygen-glucose deprivation/re-oxygenation (OGD/R). In vivo , CHK1 overexpression protected against myocardial I/R injury, while heterogenous CHK1 knockout exacerbated cardiomyopathy. In vitro , CHK1 inhibited OGD/R-induced cardiomyocyte apoptosis and bolstered cardiomyocyte survival. Mechanistically, CHK1 attenuated oxidative stress and preserved mitochondrial metabolism in cardiomyocytes under I/R. Moreover, disrupted mitochondrial homeostasis in I/R myocardium was restored by CHK1 through the promotion of mitochondrial biogenesis and mitophagy. Through mass spectrometry analysis following co-immunoprecipitation, SIRT1 was identified as a direct target of CHK1. The 266-390 domain of CHK1 interacted with the 160-583 domain of SIRT1. Importantly, CHK1 phosphorylated SIRT1 at Thr530 residue, thereby inhibiting SMURF2-mediated degradation of SIRT1. CHK1 Δ390 amino acids (aa) mutant functioned similarly to full-length CHK1 in scavenging ROS and maintaining mitochondrial dynamics. Consistently, cardiac-specific SIRT1 knockdown partially attenuated the protective role of CHK1 in I/R injury. Conclusions: Our findings revealed that CHK1 mitigates I/R injury and restores mitochondrial dynamics in cardiomyocytes through a SIRT1-dependent mechanism.

LncRNA GClnc1 promotes osteosarcoma progression by stabilizing NONO and blocking FBXW7-mediated ubiquitination

BackgroundLong non-coding RNA (lncRNA) plays a vital role in the occurrence and development of varieties of tumors. Previous studies have shown that lncRNA GClnc1 is highly expressed in osteosarcoma (OS). However, the mechanism of lncRNA GClnc1 in osteosarcoma has not been fully elucidated. In this study, we investigated the biological roles of lncRNA GClnc1 in osteosarcoma and unveiled its underlying mechanisms.MethodsThe expression of lncRNA GClnc1 in OS cells was detected by real-time quantitative PCR (qRT-PCR). The functional roles of lncRNA GClnc1 were examined by CCK8, trans-well, scratch wound healing assay, colony formation, and apoptosis assays in osteosarcoma cells upon silencing or overexpressing GClnc1. Western blot analysis, qRT-PCR, and RNA co-immunoprecipitation (RIP) assays were used to detect the interaction between lncRNA GClnc1 and NONO.ResultsThe expression of lncRNA GClnc1 was up-regulated in osteosarcoma cell lines. Knockdown of lncRNA GClnc1 suppressed the cell growth, migration, and invasion of OS cells, whereas the over-expression of GClnc1 improved the proliferation, migration, and invasion of OS cells. Mechanistically, we identified that lncRNA GClnc1 regulates the stability of NONO by blocking FBXW7-mediated ubiquitination degradation. Additionally, overexpression of NONO can reverse GClnc1 silencing exerted suppression of the cell proliferation, migration, and invasion, and vice versa.ConclusionsOur study elucidated that lncRNA GClnc1 participates in the progression of OS by regulating the NONO signal pathway. Targeting GClnc1 provides a potential target for future clinical treatment of OS.

Gastrodin reduces Aβ brain levels in an Alzheimer's disease mouse model by inhibiting P-glycoprotein ubiquitination

BackgroundStudies have demonstrated the potential of gastrodin in the treatment of Alzheimer's disease (AD), however, its mechanism of action remains elusive. Currently, the Amyloid-β (Aβ) cascade hypothesis continues to be the prevailing theory regarding AD etiology. The ubiquitination of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) contributes to the accumulation of Aβ in the brain during AD. PurposeTo investigate the mechanism of gastrodin intervention in AD. MethodsThe molecular docking, molecular dynamics simulations, and microscale thermophoresis (MST) were employed to identify the action target of gastrodin. The western blot (WB) was performed to detect the protein expression level, the ubiquitination level of P-gp was determined using co-immunoprecipitation (CO-IP) assay. P-gp transport activity was detected using an NBD-CSA fluorescence assay. Trans-Epithelial Electrical Resistance (TEER) was used to detect cell resistance. Fluorescein-labeled dextran experiments were performed to determine the individual cell permeability. The immunofluorescence (IF) was employed to detect Aβ deposition, the Morris Water Maze test was used to assess behavioral changes in APP/PS1 mice and the levels of Aβ40 and Aβ42 expression were quantified using enzyme-linked immunosorbent assay. ResultsThe FBXO15 was the target of gastrodin-mediated inhibition of P-gp ubiquitination. Gastrodin increased the P-gp expression, cell resistance, and P-gp transport activity of BEND.3 cells upon treatment with Aβ40 through mechanisms involving the reduction of FBXO15 and P-gp binding and the inhibition of P-gp ubiquitination. And gastrodin could effectively improve memory function and increase number of neurons in APP/PS1 mice, reduce the accumulation of Aβ40 and Aβ42, and enhance P-gp expression in a dose-dependent manner. ConclusionAβ40 induces the ubiquitination and proteasomal degradation of BBB P-gp, however, gastrodin inhibits the ubiquitination of P-gp by binding to FBXO15, thereby increasing P-gp protein expression and enhancing its transport function.

CDC42 enhances phagocytic activity and exhibits antimicrobial functions through the recruitment of IQGAP1 in Apostichopus japonicus

Cell Division Cycle 42 protein (CDC42) is a pivotal member of the Rho family of small GTPases, integral to cell signaling and the regulation of cell polarity. CDC42 is extensively documented to participate in host immunity, particularly in processes such as phagocytosis and cell migration. Nevertheless, the precise mechanisms by which CDC42 affects host-bacteria interactions remain unclear in Echinoderms. In this study, we have isolated and characterized the CDC42 gene from Apostichopus japonicus, referred to as AjCDC42. The complete cDNA sequence of AjCDC42 extends 1282 base pairs, including a typical classical RHO domain, spanning amino acids from 6 to 179. Spatial expression analysis demonstrated that AjCDC42 is ubiquitously expressed across all examined tissues. Following infection with Vibrio splendidus and stimulation with LPS, the protein and mRNA levels of AjCDC42 were significantly upregulated in coelomocytes. Moreover, the knockdown of AjCDC42 significantly decreased V. splendidus-induced phagocytic activity in coelomocytes, leading to a reduced intracellular load of V. splendidus, as evidenced by flow cytometry and bacterial plate counting assays. Mechanistically, AjCDC42 was identified as an interacting partner of IQ Motif Containing GTPase Activating Protein 1 (AjIQGAP1) through the use of co-immunoprecipitation, immunofluorescence, and GST pull-down assays combined with mass spectrometry techniques. Additional functional analyses revealed that the knockdown of AjIQGAP1 similarly decreased the phagocytic activity and the intracellular load of V. splendidus in coelomocytes. Furthermore, we detected that decreased levels of AjCDC42 and AjIQGAP1 impaired cytoskeletal rearrangement and delayed lysosomal clearance of V. splendidus. In summary, our findings underscore the pivotal role of the AjCDC42-AjIQGAP1 axis in regulating coelomocytes phagocytosis in A. japonicus during V. splendidus infection.

Cellular NONO protein binds to the flavivirus replication complex and promotes positive-strand RNA synthesis.

A cellular protein, non-POU-domain-containing octamer binding protein (NONO), bound to the replication complex of Japanese encephalitis virus (JEV) by directly interacting with the viral 3' UTR RNA and NS3 protein. These interactions were also identified in West Nile virus (WNV) and Zika virus (ZIKV). The infection of JEV or the expression of JEV NS3 protein in cells could induce relocation of NONO protein from the nucleus to the cytoplasm. In JEV-infected cells, the NS3, NS5, and viral RNA could be concurrently detected in the immunoprecipitation by the NONO-specific antibody, suggesting that NONO could integrate into the replication complex of JEV. Further results of co-immunoprecipitation assays showed that NONO protein interacted with NS3 helicase domains 1 and 2 by its two RNA recognize motifs (RRMs). The knockdown and knockout of NONO in cells could significantly reduce the replication of JEV and ZIKV but had no effect on the replication of vesicular stomatitis virus (VSV). The effect of NONO protein on JEV proliferation occurred during the replication stage, rather than the attachment and entry stages. The level of viral positive-strand RNA in NONO knockout cells was significantly reduced than that in wild-type cells at 12-48 h post-JEV infection. However, the level of negative-strand virus RNA had no difference between NONO knockout and wild-type cells at 12-24 h post-infection. In summary, our study identified a cellular protein that bound to the replication complex of flavivirus and facilitated the synthesis of positive-strand RNA.IMPORTANCEOver half of the world's population is at risk of flaviviruses infection, posing a serious global health concern. To date, there are no antiviral drugs or treatments for the severe symptoms caused by the infection of flaviviruses. Some cellular proteins could participate in the replication of virus, and these cellular proteins were also ideal targets in antiviral strategy. Here, we identified cellular NONO protein was recruited by flavivirus NS3 protein to the cytoplasm, serving as a "scaffold" for viral replication complex. Our findings also revealed that NONO protein was critical for flavivirus positive-strand RNA synthesis. Specific areas where NONO interacted with flavivirus NS3 proteins and viral UTRs have also been identified. These results propose a new mechanism for cellular protein to participate in flavivirus replication and also raise a new potential anti-flavivirus strategy.

GZMK Facilitates Experimental Rheumatoid Arthritis Progression by Interacting with CCL5 and Activating the ERK Signaling.

Synovial over-proliferation is a key event in the progression of rheumatoid arthritis (RA) disease. Ferroptosis may be essential for maintaining the balance between synovial proliferation and death. This study aimed to investigate the molecular mechanisms mediating the activation and ferroptosis of collagen-induced arthritis(CIA)-synovial fibroblasts (SFs). Differentially expressed genes (DEGs) in the synovial tissues of CIA rats and normal rats were screened through sequencing. The GSE115662 dataset from the GEO database was analyzed and screened for DEGs. The viability, proliferation, migration, invasion, cell cycle, and apoptosis of CIA-SFs were analyzed by cell counting kit-8, 5-ethynyl-2'-deoxyuridine, flow cytometry, transwell migration, and invasion assays. The ferroptosis of CIA-SFs was assessed using matching reagent kits to detect indicators like reactive oxygen species, ferrous iron, malondialdehyde, glutathione, and superoxide dismutase. The interaction between Granzyme K (GZMK) and C-C motif chemokine 5 (CCL5) was determined by coimmunoprecipitation assay. We found abnormal GZMK expression in the GSE115662 database and mRNA sequencing data. GZMK was overexpressed in CIA-SFs, and GZMK promoted cell proliferation, migration, invasion, inflammation, and decreased cell apoptosis and ferroptosis in CIA-SFs. GZMK could interact with CCL5 to activate the ERK signaling. GZMK and CCL5 knockdown improved by reducing arthritis scores, redness and swelling of paws, and pathological changes in joint synovium of CIA rats. CCL5 overexpression reversed the effects of GZMK silencing on CIA-SFs cell proliferation, migration, invasion, apoptosis, and ferroptosis. We confirmed that GZMK accelerated experimental rheumatoid arthritis progression by interacting with CCL5 and activating the ERK signaling.

Super-Enhancer-Driven Syndecan-4 Regulates Intercellular Communication in Hypoxic Pulmonary Hypertension.

Unveiling pro-proliferation genes involved in crosstalk between pulmonary artery endothelial cells and pulmonary artery smooth muscle cells (PASMCs) are important to improving the therapeutic outcome of pulmonary hypertension (PH). Although growing studies have shown that super-enhancers (SEs) play a pivotal role in pathological and physiological processes, the SE-associated genes in PH and their impact on PASMC proliferation remain largely unexplored. We used serotype 5 adenovirus-associated virus to interfere with syndecan-4 and constructed an SU5416 combined with hypoxia-PH model. Chromatin immunoprecipitation sequencing analysis, chromatin immunoprecipitation quantitative polymerase chain reaction, and bioinformatics were used to confirm early growth response 1 was involved in regulating syndecan-4-associated SE in PASMCs. The effects of syndecan-4 and its underlying mechanisms were subsequently elucidated using Western blot, coimmunoprecipitation, and cell coculture assays. Herein, we identified a novel SE-associated gene, syndecan-4, in hypoxia-exposed PASMCs. Syndecan-4 was transcriptionally driven by early growth response 1 via an SE and was significantly overexpressed in hypoxic PASMCs and plasma from patients with PH. Mechanism studies revealed that syndecan-4 induces PASMC proliferation by interacting and regulating protein kinase C α ubiquitination. In addition, syndecan-4 was enriched in exosomes secreted from hypoxic PASMCs, which subsequently transported and led to pulmonary artery endothelial cell dysfunction. Syndecan-4 inhibition in hypoxia by serotype 5 adenovirus-associated virus treatment attenuated the pulmonary artery remodeling and development of PH invivo. Taken together, our results demonstrate that an SE-driven syndecan-4 modulates crosstalk of PASMCs and pulmonary artery endothelial cells and promotes vascular remodeling via the protein kinase C α and exosome pathway, thus providing potential targets for the early diagnosis and treatment of hypoxic PH.

OsPIPK-FAB, A Negative Regulator in Rice Immunity Unveiled by OsMBL1 Inhibition

Phosphatidylinositol signaling system plays a crucial role in plant physiology and development, phosphatidylinositol phosphate kinases (PIPKs) are one of the essential enzymes responsible for catalyzing the synthesis of phosphatidylinositol bisphosphate (PIP2) within this signaling pathway. However, its mechanism of signal transduction remains poorly exploited in plants. OsMBL1, a jacalin-related mannose-binding lectin in rice, plays a crucial role in plant defense mechanisms, acting as a key component of the pattern-triggered immunity (PTI) pathway. Here, a rice phosphatidylinositol-phosphate kinase FAB (OsPIPK-FAB), a member of the rice PIPKs family, as an interacting protein of OsMBL1 through yeast-two-hybrid (Y2H) screening assay. And this interaction was confirmed by using co-immunoprecipitation (Co-IP) and pull-down assay techniques. Furthermore, we demonstrated that the deletion of OsPIPK-FAB gene in plant enhanced resistance against rice blast while overexpression of OsPIPK-FAB increases sensitivity to the fungal infection. Additionally, through determination and measurement of the plant inositol 1,4,5-trisphosphate (IP3) contents and the plant phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity, we revealed that OsMBL1 inhibits the PIP5K kinase activity of OsPIPK-FAB as well as the plant IP3 contents in rice. Conclusively, these findings indicated that OsPIPK-FAB serves as a novel and critical component that is negatively involved in PTI activation and was inhibited by OsMBL1.