Phosphorylation Activity Research Articles

A New Severe Congenital Neutropenia Syndrome Associated with Autosomal Recessive COPZ1 Mutations.

We have identified a new inherited bone marrow (BM) failure syndrome with severe congenital neutropenia (CN) caused by autosomal recessive mutations in the coatomer protein complex I (COPI) subunit zeta 1 (COPZ1) gene. A stop-codon COPZ1 mutation and a missense mutation were found in three patients from two unrelated families. While two affected siblings with a stop-codon COPZ1 mutation suffered from congenital neutropenia (CN) that involves other hematological lineages, and non-hematological tissues, the patient with a missense COPZ1 mutation had isolated neutropenia. Both COPZ1 mutations were localized to a highly evolutionarily conserved region. The resulting truncated COPZ1 protein was predicted to display diminished interaction with its COPI complex partner, COPG1. These findings were consistent with the observed block in retrograde protein transport from the Golgi to the ER in human fibroblasts carrying truncated COPZ1. Human CD34+ cells with truncated or missense COPZ1 had significantly impaired granulocytic differentiation and in zebrafish embryos, truncated Copz1 also resulted in defective myelopoiesis. Intracellularly, truncated COPZ1 downregulated JAK/STAT/CEBPE/G-CSFR signaling and hypoxia-responsive pathways while inducing STING, interferon-stimulated genes, stimulating oxidative phosphorylation activity, and increasing reactive oxygen species (ROS) levels in CD34+ cells. Missense COPZ1 deregulated interferon and JAK/STAT signaling but less than the truncated protein. Finally, treatment with the small molecule HIF1α activator IOX2 or transduction of cells with COPZ2 cDNA restored defective granulopoiesis in COPZ1-mutated CD34+ cells, offering potential therapeutic options.

Alzheimer's disease-like pathology induced by Porphyromonas gingivalis in middle-aged mice is mediated by NLRP3 inflammasome via the microbiota-gut-brain axis.

Porphyromonas gingivalis (P. gingivalis) has been found to enter the brain and induce inflammation, contributing to Alzheimer's disease (AD). P. gingivalis is also closely linked to gut dysbiosis. However, does P. gingivalis induce AD-like pathology through the microbiota-gut-brain axis? There is limited literature on this topic. To determine the precise causal link among P. gingivalis, intestinal inflammation, and AD-related pathology. 12- to 13-month-old female C57BL/6J mice were subjected to ligature placement and oral administration of P. gingivalis over a 24-week period. Then, cognitive performance was evaluated with behavioral tests, while AD neuropathological changes, neuroinflammation, and intestinal inflammation were assessed through qPCR, immunofluorescence, and western blot, and gut microbiota was analyzed by 16S rRNA. Mice exposed to P. gingivalis showed impaired behavior in open field test, novel object recognition, and Y-maze tests. The bacterium infiltrated their brains, increasing Aβ42, AβPP, and Aβ fragments, promoting tau phosphorylation and microglial activation, and reducing levels of ZO-1, PSD95, SYP, and NeuN proteins. Inflammatory factors like NLRP3, caspase-1, IL-1β, IL-6, and TNF-α were elevated in both brains and intestine, while ZO-1 and occludin levels decreased in intestine. P. gingivalis also altered gut microbial compositions. P. gingivalis induced gut dysbiosis and activated the NLRP3 inflammasome in the intestine and brains of mice. This led to impairment of both the intestinal and brain-blood barriers, triggering neuroinflammation and promoting the progression of AD. These findings highlight the critical role of NLRP3 inflammasome activation in the microbiota-gut-brain axis in the AD-like pathology induced by P. gingivalis.

Tumor-Associated Microglia Secrete Extracellular ATP to Support Glioblastoma Progression

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor with poor prognosis and high recurrence rates. The complex immune microenvironment of GBM is highly infiltrated by tumor-associated microglia and macrophages (TAM). TAMs are known to be heterogeneous in their functional and metabolic states and can transmit either protumoral or antitumoral signals to glioma cells. Here, we performed bulk RNA sequencing and single-cell RNA sequencing on samples from patients with GBM, which revealed increased ATP synthase expression and oxidative phosphorylation activity in TAMs located in the tumor core relative to the tumor periphery. Both in vitro and in vivo models displayed similar trends of augmented TAM mitochondrial activity, along with elevated mitochondrial fission, glucose uptake, mitochondrial membrane potential, and extracellular ATP (eATP) production by TAMs in the presence of GBM cells. Tumor-secreted factors, including GM-CSF, induced the increase in TAM eATP production. Elevated eATP in the GBM microenvironment promoted glioma growth and invasion by activating the P2X purinoceptor 7 (P2X7R) on glioma cells. Inhibition of the eATP–P2X7R axis attenuated tumor cell viability in vitro and reduced tumor size and prolonged survival in glioma-bearing mouse models. Overall, this study revealed elevated TAM-derived eATP in GBM and provided the basis for targeting the eATP–P2X7R signaling axis as a therapeutic strategy in GBM. Significance: Glioblastoma-mediated metabolic reprogramming in tumor-associated microglia increases ATP secretion that supports cancer cell proliferation and invasion by activating P2X7R, which can be inhibited to attenuate tumor growth.

Single-Cell Transcriptomics of Human Tonsils Reveals Nicotine Enhances HIV-1-Induced NLRP3 Inflammasome and Mitochondrial Activation.

HIV-1 infection, even with effective antiretroviral therapy (ART), is associated with chronic inflammation and immune dysfunction, contributing to long-term health complications. Nicotine use, prevalent among people with HIV (PWH), is known to exacerbate immune activation and disease progression, but the precise biological mechanisms remain to be fully understood. This study sought to uncover the synergistic effects of HIV-1 infection and nicotine on immune cell function, focusing on beneficial insights into NLRP3 inflammasome activation, oxidative stress, and mitochondrial pathways. Human tonsil explants were infected with HIV-1 and exposed to nicotine. Single-cell RNA sequencing was used to profile immune cell populations and gene expression linked to inflammasome activation, oxidative stress, and mitochondrial function. Gene set enrichment analysis (GSEA) and synergy assessments were conducted to investigate how nicotine modulates immune responses in the context of HIV. The combination of HIV infection and nicotine exposure significantly increased NLRP3 inflammasome activation, thioredoxin, and components of oxidative phosphorylation. This study highlights how the combined effects of HIV-1 and nicotine offer valuable insights into immune modulation, opening doors for future therapeutic strategies. Targeting the NLRP3 inflammasome and addressing nicotine use may contribute to improved outcomes for PWH.

Mitochondrial dynamics and metabolic remodeling in xenograft of IPSC-derived human neural precursors

It is well recognized that the regulation of mitochondrial functions affects the differentiation and maturation of neurons. The study of these processes is of both fundamental and practical importance for regenerative neurobiology. Aim of the study: to characterize the mitochondrial fission changes and their relation to the activation of oxidative phosphorylation (metabolic shift) during maturation of human IPSC-derived neural precursors grafted into rat striatum. Wistar rats (n = 15) were unilaterally injected into the caudate nucleus with neural precursors derived from human IPSCs. Changes in localization and expression of neuronal differentiation markers: nestin, NeuN, neuronal enolase, as well as mitochondrial outer membrane protein, ATP synthase and mitochondrial fission protein Drp1 were assessed by immunostaining. Measurements were performed on graft cells 2 weeks, 3 and 6 months after surgery. Maturation of grafted neurons was associated with fluctuations morphometric parameters of the mitochondrial fraction and Drp1 levels. Increased mitochondrial fission was detected 3 months after transplantation, before an increase in ATP synthase staining by 6th month and a switch of transplanted cells to oxidative phosphorylation. The conducted experiment demonstrated a link between mitochondrial dynamics and changes in the metabolic profile and maturation of transplanted neurons. The regulation of mitochondrial dynamics may have future implications for developing methods to improve the integration of transplanted neurons into recepient brain structures.

Abstract B011: RNA-based therapeutics to target the p53 pathway in cancer

Abstract Mutations in the TP53 gene and activation of the PI3K/AKT pathway are the two most frequent genetic alterations across multiple human tumors. We discovered a crucial mechanism of crosstalk between these two events consisting of two oncogenic lncRNAs, TROLL-2 and TROLL-3, whose expression is induced by mutant p53. We demonstrated that these lncRNAs drive tumor and metastasis formation in several orthotopic models, including breast cancer, lung adenocarcinoma, and melanoma. The investigation of these mouse models and a pan-cancer analysis of human cancers showed that these lncRNAs are markers of tumor progression and lead to the phosphorylation and subsequent activation of AKT by promoting the cytoplasmic localization of the scaffold protein WDR26. Notably, we have now proven that TROLL-2 and TROLL-3 are also involved in the response to cancer therapies. Indeed, the downregulation of these lncRNAs via siRNAs bypasses the AKT-driven resistance to chemotherapeutics (e.g. Adriamycin, docetaxel, and paclitaxel) in triple-negative breast cancer cells, to inhibitors of mutant KRAS (e.g. sotorasib) and EGFR (e.g. erlotinib) in lung adenocarcinoma cells, and to inhibitors of PARP (e.g. niraparib) in ovarian cancer cells. To target these lncRNAs in a clinically relevant manner, we designed LNA GapmeRs, also known as antisense oligonucleotides (ASOs), to downregulate these two lncRNAs directly. The efficacy of the ASOs was confirmed in ex vivo specimens of tumors and metastases of lung adenocarcinoma and ovarian cancer patients. These clinical specimens were also analyzed via single-cell RNA sequencing and phospho-proteomics to unveil the common vs. unique downstream effects of targeting each lncRNA. When administered together, the ASOs targeting TROLL-2 and TROLL-3 effectively decreased the levels of these lncRNAs, which in turn caused WDR26 sequestration in the nucleus, thus preventing the activation of the AKT pathway and the proliferation of these cancer specimens. Furthermore, we confirmed that the antitumor activities of these ASOs were also maintained in vivo, as proven by the decreased tumor proliferation observed in breast cancer xenograft models. Finally, to improve the delivery of these ASOs in vivo, the usage of non-viral systems, including lipid-based and metallic-based nanoparticles, is being tested in both ex vivo and in vivo preclinical models. Overall, our results unveiled TROLL-2 and TROLL-3 as suitable targets to counteract tumor growth and promote the efficacy of chemotherapeutics and targeted therapies in ex vivo and in vivo cancer models. Citation Format: Marco Napoli, Ashley K Lu, Avani A Deshpande, Jaden R Baldwin, Christina L Carr, Michael R Dunne, Omar K Farha, Michelle H Teplensky, Elsa R Flores. RNA-based therapeutics to target the p53 pathway in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B011.

EPCO-11. BIASES IN HEALTHY BRAIN SINGLE-CELL DATASETS: FINDING THE BEST COMPARISON FOR GLIOBLASTOMA STUDIES

Abstract Tumor cells undergo metabolic alterations that support their proliferation and simultaneously modify immune populations, resulting in an immunosuppressive tumor microenvironment. This study aims to compare the metabolic expression of immune cells within the tumor microenvironment to those in a healthy environment. However, there is a lack of single-cell transcriptomic data for normal brain cells, necessitating an analysis of different methods to compare immune cells in tumors with those in a healthy brain. To address this, we explored cell-to-cell metabolic variability using public single-cell RNA-seq (scRNA-seq) data of glioblastoma (n=338564), and from immune cells in the brains of post-mortem individuals (n=14177), fetuses (n=40000), and epilepsy patients (n=466), as well as from immune blood cells (n=329762). We employed ssGSEA to generate activity scores for 70 key metabolic pathways from KEGG for each individual cell. Known marker genes were utilized to identify normal cell populations (macrophages, T cells, oligodendrocytes) in datasets lacking cell type annotations. Our results revealed that blood cells exhibited high metabolic activity in macrophages but showed moderate activity in other cell types. Immune blood cells also demonstrated high oxidative phosphorylation (OXPHOS) but low fatty acid activity. Fetal brain cells displayed a lack of heterogeneity and showed high fatty acid activity with low OXPHOS activity. Brain cells from post-mortem individuals displayed heterogeneity but exhibited low metabolic activity across nearly all cells and pathways. Additionally, the hierarchy of pathway activity differed from the other datasets. Epilepsy data revealed a metabolic activity pattern similar to tumor-infiltrating immune cells, with comparable activity hierarchy and heterogeneity, although the sample size was limited for detailed comparison. Understanding the differences between types of datasets is important to choose the most appropriate comparison, allowing us to observe the metabolic alterations in immune cells caused by glioblastoma and to develop better immunotherapy strategies.

TMOD-29. BETA INTEGRINS REGULATE MITOCHONDRIALLY-MEDIATED CELL MIGRATION IN H3K27M-DIFFUSE MIDLINE GLIOMA

Abstract Infiltration into the brain tissue is one of the main features of diffuse midline gliomas (DMG), also characterized by the histone mutation H3K27M and overall survival of 12-15 months from diagnosis. To study which genes are critical for H3K27M-DMG tumor cell migration in the brain, we established a novel two-step pooled whole genome CRISPR-Migration screen of metastatic H3K27M-DMG (n=3) and glioblastoma (GBM, n=2) stem cells by using serum-free conditions and laminin-dependent cell attachment, mimicking cell migration on brain vasculature scaffolds. Genes involved in focal adhesion (ITGB1, CRKL, PARVA, PTK2, FERMT2) were identified as significantly restricting migration in all H3K27M-DMG lines. From those, only knockout of ITGB1 (ITGB1-KO) fully ablated in vitro migration in all models. In human tumor RNAseq, expression of ITGB1 correlated with higher glioma grade and worse survival specifically in H3K27M-DMG across pediatric brain tumors. In H3K27M-DMG scRNAseq, increased ITGB1 expression was found in cells with OPC-like/Mesenchymal cell signature associated with leading edge and microvasculature. Unexpectedly, ITGB1-KO led to the decreased expression of multiple mitochondrial NADH dehydrogenase genes, and reduced levels of glycolysis and oxidative phosphorylation (OXPHOS) activity. Preliminary mechanistic data linked this phenotype to increased mitochondrial fusion. Genetic (ITGB1-KO) and pharmacologic (anti-ITGB1 antibody AIIB2, CNS delivered) targeting of ITGB1 in a novel orthotopic model of metastatic H3K27M-DMG (UMPED83) led to reduced proliferation and significant extension of survival (p=0.0003 ITGB1-KO, and p=0.0006 AIIB2). Interestingly, ITGB1-KO in another orthotopic H3K27M-DMG model (SVZ) led to upregulation of other integrins (ITGB3 and ITGB5) without change in survival, resulting in susceptibility to treatment with the ITGB3/5 inhibitor Cilengitide (p=0.006). Treatment with AIIB2 and Cilengitide alone or in combination was ineffective in an H3WT adult GBM orthotopic model suggesting unique H3K27M-DMG targetability. In summary, targeting of integrin pathways offers untapped therapeutic opportunities in H3K27M-DMG by impairing tumor cell survival through altered mitochondrial dynamics.

The unexpected PD-L1 suppression function of celery-derived extracellular vesicles improves lung cancer chemotherapy efficacy

Aim: The article explores celery-derived extracellular vesicles (CDEVs), characterized by high cellular uptake, low immunogenicity, and high stability, as a therapeutic strategy for antitumor nanomedicines. Methods: The methods employed in this study include in vitro cell experiments such as co-culture, Western Blot, and flow cytometry. In vivo experiments were conducted in C57BL/6 tumor-bearing mice subcutaneously injected with Lewis lung carcinoma (LLC) cells. The experiments encompassed parameters such as survival rate, body weight, tumor size, flow cytometry, immunohistochemistry, and spectral live imaging system. Results: Our study revealed that CDEVs could be used as drugs to effectively downregulate the phosphorylated signal transducer and activator of transcription 3(p-STAT3)/programmed cell death ligand 1 (PD-L1) axis in lung cancer cells. In co-culture experiments, CDEVs were observed to impede the expression of PD-L1, thereby interfering with the interaction between PD-L1 and programmed death 1 (PD-1) and subsequently preventing the suppression of T cells. In in vivo distribution experiments, CDEVs loaded with paclitaxel (PTX) demonstrated better tumor targeting capabilities. Remarkably, following CDEVs-PTX treatment, CD8+ T cell levels in mice were increased, presumably leading to improved antitumor effects. Conclusion: CDEVs not only serve as drug carriers but also function as drugs themselves; as such, through a single administration of CDEVs, it is possible to combine immunotherapy and chemotherapy to achieve better effects between the two, providing a more comprehensive and effective cancer treatment strategy that promises to improve treatment outcomes and reduce the adverse effects of therapy.

TBL2 Promotes Tumorigenesis via PRMT5/WDR77-Mediated AKT Activation in Breast Cancer.

Breast cancer (BC) is a common malignancy that affects women worldwide. Although transducing beta-like 2 (TBL2), a member of the WD40 repeat protein family, has been implicated in various intracellular signaling pathways, its precise function in BC remains unclear. The expression of TBL2 is analyzed using real-time PCR, western blotting, and immunohistochemistry in BC patient specimens. Kaplan-Meier survival analysis is employed to assess its prognostic significance. Proteomic analysis, immunoprecipitation tests, and protein immunoblotting are employed to examine the impact of TBL2 on AKT phosphorylation activation. The findings reveal selective overexpression of TBL2 in BC, correlating significantly with various clinicopathological characteristics and poor survival outcomes in patients with BC. Through in vivo and in vitro experiments, it is observed that TBL2 suppression inhibits BC cell proliferation, while TBL2 overexpression has the opposite effect. Mechanistically, TBL2 is identified as a scaffolding protein that promotes PRMT5 and WDR77 interaction. This interaction enhances the methyltransferase activity of PRMT5, leading to increased AKT phosphorylation activation and promotion of breast cancer cell proliferation. In conclusion, this study uncovers a novel function of TBL2 in the activation of AKT by PRMT5 and suggests TBL2 as a potential therapeutic target for BC treatment.

The molecular consequences of FOXF1 missense mutations associated with alveolar capillary dysplasia with misalignment of pulmonary veins

BackgroundAlveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a fatal congenital lung disorder strongly associated with genomic alterations in the Forkhead box F1 (FOXF1) gene and its regulatory region. However, little is known about how FOXF1 genomic alterations cause ACD/MPV and what molecular mechanisms are affected by these mutations. Therefore, the effect of ACD/MPV patient-specific mutations in the FOXF1 gene on the molecular function of FOXF1 was studied.MethodsEpitope-tagged FOXF1 constructs containing one of the ACD/MPV-associated mutations were expressed in mammalian cell lines to study the effect of FOXF1 mutations on protein function. EMSA binding assays and luciferase assays were performed to study the effect on target gene binding and activation. Immunoprecipitation followed by SDS‒PAGE and western blotting were used to study protein‒protein interactions. Protein phosphorylation was studied using phos-tag western blotting.ResultsAn overview of the localization of ACD/MPV-associated FOXF1 mutations revealed that the G91-S101 region was frequently mutated. A three-dimensional model of the forkhead DNA-binding domain of FOXF1 showed that the G91-S101 region consists of an α-helix and is predicted to be important for DNA binding. We showed that FOXF1 missense mutations in this region differentially affect the DNA binding of the FOXF1 protein and influence the transcriptional regulation of target genes depending on the location of the mutation. Furthermore, we showed that some of these mutations can affect the FOXF1 protein at the posttranscriptional level, as shown by altered phosphorylation by MST1 and MST2 kinases.ConclusionMissense mutations in the coding region of the FOXF1 gene alter the molecular function of the FOXF1 protein at multiple levels, such as phosphorylation, DNA binding and target gene activation. These results indicate that FOXF1 molecular pathways may be differentially affected in ACD/MPV patients carrying missense mutations in the DNA-binding domain and may explain the phenotypic heterogeneity of ACD/MPV.

The structures of protein kinase A in complex with CFTR: Mechanisms of phosphorylation and noncatalytic activation

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as a model system for understanding the eukaryotic kinases. Using cryoelectron microscopy, we present complex structures of the PKA catalytic subunit (PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane conductance regulator (CFTR)-an ion channel vital to human health. CFTR gating requires phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C at two locations, establishing two "catalytic stations" near to, but not directly involving, the R domain. This configuration, coupled with the conformational flexibility of the R domain, permits transient interactions of the eleven spatially separated phosphorylation sites. Furthermore, we determined two structures of the open-pore CFTR stabilized by PKA-C, providing a molecular basis to understand how PKA-C stimulates CFTR currents even in the absence of phosphorylation.

Insights into Alkaline Phosphatase Anti-Inflammatory Mechanisms.

The endogenous ecto-enzyme and exogenously administered alkaline phosphatase (ALP) have been evidenced to significantly attenuate inflammatory conditions, including Toll-like receptor 4 (TLR4)-related signaling and cytokine overexpression, barrier tissue dysfunction and oxidative stress, and metabolic syndrome and insulin resistance, in experimental models of colitis, liver failure, and renal and cardiac ischemia-reperfusion injury. This suggests multiple mechanisms of ALP anti-inflammatory action that remain to be fully elucidated. Recent studies have contributed to a deeper comprehension of the role played by ALP in immune metabolism. This review outlines the established effects of ALP on lipopolysaccharide (LPS)-induced inflammation, including the neutralization of LPS and the modulation of purinergic signaling. The additional mechanisms of anti-inflammatory activity of ALP observed in different pathologies are proposed. The anti-inflammatory pathways of ALP may include a scavenger receptor (CD36)-mediated activation of β-oxidation and oxidative phosphorylation, caveolin-dependent endocytosis, and selective autophagy-dependent degradation.

Bioactive Porous Composite Implant Guides Mesenchymal Stem Cell Differentiation and Migration to Accelerate Bone Reconstruction.

Delayed healing and non-healing of bone defects pose significant challenges in clinical practice, with metal materials increasingly recognized for their significance in addressing these issues. Among these materials, Strontium (Sr) and Zinc (Zn) have emerged as promising agents for promoting bone repair. Building upon this insight, this research evaluates the impact of a porous Sr@Zn@SiO2 nanocomposite implant on bone regeneration, aiming to advance the field of bone repair. The preparation of the Sr@Zn@SiO2 composite implant involves various techniques such as roasting, centrifugation, and washing. The material's composition is examined, and its microstructure and element distribution are analyzed using TEM and elemental scanning technology. In vitro experiments entail the isolation and characterization of BMSCs followed by safety assessments of the implant material, evaluation of cell migration capabilities, and relevant proliferation markers. Mechanistically, this study delves into key targets associated with significant changes in the osteogenic process. In vivo experiments involve establishing a rat femur bone defect model, followed by assessment of the osteogenic potential of Sr@Zn@SiO2 using Micro-CT imaging and tissue section staining. Through in vivo and in vitro investigations, we validate the osteogenic efficacy of the Sr@Zn@SiO2 composite implant. In vitro analyses demonstrate that porous Sr@Zn@SiO2 nanocomposite materials upregulate BMP-2 expression, leading to the activation of Smad1/5/9 phosphorylation and subsequent activation of downstream osteogenic genes, culminating in BMSCs osteogenic differentiation and bone proliferation. And the migration of BMSCs is closely related to the high expression of CXCL12/CXCR4, which will also provide the conditions for osteogenesis. In vivo, the osteogenic ability of Sr@Zn@SiO2 was also confirmed in rats. In our research, the porous Sr@Zn@SiO2 composite implant displays prominent osteogenic effect and promotes the migration and differentiation of BMSCs to promote bone defect healing. This bioactive implant has surgical application potential in the future.

Linking planar polarity signalling to actomyosin contractility during vertebrate neurulation.

Actomyosin contractility represents an ancient feature of eukaryotic cells participating in many developmental and homeostasis events, including tissue morphogenesis, muscle contraction and cell migration, with dysregulation implicated in various pathological conditions, such as cancer. At the molecular level, actomyosin comprises actin bundles and myosin motor proteins that are sensitive to posttranslational modifications like phosphorylation. While the molecular components of actomyosin are well understood, the coordination of contractility by extracellular and intracellular signals, particularly from cellular signalling pathways, remains incompletely elucidated. This study focuses on WNT/planar cell polarity (PCP) signalling, previously associated with actomyosin contractility during vertebrate neurulation. Our investigation reveals that the main cytoplasmic PCP proteins, Prickle and Dishevelled, interact with key actomyosin components such as myosin light chain 9 (MLC9), leading to its phosphorylation and localized activation. Using proteomics and microscopy approaches, we demonstrate that both PCP proteins actively control actomyosin contractility through Rap1 small GTPases in relevant in vitro and in vivo models. These findings unveil a novel mechanism of how PCP signalling regulates actomyosin contractility through MLC9 and Rap1 that is relevant to vertebrate neurulation.

The Role of Changes in the Redox Status in the Pathogenesis of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia is a hemoblastosis of CD5+ B lymphocytes with lymphocytosis, damage to the lymphatic organs, occurring in the older age group, the etiology and pathogenesis of which are not fully understood. Oxidative stress is an important factor in the regulation of stem cells and the activation of intracellular survival signaling pathways in chronic lymphocytic leukemia cells. The aim of the study was to analyze the current data on the role of redox status changes in the pathogenesis of chronic lymphocytic leukemia. A review of published relevant studies 2018-2023, scientific articles in scientific electronic bibliographic databases PubMed and Social Sciences Citation Index, devoted to the pathogenesis of chronic lymphocytic leukemia and the role of free-radical oxidation processes in it was carried out. In chronic lymphocytic leukemia, oxidative stress with a systemic excess of reactive oxygen species, an imbalance in the effectiveness of antioxidant defense is caused mainly by activation of oxidative phosphorylation in mitochondria, low levels of NADPH-oxidase type 2, increased expression of heme oxygenase-1, glutathione peroxidase and glutathione recycling enzymes, superoxide dismutase-2, thioredoxins and decreased expression of catalase. One of the mechanisms of resistance to drug therapy and oxidative stress of chronic lymphocytic leukemia cells is the intracellular signaling pathway dependent on erythroid nuclear factor-2, due to the activation of expression in cells of superoxide dismutase-2, catalase, glutathione peroxidase, peroxiredoxin-3 and-5, heme oxygenase-1, thioredoxin-1 and -2, reduced glutathione, natural killer cell activity, which is associated with lifespan, chemotaxis, proliferation, and survival. FOXO family proteins are believed to suppress carcinogenesis. FOXO3a increases the expression of superoxide dismutase-2, catalase, glutathione peroxidase, peroxiredoxin-3 and -5, and the activity of natural killer cells, which promotes the survival of tumor cells. The development of new targeted pharmacological agents that are capable of accumulating reactive oxygen species and reducing antioxidant protection due to the degradation of erythroid nuclear factor-2 and activation of NADPH-quinone oxidoreductase-1 is underway, which modernizes the therapy of chronic lymphocytic leukemia.

The Influence of Aspergillus fumigatus Fatty Acid Oxygenases PpoA and PpoC on Caspofungin Susceptibility.

Aspergillus fumigatus can cause invasive pulmonary aspergillosis (IPA). Fungicidal azoles and fungistatic caspofungin (CAS) are the first- and second-line therapies, respectively, used to treat IPA. Treatment of A. fumigatus with CAS or micafungin induces the production of the oxylipin 5,8-diHODE by the fungal oxygenase PpoA. For this article, we investigated the influence of ppo genes, which encode the fatty acid oxygenases responsible for oxylipin biosynthesis, on CAS tolerance. The influence of PpoA and PpoC on CAS tolerance is mediated by MpkA phosphorylation and protein kinase A (PKA) activity. RNAseq transcriptional profiling and the label-free quantitative proteomics of the ppoA and ppoC mutants showed that differentially expressed genes and proteins are related to secondary metabolites and carbohydrate metabolism. We also characterized two clinical isolates, CM7555 and IFM61407, which decrease and increase susceptibility to CAS, respectively. CM7555 does not exhibit increased oxylipin production in the presence of CAS but oxylipin induction upon CAS exposure is increased in IFM61407, suggesting that oxylipins are not the only mechanism involved in CAS tolerance in these isolates. Upon CAS exposure, CM7555 has higher MpkA phosphorylation and PKA activity than IFM61407. Our results reveal the different aspects and genetic determinants involved in A. fumigatus CAS tolerance.

S-palmitoylation of MAP kinase is essential for fungal virulence.

S-palmitoylation is an important reversible protein post-translational modification in organisms. However, its role in fungi is uncertain. Here, we found the treatment of the rice false fungus Ustilaginoidea virens with S-palmitoylation inhibitor 2 BP resulted in a significant decrease in fungal virulence. Comprehensive identification of S-palmitoylation sites and proteins in U. virens revealed a total of 4,089 S-palmitoylation sites identified among 2,192 proteins and that S-palmitoylated proteins were involved in diverse biological processes. Among the five palmitoyltransferases, UvPfa3 and UvPfa4 were found to regulate the pathogenicity of U. virens. We then performed quantitative proteomic analysis of ∆UvPfa3 and ∆UvPfa4 mutants. Interestingly, S-palmitoylated proteins were significantly enriched in the mitogen-activated protein kinase and autophagy pathways, and MAP kinase UvSlt2 was confirmed to be an S-palmitoylated protein which was palmitoylated by UvPfa4. Mutations of S-palmitoylation sites in UvSlt2 resulted in significantly reduced fungal virulence and decreased kinase enzymatic activity and phosphorylation levels. Simulations of molecular dynamics demonstrated mutation of S-palmitoylation sites in UvSlt2 causing decreased hydrophobic solvent-accessible surface area, thereby weakening the bonding force with its substrate UvRlm1. Taken together, S-palmitoylation promotes U. virens virulence through palmitoylation of MAP kinase UvSlt2 by palmitoyltransferase UvPfa4. This enhances the enzymatic phosphorylation activity of the kinase, thereby increasing hydrophobic solvent-accessible surface area and binding activity between the UvSlt2 enzyme and its substrate UvRlm1. Our studies provide a framework for dissecting the biological functions of S-palmitoylation and reveal an important role for S-palmitoylation in regulating the virulence of the pathogen.IMPORTANCES-palmitoylation is an important post-translational lipid modification of proteins. However, its role in fungi is uncertain. In this study, we found that S-palmitoylation promotes virulence of rice false smut fungus U. virens through palmitoylation of MAP kinase UvSlt2 by palmitoyltransferase UvPfa4. This enhances the enzymatic phosphorylation activity of the kinase, thereby increasing hydrophobic solvent-accessible surface area and binding activity between the UvSlt2 enzyme and its substrate UvRlm1. Our studies provide a framework for dissecting the biological functions of S-palmitoylation and reveal an important role for S-palmitoylation in regulating the virulence of the pathogen. This is the first functional study to reveal the role of S-palmitoylation in fungal virulence.

The Biphasic Activity of Auricularia Auricula-Judae Extract on Bone Homeostasis Through Inhibition of Osteoclastogenesis and Modulation of Osteogenic Activity.

Osteoporosis arises from the disturbance of bone homeostasis, a process regulated by osteoblasts and osteoclasts. The treatment and prevention of bone metabolic disorders resulting from an imbalance in bone homeostasis require the use of agents that effectively promote both bone formation and anti-resorptive effects. Therefore, an investigation was carried out to determine the potential of the edible mushroom Auricularia auricula-judae in modulating bone remodeling by inhibiting RANKL-induced osteoclastogenesis and enhancing BMP-2-stimulated osteoblast differentiation. Moreover, this study assessed the mode of action of the Auricularia auricula-judae extracts. The staining of tartrate-resistant acid phosphatase (TRAP), a marker for osteoclast activity, demonstrated that Auricularia auricula-judae water extract (AAJWE) inhibited the formation of multinucleated osteoclasts while exhibiting no cytotoxic effects. The study demonstrated that AAJWE reduced RANKL-induced osteoclast differentiation by inhibiting c-Fos/NFATc1 through the inhibition of ERK and JNK phosphorylation during the RANKL-induced osteoclast differentiation. Moreover, AAJWE exhibited a dose-dependent induction of ALP expression in the presence of BMP-2 during osteoblast differentiation. The AAJWE strengthened BMP-2-induced osteogenesis through the activation of Runx2 and Smad phosphorylation. Therefore, AAJWE emerges as a promising candidate for both prevention and therapy owing to its biphasic effect, which aids in the preservation of bone homeostasis.

Activation patterns of intracellular signaling pathways in cardiac hypertrophy induced by different exercise modes in rats

Abstract Introduction Cardiac remodeling and hypertrophy cause as adaptive responses to physiological and pathological stimuli. Physiological cardiac hypertrophy is characterized by normal or enhanced cardiac function and myocardial metabolism, and induces concentric and eccentric hypertrophy by different exercise modes, without cardiac dysfunction and fibrosis. Each formative mechanism of cardiac hypertrophy is regulated by distinct and/or similar cellular signaling pathways. Activation of cardiac intracellular signaling pathway of phosphoinositide 3- kinase (PI3K) is involved in an underlying mechanism of developed eccentric cardiac hypertrophy by aerobic exercise training (AT). However, the activation patterns of intracellular signaling pathways of physiological cardiac hypertrophy by different exercise modes remain unclear. Purpose The purpose of this study was to investigate the activation patterns of intracellular signaling pathways in cardiac hypertrophy induced by different exercise modes, including AT, resistance training (RT), and high intensity interval training (HIIT). Methods Forty male 10-week-old Sprague-Dawley (SD) rats were divided into four groups; sedentary control (Con, n=10), AT (treadmill running, 60 min at 30 m/min, 5 days/wk for 8 wk, n=10), RT (ladder climbing, 8-10 sets/day, 3 d/wk for 8 wk, n=10), HIIT (14 repeats of 20-s swimming session with 10-s pause between sessions, 4 d/wk for 6 wk from 12-wk-old, n=10) groups. As a cardiac hypertrophic intracellular signaling pathways, PI3K protein expression level and mTOR and MAPK (ERK1/2, JNK1/2 and p38) phosphorylation levels in rat hearts were measured by Western blotting analysis. Results The hearts of rats in each training group developed cardiac hypertrophy. Moreover, the enzyme activity of oxidative phosphorylation in the skeletal muscle was promoted in the AT group, the oxidative phosphorylation and glycolytic enzyme activities were promoted in the HIIT group, and muscle hypertrophy occurred in the RT group. Levels of PI3K protein expression and mTOR phosphorylation in the heart were significantly higher in the AT, RT, and HIIT groups as compared to the Con group (each p<0.05). Additionally, p38 phosphorylation level in the heart was significantly higher in the RT group as compared to the Con, AT, and HIIT groups (each p<0.05). Furthermore, p44 ERK phosphorylation level in the heart of the RT group was significantly higher than that in the AT and HIIT groups (each p<0.05), but p42 ERK phosphorylation level did not differ among four groups. JNK1/2 (p46/p54) phosphorylation level in the heart was significantly higher in the RT group as compared to the Con group (p<0.05). Conclusion These findings suggest that the activation of PI3K-mTOR signaling pathway in the heart may be involved in the development of AT, RT and HIIT-induced cardiac hypertrophy, and the MAPK signaling activation may affect the development of cardiac hypertrophy by RT.