Screening and separation of natural anticancer active ingredients related to phospholipase C.

Based on the specific binding of drug molecules to cell membrane receptors, a screening and separation method for active compounds of natural products was established by combining phospholipase C (PLC) sensitized hollow fiber microscreening by a solvent seal with high-performance liquid chromatography technology. In the process, the factors affecting the screening were optimized. Under the optimal screening conditions, we screened honokiol (HK), magnolol (MG), negative control drug carbamazepine, and positive control drug amentoflavone, the repeatability of the method was tested. The PLC activity was determined before and after the screening. Experimental results showed that the sensitization factors of PLC of HK and MG were 61.0 and 48.5, respectively, and amentoflavone was 15.0, carbamazepine could not bind to PLC. Moreover, the molecular docking results were consistent with this measurement, indicating that HK and MG could be combined with PLC, and they were potential interacting components with PLC. This method used organic solvent to seal the PLC greatly ensuring the activity, so this method had the advantage of integrating separation, and purification with screening, it not only exhibited good reproducibility and high sensitivity but was also suitable for screening the active components in natural products by various targets in vitro.

PLC-CN-NFAT1 signaling-mediated Aß and IL-1ß crosstalk synergistically promotes hippocampal neuronal damage.

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Neuronal calcium overload plays an important role in Aß deposition and neuroinflammation, which are strongly associated with AD. However, the specific mechanisms by which calcium overload contributes to neuroinflammation and AD and the relationship between them have not been elucidated. Phospholipase C (PLC) is involved in regulation of calcium homeostasis, and CN-NFAT1 signaling is dependent on intracellular Ca2+ ([Ca2+]i) to regulate transcription of genes. Therefore, we hypothesized that the PLC-CN-NFAT1 signaling might mediate the interaction between Aß and inflammation to promote neuronal injury in AD. In this experiment, the results showed that the levels of Aß, IL-1ß and [Ca2+]i in the hippocampal primary neurons of APP/PS1 mice (APP neurons) were significantly increased. IL-1ß exposure also significantly increased Aß and [Ca2+]i in HT22 cells, suggesting a close association between Aß and IL-1ß in the development of AD. Furthermore, PLC activation induced significant calcium homeostasis imbalance, cell apoptosis, Aß and ROS production, and significantly increased expressions of CN and NFAT1, while PLC inhibitor significantly reversed these changes in APP neurons and IL-1ß-induced HT22 cells. Further results indicated that PLC activation significantly increased the expressions of NOX2, APP, BACE1, and NCSTN, which were inhibited by PLC inhibitor in APP neurons and IL-1ß-induced HT22 cells. All indications point to a synergistic interaction between Aß and IL-1ß by activating the PLC-CN-NFAT1 signal, ultimately causing a vicious cycle, resulting in neuronal damage in AD. The study may provide a new idea and target for treatment of AD.

Glucocorticoids paradoxically promote steroid resistance in B cell acute lymphoblastic leukemia through CXCR4/PLC signaling.

Glucocorticoid (GC) resistance in childhood relapsed B-cell acute lymphoblastic leukemia (B-ALL) represents an important challenge. Despite decades of clinical use, the mechanisms underlying resistance remain poorly understood. Here, we report that in B-ALL, GC paradoxically induce their own resistance by activating a phospholipase C (PLC)-mediated cell survival pathway through the chemokine receptor, CXCR4. We identify PLC as aberrantly activated in GC-resistant B-ALL and its inhibition is able to induce cell death by compromising several transcriptional programs. Mechanistically, dexamethasone (Dex) provokes CXCR4 signaling, resulting in the activation of PLC-dependent Ca2+ and protein kinase C signaling pathways, which curtail anticancer activity. Treatment with a CXCR4 antagonist or a PLC inhibitor improves survival of Dex-treated NSG mice in vivo. CXCR4/PLC axis inhibition significantly reverses Dex resistance in B-ALL cell lines (in vitro and in vivo) and cells from Dex resistant ALL patients. Our study identifies how activation of the PLC signalosome in B-ALL by Dex limits the upfront efficacy of this chemotherapeutic agent.

Unveiling the ocular battlefield: Insights into Pseudomonas aeruginosa virulence factors and their implications for multidrug resistance.

The research investigates the virulence factors of Pseudomonas aeruginosa (P. aeruginosa), a pathogen known for its ability to cause human infections by releasing various exoenzymes and virulence factors. Particularly relevant in ocular infections, where tissue degeneration can occur, even after bacterial growth has ceased due to the potential role of secreted proteins/enzymes. Clinical isolates of P. aeruginosa, both ocular (146) and non-ocular (54), were examined to determine the frequency and mechanism of virulence factors. Phenotypic characterization revealed the production of alginate, biofilm, phospholipase C, and alkaline protease, while genotypic testing using internal uniplex PCR identified the presence of Exo U, S, T, Y, and LasB genes. Results showed a significant prevalence of Exo U and Y genes in ocular isolates, a finding unique to Indian studies. Additionally, the study noted that ocular isolates often contained all four secretomes, suggesting a potential link between these factors and ocular infections. These findings contribute to understanding the pathogenesis of P. aeruginosa infections, particularly in ocular contexts, and highlights the importance of comprehensive virulence factor analysis in clinical settings.

Molecular Mechanism of Oocyte Activation in Mammals: Past, Present, and Future Directions.

During mammalian fertilization, repetitive intracellular Ca2+ increases known as Ca2+ oscillations occur. These oscillations are considered crucial for successful fertilization and subsequent embryonic development. Numerous researchers have endeavored to elucidate the factors responsible for inducing Ca2+ oscillations across various mammalian species. Notably, sperm-specific phospholipase C zeta (PLCζ) emerged as a prominent candidate capable of initiating Ca2+ oscillations, particularly in mammals. Genetic mutation of PLCζ in humans results in the absence of Ca2+ oscillations in mouse oocytes. Recent studies further underscored PLCζ's significance, revealing that sperm from PLCζ-deficient (Plcz1-/-) mice fail to induce Ca2+ oscillations upon intracytoplasmic sperm injection (ICSI). Despite these findings, observations from in vitro fertilization (IVF) experiments using Plcz1-/- sperm revealed some residual intracellular Ca2+ increases and successful oocyte activation, hinting at potential alternative mechanisms. In this review, we introduced the current hypothesis surrounding oocyte activation in mammals, informed by contemporary literature, and probed into the enigmatic mechanisms underlying mammalian fertilization-induced oocyte activation.

Nonspecific phospholipases C3 and C4 interact with PIN-FORMED2 to regulate growth and tropic responses in Arabidopsis.

The dynamic changes in membrane phospholipids affect membrane biophysical properties and cell signaling, thereby influencing numerous biological processes. Nonspecific phospholipase C (NPC) enzymes hydrolyze common phospholipids to release diacylglycerol (DAG), which is converted to phosphatidic acid (PA) and other lipids. In this study, 2 Arabidopsis (Arabidopsis thaliana) tandemly arrayed genes, NPC3 and NPC4, were identified as critical factors modulating auxin-controlled plant growth and tropic responses. Moreover, NPC3 and NPC4 were shown to interact with the auxin efflux transporter PIN-FORMED2 (PIN2). The loss of NPC3 and NPC4 enhanced the endocytosis and vacuolar degradation of PIN2, which disrupted auxin gradients and slowed gravitropic and halotropic responses. Furthermore, auxin-triggered activation of NPC3 and NPC4 is required for the asymmetric PA distribution that controls PIN2 trafficking dynamics and auxin-dependent tropic responses. Collectively, our study reveals an NPC-derived PA signaling pathway in Arabidopsis auxin fluxes that is essential for fine-tuning the balance between root growth and environmental responses.

Purinergic signaling promotes premature senescence.

Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.

Numerical Analysis of Compound Biochemical Calcium Oscillations Process in Hepatocyte Cells.

The hepatocyte cells regulate the wide range of liver function by moderating cellular activities such as lipid, protein metabolism, carbohydrate, and interact with other cells for proliferation and maintenance. In hepatocyte cells, the concentration of calcium uptake is quite extensive from various agonists such as active G α ${G_\alpha}$ subunit, active phospholipase C, free calcium in the cytosol, and endoplasmic reticulum. The overproduction and degradation of calcium signals can cause homeostasis, liver inflammation, and liver diseases. The spatiotemporal behavior of calcium oscillation reveals the physiological role of these cellular entities in understanding the process of production and degradation. No computational attempt has been registered to date on the compound calcium regulation of these cellular entities including the memory of cells. Hence, the authors proposed a fractional order compartmental model that systematically simulates the exchange of calcium intake in cellular entities. The nonlinear equations of the rate of changes in the active G α ${G_\alpha}$ subunit, active phospholipase C, free calcium in the cytosol, and endoplasmic reticulum are coupled to form a nonlinear fractional order initial value problem. The existence and uniqueness, stability analysis of the model is performed that validate the theoretical results and explore the dynamic behaviour of calcium oscillation in each compartment.

Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

Hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine, and this hydrolysis activity drives tissue damage and inflammation and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and result in secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcriptional regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional readthrough of the cerN transcription terminator. Evidence for these conclusions came from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and reverse transcriptase PCR (RT-PCR) showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observed that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to the regulation of plcH transcription as a site for the integration of multiple host-derived signals. IMPORTANCE: PlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of Pseudomonas aeruginosa. Here, we show that sphingosine, which presents itself or as a product of P. aeruginosa sphingomyelinase and ceramidase activity, leads to the induction of plcH transcription. This transcriptional induction occurs from the promoter of the upstream ceramidase gene generating a conditional operon. The transcript on which plcH resides, therefore, is different depending on which host molecule or condition leads to induction, and this may have implications for PlcH post-transcriptional regulation. This work also adds to our understanding of P. aeruginosa with host-derived sphingolipids.

5,6-diHETE lactone (EPA-L) mediates hypertensive microvascular dilation by activating the endothelial GPR-PLC-IP3 signaling pathway.

Endothelial microvascular dysfunction affects multi-organ pathologic processes that contribute to increased vascular tone and is at the base of impaired metabolic and cardiovascular diseases. The vascular dilation impaired by nitric oxide (NO) deficiency in such dysfunctional endothelium is often balanced by endothelial-derived hyperpolarizing factors (EDHFs), which play a critical role in managing vascular tone. Our latest research has uncovered a new group of lactone oxylipins produced in the polyunsaturated fatty acids (PUFAs) CYP450 epoxygenase pathway, significantly affecting vascular dilation. The lactone oxylipin, derived from arachidonic acid (5,6-diHET lactone, AA-L), has been previously shown to facilitate vasodilation dependent on the endothelium in isolated human microvessels. The administration of the lactone oxylipin derived from eicosapentaenoic acid (5,6-diHETE lactone, EPA-L) to hypertensive rats demonstrated a significant decrease in blood pressure and improvement in the relaxation of microvessels. However, the molecular signaling processes that underlie these observations were not fully understood. The current study delineates the molecular pathways through which EPA-L promotes endothelium-dependent vascular dilation. In microvessels from hypertensive individuals, it was found that EPA-L mediates endothelium-dependent vasodilation while the signaling pathway was not dependent on NO. In vitro studies on human endothelial cells showed that the hyperpolarization mediated by EPA-L relies on G-protein-coupled receptor (GPR)-phospholipase C (PLC)-IP3 signaling that further activates calcium-dependent potassium flux. The pathway was confirmed using a range of inhibitors and cells overexpressing GPR40, where a specific antagonist reduced the calcium levels and outward currents induced by EPA-L. The downstream AKT and endothelial NO synthase (eNOS) phosphorylations were non-significant. These findings show that the GPR-PLC-IP3 pathway is a key mediator in the EPA-L-triggered vasodilation of arterioles. Therefore, EPA-L is identified as a significant lactone-based PUFA metabolite that contributes to endothelial and vascular health.

Phospholipase C Zeta in Human Spermatozoa: A Systematic Review on Current Development and Clinical Application.

During fertilization, the fusion of the spermatozoa with the oocytes causes the release of calcium from the oocyte endoplasmatic reticulum. This, in turn, triggers a series of calcium ion (Ca2+) oscillations, a process known as oocyte activation. The sperm-specific factor responsible for oocyte activation is phospholipase C zeta (PLCζ). Men undergoing intracytoplasmic sperm injection (ICSI) with their spermatozoa lacking PLCζ are incapable of generating Ca2+ oscillation, leading to fertilization failure. The immunofluorescence assay is the most used technique to assess the expression and localization of PLCζ and to diagnose patients with reduced/absent ability to activate the oocytes. In these patients, the use of assisted oocyte activation (AOA) technique can help to yield successful ICSI results and shorten the time of pregnancy. However, the production of a stable PLCζ recombinant protein represents a new powerful therapeutic approach to treating individuals with this condition. We aim to conduct a systematic review focusing on the expression, level, and localization of PLCζ, discussing the novel genetic mutation associated with its impairment. In addition, we highlight the benefits of AOA, looking at new and less invasive methods to diagnose and treat cases with PLCζ dysfunction.

Cryopreservation of equine spermatozoa reduces plasma membrane integrity and phospholipase C zeta 1 content as associated with oocyte activation.

BACKGROUND: Phospholipase C zeta (PLCZ1) is considered the major sperm-borne oocyte activation factor. Cryopreserved stallion spermatozoa are commonly used for intracytoplasmic sperm injection (ICSI). However, plasma membrane damage and protein modifications caused by cryopreservation could impair sperm structure and function, leading to a reduction of PLCZ1 and oocyte activation after ICSI. OBJECTIVES: We compared membrane integrity and PLCZ1 abundance in populations for fresh, frozen, and refrozen stallion spermatozoa, either thawed and refrozen at room or low temperature; and examined the effect of relative PLCZ1 content on cleavage after ICSI. MATERIALS AND METHODS: Western blotting, ELISA, and immunofluorescence were conducted in stallion spermatozoa, freezing extenders, and detergent-extracted sperm fractions to detect and quantify PLCZ1. Retrospectively, PLCZ1 content and cleavage rate were analyzed. Fresh, frozen, and refrozen at room and low temperatures spermatozoa were evaluated for acrosomal and plasma membrane integrity and PLCZ1 content using flow cytometry. RESULTS: Western blotting, ELISA, and immunofluorescence revealed significant reduction of PLCZ1 in spermatozoa after cryopreservation and confirmed PLCZ1 detection in extenders. After detergent extraction, a PLCZ1-nonextractable fraction remained in the postacrosomal region of spermatozoa. Plasma membrane integrity was significantly reduced after freezing. Acrosomal and plasma membrane integrity were similar between frozen and refrozen samples at low temperature, but both were significantly higher than samples refrozen at room temperature. Acrosomal and plasma membrane integrity significantly correlated to PLCZ1 content. Percentages of PLCZ1-labeled spermatozoa and PLCZ1 content were reduced after freezing but not after refreezing. Relative content and localization of PLCZ1 were associated with cleavage rates after ICSI. DISCUSSION AND CONCLUSION: Sperm PLCZ1 content associates with cleavage rates after ICSI. Cryopreservation is detrimental to sperm plasma membrane integrity and PLCZ1 retention. However, refreezing did not result in additional PLCZ1 loss. Refreezing stallion spermatozoa at a low temperature resulted in better survival but did not improve PLCZ1 retention.

Membrane lipid modulations by methyl-ß-cyclodextrin uncouple the Drosophila light-activated phospholipase C from TRP and TRPL channel gating.

Sterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-ß-cyclodextrin (MßCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein-coupled phospholipase Cß (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings. Here, we studied the effects of MßCD on Drosophila phototransduction using electrophysiology while fluorescently monitoring PIP2 hydrolysis, aiming to examine the effects of sterol modulation on PIP2 hydrolysis and the ensuing light-response in the native system. Incubation of photoreceptor cells with MßCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light response. MßCD also suppressed PLC-dependent TRP/TRPL constitutive channel activity in the dark induced by mitochondrial uncouplers, but PLC-independent activation of the channels by linoleic acid was not affected. Furthermore, MßCD suppressed a constitutively active TRP mutant-channel, trpP365, suggesting that TRP channel activity is a target of MßCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptors and simultaneously monitored PIP2-hydrolysis by translocation of fluorescently tagged Tubby protein domain, from the plasma membrane to the cytosol, revealed that MßCD virtually abolished the light response when having little effect on the light-activated PLC. Together, MßCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggesting the involvement of distinct nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel gating.

Fatty Oil of Descurainia Sophia Nanoparticles Improve Monocrotaline-Induced Pulmonary Hypertension in Rats Through PLC/IP3R/Ca2+ Signaling Pathway.

Purpose: Fatty oil of Descurainia Sophia (OIL) has poor stability and low solubility, which limits its pharmacological effects. We hypothesized that fatty oil nanoparticles (OIL-NPs) could overcome this limitation. The protective effect of OIL-NPs against monocrotaline-induced lung injury in rats was studied. Methods: We prepared OIL-NPs by wrapping fatty oil with polylactic-polyglycolide nanoparticles (PLGA-NPs) and conducted in vivo and in vitro experiments to explore its anti-pulmonary hypertension (PH) effect. In vitro, we induced malignant proliferation of pulmonary artery smooth muscle cells (RPASMC) using anoxic chambers, and studied the effects of OIL-NPs on the malignant proliferation of RPASMC cells and phospholipase C (PLC)/inositol triphosphate receptor (IP3R)/Ca2+ signal pathways. In vivo, we used small animal echocardiography, flow cytometry, immunohistochemistry, western blotting (WB), polymerase chain reaction (PCR) and metabolomics to explore the effects of OIL-NPs on the heart and lung pathological damage and PLC/IP3R/Ca2+ signal pathway of pulmonary hypertension rats. Results: We prepared fatty into OIL-NPs. In vitro, OIL-NPs could improve the mitochondrial function and inhibit the malignant proliferation of RPASMC cells by inhibiting the PLC/IP3R/Ca2+signal pathway. In vivo, OIL-NPs could reduce the pulmonary artery pressure of rats and alleviate the pathological injury and inflammatory reaction of heart and lung by inhibiting the PLC/IP3R/Ca2+ signal pathway. Conclusion: OIL-NPs have anti-pulmonary hypertension effect, and the mechanism may be related to the inhibition of PLC/IP3R/Ca2+signal pathway.

Clostridium perfringens phospholipase C, an archetypal bacterial virulence factor, induces the formation of extracellular traps by human neutrophils.

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis and then escapes and spreads to deeper tissues. Understanding the role of NETs in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.

Cryopreservation, in addition to protein tyrosine phosphorylation, alters the distribution of phosphatidyl inositol bisphosphate and the localization of cytoskeletal and signaling proteins (gelsolin, tyrosine kinase c-SRC and phospholipase C-ζ) in the perinuclear theca of boar sperm.

Cryopreservation of boar spermatozoa affects the perinuclear theca (PT) and involves several proteins and molecules that play important roles during capacitation and the acrosomal reaction. The objective of the present study was to evaluate whether the deleterious effects of cryopreservation in addition to protein tyrosine phosphorylation are accompanied by changes in the distribution of phosphatidyl inositol bisphosphate (PIP2) and the localization of cytoskeletal and signaling proteins in the perinuclear theca of cryopreserved boar spermatozoa. For this purpose, by immunocytochemistry (IC) the changes in localization of phosphorylated proteins in tyrosine residues, gelsolin, c-SRC kinase and PLC-ζ, as well as in the distribution of phosphatidyl inositol bisphosphate were analyzed in thawed spermatozoa (T) non capacitated (NC), capacitated (C) and in those with acrosomal reaction (AR) and compared with fresh spermatozoa (F) under the same physiological status. Western blotting (WB) and co-immunoprecipitation were performed to confirm the presence of these proteins in PT and to determine the interaction between these molecules. IC showed that immunostaining for phosphorylated proteins significantly increased in the acrosomal region and flagellum in TNC spermatozoa (p < 0.05). The proportion of cells displaying immunolabeling for gelsolin in the acrosomal region decreased after capacitation in cryopreserved spermatozoa; the same change was found (p < 0.05) in the proportion of spermatozoa immunoreactive to PIP2 in the sperm head. c-SRC was observed in the equatorial segment and acrosomal region, subdomains that coincide with the site where phosphorylated proteins were detected. PLC-ζ immunolocalization in fresh spermatozoa underwent changes after capacitation and acrosomal reaction, with a significant increase in the equatorial segment and post-acrosomal region in cryopreserved spermatozoa (p < 0.05). WB analysis indicated the presence of gelsolin, c-SRC and PLC-ζ in PT; besides, we confirmed that gelsolin co-immunoprecipitated with c-SRC and PLC-ζ, which changes according to the physiological state of spermatozoa. As a conclusion, cryopreservation together with increased immunodetection of tyrosine phosphorylated proteins decreases the detection of PIP2 and alters the immunolocalization patterns of gelsolin, c-SRC and PLC-ζ in the PT in boar spermatozoa.

Structural basis for the unique molecular properties of broad-range phospholipase C from Listeria monocytogenes.

Listeriosis is one of the most serious foodborne diseases caused by the intracellular bacterium Listeria monocytogenes. Its two major virulence factors, broad-range phospholipase C (LmPC-PLC) and the pore-forming toxin listeriolysin O (LLO), enable the bacterium to spread in the host by destroying cell membranes. Here, we determine the crystal structure of LmPC-PLC and complement it with the functional analysis of this enzyme. This reveals that LmPC-PLC has evolved several structural features to regulate its activity, including the invariant position of the N-terminal tryptophan (W1), the structurally plastic active site, Zn2+-dependent activity, and the tendency to form oligomers with impaired enzymatic activity. We demonstrate that the enzymatic activity of LmPC-PLC can be specifically inhibited by its propeptide added in trans. Furthermore, we show that the phospholipase activity of LmPC-PLC facilitates the pore-forming activity of LLO and affects the morphology of LLO oligomerization on lipid membranes, revealing the multifaceted synergy of the two virulence factors.

Phosphatidylinositol-specific phospholipase C can decrease Müller cell viability and suppress its phagocytic activity by modulating PI3K/AKT signaling pathway.

Bacillus cereus endophthalmitis is a devastating eye infection that causes rapid blindness through the release of extracellular tissue-destructive exotoxins. The phagocytic and antibacterial functions of ocular cells are the keys to limiting ocular bacterial infections. In a previous study, we identified a new virulence gene, plcA-2 (different from the original plcA-1 gene), that was strongly associated with the plcA gene of Listeria monocytogenes. This plcA gene had been confirmed to play an important role in phagocytosis. However, how the Bc-phosphatidylinositol-specific phospholipase C (PI-PLC) proteins encoded by the plcA-1/2 genes affect phagocytes remains unclear in B. cereus endophthalmitis. Here, we found that the enzymatic activity of Bc-PI-PLC-A2 was approximately twofold higher than that of Bc-PI-PLC-A1, and both proteins inhibited the viability of Müller cells. In addition, PI-PLC proteins reduced phagocytosis of Müller cells by decreasing the phosphorylation levels of key proteins in the PI3K/AKT signaling pathway. In conclusion, we showed that PI-PLC proteins contribute to inhibit the viability of and suppress the phagocytosis of Müller cells, providing new insights into the pathogenic mechanism of B. cereus endophthalmitis.

CXCL1 enhances COX-II expression in rheumatoid arthritis synovial fibroblasts by CXCR2, PLC, PKC, and NF-κB signal pathway.

Rheumatoid arthritis (RA) is the most common autoimmune disease, affecting the joints of the hands and feet. Several chemokines and their receptors are crucial in RA pathogenesis through immune cell recruitment. C-X-C Motif Chemokine Ligand 1 (CXCL1), a chemokine for the recruitment of various immune cells, can be upregulated in patients with RA. However, the discussion on the role of CXCL1 in RA pathogenesis is insufficient. Here, we found that CXCL1 promoted cyclooxygenase-2 (COX-II) expression in a dose- and time-dependent manner in rheumatoid arthritis synovial fibroblasts (RASFs). CXCL1 overexpression in RASFs led to a significant increase in COX-II expression, while the transfection of RASFs with the shRNA plasmid resulted in a noticeable decrease in COX-II expression. Next, we delineated the molecular mechanism underlying CXCL1-promoted COX-II expression and noted that CXC chemokine receptor 2 (CXCR2), phospholipase C (PLC), and protein kinase C (PKC) signal transduction were responsible for COX-II expression after CXCL1 incubation for RASFs. Finally, we confirmed the transcriptional activation of nuclear factor κB (NF-κB) in RASFs after incubation with CXCL1. In conclusion, the current study provided a novel insight into the role of CXCL1 in RA pathogenesis.

Non-specific phospholipase C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield.

Phosphorus is a major nutrient vital for plant growth and development, with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids. Here, we report that NON-SPECIFIC PHOSPHOLIPASE C4 (NPC4) in rapeseed (Brassica napus) releases phosphate from phospholipids to promote growth and seed yield, as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated knockout of BnaNPC4 led to elevated accumulation of phospholipids and decreased growth, whereas overexpression (OE) of BnaNPC4 resulted in lower phospholipid contents and increased plant growth and seed production. We demonstrate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro, and plants with altered BnaNPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots, with a greater change in glycerolipids than sphingolipids in leaves, particularly under phosphate deficiency conditions. In addition, BnaNPC4-OE plants led to the upregulation of genes involved in lipid metabolism, phosphate release, and phosphate transport and an increase in free inorganic phosphate in leaves. These results indicate that BnaNPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.