Phospholipase C Research Articles

Silencing CaPIP5K4-1 leads to decreased male fertility in Capsicum annuum L.

Phosphatidylinositol 4-phosphate 5-kinase gene CaPIP5K4-1 is highly expressed in the pepper anthers. Virus-induced gene silencing of CaPIP5K4-1 leads to reduced male fertility in pepper. The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a pivotal enzyme in the phosphatidylinositol signaling pathway, and its crucial involvement in both plant development and stress response has been established. Here, we found that the expression of CaPIP5K4-1 in pepper was significantly higher in the fertile flower buds compared to sterile flower buds. Furthermore, its expression was validated in anthers and pollens by qRT-PCR and RNA-ISH assays, respectively. Its GFP fusion protein was mainly located on the plasma membrane. Silencing CaPIP5K4-1 in fertile pepper accessions resulted in wrinkled pollen grain cell walls, decreased pollen germination efficiency, and inhibited pollen tube growth. The transcription levels of multiple genes in the phosphatidylinositol signaling pathway were also assessed. Five phospholipase C (PLC) genes were downregulated in silenced plants. On the contrary, inositol phosphatase SAC and phosphatase and tensin homolog (PTEN) were upregulated. This study reported the role of CaPIP5K4-1 in pepper male fertility and provided insights into the regulatory mechanisms of PI signaling in pepper.

Calcium signaling in tunicate development

A comparative overview is provided of Ca2+ signaling and its potential mechanistic roles during development in tunicates. As background, the review presents an introduction to tunicate taxonomy, and then a general overview of Ca2+ signaling and methods for recording and measuring Ca2+ signals. It then covers the dynamics and implicated mechanisms of Ca2+ signals during different phases of development from oocyte to larva. These include signals arising in the unfertilized oocyte, signals associated with fertilization and meiosis, intercellular signals occurring from early cleavage stages through gastrulation, intercellular signals during organogenesis, and signals associated with early behavior. Comparisons are made among different tunicate species and where relevant to other chordate species. In many tunicate species, Ca2+ currents across the oocyte membrane are present prior to fertilization, and in the appendicularian Oikopleura dioica regular Ca2+ transients have been recorded optically prior to fertilization. Ca2+ signals at this stage have been implicated in pre-fertilization oocyte maturation events. The fertilization transient is the most well-studied Ca2+ signal and is triggered by factors from the sperm, including pivotally a phospholipase C (PLC) isoform that catalyzes the generation of IP3, which elicits release of Ca2+ from the endoplasmic reticulum. Post-fertilization signals are similarly dependent on IP3 signaling and are regulated by cyclin-dependent kinase 1 (Cdk1), and thereby linked to the meiotic divisions required for zygote formation. Ca2+ signals associated with early cleavages through gastrulation arise in blastomeres of the muscle lineage and spread from these in a coordinated fashion to other blastomeres through gap junctions. Post-gastrulation Ca2+ signals begin to show tissue-specificity in their temporal pattern as organogenesis proceeds, likely associated with loss of general gap junction transmission. Once neurulation has occurred, Ca2+ signals arise first in the nervous system and are transmitted synaptically to muscle, while Ca2+ signals arising spontaneously in the epidermis follow a separate temporal pattern. Species differences in the spatiotemporal characteristics of pre- and postgastrulation Ca+2 signals are discussed.

Neurotrophin-3 Rescues Striatal Synaptic Plasticity in Model of Neurodegeneration by PLC Signaling Activation.

Neurotrophins are essential factors for neural growth and function; they play a crucial role in neurodegenerative diseases where their expression levels are altered. Our previous research has demonstrated changes in synaptic plasticity and neurotrophin expression levels in a pharmacological model of Huntington's disease (HD) induced by 3-nitropropionic acid (3-NP). In the 3-NP-induced HD model, corticostriatal Long Term Depression (LTD) was impaired, but neurotrophin- 3 (NT-3) restored striatal LTD. This study delves into the NT-3-induced signaling pathways involved in modulating and restoring striatal synaptic plasticity in cerebral slices from 3-NPinduced striatal degeneration in mice in vivo. Phospholipase C (PLC), phosphatidylinositol-3-kinase (PI3K), and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways activated by NT-3 were analyzed by means of field electrophysiological recordings in brain slices from control and 3-NP treated in the presence of specific inhibitors of the signaling pathways. Using specific inhibitors, PLC, PI3K, and MEK/ERK signaling pathways contribute to NT-3-mediated plasticity modulation in striatal tissue slices recorded from control animals. However, in the neurodegeneration model induced by 3-NP, the recovery of striatal LTD induced by NT-3 was prevented only by the PLC inhibitor. Moreover, the PLC signaling pathway appeared to trigger downstream activation of the endocannabinoid system, evidenced by AM 251, an inhibitor of the CB1 receptor, also hindered NT-3 plasticity recovery. Our finding highlights the specific involvement of the PLC pathway in the neuroprotective effects of NT-3 in mitigating synaptic dysfunction under neurodegenerative conditions.

DEP-1 is a brain insulin receptor phosphatase that prevents the simultaneous activation of counteracting metabolic pathways

A healthy metabolism relies on precise regulation of anabolic and catabolic pathways. While insulin deficiency impairs anabolism, insulin resistance in obesity causes metabolic dysfunction, especially via altered brain insulin receptor (IR) activity. Density-enhanced phosphatase 1 (DEP-1) negatively modulates the IR in peripheral tissues. Our study shows that DEP-1 is an insulin-regulated gene, dysregulated in obesity, and uncovers its role in brain insulin signaling, impacting both anabolic and catabolic pathways. Neuro-2a cells lacking DEP-1 demonstrated heightened IR phosphorylation upon acute insulin stimulation. This coincided with simultaneous AMP-activated protein kinase (AMPK) activation, which governs catabolic pathways, due to increased phospholipase C-gamma 1 signaling. These opposing pathways in male DEP-1 forebrain-specific knockout mice resulted in elevated lipolysis in white adipose tissue and fat oxidation in brown adipose tissue, with enhanced sympathetic activation and β-adrenergic receptor expression. In conclusion, DEP-1 deficiency causes the simultaneous activation of IR and AMPK signaling in the brain, with enhanced sympathetic activity in adipose tissues.

Deciphering the Interlinked CXCR4-Mediated Feedback Loop Among Signaling Pathways in Diabetic Wound Healing

Abstract: Diabetic chronic wounds and amputations are very serious complications of diabetes mellitus (DM) that result from an integration factor, including oxygen deprivation, elevated reactive oxygen species (ROS), reduced angiogenesis, and microbial invasion. These causative factors lead to tenacious wounds in an inflammatory state, which eventually results in tissue aging and necrosis. Wound healing in DM potentially targets C-X-C chemokine receptor type 4 (CXCR4) regulates several signalling pathways. The CXCR4 signalling pathway integrated with phospholipase C (PLC)/protein kinase-C (PKC) Ca2+ pathways, stromal cell-derived factor-1 (SDF-1), and mitogen- activated protein kinases (MAPKs) pathway for enhancing cell chemotaxis, proliferation, and survival. The dysregulated CXCR4 pathway is connected with poor wound healing in DM patients. Therapeutic strategies targeting CXCR4-based molecules such as UCUF-728, UCUF-965, and AMD3100 have been shown to enhance diabetic wound healing by altering miRNA expression, promoting angiogenesis, and accelerating wound closure. This study indicates that CXCR4 participation in various signalling pathways makes it essential for Understanding the healing of diabetic wounds. Using specific compounds to target CXCR4 offers a potentially effective treatment strategy to improve wound healing in diabetes. Our understanding of CXCR4 signalling and its regulation processes will enable us to develop more potent wound care solutions for diabetic chronic wounds. This report concludes that CXCR4's potential therapeutic targeting shows improvements in diabetic wound repair. This review will demonstrate that CXCR4 plays a major role in wound healing through its various signalling pathways. Targeting CXCR4 with certain agonist molecules shows a therapeutic approach to potentially increasing wound healing in diabetes. By enhancing our understanding of the CXCR4 signalling mechanism in future studies, we can develop more potential treatments for chronic diabetic wounds.

Diet-enhanced LRG1 expression promotes insulin hypersecretion and ER stress in pancreatic beta cells.

Upregulation of serum leucine-rich α-2-glycoprotein 1 (LRG1) has been implicated in diet-induced obesity and metabolic disorders. However, its specific hormonal actions remain unclear. This study aimed to determine whether diet-enhanced serum LRG1 levels promote hyperinsulinaemia by directly stimulating insulin secretion from pancreatic beta cells. Human serum samples were obtained from individuals (both male and female) undergoing plastic surgery. Male C57BL/6 wild-type and Lrg1 whole-body knockout (Lrg1KO) mice were fed a 45% high-fat diet, with serum samples collected every 2 weeks to monitor LRG1 and insulin levels throughout diet-induced obesity. MIN6 beta cells were used to investigate the effects of LRG1 on insulin secretion and intracellular Ca2+ release. Antibodies targeting various LRG1 epitopes were used to neutralise LRG1 stimulation in MIN6 cells, and their effectiveness was tested in vivo to assess their ability to prevent LRG1-induced hyperinsulinaemia. We observed a significant positive association between human serum LRG1 levels and both age and BMI, with elevated levels observed in individuals with vs without type 2 diabetes. In mice fed a high-fat diet, LRG1 upregulation in serum was associated with hyperinsulinaemia. Lrg1 knockout protected mice from diet-induced islet hyperplasia and the loss of beta cell mass. Furthermore, neutralising LRG1 activity prevented the onset of diet-induced hyperinsulinaemia and preserved glucose tolerance. Mechanistically, LRG1 induces inositol triphosphate (IP3) production and intracellular Ca2+ release from the endoplasmic reticulum (ER) in a phospholipase C (PLC)-dependent manner, leading to excessive insulin secretion and ER stress in MIN6 beta cells. In summary, this study identifies LRG1 as a significant contributor to hyperinsulinaemia and beta cell dysfunction. Targeting LRG1 activity emerges as a promising therapeutic approach for addressing diet-induced beta cell dysfunction and managing type 2 diabetes.

The PLCG2 Inhibits Tumor Progression and Mediates Angiogenesis by VEGF Signaling Pathway in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) represents the most prevalent form of renal cell carcinoma. The management of early-stage ccRCC has a better prognosis, while patients with metastatic ccRCC have a lower five-year survival rate. Angiogenesis serves as the fundamental process underlying tumor metastasis. Therefore, it is crucial to discover new targets for angiogenesis to improve patient survival rates. The Cancer Genome Atlas database, International Cancer Genome Consortium database, Clinical Proteomic Tumor Analysis Consortium database, and a gene set of the vascular endothelial growth factor (VEGF) signaling pathway were utilized to identify differentially expressed genes. Western blot (WB), quantitative real-time polymerase chain reaction, and immunohistochemistry were employed to validate the downregulation of phospholipase C gamma 2 (PLCG2) in ccRCC tissues and cells. Cell Counting Kit-8 (CCK-8) assays, transwell assays, tube formation assays, and oil-red staining were performed to elucidate the biological functions of PLCG2 in tumor cells. Gene set enrichment analysis was applied to explore the downstream pathway. Subcutaneous tumor models and live small animal fluorescent imaging assay were utilized for in vivo investigation of the roles played by PLCG2. Our study has identified a novel biomarker, PLCG2, for ccRCC. PLCG2 is a central gene in regulating angiogenesis in ccRCC, as validated by bioinformatics analysis. The findings revealed a diminished expression of PLCG2 in both ccRCC tissues and cells. Further experiments in vivo and in vitro have demonstrated the significant roles of PLCG2 in tumor proliferation, invasion, migration, and lipid accumulation. Results of tube formation assays and WB support the role of PLCG2 in regulating VEGFA expression and angiogenesis. Our results show that PLCG2 functions as a potential biomarker and an independent prognostic indicator for ccRCC. PLCG2 may modulate angiogenesis by influencing the expression of VEGFA. Therefore, targeting PLCG2 could potentially lead to drug discovery and improved cancer treatment strategies.

Functional characterization of OR51B5 and OR1G1 in human lung epithelial cells as potential drug targets for non-type 2 lung diseases

BackgroundHypersensitivity to odorants like perfumes can induce or promote asthma with non-type 2 inflammation for which therapeutic options are limited. Cell death of primary bronchial epithelial cells (PBECs) and the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 are key in the pathogenesis. Extra-nasal olfactory receptors (ORs) can influence cellular processes involved in asthma. This study investigated the utility of ORs in epithelial cells as potential drug targets in this context.MethodsWe used the A549 cell line and primary bronchial epithelial cells using air–liquid interface culture system (ALI-PBECs). OR expression was investigated by RT-PCR, Western blot, and Immunofluorescence. Effects of OR activation by specific ligands on intracellular calcium concentration, cAMP, Phospholipase C (PLC), cell viability, and IL-6 and IL-8 secretion were analyzed by calcium imaging, enzyme immunoassays, Annexin V/ propidium iodide -based fluorescence-activated cell staining or by ELISA, respectively.ResultsBy screening A549 cells, the OR51B5 agonists Farnesol and Isononyl Alcohol and the OR1G1 agonist Nonanal increased intracellular Ca2 + . OR51B5 and OR1G1 mRNAs and proteins were detected. Both receptors showed a preferential intracellular localization. OR51B5- but not OR1G1-induced Ca2 + dependent on both cAMP and PLC signaling. Farnesol, Isononyl Alcohol, and Nonanal, all reduced cell viability and induced IL-8 and IL-6 release. The data were verified in ALI-PBECs.ConclusionORs in the lung epithelium might be involved in airway-sensitivity to odorants. Their antagonism could represent a promising strategy in treatment of odorant-induced asthma with non-type 2 inflammation.Graphical

Alzheimer’s disease-associated protective variant Plcg2-P522R modulates peripheral macrophage function in a sex-dimorphic manner

Genome-wide association studies have identified a protective mutation in the phospholipase C gamma 2 (PLCG2) gene which confers protection against Alzheimer’s disease (AD)-associated cognitive decline. Therefore, PLCG2, which is primarily expressed in immune cells, has become a target of interest for potential therapeutic intervention. The protective allele, known as P522R, has been shown to be hyper-morphic in microglia, increasing phagocytosis of amyloid-beta (Aβ), and increasing the release of inflammatory cytokines. However, the effect of this protective mutation on peripheral tissue-resident macrophages, and the extent to which sex modifies this effect, has yet to be assessed. Herein, we show that peripheral macrophages carrying the P522R mutation do indeed show functional differences compared to their wild-type (WT) counterparts, however, these alterations occur in a sex-dependent manner. In macrophages from females, the P522R mutation increases lysosomal protease activity, cytokine secretion, and gene expression associated with cytokine secretion and apoptosis. In contrast, in macrophages from males, the mutation causes decreased phagocytosis and lysosomal protease activity, modest increases in cytokine secretion, and induction of gene expression associated with negative regulation of the immune response. Taken together, these results suggest that the mutation may be conferring different effects dependent on sex and cell type, and highlight the importance of considering sex as a biological variable when assessing the effects of genetic variants and implications for potential immune system-targeted therapies.

Voltage- and Ca2+-inducible PLC activity for analyzing PI(4,5)P2 sensitivity of ion channels in Xenopus oocytes

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key membrane lipid regulating various ion channel activities. Currently, several molecular tools are used to modulate PIP2 levels, each of which has distinct advantages and drawbacks. In this study, we proposed a novel methodology using heterologous Xenopus oocytes to precisely manipulate PIP2 levels using phospholipase C (PLC)-ζ, which hydrolyzes PIP2. Xenopus oocytes injected with PLCζ exhibited notable hyperpolarization-induced Ca2+ influx driven by the increased driving force of Ca2+. High Ca2+ sensitivity of PLCζ facilitated hyperpolarization-induced PLC activity in Xenopus oocytes that was voltage- and Ca2+-dependent. This study demonstrated the regulatory capacity of PLCζ in modulating PIP2-sensitive ion channels, such as the KCNQ2/3 and GIRK channels, in a voltage- and Ca2+-dependent manner. Moreover, activation pathway of PLCζ only requires a two-electrode voltage clamp setup, making it a convenient molecular tool to manipulate PIP2 levels in combination with a voltage-sensing phosphatase (VSP). PLCζ has distinct characteristics and advantages compared to VSP: (1) Hyperpolarization, but not depolarization, reduced the PIP2 levels, (2) PIP2 levels were decreased without any increase in phosphatidylinositol 4-monophosphate (PIP) levels, and (3) PIP2 levels were reduced by Ca2+ administration. Therefore, PLCζ effectively supports understanding how PIP2 regulates ion channels, alongside VSP. Overall, this study highlights the unique characteristics of PLCζ and its distinct advantages in analyzing ion channel regulation by PIP2 and the PLC pathway in Xenopus oocytes.

Genome-Wide Identification and Analysis of Phospholipase C Gene Family Reveals Orthologs, Co-Expression Networks, and Expression Profiling Under Abiotic Stress in Sorghum bicolor.

Phospholipase C (PLC) is an essential enzyme involved in lipid signaling pathways crucial for regulating plant growth and responding to environmental stress. In sorghum, 11 PLC genes have been identified, comprising 6 PI-PLCs and 5 NPCs. Through phylogenetic and interspecies collinearity analyses, structural similarities between SbPLCs and ZmPLCs proteins have been observed, with a particularly strong collinearity between SbPLCs and OsPLCs. Promoter function analysis has shown that SbPLCs are significantly enriched under abiotic stress and hormonal stimuli, like ABA, jasmonic acid, drought, high temperature, and salt. Gene co-expression networks, constructed using a weighted gene co-expression network analysis (WGCNA), highlight distinct expression patterns of SbPLC1, SbPLC3a, and SbPLC4 in response to abiotic stress, providing further insights into the expression patterns and interactions of SbPLCs under various environmental stimuli. qRT-PCR results reveal variations in expression levels among most SbPLCs members under different stress conditions (drought, NaCl, NaHCO3), hormone treatments (ABA), and developmental stages, indicating both specific and overlapping expression patterns. This comprehensive analysis offers valuable insights into the roles of SbPLCs in sorghum, shedding light on their specific expression patterns, regulatory elements, and protein interactions across different environmental stimuli and developmental stages.

CG17192 is a Phospholipase That Regulates Signaling Lipids in the Drosophila Gut upon Infection.

The chemoproteomics technique, activity-based protein profiling (ABPP), has proven to be an invaluable tool in assigning functions to enzymes. The serine hydrolase (SH) enzyme superfamily, in particular, has served as an excellent example in displaying the versatility of various ABPP platforms and has resulted in a comprehensive cataloging of the biochemical activities associated within this superfamily. Besides SHs, in mammals, several other enzyme classes have been thoroughly investigated using ABPP platforms. However, the utility of ABPP platforms in fly models remains underexplored. Realizing this knowledge gap, leveraging complementary ABPP platforms, we reported the full array of SH activities during various developmental stages and adult tissues in the fruit fly (Drosophila melanogaster). Following up on this study, using ABPP, we mapped SH activities in adult fruit flies in an infection model and found that a gut-resident lipase CG17192 showed increased activity during infection. To assign a biological function to this uncharacterized lipase, we performed an untargeted lipidomics analysis and found that phosphatidylinositols were significantly elevated when CG17192 was depleted in the adult fruit fly gut. Next, we overexpressed this lipase in insect cells, and using biochemical assays, we show that CG17192 is a secreted enzyme that has phospholipase C (PLC) type activity, with phosphatidylinositol being a preferred substrate. Finally, we show during infection that heightened CG17192 regulates phosphatidylinositol levels and, by doing so, likely modulates signaling pathways in the adult fruit fly gut that might be involved in the resolution of this pathophysiological condition.

Induction of Ca2+ signaling and cytotoxic responses of human lung fibroblasts upon an antihistamine drug oxatomide treatment and evaluating the protective effects of Ca2+ chelating.

Oxatomide, an antihistamine drug of the diphenylmethylpiperazine family, has anti-inflammatory effects in airway disease. Because oxatomide was shown to cause diverse physiological responses in several cell models, the impact of oxatomide on Ca2+ signaling and its related physiological effects has not been explored in IMR-90 human fetal lung fibroblasts. This study assessed the effect of oxatomide on cell viability and intracellular free Ca2+ concentrations ([Ca2+]i) and examined whether oxatomide-induced cytotoxicity through Ca2+ signaling in IMR-90 cells. Cell viability was measured by the cell proliferation reagent (WST-1). [Ca2+]i was measured by the Ca2+-sensitive fluorescent dye fura-2. Oxatomide (10-40 μM) concentration dependently reduced cell viability and induced [Ca2+]i rises in IMR-90 cells. This cytotoxic effect was reversed by chelation of cytosolic Ca2+ with BAPTA-AM. In terms of Ca2+ signaling, oxatomide-caused Ca2+ entry was inhibited by modulators of store-operated Ca2+ channels (2-APB and SKF96365) and protein kinase C (PKC) inhibitor (GF109203X). Furthermore, oxatomide-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. In a Ca2+-free medium, preincubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited oxatomide-evoked [Ca2+]i rises. Conversely, treatment with oxatomide abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 also inhibited oxatomide-caused [Ca2+]i rises. In IMR-90 cells, oxatomide-induced cytotoxicity by preceding [Ca2+]i rises involving PKC-sensitive store-operated Ca2+ entry and PLC-dependent Ca2+ release from the endoplasmic reticulum. BAPTA-AM, with its Ca2+ chelating effects, may be a potential compound for preventing oxatomide-induced cytotoxicity.

Role for the PIP2‐binding protein myristoylated alanine‐rich C‐kinase substrate in vascular tissue: A novel therapeutic target for cardiovascular disease

AbstractIn vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs), phosphatidylinositol 4,5‐bisphosphate (PIP2) acts as a substrate for phospholipase C (PLC)‐ and phosphoinositol 3‐kinase (PI3K)‐mediated signaling pathways and an unmodified ligand at ion channels and other macromolecules, which are key processes in the regulation of cell physiological and pathological phenotypes. It is envisaged that these distinct roles of PIP2 are achieved by PIP2‐binding proteins, which act as PIP2 buffers to produce discrete pools of PIP2 that permits targeted release within the cell. This review discusses evidence for the expression, cell distribution, and role of myristoylated alanine‐rich C‐kinase substrate (MARCKS), a PIP2‐binding protein, in cellular signaling and function of VSMCs. The review indicates the possibilities for MARCKS as a therapeutic target for vascular disease involving dysfunctional cell proliferation and migration, endothelial barrier permeability, and vascular contractility such as atherosclerosis, systemic and pulmonary hypertension, and sepsis.

Upstream and downstream pathways of diacylglycerol kinase : Novel phosphatidylinositol turnover-independent signal transduction pathways

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprise ten isozymes (α–κ) that regulate a wide variety of physiological and pathological events. Recently, we revealed that DGK isozymes use saturated fatty acid (SFA)/monosaturated fatty acid (MUFA)-containing and docosahexaenoic acid (22:6)-containing DG species, but not phosphatidylinositol (PI) turnover-derived 18:0/20:4-DG. For example, DGKδ, which is involved in the pathogenesis of type 2 diabetes, preferentially uses SFA/MUFA-containing DG species, such as 16:0/16:0- and 16:0/18:1-DG species, in high glucose-stimulated skeletal muscle cells. Moreover, DGKδ, which destabilizes the serotonin transporter (SERT) and regulates the serotonergic system in the brain, primarily generates 18:0/22:6-PA. Furthermore, 16:0/16:0-PA is produced by DGKζ in Neuro-2a cells during neuronal differentiation. We searched for SFA/MUFA-PA- and 18:0/22:6-PA-selective binding proteins (candidate downstream targets of DGKδ) and found that SFA/MUFA-PA binds to and activates the creatine kinase muscle type, an energy-metabolizing enzyme, and that 18:0/22:6-PA interacts with and activates Praja-1, an E3 ubiquitin ligase acting on SERT, and synaptojanin-1, a key player in the synaptic vesicle cycle. Next, we searched for SFA/MUFA-DG-generating enzymes upstream of DGKδ. We found that sphingomyelin synthase (SMS)1, SMS2, and SMS-related protein (SMSr) commonly act as phosphatidylcholine (PC)-phospholipase C (PLC) and phosphatidylethanolamine (PE)-PLC, generating SFA/MUFA-DG species, in addition to SMS and ceramide phosphoethanolamine synthase. Moreover, the orphan phosphatase PHOSPHO1 showed PC- and PE-PLC activities that produced SFA/MUFA-DG. Although PC- and PE-PLC activities were first described 70–35 years ago, their proteins and genes were not identified for a long time. We found that DGKδ interacts with SMSr and PHOSPHO1, and that DGKζ binds to SMS1 and SMSr. Taken together, these results strongly suggest that there are previously unrecognized signal transduction pathways that include DGK isozymes and generate and utilize SFA/MUFA-DG/PA or 18:0/22:6-DG/PA but not PI-turnover-derived 18:0/20:4-DG/PA.

Efficient preparation of diglycerides from rapeseed oil sediment using phospholipase C in a solvent-free phospholipid hydrolysis system

Diacylglycerols (DAGs), which are involved in rapid energy supply and fat reduction, are typically produced from triglycerides, glycerol and fatty acids through lipase-catalysis. This study introduces a novel method for preparing DAGs in a solvent-free system by using phospholipase C (PLC) hydrolysis of rapeseed oil sediment, which is a byproduct of the rapeseed oil water degumming. The optimisation of DAGs production by PLC hydrolysis using Response Surface Methodology (RMS) and Box-Behnken design. Under optimal conditions of 0.6% enzyme concentration, 51 °C reaction temperature, 56% water content and 2.2 h reaction time, phosphatidylcholine (PC) and phosphatidylethanolamine in the oil sediment were almost entirely converted into DAGs. This reaction yielded an oil product containing 27.1% DAGs, with 1,2-DAGs at 24.88% ± 0.01% and 1,3-DAGs at 2.22% ± 0.01%, which is favoured in drug delivery systems, and the fatty acid composition of DAGs oil was almost consistent with rapeseed oil. Moreover, the residual phospholipids were primarily phosphatidylinositol (PI), with an amount of 69.73%, suggesting the potential for high-purity PI production. In this study, rapeseed oil sediment achieved comprehensive utilization through hydrolysis by PLC, which not only enhanced the value of the oil sediment but also provided a new approach for the synthesis of DAGs.

Impaired degradation of PLCG1 by chaperone-mediated autophagy promotes cellular senescence and intervertebral disc degeneration

ABSTRACT Defects in chaperone-mediated autophagy (CMA) are associated with cellular senescence, but the mechanism remains poorly understood. Here, we found that CMA inhibition induced cellular senescence in a calcium-dependent manner and identified its role in TNF-induced senescence of nucleus pulposus cells (NPC) and intervertebral disc degeneration. Based on structural and functional proteomic screens, PLCG1 (phospholipase C gamma 1) was predicted as a potential substrate for CMA deficiency to affect calcium homeostasis. We further confirmed that PLCG1 was a key mediator of CMA in the regulation of intracellular calcium flux. Aberrant accumulation of PLCG1 caused by CMA blockage resulted in calcium overload, thereby inducing NPC senescence. Immunoassays on human specimens showed that reduced LAMP2A, the rate-limiting protein of CMA, or increased PLCG1 was associated with disc senescence, and the TNF-induced disc degeneration in rats was inhibited by overexpression of Lamp2a or knockdown of Plcg1. Because CMA dysregulation, calcium overload, and cellular senescence are common features of disc degeneration and other age-related degenerative diseases, the discovery of actionable molecular targets that can link these perturbations may have therapeutic value. Abbreviation: ATRA: all-trans-retinoic acid; BrdU: bromodeoxyuridine; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16-INK4A: cyclin dependent kinase inhibitor 2A; CMA: chaperone-mediated autophagy; DHI: disc height index; ER: endoplasmic reticulum; IP: immunoprecipitation; IP3: inositol 1,4,5-trisphosphate; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; IVD: intervertebral disc; IVDD: intervertebral disc degeneration; KD: knockdown; KO: knockout; Leu: leupeptin; MRI: magnetic resonance imaging; MS: mass spectrometry; N/L: NH4Cl and leupeptin; NP: nucleus pulposus; NPC: nucleus pulposus cells; PI: protease inhibitors; PLC: phospholipase C; PLCG1: phospholipase C gamma 1; ROS: reactive oxygen species; RT-qPCR: real-time quantitative reverse transcription PCR; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SASP: senescence-associated secretory phenotype; STV: starvation; TMT: tandem mass tag; TNF: tumor necrosis factor; TP53: tumor protein p53; UPS: ubiquitin-proteasome system.

IGF-1 and Glucocorticoid Receptors Are Potential Target Proteins for the NGF-Mimic Effect of β-Cyclocitral from Lavandula angustifolia Mill. in PC12 Cells.

In the present study, the PC12 cells as a bioassay system were used to screen the small molecules with nerve growth factor (NGF)- mimic effect from Lavandula angustifolia Mill. The β-Cyclocitral (β-cyc) as an active compound was discovered, and its chemical structure was also determined. Furthermore, we focused on the bioactive and action mechanism of this compound to do an intensive study with specific protein inhibitors and Western blotting analysis. The β-cyc had novel NGF-mimic and NGF-enhancer effects on PC12 cells, while the insulin-like growth factor-1 receptor (IGF-1R)/phosphatidylinositol 3 kinase, (PI3K)/serine/threonine-protein kinase (AKT), and glucocorticoid receptor (GR)/phospholipase C (PLC)/protein kinase C (PKC) signaling pathways were involved in the bioactivity of β-cyc. In addition, the important role of the rat sarcoma (Ras)/protooncogene serine-threonine protein kinase (Raf) signaling pathway was observed, although it was independent of tyrosine kinase (Trk) receptors. Moreover, the non-label target protein discovery techniques, such as the cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS), were utilized to make predictions of its target protein. The stability of IGF-R and GR, proteins for temperature and protease, was dose-dependently increased after treatment of β-cyc compared with control groups, respectively. These findings indicated that β-cyc promoted the neuron differentiation of PC12 cells via targeting IGF-1R and GR and modification of downstream signaling pathways.

The activation of D2-like dopamine receptors increases NMDA currents in the dorsal raphe serotonergic neurons

The dorsal raphe nucleus (DRN) receives dopaminergic inputs from the ventral tegmental area (VTA). Also, the DRN contains a small population of cells that express dopamine (DRNDA neurons). However, the physiological role of dopamine (DA) in the DRN and its interaction with serotonergic (5-HT) neurons is poorly understood. Several works have reported moderate levels of D1, D2, and D3 DA receptors in the DRN. Furthermore, it was found that the activation of D2 receptors increased the firing of putative 5-HT neurons. Other studies have reported that D1 and D2 dopamine receptors can interact with glutamate NMDA receptors, modulating the excitability of different cell types. In the present work, we used immunocytochemical techniques to determine the kind of DA receptors in the DRN. Additionally, we performed electrophysiological experiments in brainstem slices to study the effect of DA agonists on NMDA-elicited currents recorded from identified 5-HT DRN neurons. We found that D2 and D3 but not D1 receptors are present in this nucleus. Also, we demonstrated that the activation of D2-like receptors increases NMDA-elicited currents in 5-HT neurons through a mechanism involving phospholipase C (PLC) and protein kinase C (PKC) enzymes. Possible physiological implications related to the sleep-wake cycle are discussed.

Characterization of a Novel Pathogenic PLCG2 Variant Leading to APLAIDSyndrome Responsive to a TNF Inhibitor.

Autoinflammation and phospholipase C (PLC) γ2-associated antibody deficiency and immune dysregulation (APLAID) syndrome is an autoinflammatory disease caused by gain-of-function variants in PLCG2. This study investigates the pathogenic mechanism of a novel variant of PLCG2 in a patient with APLAID syndrome. Whole-exome sequencing and Sanger sequencing were used to identify the pathogenic variant in the patient. Single-cell RNA sequencing, immunoblotting, luciferase assay, inositol monophosphate enzyme-linked immunosorbent assay, calcium flux assay, quantitative PCR, and immunoprecipitation were used to define inflammatory signatures and evaluate the effects of the PLCG2 variant on protein functionality and immune signaling. We identified a novel de novo variant, PLCG2 p.D993Y, in a patient with colitis, pansinusitis, skin rash, edema, recurrent respiratory infections, B-cell deficiencies, and hypogammaglobulinemia. The single-cell transcriptome revealed exacerbated inflammatory responses in the patient's peripheral blood mononuclear cells. Expression of the D993Y variant in HEK293T, COS-7, and PLCG2 knock-out THP-1 cell lines showed heightened PLCγ2 phosphorylation; elevated inositol-1,4,5-trisphosphate production and intracellular Ca2+ release; and activation of the MAPK, NF-κB, and NFAT signaling pathways compared with control-transfected cells. In vitro experiments indicated that the D993Y variant altered amino acid properties, disrupting the interaction between the catalytic and autoinhibitory domains of PLCγ2, resulting in PLCγ2 autoactivation. Our findings demonstrated that the PLCG2 D993Y variant is a gain-of-function mutation via impairing its autoinhibition, activating multiple inflammatory signaling pathways, thus leading to APLAID syndrome. This study further broadens the molecular underpinnings and phenotypic spectrum of PLCγ2-related disorders.