Measuring IP3 Generation in Real-Time Using a NanoBiT Luminescence Biosensor.

G protein-coupled receptors that activate Gq/11 regulate a range of physiological processes including neurotransmission, energy homeostasis, blood pressure regulation, and calcium homeostasis. Activation of Gq/11-coupled receptors stimulates the generation of inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium release from the endoplasmic reticulum. This chapter describes an assay that uses a NanoBiT-IP3 luminescent biosensor to detect increases in IP3 in live cells. It describes how to perform these assays to assess signaling by the ghrelin receptor and the calcium-sensing receptor in HEK293 cells.

Helicobacter pylori PldA modulates TNFR1-mediated p38 signaling pathways to regulate macrophage responses for its survival.

Helicobacter pylori, a dominant member of the gastric microbiota was associated with various gastrointestinal diseases and presents a significant challenge due to increasing antibiotic resistance. This study identifies H. pylori's phospholipase A (PldA) as a critical factor in modulating host macrophage responses, facilitating H. pylori 's evasion of the immune system and persistence. PldA alters membrane lipids through reversible acylation and deacylation, affecting their structure and function. We found that PldA incorporates lysophosphatidylethanolamine into macrophage membranes, disrupting their bilayer structure and impairing TNFR1-mediated p38-MK2 signaling. This disruption results in reduced macrophage autophagy and elevated RIP1-dependent apoptosis, thereby enhancing H. pylori survival, a mechanism also observed in multidrug-resistant strains. Pharmacological inhibition of PldA significantly decreases H. pylori viability and increases macrophage survival. In vivo studies corroborate PldA's essential role in H. pylori persistence and immune cell recruitment. Our findings position PldA as a pivotal element in H. pylori pathogenesis through TNFR1-mediated membrane modulation, offering a promising therapeutic target to counteract bacterial resistance.

High expression of PLA2G2A in fibroblasts plays a crucial role in the early progression of carotid atherosclerosis.

BACKGROUND: In mouse models of atherosclerosis, knockout of the PLA2G2A gene has been shown to reduce the volume of atherosclerotic plaques. Clinical trials have demonstrated the potential of using the sPLA2 inhibitor Varespladib in combination with statins to reduce lipid levels. However, this approach has not yielded the expected results in reducing the risk of cardiovascular events. Therefore, it is necessary to further investigate the mechanisms of PLA2G2A. METHODS: Single-cell transcriptome data from two sets of carotid plaques, combined with clinical patient information. were used to describe the expression characteristics of PLA2G2A in carotid plaques at different stages. In order to explore the mechanisms of PLA2G2A, we conducted enrichment analysis, cell-cell communication analysis and single-cell regulatory network inference and clustering analyses. We validated the above findings at the cellular level. RESULTS: Our findings indicate that PLA2G2A is primarily expressed in vascular fibroblasts and shows significant cell interactions with macrophages in the early-stage, especially in complement and inflammation-related pathways. We also found that serum sPLA2 levels have stronger diagnostic value in patients with mild carotid artery stenosis. Subsequent comparisons of single-cell transcriptomic data from early and late-stage carotid artery plaques corroborated these findings and predicted transcription factors that might regulate the progression of early carotid atherosclerosis (CA) and the expression of PLA2G2A. CONCLUSIONS: Our study discovered and validated that PLA2G2A is highly expressed by vascular fibroblasts and promotes plaque progression through the activation of macrophage complement and coagulation cascade pathways in the early-stage of CA.

Phospholipase D2 drives cellular lipotoxicity and tissue inflammation in alcohol-associated liver disease.

AIMS: Excessive alcohol consumption leads to alcoholic liver disease (ALD), a major contributing factor to cirrhosis and hepatocellular carcinoma. In the present study we investigated the involvement of phospholipase D2 (PLD2) in the pathogenesis of ALD. METHODS AND MATERIALS: ALD was induced in mice by chronic and binge ethanol feeding (the NIAAA model). Cellular transcriptome was examined by RNA-seq. KEY FINDINGS: Analysis of RNA-seq datasets indicated that PLD2 expression was up-regulated in liver tissues and in hepatocytes during ALD pathogenesis. Exposure of hepatocytes to ethanol treatment led to an increase in PLD2 expression. Similarly, ethanol feeding in mice stimulated PLD2 expression in the liver. On the contrary, PLD2 knockdown in hepatocytes down-regulated expression of pro-inflammatory and pro-lipogenic genes and dampened lipid accumulation. Consistently, PLD2 knockdown in mice significantly ameliorated ALD pathogenesis as evidenced by reduced steatosis and hepatic inflamamation. RNA-seq identified several metabolic pathways that were influenced by PLD2 deficiency. SIGNIFICANCE: Our data demonstrate that PLD2 is a novel regulator of ALD and suggest that small-molecule PLD2 inhibitors can be considered as a reasonable strategy for ALD treatment.

Association of plasma neurofilament light chain and Lipoprotein-related phospholipase A2 with motor subtypes of Parkinson's disease.

Neurofilament light chain (NfL) levels were reliable biomarkers of neurodegeneration in Parkinson's disease (PD). Lipoprotein-related Phospholipase A2(Lp-PLA2) levels have also been increasingly studied in PD. We aimed to explore the association of plasma NfL and Lp-PLA2 with the diagnosis, motor subtypes and disease severity of PD. Plasma NfL and Lp-PLA2 were assayed separately in 106 participants (74 PD and 32 healthy controls, HC). The motor subtypes of PD were classified according to the MDS-UPDRS components, and motor and non-motor manifestations of patients were also evaluated. Subsequently, correlation analyses were performed. The plasma NfL levels were higher in the PD than HC, and were positively correlated with age, UPDRS II, UPDRS III and the modified Hoehn and Yahr staging scale (H&Y stage) in the PD. Moreover, plasma Lp-PLA2 levels were lower in the PD than HC, and were positively correlated with Parkinson's Disease Quality of Life Questionnaire (PDQ-39) in the PD. For further distinguishing tremor-dominant (TD) from postural instability and gait difficulty-dominant (PIGD), plasma Lp-PLA2 levels were higher in the TD than PIGD, but there was no significant difference in NfL. plasma Lp-PLA2 levels were positively correlated with UPDRS I, Hamilton Anxiety Rating Scale (HAMA) and PDQ-39 in the TD. These resultssuggest that NfL and Lp-PLA2 may be potential biomarkers for the diagnosis of PD. We first demonstrated the potential utility of plasma Lp-PLA2 in differentiating motor subtypes. These findings deserve further evidence in larger PD cohorts.

A guide to selecting high-performing antibodies for PLC-gamma-2 for use in Western Blot, immunoprecipitation and immunofluorescence.

Phosphatidylinositol-specific phospholipase C gamma 2 (PLC-gamma-2) is an enzyme that regulates the function of immune cells. PLC-gamma-2 has been implicated in neurodegenerative and autoimmune disorders, yet investigation of this protein has been limited by a lack of independently characterized antibodies. Here we have characterized eleven PLC-gamma-2 commercial antibodies for use in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

The molecular mechanism of PLD2-mediated regulation of apoptosis and cell edema in pancreatic cells via the Nrf2/NF-κB pathway.

This study aimed to elucidate the molecular mechanisms by which PLD2 controls apoptosis and edema in pancreatic cells via the Nrf2/NF-κB pathway. AR42J rat pancreatic cells were treated with 10 nM mitomycin to create an in vitro pancreatitis model (model group), with a control group receiving phosphate-buffered saline. Cells were transfected with a PLD2 overexpression plasmid using Lipofectamine 3000, forming the PLD2 overexpression group. PLD2 protein expression was assessed by Western blotting, and TNF-α, IL-6, and IL-10 levels were measured by RT-qPCR. Nrf2/NF-κB protein expressions were also analyzed. Apoptosis and necrosis were evaluated using Annexin V-FITC/PI staining and the LDH release test. Cell edema was assessed by cell volume, ion content, and membrane damage. Western blotting was used to analyze pan-apoptosis-related proteins. PLD2 expression was lower in the model group compared to controls (P < 0.05) but higher in the PLD2 overexpression group (P < 0.05). TNF-α, IL-6, and IL-10 levels were elevated in the model group (P < 0.05) and reduced in the PLD2 overexpression group (P < 0.05). Nrf2 expression decreased in the model group but increased with PLD2 overexpression (P < 0.05). NF-κB expression increased in the model group but decreased with PLD2 overexpression (P < 0.05). Apoptosis and necrosis rates were higher in the model group (P < 0.05) but lower in the PLD2 overexpression group (P < 0.05). Cell volume, Na + content, and LDH release increased in the model group (P < 0.05) but decreased with PLD2 overexpression (P < 0.05). RIPK1 expression decreased in the model group (P < 0.05) but increased with PLD2 overexpression (P < 0.05). CASP8, FADD, and ZBP1 levels were higher in the model group (P < 0.05) and reduced with PLD2 overexpression (P < 0.05). PLD2 exerts a protective effect in acute pancreatitis by activating Nrf2 and inhibiting NF-κB, reducing apoptosis, cell swelling, and membrane damage. This highlights potential therapeutic targets for pancreatic inflammation.

A noninvasive method of diagnosing membranous nephropathy using exosomes derived from urine.

Background: Membranous nephropathy (MN) is a specific autoimmune disease affecting kidneys. It is characterized by the accumulation of immune complexes in the glomerular basement membrane. Renal biopsy is currently the standard procedure to confirm the diagnosis, although the presence of autoantibodies against the phospholipase A2 receptor (PLA2R) can also help diagnose. In this study, we aimed to investigate the potential of urinary exosomes as noninvasive markers for diagnosing MN. Methods: Exosomes were extracted from urine samples of five patients with MN and four healthy controls. The concentration of PLA2R was measured in both urine and isolated exosomes using enzyme-linked immunosorbent assay techniques. The measurements were adjusted based on the urine creatinine (UCr) level of each participant. Results: The levels of PLA2R/UCr were investigated in urine and urine-derived exosomes from patients and controls. Results of the analysis revealed significantly higher expression of PLA2R/UCr in patients compared to the control group (p < 0.05). Furthermore, the expression level of PLA2R/UCr was higher in urine-derived exosomes than in urine samples. Additionally, a positive correlation was observed between the expression levels of PLA2R/UCr and the urine protein-to-creatinine ratio, with urine-derived exosomes exhibiting a stronger correlation than urine samples. Conclusion: Studies have indicated that measuring exosomal PLA2R/UCr levels in urine could be a noninvasive method for diagnosing MN. Using urine-derived exosomes could also reduce the burden of performing a biopsy on patients and facilitate follow-up treatment, such as monitoring for future recurrence.

Glycerophospholipid-driven lipid metabolic reprogramming as a common key mechanism in the progression of human primary hepatocellular carcinoma and cholangiocarcinoma.

Metabolic reprogramming, a key mechanism regulating the growth and recurrence of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), still lacks effective clinical strategies for its integration into the precise screening of primary liver cancer. This study utilized ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry to conduct a comprehensive, non-targeted metabolomics analysis, revealing significant upregulation of lipid metabolites such as phosphatidylcholine and lysophosphatidylcholine in patients with HCC and CCA, particularly within the glycerophospholipid metabolic pathway. Hematoxylin and eosin and immunohistochemical staining demonstrated marked upregulation of phospholipase A2 in tumor tissues, further emphasizing the potential of lipid metabolism as a therapeutic target and its important part in the course of cancer. This work provides a new viewpoint for addressing the clinical challenges associated with HCC and CCA, laying the groundwork for the broad application of early diagnosis and personalized treatment strategies, and ultimately aiming to provide tailored and precise therapeutic options for patients.

Secretory Phospholipase A2 as a Promising Biomarker for Predicting Acute Chest Syndrome in Children With Sickle Cell Disease: A Systematic Review and Meta-Analysis.

Acute chest syndrome (ACS) is a severe and potentially life-threatening complication of sickle cell disease (SCD). Early identification of patients at risk for ACS is crucial for timely intervention. There is a potential association between ACS and elevated levels of secretory phospholipase A2 (sPLA2), an enzyme involved in the breakdown of phospholipids. sPLA2 has emerged as a promising biomarker for predicting ACS. This systematic review and meta-analysis aimed to assess the diagnostic value of PLA2 in predicting ACS among children with SCD. A comprehensive search was conducted across multiple databases, including MEDLINE, Embase, Cochrane Library, PubMed, and Web of Science. Studies assessing the relationship between sPLA2 levels and ACS in SCD patients were included. Pooled sensitivity, specificity, likelihood ratios, and the area under the receiver operating characteristic curve (AUC) were calculated to assess sPLA2's diagnostic accuracy. There is a potential association between significant association between elevated sPLA2 levels and increased ACS risk in SCD patients. The pooled sensitivity of sPLA2 for predicting ACS was 0.766 (95% CI: 0.620-0.877), with a pooled specificity of 0.736 (95% CI: 0.680-0.787). The AUC of the summary receiver operating characteristic (SROC) curve was 0.84, indicating good discriminatory ability. sPLA2 emerges as a promising biomarker for predicting ACS in SCD patients, potentially guiding risk stratification and early intervention strategies to enhance patient outcomes. Nonetheless, further prospective studies are warranted to validate its clinical utility and standardize sPLA2 assay protocols.

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.

Cerebellar Molecular Signatures in Non-Human Primates.

Cerebellar molecular signatures in primates remain largely unexplored. Here, we investigated the immunoreactivity of neuroplasticity-related molecular markers, including aldolase C (Aldoc), phospholipase C beta 3 (PLCB3), and phospholipase C beta 4 (PLCB4) in the cerebellar cortex and associated nuclei of rhesus macaque monkeys (Macaca mulatta). Our main findings are as follows: First, the cerebellar vermis in macaques exhibited striped compartmentalization for all markers, with the striped expression boundary of PLCB3 being less distinct than those of Aldoc and PLCB4. Second, the striped pattern was less pronounced in the cerebellar hemisphere compared to the vermis, with signals in the hemisphere being predominantly intense throughout. Third, distinct zonal patterns and elevated signals for Aldoc and PLCB3 were observed in the cerebellar deep nuclei. Specifically, the fastigial nucleus displayed intense Aldoc signals in both caudal and rostral regions, while the dentate nucleus displayed strong Aldoc signals in both ventral and dorsal regions. Compared to previous rodent studies, the macaque cerebellum demonstrated a higher proportion of intense signal areas and distinct compartmentalization patterns in both cortical and deep nuclei. These findings offer crucial insights into the unique molecular organization of the primate cerebellum, enhancing our understanding of the advanced neuroplasticity, cognitive, and motor capabilities in primates.

Sphingomyelin Inhibits Hydrolytic Activity of Heterodimeric PLA2 in Model Myelin Membranes: Pharmacological Relevance.

In this work, the heterodimeric phospholipase A2, HDP-2, from viper venom was investigated for its hydrolytic activity in model myelin membranes as well as for its effects on intermembrane exchange of phospholipids (studied by phosphorescence quenching) and on phospholipid polymorphism (studied by 1H-NMR spectroscopy) to understand the role of sphingomyelin (SM) in the demyelination of nerve fibers. By using well-validated in vitro approaches, we show that the presence of SM in model myelin membranes leads to a significant inhibition of the hydrolytic activity of HDP-2, decreased intermembrane phospholipid exchange, and reduced phospholipid polymorphism. Using AutoDock software, we show that the NHδ+ group of the sphingosine backbone of SM binds to Tyr22(C=Opbδ-) of HDP-2 via a hydrogen bond which keeps only the polar head of SM inside the HDP-2's active center and positions the sn-2 acyl ester bond away from the active center, thus making it unlikely to hydrolyze the alkyl chains at the sn-2 position. This observation strongly suggests that SM inhibits the catalytic activity of HDP-2 by blocking access to other phospholipids to the active center of the enzyme. Should this observation be verified in further studies, it would offer a tantalizing opportunity for developing effective pharmaceuticals to stop the demyelination of nerve fibers by aberrant PLA2s with overt activity - as observed in brain degenerative diseases - by inhibiting SM hydrolysis and/or facilitating SM synthesis in the myelin sheath membrane.

Phase-Specific Immobilization of Phospholipase D as an Efficient Pickering Emulsion Interfacial Catalyst for Converting Antarctic Krill Oil Phospholipid.

Phosphatidylserine (PS), a rare phospholipid in Antarctic krill oil (AKO) critical for brain development, can be produced from the abundant phosphatidylcholine (PC) using phospholipase D (PLD) in Pickering emulsion interfacial catalysis (PIC) systems. However, the exposure of PLD to organic solvent around the emulsion interface diminished PLD activity, limiting the conversion efficiency of PS. In this study, we proposed a strategy to fabricate a PIC system with high efficiency and stability by immobilizing PLD in a specific phase on the emulsion interface, based on investigating the effect of the interfacial microenvironment on PLD activity. Janus-poly(acrylic acid)/polystyrene (JPP) and Janus-polyethylenimine/octadecane (JPO) particles were fabricated as carriers to realize the specific-phase immobilization of PLD. The highest activity was observed when PLD was immobilized on the hydrophilic side of JPP (PLD@JPP(W)), 1.9-fold that of free PLD. The catalytic efficiency of PLD@JPP(W) was 1.7-fold that of free PLD, confirmed by the kcat/Km value enhancement. Immobilization on the hydrophilic side also enhanced the thermal stability of PLD. The half-lives of PLD were extended from 4 to 36 h at 40 °C and from 6 to 28 days at 4 °C. Importantly, PLD@JPP(W) showed excellent catalytic efficiency as a PIC system, achieving a PS productivity of 93% within a short time of 2 h at an enzyme dosage of 0.05 mg. PLD@JPP(W) exhibited a 3.6 times higher yield than free PLD in the production of PS from PC rich in Antarctic krill oil. The strategy in this work could also be applied to other lipases, providing a promising method for the efficient conversion of functional lipids.

Preparation and characterization of niosomes for the delivery of a lipophilic model drug: comparative stability study with liposomes against phospholipase-A2.

Vesicular nanocarriers like niosomes and liposomes are widely researched for controlled drug delivery systems, with niosomes emerging as promising alternatives due to their higher stability and ease of manufacturing. This study aimed to develop and characterize a niosomal formulation for the encapsulation and sustained release of temozolomide (TMZ), a model lipophilic drug, and to compare the stability of niosomes and liposomes, with a particular focus on the behavior of their lipid bilayers. Niosomes were prepared using the thin-film hydration method, composed of Span 60 (Sorbitan monostearate), cholesterol, and soy lecithin in varying molar ratios. The study investigated critical properties such as drug loading capacity, release kinetics, and resistance to enzymatic degradation. The optimized formulation was analyzed for drug entrapment efficiency and stability against phospholipase A2 (PLA2) degradation. The optimized niosomal formulation, with a 4:2:1 molar ratio of Span 60: cholesterol, achieved a high TMZ entrapment efficiency of 73.23 ± 1.02% and demonstrated sustained drug release over 24 hours. In comparison, liposomes released their TMZ payload within 4 hours upon exposure to PLA2, while the niosomes maintained their release profile, indicating superior stability. Spectroscopic and thermal analysis confirmed successful drug encapsulation with no component incompatibilities.

Evaluation of Microbial Phospholipase and Lipase Activity Through the Chromatographic Analysis of Crude, Degummed, and Transesterified Soybean Oil for Biodiesel Production.

The present study aimed at synthesizing fatty acid methyl esters in a combined enzymatic method by applying degumming and transesterification of soybean oil. A soluble lipase from Serratia sp. W3 and a recombinant phosphatidylcholine-preferring phospholipase C (PC-PLC) from Bacillus thuringiensis were used in a consecutive manner for phosphorus removal and conversion into methyl esters. By applying 1% of recombinant PC-PLC almost 83% of phosphorus was removed (final content of 21.01 mg/kg). Moreover, a sensitive and selective high-performance liquid chromatography method coupled to tandem mass spectrometry was applied to obtain a comprehensive lipid profile for the simultaneous evaluation of phospholipids removal and diacylglycerol (DAG) increase. A significant increase for all the monitored DAG species, up to 138.42%, was observed by using the enzymatic degumming, in comparison to the crude sample, resulting in an increased oil yield. Serratia sp. W3 lipase was identified as a suitable biocatalyst for biodiesel production, converting efficiently the acylglycerols. The results regarding the physical-chemical characteristics show that the cetane level, density and pour point of the obtained biodiesel are close to current regulation requirements. These findings highlight the potential of a two-step process implementation, based on the combination of lipase and phospholipase, as a suitable alternative for biodiesel production.

Role of PNPLA3 in Hepatic Stellate Cells and Hepatic Cellular Crosstalk.

AIMS: Since its discovery, the patatin-like phospholipase domain containing 3 (PNPLA3) (rs738409 C>G p.I148M) variant has been studied extensively to unravel its molecular function. Although several studies proved a causal relationship between the PNPLA3 I148M variant and MASLD development and particularly fibrosis, the pathological mechanisms promoting this phenotype have not yet been fully clarified. METHODS: We summarise the latest data regarding the PNPLA3 I148M variant in hepatic stellate cells (HSCs) activation and macrophage biology or the path to inflammation-induced fibrosis. RESULTS: Elegant but contradictory studies have ascribed PNPLA3 a hydrolase or an acyltransferase function. The PNPLA3 I148M results in hepatic lipid accumulation, which predisposes the hepatocyte to lipotoxicity and lipo-apoptosis, producing DAMPs, cytokines and chemokines leading to recruitment and activation of macrophages and HSCs, propagating fibrosis. Recent studies showed that the PNPLA3 I148M variant alters HSCs biology via attenuation of PPARγ, AP-1, LXRα and TGFß activity and signalling. CONCLUSIONS: The advent of refined techniques in isolating HSCs has made PNPLA3's direct role in HSCs for liver fibrosis development more apparent. However, many other mechanisms still need detailed investigations.

Effects of phospholipase D1 inhibitory peptide on the growth and metastasis of gastric cancer cells.

Phospholipase D1 (PLD1) contributes to cancer development and progression through its effects on cell proliferation, survival, invasion, metastasis, angiogenesis, drug resistance, and modulation of the tumor microenvironment. Its central role in these processes makes it a promising target for novel cancer treatments aimed at inhibiting its activity and disrupting the signaling pathways it regulates. In this study, we aimed to investigate the effect of PLD1 inhibition on gastric cancer cell growth using a novel peptide inhibitor, TAT-TVTSP. PLD1, which plays a role in cancer progression, catalyzes the conversion of phosphatidylcholine into choline and phosphatidic acid through hydrolysis. To effectively target PLD1 in cells, we engineered TAT-TVTSP by fusing a PLD1-inhibitory peptide (TVTSP) with a cell-penetrating peptide (TAT). We observed that TAT-TVTSP effectively inhibited PLD1 activity in AGS gastric cancer cells. Moreover, TAT-TVTSP significantly inhibited the mammalian target of the rapamycin signaling pathway, including the phosphorylation of key downstream targets such as S6K1, AKT, S473, glycogen synthase kinase-3b, and forkhead box O1. TAT-TVTSP did not induce cell death, but it triggered cell cycle arrest by activating p21 and p27 via AKT phosphorylation. Functional assays revealed that TAT-TVTSP significantly impaired the colony-forming ability of AGS cells, thus inhibiting cell proliferation. Transwell and wound-healing assays revealed that this peptide disrupted the cellular behaviors critical to cancer progression, such as migration and invasion. In vivo, TAT-TVTSP significantly reduced tumor growth in the xenograft model of gastric cancer without any toxicity. Overall, our results suggest that TAT-TVTSP is a novel therapeutic agent for PLD1-mediated cancers.

Genome-Wide Analysis of the Phospholipase Ds in Perennial Ryegrass Highlights LpABFs-LpPLDδ3 Cascade Modulated Osmotic and Heat Stress Responses.

The phospholipase Ds (PLDs) are crucial for cellular signalling and play roles in plant abiotic stress response. In this study, we identified 12 PLD genes from the genome data of perennial ryegrass (Lolium perenne), which is widely used as forage and turfgrass. Among them, LpPLDδ3 was significantly repressed by ABA treatment, and induced by drought stress and heat stress treatments. The ectopic overexpression (OE) of LpPLDδ3 in Arabidopsis enhanced plant tolerance to osmotic and heat stress as demonstrated by an increased survival rate and reduced malondialdehyde (MDA) accumulation and electrolyte leakage (EL). Arabidopsis endogenous ABA RESPONSIVE ELEMENT BINDING FACTORs (ABFs) and heat stress responsive genes were elevated in LpPLDδ3 OE lines under osmotic and heat stress treatments. Additionally, overexpression of LpPLDδ3 in perennial ryegrass protoplasts could increase heat stress tolerance and elevate expression level of heat stress responsive genes. Moreover, LpABF2 and LpABF4 depressed the LpPLDδ3 expression by directly binding to its ABRE core-binding motif of promoter region. In summary, LpPLDδ3 was repressed by LpABF2 and LpABF4 and positively involved in perennial ryegrass osmotic and heat stress responses.

Development and drought escape response in Arabidopsis thaliana are regulated by AtPLC1 in response to abscisic acid.

MAIN CONCLUSION: AtPLC1 plays a critical role in plant growth, development, and response to drought stress. Phosphoinositide-specific phospholipase C (PI-PLC) hydrolyzes substrates to generate secondary messengers crucial for plant growth, development, and stress responses. Drought escape (DE) response is an adaptive strategy that plants employ under drought conditions. The expression levels of the flower meristem-specific gene APETALA 1 and flowering regulatory genes FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 were downregulated in plc1, and FLOWERING LOCUS C was upregulated. The flowering time of the plc1flc double mutant was earlier than that of the wild type. Transcriptome analysis revealed that the Gene Ontology of differentially expressed genes (DEGs) was enriched in abscisic acid (ABA) response signaling, and Kyoto Encyclopedia of Genes and Genomes analysis revealed differential gene expression annotated to plant hormone signaling pathways. Our experiments show that AtPLC1 is upregulated by ABA in Arabidopsis. Under ABA induction and water stress, wild-type plants exhibit a DE response, and the DE response in plc1 disappears. Expression levels of ABA signaling pathway transcription factors ABA-responsive element-binding factors 3 (ABF3) and ABF4 were downregulated in plc1. In conclusion, our study suggests that AtPLC1 participates in regulating plant growth and development and participates in the DE response through the regulation of ABA signaling pathway transcription factors ABF3/ABF4. The study enhances our comprehension of the role of AtPLC1 in plant development and drought stress, providing a theoretical foundation for further investigation into DE responses.