Activation Of Phospholipase Research Articles

Biological Role of the Intercellular Transfer of Glycosylphosphatidylinositol-Anchored Proteins: Stimulation of Lipid and Glycogen Synthesis.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs), which are anchored at the outer leaflet of plasma membranes (PM) only by a carboxy-terminal GPI glycolipid, are known to fulfill multiple enzymic and receptor functions at the cell surface. Previous studies revealed that full-length GPI-APs with the complete GPI anchor attached can be released from and inserted into PMs in vitro. Moreover, full-length GPI-APs were recovered from serum, dependent on the age and metabolic state of rats and humans. Here, the possibility of intercellular control of metabolism by the intercellular transfer of GPI-APs was studied. Mutant K562 erythroleukemia (EL) cells, mannosamine-treated human adipocytes and methyl-ß-cyclodextrin-treated rat adipocytes as acceptor cells for GPI-APs, based on their impaired PM expression of GPI-APs, were incubated with full-length GPI-APs, prepared from rat adipocytes and embedded in micelle-like complexes, or with EL cells and human adipocytes with normal expression of GPI-APs as donor cells in transwell co-cultures. Increases in the amounts of full-length GPI-APs at the PM of acceptor cells as a measure of their transfer was assayed by chip-based sensing. Both experimental setups supported both the transfer and upregulation of glycogen (EL cells) and lipid (adipocytes) synthesis. These were all diminished by serum, serum GPI-specific phospholipase D, albumin, active bacterial PI-specific phospholipase C or depletion of total GPI-APs from the culture medium. Serum inhibition of both transfer and glycogen/lipid synthesis was counteracted by synthetic phosphoinositolglycans (PIGs), which closely resemble the structure of the GPI glycan core and caused dissociation of GPI-APs from serum proteins. Finally, large, heavily lipid-loaded donor and small, slightly lipid-loaded acceptor adipocytes were most effective in stimulating transfer and lipid synthesis. In conclusion, full-length GPI-APs can be transferred between adipocytes or between blood cells as well as between these cell types. Transfer and the resulting stimulation of lipid and glycogen synthesis, respectively, are downregulated by serum proteins and upregulated by PIGs. These findings argue for the (patho)physiological relevance of the intercellular transfer of GPI-APs in general and its role in the paracrine vs. endocrine (dys)regulation of metabolism, in particular. Moreover, they raise the possibility of the use of full-length GPI-APs as therapeutics for metabolic diseases.

Differential Effects of the G-Protein-Coupled Estrogen Receptor (GPER) on Rat Embryonic (E18) Hippocampal and Cortical Neurons.

Estrogen plays fundamental roles in nervous system development and function. Traditional studies examining the effect of estrogen in the brain have focused on the nuclear estrogen receptors (ERs), ERα and ERβ. Studies related to the extranuclear, membrane-bound G-protein-coupled ER (GPER/GPR30) have revealed a neuroprotective role for GPER in mature neurons. In this study, we investigated the differential effects of GPER activation in primary rat embryonic day 18 (E18) hippocampal and cortical neurons. Microscopy imaging, multielectrode array (MEA), and Ca2+ imaging experiments revealed that GPER activation with selective agonist, G-1, and nonselective agonist, 17β-estradiol (E2), increased neural growth, neural firing activity, and intracellular Ca2+ more profoundly in hippocampal neurons than in cortical neurons. The GPER-mediated Ca2+ rise in hippocampal neurons involves internal Ca2+ store release via activation of phospholipase C (PLC) and extracellular entry via Ca2+ channels. Immunocytochemistry results revealed no observable difference in GPER expression/localization in neurons, yet real-time qPCR (RT-qPCR) and Western blotting showed a higher GPER expression in the cortex than hippocampus, implying that GPER expression level may not fully account for its robust physiological effects in hippocampal neurons. We used RNA sequencing data to identify distinctly enriched pathways and significantly expressed genes in response to G-1 or E2 in cultured rat E18 hippocampal and cortical neurons. In summary, the identification of differential effects of GPER activation on hippocampal and cortical neurons in the brain and the determination of key genes and molecular pathways are instrumental toward an understanding of estrogen’s action in early neuronal development.

In Vitro Production of Virulence Factors and Antifungal Susceptibility Pattern of Aspergillus Isolates from Clinical Samples in a Tertiary Care Center.

Objectives This study was aimed to investigate the association between virulence factors and antifungal susceptibility pattern among Aspergillus species. Materials and Methods This study was carried out in the Department of Microbiology, from May 2018 to June 2019. A total of 52 Aspergillus isolates obtained from various clinical samples were speciated based on microscopic identification by lacto phenol cotton blue (LPCB) mount and slide culture technique. The production virulence factors such as biofilm, lipase, phospholipase, amylase, and hemolysin were detected using standard phenotypic methods with Aspergillus niger ATCC (American Type Culture Collection) 6275 as the control strain. Antifungal susceptibility patterns of all Aspergillus isolates to amphotericin B, itraconazole, voriconazole, and posaconazole were evaluated in line with the Clinical Laboratory Standards Institute (CLSI) M38-A2 guidelines. Results The percentage of resistance was the highest in itraconazole (48.08%), followed by amphotericin B (28.85%) and voriconazole (9.62%). All amphotericin B-resistant isolates produced biofilm, itraconazole-resistant isolates exhibited phospholipase activity, and voriconazole-resistant isolates produced biofilm and demonstrated phospholipase and hemolytic activities. Regardless of the virulence factors produced, all isolates were susceptible to posaconazole. Conclusion Understanding the relationship between virulence factors and antifungal resistance aids in the development of new therapeutic approaches involving virulence mechanisms as potential targets for effective antifungal drug development.

Ceramide levels in blood plasma correlate with major depressive disorder severity and its neutralization abrogates depressive behavior in mice

Major depressive disorder (MDD) is a severe disease of unknown pathogenesis that will affect ∼10% of people during their lifetime. Therapy for MDD requires prolonged treatment and often fails, predicating a need for novel treatment strategies. Here, we report increased ceramide levels in the blood plasma of MDD patients and in murine stress-induced models of MDD. These blood plasma ceramide levels correlated with the severity of MDD in human patients and were independent of age, sex, or body mass index. In addition, intravenous injection of anti-ceramide antibodies or neutral ceramidase rapidly abrogated stress-induced MDD, and intravenous injection of blood plasma from mice with MDD induced depression-like behavior in untreated mice, which was abrogated by ex vivo preincubation of the plasma with anti-ceramide antibodies or ceramidase. Mechanistically, we demonstrate that ceramide accumulated in endothelial cells of the hippocampus of stressed mice, evidenced by the quantitative measurement of ceramide in purified hippocampus endothelial cells. We found ceramide inhibited the activity of phospholipase D (PLD) in endothelial cells in vitro and in the hippocampus in vivo and thereby decreased phosphatidic acid in the hippocampus. Finally, we show intravenous injection of PLD or phosphatidic acid abrogated MDD, indicating the significance of this pathway in MDD pathogenesis. Our data indicate that ceramide controls PLD activity and phosphatidic acid formation in hippocampal endothelial cells and thereby mediates MDD. We propose that neutralization of plasma ceramide could represent a rapid-acting targeted treatment for MDD.

Characterization of the membrane interactions of phospholipase Cγ reveals key features of the active enzyme.

PLCγ enzymes are autoinhibited in resting cells and form key components of intracellular signaling that are also linked to disease development. Insights into physiological and aberrant activation of PLCγ require understanding of an active, membrane-bound form, which can hydrolyze inositol-lipid substrates. Here, we demonstrate that PLCγ1 cannot bind membranes unless the autoinhibition is disrupted. Through extensive molecular dynamics simulations and experimental evidence, we characterize membrane binding by the catalytic core domains and reveal previously unknown sites of lipid interaction. The identified sites act in synergy, overlap with autoinhibitory interfaces, and are shown to be critical for the phospholipase activity in cells. This work provides direct evidence that PLCγ1 is inhibited through obstruction of its membrane-binding surfaces by the regulatory region and that activation must shift PLCγ1 to a conformation competent for membrane binding. Knowledge of the critical sites of membrane interaction extends the mechanistic framework for activation, dysregulation, and therapeutic intervention.

Role of Oxidative Stress in Cardiac Dysfunction and Subcellular Defects Due to Ischemia-Reperfusion Injury.

Ischemia-reperfusion (I/R) injury is well-known to be associated with impaired cardiac function, massive arrhythmias, marked alterations in cardiac metabolism and irreversible ultrastructural changes in the heart. Two major mechanisms namely oxidative stress and intracellular Ca2+-overload are considered to explain I/R-induced injury to the heart. However, it is becoming apparent that oxidative stress is the most critical pathogenic factor because it produces myocardial abnormalities directly or indirectly for the occurrence of cardiac damage. Furthermore, I/R injury has been shown to generate oxidative stress by promoting the formation of different reactive oxygen species due to defects in mitochondrial function and depressions in both endogenous antioxidant levels as well as regulatory antioxidative defense systems. It has also been demonstrated to adversely affect a wide variety of metabolic pathways and targets in cardiomyocytes, various resident structures in myocardial interstitium, as well as circulating neutrophils and leukocytes. These I/R-induced alterations in addition to myocardial inflammation may cause cell death, fibrosis, inflammation, Ca2+-handling abnormalities, activation of proteases and phospholipases, as well as subcellular remodeling and depletion of energy stores in the heart. Analysis of results from isolated hearts perfused with or without some antioxidant treatments before subjecting to I/R injury has indicated that cardiac dysfunction is associated with the development of oxidative stress, intracellular Ca2+-overload and protease activation. In addition, changes in the sarcolemma and sarcoplasmic reticulum Ca2+-handling, mitochondrial oxidative phosphorylation as well as myofibrillar Ca2+-ATPase activities in I/R hearts were attenuated by pretreatment with antioxidants. The I/R-induced alterations in cardiac function were simulated upon perfusing the hearts with oxyradical generating system or oxidant. These observations support the view that oxidative stress may be intimately involved in inducing intracellular Ca2+-overload, protease activation, subcellular remodeling, and cardiac dysfunction as a consequence of I/R injury to the heart.

Future scope and challenges for congestive heart failure: moving toward development of pharmacotherapy.

Heart failure is invariably associated with cardiac hypertrophy and impaired cardiac performance. Although several drugs have been developed to delay the progression of heart failure, none of the existing interventions have shown beneficial effects in reducing morbidity and mortality. To determine specific targets for future drug development, we have discussed different mechanisms involving both cardiomyocytes and nonmyocyte extracellular matrix (ECM)) alterations for the transition of cardiac hypertrophy to heart failure as well as for the progression of heart failure. We have emphasized the role of oxidative stress, inflammatory cytokines, metabolic alterations, and Ca2+-handling defects in adverse cardiac remodeling and heart dysfunction in hypertrophied myocardium. Alterations in the regulatory process due to several protein kinases, as well as the participation of mitochondrial Ca2+ overload, activation of proteases and phospholipases, and changes in gene expression for subcellular remodeling have also been described for the occurrence of cardiac dysfunction. Association of cardiac arrhythmia with heart failure has been explained as a consequence of catecholamine oxidation products. Since these multifactorial defects in ECM and cardiomyocytes are evident in the failing heart, it is a challenge for experimental cardiologists to develop appropriate combination drug therapy for improving cardiac function in heart failure.

A Metabolomic Profiling of Intra-Uterine Growth Restriction in Placenta and Cord Blood Points to an Impairment of Lipid and Energetic Metabolism.

(1) Background: Intrauterine growth restriction (IUGR) involves metabolic changes that may be responsible for an increased risk of metabolic and cardiovascular diseases in adulthood. Several metabolomic profiles have been reported in maternal blood and urine, amniotic fluid, cord blood and newborn urine, but the placenta has been poorly studied so far. (2) Methods: To decipher the origin of this metabolic reprogramming, we conducted a targeted metabolomics study replicated in two cohorts of placenta and one cohort of cord blood by measuring 188 metabolites by mass spectrometry. (3) Results: OPLS-DA multivariate analyses enabled clear discriminations between IUGR and controls, with good predictive capabilities and low overfitting in the two placental cohorts and in cord blood. A signature of 25 discriminating metabolites shared by both placental cohorts was identified. This signature points to sharp impairment of lipid and mitochondrial metabolism with an increased reliance on the creatine-phosphocreatine system by IUGR placentas. Increased placental insulin resistance and significant alteration of fatty acids oxidation, together with relatively higher phospholipase activity in IUGR placentas, were also highlighted. (4) Conclusions: Our results show a deep lipid and energetic remodeling in IUGR placentas that may have a lasting effect on the fetal metabolism.

Neutralization of “Chaco eagle” (Buteogallus coronatus) serum on some activities of Bothrops spp. venoms

Several species of reptiles and mammals have components in their sera that can neutralize toxic components present in snake venoms. In this manuscript, we studied the neutralizing capacity of Chaco eagle's (Buteogallus coronatus) serum. This South American bird of prey eats snakes as a regular part of its diet and has anatomical features that protect from snakes’ bites. The neutralizing potency of the Chaco eagle's serum was tested on lethal, hemorrhagic, procoagulant, and phospholipase activities of the venom of “yarará grande” (Bothrops alternatus) and on phospholipase activity of “yarará ñata” (Bothrops ammodytoides) venom; both snakes are known to be the prey of Chaco eagle. Sera of crested caracara (Caracara plancus-a scavenger, omnivorous pan-American bird of prey), secretary bird (Saggitarius serpentarius-an omnivorous bird of prey from Africa that can include venomous snakes in its diet), common hen (Gallus gallus), rat (Rattus norvegicus), mouse (Mus musculus), horse (Equus caballus), and dog (Canis lupus familiaris) were also tested to compare the inhibitory capacity of neutralization. To test isologous and xenologous neutralization, sera from Bothrops alternatus and white-eared opossum (Didelphis albiventris), respectively, were used due to their known inhibitory activity on Bothrops venoms. As a control for the neutralization activity, antibothropic antivenom was used. Chaco eagle's serum neutralized hemorrhagic and phospholipasic activity and slightly neutralized the coagulation and the lethal activity of Bothrops spp. venom. The neutralizing capacity was present in the non-immunoglobulin fraction of the serum, which showed components of acidic characteristics and lower molecular weight than IgY, in correspondence with the characteristics of PLA2s and SVMPs inhibitors described in sera from some snakes and mammals. These studies showed that Chaco eagle's serum neutralizes all toxic activities tested at a higher level than sera from animal species in which inhibitors of snake venoms have not been described (p < 0.05), while it is lower or similar in neutralizing capacity to white-eared opossum and B. alternatus sera.

Crotoxin B: Heterologous Expression, Protein Folding, Immunogenic Properties, and Irregular Presence in Crotalid Venoms.

Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA2) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR®2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA2 from C. tzabcan. The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini, and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.

The Phospholipase Activity of Ammodytoxin, a Prototype Snake Venom β-Neurotoxin, Is Not Obligatory for Cell Internalisation and Translocation to Mitochondria.

β-Neurotoxins are secreted phospholipase A2 molecules that inhibit transmission in neuromuscular synapses by poisoning the motor neurons. These toxins specifically and rapidly internalise into the nerve endings of motor neurons. Ammodytoxin (Atx) is a prototype β-neurotoxin from the venom of the nose-horned viper (Vipera ammodytes ammodytes). Here, we studied the relevance of the enzymatic activity of Atx in cell internalisation and subsequent intracellular movement using transmission electron microscopy (TEM). We prepared a recombinant, enzymatically inactive mutant of Atx, Atx(D49S), labelled with gold nanoparticles (GNP), and incubated this with PC12 cells, to analyse its localisation by TEM. Atx(D49S)-GNP internalised into the cells. Inside the cells, Atx(D49S)-GNP was detected in different vesicle-like structures, cytosol, endoplasmic reticulum and mitochondria, where it was spotted in the intermembrane space and matrix. Co-localization of fluorescently labelled Atx(D49S) with mitochondria in PC12 cells by confocal fluorescence microscopy confirmed the reliability of results generated using Atx(D49S)-GNP and TEM and allowed us to conclude that the phospholipase activity of Atx is not obligatory for its cell internalisation and translocation into the mitochondrial intermembrane space and matrix.

The Threat Called Candida haemulonii Species Complex in Rio de Janeiro State, Brazil: Focus on Antifungal Resistance and Virulence Attributes.

Although considered rare, the emergent Candida haemulonii species complex, formed by C. haemulonii sensu stricto (Ch), C. duobushaemulonii (Cd) and C. haemulonii var. vulnera (Chv), is highlighted due to its profile of increased resistance to the available antifungal drugs. In the present work, 25 clinical isolates, recovered from human infections during 2011–2020 and biochemically identified by automated system as C. haemulonii, were initially assessed by molecular methods (amplification and sequencing of ITS1-5.8S-ITS2 gene) for precise species identification. Subsequently, the antifungal susceptibility of planktonic cells, biofilm formation and susceptibility of biofilms to antifungal drugs and the secretion of key molecules, such as hydrolytic enzymes, hemolysins and siderophores, were evaluated by classical methodologies. Our results revealed that 7 (28%) isolates were molecularly identified as Ch, 7 (28%) as Chv and 11 (44%) as Cd. Sixteen (64%) fungal isolates were recovered from blood. Regarding the antifungal susceptibility test, the planktonic cells were resistant to (i) fluconazole (100% of Ch and Chv, and 72.7% of Cd isolates), itraconazole and voriconazole (85.7% of Ch and Chv, and 72.7% of Cd isolates); (ii) no breakpoints were defined for posaconazole, but high MICs were observed for 85.7% of Ch and Chv, and 72.7% of Cd isolates; (iii) all isolates were resistant to amphotericin B; and (iv) all isolates were susceptible to echinocandins (except for one isolate of Cd) and to flucytosine (except for two isolates of Cd). Biofilm is a well-known virulence and resistant structure in Candida species, including the C. haemulonii complex. Herein, we showed that all isolates were able to form viable biofilms over a polystyrene surface. Moreover, the mature biofilms formed by the C. haemulonii species complex presented a higher antifungal-resistant profile than their planktonic counterparts. Secreted molecules associated with virulence were also detected in our fungal collection: 100% of the isolates yielded aspartic proteases, hemolysins and siderophores as well as phospholipase (92%), esterase (80%), phytase (80%), and caseinase (76%) activities. Our results reinforce the multidrug resistance profile of the C. haemulonii species complex, including Brazilian clinical isolates, as well as their ability to produce important virulence attributes such as biofilms and different classes of hydrolytic enzymes, hemolysins and siderophores, which typically present a strain-dependent profile.

Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Phosphatidylserine (PS) has been widely used in the fields of food and medicine, among others, owing to its unique chemical structure and health benefits. However, the phospholipase D (PLD)-mediated enzymatic production of PS remains a challenge due to the low transphosphatidylation activity of PLD. Therefore, in the present study, we designed a maltose-binding protein (MBP) tag and a PLD co-expression method to achieve the expression of soluble PLD in Escherichia coli. A "reconstruct substrate pocket" strategy was then proposed based on the catalytic mechanism and molecular dynamics simulation, expanding the substrate pocket and manipulating the coordination of l-Ser within the active site. The best mutant (SrMBPPLDMu6) exhibited a 2.04-fold higher transphosphatidylation/hydrolysis ratio than the wild-type Furthermore, under optimal conditions, Mu6 produced 58.6 g/L PS with 77.2% conversion, within 12 h on a 3 L scale, which demonstrates the potential of the proposed method for industrial application.

A pyroptosis nanotuner for cancer therapy.

Pyroptosis is a gasdermin-mediated programmed necrosis that occurs via membrane perforation and that can be exploited for biomedical applications in cancer therapy. However, inducing specific pyroptotic cancer cell death while sparing normal cells is challenging. Here, we report an acid-activatable nanophotosensitizer library that can be used to spatiotemporally target distinct stages of endosomal maturation, enabling tunable cellular pyroptosis. Specific activation of phospholipase C signalling transduction in early endosomes triggers gasdermin-E-mediated pyroptosis, which is dramatically reduced when acid-activatable nanophotosensitizers are transported into late endosomes/lysosomes. This nanotuner platform induces pyroptotic cell death with up to 40-fold tunability in various gasdermin-E-positive human cancers, resulting in enhanced anti-tumour efficacy and minimized systemic side effects. This study offers new insights into how to engineer nanomedicines with tunable pyroptosis activity through specific targeting of distinct endocytic signalling for biomedical applications.

Characterization of lncRNA Profiles of Plasma-Derived Exosomes From Type 1 Diabetes Mellitus.

BackgroundsExosomes contain several types of transcripts, including long non-coding RNAs (lncRNAs), and have been shown to exert important effects in human diseases. However, the roles of exosomal lncRNAs in type 1 diabetes mellitus (T1DM) have not been well investigated. In the present study, we characterized the plasma-derived exosomal lncRNAs expression profiles of T1DM and predict their potential function in the pathogenesis of T1DM.Material and MethodsExosomal lncRNA expression profiles were detected by Illumina Hiseq platform (T1DM subjects N=10; age-, sex- matched Control subjects N=10). Six exosomal lncRNAs were selected to validate their expression level by using quantitative real-time PCR (qRT-PCR) (T1DM subjects N=30; age-, sex- matched Control subjects N=30). Bioinformatics analysis approaches were carried out to explore the potential biological function of differentially expressed lncRNAs.ResultsA total of 162 differentially expressed exosomal lncRNAs were identified in T1DM patients compared with control subjects, among which 77 up-regulated and 85 down-regulated. The expression level of the selected six lncRNAs didn’t show significant difference in the following qRT-PCR analysis. Gene Ontology analysis enriched terms such as activation of phospholipase D activity, neuronal cell body membrane, and calcium sensitive guanylate cyclase activator activity for cis-acting genes of lncRNAs, and metal ion binding for trans-acting genes. The most enriched Kyoto Encyclopedia of Genes and Genomes pathways for the lncRNAs were associated with oxidative phosphorylation and Parkinson’s disease for cis-acting genes, and pathways in cancer as well as focal adhesion for trans-acting genes.ConclusionsThis study characterized the lncRNA profiles of plasma-derived exosomes from T1DM for the first time and these results highlighted the potential role of exosomal lncRNAs in T1DM pathogenesis. A better understanding of exosomal lncRNA profiling will provide novel insights into its molecular mechanisms.

Lipidomics Revealed Aberrant Lipid Metabolism Caused by Inflammation in Cardiac Tissue in the Early Stage of Systemic Lupus Erythematosus in a Murine Model.

Cardiac involvement, displayed as premature cardiovascular disease (CVD), is one of common clinical symptoms of patients with systemic lupus erythematosus (SLE), contributing to mortality of the disease. The precise underlying pathological mechanism(s) for the cardiac involvement in lupus remains poorly understood. Lipids and their metabolites are directly involved in atherosclerosis development, oxidative stress, and inflammation, which are closely related to the development of CVD. In the study, shotgun lipidomics was exploited to quantitatively analyze cellular lipidomes in the cardiac tissue of MRL/lpr mice at two different time points (i.e., pre-lupus and lupus state) with/without treatment with glucocorticoids (GCs). Urine protein, spleen index, and renal histopathological evaluation of the mice were also performed for assessment of SLE onset and/or outcome. Lipidomics analysis revealed that the deposition of cholesterol and the aberrant metabolism of lipids caused by the increased energy metabolism and the enhanced activation of phospholipases, both of which were originally induced by inflammation, were already present in cardiac tissues from lupus-prone mice even at pre-lupus state. These lipid alterations could further induce inflammation and autoimmune responses, accelerating the process of CVD. In addition, the present study also demonstrated that GCs therapy could not only delay the progression of SLE, but also partially corrected these alterations of lipid species in cardiac tissue due to their anti-inflammatory effect. Thus, the medications with better anti-inflammatory effect might be a useful therapeutic method for premature CVD of SLE.

Chemical Tools for Understanding Phosphatidic Acid Signaling

Phosphatidic acid (PA) is an important phospholipid biosynthetic intermediate and a lipid second messenger. Both the metabolism of PA and the interactions of PA with its effector proteins can occur on numerous organelle membranes, raising important questions about how cells direct specificity into the outcomes of PA signaling. We have endeavored to ‘uncouple the pleiotropy’ of PA signaling through the development of two complementary types of chemical biology toolsets for, respectively, visualizing and manipulating PA metabolism and signaling. The first of these, IMPACT, harnesses bioorthogonal chemistry to generate fluorescent lipids whose localization and abundance report on the activity of phospholipase D (PLD) enzymes that generate ‘signaling pools’ of PA. The second tool, optoPLD, involves the use of light‐controlled, or optogenetic, PLD enzymes capable of generating pools of PA at precise organelle locations upon blue light illumination. We will describe the development of these tools, including newer, second‐generation IMPACT probes and optimized ‘superPLD’ enzymes with much better catalytic properties than our original optoPLD systems. Further, we will describe select applications of IMPACT and optoPLD. These include the use of IMPACT as a readout in genome‐wide CRISPR screening to discover new regulators of PLD signaling and the use of these tools to examine the spatiotemporal dynamics of important pathways that intersect with PLD activation, such as GPCR signaling and the Hippo pathway. Collectively, this work highlights the power of applying diverse chemical biology approaches to shed light on cellular lipid signaling pathways.

Activity‐based chemoproteomics reveals targets of phospholipase D‐derived phosphatidyl ethanol lipids

Alcohol consumption leads to formation of phosphatidylethanol (PEth) via the transphosphatidylation activity of phospholipase D (PLD) enzymes. Though this non‐natural phospholipid routinely serves as a biomarker of chronic alcoholism, its pathophysiological roles remain unknown. We use a minimalist diazirine alkyne alcohol as an ethanol surrogate to generate clickable, photoaffinity lipid reporters of PEth localization and lipid–protein interactions via PLD‐mediated transphosphatidylation. We use these tools to visualize phosphatidyl alcohols in a manner compatible with standard permeabilization and immunofluorescence methods. We also use click chemistry tagging, enrichment, and proteomics analysis to define the phosphatidyl alcohol interactome. Our analysis reveals an enrichment of putative interactors at various membrane locations, and we validate one such interaction with the single‐pass transmembrane protein basigin/CD147. Further, we identify binding sites of phosphatidyl alcohols to basigin/CD147 via both proteomics and targeted mutagenesis. This study provides a comprehensive view of the molecular interactions of phosphatidyl alcohols with the cellular proteome and points to future work to connect such interactions to potential pathophysiological roles of PEth.

Identifying Novel Signaling Mechanisms Downstream of Gq‐Coupled Receptors

The G protein alpha subunit G□q is a critical mediator of cells’ response to a variety of external stimuli as a result of activation of Gq‐coupled GPCRs. G□q‐dependent signaling influences an array of physiological processes, including neurotransmission and vasoconstriction; additionally, dysregulated G□q‐dependent signaling has been firmly linked to several pathologies, including uveal melanoma and maladaptive cardiomyocyte hypertrophy. One established mechanism by which activated G□q subunits might promote these processes involves activation of phospholipase C beta isoforms (PLC□), which catalyze the hydrolysis of PI(4,5)P2 into inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG), ultimately leading to Ca2+ mobilization and protein kinase C activation. However, G□q subunits also signal independently of PLC□, through p63RhoGEF and Trio, for example.To identify novel G□q targets that might be involved in Gq‐driven pathologies, we conducted a proximity biotinylation proteomic screen in HEK293A cells comparing wild‐type G□q and constitutively active G□q Q209L each fused to TurboID, a promiscuous biotin ligase. This approach enabled the high‐confidence identification of numerous proteins that were selectively enriched in cells expressing G□q‐Q209L‐TurboID compared to cells expressing G□q‐WT‐TurboID. These enriched proteins included known G□q interactors (PLC□, Trio, and GRK2); however, scattered among these known interactors were several proteins that have not been previously shown to interact with active G□q, including SPRED1, SMARCD3, YAP1, BCAS2, and HDAC9.Our initial efforts have focused on SMARCD3, a regulatory component of the SWI/SNF chromatin remodeling complex. To test for SMARCD3 interactions with G□q, we expressed SMARCD3 in COS‐7 cells and found that SMARCD3 selectively inhibits G□q‐stimulated, but not G□□‐stimulated, PLC□ activity, indicating that SMARCD3 competes with PLC□ for access to active G□q. In addition, we expressed SMARCD3 in HEK293A cells and found that (1) SMARCD3 co‐immunoprecipitates with G□q and (2) interacts with G□q in a nanoluciferase fragment complementation assay.SWI/SNF regulates gene transcription by modulating chromatin accessibility. Given that Gq signaling regulates gene expression in both physiological and pathological systems, we will assess the impact of SMARCD3 knockdown in cellular models of G□q‐driven transcription and cell function. Ultimately, we will expand this approach to test other top hits' ability to interact with and potentiate signaling downstream of active G□q. Through these endeavors, we aim to uncover novel G□q signaling mechanisms and inform therapeutic strategies for combatting pathologies marked by aberrant G□q signaling.

Regulation of PIP2 homeostasis at ER‐plasma membrane contacts by Nir proteins

Phosphatidylinositol 4,5‐bisphosphate (PIP2) in the plasma membrane is a critical lipid molecule that regulates nearly all functional processes occurring at the cell surface. Additionally, PIP2 is hydrolyzed following receptor‐induced activation of phospholipase C to generate second messengers for signal transduction. It is crucial to quickly replenish PIP2 in the plasma membrane following receptor‐induced hydrolysis to sustain signaling outputs and to maintain PIP2‐dependent cellular functions. Replenishment of plasma membrane PIP2 in receptor‐stimulated cells involves transport of phosphatidylinositol (PI) from the endoplasmic reticulum (ER) to the plasma membrane for PIP2 re‐synthesis. ER‐plasma membrane contacts are subcellular loci characterized by the close apposition of the ER to the plasma membrane. Recent findings revealed that the PI transfer proteins, Nir2 and Nir3, dynamically localize at ER‐plasma membrane contacts to mediate replenishment of PIP2 hydrolyzed following receptor stimulation. Deficiency in Nir2 and Nir3 suppressed replenishment of PIP2 in the plasma membrane and Ca2+ signaling in receptor‐stimulated cells. A third mammalian Nir protein, Nir1, has been linked to several human diseases including retinal dystrophy. Unlike Nir2 and Nir3, Nir1 lacks a PI transfer protein domain and readily localizes at ER‐plasma membrane contacts in resting cells. The presence of Nir1 promotes Nir2 localization at ER‐plasma membrane contacts to mediate PIP2 replenishment following receptor stimulation. Together, Nir proteins maintain plasma membrane PIP2 homeostasis at ER‐plasma membrane contacts to support cell signaling and plasma membrane functions in receptor‐stimulated cells.