Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation.

Phospholipase D (PLD) is an enzyme with many functions, one of which is the synthesis of phosphatidic acid (PA), a molecule with a myriad of effects on various organ systems and processes. These numerous roles make it hard to understand the true action of PA in cellular and bodily processes. Imaging PLD activity is one way to better understand the synthesis of PA and start to elucidate its function. However, many of the current imaging techniques for PLD come with limitations. This chapter presents a thorough methodology of a new imaging technique for PLD activity with clickable alcohols via transphosphatidylation (IMPACT) and Real-Time IMPACT (RT-IMPACT) that takes advantage of clickable chemistry to overcome current limitations. Using strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron-demand Diels-Alder (IEDDA), and the synthesis of various organic compounds, this chapter will explain a step-by-step procedure of how to perform the IMPACT and RT-IMPACT method(s).

Synthesis of Azo Analogs for Investigating Phosphatidic Acid-Mediated Signaling.

Phosphatidic acid (PA) is a key signaling lipid that plays a crucial role in regulating various cellular processes. Studies have shown that azobenzene-containing PA analogues can be used as an all-chemical strategy for light-mediated control of PA signaling. These photoswitchable lipids offer a solution to the limitations of traditional bulk dosing methods by allowing for light- and shape-dependent interactions with protein effectors and lipid-metabolizing enzymes. This chapter describes how to synthesize AzoPA and dAzoPA.

Nuclear lipid droplet: Guardian of nuclear membrane lipid homeostasis?

Lipid droplets (LDs) are cytoplasmic organelles, but they are also found within the nucleus in small numbers. Nuclear LDs that form at the inner nuclear membrane (INM) often increase in response to perturbation in phosphatidic acid (PA) and/or diacylglycerol (DAG), both implicated in various INM functions. Nuclear LDs also increase upon downregulation of seipin, a protein that can trap PA and DAG in the endoplasmic reticulum. Notably, both PA and DAG appear to be more densely distributed on the surface of nuclear LDs than in the INM. I propose that nuclear LDs play a role in regulating the PA and DAG level in the INM, thereby contributing to the lipid homeostasis in this compartment.

Spatially resolved lipidomics shows conditional transfer of lipids produced by Bacteroides thetaiotaomicron into the mouse gut.

The extent to which bacterial lipids produced by the gut microbiota penetrate host tissues is unclear. Here, we combined mass spectrometry approaches to identify lipids produced by the human gut symbiont Bacteroides thetaiotaomicron (B. theta) and spatially track these bacterial lipids in the mouse colon. We characterize 130 B. theta lipids by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using wild-type and mutant B. theta strains to confidently identify lipid structures and their interconnected pathways in vitro. Of these, 103 B. theta lipids can be detected and spatially mapped in a single MALDI mass spectrometry imaging run. We map unlabeled bacterial lipids across colon sections of germ-free and specific-pathogen-free (SPF) mice and mice mono-colonized with wild-type or sphingolipid-deficient (BTMUT) B. theta. We observe co-localization of bacterially derived phosphatidic acid with host tissues in BTMUT mice, consistent with lipid penetration into host tissues. These results indicate limited and selective transfer of bacterial lipids to the host.

Phosphatidic acid-dependent recruitment of microtubule motors to spherical supported lipid bilayers for in vitro motility assays.

Motor proteins transport diverse membrane-bound vesicles along microtubules inside cells. How specific lipids, particularly rare lipids, on the membrane recruit and activate motors is poorly understood. To address this, we prepare spherical supported lipid bilayers (SSLBs) consisting of a latex bead enclosed within a membrane of desired lipid composition. SSLBs containing phosphatidic acid recruit dynein when incubated with Dictyostelium fractions but kinesin-1 when incubated with rat brain fractions. These SSLBs allow controlled biophysical investigation of membrane-bound motors along with their regulators at the single-cargo level in vitro. Optical trapping of single SSLBs reveals that motor-specific inhibitors can "lock" a motor to a microtubule, explaining the paradoxical arrest of overall cargo transport by such inhibitors. Increasing their size causes SSLBs to reverse direction more frequently, relevant to how large cargoes may navigate inside cells. These studies are relevant to understand how unidirectional or bidirectional motion of vesicles might be generated.

DGK5-mediated phosphatidic acid homeostasis interplays with reactive oxygen species in plant immune signaling.

Reactive oxygen species (ROS) and phosphatidic acid (PA) are important second messengers in plant immunity. PA binding to RBOHD, an NADPH oxidase responsible for ROS production, enhances RBOHD stability and promotes ROS production. Distinct phosphorylation of the lipid kinase DGK5 optimizes the PA burst in regulating ROS production.

Lysosomal diacylglycerol pyrophosphate phosphatase is not essential in Trypanosoma brucei.

Mg2+-independent phosphatidic acid phosphatase (PAP2), diacylglycerol pyrophosphate phosphatase 1 (Dpp1) is a membrane-associated enzyme in Saccharomyces cerevisiae. The enzyme is responsible for inducing the breakdown of ß-phosphate from diacylglycerol pyrophosphate (DGPP) into phosphatidate (PA) and then removes the phosphate from PA to give diacylglycerol (DAG). In this study through RNAi suppression, we have demonstrated that Trypanosoma brucei diacylglycerol pyrophosphate phosphatase 1 (TbDpp1) procyclic form production is not required for parasite survival in culture. The steady-state levels of triacylglycerol (TAG), the number of lipid droplets, and the PA content are all maintained constant through the inducible down-regulation of TbDpp1. Furthermore, the localization of C-terminally tagged variants of TbDpp1 in the lysosome was demonstrated by immunofluorescence microscopy.

Identification of a 1-acyl-glycerol-3-phosphate acyltransferase from Mycobacterium tuberculosis, a key enzyme involved in triacylglycerol biosynthesis.

Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol-3-phosphate, wherein the second acylation step is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane-bound enzyme, and functions as 1-acylglycerol-3-phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol-3-phosphate (LPA) from monounsaturated fatty acyl-coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant in vivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA in vitro and were not able to rescue the growth defect of E. coli PlsC mutant in vivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.

A light-controlled phospholipase C for imaging of lipid dynamics and controlling neural plasticity.

Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCß (opto-PLCß). Opto-PLCß uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCß triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCß can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCß offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo.

Therapeutic potential of lipin inhibitors for the treatment of cancer.

Lipins are phosphatidic acid phosphatases (PAP) that catalyze the conversion of phosphatidic acid (PA) to diacylglycerol (DAG). Three lipin isoforms have been identified: lipin-1, -2 and -3. In addition to their PAP activity, lipin-1 and -2 act as transcriptional coactivators and corepressors. Lipins have been intensely studied for their role in regulation of lipid metabolism and adipogenesis; however, lipins are hypothesized to mediate several pathologies, such as those involving metabolic diseases, neuropathy and even cognitive impairment. Recently, an emerging role for lipins have been proposed in cancer. The study of lipins in cancer has been hampered by lack of inhibitors that have selectivity for lipins, that differentiate between lipin family members, or that are suitable for in vivo studies. Such inhibitors have the potential to be extremely useful as both molecular tools and therapeutics. This review describes the expression and function of lipins in various tissues and their roles in several diseases, but with an emphasis on their possible role in cancer. The mechanisms by which lipins mediate cancer cell growth are discussed and the potential usefulness of selective lipin inhibitors is hypothesized. Finally, recent studies reporting the crystallization of lipin-1 are discussed to facilitate rational design of novel lipin inhibitors.

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

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

DGK5ß-derived phosphatidic acid regulates ROS production in plant immunity by stabilizing NADPH oxidase.

In plant immunity, phosphatidic acid (PA) regulates reactive oxygen species (ROS) by binding to respiratory burst oxidase homolog D (RBOHD), an NADPH oxidase responsible for ROS production. Here, we analyze the influence of PA binding on RBOHD activity and the mechanism of RBOHD-bound PA generation. PA binding enhances RBOHD protein stability by inhibiting vacuolar degradation, thereby increasing chitin-induced ROS production. Mutations in diacylglycerol kinase 5 (DGK5), which phosphorylates diacylglycerol to produce PA, impair chitin-induced PA and ROS production. The DGK5 transcript DGK5ß (but not DGK5α) complements reduced PA and ROS production in dgk5-1 mutants, as well as resistance to Botrytis cinerea. Phosphorylation of S506 residue in the C-terminal calmodulin-binding domain of DGK5ß contributes to the activation of DGK5ß to produce PA. These findings suggest that DGK5ß-derived PA regulates ROS production by inhibiting RBOHD protein degradation, elucidating the role of PA-ROS interplay in immune response regulation.

Phosphatidic acid interacts with an HD-ZIP transcription factor GhHOX4 to influence its function in fiber elongation of cotton (Gossypium hirsutum).

Upland cotton, the mainly cultivated cotton species in the world, provides over 90% of natural raw materials (fibers) for the textile industry. The development of cotton fibers that are unicellular and highly elongated trichomes on seeds is a delicate and complex process. However, the regulatory mechanism of fiber development is still largely unclear in detail. In this study, we report that a homeodomain-leucine zipper (HD-ZIP) IV transcription factor, GhHOX4, plays an important role in fiber elongation. Overexpression of GhHOX4 in cotton resulted in longer fibers, while GhHOX4-silenced transgenic cotton displayed a "shorter fiber" phenotype compared with wild type. GhHOX4 directly activates two target genes, GhEXLB1D and GhXTH2D, for promoting fiber elongation. On the other hand, phosphatidic acid (PA), which is associated with cell signaling and metabolism, interacts with GhHOX4 to hinder fiber elongation. The basic amino acids KR-R-R in START domain of GhHOX4 protein are essential for its binding to PA that could alter the nuclear localization of GhHOX4 protein, thereby suppressing the transcriptional regulation of GhHOX4 to downstream genes in the transition from fiber elongation to secondary cell wall (SCW) thickening during fiber development. Thus, our data revealed that GhHOX4 positively regulates fiber elongation, while PA may function in the phase transition from fiber elongation to SCW formation by negatively modulating GhHOX4 in cotton.

Dual phosphorylation of DGK5-mediated PA burst regulates ROS in plant immunity.

Phosphatidic acid (PA) and reactive oxygen species (ROS) are crucial cellular messengers mediating diverse signaling processes in metazoans and plants. How PA homeostasis is tightly regulated and intertwined with ROS signaling upon immune elicitation remains elusive. We report here that Arabidopsis diacylglycerol kinase 5 (DGK5) regulates plant pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). The pattern recognition receptor (PRR)-associated kinase BIK1 phosphorylates DGK5 at Ser-506, leading to a rapid PA burst and activation of plant immunity, whereas PRR-activated intracellular MPK4 phosphorylates DGK5 at Thr-446, which subsequently suppresses DGK5 activity and PA production, resulting in attenuated plant immunity. PA binds and stabilizes the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), regulating ROS production in plant PTI and ETI, and their potentiation. Our data indicate that distinct phosphorylation of DGK5 by PRR-activated BIK1 and MPK4 balances the homeostasis of cellular PA burst that regulates ROS generation in coordinating two branches of plant immunity.

Differential interactions of essential and toxic metal ions with biologically relevant phosphatidic acid and phosphatidylserine membranes.

Metal pollutants are a growing concern due to increased use in mining and other industrial processes. Moreover, the use of metals in daily life is becoming increasingly prevalent. Metals such as manganese (Mn), cobalt (Co), and nickel (Ni) are toxic in high amounts whereas lead (Pb) and cadmium (Cd) are acutely toxic at low µM concentrations. These metals are associated with system dysfunction in humans including cancer, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, and other cellular process'. One known but lesser studied target of these metals are lipids that are key membrane building blocks or serve signalling functions. It was shown that Mn, Co, Ni, Pb, and Cd cause rigidification of liposomes and increase the phase transition in membranes composed of both saturated or partly unsaturated phosphatidic acid (PA) and phosphatidylserine (PS). The selected metals showed differential effects that were more pronounced on saturated lipids. In addition, more rigidity was induced in the biologically relevant liquid-crystalline phase. Moreover, metal affinity, induced rigidification and liposome size increases also varied with the headgroup architecture, whereby the carboxyl group of PS appeared to play an important role. Thus, it can be inferred that Mn, Co, Ni, Cd, and Pb may have preferred binding coordination with the lipid headgroup, degree of acyl chain unsaturation, and membrane phase.

The wide world of non-mammalian phospholipase D enzymes.

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

Lipid signaling and proline catabolism are activated in barley roots (Hordeum vulgare L.) during recovery from cold stress.

Previous findings have shown that phospholipase D (PLD) contributes to the response to long-term chilling stress in barley by regulating the balance of proline (Pro) levels. Although Pro accumulation is one of the most prominent changes in barley roots exposed to this kind of stress, the regulation of its metabolism during recovery from stress remains unclear. Research has mostly focused on the responses to stress per se, and not much is known about the dynamics and mechanisms underlying the subsequent recovery. The present study aimed to evaluate how PLD, its product phosphatidic acid (PA), and diacylglycerol pyrophosphate (DGPP) modulate Pro accumulation in barley during recovery from long-term chilling stress. Pro metabolism involves different pathways and enzymes. The rate-limiting step is mediated by pyrroline-5-carboxylate synthetase (P5CS) in its biosynthesis, and by proline dehydrogenase (ProDH) in its catabolism. We observed that Pro levels decreased in recovering barley roots due to an increase in ProDH activity. The addition of 1-butanol, a PLD inhibitor, reverted this effect and altered the relative gene expression of ProDH. When barley tissues were treated with PA before recovery, the fresh weight of roots increased and ProDH activity was stimulated. These data contribute to our understanding of how acidic membrane phospholipids like PA help to control Pro degradation during recovery from stress.

PLAT domain protein 1 (PLAT1/PLAFP) binds to the Arabidopsis thaliana plasma membrane and inserts a lipid.

Robust agricultural yields depend on the plant's ability to fix carbon amid variable environmental conditions. Over seasonal and diurnal cycles, the plant must constantly adjust its metabolism according to available resources or external stressors. The metabolic changes that a plant undergoes in response to stress are well understood, but the long-distance signaling mechanisms that facilitate communication throughout the plant are less studied. The phloem is considered the predominant conduit for the bidirectional transport of these signals in the form of metabolites, nucleic acids, proteins, and lipids. Lipid trafficking through the phloem in particular attracted our attention due to its reliance on soluble lipid-binding proteins (LBP) that generate and solubilize otherwise membrane-associated lipids. The Phloem Lipid-Associated Family Protein (PLAFP) from Arabidopsis thaliana is generated in response to abiotic stress as is its lipid-ligand phosphatidic acid (PA). PLAFP is proposed to transport PA through the phloem in response to drought stress. To understand the interactions between PLAFP and PA, nearly 100 independent systems comprised of the protein and one PA, or a plasma membrane containing varying amounts of PA, were simulated using atomistic classical molecular dynamics methods. In these simulations, PLAFP is found to bind to plant plasma membrane models independent of the PA concentration. When bound to the membrane, PLAFP adopts a binding pose where W41 and R82 penetrate the membrane surface and anchor PLAFP. This triggers a separation of the two loop regions containing W41 and R82. Subsequent simulations indicate that PA insert into the ß-sandwich of PLAFP, driven by interactions with multiple amino acids besides the W41 and R82 identified during the insertion process. Fine-tuning the protein-membrane and protein-PA interface by mutating a selection of these amino acids may facilitate engineering plant signaling processes by modulating the binding response.

Phosphatidic acid signaling and function in nuclei.

Membrane lipidomes are dynamic and their changes generate lipid mediators affecting various biological processes. Phosphatidic acid (PA) has emerged as an important class of lipid mediators involved in a wide range of cellular and physiological responses in plants, animals, and microbes. The regulatory functions of PA have been studied primarily outside the nuclei, but an increasing number of recent studies indicates that some of the PA effects result from its action in nuclei. PA levels in nuclei are dynamic in response to stimuli. Changes in nuclear PA levels can result from activities of enzymes associated with nuclei and/or from movements of PA generated extranuclearly. PA has also been found to interact with proteins involved in nuclear functions, such as transcription factors and proteins undergoing nuclear translocation in response to stimuli. The nuclear action of PA affects various aspects of plant growth, development, and response to stress and environmental changes.

Phospholipase D1 produces phosphatidic acid at sites of secretory vesicle docking and fusion.

Phospholipase D1 (PLD1) activity is essential for the stimulated exocytosis of secretory vesicles where it acts as a lipid-modifying enzyme to produces phosphatidic acid (PA). PLD1 localizes to the plasma membrane and secretory vesicles, and PLD1 inhibition or knockdowns reduce the rate of fusion. However, temporal data resolving when and where PLD1 and PA are required during exocytosis is lacking. In this work, PLD1 and production of PA are measured during the trafficking, docking, and fusion of secretory vesicles in PC12 cells. Using fluorescently tagged PLD1 and a PA-binding protein, cells were imaged using TIRF microscopy to monitor the presence of PLD1 and the formation of PA throughout the stages of exocytosis. Single docking and fusion events were imaged to measure the recruitment of PLD1 and the formation of PA. PLD1 is present on mobile, docking, and fusing vesicles and also colocalizes with Syx1a clusters. Treatment of cells with PLD inhibitors significantly reduces fusion, but not PLD1 localization to secretory vesicles. Inhibitors also alter the formation of PA; when PLD1 is active, PA slowly accumulates on docked vesicles. During fusion, PA is reduced in cells treated with PLD1 inhibitors, indicating that PLD1 produces PA during exocytosis.