Dissecting the role of phospholipase A2 in the late stages of regulated exocytosis

This thesis is divided into five chapters. The overarching working hypothesis is published as a ‘perspective’ article in International Journal of Biochemistry and Cell biology, 2017 https://doi.org/10.1016/j.biocel.2017.01.011 and is also discussed in the last section of Chapter - 1. Hence, there are similarities in the published article and sections of Chapter - 1. The working hypothesis states that endogenous PLA2 near docking/ fusion sites provide basal levels of LPC that acts as a ‘molecular brake’ against spontaneous fusion, and FFA that aids in trans-SNARE complex formation to ensure efficient docking of secretory vesicles to the plasma membrane (PM) prior to triggering. The data in Chapters - 2 and - 3 then test the working hypothesis and are also published as original articles in cells, 2019 https://doi.org/10.3390/cells8040303 and Biochemical and Biophysical Research Communications, 2019 https://doi.org/10.1016/j.bbrc.2019.05.106, respectively. The data presented in Chapter - 2, indicate that vesicle-associated endogenous PLA2 isozymes – sPLA2 in the CV lumen and iPLA2 on the CV surface – maintain docking/priming steps in regulated exocytosis. The specific CV localization of sPLA2 and iPLA2 is also shown, using highly sensitive two-dimensional gel electrophoresis (2DE) immuno-blotting with selective antibodies on separated luminal and membrane fractions. Biochemical assays confirmed that these isozymes are catalytically active; sPLA2 showed higher efficiency in catalyzing PE than PC, while iPLA2 showed no such preference. Notably, a significant increase in the de novo metabolite TAG upon iPLA2 inhibition confirmed the role of this isozyme in maintaining membrane lipid homeostasis. Removal of CV surface proteins by treatment with trypsin, coupled with the use of a selective PLA2 substrate (PED6) also confirmed the presence of an active PLA2 isozyme in the outer CV membrane. Overall, the data presented in Chapter - 2, provide evidence in support of the working hypothesis. To test the hypothesis further, in Chapter - 3, exogenous arachidonic acid (ARA) and lysophosphatidylcholine (LPC), canonical PLA2 metabolites, are shown to impair docking/priming and actual membrane merger steps, respectively. The observed decrease in docking/priming induced by exogenous ARA is contradictory to part of the working hypothesis that was put forth based on existing literature. That exogenous LPC and ARA were to a significant extent converted into endogenous PC and FFA, respectively, indicated that endogenous enzymes maintain lipid homeostasis in the native vesicle membrane. With the intension of activating CV luminal sPLA2, Phospholipase A2 activating peptide (PLAP) - a melittin homolog, was used in Chapter – 4. The data presented indicates that PLAP inhibited CSC, and CV fusion by impairing docking/priming and also altered endogenous PE and FFA levels in the CV membrane. This suggests that PLAP caused no change in the CV luminal sPLA2 activity; an observation contrary to the earlier in vitro studies. To test further, PLAP was delivered to mouse chromaffin cells and was seen to reduce membrane capacitance and readily releasable pool (RRP) size, when triggered with depolarizing pulses. This substantiate that the PLAP blocked a common underlying mechanism associated with the late stages of Ca2+ triggered exocytosis. The Chapter - 4 is another manuscript in preparation and will be submitted to an appropriate journal. Finally, in Chapter - 5, the Discussion and Future Directions, all key finding in this thesis are reviewed and integrated with a critical evaluation of the existing literature, and prospective future experiments are outlined

The Roles of Phospholipase A₂ in Phagocytes

Phagocytic cells, such as macrophages, neutrophils, and dendritic cells, ingest particles larger than about 0.5 μM and thereby clear microbial pathogens and malignant cells from the body. These phagocytic cargoes are proteolytically degraded within the lumen of phagosomes, and peptides derived from them are presented on Major Histocompatibility Complexes (MHC) for the activation of T cells. Mammalian PLA2 isozymes belong to a large family of enzymes that cleave phospholipids at the second position of the glycerol backbone, releasing a free fatty acid and a lysolipid moiety. In human macrophages, at least 15 different PLA2 forms are expressed, and expression of many of these is dependent on pathogenic stimulation. Intriguing questions are why so many PLA2 forms are expressed in macrophages, and what are the functional consequences of their altered gene expression after encountering pathogenic stimuli. In this review, we discuss the evidence of the differential roles of different forms of PLA2 in phagocytic immune cells. These roles include: lipid signaling for immune cell activation, initial phagocytic particle uptake, microbial action for the killing and degradation of ingested microbes, and the repair of membranes induced by oxygen radicals. We also discuss the roles of PLA2 in the subsequent digestion of ingested phagocytic cargoes for antigen presentation to T cells

Focal exocytosis of Syntaxin 3 and TRPML1 at pseudopodia of nascent phagosomes

Macrophages clear invading pathogens by phagocytosis. Phagocytosis is a complex mechanism involving the local expansion of the membrane, cytoskeletal remodeling, and the delivery of phagosomal proteins to the nascent phagosomes. However, the organelle trafficking events underlying this are largely unclear. Here, we show in human blood monocyte-derived macrophages that TRPML1, a calcium channel involved in the phagocytic process, is delivered to phagosomes in Syntaxin 3-positive vesicles. Syntaxin 3 is a SNARE protein previously shown to mediate the secretion of IL-6 by macrophages. Total Internal Reflection Microscopy (TIRF) revealed that Syntaxin 3 positive compartments carry TRPML1 to pseudopodia for focal exocytosis at the nascent phagosomes during E. coli uptake. Using siRNA knockdown, we show that both Syntaxin 3 and TRPML1 are required for E. coli uptake. Moreover, using TRPML1 agonists we show that increased TRPML1 activity leads to increased E. coli uptake, whereas calcium chelation decreased intracellular E. coli load. Understanding the membrane trafficking pathways is critical for understanding how macrophages clear invading pathogens

A rare case of paraneoplastic neurological syndrome with ovarian teratoma

Paraneoplastic neurological syndrome (PNS) is a neuropathological disorder of central nervous system, in this the patient presents with a tumor anywhere in the body with diverse neuropsychiatric symptoms. The clinical manifestation of the tumor may be preceded by PNS. We are reporting a case of a 23-year-old married nulliparous female who came to the gynaecology outpatient department (OPD) with the history of mass per abdomen for the past four months and also undergoing treatment for acute psychosis, memory loss and cognitive impairment for the past five months. Her symptoms were not relieved on treatment. After clinical, laboratory and radiological evaluation possibility of paraneoplastic neurological syndrome associated with ovarian teratoma was made. Patient was taken up for staging laparotomy with U/L salpingoophrectomy. After surgical treatment her psychiatric symptoms rapidly improved and her psychiatric medications doses was reduced. PNS should be suspected in female patients presenting with an acute history of neuropsychiatric symptoms. Tumor resection should be performed at the earliest to improve patient outcomes

Iron regulates contrasting toxicity of uropathogenic <i>Escherichia coli</i> in macrophages and epithelial cells

By far most urinary tract infections are caused by genetically diverse uropathogenic Escherichia coli (UPEC). Knowledge of the virulence mechanisms of UPEC is critical for drug development, but most studies focus on only a single strain of UPEC. In this study, we compared the virulence mechanisms of four antibiotic-resistant and highly pathogenic UPEC isolates in human blood monocyte-derived macrophages and a bladder epithelial cell (BEC) line: ST999, ST131, ST1981 and ST95. We found that while non-pathogenic E. coli strains are efficiently killed by macrophages in bactericidal single membrane vacuoles, the UPEC strains survive within double-membrane vacuoles. On side-by-side comparison, we found that whereas ST999 only carries Fe3+ importers, ST95 carries both Fe2+ and Fe3+ importers and the toxins haemolysin and colibactin. Moreover, we found that ST999 grows in the Fe3+ rich vacuoles of BECs and macrophages with concomitant increased expression of haem receptor chuA and the hydrogen peroxide sensor oxyR. In contrast, ST95 produces toxins in iron-depleted conditions similar to that of the urinary tract. Whereas ST95 also persist in the iron rich vacuoles of BECs, it produces colibactin in response to low Fe3+ contributing to macrophage death. Thus, iron regulates the contrasting toxicities of UPEC strains in macrophages and bladder epithelial cells due to low and high labile iron concentrations, respectively

T cell cholesterol efflux suppresses apoptosis and senescence and increases atherosclerosis in middle aged mice

Atherosclerosis is a chronic inflammatory disease driven by hypercholesterolemia. During aging, T cells accumulate cholesterol, potentially affecting inflammation. However, the effect of cholesterol efflux pathways mediated by ATP-binding cassette A1 and G1 (ABCA1/ABCG1) on T cell-dependent age-related inflammation and atherosclerosis remains poorly understood. In this study, we generate mice with T cell-specific Abca1/Abcg1-deficiency on the low-density-lipoprotein-receptor deficient (Ldlr-/-) background. T cell Abca1/Abcg1-deficiency decreases blood, lymph node, and splenic T cells, and increases T cell activation and apoptosis. T cell Abca1/Abcg1-deficiency induces a premature T cell aging phenotype in middle-aged (12-13 months) Ldlr-/- mice, reflected by upregulation of senescence markers. Despite T cell senescence and enhanced T cell activation, T cell Abca1/Abcg1-deficiency decreases atherosclerosis and aortic inflammation in middle-aged Ldlr-/- mice, accompanied by decreased T cells in atherosclerotic plaques. We attribute these effects to T cell apoptosis downstream of T cell activation, compromising T cell functionality. Collectively, we show that T cell cholesterol efflux pathways suppress T cell apoptosis and senescence, and induce atherosclerosis in middle-aged Ldlr-/- mice

Combined Targeted Omic and Functional Assays Identify Phospholipases A₂ that Regulate Docking/Priming in Calcium-Triggered Exocytosis

The fundamental molecular mechanism underlying the membrane merger steps of regulated exocytosis is highly conserved across cell types. Although involvement of Phospholipase A2 (PLA2) in regulated exocytosis has long been suggested, its function or that of its metabolites&#8212;a lyso-phospholipid and a free fatty acid&#8212;remain somewhat speculative. Here, using a combined bioinformatics and top-down discovery proteomics approach, coupled with lipidomic analyses, PLA2 were found to be associated with release-ready cortical secretory vesicles (CV) that possess the minimal molecular machinery for docking, Ca2+ sensing and membrane fusion. Tightly coupling the molecular analyses with well-established quantitative fusion assays, we show for the first time that inhibition of a CV surface calcium independent intracellular PLA2 and a luminal secretory PLA2 significantly reduce docking/priming in the late steps of regulated exocytosis, indicating key regulatory roles in the critical step(s) preceding membrane merger

Phospholipase A2 : potential roles in native membrane fusion

Membrane fusion is a fundamental molecular mechanism by which two apposed membrane bilayers coalesce in rapid, transient steps that enable the successive merging of the outer and inner leaflets allowing lipid intermixing and subsequent mixing of the two previously separate compartments. The actual membrane merger mechanism – fusion, by definition – is conceptualized to be protein- or lipid-centric. According to the widely vetted stalk-pore hypothesis, membrane fusion proceeds via high curvature lipid intermediates. By cleaving membrane phospholipids at the sn-2 position, Phospholipase A2 generates metabolites that exert spontaneous curvature stress (both negative and positive) on the membrane, thus influencing local membrane bending by altering the packing and conformation of lipids and proteins, respectively. Such changes could potentially modulate priming and attachment/docking steps that precede fusion, as well as the membrane merger steps per se

Arachidonic acid and lysophosphatidylcholine inhibit multiple late steps of regulated exocytosis

The canonical Phospholipase A2 (PLA2) metabolites lysophosphatidylcholine (LPC) and arachidonic acid (ARA) affect regulated exocytosis in a wide variety of cells and are proposed to directly influence membrane merger owing to their respective spontaneous curvatures. According to the Stalk-pore hypothesis, negative curvature ARA inhibits and promotes bilayer merger upon introduction into the distal or proximal monolayers, respectively; in contrast, with positive curvature, LPC has the opposite effects. Using fully primed, release-ready native cortical secretory vesicles (CV), well-established fusion assays and standardized lipid analyses, we show that exogenous ARA and LPC, as well as their non-metabolizable analogous, ETYA and ET-18-OCH3, inhibit the docking/priming and membrane merger steps, respectively, of regulated exocytosis