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

Abstract

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—a lyso-phospholipid and a free fatty acid—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.

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion

“…This Rab family 1 motif is largely conserved (>50%) among the Rab GTPases and across different species (Table 2), and this effector domain peptide has been previously shown to interfere with the formation of the fertilization envelope in urchin oocytes [47]. An addition of exogenous RAB peptide inhibited the extent of fusion by~63% in the modified settle assay, compared to~46% in the standard assay; this strongly indicates an effect on upstream stages of CV tethering/docking/priming [1][2][3]6,8,30,39]. With the addition of the RAB effector domain, there was no effect on Ca 2+ -sensitivity; however, after pretreatment with IA, treatment with the RAB peptide resulted in a decrease in Ca 2+ -sensitivity, mitigating the potentiation effect of IA.…”

“…Along with cholesterol-and sphingomyelin-enriched membrane domains [8][9][10][11]45,46], other lipid species, such as phosphoinositides, participate in the organization of the exocytotic machinery at release sites [4]. The generation of phospholipid metabolites from phospholipase D [3] and phospholipase A [2] can also modulate the efficiency of exocytosis via roles in upstream stages, such as docking/priming. Here, we have identified two labeled enzymes involved in lipid metabolism, long-chain-fatty-acid-CoA ligase 1 and long-chain specific acyl-CoA dehydrogenase, which may have roles in regulating local fatty acid metabolism in secretory vesicles.…”

“…Membrane fragments were isolated by centrifugation at 120,000× g. Cholesterol-enriched membrane fragments were further fractionated by density centrifugation on sucrose gradients (40%/30%/22%/10% sucrose, 25 mM Tris; pH 7), collected from the 22%/10% interface, washed in 25 mM Tris buffer and then isolated by centrifugation at 120,000× g. Membrane pellets were solubilized in 2DE sample buffer (8 M urea, 2 M thiourea, 4% CHAPS), supplemented with the broad-spectrum protease inhibitor cocktail, and protein concentration was determined using the EZQ Protein Quantitation Kit (ThermoFisher). Membrane proteins were resolved by well-established, optimized 2DE protocols, with slight modifications [2,8,40,50]. In the first dimension, proteins were resolved on immobilized nonlinear pH 3-10 gradients (3-10 NL IPG; Bio-Rad).…”

“…This involves the coordinated actions of both lipid and protein components of the secretory vesicle membrane, particularly the later steps of Ca 2+ -triggered exocytosis. There have been recent advances in understanding how the lipid matrix may modulate upstream docking/priming steps [1][2][3][4][5][6][7], regulate the Ca 2+ sensitivity and efficiency of fusion [5][6][7][8][9][10][11], and facilitate or impede the merger of biological membranes [1,6,7,9,[12][13][14]. However, there remains debate regarding the identity of proteins involved in the Ca 2+ -sensing and triggering steps that regulate the efficiency of exocytosis.…”

Proteomics of Multiple Sclerosis: Inherent Issues in Defining the Pathoetiology and Identifying (Early) Biomarkers

“…However, an independent follow-up study showed that the cleavage product was formed by degradation of the first eight N-terminal residues and was attributable to the inappropriate storage of samples (i.e., at −20 °C rather than −80 °C; [ 186 ]). Most importantly, the use of broad-spectrum inhibitor cocktails (e.g., containing protease, kinase, and phosphatase inhibitors) is essential to achieve high-confidence analyses that reflect the native proteome at the time of sampling [ 104 , 135 , 187 ]. Proteolysis in vitro is a nonspecific alteration of the native proteome being analysed [ 188 ] and the addition of broad-spectrum protease inhibitors is thus essential to block the degradation of proteoforms [ 189 ].…”