A Neurotoxic Phospholipase A2 Impairs Yeast Amphiphysin Activity and Reduces Endocytosis

Abstract

BackgroundPresynaptically neurotoxic phospholipases A2 inhibit synaptic vesicle recycling through endocytosis.Principal FindingsHere we provide insight into the action of a presynaptically neurotoxic phospholipase A2 ammodytoxin A (AtxA) on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin.ConclusionsWe identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein–protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A2 work can open new ways to regulate endocytosis.

Molecular Characterization of Lys49 and Asp49 Phospholipases A2 from Snake Venom and Their Antiviral Activities against Dengue virus

“…This is a topic that is under investigation by us. A model of the molecular mechanism of action of the neurotoxic svPLA 2 ammodytoxin (AtxA) from Vipera ammodytes ammodytes , which includes specific protein-protein interaction, and enzymatic activity has been recently reported [27]. …”

Cellular Mechanisms of Action of Snake Phospholipase A2 Toxins

“…Bulges are sites on exo-endocytic unbalance (demonstrated by accumulation of the synaptic markers VAMP2 and synaptophysin I and by the permanent exposure on the plasma membrane of a luminal epitope of the vesicular protein synaptotagmin I) (Rigoni et al 2004). A direct interaction of ammodytoxin (Atx, a group II sPLA2 from Vipera ammodytes) with the endocytic protein amphiphysin has been recently shown in yeast (Mattiazzi et al 2012). Once bound to the nerve terminal, snake PLA2 neurotoxins begin to hydrolyze phospholipids of the external layer, and this hydrolysis leads to a rapid accumulation of lysophospholipids and fatty acids.…”

Neurotoxic phospholipase A2toxicity model

“…This results in suppression of the release of acetylcholine from neurons into the synaptic cleft, causing irreversible blockade of neurotransmission to the postsynaptic muscle cell 1 . We have recently proposed a model of the molecular mechanism of action of the neurotoxic sPLA 2 ammodytoxin A (AtxA) 2 . It is based on the experimental demonstration that the AtxA-induced reduction in endocytosis is a consequence of: (1) the specific intracellular interaction of AtxA with 14-3-3 proteins, which enables the toxin molecules to concentrate on the plasma membrane near the endocytic vesicles; and of (2) the PLA 2 enzymatic activity of AtxA, which changes the shape and/or composition of the presynaptic membrane, thus inhibiting the activity of the endocytic protein amphiphysin 2 .…”

“…We have recently proposed a model of the molecular mechanism of action of the neurotoxic sPLA 2 ammodytoxin A (AtxA) 2 . It is based on the experimental demonstration that the AtxA-induced reduction in endocytosis is a consequence of: (1) the specific intracellular interaction of AtxA with 14-3-3 proteins, which enables the toxin molecules to concentrate on the plasma membrane near the endocytic vesicles; and of (2) the PLA 2 enzymatic activity of AtxA, which changes the shape and/or composition of the presynaptic membrane, thus inhibiting the activity of the endocytic protein amphiphysin 2 . The study was, however, performed in yeast, making the validity of the proposed model for mammalian cells a pivotal question.…”

“…A critical control in establishing the model in yeast was the demonstration that the introduction, by genetic engineering, of PLA 2 enzymatic activity into AtnL generated a protein capable of reducing endocytosis in the same way as AtxA 2 . This experiment provided the crucial proof for the indispensable role of PLA 2 activity in the reduction of endocytosis by AtxA in yeast.…”