For some decades, cone snail venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in venoms of mollusks. In order to shed more light on cone snail venom HMM components, the proteins of dissected and injected venom of a fish-hunting cone snail, Conus consors, were extensively assessed. HMM venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the venom duct and assigned a biological function, thus pointing to their potential role in venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups of HMM proteins passing the venom selection process – echotoxins and hyaluronidases – were clearly present in the injected venom. They are suggested to contribute to the envenomation process. This newly devised integrated HMM proteomic analysis is a big step towards identification of the protein arsenal used in a cone snail venom apparatus for venom production, maturation and function.
COBISS.SI-ID: 26042407
Presynaptically neurotoxic phospholipases A2 (PLA2) inhibit synaptic vesicle recycling through endocytosis. Here we provide insight into the action of a presynaptically neurotoxic PLA2 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. We 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 PLA2s work can open new ways to regulate endocytosis.
COBISS.SI-ID: 5026074
APS12-2, a non-competitive acetylcholinesterase inhibitor, is a synthetic analog of polymeric alkylpyridinium salts (poly-APS). APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC(50)=0.74 μM). The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1(2)β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC(50)=0.0005 μM), indicating a higher affinity of the compound for Torpedo (α1(2)β1γδ) than for the mouse (α1(2)β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction.
COBISS.SI-ID: 3587706
Listeriolysin O (LLO) is the major factor implicated in the escape of the intracellular pathogenic bacterium, Listeria monocytogenes, from the phagolysosome. Despite intense studies of LLO pH-dependence, this feature of the toxin still remains incompletely explained. We showed that the structure and membrane permeabilizing activity of LLO is not significantly affected by pH. However, alkaline pH and elevated temperatures caused rapid denaturation of LLO, and appearance of the toxin aggregates resembling amyloids. We therefore suggest that LLO spontaneously aggregates at the neutral pH found in the host cell cytosol and that this is a major mechanism of LLO inactivation.
COBISS.SI-ID: 4881690
The role of secreted phospholipase A2 (sPLA2) enzymatic activity, including arachidonic acid (AA) release, in the induction of motoneuronal apoptosis has been studied by ammodytoxin A (AtxA) and homologous recombinant sPLA2s with different enzymatic properties. We analysed the effects of an AtxA mutant (V31W) with very high enzymatic activity, enzymatically inactive S49-sPLA2 (ammodytin L, AtnL), its mutant (LW) with restored enzymatic activity, and non-toxic, enzymatically active sPLA2 (AtnI2). Addition of AA, AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, to motoneuronal cells resulted in caspase-3 activation, DNA fragmentation and disruption of mitochondrial membrane potential, leading to a significant and rapid decrease in motoneuronal cell viability that was not observed in (control) mouse myoblast and human embryonic kidney cells. AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, also liberated large amounts of AA specifically from motoneuronal cells, and this ability correlated well with the ability to induce apoptotic changes and decrease cell viability. The enzymatic activity of AtxA and similar sPLA2s is thus necessary, but not sufficient, for inducing motoneuronal apoptosis. These results suggests that specific binding to the motoneuronal cell surface, followed by internalization and enzymatic activity-dependent induction of apoptosis, possibly as a consequence of both extensive extra- and intracellular AA release, is necessary for Atx-induced motoneuronal cell death.
COBISS.SI-ID: 26387239