Elucidating the transcriptional and post-translational systems that regulate snake venom assembly and that control autolysis and self-toxicity

Viper venom consists of a mixture of nature's most tissue-destructive proteins. These enzymes cause rapid and catastrophic cardiovascular collapse in envenomed rodent prey and, in humans, life-threatening bleeding and non-clotting blood. Yet while this lethal toxin cocktail is stored in the snake's venom gland it is somehow controlled so that the snakes' own tissues are unaffected. This extraordinary biological phenomenon is very poorly understood but is clearly central to the successful evolutionary development of venom as a predation and defensive/offensive survival strategy for venomous snakes. The control of viper protein toxicity has been very little studied because the appropriate gene and protein identification technologies have only recently become available. Previously, using basic biochemical techniques we and others have shown that one group of venom enzymes are kept in a quiescent state until activated by other, unknown, venom enzymes in an equally unknown time frame. It is also believed venom contains inhibitors to three key viper venom toxins but this has yet to be experimentally verified. We have recently developed expertise and resources in the application of new high throughput methods to identify new viper venom genes. Here, in the first study of its kind, we aim to provide the first holistic view of how sets of toxin and toxin-regulating venom gland genes are orchestrated during venom synthesis. Assisted by new gene and protein quantification technologies, we will first identify the genes and their protein products that constitute a model viper venom. Next, we will monitor the changes in the levels of these genes and proteins at several intervals spanning the time from when the snake ejects venom to the time it re-synthesises a fully competent arsenal of new venom. Using computational techniques, we will distinguish between toxins and proteins predicted to regulate their toxic activity. We will use molecular techniques to identify whether the genetic machinery controlling the production of (i) the toxins and (ii) the toxin-regulatory proteins, are co-ordinated to ensure that the regulatory proteins are produced before the respective toxin. The results of this study will greatly improve our understanding of the dynamics and the fundamental biological principles regulating venom assembly and venom toxicity.

Venom has evolved as a predation/defence strategy in many animal species. In snakes, a complex arsenal of enzymatic toxins is synthesised and maintained for some time (weeks) in a functionally inert state; non-injurious to the snake but whose toxicity can be instantly exerted when required for predation or defence. Our nearly complete lack of understanding of the molecular processes regulating initial venom synthesis and suppression of venom toxicity is a clear gap in our understanding of this fundamental evolutionary adaptation. Advances in molecular and proteomic technologies offer a timely opportunity to address this knowledge gap. We have recent data showing that venom protein profiles differ significantly over time during synthesis and that viper venom contains high concentrations of inhibitors not previously described. This data strongly indicates that venom synthesis is tightly regulated and involves coordinated expression of distinct toxin and inhibitor combinations. We will use the puff adder as a model system. We will first define the mature venom proteome by characterising the processed N-termini of each secreted venom component and correlate this data with the respective cDNA precursors acquired from EST sequencing of existing venom gland cDNA libraries. We will next analyse samples of venom during re-synthesis, using initially using SDS-PAGE, then iTRAQ and qPCR, to identify toxins and toxin-regulating proteins that cluster into groups showing early, intermediate, late or constitutive expression profiles. Focussing on co-ordinated or differentially expressed genes encoding toxin and toxin-regulating proteins, we will perform molecular and bioinformatic analysis of promotors to predict the elements responsible for regulating their transcription. The results will define the operational control of this complex biological system and provide the foundation for future studies ranging from protein-protein interactions to predatory-prey behavioural strategies.