New approaches to modulate VPS34 PI 3-kinase activity

Phosphoinositide 3-kinases (PI3Ks) regulate signalling and vesicular trafficking [1-3]. Mammals have eight PI3K isoforms divided in three classes. Class I PI3Ks, frequently mutated in different pathologies [4-6], are the most studied PI3Ks and regulate signalling downstream of membrane receptors and monomeric small GTPases. In contrast, class II and III are involved in vesicular trafficking and their signalling roles have recently started to be uncovered. VPS34, the sole class III PI3K member, is found in different protein complexes that regulate autophagy, endosomal sorting, phagocytosis and signalling [7]. VPS34 functions have been studied using approaches that deplete VPS34.Those do not distinguish its scaffolding versus kinase-dependent functions, and result in VPS34-associated protein depletion, which has confounded the interpretation of the phenotypes. The aim of this study was to develop different ways of modulating VPS34 activity using two models. First, using a genetic approach, the host laboratory developed a knock-in strategy to inactivate VPS34 lipid kinase activity in mice while preserving its scaffolding function. This was a theoretical targeting approach based on VPS34 protein structure, but the resulting knock-in allele remained functionally uncharacterised. Using mammalian cells, I characterise this truncated version of VPS34 whereby exon21 is deleted and showed that is expressed and lacks lipid kinase activity. The second approach was using the nanobodies’ properties to stabilise VPS34 complexes in different conformational states to obtain VPS34 complex-specific binders, stabilizing them in active or inactive state. This is particularly relevant since pharmacological inhibition of VPS34 kinase activity disables the functions of all VPS34 complexes. Here, by overexpression in mammalian cells, I characterise nanobodies that can bind different VPS34 complexes. I focus on CI60, a VPS34 complex I-specific binder nanobody and uncover its potential as research tool to reveal unknown complex I functions and as autophagy inducer, although further experiments are required to fully address this function