Regulation of autophagy induction by the Vps34 complex: structural and functional studies.

This work will determine the mechanism by which the lipid kinase Vps34 and its lipid product PI3P regulate autophagy. Autophagy (from the Greek self-eating) is a way for cells to generate nutrients from their own material during times of starvation. The pathway of autophagy is very important for the health of an organism for several reasons. Autophagy underpins the normal physiology of all cell types, and is a critical contributor to life-span extension. Cellular components that are damaged become substrates for autophagy and are efficiently eliminated. This leads to a periodic clean-up of the cell interior and allows for regeneration of important proteins and organelles. Of note, when autophagy is suppressed cells exhibit signs of oxidative damage because their dysfunctional mitochondria cannot be removed and continue to produce reactive oxygen species. Similarly, suppression of autophagy contributes in a significant way to the build-up of mutant aggregate-prone proteins (which are normally substrates of autophagic degradation) that cause neuro-degenerative disorders. Autophagy is also critical for the neo natal period: animals that cannot mount autophagy die soon after birth because they cannot generate nutrients from their own resources during a period where they do not obtain nutrients from the outside.
The pathway of autophagy starts when a novel double membrane vesicle called autophagosome is formed in the cell interior. These autophagosomes (which can be hundreds in number at any one time) enclose cellular material and deliver it to a degradative organelle called lysosome for digestion. There is currently high interest in finding ways to enhance or suppress autophagy for various health-related reasons and the best place to start is to undesratnd how autophagosomes are formed.
Our previous work has shown that one of the signals for formation of autophagosomes is the synthesis of a lipid called PI3P by the activity of the Vps34 enzyme. This results in the formation of a series of intermediate structures that eventually give rise to an autophagosome. However exactly how this happens and how the intermediate structures eventually lead to an autophagosome is a mystery and will be tackled by our proposed work.
We will combine expertise in cell biology and structural biology in order to find the structure of the Vps34 comlex that regulates the start of autophagy, and we will use biochemistry and microscopy to describe in detail the pathway that leads from PI3P to autophagosome formation.

Autophagy, a critical component of nutrient sensing in all eukaryotic cells, is induced when the protein kinase mTOR is inactivated under low nutrients. As a consequence, novel double membrane vesicles termed autophagosomes are formed, which engulf cytosolic material and deliver it to lysosomes for degradation. Two protein complexes regulate the induction of autophagy. One consists of the lipid kinase Vps34 (and its regulatory subunits), which synthesises PI3P whereas the other consists of the protein kinase ULK1 and its associated proteins. For at least some types of autophagosomes the Vps34/PI3P requirement leads to formation of omegasomes, membrane extensions of the endoplasmic reticulum (ER) enriched in PI3P within which autophagosomes form. In addition to its essential role during autophagy, Vps34 and effectors of its lipid product regulate a number of cellular pathways including endosomal sorting and fusion, phagocytosis and cytokinesis. Our labs at Babraham (NTK) and the LMB (RLW) have been working on several aspects of the biochemistry and cell biology of Vps34, and our work has highlighted important properties of the protein including the discovery of omegasomes and the elucidation of the first crystal structure of Vps34. Based on recent unpublished work we hypothesise that activation of Vps34 during autophagy depends on its binding partners and on the physical shape of the membranes on which it acts. We will explore this in a collaboration between the two labs in order to understand how Vps34 leads to formation of early autophagy intermediates. Our specific objectives are: (1) Determine the 3-dimensional structure of the Vps34 autophagy complex and its dynamic regulation. (2) With the pure complex at hand, determine how it responds to and generates membrane curvature. (3) Reconstitute omegasome-related structures on liposomes and in perforated cells using purified proteins. (4) Exploit our findings in intact yeast and in mammalian cells.

Impact summary
Our results will impact two scientific communities: one engaged in the study of autophagy in a variety of organisms, and a broader scientific community concerned with regulation of cell decisions by lipid signals. For both of these communities, our work will provide important insights, which can then be used to model a functional cell and to try to predict its behavior under a variety of inputs.

Methods of studying protein complexes on membranes
An element of our work that will benefit a wide circle of scientists has to do with our development of strategies for combining HDX-MS, cross-linking MS, hydroxyl radical footprinting MS, and X-ray crystallography for understanding the structures and dynamics of activation and inhibition of large complexes working at the membrane surface. The work on Vps34 Complex I is the largest complex that has been studied by this hybrid approach, and the work outlined in the proposal will lead to more robust methods and automation that will help to make it a routine approach that is commonly applied. This will provide the UK with an internationally recognized edge in protein membrane biology and will facilitate work on a vast network of protein complexes functioning at membrane interfaces.

Pharmaceutical development
A direct beneficiary of our work will be pharmaceutical companies investigating ways to alter autophagy for health benefit. To our direct knowledge, several large pharmaceutical companies are currently engaged in developing drugs that will enhance or inhibit autophagy. The attractiveness of targeting autophagy is that it appears to modulate a myriad of health conditions, from cancer and infections to neural degeneration and healthy ageing. The Vps34 protein is central to the initiation of autophagy, and it belongs to a family of lipid kinases (the PI 3-kinases) for which various drugs are in clinical trials. Therefore, targeting it for health benefit does not represent a leap of faith but a road already taken. However, as was made clear in our proposal, targeting of Vps34 has been difficult due to the geometry of its active site and its complicated job description within cells. Our work will solve some of these issues and will provide critical insights on best ways (and sites) for targeting Vps34. More specifically, we will be able to understand how the activity of Vs34 is modified by the presence of its protein partners and lipid membranes. This could provide a basis for developing a new generation of Vps34 inhibitors that target regions of the enzyme that undergo conformational changes upon activation by highly curved membranes.

Healthy ageing
We wish to further highlight how work on autophagy modulators can enhance healthy ageing, a major current focus of the BBSRC and a critical component of the future economic competitiveness of the UK. Autophagy removes protein aggregates that can lead to dementia, and genetic inhibition of autophagy in mouse models leads to an increase in protein aggregates. In addition, enhancing autophagy via inhibition of mTOR with rapamycin has been shown to increase lifespan in mammals. The work described in this proposal could guide strategies to stimulate autophagy in anti-ageing and anti-dementia applications by modulating the activity of Vps34. Indeed, the structural basis for the stimulated activity of the Vps34 complex on curved membranes could help inform these efforts.

Autophagy in infectious diseases
Autophagy can augment the degradation of bacteria and viruses. As such it has an important role in the innate immune defences against infections diseases. Some pathogens such as picornaviruses stimulate the production of autophagosomes as an essential part of their life cycle. The work outlined in this proposal may help understand how pathogens are able to modify sorting machinery to circumvent the protective role of autophagy.