Periodic Reporting for period 3 - ATG9_SOLVES_IT (In vitro high resolution reconstitution of autophagosome nucleation and expansion catalyzed byATG9)
Reporting period: 2021-07-01 to 2022-12-31
Human health and lifespan depends on the ability of the individual cells in each of the organs which make up our body to keep themselves working properly. This means each cell itself must be clean, free of contaminats and infections and fit. To do this it removes damaged or damaging parts, (proteins for example can become damaged and clump together in an damaging ball), as well as removing invaders such as viruses or bacteria (pathogens). The cell uses a tool called autophagy (self-eating) to keep itself clean. This tool, autophagy, works as a waste disposal system, finding garbage (balls of damaged proteins or pathogens) and breaking them down to dipose of them inside itself, hence "self-eating". Autophagy starts when it gets a signal alerting it to the presence of damaged part or invaders, and targets these damaged proteins or pathogens for elimination. The damaged proteins or pathogens are engulfed by membrane sacs or vesicles, called autophagosomes, and these autophagosomes deliver the eaten material to the cell’s waste disposal system. the lysosome (see Figure). Autophagy is needed for survival in humans, other mammals like mice, worms, fish and even bakers yeast showing how important it is to many living creatures. Its important role in human health is revealed when it doesn’t work. Diseases caused by the accumulation inside the cells of damaged proteins, for example in brain cells, give rise to Alzheimers disease, Parkinsons disease and other neurodegenerative diseases are a major global concern. Thus, it is of fundamental importance we understand how autophagy works.
The membrane sac, the autophagosome, is made up of lipids (oily molecules which are easy shaped into sacs or vesicles), and proteins which give the vesicle an identity, a bar codes or information on what the vesicle should and can do). The autophagosome forms and grows by obtaining both lipids and proteins from other vesicles coming from other parts of the cell. These vesicles contact the autophagosome formation site and deliver the necessary lipids and proteins. This type of delivery enables the whole cell to pool its resources to build an autophagosome after it gets an alert to damage or invasion. Our data show that vesicles carry a protein called ATG9A are amongst the most important delivery vesicles. However, we have not yet understood the function of ATG9A. It is a multi-spanning membrane protein which means large parts of it are buried in the lipid of the vesicle making it particularly hard to work with.
The ERC project “ATG9_SOLVES_IT” aims to understand how ATG9A works, and thus provide a way forward to better understand how autophagosomes are formed, and how autophagy works. This will benefit society as we will provide information for clinicians, industry and others to develop ways of modulating autophagy to improve disease and infection.
The overall objectives to reach this goal of developing knowledge for eventual translational science are: 1) to elucidate the function of ATG9, and 2) to understand how the autophagosome forms. The first objective is being addressed by identifying the composition of ATG9 vesicles (both proteins and lipids) and determining the activity of the components. In line with this aim we are isolating and determining the structure of ATG9 to get a handle of how it works. The second objective is being approached in two ways, first by isolating and characterizing the autophagosome vesicles before the ATG9 vesicle reaches it, this we call the initiation site, and secondly by using the information about initiation site to build an artificial system capable of replicating the vents in the cells so we can better understand how it works.
The membrane sac, the autophagosome, is made up of lipids (oily molecules which are easy shaped into sacs or vesicles), and proteins which give the vesicle an identity, a bar codes or information on what the vesicle should and can do). The autophagosome forms and grows by obtaining both lipids and proteins from other vesicles coming from other parts of the cell. These vesicles contact the autophagosome formation site and deliver the necessary lipids and proteins. This type of delivery enables the whole cell to pool its resources to build an autophagosome after it gets an alert to damage or invasion. Our data show that vesicles carry a protein called ATG9A are amongst the most important delivery vesicles. However, we have not yet understood the function of ATG9A. It is a multi-spanning membrane protein which means large parts of it are buried in the lipid of the vesicle making it particularly hard to work with.
The ERC project “ATG9_SOLVES_IT” aims to understand how ATG9A works, and thus provide a way forward to better understand how autophagosomes are formed, and how autophagy works. This will benefit society as we will provide information for clinicians, industry and others to develop ways of modulating autophagy to improve disease and infection.
The overall objectives to reach this goal of developing knowledge for eventual translational science are: 1) to elucidate the function of ATG9, and 2) to understand how the autophagosome forms. The first objective is being addressed by identifying the composition of ATG9 vesicles (both proteins and lipids) and determining the activity of the components. In line with this aim we are isolating and determining the structure of ATG9 to get a handle of how it works. The second objective is being approached in two ways, first by isolating and characterizing the autophagosome vesicles before the ATG9 vesicle reaches it, this we call the initiation site, and secondly by using the information about initiation site to build an artificial system capable of replicating the vents in the cells so we can better understand how it works.
To date we have: 1) refined our analysis of ATG9 vesicles and are examining additional proteins components, 2) determining the lipid composition of the Atg9 vesicles, 3) purifying ATG9 from human cells and started to collect high resolution data, 4) identified an interaction of ATG9 with ATG2 which revelas regulation of ATG9 function,5) developed labelling techniques to label the initiation site within a rapid time course, 6) developed a system to create artifical vesicles (GUVs) and bind key proteins required to grow the autophagosome.
The progress we have made so far is state of the art biochemistry and protein production and purification. We aim to go beyond the state of the art with high resolution microscopy of the ATG9 proteins and initiation site. Development of an artificial system is also currently using state of the art systems, and to go beyond we are exploring unique lipid compositions which have not so far been used in reconstitution systems but most likely will mimic the real membrane more accurately than the existing ones.