Periodic Reporting for period 1 - LYSOKIN (Architecture and regulation of PI3KC2β lipid kinase complex for nutrient signaling at the lysosome)
Okres sprawozdawczy: 2020-07-01 do 2022-06-30
Communication is paramount for the normal function of cells, which must relay many messages from the outside environment and within the cell to determine their actions. When communication networks break down, this can lead to human disease. One of the messages cells require is whether they have enough nutrients and should grow and multiply, or whether they should conserve resources to survive when nutrients are low. One of the ways the cell can conserve resources is through recycling molecules into their building blocks. The detection of low nutrients and the breaking down of molecules is coordinated by machinery in the cell that is situated at the cell “recycling center”, known as the lysosome. When nutrients are low, a protein called PI3KC2B moves to the lysosome and produces a lipid molecule [PI(3,4)P2] that switches off a multi-protein complex, mTORC1. How the function and location of PI3KC2B in the cell is controlled is currently not well understood. Previous data have shown that PI3KC2B interacts with mTORC1 and other lysosome resident proteins. Improper function of the lysosome leads to many human diseases such as cancer, diabetes, and neurological dysfunction, so understanding how the lysosome coordinates these functions is crucial to identifying new therapies for these diseases. To better understand how PI3KC2B and mTORC1/other proteins work in concert to control lysosome function, we set out to map the interfaces of the complexes formed between these proteins using the 2017 Nobel Prize winning technique cryo electron microscopy in combination with cellular imaging to investigate how interrupting these relationships in cells affects lysosome function and cellular communication. To date, we have identified a new interaction partner of PI3KC2B and found new aspects of how the structure of PI3KC2B mediates its own function. the knowledge we gain from this research will allow a better understanding of the lysosome. Over the next two years, we aim to complete our investigation of how PI3KC2B interacts with mTORC1 and other proteins to communicate nutrient availability within the cell.
The work performed during the project aimed to identify the precise relationship between PI3KC2B and mTORC1 or other proteins and how these relationships were important for the communication of nutrient availability at the lysosome. This work was split into three key areas: Using biochemical and proteomic methods to characterise interactions, using electron microscopy methods including cryo EM to define the interface between PI3KC2B and other proteins in depth, and using cell biology and biochemistry techniques to understand the biological outcome of these relationships. Our biggest findings thus far include identifying a new interaction partner of PI3KC2B and alternate ways that PI3KC2B can be self-regulated. Using a mass spectrometry screen on cells expressing different variants of PI3KC2B, we were able to identify a new interaction partner of PI3KC2B. This interaction was characterised using biochemical assays and a specialist technique called hydrogen-deuterium exchange mass spectrometry (HDX-MS), which can map interfaces between proteins and structural changes in the proteins that occur when they interact. Proteins are like machines, that often undergo movements to control their function and using HDX-MS to identify these movements can lead to a better understanding of how the proteins functions are regulated. To date, we have made progress in all three areas and will continue this work over the next two years.
The progress in this project has led to the transfer of knowledge of new techniques, reagents, and resources in the host lab and the research campus in general. Establishment of HDX-MS facility has also led to many collaborations within the research campus and the German scientific community. The results from this project have also instigated a new line of research in the host lab, and the manuscripts that come from this project will be of great benefit for the broader field both in the biological implications and the methodology.