Structural proteomics surveys protein instability in cancer
- Reference number
- ICA16-0023
- Start and end dates
- 170901-210831
- Amount granted
- 4 000 000 SEK
- Administrative organization
- Karolinska Institutet
- Research area
- Life Sciences
Summary
Virtually all cellular processes are mediated by interactions between natively folded proteins. Consequently, aberrant interactions that arise when proteins lose their correct fold and self-assemble into insoluble aggregates can lead to failure of entire regulatory networks, as recently shown for aggregation of the tumour suppressor p53. However, the difficult chemical nature of the underlying interactions makes structural studies notoriously challenging and has hampered the development of drugs that target instable tumour suppressors. This proposal describes the development and application of a mass spectrometry (MS) toolbox for the study of molecular and cellular effects of protein aggregation in cancer. Firstly, I propose to use native MS to compare the stabilities and interactions of the natively unstable p14ARF and p53 carrying destabilizing mutations. Secondly, I will use hydrogen/deuterium exchange, which measures the structure-dependent incorporation of chemical labels into the protein, to reveal aggregation-prone regions and target sites for chemical stabilization. Thirdly, the stabilizing effect of interactions between native proteins and intracellular aggregates will be exploited using cellular thermal shift assays (CETSA) and MS proteomics to reveal the interactome of misfolded proteins in cancer. This project will provide new insights into the role of protein aggregation in cancer and help to establish previously unavailable structural MS in Sweden.
Popular science description
Proteins are composed of amino acid long chains that fold into specific shapes in an origami-like fashion. Once they are folded correctly, they are able to perform specific tasks inside to cell to ensure its integrity and function. However, some proteins are unstable and can also fold incorrectly, which causes them to stick together and form non-functional bundles instead. These bundles act like flycatchers for other proteins, effectively preventing them from performing their tasks, and eventually resulting in the collapse and death of the entire cell. This is particularly problematic if proteins that control cell growth and self-repair are affected, as the breakdown of their functions leads to uncontrolled cell growth and cancer. The aim of this project is to find ways to identify and capture defect proteins and restore the growth control mechanism that prevent cancer from developing. This requires detailed knowledge about how a protein folds the wrong way, and how this affects the way the protein performs its task. The key to this information is mass spectrometry (MS), a technique that measures the exact weight of individual molecules. It allows us to monitor how defect proteins attach to each other (as this increases their weight), and whether small molecules prevent this from happening. In this manner, we can compare the ability of different drugs to catch wrongly folded proteins. To find out what parts of the protein are defect, the surface of the protein can be labelled, i.e. chemically painted, so that the areas that form contacts with other proteins or drugs will remain label-free. By cutting the protein into small pieces and measuring their weight by mass spectrometry, it is then possible to map how each part of the protein is folded. This technique, called HDX-MS, provides a detailed picture of the contacts between defect and intact proteins and drugs. We then test how the accumulation of defect proteins affects other proteins inside the cell. We expliot the fact that defect protein bundles are very stable to identify what proteins get caught up in them. After heating the cells with wrongly folded proteins inside, we use MS to find out what other proteins have been trapped and become more stable, too. This tells us which functions in the cell are affected. We can then treat cells with drugs that catch defect proteins and prevent them from destroying the cell’s control mechanism, thus stopping cancer from developing.