Dissecting the Ubiquitination function of the metastasis-promoting protein Podocalyxin


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Mohammed A. Mansour1
1Cancer Research UK Beatson Institute

Abstract

Background

An important feature of cancer is that the first tumour rarely causes mortality. If this tumour spreads, it is a very poor outlook for patients. If we can stop cancer spreading, it is a much better outlook, as some tumours can be surgically removed. If tumour cells have high levels of the protein Podocalyxin on their cell surface, they are exceptionally good at spreading.

Method

We ‘rescued’ CRISPR-based knockout of Podxl by expressing CRISPR- resistant Podxl Ub mutants to determine the minimal lysine residues required for Podxl Ub. We probed how the minimal Ub mutant compromises Podxl function. We examined surface levels of Podxl using the cell sorting. We examined localisation of mutant Podxl and co-labelling with a variety of intracellular markers. We examined endocytosis, recycling, and degradation of Podxl via surface biotinylation assays. Finally, we examined live imaging of trafficking of Podxl using photoconvertible Podxl (WT vs Ub mutant), using the photoconvertible protein mMaple3.

Results

We have found that it is not whether Podxl is expressed in cells, but where it is expressed that confers its pro-invasive function. After performing in-depth proteomics to compare the interactors of wild-type Podxl to the invasion-deficient Ub mutant, we have identified several Podxl interactors like Galectin-3 that might be responsible for Podxl proper localization to the cell surface.

Conclusion

These results and our current efforts shall uncover vulnerabilities in cells that we can utilize for future development of drugs to stop cancer from spreading.