Konpartimentu-espezifikoko gertuko biotinilazioaXPO1en esportazio-kargoak identifikatzeko hurbilketa berria

Resumen

A major feature of eukaryotic cells is the separation between nucleus and cytoplasm. Although physically separated, these two compartments are in permanent communication through a transport system that must be precisely regulated to maintain cell homeostasis and avoid serious diseases, such as cancer. A crucial element in this transport system is the exportin XPO1, which exports many proteins (so-called cargoes) from the nucleus to cytoplasm. XPO1 alteration has been frequently associated with cancer and XPO1 is an important therapeutic target. The cellular effect of XPO1 inhibition is expected to be mediated by changes in the subcellular distribution of its cargoes. However, while many of XPO1 cargoes have been already identified, the complete set remains uncharacterized, making XPO1 a very interesting candidate for proteomic studies. Thus, we have designed a novel proteomics strategy, based on compartment-specific proximity biotinylation using the APEX2 peroxidase, to search for XPO1 cargos. To this end, we have targeted APEX2 to cytoplasm and nucleus, isolated the biotinylated proteins by affinity purification, and identified them by mass spectrometry. The results of a proof-of concept experiment reported here show that this strategy, combined with specific XPO1 inhibition, can lead to the identification of XPO1 cargoes. This novel approach, therefore, may advance our understanding of XPO1-dependent nuclear export by facilitating the identification of novel cargoes, and may also contribute to better characterize the cellular effect of therapeutically used XPO1 inhibitors.