Konpartimentu-espezifikoko gertuko biotinilazioa: XPO1en esportazio-kargoak identifikatzeko hurbilketa berria

Abstract

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.; Nukleo eta zitoplasmaren arteko banaketa da zelula eukariotoen ezaugarririk bereizgarriena. Konpartimentuok fisikoki banaturik egon arren, elkarren arteko komunikazioa etengabea da, eta horretarako estuki erregulaturiko garraio-sistema dago zeina zelularen homeostasia mantendu eta minbizia bezalako gaixotasunak ekiditeko ezinbestekoa den. Garraio-sistema honetako pieza gakoa XPO1 esportina da zeinak kargo deritzen proteina askoren nukleotik zitoplasmaranzko esportazioa egikaritzen duen. Esportina honen eta minbiziaren arteko lotura maiz aipatu izan da, eta badira XPO1 inhibitzen duten agente terapeutikoak. XPO1en inhibizioak bere kargoen banaketa azpizelularrean aldaketak eragingo dituela espero daiteke. Alabaina, XPO1en kargo asko ezagunak badira ere, bere kargo bilduma osoa ez da zehaztu oraindik, eta honek XPO1 ikerketa proteomikoetarako kandidatu oso interesgarri bilakatzen du. Hau honela, estrategia proteomiko berri bat diseinatu dugu, zeinetan APEX2 peroxidasa erabiliz konpartimentu-espezifikoko gertuko biotinilazioa burutu dugun XPO1en kargo berriak bilatzeko. Horretarako, APEX2 zitoplasma eta nukleora ituratu dugu eta, biotinilatutako proteinak afinitate-purifikazioz arrantzatu ostean, masa-espektrometriaz identifikatu ditugu. Lan honetan azaldutako kontzeptu-froga esperimentuaren emaitzek erakusten dute estrategia hau, XPO1en inhibizio espezifikoarekin konbinatuz, kargo berriak identifikatzeko baliagarria izan daitekeela. Hurbilketa berri honek beraz, kargo gehiagoren identifikazioa erraztearekin batera, XPO1en mendeko garraioan sakondu eta terapeutikoki erabiltzen diren XPO1en inhibitzaileek zelula mailan duten eragina argitzeko balio dezake ere.