Dynein Activity Induced by a Kunitz‐type Molecule Acting on the Proteasome

Abstract

MethodsCell culture: Mia PaCa‐2 and SK‐Mel‐28 tumor cells. Western blotting: Proteins were analyzed via chemiluminescence. Confocal microscopy: Live or fixed cells were analyzed using labeled Amblyomin‐X or specific antibodies. Proteasome assay: Trypsin and chymotrypsin‐like activities were accessed using chromogenic substrates. Co‐IP: Cell lysates were submitted to Co‐IP and injected into a mass spectrometer.ResultsProteasome inhibition by Amblyomin‐X and its cellular uptake was abolished when the cells were pretreated with a dynein inhibitor. Amblyomin‐X increased Rab11A protein levels and induced its co‐localization with dynein. Several proteins were found bound to dynein, such as receptors, signaling transducers, early and recycling endosome components, proteasome, aggresome, ubiquitin, cell cycle, apoptosis and extracellular matrix components.ConclusionsDynein was required for Amblyomin‐X uptake and may transport the endosome towards the endocytic recycling compartment, because Rab11A was overexpressed, co‐localized with dynein; and Rab11A and Rab20 but not late endosome markers were bound to dynein. In addition, dynein was required for proteasome inhibition induced by Amblyomin‐X, thus suggesting its transportation to the molecular target. Some proteins, such as Bag3 and Raptor were found bound to dynein and this data corroborates with the aggresome formation and autophagy inhibition induced by Amblyomin‐X, as previously reported. Furthermore, dynein ligands indicated a possible involvement of the lipid rafts, intracellular pathways, transcription factors and membrane receptors. Those findings distinguish this molecule from proteasome inhibitors, such as bortezomib and carlfizomib. Supported by FAPESP.