Biology

Dr. Arnab Gupta
SN Pradhan Centre for Neurosciences, University of Calcutta

Normal copper homeostasis is essential for human growth and development. Copper serves as essential cofactors for many enzymes; however excess copper is associated with morphological and metabolic changes in tissues and, if untreated, eventual death. Therefore, the level of copper in cells and tissue must be tightly regulated. The ATP-driven copper transporters ATP7B play a central role in this process. Inactivation of their transport activity is associated with diminished copper efflux from cells. Mutation in ATP7B gene causes Wilson disease (WD) characterized by tremendous copper overload in the liver and brain. ATP7B localizes to the Trans Golgi Network under basal copper. When copper is in excess, ATP7B sequesters copper within vesicles that traffic to the canalicular (apical) membrane of the liver, where ATP7B facilitates the extrusion of excess copper into the bile. The present talk will focus on identifying and characterizing the proteins that regulate ATP7B’s exit and entry at these sub-steps of its copper-dependent trafficking route. Using, novel vesicle/sub-compartment isolation techniques, Isobaric tags for relative and absolute quantitation (iTRAQ) and high resolution microscopy, we have identified ~80 unique proteins with their relative abundanceassociated with ATP7B vesicles as it traffics through the apical pathway. Our present effort is directed towards understanding the mechanism by which each of these identified trafficking regulators (individually and synchronously) affect sub-steps of ATP7B trafficking. We have tested the putative role and characterized of one of the candidates, Myosin Vb in regulation of ATP7B trafficking. We demonstrate that myosin Vb regulates the Copper-stimulated delivery of ATP7B to the apical domain of polarized hepatic cells, and that disruption of the ATP7B myosin Vb interaction reduces the apical surface expression of ATP7B. Overexpression of the myosin Vb tail, which competes for binding of subapical cargos to myosin Vb bound to subapical actin, disrupted the surface expression of ATP7B, leading to reduced cellular Copper export. The myosin-Vb- dependent targeting step occurred in parallel with hepatocyte-like polarity. If the myosin Vb tail was expressed acutely in cells just prior to the establishment of polarity, it appeared as part of an intracellular apical compartment, centered on γ-tubulin. ATP7B became selectively arrested in this compartment at high copper in the presence of myosin Vb tail, suggesting that these compartments are precursors of donor acceptor transfer stations for apically targeted cargos of myosin Vb. Our data suggest that reduced hepatic Copper clearance in idiopathic non-Wilsonian types of disease might be associated with the loss of function of myosin Vb. Parallel to the Myosin Vb studies, my lab is pursuing the role of retromers in sorting and copper induced trafficking of ATP7B.