Wilson’s disease (WD) is an inherited disorder of copper metabolism characterized by liver disease and/or neurologic and psychiatric pathology. The disease is a result of mutation in ATP7B, which encodes the ATP7B copper transporting ATPase. Loss of copper transport function by ATP7B results in copper accumulation primarily in the liver, but also in other organs including the brain. Studies in the Atp7b−/− mouse model of WD revealed specific transcript and metabolic changes that precede development of liver pathology, most notably downregulation of transcripts in the cholesterol biosynthetic pathway. In order to gain insight into the molecular mechanisms of transcriptomic and metabolic changes, we used a systems approach analysing the pre-symptomatic hepatic nuclear proteome and liver metabolites. We found that ligand-activated nuclear receptors FXR/NR1H4 and GR/NR3C1 and nuclear receptor interacting partners are less abundant in Atp7b−/− hepatocyte nuclei, while DNA repair machinery and the nucleus-localized glutathione peroxidase, SelH, are more abundant. Analysis of metabolites revealed an increase in polyol sugar alcohols, indicating a change in osmotic potential that precedes hepatocyte swelling observed later in disease. This work is the first application of quantitative Multidimensional Protein Identification Technology (MuDPIT) to a model of WD to investigate protein-level mechanisms of WD pathology. The systems approach using “shotgun” proteomics and metabolomics in the context of previous transcriptomic data reveals molecular-level mechanisms of WD development and facilitates targeted analysis of hepatocellular copper toxicity.
Copper-responsive intracellular ATP7B trafficking is critical to maintain copper balance in mammalian hepatocytes and thus organismal copper levels. The COMMD1 protein binds both the ATP7B copper transporter and phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2), while COMMD1 loss causes hepatocyte copper accumulation. Although it is clear that COMMD1 is localized to endocytic trafficking complexes, a direct function for COMMD1 in ATP7B trafficking is not defined. In this study, experiments using quantitative colocalization analysis reveal that COMMD1 modulates the copper-responsive ATP7B trafficking through recruitment to PtdIns(4,5)P2. Decreased COMMD1 abundance results in loss of ATP7B from lysosomes and the trans-Golgi network (TGN) in high copper conditions, while excess expression of COMMD1 also disrupts ATP7B trafficking and TGN structure. Overexpression of COMMD1 mutated to inhibit PtdIns(4,5)P2 binding has little impact on ATP7B trafficking. A mechanistic PtdIns(4,5)P2-mediated function for COMMD1 is proposed that is consistent with decreased cellular copper export due to disruption of the ATP7B trafficking itinerary and early endosome accumulation when COMMD1 is depleted. PtdIns(4,5)P2 interaction with COMMD1 as well as COMMD1 abundance may both be important in maintenance of specific membrane protein trafficking pathways.
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