Copper has been associated with humankind since ages with its beneficial effect on health associated with it across civilizations. As we made progress in the scientific field, our understanding of the role of copper in human disease such as cancer, neurodegeneration, Wilson and Menke’s disease started to unfold. These diseases pose a serious concern since the existing drugs are not specific to individuals as the metabolism, and metabolic rate varies from people to people. In order to address this differential responsiveness and facilitate rational drug design, we have to understand the fundamental of protein function, especially metal-transport proteins, since proteins are involved in metabolic processes. Fe transport in eukaryotes is dependent on Cu transport proteins. The Cu concentration in the cell is regulated via dedicated repertoire of proteins that facilitate the Cu uptake, distribution and efflux, to avoid Cu accumulation. Wilson disease is a genetic disorder that causes Cu to accumulate in the liver. The disease is caused due to mutation in Cu-transport protein ATP7B. If untreated, the disease causes neurological problems. ATP7B is a large multi-domain protein that is located in the trans-Golgi network and responsible for Cu delivery to Cu-dependent enzymes present such as ceruloplasmin (blood Cu-binding protein). To date, many mutations has been identified in different region of the protein. There are several domains (a section of the protein which has its own function in the protein) in the ATP7B protein. It is unclear how ATP7B protein receives Cu, how the Cu is released from the protein, and why several domains are present in the protein.
In this thesis, we have developed a yeast system for studying human Cu transport proteins. Using this system, we studied the Cu transport function of ATP7B. This yeast system requires active Cu transport for yeast growth. We created different variants of ATP7B protein by truncating domains one by one, mutating amino acid residues, and introduced WD mutations; and then incorporated them into our yeast system to understand the protein function in terms of Cu transport efficiency. This system helped us to understand the importance of crucial domains in the protein, Cu release, and Cu transport efficiency of some important WD causing mutations. In future, our yeast system can be used to study various human Cu transport proteins as well as diseases linked to malfunctioning of Cu metabolism.