Engineering Yeast for Functional Analysis of Human Cu Transport Proteins
Doctoral thesis, 2019
ABSTRACT
Copper (Cu) is an important trace element that plays a vital role in several biological processes. Many proteins that are involved in various biochemical pathways require Cu as a cofactor. In human cells, Cu uptake is mediated by the high-affinity Cu uptake protein Ctr1, followed by the cytoplasmic Cu chaperone Atox1, which shuttles Cu from the plasma membrane to the Wilson disease protein ATP7B, a P1B-type ATPase located at the Golgi compartment. ATP7B incorporates Cu to Cu-dependent enzymes in the secretory pathway and also export excess Cu from the cells. Genetic defects in ATP7B may lead to a nonfunctional protein where Cu distribution is impaired, resulting in Wilson disease (WD). ATP7B is a multi-domain membrane-spanning protein with several protruding cytoplasmic domains. In contrast to its bacterial or yeast homologs, which have one or two metal-binding domains (MBD) respectively, ATP7B has six cytosolic MBDs. Many mechanistic questions around ATP7B function remain unanswered: e.g., the reason for as many as six MBDs and how Cu is released in the Golgi lumen.
In this thesis, the development of a new yeast model system for investigating Cu transport by human proteins is described. The system probes Cu shuttling from Atox1 to ATP7B, and within ATP7B, with yeast growth as the readout. Using this system, combined with biophysical methods such as NMR and MD simulations in some studies, we investigated the roles of the six MBDs in ATP7B (Paper I and III), examined Cu release by ATP7B in the Golgi (Paper II), and studied the effects on Cu transport due to MBD mutations causing WD (Paper IV). Our results provide new mechanistic information on how the six MBDs cooperate to move Cu along them. We also identified residues important for Cu release from ATP7B to the Golgi lumen. The collected results highlight the yeast model as a useful system for studying human Cu transport proteins, in particular when combined with other experimental methods. The yeast model system may be utilized to gain further understanding of various human Cu transport proteins as well as of disorders related to Cu mismetabolism, such as cancer and neurodegeneration.
Copper (Cu) is an important trace element that plays a vital role in several biological processes. Many proteins that are involved in various biochemical pathways require Cu as a cofactor. In human cells, Cu uptake is mediated by the high-affinity Cu uptake protein Ctr1, followed by the cytoplasmic Cu chaperone Atox1, which shuttles Cu from the plasma membrane to the Wilson disease protein ATP7B, a P1B-type ATPase located at the Golgi compartment. ATP7B incorporates Cu to Cu-dependent enzymes in the secretory pathway and also export excess Cu from the cells. Genetic defects in ATP7B may lead to a nonfunctional protein where Cu distribution is impaired, resulting in Wilson disease (WD). ATP7B is a multi-domain membrane-spanning protein with several protruding cytoplasmic domains. In contrast to its bacterial or yeast homologs, which have one or two metal-binding domains (MBD) respectively, ATP7B has six cytosolic MBDs. Many mechanistic questions around ATP7B function remain unanswered: e.g., the reason for as many as six MBDs and how Cu is released in the Golgi lumen.
In this thesis, the development of a new yeast model system for investigating Cu transport by human proteins is described. The system probes Cu shuttling from Atox1 to ATP7B, and within ATP7B, with yeast growth as the readout. Using this system, combined with biophysical methods such as NMR and MD simulations in some studies, we investigated the roles of the six MBDs in ATP7B (Paper I and III), examined Cu release by ATP7B in the Golgi (Paper II), and studied the effects on Cu transport due to MBD mutations causing WD (Paper IV). Our results provide new mechanistic information on how the six MBDs cooperate to move Cu along them. We also identified residues important for Cu release from ATP7B to the Golgi lumen. The collected results highlight the yeast model as a useful system for studying human Cu transport proteins, in particular when combined with other experimental methods. The yeast model system may be utilized to gain further understanding of various human Cu transport proteins as well as of disorders related to Cu mismetabolism, such as cancer and neurodegeneration.
Cu transport
yeast model
Atox1
genetic engineering
Wilson disease protein
ATP7B
complementation assay
yeast growth
metal-binding domain
mechanisms.
Lecture Hall : KC kemihuset, kemigården 4
Opponent: Prof. Gerhard Gröbner, Umeå University
Author
Kumaravel Ponnandai Schanmugavel
Chalmers, Biology and Biological Engineering, Chemical Biology
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.
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.
Subject Categories
Biochemistry and Molecular Biology
Biophysics
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
ISBN
978-91-7905-200-3
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4667
Publisher
Chalmers
Lecture Hall : KC kemihuset, kemigården 4
Opponent: Prof. Gerhard Gröbner, Umeå University