Human and viral Golgi anti-apoptotic protein (GAAP); a structural and functional study of its mechanism of action
Lead Research Organisation:
University of Cambridge
Department Name: Pathology
Abstract
The life or death of a cell is very strictly regulated and is an essential component in the life of multicellular organisms, including humans. Programmed cell death, called apoptosis, is critical in many processes. For instance, during development, many cells must die to create properly formed organ structures. Cell death also occurs throughout an organism?s life and is crucial to eliminate cells that are functioning incorrectly, including cancer cells or cells infected by a virus. We discovered a new human protein, termed h-GAAP, which controls cell death and that has been conserved during evolution all the way from plants to humans and poxviruses. We studied h-GAAP because vaccinia virus, the vaccine used to eradicate smallpox, makes a very similar protein called v-GAAP.
We intend to determine the structure of h-GAAP and v-GAAP, and identify the parts of GAAPs that are needed to i) direct it to its particular location within the cell called the Golgi complex, ii) mediate its interaction with a cell protein called IP3R that regulates calcium and iii) protect cells from apoptosis. The location of h-GAAP in the Golgi complex is particularly interesting because the Golgi is not often associated with the control of cell death. It is important to increase our understanding of this important protein, because h-GAAP is likely to have a role in development, cancer and infectious diseases. We will also study the role of v-GAAP during vaccinia virus infection and compare the role of v-GAAP and other vaccinia virus proteins that inhibit apoptosis.
We intend to determine the structure of h-GAAP and v-GAAP, and identify the parts of GAAPs that are needed to i) direct it to its particular location within the cell called the Golgi complex, ii) mediate its interaction with a cell protein called IP3R that regulates calcium and iii) protect cells from apoptosis. The location of h-GAAP in the Golgi complex is particularly interesting because the Golgi is not often associated with the control of cell death. It is important to increase our understanding of this important protein, because h-GAAP is likely to have a role in development, cancer and infectious diseases. We will also study the role of v-GAAP during vaccinia virus infection and compare the role of v-GAAP and other vaccinia virus proteins that inhibit apoptosis.
Technical Summary
A new inhibitor of apoptosis was discovered in camelpox virus and some strains of vaccinia virus (VACV) that has a very closely related protein (73% aa identity) in humans. The human and viral proteins have 7 transmembrane domains, are resident in the Golgi and inhibit apoptosis in response to a wide range of pro-apoptotic stimuli. They have been called v-GAAP and h-GAAP for viral and human Golgi anti-apoptotic protein. Closely related proteins are present across eukaryotes implying an ancient and conserved function. Functional analyses have shown that the human protein is expressed in all human tissues and is essential for cell viability. When h-GAAP is knocked down by siRNA cells die by apoptosis, and conversely, over-expression renders cells resistant to apoptosis. h-GAAP is also dys-regulated in many human cancers. v-GAAP is non-essential for virus replication, but affects the outcome of infection in vivo. Unpublished data indicate that h-GAAP and v-GAAP bind to the inositol 1,4,5-triphosphate receptor (IP3R) and modulate the release of intracellular calcium. This project concerns i) the structure and mechanism of action of h-GAAP and v-GAAP, and ii) the contribution of v-GAAP to inhibition of apoptosis by VACV that expresses other anti-apoptotic proteins.
The study of h- and v-GAAP provides an opportunity to study the role of the Golgi in sensing pro-apoptotic stimuli and how modulation of the IP3R leads to alterations in intracellular calcium and resistance to apoptosis. Golgi proteins that regulate apoptosis are rare and so we intend to map the functional domains of h- and v-GAAP to determine if a) Golgi localisation and b) interaction with IP3R are required for their anti-apoptotic ability. The lack of v-GAAP in uninfected cells makes it ideal for analysis. We will continue to study h-GAAP because viral proteins often have subtle differences from their cellular counterparts and h-GAAP is clearly a key regulator of mammalian cell death. In addition, we will determine the structure of h-GAAP and v-GAAP and investigate if h-GAAP or v-GAAP can form a membrane pore. In parallel, we will obtain a transgenic mouse lacking mouse GAAP to study the role of m-GAAP in development and if GAAP-/- mice are viable they may also be valuable for studying the contribution of GAAP to development of cancer. An increased understanding of how IP3R function and calcium mobilisation are controlled and how this influences apoptosis is very important to many cellular processes.
The study of h- and v-GAAP provides an opportunity to study the role of the Golgi in sensing pro-apoptotic stimuli and how modulation of the IP3R leads to alterations in intracellular calcium and resistance to apoptosis. Golgi proteins that regulate apoptosis are rare and so we intend to map the functional domains of h- and v-GAAP to determine if a) Golgi localisation and b) interaction with IP3R are required for their anti-apoptotic ability. The lack of v-GAAP in uninfected cells makes it ideal for analysis. We will continue to study h-GAAP because viral proteins often have subtle differences from their cellular counterparts and h-GAAP is clearly a key regulator of mammalian cell death. In addition, we will determine the structure of h-GAAP and v-GAAP and investigate if h-GAAP or v-GAAP can form a membrane pore. In parallel, we will obtain a transgenic mouse lacking mouse GAAP to study the role of m-GAAP in development and if GAAP-/- mice are viable they may also be valuable for studying the contribution of GAAP to development of cancer. An increased understanding of how IP3R function and calcium mobilisation are controlled and how this influences apoptosis is very important to many cellular processes.
