March 8, 2010
3D model of cellular “pump” may hold clues to developing new treatments for diseases
Researchers at The Hospital for Sick Children (SickKids) have developed a new three-dimensional model of the physical structure of Vacuolar-type ATPase (V-ATPase). V-ATPase is a molecular complex that uses energy from a chemical called ATP to control the acidity within parts of the cell. Understanding the structure of V-ATPase provides a better understanding of how it works in normal cells, and also provides insights into how it can be controlled in cells affected by disease. The study is published in the February 2010 issue of Proceedings of the National Academy of Sciences.
V-ATPase is a referred to as a “pump” because it pumps protons across membranes. Normally, V-ATPases are used by healthy cells to maintain a carefully controlled internal environment. In some types of cancer, the pumps are “hijacked” to acidify the external environment of tumours, allowing the cancer to invade surrounding tissues and spread throughout the body. The cells that take up bone minerals also use V-ATPases to dissolve bone, a process that must be limited in treating osteoporosis.
“Understanding the molecular structure of this pump is the first step in designing strategies to prevent the pump from enabling tumour invasion and the spread of cancer,” explains Dr. John Rubinstein, a scientist in the Molecular Structure & Function Program at SickKids Research Institute and an assistant professor in the Department of Biochemistry at the University of Toronto.
In their research, graduate student Wilson Lau and Rubinstein determined how the nine different components of one type of V-ATPase are arranged into a molecular pump. This model is the most detailed created for any V-ATPase to date. "Although the structures of some of the individual components of the complex were known, this is the first time we have been able to clearly visualize how they fit together, and create a three-dimensional model that suggests how the intact assembly might function,” says Rubinstein.
To create the model Lau combined three powerful technologies: protein purification, high-resolution imaging and computational image analysis. Large amounts of a simple and stable form of V-ATPase were isolated from a bacterium and then frozen. Single particle electron cryomicroscopy (electron microscopy performed at liquid nitrogen temperatures) was then used to image the molecules. Finally, powerful computational tools put the pictures together to create a three-dimensional model of the pump. Two videos that show what the pump actually looks like are available at: http://www.pnas.org/content/suppl/2010/01/06/0911085107.DCSupplemental.
Now that they have determined the structure of the bacterial V- ATPase, Rubinstein and his team will attempt to refine their approach to obtain an even higher-resolution model of the complex. They have also begun work on purifying the yeast V- ATPase, which is very similar to the human version. "We anticipate that by understanding how this pump works at the chemical level, we will be able discover very specific approaches to turn it on or off in the cell", stated Rubinstein. "This may have therapeutic implications, not only for cancer, but for other diseases such as osteoporosis."
This research was funded by the Canadian Institutes of Health Research, the Ministry of Research and Innovation, and SickKids Foundation.