The long-term goal of this proposal is to understand the fundamental mechanisms of autophagosome- lysosome fusion. Autophagosome-lysosome fusion is needed for completion of autophagy, a cellular stress response in which cytosolic material is packaged within membrane-enclosed structures termed autophagosomes. Autophagosome fuse with lysosomes, where cytosolic cargo is degraded and recycled. The resulting nutrients, e.g. amino acids and lipids, permit cells to survive stresses like hypoxia and starvation. Autophagy is increasingly recognized as being key both to the initiation and progression of cancer. Autophagy prevents cellular transformation. On the other hand, autophagy is needed for survival of already cancerous cells. Moreover, autophagy is induced in tumor cells by chemotherapy and radiation therapy. Thus, improved tools for investigating autophagy will lead to (1) a deeper understanding of the role that autophagy plays in cancer; and (2) novel anticancer therapies that block autophagy and thus kill cancerous cells. The 6-subunit HOPS complex mediates autophagosome-lysosome fusion, and thus is required for completion of autophagy. In past work, we purified yeast HOPS and biochemically reconstituted its activity in an in vitro membrane fusion reaction. However, yeast HOPS is a poor model for human HOPS because sequence identity between yeast and human HOPS subunits is at most 27%. Thus, we aim to biochemically reconstitute autophagosome-lysosome fusion using human HOPS and the other components of the human autophagosome-lysosome fusion machinery. As a first step towards reconstitution of human autophagosome-lysosome fusion, this proposal has just one specific aim: express and purify human HOPS complex. We will express human HOPS in insect cells, using a single baculovirus for simultaneously expressing all six human HOPS subunits. As proof of principle for this effort, we have used a single baculovirus to express yeast HOPS in insect cells. Yeast HOPS expressed in and purified from insect cells is equally active to yeast HOPS purified via our previous method, i.e. directly from yeast. Furthermore, we have created a baculovirus for expressing two human HOPS subunits. These subunits express well in insect cells and assemble into a sub-complex, as assessed by co-purification via an affinity tag. Thus, we are ready, immediately, to generate a six-subunit baculovirus for expressing human HOPS. In future work, we will purify the remainder of the human autophagosome-lysosome fusion machinery (expression systems for these components already exist) and reconstitute human autophagosome-lysosome fusion in vitro. We then plan two sets of studies: (1) biochemical, biophysical, and structural experiments to determine the fundamental mechanisms of autophagosome-lysosome fusion; and (2) high-throughput screening for inhibitors of human HOPS with therapeutic potential for treating cancer.
The goal of this proposal is to develop a novel research tool for investigating a cellular response to cancer termed autophagy. Autophagy is needed for cancerous cells to survive chemotherapy and radiation therapy. Thus, a deeper understanding of autophagy will lead to improved treatments for cancer.
