Eukaryotic cells adapt to environmental changes by altering their protein complements through synthesis and degradation. Cells that adapt poorly or improperly may either cease to exist (e.g., apoptosis) or become neoplastic (e.g., hepatoma). Cells adapt to low levels of amino acids by sequestering proteins and organelles for lysosomal degradation via a process called autophagy. The data from many laboratories suggest that autophagy is turned on during apoptosis and turned off during neoplastic growth implicating a role for autophagy in cancer suppression. In addition, it appears that some bacteria utilize the autophagy pathway of the host cell to escape lysosomal destruction and thereby replicating within the host. Our long term goals are to characterize the molecular aspects of the regulation and mechanisms of cellular autophagy. The molecular events of autophagy have yet to be defined given the difficulty in manipulating mammalian genetics. In order to overcome the shortcomings of the mammalian system, we have characterized a yeast system (Pichia pastoris) by which to investigate the selective degradation of peroxisomes (P) by the vacuole (V) during nutrient adaptation. We have shown that during adaptation peroxisomes are sequestered selectively into the yeast vacuole by a process analogous to autophagy. However, during nutrient-starvation autophagic sequestration is nonselective. Selective constitutive autophagy and nonselective regulated autophagy has been characterized in S. cerevisiae. Of the ten genes that we have identified are required for selective peroxisome autophagy, four have been shown to be required for constitutive and regulated autophagy in S. cerevisiae and one is required for selective autophagy but not nonselective autophagy in both P. pastoris and S. cerevisiae. Therefore, it appears that at least some of the molecular machinery we have defined for peroxisome autophagy in P. pastoris is the same for constitutive and regulated autophagy in S. cerevisiae. Our long term goals are to utilize the genetic opportunities offered by these yeast models to define the molecular mechanisms of selective and nonselective autophagy. The identification of those proteins that are required for autophagy will allow us to better determine why this process is shut down in neoplastic tissue and turned on during bacterial invasion. In addition, with our new understanding of the molecular events of autophagy, we can begin to design clinical approaches by which to turn on autophagy and arrest neoplastic growth or turn off autophagy and suppress bacterial replication.

Molecular Characterization of Autophagy: We are utilizing a multidisciplinary approach of cell, molecular, and genetic procedures to establish the functional roles of specific cellular proteins in selective and nonselective autophagy in yeast and mammalian cells.

The Role of Autophagy in Apoptosis: We are utilizing a cell and molecular approach to evaluate the role of autophagy in the drug-induced cell death of liver and breast cancer cells in culture.

Interactions Between Oral Pathogens and Vascular Cells: In colaboration with Dr. Ann Progulske-Fox (Dept. of Oral Biology, College of Dentistry), we are utilizing both morphologic and genetic approaches to characterize the events of bacterial invasion in vascular endothelial and smooth muscle cells.