Most toxicants induce apoptosis by triggering the activation of caspases (cysteinyl aspartate-specific proteases). Initiator caspases are activated through their association with specific adapter proteins, and in turn, activate effector caspases that cleave cellular proteins and dismantle the cell. Thus, caspases are activated through unique signal transduction pathways, and our general goal is to understand, in molecular detail, how these caspases are activated and how their activities are modulated. We are particularly interested in a multimeric complex known as the apoptosome. This large complex--composed of seven Apaf-1 (apoptosis protease-activating factor-1) proteins and formed in response to mitochondrial stress--binds to the initiator caspase-9 to form a holoenzyme that subsequently activates the effector caspases-3 and -7. We are currently studying the regulatory mechanisms that control the activity of this complex in vitro and in vivo.
Inhibitor of Apoptosis (IAP) Proteins and IAP Antagonists
IAPs are important antiapoptotic proteins present throughout nature. In humans and rodents, at least one family member, XIAP (X-linked IAP), is an established inhibitor of caspases-9, -3 and -7. However, it remains unclear how other family members suppress apoptosis. We are currently investigating the mechanisms whereby other IAPs, such as cellular IAP1 (cIAP1) and cIAP2, as well as Drosophila IAP-1 (DIAP1) and DIAP2, suppress apoptosis in human and fly models of cell death. The fly IAP antagonists, Reaper, Hid (head involution defective), Grim, Sickle, and dOmi, are thought to induce apoptosis during development by antagonizing DIAP1. Recent studies in our laboratory and others, however, suggest that these IAP antagonists may promote cell death through mechanisms that do not involve antagonism of DIAP1 per se. Thus, we are actively pursuing these alternative pathways to death.
Heat Shock-induced Apoptosis
Hyperthermia or heat shock therapy is currently being utilized in phase II/III clinical trials, either alone or in combination with radiation or chemotherapy, for the treatment of various cancers. Unfortunately, though intense heat shock induces apoptosis, the underlying mechanisms remain controversial and unclear. We have recently shown that heat shock induces caspase-dependent apoptosis, but does so through mechanisms that do not require any of the known initiator caspases or their activating complexes. We are currently working to identify the mechanisms responsible for heat shock-induced apoptosis, including the identification and characterization of the apical protease(s) responsible for initiating the caspase cascade.
Heat shock can induce apoptosis. We hypothesize that heat shock induces cell death, in part by stimulating rapid endo-lysosomal membrane permeabilization (ELMP), which coincides with cytosolic acidification and release of cathepsins into the cytoplasm. In the image to the left, we have used LAMP1 as a marker of lysosomal membranes, and we can study the release of cathepsin B, a cysteine protease stored in the lysosome, to study the effects of heat shock.
TRAIL-induced Apoptosis and Resistance in Prostate Cancer
Tumor cells are normally removed from the body, in part, through the activation of death receptors located on tumor cells, such as Fas/CD95 or death receptors-4 and 5 (DR4/5). These receptors are activated by their cognate ligands, FasL and Tumor necrosis factor- related apoptosis-inducing ligand (TRAIL), respectively, and TRAIL has potential therapeutic value due to its capacity to induce apoptosis selectively in cancer cells. In fact, recombinant TRAIL and agonistic antibodies to DR4/5 are currently in clinical trials for the treatment of various cancers. Unfortunately, ~50% of tumors exhibit resistance to TRAIL. We are investigating the primary mechanisms of TRAIL resistance in prostate cancer using both in vitro and animal models.
Assembly and Formation of the Autophagosome
Lysosomes and proteasomes serve as the two primary sites for degradation within the cell. Macroautophagy, commonly referred to as just autophagy, involves the formation of double-membrane vesicles, called ‘autophagosomes’, that envelop cytoplasmic material and fuse with lysosomes resulting in the degradation of the sequestered material by lysosomal proteases. Formation of the autophagosome requires numerous autophagy-related (ATG) proteins including ATG12, ATG5, and ATG16L1, which together form a complex that catalyzes the ubiquitin-like conjugation of LC3 directly to the expanding membrane of the autophagosome precursor, termed the ‘phagophore’. Formation of this complex requires another ubiquitin-like conjugation reaction resulting in the covalent linkage of ATG12 to ATG5. We are actively interested in the mechanisms regulating these essential ubiquitin-like conjugation reactions, as well as specifically how the ATG12–ATG5-ATG16L1 complex is assembled and disassembled.
The Role of ATG Genes and Autophagy in Prostate Cancer
While the ubiquitin-proteasome system is generally limited to targeting short-lived soluble proteins, autophagy can eliminate excess or damaged organelles, large protein aggregates or invading pathogens in order to maintain basal homeostasis or cell survival in response to nutrient, oxidative, chemotherapeutic and many other types of stress. These diverse functions mean that autophagy plays a complex and contextually specific role in tumorigenesis, tumor progression and metastasis that is incompletely understood. We are currently investigating the regulatory mechanisms and the specific functions of ATG genes and autophagy in both human and murine prostate cancers.