Introduction

This laboratory is investigating the central mechanisms of apoptosis (programmed cell death). Apoptosis is a pivotal process in normal physiology, as it permits the organism to regulate the numbers of various types of cells and to eliminate cells whose presence would be deleterious. Also, diseases, such as cancer, AIDS, and autoimmunity, can involve the improper death or survival of certain types of cells.

In apoptotic cell death, a highly choreographed pre-existing program is activated within the dying cell. Although the stimuli that set this process in motion can be highly diverse, the program itself is widely conserved among higher eukaryotes. Some of the microscopically observable features of the apoptotic process include chromatin condensation, nuclear shrinkage and fragmentation, and rapid blebbing of the plasma membrane. Apoptotic cells display surface markers that are recognized by surrounding cells, which then engulf the dying cell. This prevents the release of the cell's contents into the surrounding milieu, which would elicit an undesirable inflammatory response.

Although apoptotic cell death has been studied extensively, the mechanisms of this process are still incompletely understood. However, several of the molecules that regulate apoptosis have been identified. Of particular importance are cysteine proteases belonging to the caspase family. These enzymes cleave polypeptide chains at specific sites, following aspartate residues. Also important in regulating apoptosis is another family of proteins related to Bcl-2. The functions of these proteins, as well as the caspases, are to some extent conserved across a range of species from the nematode worm, Caenorhabditis elegans, to humans.

Taking advantage of this interspecies consistency, we have been investigating the biochemical mechanisms of apoptosis, using a novel cell-free system. This system is based on extracts derived from eggs of the South African clawed frog, Xenopus laevis. Xenopus egg extracts can reproduce many of the intracellular events typically associated with apoptosis. The most obvious of these events include striking changes in nuclear morphology, involving nuclear shrinkage, and the condensation and fragmentation of chromatin. Concomitant with these morphological changes, a number of biochemical events are observed, such as the fragmentation of genomic DNA and the cleavage of certain protein substrates (e.g., PARP, fodrin, and lamin B) whose proteolysis is characteristic of cells undergoing apoptosis.

In many ways, the Xenopus system has proved to be a faithful model of the apoptotic process occurring in whole cells. For example, the apoptotic events in the Xenopus system are blocked by the Bcl-2 protein and certain caspase inhibitors. Most importantly, this cell-free system has been useful in uncovering novel mechanisms in the apoptotic machinery. We showed that apoptosis in the Xenopus system required the presence of a dense organelle fraction enriched in mitochondria. More recently, in collaboration with Douglas Green's laboratory at this institute, we demonstrated that mitochondria are a key control point of the apoptosis pathway. During apoptosis, mitochondria release pro-apoptotic proteins, including cytochrome c, into the soluble portion of the cytoplasm. Once cytochrome c leaves the mitochondria, it interacts with the Apaf-1 protein, leading to caspase activation and the cleavage of cellular proteins and destruction of genomic DNA which are characteristic of apoptotic cell death. The Bcl-2 protein binds to the external surface of mitochondria and somehow causes the retention of cytochrome c and other proteins within these organelles, thereby preventing caspase activation and apoptosis.

The apoptotic function of cytochrome c is quite distinct from the normal role of this protein in mitochondrial energy metabolism, which is conserved among both uni- and multi-cellular eukaryotes. The pro-apoptotic function of cytochrome c is somewhat less well conserved and may be restricted to vertebrates. For example, although insect cytochrome c can activate caspases in vertebrate cell extracts, an involvement of cytochrome c in insect cell apoptosis has yet to be demonstrated. Moreover, cytochrome c from the yeast, S. cerevisiae, is completely unable to promote caspase activation in vertebrate extracts.

Research Aims

Because of its faithfulness to the apoptotic process occurring in whole cells, the Xenopus cell-free system is a powerful tool with which to study both the intracellular mechanisms of apoptosis and the anti-apoptotic function of Bcl-2. This system has another advantage for biochemical studies, which is that Xenopus eggs permit the convenient preparation of moderately large amounts of mitochondria and concentrated cell extract. The preparation of equivalent quantities of cultured cell extract would require prohibitively large numbers of cells. Presently, the research projects in the laboratory are making use of the Xenopus system, as well as models based on apoptosis in cultured cells, to investigate some of the biochemical steps in the apoptotic process.

Ongoing studies are uncovering the mitochondrial process that allows the release of cytochrome c from the intermembrane space into the cytosol. Experiments show that this process is not specific for cytochrome c, as other proteins such as adenylate kinase and sulfite oxidase also escape from the intermembrane space into the cytosol. However, we do not detect gross changes in mitochondrial morphology or volume that accompany these changes in outer membrane permeability. Thus, our results are inconsistent with a model for cytochrome c release that involves swelling of the mitochondrial matrix and consequent rupture of the outer membrane.

Other studies in this laboratory are examining the mechanisms of action of pro-apoptotic members of the Bcl-2 family, such as Bid and Bax, which are able to permeabilize mitochondria, leading to the release of cytochrome c and other proteins from the intermembrane space. We used cell-free systems and ultimately a vesicular reconstitution from defined molecules to show that outer membrane permeabilization by Bcl-2-family proteins requires neither the mitochondrial matrix, the inner membrane, nor other proteins. Bid, or its BH3-domain peptide, activated monomeric Bax to produce membrane openings that allowed the passage of very large (2-megadalton) dextran molecules, explaining the translocation of large mitochondrial proteins during apoptosis. This process required cardiolipin and was inhibited by anti-apoptotic Bcl-xL. The results of these studies argue that mitochondrial protein release in apoptosis can be mediated by supramolecular lipid-based openings in the outer mitochondrial membrane, promoted by BH3/Bax/lipid interaction and directly inhibited by Bcl-xL.

Further studies have shown that Bid and Bax produce only a limited permeability of the outer membrane. However, following the action of Bid or Bax, another cytosolic activity, named Permeability Enhancing Factor (PEF), greatly increases the permeability of the outer membrane. PEF (whose molecular identity is still unknown) may insure that mitochondria are irreversibly damaged, thus establishing a "point of no return" for the cell death process.