TECNÓLOGO DE PLANTA

One of the most widely recognized features of apoptosis is the activation of caspases, which cleave key substrates required for normal cellular functions, such as cytoskeletal proteins, nuclear proteins, and DNA repair enzymes. Caspases involved in apoptosis are generally divided into two categories, the initiator caspases, which include caspase-2, -8, -9, and -10, and the effector caspases, which include caspase-3, -6, and -7 . All apoptotic caspases exist in normal cells as inactive enzymes analogous to the zymogens involved in the regulation of blood clotting (Srinivasula et al. 1997).

There are currently two well characterized caspase-activating cascades that regulates apoptosis: one is initiated from the cell surface death receptor (death receptor-mediated pathway) and the other is triggered by changes in mitochondrial

integrity (mitochondria-mediated pathway) (Green, 2000). The death receptor- mediated pathway, which will be detailed hereafter, leads to the activation of procaspase-8. In the mitochondria-mediated pathway, diverse proapoptotic signal transduction and damage pathways converge on mitochondrial membranes to induce their permeabilization, resulting in final activation of procaspase-9.

The role of mitochondria in apoptosis, until recently, was rather obscure, owing to the fact that mitochondria during the death process do not exhibit any prominent morphological changes. However, although mitochondria do not give any appearance of self-destruction, they actually contain all the components necessary to annihilate the cell by apoptosis. More recent studies have shown that mitochondria undergo several major changes, including alterations in their membrane integrity, even before any classical signs of apoptosis appear (Zamzami et al, 1996)

A number of mechanisms have been described to highlight the mitochondrial involvement in apoptosis. High levels of cytosolic Ca^^ and excessive concentrations of reactive oxygen species are known to contribute to the opening of the

mitochondrial permeability transition pore (PTP), which depolarizes the mitochondria. The PTP participates in the regulation of matrix Ca^" levels, pH, and volume. Adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC) are the two principal components of PTP. The VDAC operates at the inner and outer membrane contact sites and creates a channel that allows non-specific passage of ions and molecules smaller than 1.5 kDa. The opening of this channel in the inner membrane allows for equilibration of ions within the matrix and the intermembrane space. This dissipates the electrochemical gradient (A'PJ and uncouples the respiratory chain, leading to the cessation of ATP production (Kroemer et al, 1997; Marzo et al, 1998). The disruption of the which is attributed largely to the damage of the inner mitochondrial membrane, occurs before DNA fragmentation, indicating that mitochondrial injury is an early event in apoptosis.

In the low-conductance state, the opening of PTP is pH-dependent, which permits the diffusion of only small ions, and is followed by spontaneous closing. However, in the high-conductance state, the channel is stabilized in an open position and allows water and bigger molecules to enter the protein rich matrix, leading to

mitochondrial swelling. Consequently, the inner membrane, which has a far greater surface area than the outer membrane, unfolds exerting pressure on the outer membrane (Vander Heiden et al, 1997).

Several apoptosis-inducing agents are known to trigger mitochondrial uncoupling that results in a diminution of AYm. The Bcl-2 protein family includes pro- and anti- apoptotic members. The main pro-survival members, Bcl-2 and Bcl-XL, are responsible for maintaining the normal ion potential across the inner mitochondrial membrane and therefore the proper mitochondrial membrane potential (Kroemer et al, 1997; Marzo et al, 1998). Bcl-2 and other anti-apoptotic members of the Bcl-2 family are located in the outer mitochondrial membrane. Bcl-2 is especially enriched at contact sites, where the inner and outer membranes come in close proximity.

Unlike other apoptotic members of the family, which act by inhibitory interaction with Bcl-2 and Bcl-XL, Bax appears to induce apoptosis by moving from the cytosol to the mitochondrial membrane, where it forms channels that alter the normal mitochondrial physiology (Martinou and Green, 2001). Consistent with these

interpretations are the findings that the extent of Bax relocation does not correlate with the amount of Bcl-2 and Bcl-XL inserted in the mitochondrial membrane, and also that Bax-induced apoptosis is not abolished when Bax is deprived of its BH3 domain and amphipathic a-helix, which accounts for all dimérisation within the family.

In contrast with this hypothesis, Desagher documented that the appearance of Bax mitochondrial staining during apoptosis was not due to an increased level of mitochondrial Bax, which was continuously present in mitochondria, but rather to a conformational change of the protein, maybe the result of Bax proteolysis, which led to unmasking of its NH2-terminal domain (Desagher et al. 1999). The exposure of the Bax immunoreactive domain was mediated by Bid, another pro apoptotic member of the Bcl-2 family. In particular, the structural change of Bax was explained with Bid translocation to mitochondria, followed by direct binding of Bid to Bax that was observed in cells undergoing staurosporine-induced apoptosis (Desagher et al, 1999). Finally, in a different system Li and Luo (Li et al, 1998; Luo et al, 1998) reported that during Fas and TNF-mediated apoptosis. Bid (see hereafter) becomes cleaved by caspase-8 and its COOH-terminal domain translocates to mitochondria,

where it is responsible for the release of cytochrome C. The three different theories exposed above sketch out a scenario in which the roles played individually and reciprocally by Bax and Bid within apoptotic cell death appear to be still highly controversial.

SECTION 4