Análisis General de la Práctica Educativa

Caspase-8 activation in cytochrome c activated Jurkat cell extracts was found to be a good approximation of caspase-8 activation in cells following cytochrome c release. Therefore, this in vitro activation system could be used for subsequent analysis of caspase-8 activation with a degree of confidence. This system has many advantages; the cytochrome c pathway is activated in isolation, caspase activation is more rapid and synchronous and the system is cheaper for the large scale experiments described in the next chapter.

3.3.1. Cvtochrome c initiated caspase-8 activation

Caspase-8 activation downstream of cytochrome c release was defined by a series of experiments. Caspase-8 was found to be cleaved by transient interaction with an activator and it did not become part of a larger or smaller complex during activation. It was

informative to place this observation is the context of the activation of other caspases in this system. As had been observed in several other systems, a proportion of caspases -3, -7 and -9 were observed to become part of a high molecular weight apoptosome with Apaf-1 during activation (Bratton et aL, 2001; Cain et aL, 1999). The fact that similar results have been observed with several cell types, utilising different triggers of apoptosis in cells, or with caspases activated in vitro indicates that this core mechanism of apoptosis is little modulated (Beere et aL, 2000; Cain et aL, 1999; Freathy et aL, 2000; Rodriguez and Lazebnik, 1999; Saleh et aL, 2000; Zou et aL, 1999). Although not shown here, gel

filtration analysis of the apoptosome formed on Fas treated Jurkat cells was performed and also showed no differences to that formed on cytochrome c treatment of extracts.

Reaper, Hid and Grim appear to be rather different to those found in mammals, the apoptosome formation and downstream caspase activation is analogous to that found in mammals (Rodriguez et aL, 2002). This contributes to the idea that the core mechanism of apoptosis is well conserved.

3.3.2. Caspase-8 is cleaved bv trans activation: bv transient interaction with a smaller protease or proteases.

Caspase-8 was found to be cleaved by trans activation by another protease as opposed to auto activation; the prodomain of caspase-8 was not required for the activating cleavage between the large and small domains. Auto activation of caspase-8 occurs in the Fas DISC, utilising the adaptor protein FADD (Medema et aL, 1997). Auto activation has also been implicated as the mode of caspase-8 activation in a cell model of Huntington’s Disease, utilising the indirect adaptor proteins Hip and Hippi (Gervais et aL, 2002).

Caspase-8 was found to be cleaved by a smaller protease or proteases; gel filtration fractions that could support caspase-8 activation contained complexes smaller than caspase- 8. From Western blot analysis of these fractions candidate caspases could be caspase-2, -3, or -6. However, from the data presented thus far it was not possible to determine which of these caspases could activate caspase-8 directly or if all of them were involved. Caspases are not the only proteases activated during apoptosis; several members o f the calpain and cathepsin families o f proteases are also activated (Johnson, 2000). Some o f these proteases have been demonstrated to be able to cleave caspases in vitro and their role in apoptosis may be underestimated because their protease activity can be inhibited by synthetic

inhibitors which were thought to have specificity for caspases alone. At this stage the role of other proteases in caspase-8 activation was not clear.

3.3.3. Limitations of the in vitro system

Although the in vitro system used appears representative of the cytochrome c

initiated apoptosis pathway that occurs in cells, it has a number of limitiations which should be recognised.

Only proteins solubased by Dounce homogenisation o f cells in isotonic buffer are present. For example, these experiments were performed before the identification of the mitochondrial protein Smac and Htra2 which are not abundant in cell extracts prepared as described above, but are also released from the mitochondria during apoptosis (Du et aL, 2000; Suzuki et aL, 2001a; Verhagen et aL, 2000). Processed forms of both of these proteins have an N-terminal “AVP” sequence which allow binding to XIAP, freeing apoptosome caspases from repression by XIAP (Martins et aL, 2002; Srinivasula et aL, 2001). Cytochrome c induced caspase activation has been demonstrated to be more efficient in the presence o f Smac and Htra2 (Du et aL, 2000; Hegde et aL, 2002; Martins et aL, 2002; Suzuki et aL, 2001a).

In addition, apart from in Death Receptor complexes, a pool of caspase-8 has been variously described to be associated with the outer membranes of endoplasmic reticulum (Ng et aL, 1997), or the mitochondria (Stegh et aL, 2002), with Huntington binding

proteins (Gervais et aL, 2002), and with aggregates in the cytosol during activation (Ding et aL, 2000). It is a limitation of the in vitro system that some o f these components which may be relevant to cytochrome c induced caspase-8 activation in cells are not present. However, the in vitro system does contain sufficient soluble cytosolic components to activate caspase-8 and therefore can be used to investigate the proteins necessary and sufficient for cytochrome c induced caspase-8 activation.

Chapter 4 Results: Purification, identification and