Interactions in the Active Site Loops are Important for Maintaining the Active Site Environment of (Pro)caspase-3

Abstract

Apoptosis is obligatory to development and in maintaining the vital balance between cell growth and death. Commitment to apoptosis involves a proteolytic cascade by a family of cysteine proteases named caspases. Caspases are split it into two general classes, those involved in the proinflammatory response and those involved in apoptosis. Of those involved in apoptosis, there are two further subdivisions, the apical caspases and the executioner caspases. The irreversible commitment to apoptosis involves activation of executioner caspases, namely caspase-3.

Procaspase-3 exists in the cell as a dimeric zymogen, where upon limited proteolytic cleavage at specific aspartate residues, it is activated and apoptosis results. We have previously shown the (pro)caspase-3 undergoes pH dependent conformational changes monitored by fluorescence emission (Bose, et al 2003). In this study, we unambiguously assign dimer dissociation to one of the pH dependent transitions observed in this assay by using size exclusion chromatography. We have also examined the effects of breaking specific salt bridges and hydrogen bonds by mutating residues in context of caspase-3, an inactive procaspase-3(C163S) and an uncleavable procaspase-3(D3A). We show that there are a number of stabilizing contacts that are required in order to ensure proper processing during maturation, ensure enzyme fidelity and maintain overall structure. At present, the function of the prodomain of executioner caspases has elucidated researchers. We have also hypothesized that the effector caspase prodomain may have some role as an intramolecular chaperone during maturation. We show that the caspase-3 prodomain does play a role in pH dependent folding of the (pro)caspase-3 dimer.

Salt has also been well described as having effects on caspase-3 activity and stability. There is a lack of knowledge as to the direct effects that different ions have on both procaspase-3 zymogen and mature caspase-3. In this study, we describe direct effects of different cations on (pro)caspase-3 activity and active site environment. We also study stabilizing effects of salt on (pro)caspase-