Browsing by Author "Dr. Linda Hanley-Bowdoin, Committee Member"

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    The Influence of Dimer Interface Mutations Upon the Folding and Activity of Procaspase-3
    (2004-04-08) Pop, Cristina; Dr. Carla Mattos, Committee Member; Dr. Linda Hanley-Bowdoin, Committee Member; Dr. A. Clay Clark, Committee Chair; Dr. Robert Kelly, Committee Member
    Procaspase-3 is the dimeric precursor of the apoptosis-executioner caspase-3 that displays little activity in vitro. The interface of the procaspase-3 dimer plays a critical role in zymogen maturation, although the active sites are not located at the dimer interface. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with arginine or glutamate results in an increase in enzyme activity of about 25-60-fold, representing a pseudo-activation of the procaspase. In contrast, substitution of V266 with histidine abolishes the activity of the procaspase-3 as well as that of the mature caspase. This mutant can be activated by protein exposure at pH 5, followed by dialysis at neutral pH. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active sites. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate-binding pocket. In some cases, the amino acids affected by the mutations are >20 Å from the interface. We suggest that the effects of the V266E and V266R mutations upon procaspase activity are due to the formation of buried salt bridges at the dimer interface by a mechanism similar to the activation of initiator procaspases. In addition, we suggest that inactivation of V266H is mediated by residue Y197, involved in the amino acid interaction network between the interface and catalytic loops. Equilibrium unfolding studies show that the V266E mutant is a kinetic trap of procaspase-3, while the V266H mutant is remarkably more resistant to chemical denaturation than procaspase-3. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation and stability of (pro)caspase-3. Procaspase-3 dimer interface mutants can be used as therapeutic tools in cancer and neurodegenerative diseases.