The Influence of Dimer Interface Mutations Upon the Folding and Activity of Procaspase-3

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dc.contributor.advisor Dr. Carla Mattos, Committee Member en_US
dc.contributor.advisor Dr. Linda Hanley-Bowdoin, Committee Member en_US
dc.contributor.advisor Dr. A. Clay Clark, Committee Chair en_US
dc.contributor.advisor Dr. Robert Kelly, Committee Member en_US Pop, Cristina en_US 2010-04-02T18:41:54Z 2010-04-02T18:41:54Z 2004-04-08 en_US
dc.identifier.other etd-04062004-155858 en_US
dc.description.abstract 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. en_US
dc.rights I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. en_US
dc.subject apoptosis en_US
dc.subject caspase en_US
dc.subject protein folding en_US
dc.subject dimer interface en_US
dc.subject enzymology en_US
dc.title The Influence of Dimer Interface Mutations Upon the Folding and Activity of Procaspase-3 en_US PhD en_US dissertation en_US Biochemistry en_US

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