Browsing by Author "Dr. A. Clay Clark, Committee Chair"

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The importance of the dimer interface in the folding and assembly of procaspase-3.
(2009-11-16) MacKenzie, Sarah Helen; Dr. A. Clay Clark, Committee Chair; Dr. Robert Kelly, Committee Member; Dr. Paul Agris, Committee Member; Dr. Robert Rose, Committee Member
Caspases are a family of cysteine proteases that are intimately involved in apoptosis and exist in the cell as inactive zymogens prior to activation. Initiator procaspases are monomers that must dimerize for activation. Executioner procaspases, such as procaspase-3, are dimers that must be processed for activation. The chemical properties of the dimer interface are different between the two subfamilies of caspases but their structures are similar, suggesting that the interface region is important for regulation. The goal of the studies presented here is to determine the importance of the dimer interface in folding and assembly of procaspase-3. A histidine mutation was introduced into the dimer interface region, which completely abolished the activity of mature caspase-3. Equilibrium and kinetic folding studies were performed to elucidate how a mutation in the dimer interface prevents substrate turnover in the active site when the distance between the two regions is 20Ãƒâ€¦. The folding studies presented here coupled with the crystal structure show that the protein is entering a kinetic trap prior to dimerization because the histidine has to adopt an unusual rotomer to pack into a region that normally accommodates a much smaller valine residue. A hysteresis was observed by equilibrium folding studies, presumably because the time it takes to refold is different that the time it takes to unfold due to the limited conformational freedom of the histidine residues. Additionally, the hysteresis is observed to be concentration dependent suggesting that the histidine residue makes the activation mechanism of procaspase-3 more like initiator caspases that require a large local concentration of protein to promote dimerization. Kinetic refolding studies showed that the procaspase-3 monomer is becoming trapped in a conformation that is unstable and prone to aggregation prior to forming a dimerization competent species. The crystal structure of caspase-3 (V266H) revealed two separate pathways of inhibition starting from the dimer interface and culminating in the active site that could be responsible for the lack of activity in this mutant. These data, taken together, suggest that the dimer interface is a region that can be used to allosterically inhibit procaspase-3 because it is important for regulation of the enzyme. This is important because it could be used as a drug target for diseases that have too much cell death, such as neurodegenerative disorders.
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.
Interactions in the Active Site Loops are Important for Maintaining the Active Site Environment of (Pro)caspase-3
(2004-04-16) Feeney, Brett; E. Stuart Maxwell, Committee Member; Dr. A. Clay Clark, Committee Chair; Carla Mattos, Committee Member
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-