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Date of Award

5-2011

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

First Advisor

Jeanne A. Hardy

Second Advisor

Scott C. Garman

Third Advisor

Craig T. Martin

Subject Categories

Biochemistry | Biophysics

Abstract

Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's Diseases. Caspase-6 is of great interest in treatment of these neurodegenerative diseases, however the structure and molecular events of caspase-6 activation remained poorly understood prior to this thesis work.

To study the activation events and to probe the role of the prodomain and intersubunit linker on caspase-6 structure and activation, we generated a series of caspase-6 cleavage variants. Autoprocessing at D193 of the intersubunit linker proved to be crucial for activity and for subsequent processing at D179 site. For the first time we report the existence of stabilizing interactions between the prodomain and intersubunit linker of caspase-6.

We reported one of the first crystal structures of ligand-free caspase-6 with and without the intersubunit linker. Mature caspase-6 has extended 60.s and 130.s regions and the 90.s helix is rotated outwards. These striking structural features are unique to caspase-6 and are not observed in any other caspase. Caspase-6 shows a loss of helicity upon binding substrate. Therefore, binding of substrate appears to cause a conformational change, potentially converting the 60.s and 130.s helices to a canonical caspase structure. Recent crystal structure of active-site bound caspase-6, underscore our findings about the requirement of conformational changes to bind substrate.

Our work suggests that the latent state of mature caspase-6 relies on the stability and helical propensity of the 130.s region. The presence of the helical sequence in caspase-6 is more critical for formation of the extended conformation than is the helixbridging network between the 60.s and 130.s helices. This extended 130.s helix in the mature caspase-6 causes it to transition through a less stable intermediate to bind substrate. All these unique structural features reflect on the enigmatic role of caspase-6 in the cell.

The conformational changes between the apo mature and bound states are likely to be important in designing caspase-6 selective therapies. Thus our study has enhanced the current knowledge about caspase-6 structure and function and broadened our understanding of caspase activation and regulation in general.

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