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Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Jeanne A. Hardy
Eric R. Strieter
Richard W. Vachet
Scott C. Garman
Biochemistry | Biophysics | Molecular Biology | Structural Biology
Caspases are cysteine aspartate proteases involved in various cellular pathways including apoptosis, inflammation, and neurodegeneration. Caspase-9 is classified as an initiator apoptotic caspase that is activated upon intrinsic stress. Caspase-9 is composed of two domains: an N- terminal CARD domain and a catalytic core domain. We have employed hydrogen deuterium exchange mass spectrometry (H/DX-MS) to determine the 1) dynamics of the full-length caspase- 9, 2) dynamic impacts on caspase-9 upon substrate-induced dimerization, and 3) regions involved in the CARD: catalytic core domains interactions.
Upon intrinsic stress, caspase-9 activates executioners, procaspase-3 and -7 but not procaspase-6. We have employed site-directed mutagenesis technique and substrate-digestion assays to identify the factors (e.g., sequence and the local context of the cleavage sites) that facilitate caspase-9 to cleave the intersubunit linker (ISL) of procaspase-3 but not the ISL of procaspase-6.
One prime question related to caspase-9 is – Is caspase-9 only an initiator of apoptosis, or it has any others proteolytic roles? We employed reverse N-terminomics on caspase-3 and -9, and identified 124 and 906 putative substrates, respectively. We also determined which substrates are proteolyzed upon staurosporine induced apoptosis using immunoblotting.
Caspase-6, classified as an executioner of apoptosis, is involved in neurodegenerative pathways. Using a chemoproteomics study, an acyl phosphate ATP probe was shown to bind to procaspase-6 but not active caspase-6. By using site-directed mutagenesis and IC50 determination by SDS-PAGE assays, we found that 1) ATP attenuates auto-activation of procaspase-6 with an IC50 = 19 mM, and 2) dimer interface of procaspase-6 is a putative ATP-binding site.
Soni, Ishankumar V., "Investigating Structures and Functions of Apoptotic Caspases" (2022). Doctoral Dissertations. 2571.
Available for download on Saturday, May 13, 2023