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Author ORCID Identifier

https://orcid.org/0000-0003-0989-0102

AccessType

Open Access Dissertation

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2022

Month Degree Awarded

May

First Advisor

Jeanne A. Hardy

Second Advisor

Eric R. Strieter

Third Advisor

Richard W. Vachet

Fourth Advisor

Scott C. Garman

Subject Categories

Biochemistry | Biophysics | Molecular Biology | Structural Biology

Abstract

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.

DOI

https://doi.org/10.7275/28615334

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