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Development of Assays and Irreversible Inhibitors for ATG4B: A Key Cysteine Protease Implicated in the Process of Autophagy

Resource type
Thesis type
(Thesis) Ph.D.
Date created
2015-04-21
Authors/Contributors
Abstract
Autophagy is a biological process responsible for the sequestration and degradation of cellular components for the purposes of maintaining homeostasis. ATG4B is a cysteine protease that plays a key role in the initiation of autophagy by cleaving the protein proLC3 revealing a C-terminal glycine residue and to form LC3-I. This glycine then becomes lipidated with phosphatidylethanolamine (PE) to form LC3-II which is inserted into partial protomembrane fragments in the Golgi which stimulates formation of autophagosomes. ATG4B also cleaves PE from the membrane bound LC3-II to reform and recycle LC3-I and to facilitate fusion of the autophagosomes with liposomes where the contents are subsequently digested producing energy and amino acids for protein synthesis. As cancer cells frequently “hijack” autophagy as a survival and resistance mechanism against therapy, autophagy has recently emerged as a potential therapeutic target for treating cancer. The development of two different assay methods for measuring ATG4B activity are reported. The first, LC/MS-based, assay monitors the cleavage of proLC3 to LC3-I based on the ratio of peak heights detected. This method has the added utility of being able to detect enzyme integrity throughout the reaction. The second, FRET-based, assay involves a novel YFP-LC3B-EmGFP doubly fluorescent protein substrate developed for the purposes of large scale screening of compound libraries. The FRET-based assay was successfully used to screen 5000 compounds in the commercial LOPAC and KD2 compound libraries with an overall hit rate of 0.6% and 0.5% respectively. Irreversible inhibitors of ATG4B were designed and synthesized based on information from the screening results as well as previous work on putative peptide substrates conducted by Dr. Nag Kumar (Young Lab, SFU). Inhibitors of the halo-methyl ketone type were developed and tested for inhibitory activity against ATG4B. Fluorescent analogues of these irreversible inhibitors were further developed and optimized for in cellulo potency. Kinetic analysis and digestion studies revealed the inhibitors were indeed active-site directed ATG4B inhibitors.
Document
Identifier
etd8999
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Scholarly level
Supervisor or Senior Supervisor
Thesis advisor: Young, Robert
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