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Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2019

Month Degree Awarded

February

First Advisor

Jeanne A. Hardy

Subject Categories

Biochemistry

Abstract

Proteases are powerful enzymes with the intrinsic ability to proteolytically cleave other proteins, which often leads to a gain or loss of function. While proteases from the same family often share high similarity in the active site region, distal sites on the enzyme, such as allosteric sites, can differentiate closely related enzymes and allow for specific regulation. This dissertation investigates two different types of proteases, including the viral proteases from both Zika and dengue virus and the mammalian caspase proteases, and contributes significantly to the understanding of both allosteric sites and exosites in these enzymes.

Flaviviruses, including Zika virus and dengue virus, continue to threaten public health. The associated viral protease, known as NS2B-NS3pro, is essential to the viral life cycle and can interfere with host responses, making it a drug target of considerable interest. This work has made significant progress towards uncovering NS2B-NS3pro functionality on multiple fronts. After identifying 31 novel human substrates of Zika NS2B-NS3pro, we further found that the NS2B cofactor region acts as an exosite and is the principal determinant for Zika protease substrate selection. Furthermore, we identified compound MH1 that binds to an allosteric site specific to Zika NS2B-NS3pro with inhibitory capabilities unmatched against this enzyme. In addition to the work on two flaviviral proteases, this dissertation also enhanced our understanding of exosites and allosteric sites in caspases. The caspases are cysteine proteases that initiate and execute programmed cell death, or apoptosis. By engineering the active site to develop a hybrid caspase, we were able to categorize substrates that were reliant on exosites, which may allow for specific regulation. Overall, these findings add to the knowledge of both flaviviral NS2B-NS3 proteases and caspases and should help future mechanistic efforts to exploit allosteric control over these enzymes.

Available for download on Thursday, February 01, 2024

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