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Establishing a molecular connection between GDAP1 and the products of oxidative stress

Googins, Matthew (2023) Establishing a molecular connection between GDAP1 and the products of oxidative stress. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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The Ganglioside-induced Differentiation Associated Protein 1 (GDAP1) is a novel member of the glutathione-S-transferase (GST) family of detoxification enzymes. GDAP1 has the core binding domains found in GST enzymes, but has additional unique domains of unknown function. Furthermore, GDAP1 is tail anchored to the outer mitochondrial membrane, where it regulates mitochondrial fission, mitochondrial trafficking, and protects the cell from changes in redox status. The molecular interactions underpinning these functions remain elusive. GDAP1 may be defending the cell from changes in redox state like a GST enzyme, but findings are conflicted over whether GDAP1 has GST-like enzymatic activity. Additionally, research examining the unique domains of GDAP1 has identified that they contribute to its mitochondrial localization and function. To understand how GDAP1 functions on the mitochondria, I took a structural and biochemical approach to characterize the molecular interactions of GDAP1 and determine how its different domains contributed to these interactions.
The goal of my work is to determine the structure and function of GDAP1. With this focus in mind, I obtained the first crystal structure of GDAP1. My structure data indicated that GDAP1 possesses a GST-like core similar to canonical GSTs, but with specific changes that appear to prevent glutathione binding. This also explains the lack of GST-like enzymatic activity observed by our lab and others. I then determined that the GST-binding pocket of GDAP1 has the capacity to interact with 4-hydroxynonenal (4HNE), a product formed from the decomposition of peroxidated lipids. These findings forged a direct molecular connection between GDAP1 activity and oxidative stress. Interestingly, GDAP1 interacts with 4HNE through both covalent and non-covalent interactions, both of which are dependent on GDAP1’s unique α-Loop domain. Lastly, I observe interactions with other small molecules such as benzbromarone and copper. These interactions promote the formation of higher-order GDAP1 species beginning with a dimer and subsequently transitioning into larger oligomeric species. Together these findings establish that GDAP1 is using its GST-Like binding pocket in conjunction with its unique α-Loop domain to interact with the byproducts of oxidative stress on the outer mitochondrial membrane.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Googins, Matthewmrg81@pitt.edumrg810000-0002-9024-269X
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairVanDemark, Andrewandyv@pitt.eduandyv0000-0003-3424-4831
Committee MemberBerman, Andreaajb190@pitt.eduajb1900000-0003-1217-7412
Committee MemberBrodsky, Jeffreyjbrodsky@pitt.edujbrodsky0000-0002-6984-8486
Committee MemberKiselyov, Kirillkiselyov@pitt.edukiselyov0000-0001-6683-2895
Committee MemberFord, Marijnmarijn@pitt.edumarijn0000-0002-9115-4413
Date: 25 January 2023
Date Type: Publication
Defense Date: 16 November 2022
Approval Date: 25 January 2023
Submission Date: 9 December 2022
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 187
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: GDAP1, Ganglioside-induced differentiation-associated protein 1; GST, Glutathione S-transferase; GSH, glutathione; 4-hydroxy-2-nonenal, 4HNE; Malondialdehyde, MDA; CMT, Charcot-Marie Tooth; PBD, Protein Data Bank; R.M.S.D., Root Mean Square Deviation; OD, optical density; Ethacrynic Acid, EA
Date Deposited: 25 Jan 2023 16:13
Last Modified: 25 Jan 2023 16:13


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