Powers, Alyssa E
(2024)
Identification and characterization of TANGO2 protein and its interaction with metabolites.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
Pathogenic variants in the Transport and Golgi Organization 2 Homolog (TANGO2) gene lead to an autosomal recessive disorder associated with TANGO2 deficiency. The disorder can exhibit a spectrum of clinical presentations, varying in severity. Patients may experience rhabdomyolysis, acute metabolic crises, cardiomyopathy, and/or cardiac arrhythmias. Studies suggest TANGO2’s involvement in mitochondrial dysfunction and endoplasmic reticulum/Golgi trafficking, yet its exact function remains unknown. Currently, there is no known effective treatment for TANGO2 Deficiency Disorder (TDD). Recent research revealed that pantothenic acid (vitamin B5) supplementation can ameliorate clinical symptoms and rescue the phenotype in TANGO2 mutant Drosophila, though the mechanism remains unclear. Until recently, there was no confirmed crystal structure of the TANGO2 protein. I hypothesize that the determination of the TANGO2 protein three-dimensional structure and its tissue expression can shed light on its function and interaction with other proteins/metabolites.
TANGO2 cDNA with a C-terminal His-tag coding motif and wildtype TANGO2 cDNA without tag were cloned in pET-27b(+) expression plasmids. BL21, containing a plasmid expressing GroEL and GroES chaperonins, bacterial cells were transformed using both plasmids via electroporation. The TANGO2 C-His Tagged protein was purified by fast protein liquid chromatography (FPLC) using the ÄKTA Pure chromatography system, a HisTrap column, and a MonoQ column. The wildtype without tag TANGO2 was purified by ammonium sulfate fractionation, a DEAE Sepharose column using two different buffer conditions, and a MonoQ column. Elution from the column was followed using SDS-PAGE/western blot. The level of purity was assessed using SDS-PAGE gel and protein bands visualized by Coomassie blue staining. Western blotting was used for surveying TANGO2 expression in mouse tissues.
The purified C-His tagged TANGO2 protein was used to obtain the TANGO2 crystal structure as well as screening potential metabolites that interact with the TANGO2 protein. The screening of mice tissues showed universal TANGO2 expression in all tissues as well as no discernable difference between males’ and females’ tissues. The heart and brain tissue had the highest TANGO2 expression levels. This research holds public health significance because it can advance our knowledge of TANGO2 structure/function and guide future efforts to develop an effective treatment for TDD patients.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
19 August 2024 |
| Date Type: |
Publication |
| Defense Date: |
29 July 2024 |
| Approval Date: |
19 August 2024 |
| Submission Date: |
8 August 2024 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
65 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Public Health > Human Genetics |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Protein Purification
TANGO2 |
| Date Deposited: |
19 Aug 2024 19:56 |
| Last Modified: |
19 Aug 2024 19:56 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/46879 |
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