Electronic Theses and Dissertations

Esmat Fathi

Date

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biology

Committee Chair

Omar Skalli

Committee Member

Ramin Homayouni

Committee Member

Amy Abell

Committee Member

Judith Cole

Committee Member

Carlos Estrano

Abstract

Since their discovery over two decades ago, the molecular and cellular functions of the Nipsnap family of proteins (Nipsnaps) have remained elusive until recently. Nipsnaps interact with a variety of mitochondrial and cytoplasmic proteins. They have been implicated in multiple cellular processes and associated with different physiologic and pathologic conditions, including pain transmission, Parkinsons disease, and cancer. Recent evidence demonstrated a direct role for Nipsnap1 and Nipsnap2 proteins in regulation of mitophagy, a process that is critical for cellular health and maintenance. Our group has previously reported an interaction between Nipsnap1 and the Alzheimers disease amyloid precursor protein. In addition, Nipsnap1 binds to pyruvate dehydrogenase (PDH), which controls the critical step of converting pyruvate to acetyl-CoA and connects cytosolic glycolysis to mitochondrial TCA cycle. Importantly, Nipsnaps contain a 110 amino acid domain that is evolutionary conserved from mammals to bacteria. However, the molecular function of the conserved Nipsnap domain and its potential role in mitophagy and regulation of mitochondria have not been explored. Recently, using protein structure modeling and virtual ligand screening, our group identified NAD and NADP as potential ligands for the conserved Nipsnap1 domain.The goal of my studies was to investigate the physiological function of Nipsnap1 interaction with PDH and potential ligands such as NAD, NADP and ATP. In the first study, we show that Nipsnap1 deficiency does not have a significant effect on PDH protein level nor PDH enzyme activity in the brain and liver tissues of Nipsnap1 knock-down mice. In addition, overexpression of Nipsnap1 did not change PDH enzyme activity in HEK293 cells. This study suggests that Nipsnap1 may bind to PDH, possibly for targeting its localization, but does not affect PDH enzyme activity. In the second study, we show that the highly conserved C-terminal region of Nipsnap1 directly binds to NAD, NADP and ATP. These interactions were confirmed using in vitro biochemical binding assays. In addition, using site-directed mutagenesis, we show that Nipsnap1 interaction with NAD and ATP requires specific amino acid residues corresponding to a binding pocket within its conserved C-terminal domain. Dual binding of NAD and ATP to the same binding pocket is unusual. However, it raises an intriguing possibility that the highly conserved Nipsnap domain interacts with a substrate that is ubiquitously present across all species and that it might act as a sensor for mitochondrial health.

Comments

Data is provided by the student.

Library Comment

Dissertation or thesis originally submitted to ProQuest

Notes

Open Access

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