DOI
https://doi.org/10.25772/1TGF-6404
Author ORCID Identifier
https://orcid.org/0009-0004-8849-1103
Defense Date
2024
Document Type
Thesis
Degree Name
Master of Science
Department
Biochemistry
First Advisor
Can E Senkal
Second Advisor
Tomasz Kordula
Abstract
Sphingolipid dysregulation has been associated with a myriad of neurodegenerative pathologies such as lysosomal storage diseases (LSDs), hereditary sensory and autonomous neuropathy (HSAN) and amyotrophic lateral sclerosis (ALS). Within sphingolipid metabolism, ceramide has been highlighted as a vital signaling molecule, often determining the cell’s fate, by playing roles in various cell signaling pathways such as cell cycle arrest, apoptosis, senescence, stress responses and tumor metastasis. It is important to highlight that ceramides, through the action of ceramide synthases, also play a role in the regulation of autophagy, which is defined as a lysosome-based degradation system in eukaryotes critical for cellular homeostasis. In this sense, understanding autophagic flux is vital to all autophagy studies. Ferritinophagy is a specialized autophagic process whereby the iron storage protein, ferritin, is transported to the lysosome for degradation, a process necessary for maintaining iron homeostasis, and critical for the normal physiological activity of cells. Current methods of quantitating autophagic flux come with their swarm of intricacies such as altering lysosomal pH, or reproducibility issues. Here, we introduce a HaloTag-FTH1 and a tetramethylrhodamine (TMR) conjugated ligand system that allows us to quantitate the amount of ferritin being degraded in mammalian lysosomes over time, defined as ferritinophagic flux. Additionally, preliminary data from our lab showed that inhibition of ceramide synthase 1 (CerS1) prevents ferroptotic induced cell death in mammalian cell culture. Aligned with our suspicions, we show that the pharmacological CerS1 inhibitor, P053, abrogates DFO induced ferritinophagy in HeLa cells stably expressing Halotag-FTH1.
Rights
© Sachin Kumar Kempelingaiah
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-9-2024