Understanding Lysosomal Regulation in Wilson Disease Model through TFEB and its Mutants

Ashok, Borkar Aditya (2025) Understanding Lysosomal Regulation in Wilson Disease Model through TFEB and its Mutants. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Borkar Aditya Ashok (20MS212)) 20MS212_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

Lysosomes act as a center for degradation of various biomolecules, nutrient retrieval systems and signalling hubs for various physiological processes. The transcriptional regulation of lysosomes is modulated by TFEB. Transcription Factor EB (TFEB) is one of the member of bHLZ-ZIP MiT/TFE transcription factor family and a master regulator of lysosomal biogenesis and autophagy. TFEB activity is stringently regulated by phosphorylation-dependent subcellular localization. In nutrient-rich conditions, active mTORC1 phosphorylates TFEB, leading to its retention in the cytoplasm. Conversely, under nutrient starvation and lysosomal dysfunction conditions, TFEB gets dephosphorylated and translocates into the nucleus to induce target gene expression. While a number of phosphorylation sites on TFEB have been identified before, the individual role of each site in TFEB regulation is still not fully understood. Here we sought to unpack the functional role of putative phosphorylation sites on TFEB. A collection of single-point alanine replacement mutants was constructed and assayed for changed subcellular distribution by fluorescence microscopy. Six mutants (S122A, S134A, S138A, S142A, S211A, and S467A) always localized to the nucleus, implying defective cytoplasmic retention and perhaps loss of mTORC1-dependent control. To evaluate their transcriptional proficiency, we explored lysosomal biogenesis through expression of LAMP2, a lysosomal marker. Surprisingly, there were no significant variations in puncta or content of lysosomes between cells with mutants and controls, suggesting that nuclear-localizing mutants are either transcriptionally inactive or handicapped by cellular stress. Our results provide new information about TFEB regulation and suggest the complexity of control of its activity by phosphorylation, which may lead to improved lysosome-related cellular homeostasis under physiological and stressful conditions.

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