Maria Arshad, Nhung Hong Vu and Eiríkur Steingrímsson
Introduction: TFEB and TFE3, members of the MiT/TFE family of bHLHZip transcription factors, contain large intrinsically disordered regions (IDRs) at their C-termini. These regions have multiple sites for post-translational modifications (PTMs) that potentially affect the proteins’ stability, localization, and function. While TFEB/3 overlap in roles such as autophagy, lysosomal biogenesis, and oncogenesis, their IDR-mediated structural dynamics and PTM regulation are not fully understood. This study aims to elucidate the structure-function relationship of IDRs in TFEB/3, focusing on their roles in subcellular localization and stability. Additionally, it seeks to identify interacting proteins affected by PTMs to pinpoint specific regions and residues governing TFEB/3 dynamics.
Methods and data: Constructs with C-termini removed and PTM sites mutated were transfected into A375P cells. Subcellular localization was determined using western blots of nuclear and cytoplasmic fractions. Likewise, cycloheximide was used to assess protein stability. Protein interactions will be determined using mass spectrometry,. The shortlisted candidates will be further characterized for protein-protein interactions using co-immunoprecipitation and co-localization studies as well as functional assays to determine role of the interactions.
Results: C-terminal truncated TFEB/3 are found to be accumulated in the nucleus more than their full-length counterparts, with truncated TFEB showing reduced stability also. These findings suggest the C-terminus plays a crucial role in the subcellular localization, stability, and transcriptional activity of TFEB/3.
Conclusion: This study will provide insights into the regulatory mechanisms of TFEB/3, contributing to our understanding of disease mechanisms and the development of targeted therapies.
