Lysosomal gene transcription and protein expression

• Applicants: Professor Dr. Thomas Braulke; Dr. Dominic Winter
• Subject Area: Cell Biology
• Term: since 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 323732846

Proteins related to lysosomal biogenesis and the autophagy pathway are subject to global transcriptional regulation, in which the transcription factor EB (TFEB) and several repressors play key roles. The transcriptional activity of TFEB is regulated by phosphorylation/ dephosphorylation of multiple conserved serine residues which determine the localization of TFEB to be either at the surface of lysosomes, in the cytoplasm, or the nucleus. In the first funding period, we established a variety of mass spectrometry (MS) tools for the identification and quantification of TFEB posttranslational modifications (PTMs) and the targeted absolute quantification of 144 verified murine lysosomal proteins from any type of mouse sample. These approaches led to the identification of 30 phosphorylation and 7 acetylation sites in TFEB resulting in >30 isoforms which were visualized by 2D gel electrophoretic separation. Substitutions of various TFEB acetylation sites completely inhibited its transcriptional activity, which was due to cytosolic retention by unknown mechanisms. In the second funding period, we aim to analyze whether mutated acetylation sites cause secondary changes in the phosphorylation pattern. Additionally, we will define PTM patterns for individual TFEB isoforms, followed by combinatorial site-specific substitutions, analysis of subcellular localization, transcriptional activity, and the effect of these mutants on the proteome of lysosomes and their subpopulations. Furthermore, we plan to identify interaction partners of acetylated and non-acetylated TFEB and their role in transcriptional activation of lysosomal genes using co-immunoprecipitation and proximity biotinylation approaches in combination with MS-based proteomics. Finally, TFEB-independent mechanisms of transcriptional suppression of multiple lysosomal genes will be analyzed in lysosomal disease-related cells. These studies will provide deeper insight into the role of TFEB PTMs for autophagy and lysosomal function as well as the interplay between transcriptional activators and repressors.

• DFG Programme: Research Units
• Subproject of: FOR 2625: Mechanisms of Lysosomal Homeostasis