Quantitative determination of the effect of transcription factors TFEB and TFE3 on the expression of lysosomal proteins

• Applicants: Professor Dr. Volkmar Gieselmann; Dr. Dominic Winter
• Subject Area: Cell Biology
• Term: from 2017 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 323732846

Studies investigating lysosomal proteins usually focus either on a single protein or on a representative group of several proteins, dependent on their ease of detection in Western blot or qPCR experiments. Using these well-established methods, it is very intricate to investigate all confirmed lysosomal proteins in parallel. Furthermore, it is impossible to determine their absolute amounts, their copy numbers or the stoichiometry at which these proteins are present in the lysosome. In order to investigate this, we designed synthetic standard proteins - so-called QConCats (Pratt et. al 2006) - which allow for absolute quantification of 144 out of 153 confirmed lysosomal proteins from any sample of mouse origin. QConCats are artificial proteins which consist of tryptic peptides from several proteins which are to be quantified. The QConCats are expressed in stable isotope labeled medium and can therefore be added to the sample allowing for absolute quantification of the proteins of interest. Upon tryptic digestion, the QConCat releases the isotope labelled peptides and each of them serves as internal standard for the non-labelled peptide produced by the endogenous protein. This allows to perform absolute quantification of the peptides, and therefore the correlating proteins. This is achieved using so-called triple quadrupole mass spectrometers operated in the multiple reaction monitoring (MRM) mode. Once established, these assays are highly robust, linear and allow to quantify the desired peptides (and therefore proteins) from highly complex mixtures (whole cell or tissue lysates) without prior enrichment or fractionation. We generated and verified 12 heavy isotope labelled QConCats containing representative peptides for 12 lysosomal proteins each, allowing for the absolute quantification of 144 currently known lysosomal proteins. We call these standards Lysosomal QConCats (LysoQCCs). The project will establish the conditions under which these LysoQCCs can be used to determine the absolute amounts of lysosomal proteins from murine whole cell lysates or tissues. The technique will be used to quantify the regulation of the expression levels of the 144 confirmed lysosomal proteins by the transcription factor EB (TFEB), which is considered as a master regulator of lysosomal biogenesis, and its close relative TFE3. Extensive posttranscriptional modifications (PTMs) of TFEB lead to an array of TFEB isoforms. We will analyze the PTMS of these various isoforms and in how far they are influenced by ERK, mTORC1, GSK3ß and PKC signal transduction pathways. Based on LysoQCC quantification of lysosomal proteins in these samples, we will furthermore determine how selected posttranslational modifications will affect the spectrum of lysosomal proteins controlled by TFEB.

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