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

研究溶酶体蛋白的研究通常集中于单个蛋白质或几种具有代表性的蛋白质组，这取决于它们在Western blot或qPCR实验中的检测难易程度。使用这些成熟的方法，并行研究所有已确认的溶酶体蛋白是非常复杂的。此外，不可能确定它们的绝对数量，它们的拷贝数或这些蛋白质存在于溶酶体中的化学计量。为了研究这一点，我们设计了合成标准蛋白——所谓的QConCats （Pratt et. al . 2006）——它允许从任何小鼠来源的样品中对153个已确认的溶酶体蛋白中的144个进行绝对定量。qconcat是一种人工蛋白质，由几种待定量蛋白质的色氨酸组成。QConCats在稳定同位素标记的培养基中表达，因此可以添加到样品中，允许对感兴趣的蛋白质进行绝对定量。在胰蛋白酶消化后，QConCat释放同位素标记的肽，它们中的每一个都作为内源性蛋白产生的未标记肽的内标。这允许执行肽的绝对定量，因此相关的蛋白质。这是通过所谓的三重四极杆质谱仪在多反应监测（MRM）模式下运行来实现的。一旦建立，这些检测是高度稳健的，线性的，并且允许从高度复杂的混合物（整个细胞或组织裂解物）中定量所需的肽（因此是蛋白质），而无需事先富集或分离。我们生成并验证了12个重同位素标记的qconcat，每个qconcat含有12种溶酶体蛋白的代表肽，允许对144种目前已知的溶酶体蛋白进行绝对定量。我们称这些标准为溶酶体QConCats （lysoqcc）。该项目将建立条件，在此条件下，这些lysoqcc可用于确定小鼠全细胞裂解物或组织中溶酶体蛋白的绝对数量。该技术将用于量化转录因子EB （TFEB）及其近亲TFE3对144种已确认的溶酶体蛋白表达水平的调控。转录因子EB被认为是溶酶体生物发生的主要调控因子。广泛的TFEB转录后修饰（ptm）导致一系列TFEB同种异构体。我们将分析这些不同亚型的PTMS，以及它们受ERK、mTORC1、GSK3ß和PKC信号转导途径的影响程度。基于这些样品中溶酶体蛋白的LysoQCC定量，我们将进一步确定选择的翻译后修饰如何影响TFEB控制的溶酶体蛋白谱。

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.