Investigation of the <em>in vivo</em> Lysosomal Proteome Changes in Different Starvation Periods

Abstract

Nutrient deprivation can change the activity and subcellular localization of a protein. The cellular response to starvation includes the autophagy/lysosomal degradation pathway and the nutrient signaling pathway via the mechanistic target of rapamycin complex 1 (mTORC1) as the regulator of cell growth. The current study investigates the impact of <em>in vivo</em> starvation on the lysosomal proteome and on cellular metabolism. Before analyzing the lysosomal proteome, the activity of mTORC1 displayed different activity patterns in three mouse metabolic organs. It was deactivated/reactivated after short/prolonged starvation periods, respectively, in the liver, gradually deactivated in the skeletal muscle, and continuously activated in the brain. <br /> In the second part of this study, the two selected starvation periods with distinct molecular effects in the liver had different impacts on the body metabolism after measuring the levels of essential metabolites. Glucose levels were stable at 6 hours of starvation, while the ketogenesis started to occur. Furthermore, the decrease in glucose levels after 24 hours was accompanied by a significant increase in ketogenesis. Moreover, the levels of long-chain acylcarnitines significantly increased after prolonged starvation as a marker of potential fatty acid beta-oxidation. In addition, the increased levels of muscle protein breakdown biomarkers indicated an activation of gluconeogenesis. <br /> The last part of the thesis focused on the analysis of starvation-induced changes in liver lysosomal/non-lysosomal protein amounts by quantitative mass spectrometry of enriched lysosomes. The proteomic analysis quantified several lysosomal downregulated proteins linked to the mTOR signaling pathway. Western blot analysis depicted a decreased abundance of mTORC1 and Rag complex after short starvation and an increase after 24 hours of starvation, while a continuous decrease in LAMTOR complex levels was monitored during starvation, suggesting a Ragulator-independent mTORC1 reactivation. Moreover, non-lysosomal proteins were also quantified and the selected proteins included biomarkers of glycogen metabolism and gluconeogenesis. In addition, the data depicted a persistent decreased levels of the endosomal WASH complex in the lysosomal fraction during starvation. Moreover, the results showed a significant upregulation in 12 proteasome subunits during starvation indicating a continuously induced proteaphagy. In contrast, starvation-induced autophagy displayed a significant upregulation of 12 peroxisomal proteins and 54 mitochondrial proteins after short and prolonged starvation, respectively, hypothesizing an early pexophagy and a late mitophagy, and therefore an ordered organelle degradation.