Regulation mechanisms and signaling pathways of autophagy

Regulation mechanisms and signaling pathways of autophagy. membrane-bound organelles present in all cell types. Their role in degradation and recycling processes has been extensively characterized1C3. The lysosomal lumen acidic pH with the presence of a broad variety of hydrolases able to degrade an sufficient spectrum of substrates, make these organelles remarkable machineries for the recycling of cellular waste. Extracellular substrates reach the lysosome mainly via the endocytic and phagocytic pathways, while intracellular substrates are the delivered to the lysosome by the autophagic pathway via the fusion of autophagosomes with lysosomes4, 5. Thus, lysosomes are the terminal end of most cellular catabolic pathways. The role of the lysosomes in degradation and recycling processes has always been considered as a cellular housekeeping function and little attention has been paid to the regulation of these processes and to the possible influence of environmental cues, such as starvation and physical exercise. The discovery that this Transcription Factor EB (TFEB) is usually a grasp regulator of lysosomal and autophagic function and of energy metabolism6C8 suggested that environmental cues may control lysosomal function via the induction of a broad transcriptional program. TFEB activity is usually regulated by phosphorylation9C13, which keeps TFEB inactive in the cytoplasm, while dephosphorylated TFEB travels to the nucleus to activate transcriptional target genes. TFEB phosphorylation is usually mediated by mTORC1, a major kinase complex that positively regulates cell growth and negatively regulates autophagy. Interestingly, it is known mTORC1 exerts its activity around the lysosomal surface and is positively regulated by lysosomal nutrients14, 15. The regulation of TFEB by lysosomal mTORC1 and the shuttling of TFEB to AMG 548 the nucleus revealed a lysosome-to-nucleus signaling mechanism9. Thus, these studies identify the lysosome as a signaling hub that controls cellular homeostasis via both post-translational and transcriptional mechanisms14C17. Another aspect of lysosomal function underestimated in the past is the ability of lysosomes to store Ca2+ and to participate to calcium signaling processes. Several calcium channels reside around the lysosomal membrane. Recent studies have investigated the role of these lysosomal calcium channels in fundamental cellular processes and their involvement in disease mechanisms18. In addition, the recent discoveries of calcium microdomains, which mediate local calcium signals from several intracellular compartments (e.g. mitochondria)19, further suggest the involvement of the lysosome in intracellular calcium signaling. In the present study, while searching for a phosphatase that de-phosphorylates TFEB, we discovered another example of lysosomal signaling. We revealed a calcium signaling mechanism that starts at the lysosome and controls autophagy via calcineurin-mediated induction of TFEB. Calcineurin modulates TFEB subcellular localization Previous studies exhibited that mTOR-mediated phosphorylation of TFEB serine residues Ser142 and Speer4a Ser211 promotes the conversation of TFEB with the 14-3-3 protein and results in a cytoplasmic localization. Conversely, conditions that lead to mTOR inhibition, such as starvation AMG 548 and lysosomal stress, promote TFEB nuclear translocation and transcriptional activation of lysosomal and autophagic genes6, 7, 9, 14, 15, 17. While the role AMG 548 of the kinases that mediate TFEB phosphorylation has been defined by previous studies9C13, the phosphatase(s) involved in its de-phosphorylation have remained AMG 548 elusive. To identify the phosphatase(s) that de-phosphorylate(s) TFEB we performed a High Content (HC) screening of a phosphatase siRNA library using a cellular assay based on cytoplasm-to-nucleus shuttling of TFEB during starvation9. We tested the effects of the specific inhibition of each of 231 AMG 548 phosphatases or putative phosphatases on TFEB subcellular localization. The most significant hit recognized by the primary screening was the calcineurin catalytic subunit isoform beta (PPP3CB; Gene ID:5532)20, thus we focused subsequent studies exclusively on this phosphatase. Fig. 1a shows that inhibition of PPP3CB suppressed starvation-induced nuclear translocation of TFEB. The ability.

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