br STAR Methods br Acknowledgments We thank
Acknowledgments We thank the Shanghai Synchrotron Radiation Facility (SSRF) BL18U for help with X-ray data collection. We thank Dr. Thomas F.J. Martin for providing the pcDNA3.1-CAPS-1 plasmid and Dr. Xiaofei Yang for providing the pFHUUIG_shortU6 (l309) plasmid. We thank Dr. Shun Zhao for helping to optimize the structure and Yaru Hu for helping with CD data collection. This work was supported by grants from the National Natural Science Foundation of China (31670846, 31670850, and 31721002), the National Key Basic Research Program of China (2015CB910800), and funds from Huazhong University of Science and Technology, China.
Introduction Production of reactive oxygen species (ROS) normally accompanies mitochondrial and cytoplasmic enzymatic activities , . It can be accelerated by mitochondrial damage and by toxic compounds that promote ROS production . The cellular defense system comprising catalases and superoxide dismutases effectively fights ROS at near-physiological conditions , , , , , . However, a significant imbalance towards the ROS accumulation leads to oxidative stress and to organellar and cytoplasmic damage. Oxidative stress causes lipid peroxidation , , , and the ensuing loss of membrane integrity damages membrane organelles. The injured mitochondria and lysosomes accelerate the damage by stimulating ROS production and by leaking the digestive enzymes and pro-apoptotic factors , , , , , , . The repair of oxidative damage necessitates a rapid elimination of the damaged organelles by autophagy, which depends upon proper lysosomal function , , , , , . Oxidative stress damages the cytoplasmic proteins as well, leading to protein aggregation. Elimination of such GSK1363089 depends on autophagy, which, in turn, depends on lysosomes as well. The endocytic/autophagic/lysosomal roles in cell health are not limited to degradation, but also involve the capture and rapid evacuation of organelles and cytoplasmic compartments. Indeed, lysosomal and autophagic exocytosis has recently emerged as an important cytoprotective mechanism , , , , , , , , , , . Lysosomal exocytosis was observed more than 50 years ago as a mechanism to limit focal cellular injuries by sequestration followed by digestion or extrusion . Although early studies focused upon its role in a plasma-membrane repair , exocytosis has recently re-emerged as an important component of the cellular clearance pathway. Upregulation of lysosomal exocytosis is proposed to underlie the therapeutic effect of TFEB overexpression in several disease models , , . The lysosomal divalent cation channel TRPML1 was proposed to drive lysosomal exocytosis because of suppression of lysosomal exocytosis observed in mucolipidosis type IV models , . The proposed underlying mechanisms involves releasing the calcium ions stored in lysosomes , which actuates the conformational change in SNARE that drive the fusion of the lysosomes with the plasma membrane. A role of TRPML1 in lysosomal traffic and positioning  is likely to contribute to the exocytosis process as well. Finally, TRPML1 has been proposed to facilitate the expulsion of the copper-filled lysosomes , a process that is crucial for limiting oxidative stress , . The recent evidence of TRPML1 activation by oxidative stress  raises the possibility that lysosomal, exocytosis responds to oxidative stress as well. Such a response would modulate the cellular clearance pathways in a manner integrating signals from oxidative stress and cellular energy sending pathway due to the regulation of TRPML1 expression via mTORC1 and TFEB/TFE3. As the details of the lysosomal exocytosis mechanisms are emerging, the complexity of the machinery that drives it is becoming apparent. This provides opportunities for pharmacological interventions into many diseases, as many additional targets of pathologies can now be recognized. We have recently shown that lysosomal exocytosis limits the oxidative damage caused by transition metal exposure , . A surprising finding that emerged in the course of our previous studies is that while short-term exposure to transition metals stimulated lysosomal exocytosis, long-term exposure had an inhibitory effect , . Since the long-term metal exposure is associated with oxidative stress , , here we investigate the role of oxidative stress in modulating lysosomal exocytosis and provide novel insights into its mechanisms.