板倉 英祐

The regulation of cellular metabolism is crucial for cell survival, with Sch9 in Saccharomyces cerevisiae serving a key role as a substrate of TORC1. Sch9 localizes to the vacuolar membrane through binding to PI(3,5)P2, which is necessary for TORC1-dependent phosphorylation. This study demonstrates that cytosolic pH regulates Sch9 localization. Under stress conditions that induce cytosolic acidification, Sch9 detached from the vacuolar membrane. In vitro experiments confirmed that Sch9's affinity for PI(3,5)P2 is pH-dependent. This pH-dependent localization switch is essential for regulating the TORC1-Sch9 pathway. Impairment of the dissociation of Sch9 from the vacuolar membrane in response to cytosolic acidification resulted in the deficient induction of stress response gene expression and delayed the adaptive response to acetic acid stress. These findings indicate the importance of proper Sch9 localization for metabolic reprogramming and stress response in yeast cells.

The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including α-synuclein (αSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble αSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.

The endosomal-lysosomal system represents a crucial degradation pathway for various extracellular substances, and its dysfunction is linked to cardiovascular and neurodegenerative diseases. This degradation process involves multiple steps: (1) the uptake of extracellular molecules, (2) transport of cargos to lysosomes, and (3) digestion by lysosomal enzymes. While cellular uptake and lysosomal function are reportedly regulated by the mTORC1-TFEB axis, the key regulatory signal for cargo transport remains unclear. Notably, our previous study discovered that isorhamnetin, a dietary flavonoid, enhances endosomal-lysosomal proteolysis in the J774.1 cell line independently of the mTORC1-TFEB axis. This finding suggests the involvement of another signal in the mechanism of isorhamnetin. This study analyzes the molecular mechanism of isorhamnetin using transcriptome analysis and reveals that the transcription factor GATA3 plays a critical role in enhanced endosomal-lysosomal degradation. Our data also demonstrate that mTORC2 regulates GATA3 nuclear translocation, and the mTORC2-GATA3 axis alters endosomal formation and maturation, facilitating the efficient transport of cargos to lysosomes. This study suggests that the mTORC2-GATA3 axis might be a novel target for the degradation of abnormal substances.

Lysosomes are intracellular organelles responsible for degrading diverse macromolecules delivered from several pathways, including the endo-lysosomal and autophagic pathways. Recent reports have suggested that lysosomes are essential for regulating neural stem cells in developing, adult and aged brains. However, the activity of these lysosomes has yet to be monitored in these brain tissues. Here, we report the development of a new probe to measure lysosomal protein degradation in brain tissue by immunostaining. Our results indicate that lysosomal protein degradation fluctuates in neural stem cells of the hippocampal dentate gyrus, depending on age and brain disorders. Neural stem cells increase their lysosomal activity during hippocampal development in the dentate gyrus, but aging and aging-related disease reduce lysosomal activity. In addition, physical exercise increases lysosomal activity in neural stem cells and astrocytes in the dentate gyrus. We therefore propose that three different stages of lysosomal activity exist: the state of increase during development, the stable state during adulthood and the state of reduction due to damage caused by either age or disease.

The spatiotemporal sequestration of misfolded proteins is a mechanism by which cells counterbalance proteome homeostasis upon exposure to various stress stimuli. Chronic inhibition of proteasomes results in a large, juxtanuclear, membrane-less inclusion, known as the aggresome. Although the molecular mechanisms driving its formation, clearance, and pathophysiological implications are continuously being uncovered, the biophysical aspects of aggresomes remain largely uncharacterized. Using fluorescence recovery after photobleaching and liquid droplet disruption assays, we found that the aggresomes are a homogeneously blended condensates with liquid-like properties similar to droplets formed via liquid-liquid phase separation. However, unlike fluidic liquid droplets, aggresomes have more viscosity and hydrogel-like characteristics. We also observed that the inhibition of aggresome formation using microtubule-disrupting agents resulted in less soluble and smaller cytoplasmic speckles, which was associated with marked cytotoxicity. Therefore, the aggresome appears to be cytoprotective and serves as a temporal reservoir for dysfunctional proteasomes and substrates that need to be degraded. Our results suggest that the aggresome assembles through distinct and potentially sequential processes of energy-dependent retrograde transportation and spontaneous condensation into a hydrogel.