TFEB Knockout Cell Line (HEK293)

Induction of autophagy is an ancient function of the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway through which autophagic cargoes are delivered to lysosomes for degradation. However, whether lysosome function is also modulated by the cGAS-STING pathway remains unknown. Here, we discovered that the cGAS-STING pathway upregulated lysosomal activity by stimulating lysosome biogenesis independently of the downstream protein kinase TANK-binding kinase 1 (TBK1). STING activation enhanced lysosome biogenesis through inducing the nuclear translocation of transcription factor EB (TFEB) as well as its paralogs transcription factor E3 (TFE3) and microphthalmia-associated transcription factor (MITF). STING-induced lipidation of GABA type A receptor-associated protein (GABARAP), an autophagy-related protein, on STING vesicles was responsible for TFEB activation. Membrane-bound GABARAP sequestered the GTPase-activating protein folliculin (FLCN) and FLCN-interacting protein (FNIP) complex to block its function toward the Rag GTPases Ras-related GTP-binding C and D (RagC and RagD), abolishing mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent phosphorylation and inactivation of TFEB. Functionally, STING-induced lysosome biogenesis within cells facilitated the clearance of cytoplasmic DNA and invading pathogens. Thus, our findings reveal that induction of lysosome biogenesis is another important function of the cGAS-STING pathway.

Stimulator of interferon genes (STING) is activated in many pathophysiological conditions, leading to TBK1-dependent interferon production in higher organisms. However, primordial functions of STING independent of TBK1 are poorly understood. Here, through proteomics and bioinformatics approaches, we identify lysosomal biogenesis as an unexpected function of STING. Transcription factor EB (TFEB), an evolutionarily conserved regulator of lysosomal biogenesis and host defense, is activated by STING from multiple species, including humans, mice, and frogs. STING-mediated TFEB activation is independent of TBK1, but it requires STING trafficking and its conserved proton channel. GABARAP lipidation, stimulated by the channel of STING, is key for STING-dependent TFEB activation. STING stimulates global upregulation of TFEB-target genes, mediating lysosomal biogenesis and autophagy. TFEB supports cell survival during chronic sterile STING activation, a common condition in aging and age-related diseases. These results reveal a primordial function of STING in the biogenesis of lysosomes, essential organelles in immunity and cellular stress resistance.

Oxidative stress underlies a number of pathological conditions, including cancer, neurodegeneration, and aging. Antioxidant-rich foods help maintain cellular redox homeostasis and mitigate oxidative stress, but the underlying mechanisms are not clear. For example, sulforaphane (SFN), an electrophilic compound that is enriched in cruciferous vegetables such as broccoli, is a potent inducer of cellular antioxidant responses. NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2), a transcriptional factor that controls the expression of multiple detoxifying enzymes through antioxidant response elements (AREs), is a proposed target of SFN. is a target gene of TFEB (transcription factor EB), a master regulator of autophagic and lysosomal functions, which we show here to be potently activated by SFN. SFN induces TFEB nuclear translocation via a Ca-dependent but MTOR (mechanistic target of rapamycin kinase)-independent mechanism through a moderate increase in reactive oxygen species (ROS). Activated TFEB then boosts the expression of genes required for autophagosome and lysosome biogenesis, which are known to facilitate the clearance of damaged mitochondria. Notably, TFEB activity is required for SFN-induced protection against both acute oxidant bursts and chronic oxidative stress. Hence, by simultaneously activating macroautophagy/autophagy and detoxifying pathways, natural compound SFN may trigger a self-defense cellular mechanism that can effectively mitigate oxidative stress commonly associated with many metabolic and age-related diseases. ANOVA: analyzes of variance; AREs: antioxidant response elements; Baf-A1: bafilomycin A; BHA: butylhydroxyanisole; CAT: catechin hydrate; CCCP: carbonyl cyanide m- chlorophenylhydrazone; CLEAR: coordinated lysosomal expression and regulation; DCFH-DA: 2',7'-dichlorofluorescin diacetate; FBS: fetal bovine serum; GFP: green fluorescent protein; HMOX1/HO-1: heme oxygenase 1; KD: knockdown; KEAP1: kelch like ECH associated protein 1; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MCOLN1/TRPML1: mucolipin 1; ML-SA1: mucolipin-specific synthetic agonist 1; ML-SI3: mucolipin-specific synthetic inhibitor 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NFE2L2/NRF2: nuclear factor: erythroid 2 like 2; NPC: Niemann-Pick type C; PBS: phosphate-buffered saline; PPP2/PP2A: protein phosphatase 2; Q-PCR: real time polymerase chain reaction; ROS: reactive oxygen species; RPS6KB1/S6K1/p70S6K: ribosomal protein S6 kinase B1; SFN: sulforaphane; TFEB: transcription factor EB; WT, wild-type.

Spastic paraplegia 21 is a neurodegenerative disease characterized by the degeneration of corticospinal axons. It is caused by mutations in the SPG21 gene, which encodes maspardin, a cytosolic protein of unknown function that associates with the late endosomal/lysosomal membrane. Intriguingly, we found that the phosphorylation level of the transcription factor EB (TFEB), a master regulator of the CLEAR gene network, is decreased in SPG21 knockout cells, leading to TFEB nuclear translocation. Our investigations revealed that the Rag-mediated presentation of TFEB to the mTOR kinase and its subsequent phosphorylation is disturbed by a delocalization of the RAB7 GTPase, a maspardin-binding partner, from retromer-positive late endosomes to lysosomes. This redistribution decreases the interaction between RAB7 and its GTPase-activating protein (GAP), TBC1D5. Consequently, RAB7 remains primarily GTP-bound, recruiting more FYCO1 to lysosomes and promoting the anterograde movement of these organelles along microtubules. These findings identify maspardin as a newly discovered RAB7 effector and shed light on several consequences of its deficiency.