Structural basis for growth factor and nutrient signal integration on the lysosomal membrane by mTORC1.

Mechanistic target of rapamycin complex 1 (mTORC1), which consists of mTOR, Raptor, and mLST8, receives signaling inputs from growth factor signals and nutrients. These signals are mediated by the Rheb and Rag small GTPases, respectively, which activate mTORC1 on the cytosolic face of the lysosome membrane. We biochemically reconstituted the activation of mTORC1 on membranes by physiological submicromolar concentrations of Rheb, Rags, and Ragulator.

A SPLICS reporter reveals [Formula: see text]-synuclein regulation of lysosome-mitochondria contacts which affects TFEB nuclear translocation.

Mitochondrial and lysosomal activities are crucial to maintain cellular homeostasis: optimal coordination is achieved at their membrane contact sites where distinct protein machineries regulate organelle network dynamics, ions and metabolites exchange. Here we describe a genetically encoded SPLICS reporter for short- and long- juxtapositions between mitochondria and lysosomes. We report the existence of narrow and wide lysosome-mitochondria contacts differently modulated by mitophagy, autophagy and genetic manipulation of tethering factors.

TFEB and TFE3 control glucose homeostasis by regulating insulin gene expression.

To fulfill their function, pancreatic beta cells require precise nutrient-sensing mechanisms that control insulin production. Transcription factor EB (TFEB) and its homolog TFE3 have emerged as crucial regulators of the adaptive response of cell metabolism to environmental cues. Here, we show that TFEB and TFE3 regulate beta-cell function and insulin gene expression in response to variations in nutrient availability.

Structure of the lysosomal mTORC1-TFEB-Rag-Ragulator megacomplex.

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates.

Phosphorylation of FAM134C by CK2 controls starvation-induced ER-phagy.

Selective degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is initiated by ER-phagy receptors, which facilitate the incorporation of ER fragments into autophagosomes. FAM134 reticulon family proteins (FAM134A, FAM134B, and FAM134C) are ER-phagy receptors with structural similarities and nonredundant functions. Whether they respond differentially to the stimulation of ER-phagy is unknown.

Non-canonical mTORC1 signaling at the lysosome.

The mechanistic target of rapamycin complex 1 (mTORC1) signaling hub integrates multiple environmental cues to modulate cell growth and metabolism. Over the past decade considerable knowledge has been gained on the mechanisms modulating mTORC1 lysosomal recruitment and activation. However, whether and how mTORC1 is able to elicit selective responses to diverse signals has remained elusive until recently.

Fluoxetine ameliorates mucopolysaccharidosis type IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is an autosomal recessive disorder caused by mutations in SGSH involved in the degradation of heparan sulfate. MPS-IIIA presents severe neurological symptoms such as progressive developmental delay and cognitive decline, for which there is currently no treatment. Brain targeting represents the main challenge for therapeutics to treat MPS-IIIA, and the development of small-molecule-based treatments able to reach the CNS could be a relevant advance for therapy.

Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype.

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC).

The atypical small GTPase GEM/Kir is a negative regulator of the NADPH oxidase and NETs production through macroautophagy.

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS).

c-Abl Inhibition Activates TFEB and Promotes Cellular Clearance in a Lysosomal Disorder.

The transcription factor EB (TFEB) has emerged as a master regulator of lysosomal biogenesis, exocytosis, and autophagy, promoting the clearance of substrates stored in cells. c-Abl is a tyrosine kinase that participates in cellular signaling in physiological and pathophysiological conditions. In this study, we explored the connection between c-Abl and TFEB.

LC3 lipidation is essential for TFEB activation during the lysosomal damage response to kidney injury.

Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)-a master transcriptional regulator of lysosomal biogenesis and autophagy-is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation.

A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases.

The CLN3 gene and protein: What we know.

Background: One of the most important steps taken by Beyond Batten Disease Foundation in our quest to cure juvenile Batten (CLN3) disease is to understand the State of the Science. We believe that a strong understanding of where we are in our experimental understanding of the CLN3 gene, its regulation, gene product, protein structure, tissue distribution, biomarker use, and pathological responses to its deficiency, lays the groundwork for determining therapeutic action plans.

Objectives: To present an unbiased comprehensive reference tool of the experimental understanding of the CLN3 gene and gene product of the same name.

Polyamines Control eIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell Senescence.

Failure to make adaptive immune responses is a hallmark of aging. Reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. Here we show that reduced autophagy is a central molecular mechanism underlying immune senescence.

Cross-regulation of defective endolysosome trafficking and enhanced autophagy through TFEB in UNC13D deficiency.

Several lines of evidence support the occurrence of cross-regulation between the endocytic pathway and autophagy, but the molecular mechanisms regulating this process are not well-understood. Here, we show that the calcium sensor UNC13D regulates the molecular mechanism of late endosomal trafficking and endosomal maturation, and defects in UNC13D lead to macroautophagy upregulation. -null cells showed impaired endosomal trafficking and defective endocytic flux.

TFEB-driven endocytosis coordinates MTORC1 signaling and autophagy.

The mechanistic target of rapamycin kinase complex 1 (MTORC1) is a central cellular kinase that integrates major signaling pathways, allowing for regulation of anabolic and catabolic processes including macroautophagy/autophagy and lysosomal biogenesis. Essential to these processes is the regulatory activity of TFEB (transcription factor EB). In a regulatory feedback loop modulating transcriptional levels of RRAG/Rag GTPases, TFEB controls MTORC1 tethering to membranes and induction of anabolic processes upon nutrient replenishment.

mTOR-dependent phosphorylation controls TFEB nuclear export.

During starvation the transcriptional activation of catabolic processes is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient refeeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step.

Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A.

The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells.

TFEB at a glance.

The transcription factor EB (TFEB) plays a pivotal role in the regulation of basic cellular processes, such as lysosomal biogenesis and autophagy. The subcellular localization and activity of TFEB are regulated by mechanistic target of rapamycin (mTOR)-mediated phosphorylation, which occurs at the lysosomal surface. Phosphorylated TFEB is retained in the cytoplasm, whereas dephosphorylated TFEB translocates to the nucleus to induce the transcription of target genes.

Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells.

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome.