Ragulator Complex Research Articles

Starvation-induced metabolic rewiring affects mTORC1 composition in vivo.

Lysosomes play a crucial role in metabolic adaptation to starvation, but detailed in vivo studies are scarce. Therefore, we investigated the changes of the proteome of liver lysosomes in mice starved short-term for 6h or long-term for 24h. We verified starvation-induced catabolism by weight loss, ketone body production, drop in blood glucose and an increase of 3-methylhistidine. Deactivation of mTORC1 in vivo after short-term starvation causes a depletion of mTORC1 and the associated Ragulator complex in hepatic lysosomes, resulting in diminished phosphorylation of mTORC1 target proteins. While mTORC1 lysosomal protein levels and activity in liver were restored after long-term starvation, the lysosomal levels of Ragulator remained constantly reduced. To determine whether this mTORC1 activity pattern may be organ-specific, we further investigated the key metabolic organs muscle and brain. mTORC1 inactivation, but not re-activation, occurred in muscle after a starvation of 12 h or longer. In brain, mTORC1 activity remained unchanged during starvation. As mTORC1 deactivation is known to induce autophagy, we further investigated the more than 150 non-lysosomal proteins enriched in the lysosomal fraction upon starvation. Proteasomal, cytosolic and peroxisomal proteins dominated after short-term starvation, while after long-term starvation, mainly proteasomal and mitochondrial proteins accumulated, indicating ordered autophagic protein degradation.

Rag-Ragulator is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway.

The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates cell growth and metabolism in response to many environmental cues, including nutrients. Amino acids signal to mTORC1 by modulating the guanine nucleotide loading states of the heterodimeric Rag GTPases, which bind and recruit mTORC1 to the lysosomal surface, its site of activation. The Rag GTPases are tethered to the lysosome by the Ragulator complex and regulated by the GATOR1, GATOR2, and KICSTOR multiprotein complexes that localize to the lysosomal surface through an unknown mechanism(s). Here, we show that mTORC1 is completely insensitive to amino acids in cells lacking the Rag GTPases or the Ragulator component p18. Moreover, not only are the Rag GTPases and Ragulator required for amino acids to regulate mTORC1, they are also essential for the lysosomal recruitment of the GATOR1, GATOR2, and KICSTOR complexes, which stably associate and traffic to the lysosome as the "GATOR" supercomplex. The nucleotide state of RagA/B controls the lysosomal association of GATOR, in a fashion competitively antagonized by the N terminus of the amino acid transporter SLC38A9. Targeting of Ragulator to the surface of mitochondria is sufficient to relocalize the Rags and GATOR to this organelle, but not to enable the nutrient-regulated recruitment of mTORC1 to mitochondria. Thus, our results reveal that the Rag-Ragulator complex is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway and underscore that mTORC1 activation requires signal transduction on the lysosomal surface.

The eukaryotic-like characteristics of small GTPase, roadblock and TRAPPC3 proteins from Asgard archaea

Membrane-enclosed organelles are defining features of eukaryotes in distinguishing these organisms from prokaryotes. Specification of distinct membranes is critical to assemble and maintain discrete compartments. Small GTPases and their regulators are the signaling molecules that drive membrane-modifying machineries to the desired location. These signaling molecules include Rab and Rag GTPases, roadblock and longin domain proteins, and TRAPPC3-like proteins. Here, we take a structural approach to assess the relatedness of these eukaryotic-like proteins in Asgard archaea, the closest known prokaryotic relatives to eukaryotes. We find that the Asgard archaea GTPase core domains closely resemble eukaryotic Rabs and Rags. Asgard archaea roadblock, longin and TRAPPC3 domain-containing proteins form dimers similar to those found in the eukaryotic TRAPP and Ragulator complexes. We conclude that the emergence of these protein architectures predated eukaryogenesis, however further adaptations occurred in proto-eukaryotes to allow these proteins to regulate distinct internal membranes.

Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity.

Lysosomes are active sites to integrate cellular metabolism and signal transduction. A collection of proteins associated with the lysosome mediate these metabolic and signaling functions. Both lysosomal metabolism and lysosomal signaling have been linked to longevity regulation; however, how lysosomes adjust their protein composition to accommodate this regulation remains unclear. Using deep proteomic profiling, we systemically profiled lysosome-associated proteins linked with four different longevity mechanisms. We discovered the lysosomal recruitment of AMP-activated protein kinase and nucleoporin proteins and their requirements for longevity in response to increased lysosomal lipolysis. Through comparative proteomic analyses of lysosomes from different tissues and labeled with different markers, we further elucidated lysosomal heterogeneity across tissues as well as the increased enrichment of the Ragulator complex on Cystinosin-positive lysosomes. Together, this work uncovers lysosomal proteome heterogeneity across multiple scales and provides resources for understanding the contribution of lysosomal protein dynamics to signal transduction, organelle crosstalk, and organism longevity.

Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

The CLN1-disease, formerly called infantile neuronal ceroid lipofuscinosis (INCL), is a fatal hereditary neurodegenerative lysosomal storage disorder. This disease is caused by loss-of-function mutations in the CLN1 gene, encoding palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes depalmitoylation of S-palmitoylated proteins for degradation and clearance by lysosomal hydrolases. Numerous proteins, especially in the brain, require dynamic S-palmitoylation (palmitoylation-depalmitoylation cycles) for endosomal trafficking to their destination. While 23 palmitoyl-acyl transferases in the mammalian genome catalyze S-palmitoylation, depalmitoylation is catalyzed by thioesterases such as PPT1. Despite these discoveries, the pathogenic mechanism of CLN1-disease has remained elusive. Here we report that in the brain of Cln1-/- mice, which mimic CLN1-disease, the mechanistic target of rapamycin complex-1 (mTORC1)-kinase is hyperactivated. The activation of mTORC1 by nutrients requires its anchorage to lysosomal limiting membrane by Rag GTPases and Ragulator complex. These proteins form the lysosomal nutrient sensing scaffold to which mTORC1 must attach to activate. We found that in Cln1-/- mice, two constituent proteins of the Ragulator complex (vATPase and Lamtor1) require dynamic S-palmitoylation for endosomal trafficking to the lysosomal limiting membrane. Intriguingly, Ppt1-deficiency in Cln1-/- mice misrouted these proteins to the plasma membrane disrupting the lysosomal nutrient sensing scaffold. Despite this defect, mTORC1 was hyperactivated via the IGF1/PI3K/Akt-signaling pathway, which suppressed autophagy contributing to neuropathology. Importantly, pharmacological inhibition of PI3K/Akt suppressed mTORC1-activation, restored autophagy, and ameliorated neurodegeneration in Cln1-/- mice. Our findings reveal a previously unrecognized role of Cln1/Ppt1 in regulating mTORC1-activation and suggest that IGF1/PI3K/Akt may be a targetable pathway for CLN1 disease.

The lysosomal LAMTOR / Ragulator complex is essential for nutrient homeostasis in brown adipose tissue

ObjectiveIn brown adipose tissue (iBAT), the balance between lipid/glucose uptake and lipolysis is tightly regulated by insulin signaling. Downstream of the insulin receptor, PDK1 and mTORC2 phosphorylate AKT, which activates glucose uptake and lysosomal mTORC1 signaling. The latter requires the late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) complex, which serves to translate the nutrient status of the cell to the respective kinase. However, the role of LAMTOR in metabolically active iBAT has been elusive. MethodsUsing an AdipoqCRE-transgenic mouse line, we deleted LAMTOR2 (and thereby the entire LAMTOR complex) in adipose tissue (LT2 AKO). To examine the metabolic consequences, we performed metabolic and biochemical studies in iBAT isolated from mice housed at different temperatures (30 °C, room temperature and 5 °C), after insulin treatment, or in fasted and refed condition. For mechanistic studies, mouse embryonic fibroblasts (MEFs) lacking LAMTOR 2 were analyzed. ResultsDeletion of the LAMTOR complex in mouse adipocytes resulted in insulin-independent AKT hyperphosphorylation in iBAT, causing increased glucose and fatty acid uptake, which led to massively enlarged lipid droplets. As LAMTOR2 was essential for the upregulation of de novo lipogenesis, LAMTOR2 deficiency triggered exogenous glucose storage as glycogen in iBAT. These effects are cell autonomous, since AKT hyperphosphorylation was abrogated by PI3K inhibition or by deletion of the mTORC2 component Rictor in LAMTOR2-deficient MEFs. ConclusionsWe identified a homeostatic circuit for the maintenance of iBAT metabolism that links the LAMTOR-mTORC1 pathway to PI3K-mTORC2-AKT signaling downstream of the insulin receptor.

Hierarchical inhibition of mTORC1 by glucose starvation-triggered AXIN lysosomal translocation and by AMPK

Abstract When glucose is replete, mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is active and anchored to the lysosomal surface via the two GTPases, Ras-related GTPase (RAG) and Ras homolog enriched in brain (Rheb), which are regulated by Ragulator and tuberous sclerosis complex 2 (TSC2), respectively. When glucose is low, aldolase senses low fructose-1,6-bisphosphate level and promotes the translocation of AXIN−liver kinase B1 (LKB1) to the lysosomal surface, which leads to the activation of AMP-activated protein kinase (AMPK) and the inhibition of RAGs, sundering mTORC1 from the lysosome and causing its inactivation. AMPK can also inactivate mTORC1 by phosphorylating Raptor and TSC2. However, the hierarchy of AXIN- and AMPK-mediated inhibition of mTORC1 remains poorly defined. Here, we show that AXIN translocation does not require AMPK expression or activity. In glucose starvation conditions, knockout of AXIN extended the half-life of mTORC1 inhibition from 15 to 60 min, whereas knockout of AMPK only extended it to 30 min. RAGBGTP (constitutively active RAGB) almost entirely blocked the lysosomal dissociation and inhibition of mTORC1 under glucose starvation, but it did not inhibit AMPK, indicating that under these conditions, it is AXIN lysosomal translocation that inhibits mTORC1, and it does so via inhibition of RAGs. 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a mimetic of AMP, which activates both cytosolic AMPK and lysosomal AMPK, fully inhibited mTORC1 even when it is stably anchored to the lysosome by RAGBGTP, whereas glucose starvation mildly inhibited such anchored mTORC1. Together, we demonstrate that the lysosomal translocation of AXIN plays a primary role in glucose starvation-triggered inhibition of mTORC1 by inhibiting RAGs, and that AMPK activity inhibits mTORC1 through phosphorylating Raptor and TSC2, especially under severe stress.

Structural basis for mTORC1-dependent regulation of the lysosomal and autophagic transcription factor EB.

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 GAP FLCN. TFEB lacks the TOR signaling (TOS) motif responsible for the recruitment of other mTORC1 substrates. We used cryo-electron microscopy (cryo-EM) to determine the structure of TFEB as presented to mTORC1 for phosphorylation. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first via a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB in a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. Mutation of the clamp drives TFEB constitutively into the nucleus whilst having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. This structure presents the phosphorylatable Ser residues of TFEB to the mTORC1 active site in a suitable geometry for their phosphorylation. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

Structure of the lysosomal mTORC1–TFEB–Rag–Ragulator megacomplex

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy1. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1)2–5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN6,7. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the ‘megacomplex’. Two full Rag–Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag–Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag–Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

USP32-regulated LAMTOR1 ubiquitination impacts mTORC1 activation and autophagy induction.

The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.

The lysosomal Ragulator complex activates NLRP3 inflammasome invivo via HDAC6.

The cellular activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but whether lysosomes contribute to this process remains unclear. Here, we show the vital role of the lysosomal membrane-tethered Ragulator complex inNLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophages and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed marked attenuation of NLRP3-associated inflammatory disease severity, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with both NLRP3 and histone deacetylase 6 (HDAC6). HDAC6 enhances the interaction between Lamtor1 and NLRP3, resulting in NLRP3 inflammasome activation. DL-all-rac-α-tocopherol, a synthetic form of vitamin E, inhibited the Lamtor1-HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. Further, DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. These results provide novel insights into the role of lysosomes in the activation of NLRP3 inflammasomes by the Ragulator complex.

Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages.

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that complement components react with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage interactions with fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc, the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during Hc infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

Hbxip is essential for murine embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1.

HBXIP, also named LAMTOR5, has been well characterized as a transcriptional co-activator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5, and that depletion of Hbxip compromises the self-renewal of embryonic stem cells (ESCs), with reduced expression of pluripotency genes, reduced cell proliferation and decreased colony-forming capacity. In addition, both Hbxip-/- ESCs and E7.5 embryos displayed defects in ectodermal and mesodermal differentiation. Mechanistically, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, displayed differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/- and p18-/- mice, lacking subunits of the Ragulator complex, also shared similar phenotypes, embryonic lethality and retarded growth around E7-E8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

Cryo-EM structures of the human GATOR1-Rag-Ragulator complex reveal a spatial-constraint regulated GAP mechanism

mTORC1 controls cellular metabolic processes in response to nutrient availability. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which are localized on the lysosomal surface by the Ragulator complex. The Rag GTPases receive amino acid signals from multiple upstream regulators. One negative regulator, GATOR1, is a GTPase activating protein (GAP) for RagA. GATOR1 binds to the Rag GTPases via two modes: an inhibitory mode and a GAP mode. How these two binding interactions coordinate to process amino acid signals is unknown. Here, we resolved three cryo-EM structural models of the GATOR1-Rag-Ragulator complex, with the Rag-Ragulator subcomplex occupying the inhibitory site, the GAP site, and both binding sites simultaneously. When the Rag GTPases bind to GATOR1 at the GAP site, both Rag subunits contact GATOR1 to coordinate their nucleotide loading states. These results reveal a potential GAP mechanism of GATOR1 during the mTORC1 inactivation process.

The Ragulator complex serves as a substrate-specific mTORC1 scaffold in regulating the nuclear translocation of transcription factor EB

The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by tenfold in the Ragulator-deficient clone compared with that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling.

Abstract P5-08-15: Overcoming mTOR inhibitor resistance by targeting the lysosomal deregulation

Abstract Increasing evidence suggests that the deregulated lysosomes contribute to tumorigenesis and therapeutic resistance. However, its underlying mechanisms remain elusive. Here, we identify transmembrane protein 9 (TMEM9) as a regulator involved in lysosomal metabolism and mammary tumorigenesis. The TMEM9 gene is markedly amplified in breast cancer, correlated with its transcriptional upregulation. We uncovered that Tmem9 knock-out inhibited mammary tumorigenesis in genetically engineered mouse models. Consistently, TMEM9 depletion suppressed the proliferation of breast cancer cells highly expressing TMEM9. Conversely, the ectopic expression of TMEM9 in TMEM9 low breast cancer cells promoted cell proliferation. The lysosome purification and proteomics approach showed that TMEM9 physically and functionally interacts with LAMTOR4, a subunit of the Ragulator complex, to hyperactivate mTOR signaling in breast cancer. Moreover, the combination of TMEM9-v-ATPase inhibitors with mTOR inhibitors exhibited the synergistic growth inhibition of breast cancer cells in vitro and in vivo. Our results reveal that the TMEM9-v-ATPase axis is not only a biomarker for resistance to mTOR inhibition therapy but also a molecular target overcoming mTOR inhibitor resistance. Citation Format: Shengzhe Zhang, Sung Ho Lee, Litong Nie, Youn-Sang Jung, Sohee Jun, Jie Zhang, Esther M Lien, Junjie Chen, Jae-Il Park. Overcoming mTOR inhibitor resistance by targeting the lysosomal deregulation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-15.

LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

Lysosomes function not only as degradatory compartments but also as dynamic intracellular calcium ion stores. The transient receptor potential mucolipin 1 (TRPML1) channel mediates lysosomal Ca2+ release, thereby participating in multiple cellular functions. The pentameric Ragulator complex, which plays a critical role in the activation of mTORC1, is also involved in lysosomal trafficking and is anchored to lysosomes through its LAMTOR1 subunit. Here, we report that the Ragulator restricts lysosomal trafficking in dendrites of hippocampal neurons via LAMTOR1‐mediated tonic inhibition of TRPML1 activity, independently of mTORC1. LAMTOR1 directly interacts with TRPML1 through its N‐terminal domain. Eliminating this inhibition in hippocampal neurons by LAMTOR1 deletion or by disrupting LAMTOR1‐TRPML1 binding increases TRPML1‐mediated Ca2+ release and facilitates dendritic lysosomal trafficking powered by dynein. LAMTOR1 deletion in the hippocampal CA1 region of adult mice results in alterations in synaptic plasticity, and in impaired object‐recognition memory and contextual fear conditioning, due to TRPML1 activation. Mechanistically, changes in synaptic plasticity are associated with increased GluA1 dephosphorylation by calcineurin and lysosomal degradation. Thus, LAMTOR1‐mediated inhibition of TRPML1 is critical for regulating dendritic lysosomal motility, synaptic plasticity, and learning.

Nuciferine protects against high-fat diet-induced hepatic steatosis and insulin resistance via activating TFEB-mediated autophagy–lysosomal pathway

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment. Hyperactivation of mTOR complex 1 (mTORC1) and subsequent impairment of the transcription factor EB (TFEB)-mediated autophagy–lysosomal pathway (ALP) are implicated in the development of NAFLD. Accordingly, agents that augment hepatic TFEB transcriptional activity may have therapeutic potential against NAFLD. The objective of this study was to investigate the effects of nuciferine, a major active component from lotus leaf, on NAFLD and its underlying mechanism of action. Here we show that nuciferine activated ALP and alleviated steatosis, insulin resistance in the livers of NAFLD mice and palmitic acid-challenged hepatocytes in a TFEB-dependent manner. Mechanistic investigation revealed that nuciferine interacts with the Ragulator subunit hepatitis B X-interacting protein and impairs the interaction of the Ragulator complex with Rag GTPases, thereby suppressing lysosomal localization and activity of mTORC1, which activates TFEB-mediated ALP and further ameliorates hepatic steatosis and insulin resistance. Our present results indicate that nuciferine may be a potential agent for treating NAFLD and that regulation of the mTORC1–TFEB–ALP axis could represent a novel pharmacological strategy to combat NAFLD.

Central Suppression of the GH/IGF Axis and Abrogation of Exercise-Related mTORC1/2 Activation in the Muscle of Phenotype-Selected Male Marathon Mice (DUhTP).

The somatotropic axis is required for a number of biological processes, including growth, metabolism, and aging. Due to its central effects on growth and metabolism and with respect to its positive effects on muscle mass, regulation of the GH/IGF-system during endurance exercise is of particular interest. In order to study the control of gene expression and adaptation related to physical performance, we used a non-inbred mouse model, phenotype-selected for high running performance (DUhTP). Gene expression of the GH/IGF-system and related signaling cascades were studied in the pituitary gland and muscle of sedentary males of marathon and unselected control mice. In addition, the effects of three weeks of endurance exercise were assessed in both genetic groups. In pituitary glands from DUhTP mice, reduced expression of Pou1f1 (p = 0.002) was accompanied by non-significant reductions of Gh mRNA (p = 0.066). In addition, mRNA expression of Ghsr and Sstr2 were significantly reduced in the pituitary glands from DUhTP mice (p ≤ 0.05). Central downregulation of Pou1f1 expression was accompanied by reduced serum concentrations of IGF1 and coordinated downregulation of multiple GH/IGF-signaling compounds in muscle (e.g., Ghr, Igf1, Igf1r, Igf2r, Irs1, Irs2, Akt3, Gskb, Pik3ca/b/a2, Pten, Rictor, Rptor, Tsc1, Mtor; p ≤ 0.05). In response to exercise, the expression of Igfbp3, Igfbp 4, and Igfbp 6 and Stc2 mRNA was increased in the muscle of DUhTP mice (p ≤ 0.05). Training-induced specific activation of AKT, S6K, and p38 MAPK was found in muscles from control mice but not in DUhTP mice (p ≤ 0.05), indicating a lack of mTORC1 and mTORC2 activation in marathon mice in response to physical exercise. While hormone-dependent mTORC1 and mTORC2 pathways in marathon mice were repressed, robust increases of Ragulator complex compounds (p ≤ 0.001) and elevated sirtuin 2 to 6 mRNA expression were observed in the DUhTP marathon mouse model (p ≤ 0.05). Activation of AMPK was not observed under the experimental conditions of the present study. Our results describe coordinated downregulation of the somatotropic pathway in long-term selected marathon mice (DUhTP), possibly via the pituitary gland and muscle interaction. Our results, for the first time, demonstrate that GH/IGF effects are repressed in a context of superior running performance in mice.

The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II

Lysosomes are involved in nutrient sensing via the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is tethered to lysosomes by the Ragulator complex, a heteropentamer in which Lamtor1 wraps around Lamtor2–5. Although the Ragulator complex is required for cell migration, the mechanisms by which it participates in cell motility remain unknown. Here, we show that lysosomes move to the uropod in motile cells, providing the platform where Lamtor1 interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and MYPT1, a subunit of myosin light chain phosphatase (MLCP), thereby increasing myosin II–mediated actomyosin contraction. Additionally, formation of the complete Ragulator complex is required for leukocyte migration and pathophysiological immune responses. Together, our findings demonstrate that the lysosomal Ragulator complex plays an essential role in leukocyte migration by activating myosin II through interacting with MPRIP.