bims-lysosi 2021-01-24 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021–01–24
35 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

The rapidly evolving view of lysosomal storage diseases.
SVIP is a molecular determinant of lysosomal dynamic stability, neurodegeneration and lifespan.
Snd3 controls nucleus-vacuole junctions in response to glucose signaling.
Methionine controls insulin/mammalian target of rapamycin complex 1 activity by modulating tuberous sclerosis complex 2 stability.
Autophagy activation, lipotoxicity and lysosomal membrane permeabilization synergize to promote pimozide- and loperamide-induced glioma cell death.
TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast.
Arsenic trioxide induces macrophage autophagy and atheroprotection by regulating ROS-dependent TFEB nuclear translocation and AKT/mTOR pathway.
A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3.
The anti-tubercular activity of simvastatin is mediated by cholesterol-driven autophagy via the AMPK-mTORC1-TFEB axis.
mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.
Persistently elevated CK and lysosomal storage myopathy associated with mucolipin 1 defects.
The phosphatidylinositol 3-phosphate binding protein SNX4 controls ATG9A recycling and autophagy.
Proteomics analysis of a human brain sample from a mucolipidosis type IV patient reveals pathophysiological pathways.
Fibroblasts from idiopathic Parkinson's disease exhibit deficiency of lysosomal glucocerebrosidase activity associated with reduced levels of the trafficking receptor LIMP2.
Rag GTPases and phosphatidylinositol 3-phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex.
Lysosomal calcium is modulated by STIM1/TRPML1 interaction which participates to neuronal survival during ischemic preconditioning.
FUS contributes to mTOR-dependent inhibition of translation.
mTORC1 and mTORC2 coordinate early NK cell development by differentially inducing E4BP4 and T-bet.
Decrease of neuronal FKBP4/FKBP52 modulates perinuclear lysosomal positioning and MAPT/Tau behavior during MAPT/Tau-induced proteotoxic stress.
Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program.
ABHD5 suppresses cancer cell anabolism through lipolysis-dependent activation of the AMPK/mTORC1 pathway.
Crosstalk of the Caspase Family and Mammalian Target of Rapamycin Signaling.
Fibrillar α-synuclein toxicity depends on functional lysosomes.
Nutrient Sensor mTOR and OGT: Orchestrators of Organelle Homeostasis in Pancreatic β-Cells.
Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells.
Autophagy regulation by acetylation-implications for neurodegenerative diseases.
Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction.
Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice.
Rapamycin relieves the cataract caused by ablation of Gja8b through stimulating autophagy in zebrafish.
Thioxothiazolidin derivative, 4-OST, inhibits melanogenesis by enhancing the specific recruitment of tyrosinase-containing vesicles to lysosome.
The ER-embedded UBE2J1/RNF26 ubiquitylation complex exerts spatiotemporal control over the endolysosomal pathway.
The metabolism of cancer cells during metastasis.
PIWIL2 interacting with IKK to regulate autophagy and apoptosis in esophageal squamous cell carcinoma.
A cell-nonautonomous mechanism of yeast chronological aging regulated by caloric restriction and one-carbon metabolism.
Endonuclease G promotes autophagy by suppressing mTOR signaling and activating the DNA damage response.

EMBO Mol Med. 2021 Jan 18. e12836

The rapidly evolving view of lysosomal storage diseases.

Giancarlo Parenti, Diego L Medina, Andrea Ballabio.

  Lysosomal storage diseases are a group of metabolic disorders caused by deficiencies of several components of lysosomal function. Most commonly affected are lysosomal hydrolases, which are involved in the breakdown and recycling of a variety of complex molecules and cellular structures. The understanding of lysosomal biology has progressively improved over time. Lysosomes are no longer viewed as organelles exclusively involved in catabolic pathways, but rather as highly dynamic elements of the autophagic-lysosomal pathway, involved in multiple cellular functions, including signaling, and able to adapt to environmental stimuli. This refined vision of lysosomes has substantially impacted on our understanding of the pathophysiology of lysosomal disorders. It is now clear that substrate accumulation triggers complex pathogenetic cascades that are responsible for disease pathology, such as aberrant vesicle trafficking, impairment of autophagy, dysregulation of signaling pathways, abnormalities of calcium homeostasis, and mitochondrial dysfunction. Novel technologies, in most cases based on high-throughput approaches, have significantly contributed to the characterization of lysosomal biology or lysosomal dysfunction and have the potential to facilitate diagnostic processes, and to enable the identification of new therapeutic targets.

Keywords:  autophagy; lysosomal biology; lysosomal storage diseases; lysosomes

DOI:  https://doi.org/10.15252/emmm.202012836
Nat Commun. 2021 01 21. 12(1): 513

SVIP is a molecular determinant of lysosomal dynamic stability, neurodegeneration and lifespan.

Alyssa E Johnson, Brian O Orr, Richard D Fetter, Armen J Moughamian, Logan A Primeaux, Ethan G Geier, Jennifer S Yokoyama, Bruce L Miller, Graeme W Davis.

Missense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson's disease. Here, we characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal-lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. We also establish multiple links between SVIP and VCP-dependent disease in our Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, we identify a human SVIP mutation and confirm the pathogenicity of this mutation in our Drosophila model. We propose a model for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles.

DOI: https://doi.org/10.1038/s41467-020-20796-8
Cell Rep. 2021 Jan 19. pii: S2211-1247(20)31626-0. [Epub ahead of print]34(3): 108637

Snd3 controls nucleus-vacuole junctions in response to glucose signaling.

Sergi Tosal-Castano, Carlotta Peselj, Verena Kohler, Lukas Habernig, Lisa Larsson Berglund, Mahsa Ebrahimi, F-Nora Vögtle, Johanna Höög, Claes Andréasson, Sabrina Büttner.

  Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites.

Keywords:  Nvj1; SND pathway; Saccharomyces cerevisiae; Snd3; Tsc13; Vac8; glucose metabolism; interorganellar connectivity; membrane contact sites; nucleus-vacuole junction

DOI:  https://doi.org/10.1016/j.celrep.2020.108637
Biochem Biophys Res Commun. 2021 Jan 19. pii: S0006-291X(21)00068-1. [Epub ahead of print]541 84-89

Methionine controls insulin/mammalian target of rapamycin complex 1 activity by modulating tuberous sclerosis complex 2 stability.

Seishu Gen, Yu Matsumoto, Tsukasa Suzuki, Jun Inoue, Yuji Yamamoto.

  Tuberous sclerosis complex 2 (TSC2) is a tumor-suppressor protein that is partially regulated by insulin, energy, oxygen, and growth factors. Mutations in the TSC2 gene and loss of TSC2 promote cell growth by the mammalian target of rapamycin complex 1 (mTORC1) activation. Furthermore, S-adenosylmethionine (SAM) sensor upstream of mTORC1 indirectly inhibits mTORC1 activity via the methionine metabolite SAM. Here, we investigated the effects of methionine on insulin/TSC2/mTORC1 activity. Our results showed that methionine affected TSC2 stability and abolished TSC2 localization to the lysosome. Moreover, activation of insulin signaling contributed to TSC2 degradation in a methionine deprivation-dependent manner. Thus, methionine and insulin crosstalk occurred via TSC2.

Keywords:  AKT; Amino acid; Mammalian target of rapamycin complex 1; Methionine; S-adenosylmethionine; Tuberous sclerosis complex 2

DOI:  https://doi.org/10.1016/j.bbrc.2021.01.033
Autophagy. 2021 Jan 19. 1-20

Autophagy activation, lipotoxicity and lysosomal membrane permeabilization synergize to promote pimozide- and loperamide-induced glioma cell death.

Nina Meyer, Lisa Henkel, Benedikt Linder, Svenja Zielke, Georg Tascher, Sandra Trautmann, Gerd Geisslinger, Christian Münch, Simone Fulda, Irmgard Tegeder, Donat Kögel.

  Increasing evidence suggests that induction of lethal macroautophagy/autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7 (autophagy related 5 and 7)-dependent type of cell death that is significantly reduced with cathepsin inhibitors and the lipid reactive oxygen species (ROS) scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of SMPD1/ASM (sphingomyelin phosphodiesterase 1) and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of CTSB (cathepsin B) into the cytosol in MZ-54 wild-type (WT) cells. Whereas LMP and cell death were significantly attenuated in ATG5 and ATG7 knockout (KO) cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM. Abbreviations: ACD: autophagic cell death; AKT1: AKT serine/threonine kinase 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; CERS1: ceramide synthase 1; CTSB: cathepsin B; CYBB/NOX2: cytochrome b-245 beta chain; ER: endoplasmatic reticulum; FBS: fetal bovine serum; GBM: glioblastoma; GO: gene ontology; HTR7/5-HT7: 5-hydroxytryptamine receptor 7; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; LMP: lysosomal membrane permeabilization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; RB1CC1: RB1 inducible coiled-coil 1; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SMPD1/ASM: sphingomyelin phosphodiesterase 1; VCP/p97: valosin containing protein; WT: wild-type.

Keywords:  Acid sphingomyelinase; autophagy-dependent cell death; brain tumors; cholesterol metabolism; drug repurposing; er stress; lysophagy

DOI:  https://doi.org/10.1080/15548627.2021.1874208
J Biol Chem. 2020 Aug 21. pii: S0021-9258(17)50066-6. [Epub ahead of print]295(34): 12028-12044

TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast.

Oliver Schmidt, Yannick Weyer, Simon Sprenger, Michael A Widerin, Sebastian Eising, Verena Baumann, Mihaela Angelova, Robbie Loewith, Christopher J Stefan, Michael W Hess, Florian Fröhlich, David Teis.

  The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (Saccharomyces cerevisiae) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis.

Keywords:  ORMDL family; ORMDL-family; TORC2; calcineurin; endosomal sorting complexes required for transport (ESCRT); endosome and Golgi-associated degradation (EGAD); mTOR complex (mTORC); membrane; membrane stress; sphingolipid; stress

DOI:  https://doi.org/10.1074/jbc.RA120.013222
Cell Death Dis. 2021 Jan 18. 12(1): 88

Arsenic trioxide induces macrophage autophagy and atheroprotection by regulating ROS-dependent TFEB nuclear translocation and AKT/mTOR pathway.

Shaohong Fang, Xin Wan, Xiaoyi Zou, Song Sun, Xinran Hao, Chenchen Liang, Zhenming Zhang, Fangni Zhang, Bo Sun, Hulun Li, Bo Yu.

Inducing autophagy and inhibiting apoptosis may provide a therapeutic treatment for atherosclerosis (AS). For the treatment of progressive AS, arsenic trioxide (ATO) has been used to coat vascular stents. However, the effect of ATO on autophagy of macrophages is still unknown. Therefore, the aims of this study were to characterize the effects and the mechanism of actions of ATO on autophagy in macrophages. Our results showed that ATO-induced activation of autophagy was an earlier event than ATO-induced inhibition of the expression of apoptosis markers in macrophages and foam cells. Nuclear transcription factor EB (TFEB) prevents atherosclerosis by activating macrophage autophagy and promoting lysosomal biogenesis. Here, we report that ATO triggered the nuclear translocation of TFEB, which in turn promoted autophagy and autophagosome-lysosome fusion. Both the latter events were prevented by TFEB knockdown. Moreover, ATO decreased the p-AKT and p-mTOR in the PI3K/AKT/mTOR signaling pathway, thus inducing autophagy. Correspondingly, treatment with the autophagy inhibitor 3-methyladenine (3-MA) abolished the autophagy-inducing effects of ATO. Meanwhile, PI3K inhibitor (LY294002) and mTOR inhibitor (rapamycin) cooperated with ATO to induce autophagy. Furthermore, reactive oxygen species (ROS) were generated in macrophages after treatment with ATO. The ROS scavenger N-acetyl-1-cysteine (NAC) abolished ATO-induced nuclear translocation of TFEB, as well as changes in key molecules of the AKT/mTOR signaling pathway and downstream autophagy. More importantly, ATO promoted autophagy in the aorta of ApoE-/- mice and reduced atherosclerotic lesions in early AS, which were reversed by 3-MA treatment. In summary, our data indicated that ATO promoted ROS induction, which resulted in nuclear translocation of TFEB and inhibition of the PI3K/AKT/mTOR pathway. These actions ultimately promoted macrophage autophagy and reduced atherosclerotic lesions at early stages. These findings may provide a new perspective for the clinical treatment of early-stage atherosclerosis and should be further studied.

DOI: https://doi.org/10.1038/s41419-020-03357-1
J Biol Chem. 2020 Aug 21. pii: S0021-9258(17)50084-8. [Epub ahead of print]295(34): 12262-12278

A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3.

Surya P Manandhar, Ikha M Siddiqah, Stephanie M Cocca, Editte Gharakhanian.

  Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast (Saccharomyces cerevisiae) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (328SRFS331). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3-Env7 kinase cascade.

Keywords:  CK1 Yck3; Env7; Saccharomyces cerevisiae; lysosomal vacuole; lysosome; membrane fusion; protein phosphorylation; regulation of membrane dynamics; serine/threonine protein kinase; subcellular organelle; vacuolar membrane dynamics; vacuole

DOI:  https://doi.org/10.1074/jbc.RA119.012346
J Lipid Res. 2020 Dec;pii: S0022-2275(20)60023-2. [Epub ahead of print]61(12): 1617-1628

The anti-tubercular activity of simvastatin is mediated by cholesterol-driven autophagy via the AMPK-mTORC1-TFEB axis.

Natalie Bruiners, Noton K Dutta, Valentina Guerrini, Hugh Salamon, Ken D Yamaguchi, Petros C Karakousis, Maria L Gennaro.

  The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

Keywords:  Mycobacterium tuberculosis; adenosine 5′-monophosphate-activated protein kinase-mechanistic target of rapamycin complex 1-transcription factor EB axis; immunology; lipids; macrophages/monocytes; mechanistic target of rapamycin complex 1 regulation; statins

DOI:  https://doi.org/10.1194/jlr.RA120000895
J Biol Chem. 2020 Dec 09. pii: S0021-9258(20)00150-7. [Epub ahead of print]296 100157

mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.

Roberta L Beauchamp, Serkan Erdin, Luke Witt, Justin T Jordan, Scott R Plotkin, James F Gusella, Vijaya Ramesh.

  Meningiomas (MNs), arising from the arachnoid/meningeal layer, are nonresponsive to chemotherapies, with ∼50% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously, we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mechanistic target of rapamycin complex 2 (mTORC2) signaling, leading to clinical trials for NF2 and MN. Recently our omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly neuregulin-1/heregulin (NRG1), and confirm increased NRG1 secretion and activation of V-ERB-B avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3) receptor kinase. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2, and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a mechanism independent of NRG1-ERBB3 but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of insulin-like growth factor receptor 1 (IGF1R). Drug treatment co-targeting mTORC1/2 and IGF1R/insulin receptor attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion/activation of NRG1-ERBB3 signaling. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/insulin receptor and co-targeting these pathways may prove effective for treatment of NF2-deficient MN.

Keywords:  Akt PKB; NF2; NRG1-ERBB3; brain tumor; dual mTORC1/mTORC2 inhibition; insulin-like growth factor (IGF) receptor 1; mammalian target of rapamycin (mTOR); meningioma; signaling; tumor suppressor gene

DOI:  https://doi.org/10.1074/jbc.RA120.014960
Neuromuscul Disord. 2021 Jan 06. pii: S0960-8966(20)30703-3. [Epub ahead of print]

Persistently elevated CK and lysosomal storage myopathy associated with mucolipin 1 defects.

Genomics England Research Consortium

  Mucolipidosis type IV is a rare autosomal recessive lysosomal storage disorder caused by bi-allelic pathogenic variants in the gene MCOLN1. This encodes for mucolipin-1 (ML1), an endo-lysosomal transmembrane Ca++ channel involved in vesicular trafficking. Although experimental models suggest that defects in mucolipin-1 can cause muscular dystrophy, putatively due to defective lysosomal-mediated sarcolemma repair, the role of mucolipin-1 in human muscle is still poorly deciphered. Elevation of creatine kinase (CK) had been reported in a few cases in the past but comprehensive descriptions of muscle pathology are lacking. Here we report a 7-year-old boy who underwent muscle biopsy due to persistently elevated CK levels (780-15,000 UI/L). Muscle pathology revealed features of a lysosomal storage myopathy with mild regenerative changes. Next generation sequencing confirmed homozygous nonsense variants in MCOLN1. This is a comprehensive pathological description of ML1-related myopathy, supporting the role of mucolipin-1 in muscle homoeostasis.

Keywords:  Lysosome; Mucolipidosis IV; Mucolipin-1; Myopathy; Sarcolemma repair

DOI:  https://doi.org/10.1016/j.nmd.2020.12.009
J Cell Sci. 2021 Jan 19. pii: jcs.250670. [Epub ahead of print]

The phosphatidylinositol 3-phosphate binding protein SNX4 controls ATG9A recycling and autophagy.

Anthony Ravussin, Andreas Brech, Sharon A Tooze, Harald Stenmark.

  Late endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Whereas these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the phosphatidylinositol 3-phosphate (PI3P) binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the lipid scramblase ATG9A, which drives expansion of nascent autophagosome membranes, from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. We propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.

Keywords:  Autophagy; Endosome; Phosphoinositide; Recycling

DOI:  https://doi.org/10.1242/jcs.250670
Orphanet J Rare Dis. 2021 Jan 21. 16(1): 39

Proteomics analysis of a human brain sample from a mucolipidosis type IV patient reveals pathophysiological pathways.

Ayelet Vardi, Amir Pri-Or, Noa Wigoda, Yulia Grishchuk, Anthony H Futerman.

   BACKGROUND: Mucolipidosis type IV (MLIV), an ultra-rare neurodevelopmental and neurodegenerative disorder, is caused by mutations in the MCOLN1 gene, which encodes the late endosomal/lysosomal transient receptor potential channel TRPML1 (mucolipin 1). The precise pathophysiogical pathways that cause neurological disease in MLIV are poorly understood. Recently, the first post-mortem brain sample became available from a single MLIV patient, and in the current study we performed mass spectrometry (MS)-based proteomics on this tissue with a view to delineating pathological pathways, and to compare with previously-published data on MLIV, including studies using the Mcoln1-/- mouse.
RESULTS: A number of pathways were altered in two brain regions from the MLIV patient, including those related to the lysosome, lipid metabolism, myelination, cellular trafficking and autophagy, mTOR and calmodulin, the complement system and interferon signaling. Of these, levels of some proteins not known previously to be associated with MLIV were altered, including APOD, PLIN4, ATG and proteins related to interferon signaling. Moreover, when proteins detected by proteomics in the human brain were compared with their orthologs detected in the Mcoln1-/- mouse by RNAseq, the results were remarkably similar. Finally, analysis of proteins in human and mouse CSF suggest that calbindin 1 and calbindin 2 might be useful as biomarkers to help chart the course of disease development.
CONCLUSIONS: Despite the sample size limitations, our findings are consistent with the relatively general changes in lysosomal function previously reported in MLIV, and shed light on new pathways of disease pathophysiology, which is required in order to understand the course of disease development and to determine the efficacy of therapies when they become available for this devastating disease.

Keywords:  Autophagy; Brain; Calbindin; Lysosome; Neuroinflammation; Neuropathology; TRPML1

DOI:  https://doi.org/10.1186/s13023-021-01679-7
Mol Brain. 2021 Jan 19. 14(1): 16

Fibroblasts from idiopathic Parkinson's disease exhibit deficiency of lysosomal glucocerebrosidase activity associated with reduced levels of the trafficking receptor LIMP2.

Ria Thomas, Elizabeth B Moloney, Zachary K Macbain, Penelope J Hallett, Ole Isacson.

  Lysosomal dysfunction is a central pathway associated with Parkinson's disease (PD) pathogenesis. Haploinsufficiency of the lysosomal hydrolase GBA (encoding glucocerebrosidase (GCase)) is one of the largest genetic risk factors for developing PD. Deficiencies in the activity of the GCase enzyme have been observed in human tissues from both genetic (harboring mutations in the GBA gene) and idiopathic forms of the disease. To understand the mechanisms behind the deficits of lysosomal GCase enzyme activity in idiopathic PD, this study utilized a large cohort of fibroblast cells from control subjects and PD patients with and without mutations in the GBA gene (N370S mutation) (control, n = 15; idiopathic PD, n = 31; PD with GBA N370S mutation, n = 6). The current data demonstrates that idiopathic PD fibroblasts devoid of any mutations in the GBA gene also exhibit reduction in lysosomal GCase activity, similar to those with the GBA N370S mutation. This reduced GCase enzyme activity in idiopathic PD cells was accompanied by decreased expression of the GBA trafficking receptor, LIMP2, and increased ER retention of the GBA protein in these cells. Importantly, in idiopathic PD fibroblasts LIMP2 protein levels correlated significantly with GCase activity, which was not the case in control subjects or in genetic PD GBA N370S cells. In conclusion, idiopathic PD fibroblasts have decreased GCase activity primarily driven by altered LIMP2-mediated transport of GBA to lysosome and the reduced GCase activity exhibited by the genetic GBA N370S derived PD fibroblasts occurs through a different mechanism.

Keywords:  GBA; Idiopathic PD fibroblasts; LIMP2; Lysosomal dysfunction

DOI:  https://doi.org/10.1186/s13041-020-00712-3
J Cell Biol. 2021 Feb 01. pii: e202002075. [Epub ahead of print]220(2):

Rag GTPases and phosphatidylinositol 3-phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex.

Jennifer Hirst, Geoffrey G Hesketh, Anne-Claude Gingras, Margaret S Robinson.

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.

DOI: https://doi.org/10.1083/jcb.202002075
FASEB J. 2021 Feb;35(2): e21277

Lysosomal calcium is modulated by STIM1/TRPML1 interaction which participates to neuronal survival during ischemic preconditioning.

Valentina Tedeschi, Maria José Sisalli, Tiziana Petrozziello, Lorella Maria Teresa Canzoniero, Agnese Secondo.

  A robust activity of the lysosomal Ca2+ channel TRPML1 is sufficient to correct cellular defects in neurodegeneration. Importantly, lysosomes are refilled by the endoplasmic reticulum (ER). However, it is unclear how TRPML1 function could be modulated by the ER. Here, we deal with this issue in rat primary cortical neurons exposed to different oxygen conditions affecting neuronal survival. Under normoxic conditions, TRPML1: (1) showed a wide distribution within soma and along neuronal processes; (2) was stimulated by the synthetic agonist ML-SA1 and the analog of its endogenous modulator, PI(3,5)P2 diC8; (3) its knockdown by siRNA strategy produced an ER Ca2+ accumulation; (4) co-localized and co-immunoprecipitated with the ER-located Ca2+ sensor stromal interacting molecule 1 (STIM1). In cortical neurons lacking STIM1, ML-SA1 and PI(3,5)P2 diC8 failed to induce Ca2+ release and, more deeply, they induced a negligible Ca2+ passage through the channel in neurons transfected with the genetically encoded Ca2+ indicator GCaMP3-ML1. Moreover, TRPML1/STIM1 interplay changed at low-oxygen conditions: both proteins were downregulated during the ischemic preconditioning (IPC) while during IPC followed by 1 hour of normoxia, at which STIM1 is upregulated, TRPML1 protein was reduced. However, during oxygen and glucose deprivation (OGD) followed by reoxygenation, TRPML1 and STIM1 proteins peaked at 8 hours of reoxygenation, when the proteins were co-immunoprecipitated and reactive oxygen species (ROS) hyperproduction was measured in cortical neurons. This may lead to a persistent TRPML1 Ca2+ release and lysosomal Ca2+ loss. Collectively, we showed a new modulation exerted by STIM1 on TRPML1 activity that may differently intervene during hypoxia to regulate organellar Ca2+ homeostasis.

Keywords:  TRPML1; ischemic preconditioning; lysosome/ER interplay; neuronal survival; organellar Ca2+ homeostasis; oxygen and glucose deprivation followed by reoxygenation; primary cortical neurons

DOI:  https://doi.org/10.1096/fj.202001886R
J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50712-7. [Epub ahead of print]295(52): 18459-18473

FUS contributes to mTOR-dependent inhibition of translation.

Myriam Sévigny, Isabelle Bourdeau Julien, Janani Priya Venkatasubramani, Jeremy B Hui, Paul A Dutchak, Chantelle F Sephton.

  The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)-linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.

Keywords:  RNA-binding protein; Torin1; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); fragile X mental retardation (FMRP); frontotemporal degeneration (FTD); fused in sarcoma (FUS); mRNA translation; mTORC1; mTORC2; mammalian target of rapamycin (mTOR); neurodegeneration; polyribosome; protein synthesis; rapamycin; ribosome; translation regulation

DOI:  https://doi.org/10.1074/jbc.RA120.013801
Cell Death Differ. 2021 Jan 18.

mTORC1 and mTORC2 coordinate early NK cell development by differentially inducing E4BP4 and T-bet.

Dan Li, Yuande Wang, Meixiang Yang, Zhongjun Dong.

Natural killer (NK) cell development is a multistep process that requires a variety of signals and transcription factors. The lack of mammalian target of rapamycin (mTOR) kinase severely impairs NK cell development in mice. mTOR binds to Raptor and Rictor to form two complexes, mTORC1 and mTORC2, respectively. How mTOR and its two complexes regulate NK cell development is not fully understood. Here, we developed two methods to inactivate mTOR, Raptor, or Rictor in early stage NK cells (using CD122-Cre) or in late-stage NK cells (using Ncr1-CreTg). First, we found that when mTOR was deleted by CD122-Cre during and after NK cell commitment, NK cell development was severely impaired, while Ncr1-CreTg mediated mTOR deletion slightly affected NK cell terminal differentiation, suggesting that mTOR is essential for early NK cell differentiation. Second, we found that CD122-mediated deletion of Raptor significantly limited the differentiation of CD27+CD11b- immature NK (iNK) cell into mature NK cells. In contrast, the absence of Rictor significantly interfered with the differentiation of CD27-CD11b- early iNK cells. Third, Ncr1-mediated deletion of Raptor, rather than Rictor, moderately affected NK cell terminal differentiation. In terms of mechanism, mTORC1 mainly promotes the expression of NK cell-specific transcription factor E4 promoter-binding protein 4 (E4BP4), while both mTORC1 and mTORC2 can enhance the expression of T-bet. Therefore, mTORC1 and mTORC2 subtly coordinate NK cell development by differentially inducing E4BP4 and T-bet.

DOI: https://doi.org/10.1038/s41418-020-00715-6
Autophagy. 2021 Jan 18.

Decrease of neuronal FKBP4/FKBP52 modulates perinuclear lysosomal positioning and MAPT/Tau behavior during MAPT/Tau-induced proteotoxic stress.

Béatrice Chambraud, Corentin Daguinot, Kevin Guillemeau, Melanie Genet, Omar Dounane, Geri Meduri, Christian Poüs, Etienne Emile Baulieu, Julien Giustiniani.

  Defects of autophagy-lysosomal protein degradation are thought to contribute to the pathogenesis of several neurodegenerative diseases, and the accumulation of aggregation prone proteins such as MAPT/Tau in Alzheimer disease (AD). We previously showed the localization of the immunophilin FKBP4/FKBP52 in the lysosomal system of healthy human neurons suggesting its possible role in lysosome function. We also showed that decreased FKBP4 levels in AD brain neurons correlate with abnormal MAPT accumulation and aggregation. In this study, we demonstrate that FKBP4 decrease in a human neuronal cell line (SH-SY5Y) and in dorsal root ganglion (DRG) neurons from human MAPTP301S transgenic mice affected the function of the autophagy-lysosomal system under MAPT induced proteotoxic stress conditions. We show that acute MAPT accumulation in SH-SY5Y cells induced perinuclear clustering of lysosomes, triggered FKBP4 localization around the clusters and its colocalization with MAPT and MAP1LC3/LC3-positive autophagic vesicles; a similar FKBP4 localization was detected in some AD brain neurons. We demonstrate that FKBP4 decrease altered lysosomal clustering along with MAPT and MAP1LC3 secretion increase. Although ectopic FKBP4 expression could not induce autophagy under our experimental conditions, it prevented MAPT secretion after MAPT accumulation in SH-SY5Y cells implying a regulatory role of FKBP4 on MAPT secretion. Finally, we observe that FKBP4 deficiency decreased MAP1LC3-II expression and provoked MAPT accumulation during long-term stress in mouse DRG neurons. We hypothesize that the abnormal FKBP4 decrease observed in AD brain neurons might hinder autophagy efficiency and contribute to the progression of the tauopathy by modulating MAPT secretion and accumulation during MAPT pathogenesis.

Keywords:  Alzheimer disease; FKBP52; Tau; autophagy; lysosomes; proteotoxic stress

DOI:  https://doi.org/10.1080/15548627.2021.1875611
J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50707-3. [Epub ahead of print]295(52): 18390-18405

Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program.

Adhish S Walvekar, Ganesh Kadamur, Sreesa Sreedharan, Ritu Gupta, Rajalakshmi Srinivasan, Sunil Laxman.

  Methionine, through S-adenosylmethionine, activates a multifaceted growth program in which ribosome biogenesis, carbon metabolism, and amino acid and nucleotide biosynthesis are induced. This growth program requires the activity of the Gcn4 transcription factor (called ATF4 in mammals), which facilitates the supply of metabolic precursors that are essential for anabolism. However, how Gcn4 itself is regulated in the presence of methionine is unknown. Here, we discover that Gcn4 protein levels are increased by methionine, despite conditions of high cell growth and translation (in which the roles of Gcn4 are not well-studied). We demonstrate that this mechanism of Gcn4 induction is independent of transcription, as well as the conventional Gcn2/eIF2α-mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 phosphorylation is decreased, which reduces its ubiquitination and therefore degradation. Gcn4 is dephosphorylated by the protein phosphatase 2A (PP2A); our data show that when methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the balance of Gcn4 toward a dephosphorylated, stable state. The absence of Ppm1 or the loss of the PP2A methylation destabilizes Gcn4 even when methionine is abundant, leading to collapse of the Gcn4-dependent anabolic program. These findings reveal a novel, methionine-dependent signaling and regulatory axis. Here methionine directs the conserved methyltransferase Ppm1 via its target phosphatase PP2A to selectively stabilize Gcn4. Through this, cells conditionally modify a major phosphatase to stabilize a metabolic master regulator and drive anabolism.

Keywords:  ATF4; Gcn4; S-adenosylmethionine (SAM); Saccharomyces cerevisiae; amino acid; anabolism; cell growth; methionine; methyltransferase; nucleotide; nucleotide metabolism; protein methylation; protein phosphatase 2 (PP2A)

DOI:  https://doi.org/10.1074/jbc.RA120.014248
J Biol Chem. 2020 Nov 27. pii: S0021-9258(20)00094-0. [Epub ahead of print]296 100104

ABHD5 suppresses cancer cell anabolism through lipolysis-dependent activation of the AMPK/mTORC1 pathway.

Guohua Chen, Guoli Zhou, Aaron Lotvola, James G Granneman, Jian Wang.

  ABHD5 is an essential coactivator of ATGL, the rate-limiting triglyceride (TG) lipase in many cell types. Importantly, ABHD5 also functions as a tumor suppressor, and ABHD5 mRNA expression levels correlate with patient survival for several cancers. Nevertheless, the mechanisms involved in ABHD5-dependent tumor suppression are not known. We found that overexpression of ABHD5 induces cell cycle arrest at the G1 phase and causes growth retardation in a panel of prostate cancer cells. Transcriptomic profiling and biochemical analysis revealed that genetic or pharmacological activation of lipolysis by ABHD5 potently inhibits mTORC1 signaling, leading to a significant downregulation of protein synthesis. Mechanistically, we found that ABHD5 elevates intracellular AMP content, which activates AMPK, leading to inhibition of mTORC1. Interestingly, ABHD5-dependent suppression of mTORC1 was abrogated by pharmacological inhibition of DGAT1 or DGAT2, isoenzymes that re-esterify fatty acids in a process that consumes ATP. Collectively, this study maps out a novel molecular pathway crucial for limiting cancer cell proliferation, in which ABHD5-mediated lipolysis creates an energy-consuming futile cycle between TG hydrolysis and resynthesis, leading to inhibition of mTORC1 and cancer cell growth arrest.

Keywords:  AMP-activated protein kinase (AMPK); cancer metabolism; lipolysis; mTOR; αβ hydrolase domain containing 5 (ABHD5)

DOI:  https://doi.org/10.1074/jbc.RA120.014682
Int J Mol Sci. 2021 Jan 15. pii: E817. [Epub ahead of print]22(2):

Crosstalk of the Caspase Family and Mammalian Target of Rapamycin Signaling.

Junfang Yan, Yi Xie, Jing Si, Lu Gan, Hongyan Li, Chao Sun, Cuixia Di, Jinhua Zhang, Guomin Huang, Xuetian Zhang, Hong Zhang.

  Cell can integrate the caspase family and mammalian target of rapamycin (mTOR) signaling in response to cellular stress triggered by environment. It is necessary here to elucidate the direct response and interaction mechanism between the two signaling pathways in regulating cell survival and determining cell fate under cellular stress. Members of the caspase family are crucial regulators of inflammation, endoplasmic reticulum stress response and apoptosis. mTOR signaling is known to mediate cell growth, nutrition and metabolism. For instance, over-nutrition can cause the hyperactivation of mTOR signaling, which is associated with diabetes. Nutrition deprivation can inhibit mTOR signaling via SH3 domain-binding protein 4. It is striking that Ras GTPase-activating protein 1 is found to mediate cell survival in a caspase-dependent manner against increasing cellular stress, which describes a new model of apoptosis. The components of mTOR signaling-raptor can be cleaved by caspases to control cell growth. In addition, mTOR is identified to coordinate the defense process of the immune system by suppressing the vitality of caspase-1 or regulating other interferon regulatory factors. The present review discusses the roles of the caspase family or mTOR pathway against cellular stress and generalizes their interplay mechanism in cell fate determination.

Keywords:  cell fate; interplay; mTOR signaling; the caspase family

DOI:  https://doi.org/10.3390/ijms22020817
J Biol Chem. 2020 Dec 18. pii: S0021-9258(17)50636-5. [Epub ahead of print]295(51): 17497-17513

Fibrillar α-synuclein toxicity depends on functional lysosomes.

Stephanie J Guiney, Paul A Adlard, Peng Lei, Celeste H Mawal, Ashley I Bush, David I Finkelstein, Scott Ayton.

  Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron-derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.

Keywords:  Parkinson disease; Parkinson's disease; alpha-synuclein; alpha-synuclein (a-synuclein); cell death; cell viability; iron; lysosome; lysosomes

DOI:  https://doi.org/10.1074/jbc.RA120.013428
J Diabetes Res. 2020 ;2020 8872639

Nutrient Sensor mTOR and OGT: Orchestrators of Organelle Homeostasis in Pancreatic β-Cells.

Nicholas Esch, Seokwon Jo, Mackenzie Moore, Emilyn U Alejandro.

The purpose of this review is to integrate the role of nutrient-sensing pathways into β-cell organelle dysfunction prompted by nutrient excess during type 2 diabetes (T2D). T2D encompasses chronic hyperglycemia, hyperlipidemia, and inflammation, which each contribute to β-cell failure. These factors can disrupt the function of critical β-cell organelles, namely, the ER, mitochondria, lysosomes, and autophagosomes. Dysfunctional organelles cause defects in insulin synthesis and secretion and activate apoptotic pathways if homeostasis is not restored. In this review, we will focus on mTORC1 and OGT, two major anabolic nutrient sensors with important roles in β-cell physiology. Though acute stimulation of these sensors frequently improves β-cell function and promotes adaptation to cell stress, chronic and sustained activity disturbs organelle homeostasis. mTORC1 and OGT regulate organelle function by influencing the expression and activities of key proteins, enzymes, and transcription factors, as well as by modulating autophagy to influence clearance of defective organelles. In addition, mTORC1 and OGT activity influence islet inflammation during T2D, which can further disrupt organelle and β-cell function. Therapies for T2D that fine-tune the activity of these nutrient sensors have yet to be developed, but the important role of mTORC1 and OGT in organelle homeostasis makes them promising targets to improve β-cell function and survival.

DOI: https://doi.org/10.1155/2020/8872639
Elife. 2021 Jan 18. pii: e61630. [Epub ahead of print]10

Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells.

Iris E Glykofridis, Jaco C Knol, Jesper A Balk, Denise Westland, Thang V Pham, Sander R Piersma, Sinéad M Lougheed, Sepide Derakhshan, Puck Veen, Martin A Rooimans, Saskia E van Mil, Franziska Böttger, Pino J Poddighe, Irma van de Beek, Jarno Drost, Fried Jt Zwartkruis, Renee X de Menezes, Hanne Ej Meijers-Heijboer, Arjan C Houweling, Connie R Jimenez, Rob Mf Wolthuis.

  Germline inactivating mutations in Folliculin (FLCN) cause Birt-Hogg-Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing to kidney tumors. FLCN is a conserved, essential gene linked to diverse cellular processes but the mechanisms by which FLCN prevents kidney cancer remain unknown. Here we show that deleting FLCN activates TFE3, upregulating its downstream E-box genes in human renal tubular epithelial cells (RPTEC/TERT1), including RRAGD and GPNMB, without modifying mTORC1 activity. Surprisingly, deletion of FLCN or its binding partners FNIP1/FNIP2 also induces interferon response genes, but independently of interferon. Mechanistically, FLCN loss promotes STAT2 recruitment to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 signaling as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence the immune response. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint candidate prognostic biomarkers.

Keywords:  cancer biology; genetics; genomics; human

DOI:  https://doi.org/10.7554/eLife.61630
Exp Mol Med. 2021 Jan 22.

Autophagy regulation by acetylation-implications for neurodegenerative diseases.

Sung Min Son, So Jung Park, Marian Fernandez-Estevez, David C Rubinsztein.

Posttranslational modifications of proteins, such as acetylation, are essential for the regulation of diverse physiological processes, including metabolism, development and aging. Autophagy is an evolutionarily conserved catabolic process that involves the highly regulated sequestration of intracytoplasmic contents in double-membrane vesicles called autophagosomes, which are subsequently degraded after fusing with lysosomes. The roles and mechanisms of acetylation in autophagy control have emerged only in the last few years. In this review, we describe key molecular mechanisms by which previously identified acetyltransferases and deacetylases regulate autophagy. We highlight how p300 acetyltransferase controls mTORC1 activity to regulate autophagy under starvation and refeeding conditions in many cell types. Finally, we discuss how altered acetylation may impact various neurodegenerative diseases in which many of the causative proteins are autophagy substrates. These studies highlight some of the complexities that may need to be considered by anyone aiming to perturb acetylation under these conditions.

DOI: https://doi.org/10.1038/s12276-021-00556-4
Proc Natl Acad Sci U S A. 2021 Jan 26. pii: e2022120118. [Epub ahead of print]118(4):

Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction.

Kendall J Condon, Jose M Orozco, Charles H Adelmann, Jessica B Spinelli, Pim W van der Helm, Justin M Roberts, Tenzin Kunchok, David M Sabatini.

  In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.

Keywords:  CRISPR-Cas9 screen; mTORC1; mitochondria

DOI:  https://doi.org/10.1073/pnas.2022120118
Biology (Basel). 2021 Jan 05. pii: E29. [Epub ahead of print]10(1):

Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice.

Raghubendra Singh Dagur, Moses New-Aaron, Murali Ganesan, Weimin Wang, Svetlana Romanova, Srivatsan Kidambi, Kusum K Kharbanda, Larisa Y Poluektova, Natalia A Osna.

   BACKGROUND: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction.
METHODS: The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus.
RESULTS: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol-HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs' generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet.
CONCLUSION: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

Keywords:  ethanol metabolism; humanized mice; liver disease; next-generation RNA sequencing; small EVs

DOI:  https://doi.org/10.3390/biology10010029
Autophagy. 2021 Jan 20. 1-15

Rapamycin relieves the cataract caused by ablation of Gja8b through stimulating autophagy in zebrafish.

Xiyuan Ping, Jiancheng Liang, Kexin Shi, Jing Bao, Jing Wu, Xiaoning Yu, Xiajing Tang, Jian Zou, Xingchao Shentu.

  Macroautophagy/autophagy is known to be important for intracellular quality control in the lens. GJA8 is a major gap junction protein in vertebrate lenses. Mutations in GJA8 cause cataracts in humans. The well-known cataractogenesis mechanism is that mutated GJA8 leads to abnormal assembly of gap junctions, resulting in defects in intercellular communication among lens cells. In this study, we observed that ablation of Gja8b (a homolog of mammalian GJA8) in zebrafish led to severe defects in organelle degradation, an important cause of cataractogenesis in developing lens. The role of autophagy in organelle degradation in lens remains disputable. Intriguingly, we also observed that ablation of Gja8b induced deficient autophagy in the lens. More importantly, in vivo treatment of zebrafish with rapamycin, an autophagy activator that inhibits MAPK/JNK and MTORC1 signaling, stimulated autophagy in the lens and relieved the defects in organelle degradation, resulting in the mitigation of cataracts in gja8b mutant zebrafish. Conversely, inhibition of autophagy by treatment with the chemical reagent 3-MA blocked these recovery effects, suggesting the important roles of autophagy in organelle degradation in the lens in gja8b mutant zebrafish. Further studies in HLE cells revealed that GJA8 interacted with ATG proteins. Overexpression of GJA8 stimulated autophagy in HLE cells. These data suggest an unrecognized cataractogenesis mechanism caused by ablation of Gja8b and a potential treatment for cataracts by stimulating autophagy in the lens. Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy related; AV: autophagic vacuoles; Dpf: days post fertilization; GJA1: gap junction protein alpha 1; GJA3: gap junction protein alpha 3; GJA8: gap junction protein alpha 8; Hpf: hours post fertilization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; WT: wild type.

Keywords:  3-MA; GJA8; Gja8b; autophagy; cataract; lens; organelle degradation; rapamycin

DOI:  https://doi.org/10.1080/15548627.2021.1872188
J Cell Biochem. 2021 Jan 22.

Thioxothiazolidin derivative, 4-OST, inhibits melanogenesis by enhancing the specific recruitment of tyrosinase-containing vesicles to lysosome.

Kenta Isogawa, Masataka Asano, Masumi Hayazaki, Kenichi Koga, Miyu Watanabe, Keiichi Suzuki, Takahiro Kobayashi, Kyoka Kawaguchi, Akane Ishizuka, Shinya Kato, Hironari Ito, Akie Hamamoto, Hiroko Koyama, Kyoji Furuta, Hiroshi Takemori.

  Tyrosinase catalyzes the rate-limiting step in melanin synthesis. Melanin is synthesized from l-tyrosin in the melanosomes, where tyrosinase and other melanogenic factors are recruited via the vesicle transport system. Genetic and biochemical approaches have revealed a correlation between impairments in the vesicle transport system and albinism. However, the specificity of the individual transport systems for the corresponding melanogenic factors has not been well elucidated yet. Here, we report that the thioxothiazolidin derivative, 4-OST (4-[(5E)-5-[(4-fluorophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]-4-azatricyclo [5.2.1.02 ,6]dec-8-ene-3,5-dione: CAS RN. 477766-87-3) strongly inhibited melanogenesis in mouse melanoma B16F10 cells. 4-OST reduces tyrosinase protein levels without affecting its messenger RNA levels or enzymatic activity. Although a reduction in tyrosinase protein level was observed in the presence of a protein synthesis inhibitor, the reduction may be coupled with protein synthesis. Similarly, GIF-2202 (a derivative of 4-OST) lowers tyrosinase protein levels without affecting the levels of another melanogenic enzyme, tyrosinase-related protein 1 (TYRP1) level. The reduction in tyrosinase protein level is associated with an increase in the levels of the lysosomal proteinase cathepsin S. Chloroquine, a lysosome inhibitor, restored the tyrosinase protein level downregulated by GIF-2202, although no effects of other inhibitors (against proteasome, autophagy, or exocytosis) were observed. In addition, GIF-2202 segregated the immunofluorescence signals of tyrosinase from those of TYRP1. Chloroquine treatment resulted in co-localization of tyrosinase and cathepsin S signals near the perinuclear region, suggesting that 4-OST and GIF-2202 may alter the destination of the tyrosinase vesicle from the melanosome to the lysosome. 4-OST and GIF-2202 can be new tools for studying the tyrosinase-specific vesicle transport system.

Keywords:  chemical biology; melanocytes; melanogenesis; tyrosinase; vesicle transport

DOI:  https://doi.org/10.1002/jcb.29895
Cell Rep. 2021 Jan 19. pii: S2211-1247(20)31648-X. [Epub ahead of print]34(3): 108659

The ER-embedded UBE2J1/RNF26 ubiquitylation complex exerts spatiotemporal control over the endolysosomal pathway.

Tom Cremer, Marlieke L M Jongsma, Fredrik Trulsson, Alfred C O Vertegaal, Jacques Neefjes, Ilana Berlin.

  The endolysosomal system fulfills a wide variety of cellular functions, many of which are modulated through interactions with other organelles. In particular, the ER exerts spatiotemporal constraints on the organization and motility of endosomes and lysosomes. We have recently described the ER transmembrane E3 ubiquitin ligase RNF26 as a regulator of endolysosomal perinuclear positioning and transport dynamics. Here, we report that the ubiquitin conjugating enzyme UBE2J1, also anchored in the ER membrane, partners with RNF26 in this context, and that the cellular activity of the resulting E2/E3 pair is localized in a perinuclear ER subdomain and supported by transmembrane interactions. Through modification of SQSTM1/p62 on lysine 435, the ER-embedded UBE2J1/RNF26 ubiquitylation complex recruits endosomal adaptors to immobilize their cognate vesicles in the perinuclear region of the cell. The resulting spatiotemporal compartmentalization promotes the trafficking of activated EGFR to lysosomes and facilitates the termination of EGF-induced AKT signaling.

Keywords:  Akt; EGFR; RNF26; UBE2J1; endoplasmic reticulum; endosomes; p62; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2020.108659
Nat Rev Cancer. 2021 Jan 18.

The metabolism of cancer cells during metastasis.

Gabriele Bergers, Sarah-Maria Fendt.

Metastasis formation is the major cause of death in most patients with cancer. Despite extensive research, targeting metastatic seeding and colonization is still an unresolved challenge. Only recently, attention has been drawn to the fact that metastasizing cancer cells selectively and dynamically adapt their metabolism at every step during the metastatic cascade. Moreover, many metastases display different metabolic traits compared with the tumours from which they originate, enabling survival and growth in the new environment. Consequently, the stage-dependent metabolic traits may provide therapeutic windows for preventing or reducing metastasis, and targeting the new metabolic traits arising in established metastases may allow their eradication.

DOI: https://doi.org/10.1038/s41568-020-00320-2
Cell Death Differ. 2021 Jan 19.

PIWIL2 interacting with IKK to regulate autophagy and apoptosis in esophageal squamous cell carcinoma.

Xu Zhao, Lian Huang, Yilu Lu, Wenhao Jiang, Yue Song, Bojun Qiu, Dachang Tao, Yunqiang Liu, Yongxin Ma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies and cause of death from cancer in China. Previous studies showed that autophagy and apoptosis inhibition are critical for the survival of ESCC cells. However, the underlying mechanisms remain to be clarified. Recently, we found that PIWIL2, a novel cancer testis protein, is highly expressed in ESCC and associated with high T-stage and poor 5-year survival rate in patients. Our further study showed that PIWIL2 can directly bind to IKK and promote its phosphorylation, leading to phosphorylation of IκB and subsequently nuclear translocation of NF-κB for apoptosis inhibition. Meanwhile, PIWIL2 competitively inhibits binding of IKK to TSC1, and thus deactivate mTORC1 pathway which suppresses ULK1 phosphorylation and initiation of autophagy. The mouse xenograft model suggested that PIWIL2 can promote ESCC growth in an IKK-dependent manner. This present work firstly revealed that PIWIL2 can play a role in regulating autophagy and apoptosis, and is associated with poor prognosis in ESCC patients, providing novel insights into the roles of PIWIL2 in tumorigenesis.

DOI: https://doi.org/10.1038/s41418-020-00725-4
J Biol Chem. 2020 Dec 02. pii: S0021-9258(20)00116-7. [Epub ahead of print]296 100125

A cell-nonautonomous mechanism of yeast chronological aging regulated by caloric restriction and one-carbon metabolism.

Elisa Enriquez-Hesles, Daniel L Smith, Nazif Maqani, Margaret B Wierman, Matthew D Sutcliffe, Ryan D Fine, Agata Kalita, Sean M Santos, Michael J Muehlbauer, James R Bain, Kevin A Janes, John L Hartman, Matthew D Hirschey, Jeffrey S Smith.

  Caloric restriction (CR) improves health span and life span of organisms ranging from yeast to mammals. Understanding the mechanisms involved will uncover future interventions for aging-associated diseases. In budding yeast, Saccharomyces cerevisiae, CR is commonly defined by reduced glucose in the growth medium, which extends both replicative and chronological life span (CLS). We found that conditioned media collected from stationary-phase CR cultures extended CLS when supplemented into nonrestricted (NR) cultures, suggesting a potential cell-nonautonomous mechanism of CR-induced life span regulation. Chromatography and untargeted metabolomics of the conditioned media, as well as transcriptional responses associated with the longevity effect, pointed to specific amino acids enriched in the CR conditioned media (CRCM) as functional molecules, with L-serine being a particularly strong candidate. Indeed, supplementing L-serine into NR cultures extended CLS through a mechanism dependent on the one-carbon metabolism pathway, thus implicating this conserved and central metabolic hub in life span regulation.

Keywords:  Saccharomyces cerevisiae; aging; amino acids; caloric restriction; cell-nonautonomous; chronological life span; one-carbon metabolism; serine

DOI:  https://doi.org/10.1074/jbc.RA120.015402
Nat Commun. 2021 01 20. 12(1): 476

Endonuclease G promotes autophagy by suppressing mTOR signaling and activating the DNA damage response.

Wenjun Wang, Jianshuang Li, Junyang Tan, Miaomiao Wang, Jing Yang, Zhi-Min Zhang, Chuanzhou Li, Alexei G Basnakian, Hong-Wen Tang, Norbert Perrimon, Qinghua Zhou.

Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response.

DOI: https://doi.org/10.1038/s41467-020-20780-2