bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒02‒02
twenty-nine papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2020 Jan 26. 1-18
      Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMP-dependent protein kinase (AMPK) complex and ULK1 kinase. In normal culture conditions, PRKDC interacted with the AMPK complex and phosphorylated its nucleotide-sensing γ1 subunit PRKAG1/AMPKγ1 at Ser192 and Thr284, both events being significantly reduced upon the activation of the AMPK complex. Alanine substitutions of PRKDC phosphorylation sites in PRKAG1 reduced AMPK complex activation without affecting its nucleotide sensing capacity. Instead, the disturbance of PRKDC-mediated phosphorylation of PRKAG1 inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with STK11 (serine/threonine kinase 11). Taken together, our data suggest that PRKDC-mediated phosphorylation of PRKAG1 primes AMPK complex to the lysosomal activation by STK11 in cancer cells thereby linking DNA damage response to autophagy and cellular metabolism.Abbreviations: AXIN1: axin 1; 3-MA: 3-methyladenine; 5-FU: 5-fluorouracil; AA mutant: double alanine mutant (S192A, T284A) of PRKAG1; ACACA: acetyl-CoA carboxylase alpha; AICAR: 5-Aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ATM: ataxia telangiectasia mutated; ATR: ATM serine/threonine kinase; AV: autophagic vacuole; AVd: degradative autophagic vacuole; AVi: initial autophagic vacuole; BECN1: beclin 1; BSA: bovine serum albumin; CBS: cystathionine beta-synthase; CDK7: cyclin dependent kinase 7; CDKN1A: cyclin dependent kinase inhibitor 1A; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S transferase; H2AX/H2AFX: H2A.X variant histone; HBSS: Hanks balanced salt solution; IP: immunopurification; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K9: mitogen-activated protein kinase kinase kinase 9; mRFP: monomeric red fluorescent protein; mCh: mCherry; MCM7: minichromosome maintenance complex component 7; MTORC1: mechanistic target of rapamycin kinase complex 1; NHEJ: non-homologous end joining; NRBP2: nuclear receptor binding protein 2; NTC: non-targeting control; NUAK1: NUAK family kinase 1; PBS: phosphate-buffered saline; PIK3AP1: phosphoinositide-3-kinase adaptor protein 1; PIK3CA: phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha; PIKK: phosphatidylinositol 3-kinase-related kinase; PRKAA: protein kinase AMP-activated catalytic subunit alpha; PRKAB: protein kinase AMP-activated non-catalytic subunit beta; PRKAG: protein kinase AMP-activated non-catalytic subunit gamma; PRKDC: protein kinase, DNA-activated, catalytic subunit; RLuc: Renilla luciferase; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TP53: tumor protein p53; TSKS: testis specific serine kinase substrate; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.
    Keywords:  AMPK; PRKDC; autophagy; lysosome; metabolism; signaling
  2. Mol Cell. 2020 Jan 20. pii: S1097-2765(19)30956-6. [Epub ahead of print]
      Clearance of biomolecular condensates by selective autophagy is thought to play a crucial role in cellular homeostasis. However, the mechanism underlying selective autophagy of condensates and whether liquidity determines a condensate's susceptibility to degradation by autophagy remain unknown. Here, we show that the selective autophagic cargo aminopeptidase I (Ape1) undergoes phase separation to form semi-liquid droplets. The Ape1-specific receptor protein Atg19 localizes to the surface of Ape1 droplets both in vitro and in vivo, with the "floatability" of Atg19 preventing its penetration into droplets. In vitro reconstitution experiments reveal that Atg19 and lipidated Atg8 are necessary and sufficient for selective sequestration of Ape1 droplets by membranes. This sequestration is impaired by mutational solidification of Ape1 droplets or diminished ability of Atg19 to float. Taken together, we propose that cargo liquidity and the presence of sufficient amounts of autophagic receptor on cargo are crucial for selective autophagy of biomolecular condensates.
    Keywords:  Cvt pathway; autophagic receptor; autophagy; biomolecular condensates; floatability; liquidity; phase separation; selective autophagy
  3. Cell Rep. 2020 Jan 28. pii: S2211-1247(19)31751-6. [Epub ahead of print]30(4): 1101-1116.e5
      Although typically upregulated upon cellular stress, autophagy can also be utilized under homeostatic conditions as a quality control mechanism or in response to developmental cues. Here, we report that autophagy is required for the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis. Disruption of autophagy in CySCs and early cyst cells (CCs) by the depletion of autophagy-related (Atg) genes reduced early CC numbers and affected CC function, resembling decreased epidermal growth factor receptor (EGFR) signaling. Indeed, our data indicate that EGFR acts to stimulate autophagy to preserve early CC function, whereas target of rapamycin (TOR) negatively regulates autophagy in the differentiating CCs. Finally, we show that the EGFR-mediated stimulation of autophagy regulates lipid levels in CySCs and CCs. These results demonstrate a key role for autophagy in regulating somatic stem cell behavior and tissue homeostasis by integrating cues from both the EGFR and TOR signaling pathways to control lipid metabolism.
    Keywords:  Drosophila; EGFR; TOR; autophagy; lipids; somatic stem cells; testis
  4. Mol Cell. 2020 Jan 22. pii: S1097-2765(19)30958-X. [Epub ahead of print]
      AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.
    Keywords:  ESCRT; LKB1; TAK1; TOR; TRAIL; autophagosome; diabetes; metabolism; metformin; tuberculosis
  5. Ann N Y Acad Sci. 2020 Jan 27.
      Macroautophagy is a lysosomal degradative pathway or recycling process that maintains cellular homeostasis. This autophagy involves a series of sequential processing events, such as initiation; elongation and nucleation of the isolation membrane; cargo recruitment and maturation of the autophagosome (AP); transport of the AP; docking and fusion of the AP with a late endosome or lysosome; and regeneration of the lysosome by the autophagic lysosomal reformation cycle. These events are critically coordinated by the action of a set of several key components, including autophagy-related proteins (Atg), and regulated by intricate networks, such as mechanistic target of rapamycin (mTOR), a master regulator of autophagy, as well as mTOR-independent signaling pathways. Among mTOR-independent pathways, the transient receptor potential (TRP) calcium ion channel TRPML (mucolipin) subfamily is emerging as an important signaling channel to modulate lysosomal biogenesis and autophagy. This review discusses the recent advances in elucidating the molecular mechanisms and regulation of the autophagy process. Understanding these mechanisms may ultimately allow scientists and clinicians to control this process in order to improve human health.
    Keywords:  TRPML; amino acid sensing; autophagy regulation; calcium channel; mTOR signaling; macroautophagy; miRNA
  6. Free Radic Biol Med. 2020 Jan 23. pii: S0891-5849(19)32564-X. [Epub ahead of print]
      Leucine, nutrient signal and substrate for the branched chain aminotransferase (BCAT) activates the mechanistic target of rapamycin (mTORC1) and regulates autophagic flux, mechanisms implicated in the pathogenesis of neurodegenerative conditions such as Alzheimer's disease (AD). BCAT is upregulated in AD, where a moonlighting role, imparted through its redox-active CXXC motif, has been suggested. Here we demonstrate that the redox state of BCAT signals differential phosphorylation by protein kinase C (PKC) regulating the trafficking of cellular pools of BCAT. We show inter-dependence of BCAT expression and proteins associated with the P13K/Akt/mTORC1 and autophagy signalling pathways. In response to insulin or an increase in ROS, BCATc is trafficked to the membrane and docks via palmitoylation, which is associated with BCATc-induced autophagy through PKC phosphorylation. In response to increased levels of BCATc, as observed in AD, amyloid β (Aβ) levels accumulate due to a shift in autophagic flux. This effect was diminished when incubated with leucine, indicating that dietary levels of amino acids show promise in regulating Aβ load. Together these findings show that increased BCATc expression, reported in human AD brain, will affect autophagy and Aβ load through the interdependence of its redox-regulated phosphorylation offering a novel target to address AD pathology.
    Keywords:  Autophagy; Aβ; BCAT; Insulin signalling; PKC; Phosphorylation; Redox regulated
  7. Autophagy. 2020 Jan 26.
      Mitochondria operate as a central hub for many metabolic processes by sensing and responding to the cellular environment. Developmental cues from the environment have been implicated in selective autophagy, or mitophagy, of mitochondria during cell differentiation and tissue development. Mitophagy occurring in this context, termed programmed mitophagy, responds to cell state rather than mitochondrial damage and is often accompanied by a metabolic transition. However, little is known about the mechanisms that engage and execute mitophagy under physiological or developmental conditions. As the mammary gland undergoes post-natal development and lactation challenges mitochondrial homeostasis, we investigated the contribution of mitochondria to differentiation of mammary epithelial cells (MECs). Using lactogenic differentiation of the HC11 mouse MEC line, we demonstrated that HC11 cells transition to a highly energetic state during differentiation by engaging both oxidative phosphorylation and glycolysis. Interestingly, this transition was lost when autophagy was inhibited with bafilomycin A1 or knockdown of Atg7 (autophagy related 7). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover in vitro with the fluorescent probe, pMitoTimer. Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of Prkn/parkin in HC11 cells. We found that MEC differentiation was impaired in shPrkn cells, implying that PRKN is required for MEC differentiation. These studies suggest novel regulation of MEC differentiation through programmed mitophagy and provide a foundation for future studies of development and disease associated with mitochondrial function in the mammary gland.
    Keywords:  Development; differentiation; mammary gland; mitochondria; mitophagy; parkin
  8. Nat Commun. 2020 Jan 31. 11(1): 645
      Autophagy malfunctioning occurs in multiple human disorders, making attractive the idea of chemically modulating it with therapeutic purposes. However, for many types of autophagy, a clear understanding of tissue-specific differences in their activity and regulation is missing because of lack of methods to monitor these processes in vivo. Chaperone-mediated autophagy (CMA) is a selective type of autophagy that until now has only been studied in vitro and not in the tissue context at single cell resolution. Here, we develop a transgenic reporter mouse that allows dynamic measurement of CMA activity in vivo using image-based procedures. We identify previously unknown spatial and temporal differences in CMA activity in multiple organs and in response to stress. We illustrate the versatility of this model for monitoring CMA in live animals, organotypic cultures and cell cultures from these mice, and provide practical examples of multiorgan response to drugs that modulate CMA.
  9. FEBS Lett. 2020 Jan 29.
      Autophagy is an intracellular process that regulates the degradation of cytosolic proteins and organelles. Dying cells often accumulate autophagosomes. However, the mechanisms by which necroptotic stimulation induces autophagosomes are not defined. Here, we demonstrate that activation of necroptosis with TNFα plus the cell-permeable pan-caspase inhibitor Z-VAD induces LC3-II and LC3 puncta, markers of autophagsomes, via the receptor-interacting protein kinase 3 (RIPK3) in intestinal epithelial cells. Surprisingly, necroptotic stimulation reduces autophagic activity, as evidenced by enlarged puncta of the autophagic substrate SQSTM1/p62 and its increased colocalization with LC3. However, necroptotic stimulation does not induce the lysosome-associated membrane protein 1 (LAMP1) nor syntaxin 17, which mediates autophagosome-lysosome fusion, to colocalize with LC3. These data indicate that necroptosis attenuates autophagic flux before the lysosome fusion step. Our findings may provide insights into human diseases involving necroptosis.
    Keywords:  RIPK1; RIPK3; SQSTM1/p62; STX17; autophagy; necroptotic stimulation; necrostatin-1
  10. Dev Cell. 2020 Jan 27. pii: S1534-5807(19)31067-6. [Epub ahead of print]52(2): 236-250.e7
      Regulation of embryonic diapause, dormancy that interrupts the tight connection between developmental stage and time, is still poorly understood. Here, we characterize the transcriptional and metabolite profiles of mouse diapause embryos and identify unique gene expression and metabolic signatures with activated lipolysis, glycolysis, and metabolic pathways regulated by AMPK. Lipolysis is increased due to mTORC2 repression, increasing fatty acids to support cell survival. We further show that starvation in pre-implantation ICM-derived mouse ESCs induces a reversible dormant state, transcriptionally mimicking the in vivo diapause stage. During starvation, Lkb1, an upstream kinase of AMPK, represses mTOR, which induces a reversible glycolytic and epigenetically H4K16Ac-negative, diapause-like state. Diapause furthermore activates expression of glutamine transporters SLC38A1/2. We show by genetic and small molecule inhibitors that glutamine transporters are essential for the H4K16Ac-negative, diapause state. These data suggest that mTORC1/2 inhibition, regulated by amino acid levels, is causal for diapause metabolism and epigenetic state.
    Keywords:  H4K16Ac; LKB1; amino acids; diapause; epigenetics; glutamine transporter; lipolysis; mTOR; metabolism; pluripotent stem cells
  11. Cell Death Differ. 2020 Jan 27.
      Macroautophagy (autophagy) delivers intracellular constituents to the lysosome to promote catabolism. During development in multiple organisms, autophagy mediates various cellular processes, including survival during starvation, programmed cell death, phagocytosis, organelle elimination, and miRNA regulation. Our current understanding of autophagy has been enhanced by developmental biology research during the last quarter of a century. Through experiments that focus on animal development, fundamental mechanisms that control autophagy and that contribute to disease were elucidated. Studies in embryos revealed specific autophagy molecules that mediate the removal of paternally derived mitochondria, and identified autophagy components that clear protein aggregates during development. Importantly, defects in mtDNA inheritance, or removal of paternal mtDNA via mitochondrial autophagy, can contribute to mitochondrial-associated disease. In addition, impairment of the clearance of protein aggregates by autophagy underlies neurodegenerative diseases. Experiments in multiple organisms also reveal conserved mechanisms of tissue remodeling that rely on the cooperation between autophagy and apoptosis to clear cell corpses, and defects in autophagy and apoptotic cell clearance can contribute to inflammation and autoimmunity. Here we provide an overview of key developmental processes that are mediated by autophagy in multiple animals.
  12. Neuron. 2020 Jan 24. pii: S0896-6273(20)30004-0. [Epub ahead of print]
      Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington's disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways.
    Keywords:  Huntington’s disease; Nme1; genome-wide screening; neuronal essential genes
  13. J Mater Chem B. 2020 Jan 29.
      Autophagy plays crucial roles in maintaining normal intracellular homeostasis. Molecular probes capable of monitoring lysosomal pH changes during autophagy are still highly required yet challenging to develop. Here, a lysosome-targeting fluorescent pH probe, RML, is presented by introducing a methylcarbitol unit as the lysosome-targeting group to rhodamine B, which is highly sensitive to pH changes. RML exhibits remarkable pH-dependent behavior at 583 nm with a fluorescent enhancement of more than 148-fold. The pKa value is determined as 4.96, and the linear response with pH changes from 4.50-5.70, which is favorable for lysosomal pH imaging. We also confirm that RML diffuses selectively into lysosomes using confocal fluorescence microscopy. Using RML, we have successfully visualized autophagy by monitoring the lysosomal pH changes.
  14. J Lipid Res. 2020 Jan 27. pii: jlr.RA119000571. [Epub ahead of print]
      Niemann-Pick type C (NPC) disease is a lipid storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-β-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD's mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the endoplasmic reticulum (ER) are most consistent with increased ER membrane cholesterol.  To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol traffics to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and re-esterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during restoration of cholesterol homeostatic responses.
    Keywords:  Cholesterol; Cholesterol/Trafficking; Drug therapy; Lipoproteins; Niemann-Pick disease; cyclodextrin; stable isotope tracers
  15. Nat Commun. 2020 Jan 29. 11(1): 578
      Lipid droplets (LDs) are key subcellular organelles for regulating lipid metabolism. Although several subcellular organelles participate in lipid metabolism, it remains elusive whether physical contacts between subcellular organelles and LDs might be involved in lipolysis upon nutritional deprivation. Here, we demonstrate that peroxisomes and peroxisomal protein PEX5 mediate fasting-induced lipolysis by stimulating adipose triglyceride lipase (ATGL) translocation onto LDs. During fasting, physical contacts between peroxisomes and LDs are increased by KIFC3-dependent movement of peroxisomes toward LDs, which facilitates spatial translocations of ATGL onto LDs. In addition, PEX5 could escort ATGL to contact points between peroxisomes and LDs in the presence of fasting cues. Moreover, in adipocyte-specific PEX5-knockout mice, the recruitment of ATGL onto LDs was defective and fasting-induced lipolysis is attenuated. Collectively, these data suggest that physical contacts between peroxisomes and LDs are required for spatiotemporal translocation of ATGL, which is escorted by PEX5 upon fasting, to maintain energy homeostasis.
  16. Sci Rep. 2020 Jan 30. 10(1): 1465
      Mitophagy plays an important role in the maintenance of mitochondrial homeostasis. PTEN-induced kinase (PINK1), a key regulator of mitophagy, is degraded constitutively under steady-state conditions. During mitophagy, it becomes stabilized in the outer mitochondrial membrane, particularly under mitochondrial stress conditions, such as in treatment with uncouplers, generation of excessive mitochondrial reactive oxygen species, and formation of protein aggregates in mitochondria. Stabilized PINK1 recruits and activates E3 ligases, such as Parkin and mitochondrial ubiquitin ligase (MUL1), to ubiquitinate mitochondrial proteins and induce ubiquitin-mediated mitophagy. Here, we found that the anticancer drug gemcitabine induces the stabilization of PINK1 and subsequent mitophagy, even in the absence of Parkin. We also found that gemcitabine-induced stabilization of PINK1 was not accompanied by mitochondrial depolarization. Interestingly, the stabilization of PINK1 was mediated by MUL1. These results suggest that gemcitabine induces mitophagy through MUL1-mediated stabilization of PINK1 on the mitochondrial membrane independently of mitochondrial depolarization.
  17. Genes Dev. 2020 Jan 30.
      Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown. Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction. Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP.
    Keywords:  cytoplasmic chromatin; inflammation; mitochondria; senescence
  18. Autophagy. 2020 Jan 27. 1-15
      Although macroautophagy/autophagy is involved in hepatocellular carcinoma (HCC) initiation and development and has been identified as a mechanism of HCC therapy resistance, the role of ULK1 (unc-51 like autophagy activating kinase 1) in HCC remains unclear. Here, we report that both knockdown and knockout of ULK1 inhibited human HCC cell proliferation and invasion, and Ulk1 deletion abrogated tumor growth in a xenograft mouse model. Furthermore, ULK1 ablation in combination with sorafenib significantly inhibited HCC progression compared with sorafenib treatment alone or vehicle control. To identify candidate ULK1 inhibitors, we used a structure-based virtual docking approach to screen 3428 compounds. Among these compounds, XST-14 showed the highest affinity for the ULK1 protein and specifically blocked ULK1 kinase activity. Moreover, the Lys46, Tyr94 and Asp165 amino acid residues of ULK1 were required for its binding to XST-14 according to molecular docking and mutagenesis experiments. Functional assays revealed that XST-14 blocked autophagy and subsequently induced apoptosis and inhibited growth in HCC cells. More importantly, XST-14 acted synergistically with sorafenib to attenuate HCC progression by inhibiting sorafenib-induced autophagy activation both in vitro and in vivo. In addition, XST-14 was well tolerated and exhibited favorable drug metabolism and pharmacokinetic properties and low toxicity in mice. In summary, our study determined that ULK1 may represent a new therapeutic target for HCC and that targeting ULK1 in combination with sorafenib treatment may serve as a promising interventional strategy for treating HCC.Abbreviations: 3MA: 3-methyladenine; ADV: AutoDock Vina; ATP: adenosine triphosphate; EdU: 5-ethynyl-2'-deoxyuridine; ESI: electrospray ionization; HCC: hepatocellular carcinoma; IC50: half maximal inhibitory concentration; KD: kinase domain; q.o.d., every other day; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPR, surface plasmon resonance.
    Keywords:  Chemotherapy; HCC; ULK1; combination therapy; small molecule therapeutics; sorafenib
  19. Dev Cell. 2020 Jan 27. pii: S1534-5807(19)31040-8. [Epub ahead of print]52(2): 141-151.e5
      We explored the potential for autophagy to regulate budding yeast meiosis. Following pre-meiotic DNA replication, we blocked autophagy by chemical inhibition of Atg1 kinase or engineered degradation of Atg14 and observed homologous chromosome segregation followed by sister chromatid separation; cells then underwent additional rounds of spindle formation and disassembly without DNA re-replication, leading to aberrant chromosome segregation. Analysis of cell-cycle regulators revealed that autophagy inhibition prevents meiosis II-specific expression of Clb3 and leads to the aberrant persistence of Clb1 and Cdc5, two substrates of a meiotic ubiquitin ligase activated by Ama1. Lastly, we found that during meiosis II, autophagy degrades Rim4, an amyloid-like translational repressor whose timed clearance regulates protein production from its mRNA targets, which include CLB3 and AMA1. Strikingly, engineered Clb3 or Ama1 production restored meiotic termination in the absence of autophagy. Thus, autophagy destroys a master regulator of meiotic gene expression to enable irreversible meiotic exit.
    Keywords:  APC/C; Ama1; Atg1; Atg14; Clb3; Rim4; autophagy; meiosis; sporulation; translational repression
  20. Nat Med. 2020 Jan 27.
      Young-onset Parkinson's disease (YOPD), defined by onset at <50 years, accounts for approximately 10% of all Parkinson's disease cases and, while some cases are associated with known genetic mutations, most are not. Here induced pluripotent stem cells were generated from control individuals and from patients with YOPD with no known mutations. Following differentiation into cultures containing dopamine neurons, induced pluripotent stem cells from patients with YOPD showed increased accumulation of soluble α-synuclein protein and phosphorylated protein kinase Cα, as well as reduced abundance of lysosomal membrane proteins such as LAMP1. Testing activators of lysosomal function showed that specific phorbol esters, such as PEP005, reduced α-synuclein and phosphorylated protein kinase Cα levels while increasing LAMP1 abundance. Interestingly, the reduction in α-synuclein occurred through proteasomal degradation. PEP005 delivery to mouse striatum also decreased α-synuclein production in vivo. Induced pluripotent stem cell-derived dopaminergic cultures reveal a signature in patients with YOPD who have no known Parkinson's disease-related mutations, suggesting that there might be other genetic contributions to this disorder. This signature was normalized by specific phorbol esters, making them promising therapeutic candidates.
  21. Front Cell Dev Biol. 2019 ;7 376
    Keywords:  autophagy; data; disease; screening; xenophagy
  22. Mol Cell. 2020 Jan 15. pii: S1097-2765(20)30003-4. [Epub ahead of print]
      The lysosome is an acidic multi-functional organelle with roles in macromolecular digestion, nutrient sensing, and signaling. However, why cells require acidic lysosomes to proliferate and which nutrients become limiting under lysosomal dysfunction are unclear. To address this, we performed CRISPR-Cas9-based genetic screens and identified cholesterol biosynthesis and iron uptake as essential metabolic pathways when lysosomal pH is altered. While cholesterol synthesis is only necessary, iron is both necessary and sufficient for cell proliferation under lysosomal dysfunction. Remarkably, iron supplementation restores cell proliferation under both pharmacologic and genetic-mediated lysosomal dysfunction. The rescue was independent of metabolic or signaling changes classically associated with increased lysosomal pH, uncoupling lysosomal function from cell proliferation. Finally, our experiments revealed that lysosomal dysfunction dramatically alters mitochondrial metabolism and hypoxia inducible factor (HIF) signaling due to iron depletion. Altogether, these findings identify iron homeostasis as the key function of lysosomal acidity for cell proliferation.
    Keywords:  CRISPR; Chelation; Genetic Screens; Iron Depletion; Iron Homeostasis; Iron Sulfur Clusters; Lysosomal Acidity; Lysosomal Dysfunction; Organelle Metabolism; v-ATPase
  23. Nat Metab. 2020 Jan;2(1): 41-49
      Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK)1,2, dysregulation of which are implicated in pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex-1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly down-regulates AMPK signalling by phosphorylating the evolutionarily conserved residue Ser367 in the fission yeast AMPK catalytic subunit Ssp2, and AMPK α1Ser347/α2Ser345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop Thr172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP:ATP ratios; under nutrient stress conditions this was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental, bi-directional regulation between two major metabolic signalling networks and uncover new opportunity for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumor microenvironment.
  24. Nat Commun. 2020 Jan 29. 11(1): 575
      mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.
  25. Cell Commun Signal. 2020 Jan 27. 18(1): 12
      BACKGROUND: Cell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood. To gain more insight into these mechanisms, the ER stressor thapsigargin (Tg) is an instrumental experimental tool. Additionally, Tg forms the basis for analog prodrugs designed for cell killing in targeted cancer therapy. Tg induces apoptosis via the unfolded protein response (UPR), but how apoptosis is initiated, and how individual effects of the various UPR components are integrated, is unclear. Furthermore, the role of autophagy and autophagy-related (ATG) proteins remains elusive.METHODS: To systematically address these key questions, we analyzed the effects of Tg and therapeutically relevant Tg analogs in two human cancer cell lines of different origin (LNCaP prostate- and HCT116 colon cancer cells), using RNAi and inhibitory drugs to target death receptors, UPR components and ATG proteins, in combination with measurements of cell death by fluorescence imaging and propidium iodide staining, as well as real-time RT-PCR and western blotting to monitor caspase activity, expression of ATG proteins, UPR components, and downstream ER stress signaling.
    RESULTS: In both cell lines, Tg-induced cell death depended on death receptor 5 and caspase-8. Optimal cytotoxicity involved a non-autophagic function of MAP1LC3B upstream of procaspase-8 cleavage. PERK, ATF4 and CHOP were required for Tg-induced cell death, but surprisingly acted in parallel rather than as a linear pathway; ATF4 and CHOP were independently required for Tg-mediated upregulation of death receptor 5 and MAP1LC3B proteins, whereas PERK acted via other pathways. Interestingly, IRE1 contributed to Tg-induced cell death in a cell type-specific manner. This was linked to an XBP1-dependent activation of c-Jun N-terminal kinase, which was pro-apoptotic in LNCaP but not HCT116 cells. Molecular requirements for cell death induction by therapy-relevant Tg analogs were identical to those observed with Tg.
    CONCLUSIONS: Together, our results provide a new, integrated understanding of UPR signaling mechanisms and downstream mediators that induce cell death upon Tg-triggered, unmitigated ER stress. Video Abstract.
    Keywords:  ATF4; Apoptosis; Autophagy; CHOP; Caspase-8; Cell death; DR5; IRE1; JNK; LC3B; PERK; SERCA; Thapsigargin; Unfolded protein response; XBP1s
  26. Mol Cell. 2020 Jan 20. pii: S1097-2765(19)30954-2. [Epub ahead of print]
      Ribosome-associated quality control (RQC) purges aberrant mRNAs and nascent polypeptides in a multi-step molecular process initiated by the E3 ligase ZNF598 through sensing of ribosomes collided at aberrant mRNAs and monoubiquitination of distinct small ribosomal subunit proteins. We show that G3BP1-family-USP10 complexes are required for deubiquitination of RPS2, RPS3, and RPS10 to rescue modified 40S subunits from programmed degradation. Knockout of USP10 or G3BP1 family proteins increased lysosomal ribosomal degradation and perturbed ribosomal subunit stoichiometry, both of which were rescued by a single K214R substitution of RPS3. While the majority of RPS2 and RPS3 monoubiquitination resulted from ZNF598-dependent sensing of ribosome collisions initiating RQC, another minor pathway contributed to their monoubiquitination. G3BP1 family proteins have long been considered RNA-binding proteins, however, our results identified 40S subunits and associated mRNAs as their predominant targets, a feature shared by stress granules to which G3BP1 family proteins localize under stress.
    Keywords:  RNA-binding proteins; lysosomal degradation; mRNA surveillance; regulatory protein (de-)ubiquitination; ribophagy; ribosome quality control; stress granule formation; translation
  27. Nature. 2020 Jan 29.
      ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia1-and early-onset Parkinson's disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.
  28. Mol Cell Oncol. 2020 ;7(1): 1682309
      Implication of autophagy in the downregulation of immune signaling pathways through the degradation of their components constitutes an emerging field of investigation. Our work showed that the selective interaction of Drosophila protein Kenny/IKKγ (CG16910) with the autophagic machinery is required for the degradation of the I-kappa B kinase complex. This regulatory mechanism is essential for the downregulation of the immune deficiency (IMD) pathway in response to commensal microbiota to prevent inflammation.
  29. Stem Cell Reports. 2020 Jan 13. pii: S2213-6711(19)30451-5. [Epub ahead of print]
      Long-term culture of primary cells is characterized by functional and secretory changes, which ultimately result in replicative senescence. It is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. We have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and induced pluripotent stem cell-derived MSCs (iMSCs) until they reached growth arrest. Both cell types acquired similar changes in morphology, in vitro differentiation potential, senescence-associated β-galactosidase, and DNA methylation. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes, particularly in functional categories related to metabolic processes. We subsequently compared the metabolomes of MSCs and iMSCs and observed overlapping senescence-associated changes in both cell types, including downregulation of nicotinamide ribonucleotide and upregulation of orotic acid. Taken together, replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging.
    Keywords:  DNA methylation; induced pluripotent stem cells; mesenchymal stromal cells; metabolomics; replicative senescence; transcriptomics