bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒08‒23
thirty-four papers selected by
Viktor Korolchuk
Newcastle University


  1. Stem Cells Transl Med. 2020 Aug 20.
    Cerniauskas E, Kurzawa-Akanbi M, Xie L, Hallam D, Moya-Molina M, White K, Steel D, Doherty M, Whitfield P, Al-Aama J, Armstrong L, Kavanagh D, Lambris JD, Korolchuk VI, Harris C, Lako M.
      Age-related macular degeneration (AMD) is a multifactorial disease, which is characterized by loss of central vision, affecting one in three people by the age of 75. The Y402H polymorphism in the complement factor H (CFH) gene significantly increases the risk of AMD. We show that Y402H-AMD-patient-specific retinal pigment epithelium (RPE) cells are characterized by a significant reduction in the number of melanosomes, an increased number of swollen lysosome-like-vesicles with fragile membranes, Cathepsin D leakage into drusen-like deposits and reduced lysosomal function. The turnover of C3 is increased significantly in high-risk RPE cells, resulting in higher internalization and deposition of the Terminal Complement Complex C5b-9 at the lysosomes. Inhibition of C3 processing via the compstatin analogue Cp40 reverses the disease phenotypes by relieving the lysosomes of their overburden and restoring their function. These findings suggest that modulation of the complement system represents a useful therapeutic approach for AMD patients associated with complement dysregulation.
    Keywords:  C3; C3b; C5b-9; Y402H polymorphism; autophagy; complement activation; complement factor H; human induced pluripotent stem cells; lysosome; retinal pigment epithelium
    DOI:  https://doi.org/10.1002/sctm.20-0211
  2. Nat Cell Biol. 2020 Aug 17.
    Nowosad A, Jeannot P, Callot C, Creff J, Perchey RT, Joffre C, Codogno P, Manenti S, Besson A.
      Autophagy is a catabolic process whereby cytoplasmic components are degraded within lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several studies reported that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy by an unknown mechanism. Here we find that p27 controls autophagy via an mTORC1-dependent mechanism in amino acid-deprived cells. During prolonged starvation, a fraction of p27 is recruited to lysosomes, where it interacts with LAMTOR1, a component of the Ragulator complex required for mTORC1 activation. Binding of p27 to LAMTOR1 prevents Ragulator assembly and mTORC1 activation, promoting autophagy. Conversely, p27-/- cells exhibit elevated mTORC1 signalling as well as impaired lysosomal activity and autophagy. This is associated with cytoplasmic sequestration of TFEB, preventing induction of the lysosomal genes required for lysosome function. LAMTOR1 silencing or mTOR inhibition restores autophagy and induces apoptosis in p27-/- cells. Together, these results reveal a direct coordinated regulation between the cell cycle and cell growth machineries.
    DOI:  https://doi.org/10.1038/s41556-020-0554-4
  3. J Biomed Sci. 2020 Aug 17. 27(1): 87
    Takahara T, Amemiya Y, Sugiyama R, Maki M, Shibata H.
      The mechanistic target of rapamycin complex 1 (mTORC1) is an essential regulator of cell growth and metabolism through the modulation of protein and lipid synthesis, lysosome biogenesis, and autophagy. The activity of mTORC1 is dynamically regulated by several environmental cues, including amino acid availability, growth factors, energy levels, and stresses, to coordinate cellular status with environmental conditions. Dysregulation of mTORC1 activity is closely associated with various diseases, including diabetes, cancer, and neurodegenerative disorders. The discovery of Rag GTPases has greatly expanded our understanding of the regulation of mTORC1 activity by amino acids, especially leucine and arginine. In addition to Rag GTPases, other factors that also contribute to the modulation of mTORC1 activity have been identified. In this review, we discuss the mechanisms of regulation of mTORC1 activity by particular amino acids.
    Keywords:  Amino acids; Intracellular Ca2+ concentration; Rag GTPases; Rheb GTPase; mTOR; mTORC1
    DOI:  https://doi.org/10.1186/s12929-020-00679-2
  4. Nat Commun. 2020 Aug 18. 11(1): 4150
    Yamamuro T, Kawabata T, Fukuhara A, Saita S, Nakamura S, Takeshita H, Fujiwara M, Enokidani Y, Yoshida G, Tabata K, Hamasaki M, Kuma A, Yamamoto K, Shimomura I, Yoshimori T.
      The systemic decline in autophagic activity with age impairs homeostasis in several tissues, leading to age-related diseases. A mechanistic understanding of adipocyte dysfunction with age could help to prevent age-related metabolic disorders, but the role of autophagy in aged adipocytes remains unclear. Here we show that, in contrast to other tissues, aged adipocytes upregulate autophagy due to a decline in the levels of Rubicon, a negative regulator of autophagy. Rubicon knockout in adipocytes causes fat atrophy and hepatic lipid accumulation due to reductions in the expression of adipogenic genes, which can be recovered by activation of PPARγ. SRC-1 and TIF2, coactivators of PPARγ, are degraded by autophagy in a manner that depends on their binding to GABARAP family proteins, and are significantly downregulated in Rubicon-ablated or aged adipocytes. Hence, we propose that age-dependent decline in adipose Rubicon exacerbates metabolic disorders by promoting excess autophagic degradation of SRC-1 and TIF2.
    DOI:  https://doi.org/10.1038/s41467-020-17985-w
  5. Autophagy. 2020 Aug 19.
    Han S, Jeong YY, Sheshadri P, Cai Q.
      Mitochondria sustain various essential functions at synaptic terminals. Synaptic mitochondria deficits have been implicated in early Alzheimer disease (AD) pathophysiology. Mitophagy, a selective autophagy for removal of damaged mitochondria, plays a key role in mitochondrial quality control in neurons. However, fundamental questions remain unanswered as to whether mitophagy regulates synaptic mitochondrial integrity and whether AD-associated early deficits in synaptic mitochondria are attributed to mitophagy failure. We have recently revealed that the integrity of synaptic mitochondria is maintained by a coordination of RHEB-mediated mitophagy with dynein- and SNAPIN-driven retrograde transport. We demonstrate that increased mitophagy initiation, coupled with defective retrograde transport, triggers mitophagy stress at AD synapses. Excitingly, SNAPIN-enhanced retrograde transport reduces synaptic mitophagy stress and ameliorates mitochondrial deficits, thereby counteracting synaptic damage in AD mouse brains. Therefore, our study provides new mechanistic insights into how mitophagy facilitates synaptic mitochondrial maintenance and how mitophagy failure exacerbates AD-linked mitochondrial defects and synaptic degeneration.
    Keywords:  Alzheimer; Nix; PRKN; RHEB; SNAPIN; mitophagosome; retrograde transport; synaptic degeneration; synaptic mitochondrial deficits; synaptic mitophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1810919
  6. Biochem Biophys Res Commun. 2020 Sep 03. pii: S0006-291X(20)31170-0. [Epub ahead of print]529(4): 1025-1032
    Yang C, Ding H, Yang Y, Yang L, Yang Y, Fang M, Ren J, Hu R, Wang C, Geng W.
      Liver kinase B1 (LKB1), a tumour suppressor, participates in many cellular processes, including cell survival, growth, apoptosis, transformation, and metabolism. Upon performing yeast two-hybrid screening, co-immunoprecipitation, and GST pull-down, we identified that BRCA1-associated protein 1 (BAP1), a deubiquitinase, interacts with LKB1. Immunoblotting was performed to examine the effect of BAP1 on the activation of 5' AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), downstream of LKB1. The relationship between BAP1 deficiency and cancer cell proliferation was examined using cell survival assay and soft agar assay. qRT-PCR and oil red O staining were performed to evaluate lipid synthesis. Our findings reveal that BAP1 deubiquitinates LKB1, inhibits its degradation, and stabilises it, thereby affecting AMPK activation and downstream mTOR activity. BAP1 deficiency may enhance cellular proliferation as well as lipid synthesis.
    Keywords:  AMPK; BAP1; Deubiquitination; LKB1; mTOR
    DOI:  https://doi.org/10.1016/j.bbrc.2020.05.223
  7. Autophagy. 2020 Aug 20.
    Oh DS, Park JH, Jung HE, Kim HJ, Lee HK.
      Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infections in infants. Macroautophagy/autophagy is a catalytic metabolic process required for cellular homeostasis. Although intracellular metabolism is important for immune responses in dendritic cells, the link between autophagy and immunometabolism remains unknown. Here, we show that the autophagy-related protein ATG5 regulates immunometabolism. Atg5-deficient mouse dendritic cells showed increased CD8A+ T-cell response and increased secretion of proinflammatory cytokines upon RSV infection. Transcriptome analysis showed that Atg5 deficiency alters the expression of metabolism-related genes. Atg5-deficient dendritic cells also showed increased activation of glycolysis and the AKT-MTOR-RPS6KB pathway and decreased mitochondrial activity, all of which are cellular signatures for metabolic activation. These cells also showed elevated CD8A+ T-cell priming and surface major histocompatibility complex (MHC) class I expression. Our results suggested that ATG5 regulated host immune responses by modulating dendritic cell metabolism. These findings may help develop potential antiviral therapies that alter host immunity by regulating autophagy and immunometabolism.
    Keywords:  ATG5; RSV; autophagy; dendritic cell metabolism; immunometabolism
    DOI:  https://doi.org/10.1080/15548627.2020.1812218
  8. Neurobiol Dis. 2020 Aug 13. pii: S0969-9961(20)30324-7. [Epub ahead of print]144 105049
    Zhao Y, Keshiya S, Perera G, Schramko L, Halliday GM, Dzamko N.
      Kinase activating missense mutations in leucine-rich repeat kinase 2 (LRRK2) predispose to Parkinson's disease. Consequently, there is much interest in delineating LRRK2 biology, both in terms of gaining further insight into disease causes, and also determining whether or not LRRK2 is a potential Parkinson's disease therapeutic target. Indeed, many potent and selective small molecule inhibitors of LRRK2 have been developed and are currently being used for pre-clinical testing in cell and animal models. In the current study, we have obtained fibroblasts from four subjects with the common LRRK2 mutation, G2019S. Fibroblasts were reprogrammed to induced pluripotent stem cells and then to neural stem cells and ultimately neurons. Two clones for each of the human neural cell lines were then chronically treated with and without either of two distinct inhibitors of LRRK2 and effects on toxicity and Parkinson's disease related phenotypes were assessed. Cells with the G2019S mutation had a propensity to accumulate the pathological Parkinson's disease protein α-synuclein. Moreover, α-synuclein accumulation in the G2019S cells was significantly reduced with both LRRK2 inhibitors in seven of the eight cell lines studied. LRRK2 inhibitors also improved the nuclear morphology of G2019S cells and impacted on measures of autophagy and endoplasmic reticulum stress. Lastly, we did not find evidence of inhibitor toxicity under the chronic treatment conditions. These results add to evidence that LRRK2 inhibitors may have utility in the treatment of Parkinson's disease via reducing α-synuclein.
    Keywords:  Kinase inhibitors; LRRK2; Lysosome; Neuron; Parkinson's disease; Pluripotent stem cells; α-synuclein
    DOI:  https://doi.org/10.1016/j.nbd.2020.105049
  9. Sci Adv. 2020 Aug;6(32): eaba1306
    Zou K, Rouskin S, Dervishi K, McCormick MA, Sasikumar A, Deng C, Chen Z, Kaeberlein M, Brem RB, Polymenis M, Kennedy BK, Weissman JS, Zheng J, Ouyang Q, Li H.
      Caloric restriction (CR) is known to extend life span across species; however, the molecular mechanisms are not well understood. We investigate the mechanism by which glucose restriction (GR) extends yeast replicative life span, by combining ribosome profiling and RNA-seq with microfluidic-based single-cell analysis. We discovered a cross-talk between glucose sensing and the regulation of intracellular methionine: GR down-regulated the transcription and translation of methionine biosynthetic enzymes and transporters, leading to a decreased intracellular methionine concentration; external supplementation of methionine cancels the life span extension by GR. Furthermore, genetic perturbations that decrease methionine synthesis/uptake extend life span. These observations suggest that intracellular methionine mediates the life span effects of various nutrient and genetic perturbations, and that the glucose-methionine cross-talk is a general mechanism for coordinating the nutrient status and the translation/growth of a cell. Our work also implicates proteasome as a downstream effector of the life span extension by GR.
    DOI:  https://doi.org/10.1126/sciadv.aba1306
  10. Cell Death Differ. 2020 Aug 17.
    Hattori T, Takahashi Y, Chen L, Tang Z, Wills CA, Liang X, Wang HG.
      Autophagosomal membranes can serve as activation platforms for intracellular death-inducing signaling complexes (iDISCs) to initiate Caspase-8-dependent apoptosis. In this study, we explore the impact of ESCRT-III-dependent phagophore closure on iDISC assemblies and cell death in osteosarcoma and neuroblastoma cells. Inhibition of phagophore closure by conditional depletion of CHMP2A, an ESCRT-III component, stabilizes iDISCs on immature autophagosomal membranes and induces Caspase-8-dependent cell death. Importantly, suppression of the iDISC formation via deletion of ATG7, an E1 enzyme for ubiquitin-like autophagy-related proteins, blocks Caspase-8 activation and cell death following CHMP2A depletion. Although DR5 expression and TRAIL-induced apoptosis are enhanced in CHMP2A-depleted cells, the canonical extrinsic pathway of apoptosis is not responsible for the initiation of cell death by CHMP2A depletion. Furthermore, the loss of CHMP2A impairs neuroblastoma tumor growth associated with decreased autophagy and increased apoptosis in vivo. Together, these findings indicate that inhibition of the ESCRT-III-dependent autophagosome sealing process triggers noncanonical Caspase-8 activation and apoptosis, which may open new avenues for therapeutic targeting of autophagy in cancer.
    DOI:  https://doi.org/10.1038/s41418-020-00610-0
  11. Redox Biol. 2020 Aug 11. pii: S2213-2317(20)30884-3. [Epub ahead of print]36 101679
    Ornatowski W, Lu Q, Yegambaram M, Garcia AE, Zemskov EA, Maltepe E, Fineman JR, Wang T, Black SM.
      The autophagic pathway involves the encapsulation of substrates in double-membraned vesicles, which are subsequently delivered to the lysosome for enzymatic degradation and recycling of metabolic precursors. Autophagy is a major cellular defense against oxidative stress, or related conditions that cause accumulation of damaged proteins or organelles. Selective forms of autophagy can maintain organelle populations or remove aggregated proteins. Dysregulation of redox homeostasis under pathological conditions results in excessive generation of reactive oxygen species (ROS), leading to oxidative stress and the associated oxidative damage of cellular components. Accumulating evidence indicates that autophagy is necessary to maintain redox homeostasis. ROS activates autophagy, which facilitates cellular adaptation and diminishes oxidative damage by degrading and recycling intracellular damaged macromolecules and dysfunctional organelles. The cellular responses triggered by oxidative stress include the altered regulation of signaling pathways that culminate in the regulation of autophagy. Current research suggests a central role for autophagy as a mammalian oxidative stress response and its interrelationship to other stress defense systems. Altered autophagy phenotypes have been observed in lung diseases such as chronic obstructive lung disease, acute lung injury, cystic fibrosis, idiopathic pulmonary fibrosis, and pulmonary arterial hypertension, and asthma. Understanding the mechanisms by which ROS regulate autophagy will provide novel therapeutic targets for lung diseases. This review highlights our current understanding on the interplay between ROS and autophagy in the development of pulmonary disease.
    Keywords:  Autophagy; Mitochondria; Mitophagy; Oxidative stress; Pulmonary disease; Reactive oxygen species
    DOI:  https://doi.org/10.1016/j.redox.2020.101679
  12. Trends Neurosci. 2020 Aug 13. pii: S0166-2236(20)30167-3. [Epub ahead of print]
    Fleming A, Rubinsztein DC.
      Autophagy is a highly conserved intracellular clearance pathway in which cytoplasmic contents are trafficked to the lysosome for degradation. Within neurons, it helps to remove damaged organelles and misfolded or aggregated proteins and has therefore been the subject of intense research in relation to neurodegenerative disease. However, far less is understood about the role of autophagy in other aspects of neuronal physiology. Here we review the literature on the role of autophagy in maintaining neuronal stem cells and in neuronal plasticity in adult life and we discuss how these contribute to structural and functional deficits observed in a range of human disorders.
    Keywords:  autophagy; memory; neurogenesis; neuronal stem cell (NSC); plasticity; psychiatric disease
    DOI:  https://doi.org/10.1016/j.tins.2020.07.003
  13. Autophagy. 2020 Aug 18. 1-2
    Tsang YH, Mills GB.
      The melanoma-associated antigen family A (MAGEA) antigens are expressed in a wide variety of malignant tumors but not in adult somatic cells, rendering them attractive targets for cancer immunotherapy. Recent studies uncovered a role for MAGEA6 in suppression of macroautophagy/autophagy implicating MAGEA6 in tumorigenesis. The impact of cancer-associated MAGEA6 mutations on tumor pathophysiology are less well explored. In pancreatic cancer cell models, MAGEA6 inhibits autophagy, facilitating pancreatic cancer initiation. However, autophagy places a brake on cancer progression and is released upon MAGEA6 degradation, which can be induced by nutrient deficiency or by acquisition of cancer-associated mutations that reinstitute autophagy. Further cancer-associated mutations of the broader MAGEA genes frequently result in degradation of the corresponding protein products by proteasome-dependent machinery, potentially jeopardizing the utility of MAGEA genes as immunotherapeutic targets. Altogether, our findings provide mechanistic insight into the divergent roles of MAGEA6 during pancreatic cancer initiation and progression, and could inform cancer immunotherapeutic strategies for targeting MAGEA antigens.
    Keywords:   MAGEA6 ; Autophagy; cancer; degradation; mutation; oncogene; pancreatic ductal adenocarcinoma; proteasome and tumor suppressor
    DOI:  https://doi.org/10.1080/15548627.2020.1802091
  14. Cell Cycle. 2020 Aug 18. 1-10
    Tang F, Christofori G.
      Organ development is precisely guided by spatiotemporal cross-talks between a variety of signaling pathways regulating cell differentiation, proliferation, growth arrest and physiological cell death. Aberrant signaling inputs invariably lead to tissue dysfunction and to certain conditions, even malignant transformation. In this review, we focus on the functional interplay between the Hippo signaling pathway and autophagy in normal tissue homeostasis and in malignant tumor progression. Mounting experimental evidence for the regulation of cancer cell malignancy and therapy resistance by the functional cross-talk between Hippo signaling and autophagy highlights this signaling axis as a suitable therapeutic target to combat cancer.
    Keywords:  Autophagy; Beclin-1; Hippo signaling; therapy response
    DOI:  https://doi.org/10.1080/15384101.2020.1806450
  15. Theranostics. 2020 ;10(20): 9280-9302
    Zhou K, Zheng Z, Li Y, Han W, Zhang J, Mao Y, Chen H, Zhang W, Liu M, Xie L, Zhang H, Xu H, Xiao J.
      Background and Aim: Increasing evidence suggests that spinal cord injury (SCI)-induced defects in autophagic flux may contribute to an impaired ability for neurological repair following injury. Transcription factor E3 (TFE3) plays a crucial role in oxidative metabolism, lysosomal homeostasis, and autophagy induction. Here, we investigated the role of TFE3 in modulating autophagy following SCI and explored its impact on neurological recovery. Methods: Histological analysis via HE, Nissl and Mason staining, survival rate analysis, and behavioral testing via BMS and footprint analysis were used to determine functional recovery after SCI. Quantitative real-time polymerase chain reaction, Western blotting, immunofluorescence, TUNEL staining, enzyme-linked immunosorbent assays, and immunoprecipitation were applied to examine levels of autophagy flux, ER-stress-induced apoptosis, oxidative stress, and AMPK related signaling pathways. In vitro studies using PC12 cells were performed to discern the relationship between ROS accumulation and autophagy flux blockade. Results: Our results showed that in SCI, defects in autophagy flux contributes to ER stress, leading to neuronal death. Furthermore, SCI enhances the production of reactive oxygen species (ROS) that induce lysosomal dysfunction to impair autophagy flux. We also showed that TFE3 levels are inversely correlated with ROS levels, and increased TFE3 levels can lead to improved outcomes. Finally, we showed that activation of TFE3 after SCI is partly regulated by AMPK-mTOR and AMPK-SKP2-CARM1 signaling pathways. Conclusions: TFE3 is an important regulator in ROS-mediated autophagy dysfunction following SCI, and TFE3 may serve as a promising target for developing treatments for SCI.
    Keywords:  AMPK signaling pathways; Autophagy; ER stress-induced apoptosis; Spinal cord injury; TFE3
    DOI:  https://doi.org/10.7150/thno.46566
  16. EMBO J. 2020 Aug 16. e106162
    Radulovic M, Stenmark H.
      Mutations in several genes encoding for lysosomal proteins are involved in Parkinson's disease (PD). In this issue, Herbst et al (2020) show that PD-related leucine-rich repeat kinase 2 (LRRK2) is activated in response to pathogen or membranolytic drug-induced damage of phagolysosomes and lysosomes in macrophages, and regulates endolysosomal homeostasis by controlling the balance between membrane repair and degradation.
    DOI:  https://doi.org/10.15252/embj.2020106162
  17. Cancers (Basel). 2020 Aug 13. pii: E2266. [Epub ahead of print]12(8):
    Colella B, Colardo M, Iannone G, Contadini C, Saiz-Ladera C, Fuoco C, Barilà D, Velasco G, Segatto M, Di Bartolomeo S.
      Epidermal Growth Factor receptor (EGFR) is a tyrosine kinase receptor widely expressed on the surface of numerous cell types, which activates several downstream signalling pathways involved in cell proliferation, migration and survival. EGFR alterations, such as overexpression or mutations, have been frequently observed in several cancers, including glioblastoma (GBM), and are associated to uncontrolled cell proliferation. Here we show that the inhibition of mammalian target of Rapamycin (mTOR) mediates EGFR delivery to lysosomes for degradation in GBM cells, independently of autophagy activation. Coherently with EGFR internalisation and degradation, mTOR blockade negatively affects the mitogen activated protein/extracellular signal-regulated kinase (MAPK)/ERK pathway. Furthermore, we provide evidence that Src kinase activation is required for EGFR internaliation upon mTOR inhibition. Our results further support the hypothesis that mTOR targeting may represent an effective therapeutic strategy in GBM management, as its inhibition results in EGFR degradation and in proliferative signal alteration.
    Keywords:  EGFR; autophagy; glioma; mTOR
    DOI:  https://doi.org/10.3390/cancers12082266
  18. J Lipid Atheroscler. 2020 Jan;9(1): 8-22
    Yeh HS, Yong J.
      Post-transcriptional regulations of mRNA transcripts such as alternative splicing and alternative polyadenylation can affect the expression of genes without changing the transcript levels. Recent studies have demonstrated that these post-transcriptional events can have significant physiological impacts on various biological systems and play important roles in the pathogenesis of a number of diseases, including cancers. Nevertheless, how cellular signaling pathways control these post-transcriptional processes in cells are not very well explored in the field yet. The mammalian target of rapamycin complex 1 (mTORC1) pathway plays a key role in sensing cellular nutrient and energy status and regulating the proliferation and growth of cells by controlling various anabolic and catabolic processes. Dysregulation of mTORC1 pathway can tip the metabolic balance of cells and is associated with a number of pathological conditions, including various types of cancers, diabetes, and cardiovascular diseases. Numerous reports have shown that mTORC1 controls its downstream pathways through translational and/or transcriptional regulation of the expression of key downstream effectors. And, recent studies have also shown that mTORC1 can control downstream pathways via post-transcriptional regulations. In this review, we will discuss the roles of post-transcriptional processes in gene expression regulations and how mTORC1-mediated post-transcriptional regulations contribute to cellular physiological changes. We highlight post-transcriptional regulation as an additional layer of gene expression control by mTORC1 to steer cellular biology. These emphasize the importance of studying post-transcriptional events in transcriptome datasets for gaining a fuller understanding of gene expression regulations in the biological systems of interest.
    Keywords:  Alternative splicing; Gene expression; Mammalian target of rapamycin; Polyadenylation; Transcriptome
    DOI:  https://doi.org/10.12997/jla.2020.9.1.8
  19. Proc Natl Acad Sci U S A. 2020 Aug 19. pii: 202006997. [Epub ahead of print]
    Li Y, Cheng X, Li M, Wang Y, Fu T, Zhou Z, Wang Y, Gong X, Xu X, Liu J, Pan L.
      Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.
    Keywords:  GABARAP; SNARE; Syntaxin17; autophagosome–lysosome fusion; autophagy
    DOI:  https://doi.org/10.1073/pnas.2006997117
  20. Hepatology. 2020 Aug 16.
    Davuluri G, Welch N, Sekar J, Gangadhariah M, Alsabbagh Alchirazi K, Mohan ML, Kumar A, Kant S, Thapaliya S, Stine M, McMullen MR, McCullough RL, Stark GR, Nagy LE, Naga Prasad SV, Dasarathy S.
      BACKGROUND AND AIMS: Despite the high clinical significance of sarcopenia in alcoholic cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol-induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and AMP kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown).APPROACH AND RESULTS: Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from alcoholic cirrhotics were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue ATP indicating a "pseudofed" state. We identified protein phosphatase 2A (PP2A) as a key mediator of ethanol-induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A (I2-PP2A) to PP2A resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3-kinase-γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol-induced molecular and phenotypic perturbations in wild type mice were observed in PI3Kγ-/- mice even at baseline. Importantly, overexpressing kinase-active PI3Kγ but not the kinase-dead mutant reversed ethanol-induced molecular perturbations.
    CONCLUSIONS: Our study describes the mechanistic underpinnings for previously unrecognized ethanol-mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ-PP2A axis.
    Keywords:  Signaling; ethanol; phosphoinositide-3-kinase-gamma; protein homeostasis; skeletal muscle
    DOI:  https://doi.org/10.1002/hep.31524
  21. Proc Natl Acad Sci U S A. 2020 Aug 21. pii: 202000417. [Epub ahead of print]
    Li XJ, Doetzlhofer A.
      Mechano-sensory hair cells within the inner ear cochlea are essential for the detection of sound. In mammals, cochlear hair cells are only produced during development and their loss, due to disease or trauma, is a leading cause of deafness. In the immature cochlea, prior to the onset of hearing, hair cell loss stimulates neighboring supporting cells to act as hair cell progenitors and produce new hair cells. However, for reasons unknown, such regenerative capacity (plasticity) is lost once supporting cells undergo maturation. Here, we demonstrate that the RNA binding protein LIN28B plays an important role in the production of hair cells by supporting cells and provide evidence that the developmental drop in supporting cell plasticity in the mammalian cochlea is, at least in part, a product of declining LIN28B-mammalian target of rapamycin (mTOR) activity. Employing murine cochlear organoid and explant cultures to model mitotic and nonmitotic mechanisms of hair cell generation, we show that loss of LIN28B function, due to its conditional deletion, or due to overexpression of the antagonistic miRNA let-7g, suppressed Akt-mTOR complex 1 (mTORC1) activity and renders young, immature supporting cells incapable of generating hair cells. Conversely, we found that LIN28B overexpression increased Akt-mTORC1 activity and allowed supporting cells that were undergoing maturation to de-differentiate into progenitor-like cells and to produce hair cells via mitotic and nonmitotic mechanisms. Finally, using the mTORC1 inhibitor rapamycin, we demonstrate that LIN28B promotes supporting cell plasticity in an mTORC1-dependent manner.
    Keywords:  LIN28; hair cell regeneration; inner ear cochlea; let-7 miRNA; mTOR pathway
    DOI:  https://doi.org/10.1073/pnas.2000417117
  22. J Neurovirol. 2020 Aug 17.
    Andhavarapu S, Katuri A, Bryant J, Patel V, Gupta U, Asemu G, Makar TK.
      HIV-associated neurocognitive disorder (HAND) is a collective term describing the spectrum of neurocognitive deficits that arise from HIV infection. Although the introduction to highly active antiretroviral therapy (HAART) has prolonged the lifespan of HIV patients, neurocognitive impairments remain prevalent, as patients are left perpetually with HIV. Currently, physicians face a challenge in treating HAND patients, so a greater understanding of the mechanisms underlying HAND pathology has been a growing focus in HIV research. Recent research has revealed the role disrupted calcium homeostasis in HIV-mediated neurotoxicity. Calcium plays a well-established role in the crosstalk between the mitochondrion and ER as well as in regulating autophagy, and ER stress, mitochondrial dysfunction, and impaired autophagic activity are considered hallmarks in several neurodegenerative and neurocognitive disorders. Therefore, it is paramount that the intricate inter-organelle signaling in relation to calcium homeostasis during HIV infection and the development of HAND is elucidated. This review consolidates current knowledge regarding the neuropathology of neurocognitive disorders and HIV infection with a focus on the underlying role of calcium during ER stress, mitochondrial dysfunction, and autophagy associated with the progression of HAND. The details of this intricate crosstalk during HAND remain relatively unknown; further research in this field can potentially aid in the development of improved therapy for patients suffering from HAND.
    Keywords:  Autophagy; Calcium; Endoplasmic reticulum stress; HIV-associated neurocognitive disorder; Mitochondrial dysfunction
    DOI:  https://doi.org/10.1007/s13365-020-00861-0
  23. Cells. 2020 Aug 20. pii: E1928. [Epub ahead of print]9(9):
    Kang HJ, Yoo EJ, Lee HH, An SM, Park H, Lee-Kwon W, Choi SY, Kwon HM.
      The endoplasmic reticulum (ER) stress response and autophagy are important cellular responses that determine cell fate and whose dysregulation is implicated in the perturbation of homeostasis and diseases. Tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) is a pleiotropic stress protein that mediates both protective and pathological cellular responses. Here, we examined the role of TonEBP in β-cell survival under ER stress. We found that TonEBP increases β-cell survival under ER stress by enhancing autophagy. The level of TonEBP protein increased under ER stress due to a reduction in its degradation via the ubiquitin-proteasome pathway. In response to ER stress, TonEBP increased autophagosome formations and suppressed the accumulation of protein aggregates and β-cell death. The Rel-homology domain of TonEBP interacted with FIP200, which is essential for the initiation of autophagy, and was required for autophagy and cell survival upon exposure to ER stress. Mice in which TonEBP was specifically deleted in pancreatic endocrine progenitor cells exhibited defective glucose homeostasis and a loss of islet mass. Taken together, these findings demonstrate that TonEBP protects against ER stress-induced β-cell death by enhancing autophagy.
    Keywords:  FIP200; NFAT5; UPR; autophagy initiation; islet; unfolded protein response
    DOI:  https://doi.org/10.3390/cells9091928
  24. Genes (Basel). 2020 Aug 19. pii: E956. [Epub ahead of print]11(9):
    Otsubo Y, Kamada Y, Yamashita A.
      Target of rapamycin (TOR) is a serine/threonine kinase that modulates cell growth and metabolism in response to environmental changes. Transfer RNA (tRNA) is an abundant and ubiquitous small non-coding RNA that is essential in the translation of mRNAs. Beyond its canonical role, it has been revealed that tRNAs have more diverse functions. TOR complex 1 (TORC1), which is one of the two TOR complexes, regulates tRNA synthesis by controlling RNA polymerase III. In addition to tRNA synthesis regulation, recent studies have revealed hidden connections between TORC1 and tRNA, which are both essential players in eukaryotic cellular activities. Here, we review the accumulating findings on the regulatory links between TORC1 and tRNA-particularly those links in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe.
    Keywords:  TORC1; budding yeast; fission yeast; tRNA
    DOI:  https://doi.org/10.3390/genes11090956
  25. J Invest Dermatol. 2020 Sep;pii: S0022-202X(19)33570-5. [Epub ahead of print]140(9): 1691-1697
    Jeong D, Qomaladewi NP, Lee J, Park SH, Cho JY.
      Human skin acts as a barrier to protect our bodies from UV rays and external pathogens and to prevent water loss. Phenotypes of aging, or natural aging due to chronic damage, include wrinkles and the reduction of skin thickness that occur because of a loss of skin cell function. The dysregulation of autophagy, a lysosome-related degradation pathway, can lead to cell senescence, cancer, and various human diseases due to abnormal cellular homeostasis. Here, we discuss the roles and molecular mechanisms of autophagy involved in the anti-aging effects of autophagy and the relationship between autophagy and aging in skin cells.
    DOI:  https://doi.org/10.1016/j.jid.2019.11.023
  26. Mol Biol Cell. 2020 Aug 20. mbcE20010068
    Rogers AM, Egan MJ.
      The chaperone-mediated sequestration of misfolded proteins into specialized quality control compartments represents an important strategy for maintaining protein homeostasis in response to stress. However, precisely how this process is controlled in time and subcellular space and integrated with the cell's protein refolding and degradation pathways, remains unclear. We set out to understand how aggregated proteins are managed during infection-related development by a globally devastating plant pathogenic fungus, and to determine how impaired protein quality control impacts upon cellular differentiation and pathogenesis in this system. Here we show that in the absence of Hsp104 disaggregase activity aggregated proteins are spatially sequestered into quality control compartments within conidia, but not within terminally differentiated infection cells, and thus spatial protein quality control is cell-type dependent. We demonstrate that impaired aggregate resolution results in a short-term developmental penalty but has no significant impact upon appressorium function. Lastly, we show that, somewhat unexpectedly, the autophagy machinery is necessary for the normal formation and compartmentalization of protein aggregates. Taken together, our findings provide important new insight into spatial protein quality control during the process of terminal cellular differentiation by a globally important model eukaryote and reveals a new level of interplay between major proteostasis pathways. [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E20-01-0068
  27. Cancers (Basel). 2020 Aug 21. pii: E2371. [Epub ahead of print]12(9):
    Bernard M, Cardin GB, Cahuzac M, Ayad T, Bissada E, Guertin L, Bahig H, Nguyen-Tan PF, Filion E, Ballivy O, Soulieres D, Rodier F, Christopoulos A.
      Genomic analyses of head and neck squamous cell carcinoma (HNSCC) have highlighted alterations in the phosphatidylinositol 3-kinase (PI3K) signaling pathway, presenting a therapeutic target for multiple ongoing clinical trials with PI3K or PI3K/MTOR inhibitors. However, these inhibitors can potentially increase autophagy in HNSCC and indirectly support cancer cell survival. Here, we sought to understand the relationship between the PI3K signaling pathway and autophagy during their dual inhibition in a panel of HNSCC cell lines. We used acridine orange staining, immunoblotting, and tandem sensor Red Fluorescent Protein- Green Fluorescent Protein-, microtubule-associated protein 1 light chain 3 beta (RFP-GFP-LC3B) expression analysis to show that PI3K inhibitors increase autophagosomes in HNSCC cells, but that chloroquine treatment effectively inhibits the autophagy that is induced by PI3K inhibitors. Using the Bliss independence model, we determined that the combination of chloroquine with PI3K inhibitors works in synergy to decrease cancer cell proliferation, independent of the PIK3CA status of the cell line. Our results indicate that a strategy focusing on autophagy inhibition enhances the efficacy of therapeutics already in clinical trials. Our results suggest a broader application for this combination therapy that can be promptly translated to in vivo studies.
    Keywords:  HNSCC; PI3K inhibitor; PI3K signaling pathway; autophagy; buparlisib; cancer; chloroquine; combination therapy; omipalisib; oral tongue
    DOI:  https://doi.org/10.3390/cancers12092371
  28. Biochem Biophys Res Commun. 2020 Aug 12. pii: S0006-291X(20)31446-7. [Epub ahead of print]
    Yokota A, Hiramoto M, Hino H, Tokuhisa M, Miyazaki M, Kazama H, Takano N, Miyazawa K.
      Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.
    Keywords:  Argonaute 2; Breast cancer; Progesterone receptor; Prognosis; Sequestosome 1; Subtype discordance
    DOI:  https://doi.org/10.1016/j.bbrc.2020.07.058
  29. Life Sci. 2020 Aug 14. pii: S0024-3205(20)30999-1. [Epub ahead of print] 118247
    Wang N, Zhu P, Huang R, Wang C, Sun L, Lan B, He Y, Zhao H, Gao Y.
      PTEN-induced putative kinase 1 (PINK1) performs many important functions in cells and has been highlighted for its role in early-onset Parkinson's disease. In recent years, an increasing number of studies have revealed the involvement of PINK1 in regulation of a variety of cell physiological and pathophysiological processes, of which regulation of mitochondrial function remains the most prominent. As the "energy factory" of cells, mitochondria provide energy support for various cellular activities. Changes in mitochondrial function often have a fundamental and global impact on cellular activities. Moreover, mitochondrial dysfunction has been implicated in many diseases, especially those related to aging. Thus, a comprehensive study of PINK1 will help us better understand the various cell physiological and pathophysiological processes in which PINK1 is involved, including a variety of mitochondria-related diseases such as Parkinson's disease. This article will review the structural characteristics and expression regulation of PINK1, as well as its unique role in mitochondrial quality control (MQC) systems.
    Keywords:  MQC; Mitochondria; Mitochondrial dynamics; Mitochondrial function maintenance; Mitophagy; PINK1
    DOI:  https://doi.org/10.1016/j.lfs.2020.118247
  30. Autophagy. 2020 Aug 19.
    Nath P, Jena KK, Mehto S, Chauhan NR, Sahu R, Dhar K, Srinivas K, Chauhan S, Chauhan S.
      IRGM is a genetic risk factor for several autoimmune diseases. However, the mechanism of IRGM-mediated protection in autoimmunity remains undetermined. The abnormal activation of type I interferon (IFN) response is one of the significant factors in the pathogenesis of several autoimmune diseases. In our recent study, we showed that IRGM is a master suppressor of the interferon response. We found that the depletion of IRGM results in constitutively activated CGAS-STING1, DDX58/RIG-I-MAVS, and TLR3-TICAM1/TRIF signaling pathways resulting in upregulation of almost all IFN-responsive genes. Mechanistically, IRGM utilizes a two-pronged mechanism to suppress the interferon response. First, it mediates SQSTM1/p62-dependent selective macroautophagy/autophagy of nucleic acid sensor proteins, including CGAS, DDX58/RIG-I, and TLR3. Second, it facilitates the removal of defective mitochondria by mitophagy and avoids a buildup of mito-ROS and mito-damage/danger-associated molecular patterns (DAMPs). Thus, IRGM deficiency results in increased nucleic acid sensors and DAMPs engaging a vicious cycle of aberrant activation of IFN response that is known to occur in systemic autoimmune-like conditions.
    Keywords:  Autoimmunity; DAMPs; IRGM; IRGM1; RIG-I-MAVS; autophagy; cGAS-STING; mitophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1810920
  31. Cell Death Discov. 2020 ;6 70
    Huang R, Gao S, Han Y, Ning H, Zhou Y, Guan H, Liu X, Yan S, Zhou PK.
      Authophagy and G2/M arrest are two important mechanistic responses of cells to ionizing radiation (IR), in particular the IR-induced fibrosis. However, what interplayer and how it links the autophagy and the G2/M arrest remains elusive. Here, we demonstrate that the autophagy-related protein BECN1 plays a critical role in ionizing radiation-induced G2/M arrest. The treatment of cells with autophagy inhibitor 3-methyladenine (3-MA) at 0-12 h but not 12 h postirradiation significantly sensitized them to IR, indicating a radio-protective role of autophagy in the early response of cells to radiation. 3-MA and BECN1 disruption inactivated the G2/M checkpoint following IR by abrogating the IR-induced phosphorylation of phosphatase CDC25C and its target CDK1, a key mediator of the G2/M transition in coordination with CCNB1. Irradiation increased the nuclear translocation of BECN1, and this process was inhibited by 3-MA. We confirmed that BECN1 interacts with CDC25C and CHK2, and which is mediated the amino acids 89-155 and 151-224 of BECN1, respectively. Importantly, BECN1 deficiency disrupted the interaction of CHK2 with CDC25C and the dissociation of CDC25C from CDK1 in response to irradiation, resulting in the dephosphorylation of CDK1 and overexpression of CDK1. In summary, IR induces the translocation of BECN1 to the nucleus, where it mediates the interaction between CDC25C and CHK2, resulting in the phosphorylation of CDC25C and its dissociation from CDK1. Consequently, the mitosis-promoting complex CDK1/CCNB1 is inactivated, resulting in the arrest of cells at the G2/M transition. Our findings demonstrated that BECN1 plays a role in promotion of radiation-induced G2/M arrest through regulation of CDK1 activity. Whether such functions of BECN1 in G2/M arrest is dependent or independent on its autophagy-related roles is necessary to further identify.
    Keywords:  Cancer prevention; Macroautophagy
    DOI:  https://doi.org/10.1038/s41420-020-00301-2
  32. Sci Rep. 2020 Aug 20. 10(1): 14033
    Oh SY, Hwang JR, Choi M, Kim YM, Kim JS, Suh YL, Choi SJ, Roh CR.
      Preeclampsia is one of the most serious complications of pregnancy, affecting 5-10% of parturients worldwide. Recent studies have suggested that autophagy is involved in trophoblast invasion and may be associated with defective placentation underlying preeclampsia. We thus aimed to understand the mechanistic link between autophagy and trophoblast invasion. Using the two most commonly used trophoblast cell lines, JEG-3 and HTR-8/SVneo, we inhibited autophagy by ATG5 and beclin-1 shRNA. Conversion of LC3-II was evaluated in ATG5 and beclin-1 knock-down cells in the presence of the lysosomal protease inhibitors E-64d and pepstatin A, to detect the efficiency of autophagy inhibition. Upon autophagy inhibition, we measured cell invasion, activity of NF-κB and related signaling pathways, MMP-2, MMP-9, sFlt-1, and TNF-α levels. Autophagy inhibition increased the invasiveness of these trophoblastic cell lines and increased Akt and NF-κB activity as well as p65 expression. Of note, an NF-κB inhibitor significantly attenuated the trophoblast invasion induced by autophagy inhibition. Autophagy inhibition was also associated with increased MMP-2 and MMP-9 levels and decreased the production of sFlt-1 and TNF-α. Collectively, our results indicate that autophagy regulates trophoblast invasiveness in which the NF-κB pathway and MMP-2, MMP-9, sFlt-1 and TNF-α levels are affected.
    DOI:  https://doi.org/10.1038/s41598-020-70959-2
  33. J Cell Sci. 2020 Aug 14. pii: jcs.245555. [Epub ahead of print]
    Uemura S, Mochizuki T, Amemiya K, Kurosaka G, Yazawa M, Nakamoto K, Ishikawa Y, Izawa S, Abe F.
      Mechanical stresses including high hydrostatic pressure elicit diverse physiological effects on organisms. Gtr1/Gtr2 and Ego1/Ego3, central regulators of the TOR complex 1 (TORC1) nutrient signaling pathway, are required for the growth of Saccharomyces cerevisiae cells under high pressure. Here, we showed that a pressure of 25 MPa stimulates TORC1 to promote phosphorylation of Sch9, which depends on the EGO complex (EGOC) and Pib2. Incubation of cells at this pressure aberrantly increased the glutamine and alanine levels in the ego1Δ, gtr1Δ, tor1Δ, and pib2Δ mutants, whereas the polysome profiles were unaffected. Moreover, we found that glutamine levels were reduced by combined deletions of EGO1, GTR1, TOR1, and PIB2 with GLN3 These results suggested that high pressure leads to the intracellular accumulation of amino acids. Subsequently, Pib2 loaded with glutamine stimulates the EGOC-TORC1 complex to inactivate Gln3, downregulating glutamine synthesis. Our findings illustrated the regulatory circuit that maintained the intracellular amino acid homeostasis and suggested the critical roles the EGOC-TORC1 and Pib2-TORC1 complexes played in the growth of yeast under high hydrostatic pressure.
    Keywords:  EGO complex; Glutamine; Gtr1/Gtr2; High hydrostatic pressure; Pib2; Polysome profile; TORC1
    DOI:  https://doi.org/10.1242/jcs.245555