bims-auttor 2019-08-25 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2019–08–25
ten papers selected by
Viktor Korolchuk, Newcastle University

Autophagic Degradation of the Circadian Clock Regulator Promotes Ferroptosis.
Iron overload inhibits late stage autophagic flux leading to insulin resistance.
Retromer and TBC1D5 maintain late endosomal RAB7 domains to enable amino acid-induced mTORC1 signaling.
Neuroprotective effects of memantine via enhancement of autophagy.
SQSTM1/p62 and PPARGC1A/PGC-1alpha at the Interface of Autophagy and Vascular Senescence.
USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy.
The Autophagy-Cilia Axis: An Intricate Relationship.
Suppression of p16 Induces mTORC1-Mediated Nucleotide Metabolic Reprogramming.
Polyubiquitination of p62/SQSTM1 is a prerequisite for Fas/CD95 aggregation to promote caspase-dependent apoptosis in cadmium-exposed mouse monocyte RAW264.7 cells.
The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage.

Autophagy. 2019 Aug 23.

Autophagic Degradation of the Circadian Clock Regulator Promotes Ferroptosis.

Jiao Liu, Minghua Yang, Rui Kang, Daniel J Klionsky, Daolin Tang.

  Macroautophagy (hereafter referred to as autophagy) involves a lysosomal degradation pathway and plays a context-dependent role in promoting either cell survival or cell death during stress; excessive or impaired autophagy is implicated in various types of cell death. In particular, lipid peroxidation-associated ferroptosis has recently been recognized as a type of autophagy-dependent cell death, but the mechanisms involved remain largely obscure. Our recent findings demonstrate that clockophagy, namely the selective autophagic degradation of the circadian clock regulator ARNTL/BMAL1, promotes ferroptotic cancer cell death in vitro and in vivo. Mechanically, the cargo receptor SQSTM1/p62 is responsible for the autophagic degradation of ARNTL in response to type 2 ferroptosis inducers (e.g., RSL3 and FIN56), but not type 1 ferroptosis inducers (e.g., erastin, sulfasalazine, and sorafenib). Consequently, clockophagy-mediated ARNTL degradation promotes lipid peroxidation and subsequent ferroptosis through blocking HIF1A-dependent fatty acid uptake and lipid storage. These findings highlight a novel type of selective autophagy in regulated cell death.

Keywords:  autophagy; cargo receptor; cell death; circadian rhythm; fatty acid uptake; ferroptosis; hypoxia; lipid droplets; lipid peroxidation; lipid storage

DOI:  https://doi.org/10.1080/15548627.2019.1659623
EMBO Rep. 2019 Aug 23. e47911

Iron overload inhibits late stage autophagic flux leading to insulin resistance.

James Won Suk Jahng, Reham Musaibeh Alsaadi, Rengasamy Palanivel, Erfei Song, Victoria Emily Barbosa Hipolito, Hye Kyoung Sung, Roberto Jorge Botelho, Ryan Charles Russell, Gary Sweeney.

  Iron overload, a common clinical occurrence, is implicated in the metabolic syndrome although the contributing pathophysiological mechanisms are not fully defined. We show that prolonged iron overload results in an autophagy defect associated with accumulation of dysfunctional autolysosomes and loss of free lysosomes in skeletal muscle. These autophagy defects contribute to impaired insulin-stimulated glucose uptake and insulin signaling. Mechanistically, we show that iron overload leads to a decrease in Akt-mediated repression of tuberous sclerosis complex (TSC2) and Rheb-mediated mTORC1 activation on autolysosomes, thereby inhibiting autophagic-lysosome regeneration. Constitutive activation of mTORC1 or iron withdrawal replenishes lysosomal pools via increased mTORC1-UVRAG signaling, which restores insulin sensitivity. Induction of iron overload via intravenous iron-dextran delivery in mice also results in insulin resistance accompanied by abnormal autophagosome accumulation, lysosomal loss, and decreased mTORC1-UVRAG signaling in muscle. Collectively, our results show that chronic iron overload leads to a profound autophagy defect through mTORC1-UVRAG inhibition and provides new mechanistic insight into metabolic syndrome-associated insulin resistance.

Keywords:   ALR ; autophagy; insulin resistance; iron overload; mTORC1

DOI:  https://doi.org/10.15252/embr.201947911
J Cell Biol. 2019 Aug 20. pii: jcb.201812110. [Epub ahead of print]

Retromer and TBC1D5 maintain late endosomal RAB7 domains to enable amino acid-induced mTORC1 signaling.

Arunas Kvainickas, Heike Nägele, Wenjing Qi, Ladislav Dokládal, Ana Jimenez-Orgaz, Luca Stehl, Dipak Gangurde, Qian Zhao, Zehan Hu, Jörn Dengjel, Claudio De Virgilio, Ralf Baumeister, Florian Steinberg.

Retromer is an evolutionarily conserved multiprotein complex that orchestrates the endocytic recycling of integral membrane proteins. Here, we demonstrate that retromer is also required to maintain lysosomal amino acid signaling through mTORC1 across species. Without retromer, amino acids no longer stimulate mTORC1 translocation to the lysosomal membrane, which leads to a loss of mTORC1 activity and increased induction of autophagy. Mechanistically, we show that its effect on mTORC1 activity is not linked to retromer's role in the recycling of transmembrane proteins. Instead, retromer cooperates with the RAB7-GAP TBC1D5 to restrict late endosomal RAB7 into microdomains that are spatially separated from the amino acid-sensing domains. Upon loss of retromer, RAB7 expands into the ragulator-decorated amino acid-sensing domains and interferes with RAG-GTPase and mTORC1 recruitment. Depletion of retromer in Caenorhabditis elegans reduces mTORC1 signaling and extends the lifespan of the worms, confirming an evolutionarily conserved and unexpected role for retromer in the regulation of mTORC1 activity and longevity.

DOI: https://doi.org/10.1083/jcb.201812110
Biochem Biophys Res Commun. 2019 Aug 17. pii: S0006-291X(19)31536-0. [Epub ahead of print]

Neuroprotective effects of memantine via enhancement of autophagy.

Kazuoki Hirano, Motoki Fujimaki, Yukiko Sasazawa, Akihiro Yamaguchi, Kei-Ichi Ishikawa, Kengo Miyamoto, Sanae Souma, Norihiko Furuya, Yoko Imamichi, Daisuke Yamada, Hideyuki Saya, Wado Akamatsu, Shinji Saiki, Nobutaka Hattori.

   INTRODUCTION: Chemical intervention of autophagy has been investigated in clinical trials for various age-related conditions such as sarcopenia and neurodegeneration. However, at present, no autophagy inducer has been established as a disease-modifying agent against neurodegenerative diseases.
METHODS: We screened a library consisting of 796 medicines clinically approved (in Japan) for autophagy enhancers as potential neurodegeneration therapeutics using HeLa cells stably expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) followed by an analysis of the molecular mechanisms using various neuronal models.
RESULTS: The primary screening identified 152 hits in a static cellular state. A widely available Alzheimer's disease drug, memantine, which antagonizes N-Methyl-d-aspartate receptor (NMDAR), was one of the hits. Memantine increased the levels of LC3-II in a dose-dependent and time-dependent manner, and upregulated autophagic flux. In addition, the pharmacological effects of memantine on autophagy were independent of mTORC1 activity and NMDAR activation. Furthermore, a VPS34 inhibitor suppressed the memantine-induced LC3-II upregulation, suggesting that memantine may affect VPS34 complex activity. Notably, intracellular Huntington's disease-specific aggregates of elongated huntingtin, a well-established autophagy substrate, were significantly decreased by memantine. In addition, memantine enhanced elimination of degraded mitochondrial in neurons derived from induced pluripotent stem cells of PARK2 or PARK6 patients, who exhibited defective PINK1/parkin-mediated mitophagy, suggests that memantine accelerated the clearance of damaged mitochondria.
CONCLUSION: These findings indicate that memantine may be beneficial for the treatment of neurodegeneration characterized by the abnormal accumulation of autophagy or mitophagy substrates.

Keywords:  Autophagy; Chemical screening; Memantine; Mitophagy; Neurodegenerative diseases

DOI:  https://doi.org/10.1016/j.bbrc.2019.08.025
Autophagy. 2019 Aug 23.

SQSTM1/p62 and PPARGC1A/PGC-1alpha at the Interface of Autophagy and Vascular Senescence.

Gloria Salazar, Abigail Cullen, Jingwen Huang, Yitong Zhao, Alexa Serino, Lula Hilenski, Nikolay Patrushev, Farshad Forouzandeh, Hyun Seok Hwang.

  Defective macroautophagy/autophagy and mitochondrial dysfunction are known to stimulate senescence. The mitochondrial regulator PPARGC1A (peroxisome proliferator activated receptor gamma, coactivator 1 alpha) regulates mitochondrial biogenesis, reducing senescence of vascular smooth muscle cells (VSMCs); however, it is unknown whether autophagy mediates PPARGC1A-protective effects on senescence. Using ppargc1a-/- VSMCs, we identified the autophagy receptor SQSTM1/p62 (sequestosome 1) as a major regulator of autophagy and senescence of VSMCs. Abnormal autophagosomes were observed in VSMCs in aortas of ppargc1a-/- mice. ppargc1a-/- VSMCs in culture presented reductions in LC3-II levels; in autophagosome number; and in the expression of SQSTM1 (protein and mRNA), LAMP2 (lysosomal-associated membrane protein 2), CTSD (cathepsin D), and TFRC (transferrin receptor). Reduced SQSTM1 protein expression was also observed in aortas of ppargc1a-/- mice and was upregulated by PPARGC1A overexpression, suggesting that SQSTM1 is a direct target of PPARGC1A. Inhibition of autophagy by 3-MA (3 methyladenine), spautin-1 or Atg5 (autophagy related 5) siRNA stimulated senescence. Rapamycin rescued the effect of Atg5 siRNA in Ppargc1a+/+, but not in ppargc1a-/- VSMCs, suggesting that other targets of MTOR (mechanistic target of rapamycin kinase), in addition to autophagy, also contribute to senescence. Sqstm1 siRNA increased senescence basally and in response to AGT II (angiotensin II) and zinc overload, two known inducers of senescence. Furthermore, Sqstm1 gene deficiency mimicked the phenotype of Ppargc1a depletion by presenting reduced autophagy and increased senescence in vitro and in vivo. Thus, PPARGC1A upregulates autophagy reducing senescence by a SQSTM1-dependent mechanism. We propose SQSTM1 as a novel target in therapeutic interventions reducing senescence.

Keywords:  SQSTM1; aging; autophagy; oxidative stress; senescence; vascular biology

DOI:  https://doi.org/10.1080/15548627.2019.1659612
Autophagy. 2019 Aug 21.

USP33 deubiquitinates PRKN/parkin and antagonizes its role in mitophagy.

Kaifeng Niu, Hongbo Fang, Zixiang Chen, Yuqi Zhu, Qunsong Tan, Di Wei, Yueyang Li, Adayabalam S Balajee, Yongliang Zhao.

  PRKN/parkin activation through phosphorylation of its ubiquitin and ubiquitin-like domain by PINK1 is critical in mitophagy induction for eliminating the damaged mitochondria. Deubiquitinating enzymes (DUBs) functionally reversing PRKN ubiquitination are critical in controlling the magnitude of PRKN-mediated mitophagy process. However, potential DUBs that directly target PRKN and antagonize its pro-mitophagy effect remains to be identified and characterized. Here, we demonstrated that USP33/VDU1 is localized at the outer membrane of mitochondria and serves as a PRKN DUB through their interaction. Cellular and in vitro assays illustrated that USP33 deubiquitinates PRKN in a DUB activity-dependent manner. USP33 prefers to remove K6, K11, K48 and K63-linked ubiquitin conjugates from PRKN, and deubiquitinates PRKN mainly at Lys435. Mutation of this site leads to a significantly decreased level of K63-, but not K48-linked PRKN ubiquitination. USP33 deficiency enhanced both K48- and K63-linked PRKN ubiquitination, but only K63-linked PRKN ubiquitination was significantly increased under mitochondrial depolarization. Further, USP33 knockdown increased both PRKN protein stabilization and its translocation to depolarized mitochondria leading to the enhancement of mitophagy. Moreover, USP33 silencing protects SH-SY5Y human neuroblastoma cells from the neurotoxin MPTP-induced apoptotic cell death. Our findings convincingly demonstrate that USP33 is a novel PRKN deubiquitinase antagonizing its regulatory roles in mitophagy and SH-SY5Y neuron-like cell survival. Thus, USP33 inhibition may represents an attractive new therapeutic strategy for PD patients.

Keywords:  PRKN/parkin; USP33 deubiquitinase; apoptosis; mitophagy; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2019.1656957
Cells. 2019 Aug 15. pii: E905. [Epub ahead of print]8(8):

The Autophagy-Cilia Axis: An Intricate Relationship.

Manuela Morleo, Brunella Franco.

  Primary cilia are microtubule-based organelles protruding from the surface of almost all vertebrate cells. This organelle represents the cell's antenna which acts as a communication hub to transfer extracellular signals into intracellular responses during development and in tissue homeostasis. Recently, it has been shown that loss of cilia negatively regulates autophagy, the main catabolic route of the cell, probably utilizing the autophagic machinery localized at the peri-ciliary compartment. On the other side, autophagy influences ciliogenesis in a context-dependent manner, possibly to ensure that the sensing organelle is properly formed in a feedback loop model. In this review we discuss the recent literature and propose that the autophagic machinery and the ciliary proteins are functionally strictly related to control both autophagy and ciliogenesis. Moreover, we report examples of diseases associated with autophagic defects which cause cilia abnormalities, and propose and discuss the hypothesis that, at least some of the clinical manifestations observed in human diseases associated to ciliary disfunction may be the result of a perturbed autophagy.

Keywords:  Hedgehog signaling; autophagy; ciliopathy; primary cilium

DOI:  https://doi.org/10.3390/cells8080905
Cell Rep. 2019 Aug 20. pii: S2211-1247(19)31000-9. [Epub ahead of print]28(8): 1971-1980.e8

Suppression of p16 Induces mTORC1-Mediated Nucleotide Metabolic Reprogramming.

Raquel Buj, Chi-Wei Chen, Erika S Dahl, Kelly E Leon, Rostislav Kuskovsky, Natella Maglakelidze, Maithili Navaratnarajah, Gao Zhang, Mary T Doan, Helen Jiang, Michael Zaleski, Lydia Kutzler, Holly Lacko, Yiling Lu, Gordon B Mills, Raghavendra Gowda, Gavin P Robertson, Joshua I Warrick, Meenhard Herlyn, Yuka Imamura, Scot R Kimball, David J DeGraff, Nathaniel W Snyder, Katherine M Aird.

  Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurs by activation of mTORC1 signaling, which directly mediates increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlates with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibits proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.

Keywords:  BRAF; cancer metabolism; cell cycle; melanoma; nevi; pancreatic cancer; pentose phosphate pathway; ribonucleotide reductase M2; ribose-5-phosphate isomerase A; senescence

DOI:  https://doi.org/10.1016/j.celrep.2019.07.084
Sci Rep. 2019 Aug 22. 9(1): 12240

Polyubiquitination of p62/SQSTM1 is a prerequisite for Fas/CD95 aggregation to promote caspase-dependent apoptosis in cadmium-exposed mouse monocyte RAW264.7 cells.

Ki-Tae Jung, Seon-Hee Oh.

Cadmium(Cd) induces cytotoxicity via autophagy-induced apoptosis in non-activated mouse monocytes; however, the molecular mechanism remains unclear. Here, we show that autophagy induces Fas (CD95/APO-1)-mediated apoptosis by promoting accumulation of p62/SQSTM1 in response to Cd. Cd produced tumor necrosis factor (TNF)-α, peaking at 6 h, and exhibiting a concentration-dependent increase. Immunoblot analysis revealed polyubiquitinated (polyUb) full-length Fas (antibody clone G-9) and reduced cytosolic Fas (antibody clone M-20) in Cd-exposed RAW264.7 cells. The accumulation of polyUb-Fas was transient and positively correlated with polyUb-p62 and polyUb-proteins. Autophagy inhibition via chemical and genetic modulation suppressed Cd-induced polyUb-p62, polyUb-Fas, and polyUb-protein levels, whereas the level of cytosolic Fas recovered to that of the control. Immunofluorescence (IF) staining for full-length Fas, p62, and ubiquitin revealed an aggregated pattern in Cd-induced apoptotic cells, which was inhibited by blocking autophagy. Fas colocalized with microtubule-associated protein 1 light chain (LC)-3B. IF staining and immunoprecipitation assays revealed colocalization and interaction among p62, Ub, and Fas. Knockdown of p62 reduced the binding of Ub and Fas. Together, these data suggest that polyUb-p62 targets Fas and recruits it to autophagosomes, where Fas transiently aggregates to promote apoptosis and is degraded with polyUb-p62. In conclusion, autophagy regulates C-terminal cytosolic Fas aggregation via p62 polyubiquitination, which is required for apoptosis and may play a critical role in the production of select cytokines.

DOI: https://doi.org/10.1038/s41598-019-48684-2
EMBO Rep. 2019 Aug 21. e48014

The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in response to endolysosomal damage.

Lisa Koerver, Chrisovalantis Papadopoulos, Bin Liu, Bojana Kravic, Giulia Rota, Lukas Brecht, Tineke Veenendaal, Mira Polajnar, Anika Bluemke, Michael Ehrmann, Judith Klumperman, Marja Jäättelä, Christian Behrends, Hemmo Meyer.

  The autophagic clearance of damaged lysosomes by lysophagy involves extensive modification of the organelle with ubiquitin, but the underlying ubiquitination machinery is still poorly characterized. Here, we use an siRNA screening approach and identify human UBE2QL1 as a major regulator of lysosomal ubiquitination, lysophagy, and cell survival after lysosomal damage. UBE2QL1 translocates to permeabilized lysosomes where it associates with damage sensors, ubiquitination targets, and lysophagy effectors. UBE2QL1 knockdown reduces ubiquitination and accumulation of the critical autophagy receptor p62 and abrogates recruitment of the AAA-ATPase VCP/p97, which is essential for efficient lysophagy. Crucially, it affects association of LC3B with damaged lysosomes indicating that autophagosome formation was impaired. Already in unchallenged cells, depletion of UBE2QL1 leads to increased lysosomal damage, mTOR dissociation from lysosomes, and TFEB activation pointing to a role in lysosomal homeostasis. In line with this, mutation of the homologue ubc-25 in Caenorhabditis elegans exacerbates lysosome permeability in worms lacking the lysosome stabilizing protein SCAV-3/LIMP2. Thus, UBE2QL1 coordinates critical steps in the acute endolysosomal damage response and is essential for maintenance of lysosomal integrity.

Keywords:   mTOR ; TAX1BP1; autophagy; p97; ubiquitin-conjugating enzyme

DOI:  https://doi.org/10.15252/embr.201948014