bims-auttor 2020-09-27 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2020–09–27
34 papers selected by
Viktor Korolchuk, Newcastle University

Research progress of mTOR inhibitors.
Retromer regulates the lysosomal clearance of MAPT/tau.
The anti-tumor agent, Dp44mT, promotes nuclear translocation of TFEB via inhibition of the AMPK-MTORC1 axis.
Cytoplasmic cargo receptor p62 inhibits avibirnavirus replication by mediating autophagic degradation of viral protein VP2.
SUMO promotes longevity and maintains mitochondrial homeostasis during ageing in Caenorhabditis elegans.
Coronavirus interactions with the cellular autophagy machinery.
GADD34 is a modulator of autophagy during starvation.
Proteasome malfunction activates the PPP3/calcineurin-TFEB-SQSTM1/p62 pathway to induce macroautophagy in the heart.
Cell adhesion molecule IGPR-1 activates AMPK connecting cell adhesion to autophagy.
Fluid flow-induced shear stress controls the metabolism of proximal tubule kidney epithelial cells through primary cilium-dependent lipophagy and mitochondrial biogenesis.
Cessation of electrically-induced muscle contraction activates autophagy in cultured myotubes.
Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon honokiol treatment.
Protein-carbohydrate ingestion alters Vps34 cellular localization independent of changes in kinase activity in human skeletal muscle.
Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation.
Structural Variants of poly(alkylcyanoacrylate) Nanoparticles Differentially Affect LC3 and Autophagic Cargo Degradation.
Mitochondrial Surveillance by Cdc48/p97: MAD vs. Membrane Fusion.
Lysosomal dysfunction and autophagy blockade contribute to autophagy-related cancer suppressing peptide-induced cytotoxic death of cervical cancer cells through the AMPK/mTOR pathway.
The conversion of formate into purines stimulates mTORC1 leading to CAD-dependent activation of pyrimidine synthesis.
RAB33B recruits the ATG16L1 complex to the phagophore via a noncanonical RAB binding protein.
Characterization of the complex of the lysosomal membrane transporter MFSD1 and its accessory subunit GLMP.
Autophagy in fate determination of mesenchymal stem cells and bone remodeling.
Autophagy regulates levels of tumor suppressor enzyme protein phosphatase 6.
Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90 and ROF1.
Interplay Between Autophagy and Zinc.
IKKβ Activation Promotes Amphisome Formation and Extracellular Vesicle Secretion in Tumor Cells.
Dysregulated Autophagy Mediates Sarcopenic Obesity and Its Complications via AMPK and PGC1α Signaling Pathways: Potential Involvement of Gut Dysbiosis as a Pathological Link.
Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.
TSPAN1 promotes autophagy flux and mediates cooperation between WNT-CTNNB1 signaling and autophagy via the MIR454-FAM83A-TSPAN1 axis in pancreatic cancer.
Modulating TRADD to restore cellular homeostasis and inhibit apoptosis.
Autophagy and cardiac diseases: Therapeutic potential of natural products.
The Role of Chaperone-Mediated Autophagy in Cell Cycle Control and Its Implications in Cancer.
Iron loss triggers mitophagy through induction of mitochondrial ferritin.
Translational control in aging and neurodegeneration.
Electroacupuncture Pretreatment against Cerebral Ischemia/Reperfusion Injury through Mitophagy.

Eur J Med Chem. 2020 Sep 13. pii: S0223-5234(20)30792-3. [Epub ahead of print]208 112820

Research progress of mTOR inhibitors.

Yifan Chen, Xiaoping Zhou.

  Mammalian target of rapamycin (mTOR) is a highly conserved Serine/Threonine (Ser/Thr) protein kinase, which belongs to phosphatidylinositol-3-kinase-related kinase (PIKK) protein family. mTOR exists as two types of protein complex: mTORC1 and mTORC2, which act as central controller regulating processes of cell metabolism, growth, proliferation, survival and autophagy. The mTOR inhibitors block mTOR signaling pathway, producing anti-inflammatory, anti-proliferative, autophagy and apoptosis induction effects, thus mTOR inhibitors are mainly used in cancer therapy. At present, mTOR inhibitors are divided into four categories: Antibiotic allosteric mTOR inhibitors (first generation), ATP-competitive mTOR inhibitors (second generation), mTOR/PI3K dual inhibitors (second generation) and other new mTOR inhibitors (third generation). In this article, these four categories of mTOR inhibitors and their structures, properties and some clinical researches will be introduced. Among them, we focus on the structure of mTOR inhibitors and try to analyze the structure-activity relationship. mTOR inhibitors are classified according to their chemical structure and their contents are introduced systematically. Moreover, some natural products that have direct or indirect mTOR inhibitory activities are introduced together. In this article, we analyzed the target, binding mode and structure-activity relationship of each generation of mTOR inhibitors and proposed two hypothetic scaffolds (the inverted-Y-shape scaffold and the C-shape scaffold) for the second generation of mTOR inhibitors. These findings may provide some help or reference for drug designing, drug modification or the future development of mTOR inhibitor.

Keywords:  Anticancer; Drug design; mTOR; mTOR inhibitor

DOI:  https://doi.org/10.1016/j.ejmech.2020.112820
Autophagy. 2020 Sep 22. 1-21

Retromer regulates the lysosomal clearance of MAPT/tau.

Julian M Carosi, Leanne K Hein, Mark van den Hurk, Robert Adams, Bridget Milky, Sanjna Singh, Cedric Bardy, Donna Denton, Sharad Kumar, Timothy J Sargeant.

  The macroautophagy/autophagy-lysosome axis enables the clearance and degradation of cytoplasmic components including protein aggregates, damaged organelles and invading pathogens. Protein aggregation and lysosomal system dysfunction in the brain are common features of several late-onset neurological disorders including Alzheimer disease. Spatial overlap between depletion of the endosomal-sorting complex retromer and MAPT/tau aggregation in the brain have been previously reported. However, whether retromer dysfunction plays a direct role in mediating MAPT aggregation remains unclear. Here, we demonstrate that the autophagy-lysosome axis is the primary mode for the clearance of aggregated species of MAPT using both chemical and genetic approaches in cell models of amyloid MAPT aggregation. We show that depletion of the central retromer component VPS35 causes a block in the resolution of autophagy. We establish that this defect underlies marked accumulation of cytoplasmic MAPT aggregates upon VPS35 depletion, and that VPS35 overexpression has the opposite effect. This work illustrates how retromer complex integrity regulates the autophagy-lysosome axis to suppress MAPT aggregation and spread.

Keywords:  Amyloid; VPS35; autophagy; lysosome; protein aggregation; tauopathy

DOI:  https://doi.org/10.1080/15548627.2020.1821545
Biochim Biophys Acta Mol Basis Dis. 2020 Sep 17. pii: S0925-4439(20)30318-5. [Epub ahead of print] 165970

The anti-tumor agent, Dp44mT, promotes nuclear translocation of TFEB via inhibition of the AMPK-MTORC1 axis.

S Krishan, S Sahni, D R Richardson.

  Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and its analogues are potent anti-cancer agents through their ability to target lysosomes. Considering this, it was important to understand the mechanisms involved in the Dp44mT-mediated induction of autophagy and the role of 5'-adenosine monophosphate-activated protein kinase (AMPK) as a critical autophagic regulator. As such, this investigation examined AMPK's role in the regulation of the transcription factor EB (TFEB), which transcribes genes involved in autophagy and lysosome biosynthesis. For the first time, this study demonstrated that Dp44mT induces translocation of TFEB to the nucleus. Furthermore, Dp44mT-mediated nuclear translocation of TFEB was AMPK-dependent. Considering that: (1) the mammalian target of rapamycin complex 1 (mTORC1) plays an important role in the regulation of TFEB; and (2) that AMPK is a known regulator of mTORC1, this study also elucidated the mechanisms through which Dp44mT regulates nuclear translocation of TFEB via AMPK. Silencing AMPK led to increased mTOR phosphorylation, that activates mTORC1. Since Dp44mT inhibits mTORC1 in an AMPK-dependent manner through raptor phosphorylation, Dp44mT is demonstrated to regulate TFEB translocation through dual mechanisms: AMPK activation, which inhibits mTOR, and inhibition of mTORC1 via phosphorylation of raptor. Collectively, Dp44mT-mediated activation of AMPK plays a crucial role in lysosomal biogenesis and TFEB function. As Dp44mT potently chelates copper and iron that are crucial for tumor growth, these studies provide insight into the regulatory mechanisms involved in intracellular clearance and energy metabolism that occur upon alterations in metal ion homeostasis.

Keywords:  AMPK; Anti-cancer agent; Dp44mT; Molecular pharmacology; Molecular target; pAMPK

DOI:  https://doi.org/10.1016/j.bbadis.2020.165970
J Virol. 2020 Sep 23. pii: JVI.01255-20. [Epub ahead of print]

Cytoplasmic cargo receptor p62 inhibits avibirnavirus replication by mediating autophagic degradation of viral protein VP2.

Yahui Li, Boli Hu, Gang Ji, Yina Zhang, Chenyang Xu, Jing Lei, Chan Ding, Jiyong Zhou.

Selective autophagy regulates the degradation of cytoplasmic cargos, such as damaged organelles, invading pathogens, and aggregated proteins. Furthermore, autophagy is capable of degrading avibirnavirus, but the mechanism responsible for this process is unclear. Here, we show that autophagy cargo receptor p62 regulates the degradation of the avibirnavirus capsid protein VP2. Binding of p62 to VP2 enhance autophagic induction and promotes autophagic degradation of viral protein VP2. Further study showed that the interaction of p62 with viral protein VP2 is dependent on ubiquitination at the K411 site of VP2 and the ubiquitin-associated domain of p62. Mutation analysis showed that the K411R mutation of viral protein VP2 prohibits its p62-mediated degradation. Consistently, p62 lacking the ubiquitin-associated domain or LC3-interacting region no longer promoted degradation of VP2. Virus production revealed that knockout of p62 but not overexpression of p62 promotes the replication of avibirnavirus. Collectively, our findings suggest that p62 mediates selective autophagic degradation of avibirnavirus protein VP2 in an ubiquitin-dependent manner and is an inhibitor of avibirnavirus replication.ImportanceAvibirnavirus causes severe immunosuppression and mortality in young chickens. VP2, capsid protein of avibirnavirus, is responsible for virus assembly, maturation and replication. Previous study showed that avibirnavirus particles could be engulfed into autophaogosome and degradation took apart. Selective autophagy is a highly specific and regulated degradation pathway for the clearance of damaged or unwanted cytosolic components and superfluous organelles as well as invading microbes. However, whether and how selective autophagy removes avibirnavirus capsids is largely unknown. Here, we have shown that selective autophagy specifically clears ubiquitinated avibirnavirus protein VP2 by p62 recognition and that p62 is an inhibitor of avibirnavirus replication, highlighting the role of p62 as a potential drug target for mediating the removal of ubiquitinated virus components from cells.

DOI: https://doi.org/10.1128/JVI.01255-20
Sci Rep. 2020 Sep 23. 10(1): 15513

SUMO promotes longevity and maintains mitochondrial homeostasis during ageing in Caenorhabditis elegans.

Andrea Princz, Federico Pelisch, Nektarios Tavernarakis.

The insulin/IGF signalling pathway impacts lifespan across distant taxa, by controlling the activity of nodal transcription factors. In the nematode Caenorhabditis elegans, the transcription regulators DAF-16/FOXO and SKN-1/Nrf function to promote longevity under conditions of low insulin/IGF signalling and stress. The activity and subcellular localization of both DAF-16 and SKN-1 is further modulated by specific posttranslational modifications, such as phosphorylation and ubiquitination. Here, we show that ageing elicits a marked increase of SUMO levels in C. elegans. In turn, SUMO fine-tunes DAF-16 and SKN-1 activity in specific C. elegans somatic tissues, to enhance stress resistance. SUMOylation of DAF-16 modulates mitochondrial homeostasis by interfering with mitochondrial dynamics and mitophagy. Our findings reveal that SUMO is an important determinant of lifespan, and provide novel insight, relevant to the complexity of the signalling mechanisms that influence gene expression to govern organismal survival in metazoans.

DOI: https://doi.org/10.1038/s41598-020-72637-9
Autophagy. 2020 Sep 23. 1-9

Coronavirus interactions with the cellular autophagy machinery.

Katelyn Miller, Marisa E McGrath, Zhiqiang Hu, Sohha Ariannejad, Stuart Weston, Matthew Frieman, William T Jackson.

  The COVID-19 pandemic, caused by the SARS-CoV-2 virus, is the most recent example of an emergent coronavirus that poses a significant threat to human health. Virus-host interactions play a major role in the viral life cycle and disease pathogenesis, and cellular pathways such as macroautophagy/autophagy prove to be either detrimental or beneficial to viral replication and maturation. Here, we describe the literature over the past twenty years describing autophagy-coronavirus interactions. There is evidence that many coronaviruses induce autophagy, although some of these viruses halt the progression of the pathway prior to autophagic degradation. In contrast, other coronaviruses usurp components of the autophagy pathway in a non-canonical fashion. Cataloging these virus-host interactions is crucial for understanding disease pathogenesis, especially with the global challenge of SARS-CoV-2 and COVID-19. With the recognition of autophagy inhibitors, including the controversial drug chloroquine, as possible treatments for COVID-19, understanding how autophagy affects the virus will be critical going forward. Abbreviations: 3-MA: 3-methyladenine (autophagy inhibitor); AKT/protein kinase B: AKT serine/threonine kinase; ATG: autophagy related; ATPase: adenosine triphosphatase; BMM: bone marrow macrophage; CGAS: cyclic GMP-AMP synthase; CHO: Chinese hamster ovary/cell line; CoV: coronaviruses; COVID-19: Coronavirus disease 2019; DMV: double-membrane vesicle; EAV: equine arteritis virus; EDEM1: ER degradation enhancing alpha-mannosidase like protein 1; ER: endoplasmic reticulum; ERAD: ER-associated degradation; GFP: green fluorescent protein; HCoV: human coronavirus; HIV: human immunodeficiency virus; HSV: herpes simplex virus; IBV: infectious bronchitis virus; IFN: interferon; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCoV: mouse coronavirus; MERS-CoV: Middle East respiratory syndrome coronavirus; MHV: mouse hepatitis virus; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2 (autophagy receptor that directs cargo to phagophores); nsp: non-structural protein; OS9: OS9 endoplasmic reticulum lectin; PEDV: porcine epidemic diarrhea virus; PtdIns3K: class III phosphatidylinositol 3-kinase; PLP: papain-like protease; pMEF: primary mouse embryonic fibroblasts; SARS-CoV: severe acute respiratory syndrome coronavirus; SKP2: S-phase kinase associated protein 2; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; ULK1: unc-51 like autophagy activating kinase 1; Vps: vacuolar protein sorting.

Keywords:  Autophagy; COVID-19; ERAD; MERS; SARS-CoV-2; coronavirus

DOI:  https://doi.org/10.1080/15548627.2020.1817280
Sci Adv. 2020 Sep;pii: eabb0205. [Epub ahead of print]6(39):

GADD34 is a modulator of autophagy during starvation.

Gennaro Gambardella, Leopoldo Staiano, Maria Nicoletta Moretti, Rossella De Cegli, Luca Fagnocchi, Giuseppe Di Tullio, Sara Polletti, Clarissa Braccia, Andrea Armirotti, Alessio Zippo, Andrea Ballabio, Maria Antonietta De Matteis, Diego di Bernardo.

Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.

DOI: https://doi.org/10.1126/sciadv.abb0205
Autophagy. 2020 Sep 22. 1-3

Proteasome malfunction activates the PPP3/calcineurin-TFEB-SQSTM1/p62 pathway to induce macroautophagy in the heart.

Huabo Su, Xuejun Wang.

  Proteasome inhibition (PSMI) is known to activate macroautophagy (autophagy hereafter), but the underlying mechanisms remain to be fully delineated. Here we discuss our recent work identifying an important PPP3/calcineurin-TFEB-SQSTM1/p62 pathway in mediating activation of autophagy by PSMI, a compensatory process for the heart with proteasome malfunction. Through increasing PPP3/calcineurin activity and inhibiting MTOR signaling, PSMI promotes the dephosphorylation and thereby nuclear translocation of TFEB, resulting in transactivation of genes in the autophagic-lysosomal pathway (ALP) such as Mcoln1 and Sqstm1. We have discovered that SQSTM1 is required for not only induction of autophagy but also cardiac activation of TFEB by PSMI, unveiling a novel feedforward role for SQSTM1 in TFEB activation.

Keywords:  PPP3/calcineurin; PSMC2; SQSTM1/p62; TFEB; macroautophagy

DOI:  https://doi.org/10.1080/15548627.2020.1816666
J Biol Chem. 2020 Sep 25. pii: jbc.RA120.014790. [Epub ahead of print]

Cell adhesion molecule IGPR-1 activates AMPK connecting cell adhesion to autophagy.

Razie Amraei, Tooba Alwani, Rachel Xi-Yeen Ho, Zahra Aryan, Shawn Wang, Nader Rahimi.

  Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin and lipopolysaccharide (LPS). Manipulating the IκB kinaseβ (IKKβ) activity coupled with in vivo and in vitro kinase assays demonstrated that IKKb is a key serine/threonine kinase activated by autophagy stimuli and catalyzes phosphorylation of IGPR-1 at Ser220. The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase (AMPK), which leads to phosphorylation of major pro-autophagy proteins, ULK1 and Beclin-1 (BECN1), increased LC3-II levels and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer, and suggest that IGPR-1 may serve as a promising therapeutic target.

Keywords:  AMP-activated kinase (AMPK); IGPR-1; Serine phosphorylation of IGPR-1; autophagy; cell adhesion molecule; cell surface receptor; cell-cell interaction; immunoglobulin-like domain; nutrient deprivation; post-translational modification (PTM); serine/threonine protein kinase

DOI:  https://doi.org/10.1074/jbc.RA120.014790
Autophagy. 2020 Sep 20. 1-2

Fluid flow-induced shear stress controls the metabolism of proximal tubule kidney epithelial cells through primary cilium-dependent lipophagy and mitochondrial biogenesis.

Caterina Miceli, Federica Roccio, Lucille Penalva-Mousset, Etienne Morel, Patrice Codogno, Nicolas Dupont.

  The kidney, similar to many other organs, has to face shear stress induced by biological fluids. How epithelial kidney cells respond to shear stress is poorly understood. Recently we showed in vitro and in vivo that proximal tubule epithelial cells use lipophagy to fuel mitochondria with fatty acids. Lipophagy is stimulated by a primary cilium-dependent signaling that converges at AMP kinase. AMP kinase is a central signaling hub to trigger lipophagy and also to stimulate mitochondrial biogenesis. These two pathways contribute to generate ATP needed to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis. These findings demonstrate the role of the primary cilium and selective macroautophagy/autophagy to integrate shear stress and to sustain the execution of a specific cellular program.

Keywords:  macroautophagy; metabolism; nephrology; oxidative phosphorylation

DOI:  https://doi.org/10.1080/15548627.2020.1823125
Biochem Biophys Res Commun. 2020 Sep 21. pii: S0006-291X(20)31748-4. [Epub ahead of print]

Cessation of electrically-induced muscle contraction activates autophagy in cultured myotubes.

Kiyoshi Yoshioka, Nobuaki Sasai, Yuko Kurogi, Kimihide Hayakawa, Yuta Itoh, Nobuhide Agata, Taro Murakami, Masumi Inoue-Miyazu, Masahiro Sokabe, Keisuke Kawakami.

  Exercise is known to improve skeletal muscle function. The mechanism involves muscle contraction-induced activation of the mTOR pathway, which plays a central role in protein synthesis. However, mTOR activation blocks autophagy, a recycling mechanism with a critical role in cellular maintenance/homeostasis. These two responses to muscle contraction look contradictory to the functional improvement of exercise. Herein, we investigate these paradoxical muscle responses in a series of active-inactive phases in a cultured myotube model receiving electrical stimulation to induce intermittent muscle contraction. Our model shows that (1) contractile activity induces mTOR activation and muscle hypertrophy but blocks autophagy, resulting in the accumulation of damaged proteins, while (2) cessation of muscle contraction rapidly activates autophagy, removing damaged protein, yet a prolonged inactive state results in muscle atrophy. Our findings provide new insights into muscle biology and suggest that not only muscle contraction, but also the subsequent cessation of contraction plays a substantial role for the improvement of skeletal muscle function.

Keywords:  Atrophy; Autophagy; Hypertrophy; Muscle contraction; Skeletal muscle

DOI:  https://doi.org/10.1016/j.bbrc.2020.09.009
Cell Death Discov. 2020 ;6 81

Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon honokiol treatment.

Nethaji Muniraj, Sumit Siddharth, Marey Shriver, Arumugam Nagalingam, Sheetal Parida, Juhyung Woo, Justin Elsey, Kathleen Gabrielson, Edward Gabrielson, Jack L Arbiser, Neeraj K Saxena, Dipali Sharma.

  Cancer cells hijack autophagy pathway to evade anti-cancer therapeutics. Many molecular signaling pathways associated with drug-resistance converge on autophagy induction. Honokiol (HNK), a natural phenolic compound purified from Magnolia grandiflora, has recently been shown to impede breast tumorigenesis and, in the present study, we investigated whether breast cancer cells evoke autophagy to modulate therapeutic efficacy and functional networks of HNK. Indeed, breast cancer cells exhibit increased autophagosomes-accumulation, MAP1LC3B-II/LC3B-II-conversion, expression of ATG proteins as well as elevated fusion of autophagosomes and lysosomes upon HNK treatment. Breast cancer cells treated with HNK demonstrate significant growth inhibition and apoptotic induction, and these biological processes are blunted by macroautophagy/autophagy. Consequently, inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of BECN1 and ATG7 effectively increase HNK-mediated apoptotic induction and growth inhibition. Next, we explored the functional impact of tumor suppressor STK11 in autophagy induction in HNK-treated cells. STK11-silencing abrogates LC3B-II-conversion, and blocks autophagosome/lysosome fusion and lysosomal activity as illustrated by LC3B-Rab7 co-staining and DQ-BSA assay. Our results exemplify the cytoprotective nature of autophagy invoked in HNK-treated breast cancer cells and put forth the notion that a combined strategy of autophagy inhibition with HNK would be more effective. Indeed, HNK and chloroquine (CQ) show synergistic inhibition of breast cancer cells and HNK-CQ combination treatment effectively inhibits breast tumorigenesis and metastatic progression. Tumor-dissociated cells from HNK-CQ treated tumors exhibit abrogated invasion and migration potential. Together, these results implicate that breast cancer cells undergo cytoprotective autophagy to circumvent HNK and a combined treatment with HNK and CQ can be a promising therapeutic strategy for breast cancer.

Keywords:  Breast cancer; Tumour-suppressor proteins

DOI:  https://doi.org/10.1038/s41420-020-00315-w
Exp Physiol. 2020 Sep 23.

Protein-carbohydrate ingestion alters Vps34 cellular localization independent of changes in kinase activity in human skeletal muscle.

Nathan Hodson, Jessica R Dent, Zhe Song, Mary F O'Leary, Thomas Nicholson, Simon W Jones, James T Murray, Stewart Jeromson, D Lee Hamilton, Leigh Breen, Andrew Philp.

   NEW FINDINGS: What is the central question of the study? Is Vps34 a nutrient-sensitive activator of mTORC1 in human skeletal muscle? What is the main finding and its importance? We show that altering nutrient availability, via protein-carbohydrate feeding, does not increase Vps34 kinase activity in human skeletal muscle. Instead, feeding increased Vps34-mTORC1 co-localization in parallel to increased mTORC1 activity. These findings may have important implications in the understanding nutrient-induced mTORC1 activation in skeletal muscle via interaction with Vps34.
ABSTRACT: The Class III PI3Kinase, Vps34, has recently been proposed as a nutrient sensor, essential for activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). We therefore investigated the effects of increasing nutrient availability through protein-carbohydrate (PRO-CHO) feeding on Vps34 kinase activity and cellular localization in human skeletal muscle. Eight young, healthy males (21 ± 0.5 yrs, 77.7 ± 9.9 kg, 25.9 ± 2.7 kg/m2 , mean ± SD) ingested a PRO-CHO beverage containing 20/44/1 g PRO/CHO/FAT respectively, with skeletal muscle biopsies obtained at baseline and 1 h and 3 h post-feeding. PRO-CHO feeding did not alter Vps34 kinase activity, but did stimulate Vps34 translocation toward the cell periphery (PRE (mean ± SD) - 0.273 ± 0.040, 1 h - 0.348 ± 0.061, Pearson's Coefficient (r)) where it co-localized with mTOR (PRE - 0.312 ± 0.040, 1 h - 0.348 ± 0.069, Pearson's Coefficient (r)). These alterations occurred in parallel to an increase in S6K1 kinase activity (941 ± 466% of PRE at 1 h post-feeding). Subsequent in vitro experiments in C2C12 and human primary myotubes displayed no effect of the Vps34-specific inhibitor SAR405 on mTORC1 signalling responses to elevated nutrient availability. Therefore, in summary, PRO-CHO ingestion does not increase Vps34 activity in human skeletal muscle, whilst pharmacological inhibition of Vps34 does not prevent nutrient stimulation of mTORC1 in vitro. However, PRO-CHO ingestion promotes Vps34 translocation to the cell periphery, enabling Vps34 to associate with mTOR. Therefore, our data suggests that interaction between Vps34 and mTOR, rather than changes in Vps34 activity per se may be involved in PRO-CHO activation of mTORC1 in human skeletal muscle. This article is protected by copyright. All rights reserved.

Keywords:  Vps34; lysosome; mTORC1

DOI:  https://doi.org/10.1113/EP088805
J Biol Chem. 2020 Sep 21. pii: jbc.RA120.013565. [Epub ahead of print]

Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation.

Sota Meguro, Xizhen Zhuang, Hiromi Kirisako, Hitoshi Nakatogawa.

  In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by post-translational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36-Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36-Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.

Keywords:  Atg36; Hrr25; Pex3; Saccharomyces cerevisiae; autophagy; peroxisome; pexophagy; proteasome; protein degradation; protein phosphorylation

DOI:  https://doi.org/10.1074/jbc.RA120.013565
J Biomed Nanotechnol. 2020 Apr 01. 16(4): 432-445

Structural Variants of poly(alkylcyanoacrylate) Nanoparticles Differentially Affect LC3 and Autophagic Cargo Degradation.

Tonje Sønstevold, Nikolai Engedal, Ýrr Mørch, Tore Geir Iversen, Tore Skotland, Kirsten Sandvig, Maria L Torgersen.

Nanoparticle drug carriers trigger a variety of cellular stress responses, including ER stress and antioxidant responses, but may also affect the intracellular degradative pathway autophagy. This can impose profound effects on drug delivery, cellular treatment responses, and nanoparticle cytotoxicity. We recently demonstrated that even small variations in the alkyl side chains of poly(alkylcyanoacrylate) (PACA) drug carrier nanoparticles, namely butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), differentially induce ER stress and redox imbalance in human cell lines. Here, we systematically investigate how these PACA variants affect autophagy. Interestingly, treatment with PEBCA or POCA particles led to intracellular accumulation of the autophagosome marker LC3-II, but via different mechanisms. PEBCA induced an integrated stress response-and ATF4-mediated increase in LC3B mRNA, whereas POCA blocked autophagic degradation of LC3-II and long-lived proteins in bulk. PBCA also increased LC3B mRNA via the integrated stress response and ATF4, but unlike PEBCA, it inhibited LC3 lipidation and autophagic cargo degradation. Our data demonstrate that even subtle variations in NP structure can have profoundly different impacts on autophagy, and that careful monitoring of autophagic flux and cargo degradation is critical for drawing accurate conclusions. Our findings have important implications for the choice of PACA monomer in different therapeutic settings.

DOI: https://doi.org/10.1166/jbn.2020.2906
Int J Mol Sci. 2020 Sep 18. pii: E6841. [Epub ahead of print]21(18):

Mitochondrial Surveillance by Cdc48/p97: MAD vs. Membrane Fusion.

Mafalda Escobar-Henriques, Vincent Anton.

  Cdc48/p97 is a ring-shaped, ATP-driven hexameric motor, essential for cellular viability. It specifically unfolds and extracts ubiquitylated proteins from membranes or protein complexes, mostly targeting them for proteolytic degradation by the proteasome. Cdc48/p97 is involved in a multitude of cellular processes, reaching from cell cycle regulation to signal transduction, also participating in growth or death decisions. The role of Cdc48/p97 in endoplasmic reticulum-associated degradation (ERAD), where it extracts proteins targeted for degradation from the ER membrane, has been extensively described. Here, we present the roles of Cdc48/p97 in mitochondrial regulation. We discuss mitochondrial quality control surveillance by Cdc48/p97 in mitochondrial-associated degradation (MAD), highlighting the potential pathologic significance thereof. Furthermore, we present the current knowledge of how Cdc48/p97 regulates mitofusin activity in outer membrane fusion and how this may impact on neurodegeneration.

Keywords:  Cdc48; Fzo1; MAD; Mfn1/2; VCP; fusion; mitochondria; mitofusin; p97; ubiquitin

DOI:  https://doi.org/10.3390/ijms21186841
J Exp Clin Cancer Res. 2020 Sep 22. 39(1): 197

Lysosomal dysfunction and autophagy blockade contribute to autophagy-related cancer suppressing peptide-induced cytotoxic death of cervical cancer cells through the AMPK/mTOR pathway.

Yang Yang, Qi Wang, Dongjian Song, Ruirui Zen, Lei Zhang, Yingjun Wang, Heying Yang, Da Zhang, Jia Jia, Jiao Zhang, Jiaxiang Wang.

   BACKGROUND: Autophagy is an intracellular process through which intracellular components are recycled in response to nutrient or growth factor deficiency to maintain homeostasis. We identified the peptide autophagy-related cancer-suppressing peptide (ARCSP), a potential antitumor peptide that disrupts intracellular homeostasis by blocking autophagic flux and causes cytotoxic death.
METHODS: The proliferative ability of ARCSP-treated cervical cancer cells was examined by the CCK8, EdU, and colony formation assays. The TUNEL assay was used to detect apoptosis. Mitochondrial function was evaluated based on the mitochondrial membrane potential. Autophagic flux was detected by immunofluorescence and confocal microscopy. The autophagy-related proteins AMPK, Raptor, mTOR, p62, LC3B, atg7, Rab7, LAMP1, LAMP2, and cathepsin D were detected by Immunoblotting. The antitumor effect of ARCSP was explored in vivo by establishing a transplant tumor model in nude mice.
RESULTS: The results demonstrated that ARCSP induced cell death and inhibited proliferation. ARCSP induced AMPK/mTOR activation, resulting in the accumulation of the proteins LC3B, p62 and Atg7. ARCSP also blocked autophagosome-lysosome fusion by inhibiting endosomal maturation and increasing the lysosomal pH. The accumulation of nonfused autophagosomes exacerbated cytotoxic death, whereas knocking down Atg7 reversed the cytotoxic death induced by ARCSP. ARCSP-treated cells exhibited increased cytotoxic death after cotreatment with an autophagy inhibitor (Chloroquine CQ). Furthermore, the tumors of ARCSP-treated nude mice were significantly smaller than those of untreated mice.
CONCLUSIONS: Our findings demonstrate that ARCSP, a novel lethal nonfused autophagosome inducer, might cause mitochondrial dysfunction and autophagy-related cytotoxic death and is thus a prospective agent for cancer therapy.

Keywords:  ARCSP; Autophagic flux; Autophagy-related cytotoxic death; Cervical cancer; Nonfused autophagosome

DOI:  https://doi.org/10.1186/s13046-020-01701-z
Cancer Metab. 2020 ;8 20

The conversion of formate into purines stimulates mTORC1 leading to CAD-dependent activation of pyrimidine synthesis.

Jacqueline Tait-Mulder, Kelly Hodge, David Sumpton, Sara Zanivan, Alexei Vazquez.

Background: Mitochondrial serine catabolism to formate induces a metabolic switch to a hypermetabolic state with high rates of glycolysis, purine synthesis and pyrimidine synthesis. While formate is a purine precursor, it is not clear how formate induces pyrimidine synthesis.
Methods: Here we combine phospho-proteome and metabolic profiling to determine how formate induces pyrimidine synthesis.
Results: We discover that formate induces phosphorylation of carbamoyl phosphate synthetase (CAD), which is known to increase CAD enzymatic activity. Mechanistically, formate induces mechanistic target of rapamycin complex 1 (mTORC1) activity as quantified by phosphorylation of its targets S6, 4E-BP1, S6K1 and CAD. Treatment with the allosteric mTORC1 inhibitor rapamycin abrogates CAD phosphorylation and pyrimidine synthesis induced by formate. Furthermore, we show that the formate-dependent induction of mTOR signalling and CAD phosphorylation is dependent on an increase in purine synthesis.
Conclusions: We conclude that formate activates mTORC1 and induces pyrimidine synthesis via the mTORC1-dependent phosphorylation of CAD.

DOI: https://doi.org/10.1186/s40170-020-00228-3
Autophagy. 2020 Sep 22. 1-15

RAB33B recruits the ATG16L1 complex to the phagophore via a noncanonical RAB binding protein.

Supansa Pantoom, Georgios Konstantinidis, Stephanie Voss, Hongmei Han, Oliver Hofnagel, Zhiyu Li, Yao-Wen Wu.

  Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12-ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12-ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12-ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation. Abbreviations : ATG: autophagy-related; Cα: alpha carbon; CCD: coiled-coil domain; CLEM: correlative light and electron microscopy; DTE: dithioerythritol; EBSS: Earle's balanced salt solution; EDTA: ethylenediaminetetraacetic acid; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; FLIM: fluorescence lifetime imaging microscopy; FRET: Förster resonance energy transfer; GDP: guanosine diphosphate; GOLGA2/GM130: golgin A2; GppNHp: guanosine 5'-[β,γ-imido]triphosphate; GST: glutathione S-transferase; GTP: guanosine triphosphate; GTPγS: guanosine 5'-O-[gamma-thio]triphosphate; HA (tag): hemagglutinin (tag); HEK: human embryonic kidney; HeLa: Henrietta Lacks; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); IgG: immunoglobulin G; Kd: dissociation constant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCF7: Michigan cancer foundation-7; MEF: mouse embryonic fibroblast; MEM: minimum essential medium Eagle; MST: microscale thermophoresis; NEAA: non-essential amino acids; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAS-associated binding; RB1CC1/FIP200: RB1 inducible coiled-coil protein 1; RBP: RAB-binding protein; SD: standard deviation; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TBS-T: tris-buffered saline-tween 20; WD (repeat): tryptophan-aspartic acid (repeat); WIPI2B: WD repeat domain phosphoinositide interacting 2B; WT: wild type.

Keywords:  ATG12–ATG5-ATG16L1 complex; ATG16L1; RAB33B; RAB33B-ATG16L1 complex; autophagosome formation; autophagy; crystal structure

DOI:  https://doi.org/10.1080/15548627.2020.1822629
FASEB J. 2020 Sep 22.

Characterization of the complex of the lysosomal membrane transporter MFSD1 and its accessory subunit GLMP.

David Massa López, Lea Kählau, Katharina Esther Julia Jungnickel, Christian Löw, Markus Damme.

  The two lysosomal integral membrane proteins MFSD1 and GLMP form a tight complex that confers protection of both interaction partners against lysosomal proteolysis. We here refined the molecular interaction of the two proteins and found that the luminal domain of GLMP alone, but not its transmembrane domain or its short cytosolic tail, conveys protection and mediates the interaction with MFSD1. Our data support the finding that the interaction is essential for the stabilization of the complex. These results are complemented by the observation that N-glycosylation of GLMP in general, but not the type of N-glycans (high-mannose-type or complex-type) or individual N-glycan chains, are essential for protection. We observed that the interaction of both proteins already starts in the endoplasmic reticulum, and quantitatively depends on each other. Both proteins can affect vice versa their intracellular trafficking to lysosomes in addition to the protection from proteolysis. Finally, we provide evidence that MFSD1 can form homodimers both in vitro and in vivo. Our data refine the complex interplay between an intimate couple of a lysosomal transporter and its accessory subunit.

Keywords:  GLMP; MFSD1; accessory subunit; lysosomal; transporter

DOI:  https://doi.org/10.1096/fj.202000912RR
World J Stem Cells. 2020 Aug 26. 12(8): 776-786

Autophagy in fate determination of mesenchymal stem cells and bone remodeling.

Xiao-Dan Chen, Jia-Li Tan, Yi Feng, Li-Jia Huang, Mei Zhang, Bin Cheng.

  Mesenchymal stem cells (MSCs) have been widely exploited as promising candidates in clinical settings for bone repair and regeneration in view of their self-renewal capacity and multipotentiality. However, little is known about the mechanisms underlying their fate determination, which would illustrate their effectiveness in regenerative medicine. Recent evidence has shed light on a fundamental biological role of autophagy in the maintenance of the regenerative capability of MSCs and bone homeostasis. Autophagy has been implicated in provoking an immediately available cytoprotective mechanism in MSCs against stress, while dysfunction of autophagy impairs the function of MSCs, leading to imbalances of bone remodeling and a wide range of aging and degenerative bone diseases. This review aims to summarize the up-to-date knowledge about the effects of autophagy on MSC fate determination and its role as a stress adaptation response. Meanwhile, we highlight autophagy as a dynamic process and a double-edged sword to account for some discrepancies in the current research. We also discuss the contribution of autophagy to the regulation of bone cells and bone remodeling and emphasize its potential involvement in bone disease.

Keywords:  Autophagy; Bone remodeling; Cell differentiation; Cell self-renewal; Cytoprotection; Mesenchymal stem cells

DOI:  https://doi.org/10.4252/wjsc.v12.i8.776
Cancer Sci. 2020 Sep 24.

Autophagy regulates levels of tumor suppressor enzyme protein phosphatase 6.

Nobuyuki Fujiwara, Shusaku Shibutani, Yusuke Sakai, Toshio Watanabe, Issay Kitabayashi, Hiroko Oshima, Masanobu Oshima, Hisashi Hoshida, Rinji Akada, Tatsuya Usui, Takashi Ohama, Koichi Sato.

  Protein Phosphatase 6 (PP6) is an essential serine/threonine protein phosphatase that acts as an important tumor suppressor. However, increased protein levels of PP6 have been observed in some cancer types, and correlate with poor prognosis in glioblastoma. This raises a question about how PP6 protein levels are regulated in normal and transformed cells. In this study, we show that PP6 protein levels increase in response to pharmacologic and genetic inhibition of autophagy. PP6 associates with autophagic adaptor protein p62/SQSTM1 and is degraded in a p62-dependent manner. Accordingly, protein levels of PP6 and p62 fluctuate in concert under different physiological and pathophysiological conditions. Our data reveal that PP6 is regulated by p62-dependent autophagy and suggest that accumulation of PP6 protein in tumor tissues is caused at least partially by deficiency in autophagy.

Keywords:  autophagy; cell biology; p62/SQSTM1; protein phosphatase 2A; protein phosphatase 6

DOI:  https://doi.org/10.1111/cas.14662
Autophagy. 2020 Sep 24. 1-16

Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90 and ROF1.

Venkatesh P Thirumalaikumar, Michal Gorka, Karina Schulz, Celine Masclaux-Daubresse, Arun Sampathkumar, Aleksandra Skirycz, Richard D Vierstra, Salma Balazadeh.

  In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS in Arabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of an nbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression of HSP genes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation of NBR1 resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS. Abbreviations: AIM: Atg8-interacting motif; ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; ConA: concanamycinA; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; FKBP: FK506-binding protein; FBPASE: fructose 1,6-bisphosphatase; GFP: green fluorescent protein; HS: heat stress; HSF: heat shock factor; HSFA2: heat shock factor A2; HSP: heat shock protein; HSP90: heat shock protein 90; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; 3-MA: 3-methyladenine; NBR1: next-to-BRCA1; PQC: protein quality control; RFP: red fluorescent protein; ROF1: rotamase FKBP1; TF: transcription factor; TUB: tubulin; UBA: ubiquitin-associated; YFP: yellow fluorescent protein.

Keywords:   Arabidopsis thaliana ; HSFA2; HSP90.1; NBR1; ROF1; heat stress; selective autophagy; stress memory; stress recovery

DOI:  https://doi.org/10.1080/15548627.2020.1820778
J Trace Elem Med Biol. 2020 Sep 10. pii: S0946-672X(20)30201-7. [Epub ahead of print]62 126636

Interplay Between Autophagy and Zinc.

Juan P Liuzzi, Rebecca Pazos.

  Autophagy is a conserved catabolic process that plays an important role in cellular homeostasis. The study of the interplay between autophagy and zinc has gained interest over the last years. Multiple studies have indicated that zinc stimulates autophagy and is critical for basal and induced autophagy in mammalian cells. Conversely, autophagy is induced by zinc starvation in yeast. There are no studies analyzing the role of zinc in either Microautophagy or Chaperone-Mediated-Autophagy. The mechanisms by which zinc modulates autophagy are still poorly understood. Studies examining loss of function of genes involved in cellular zinc homeostasis have provided novel insights into the role of zinc in autophagy. Autophagy may help cells adapt to changes in zinc availability in medium by controlling zinc mobilization, recycling, and secretion. Zinc is a key player in toxic and protective autophagy.

Keywords:  Autophagy; Lysosome; Metallothioneins; Mitophagy; Zinc

DOI:  https://doi.org/10.1016/j.jtemb.2020.126636
Biochim Biophys Acta Mol Cell Res. 2020 Sep 16. pii: S0167-4889(20)30215-9. [Epub ahead of print] 118857

IKKβ Activation Promotes Amphisome Formation and Extracellular Vesicle Secretion in Tumor Cells.

Xueqiang Peng, Liang Yang, Yingbo Ma, Xinyu Li, Shuo Yang, Yan Li, Bo Wu, Shilei Tang, Fuhui Zhang, Bing Zhang, Jingang Liu, Hangyu Li.

  Intracellular organelle cross-talk is a new and important research area. Under stress conditions, the coordinated action of the autophagy and endosomal systems in tumor cells is essential for maintaining cellular homeostasis and survival. The activation of the IκB kinase (IKK) complex is also involved in the regulation of stress and homeostasis in tumor cells. Here, we try to explore the effects of constitutively active IKKβ subunits (CA-IKKβ) on autophagy and endosomal system interactions. We confirm that CA-IKKβ induces accumulation of autophagosomes and their fusion with MVBs to form amphisomes in cancer cells, and also drives the release of EVs containing autophagy components through an amphisome-dependent mechanism. We further demonstrate that CA-IKKβ inhibits the expression of RAB7, thereby weakening the lysosomal-dependent degradation pathway. CA-IKKβ also induces phosphorylation of SNAP23 at Ser95 instead of Ser110, which further promotes amphisome-plasma membrane fusion and sEV secretion. These results indicate that CA-IKKβ drives the formation and transport of amphisomes, thereby regulating tumor cell homeostasis, which may illuminate a special survival mechanism in tumor cells under stress.

Keywords:  Amphisomes; Autophagy; Extracellular vesicles; IKKβ; Multivesicular bodies; Tumor cell

DOI:  https://doi.org/10.1016/j.bbamcr.2020.118857
Int J Mol Sci. 2020 Sep 19. pii: E6887. [Epub ahead of print]21(18):

Dysregulated Autophagy Mediates Sarcopenic Obesity and Its Complications via AMPK and PGC1α Signaling Pathways: Potential Involvement of Gut Dysbiosis as a Pathological Link.

Ji Yeon Ryu, Hyung Muk Choi, Hyung-In Yang, Kyoung Soo Kim.

  Sarcopenic obesity (SOB), which is closely related to being elderly as a feature of aging, is recently gaining attention because it is associated with many other age-related diseases that present as altered intercellular communication, dysregulated nutrient sensing, and mitochondrial dysfunction. Along with insulin resistance and inflammation as the core pathogenesis of SOB, autophagy has recently gained attention as a significant mechanism of muscle aging in SOB. Known as important cellular metabolic regulators, the AMP-activated protein kinase (AMPK) and the peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) signaling pathways play an important role in autophagy, inflammation, and insulin resistance, as well as mutual communication between skeletal muscle, adipose tissue, and the liver. Furthermore, AMPK and PGC-1α signaling pathways are implicated in the gut microbiome-muscle axis. In this review, we describe the pathological link between SOB and its associated complications such as metabolic, cardiovascular, and liver disease, falls and fractures, osteoarthritis, pulmonary disease, and mental health via dysregulated autophagy controlled by AMPK and/or PGC-1α signaling pathways. Here, we propose potential treatments for SOB by modulating autophagy activity and gut dysbiosis based on plausible pathological links.

Keywords:  AMPK signaling pathway; PGC-1α signaling pathway; aging; autophagy; gut axis; inflammation; insulin resistance; sarcopenic obesity

DOI:  https://doi.org/10.3390/ijms21186887
PLoS One. 2020 ;15(9): e0239625

Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.

Prasanna M Chandramouleeswaran, Manti Guha, Masataka Shimonosono, Kelly A Whelan, Hisatsugu Maekawa, Uma M Sachdeva, Gordon Ruthel, Sarmistha Mukherjee, Noah Engel, Michael V Gonzalez, James Garifallou, Shinya Ohashi, Andres J Klein-Szanto, Clementina A Mesaros, Ian A Blair, Renata Pellegrino da Silva, Hakon Hakonarson, Eishi Noguchi, Joseph A Baur, Hiroshi Nakagawa.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

DOI: https://doi.org/10.1371/journal.pone.0239625
Autophagy. 2020 Sep 25.

TSPAN1 promotes autophagy flux and mediates cooperation between WNT-CTNNB1 signaling and autophagy via the MIR454-FAM83A-TSPAN1 axis in pancreatic cancer.

Cefan Zhou, Yanyan Liang, Li Zhou, Yanan Yan, Nanxi Liu, Rui Zhang, Yuan Huang, Ming Wang, Yongfei Tang, Declan William Ali, Yefu Wang, Marek Michalak, Xing-Zhen Chen, Jingfeng Tang.

  Pancreatic cancer is one of the most aggressive tumors associated with a poor clinical prognosis, weakly effective therapeutic options. Therefore, there is a strong impetus to discover new therapeutic targets in pancreatic cancer. In the present study, we first demonstrated that TSPAN1 is upregulated in pancreatic cancer and that TSPAN1 depletion decreases pancreatic cancer cell proliferation in vitro and in vivo. TSPAN1 expression was correlated with poor overall survival of pancreatic cancer patients. Moreover, we demonstrated that TSPAN1 is a novel positive regulator of macroautophagy/autophagy characterized by decreased LC3-II and SQSTM1/p62 expressions, inhibited puncta formation of GFP-LC3 and autophagic vacuoles. We also demonstrated that tspan1 mutation impaired autophagy in the zebrafish model. Furthermore, we showed that TSPAN1 promoted autophagy maturation via direct binding to LC3 by two conserved LIR motifs. Mutations in the LIR motifs of TSPAN1 resulted in a loss of the ability to induce autophagy and promote pancreatic cancer proliferation. Second, we discovered two conservative TCF/LEF binding elements present in the promoter region of the TSPAN1 gene, which was further verified through luciferase activity and ChIP assays. Furthermore, TSPAN1 was upregulated by FAM83A through the canonical WNT-CTNNB1 signaling pathway. We further demonstrated that both TSPAN1 and FAM83A are both direct targets of MIR454 (microRNA 454). Additionally, we revealed the role of MIR454-FAM83A-TSPAN1 in the proliferation of pancreatic cancer cells in vitro and in vivo. Our findings suggest that components of the MIR454-FAM83A-TSPAN1 axis may be valuable prognosis markers or therapeutic targets for pancreatic cancer.

Keywords:   MIR454 ; FAM83A; WNT-CTNNB1; autophagy; pancreatic cancer; tetraspanin 1

DOI:  https://doi.org/10.1080/15548627.2020.1826689
Nature. 2020 Sep 23.

Modulating TRADD to restore cellular homeostasis and inhibit apoptosis.

Daichao Xu, Heng Zhao, Minzhi Jin, Hong Zhu, Bing Shan, Jiefei Geng, Slawomir A Dziedzic, Palak Amin, Lauren Mifflin, Masanori Gomi Naito, Ayaz Najafov, Jing Xing, Lingjie Yan, Jianping Liu, Ying Qin, Xinqian Hu, Huibing Wang, Mengmeng Zhang, Vica Jean Manuel, Li Tan, Zhuohao He, Zhenyu J Sun, Virginia M Y Lee, Gerhard Wagner, Junying Yuan.

Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.

DOI: https://doi.org/10.1038/s41586-020-2757-z
Med Res Rev. 2020 Sep 23.

Autophagy and cardiac diseases: Therapeutic potential of natural products.

Xiaoqian Wu, Zumei Liu, Xi-Yong Yu, Suowen Xu, Jiandong Luo.

  The global incidence of cardiac diseases is expected to increase in the coming years, imposing a substantial socioeconomic burden on healthcare systems. Autophagy is a tightly regulated lysosomal degradation mechanism important for cell survival, homeostasis, and function. Accumulating pieces of evidence have indicated a major role of autophagy in the regulation of cardiac homeostasis and function. It is well established that dysregulation of autophagy in cardiomyocytes is involved in cardiac hypertrophy, myocardial infarction, diabetic cardiomyopathy, and heart failure. In this sense, autophagy seems to be an attractive therapeutic target for cardiac diseases. Recently, multiple natural products/phytochemicals, such as resveratrol, berberine, and curcumin have been shown to regulate cardiomyocyte autophagy via different pathways. The autophagy-modifying capacity of these compounds should be taken into consideration for designing novel therapeutic agents. This review focuses on the role of autophagy in various cardiac diseases and the pharmacological basis and therapeutic potential of reported natural products in cardiac diseases by modifying autophagic processes.

Keywords:  autophagy; autophagy flux; cardiac diseases; natural products; therapeutics

DOI:  https://doi.org/10.1002/med.21733
Cells. 2020 Sep 22. pii: E2140. [Epub ahead of print]9(9):

The Role of Chaperone-Mediated Autophagy in Cell Cycle Control and Its Implications in Cancer.

Marina Andrade-Tomaz, Izadora de Souza, Clarissa Ribeiro Reily Rocha, Luciana Rodrigues Gomes.

  The cell cycle involves a network of proteins that modulate the sequence and timing of proliferation events. Unregulated proliferation is the most fundamental hallmark of cancer; thus, changes in cell cycle control are at the heart of malignant transformation processes. Several cellular processes can interfere with the cell cycle, including autophagy, the catabolic pathway involved in degradation of intracellular constituents in lysosomes. According to the mechanism used to deliver cargo to the lysosome, autophagy can be classified as macroautophagy (MA), microautophagy (MI), or chaperone-mediated autophagy (CMA). Distinct from other autophagy types, CMA substrates are selectively recognized by a cytosolic chaperone, one-by-one, and then addressed for degradation in lysosomes. The function of MA in cell cycle control, and its influence in cancer progression, are already well-established. However, regulation of the cell cycle by CMA, in the context of tumorigenesis, has not been fully addressed. This review aims to present and debate the molecular mechanisms by which CMA can interfere in the cell cycle, in the context of cancer. Thus, cell cycle modulators, such as MYC, hypoxia-inducible factor-1 subunit alpha (HIF-1α), and checkpoint kinase 1 (CHK1), regulated by CMA activity will be discussed. Finally, the review will focus on how CMA dysfunction may impact the cell cycle, and as consequence promote tumorigenesis.

Keywords:  MYC; autophagy; cancer; chaperone-mediated autophagy (CMA), cell cycle; checkpoints; hypoxia-inducible factor-1 subunit alpha (HIF-1α), checkpoint kinase 1 (CHK1)

DOI:  https://doi.org/10.3390/cells9092140
EMBO Rep. 2020 Sep 25. e50202

Iron loss triggers mitophagy through induction of mitochondrial ferritin.

Yuichi Hara, Izumi Yanatori, Atsushi Tanaka, Fumio Kishi, John J Lemasters, Sohji Nishina, Kyo Sasaki, Keisuke Hino.

  Mitochondrial quality is controlled by the selective removal of damaged mitochondria through mitophagy. Mitophagy impairment is associated with aging and many pathological conditions. An iron loss induced by iron chelator triggers mitophagy by a yet unknown mechanism. This type of mitophagy may have therapeutic potential, since iron chelators are clinically used. Here, we aimed to clarify the mechanisms by which iron loss induces mitophagy. Deferiprone, an iron chelator, treatment resulted in the increased expression of mitochondrial ferritin (FTMT) and the localization of FTMT precursor on the mitochondrial outer membrane. Specific protein 1 and its regulator hypoxia-inducible factor 1α were necessary for deferiprone-induced increase in FTMT. FTMT specifically interacted with nuclear receptor coactivator 4, an autophagic cargo receptor. Deferiprone-induced mitophagy occurred selectively for depolarized mitochondria. Additionally, deferiprone suppressed the development of hepatocellular carcinoma (HCC) in mice by inducing mitophagy. Silencing FTMT abrogated deferiprone-induced mitophagy and suppression of HCC. These results demonstrate the mechanisms by which iron loss induces mitophagy and provide a rationale for targeting mitophagic activation as a therapeutic strategy.

Keywords:  hepatocellular carcinoma; iron chelator; mitochondria; mitochondrial ferritin; mitophagy

DOI:  https://doi.org/10.15252/embr.202050202
Wiley Interdiscip Rev RNA. 2020 Sep 20. e1628

Translational control in aging and neurodegeneration.

Geena Skariah, Peter K Todd.

  Protein metabolism plays central roles in age-related decline and neurodegeneration. While a large body of research has explored age-related changes in protein degradation, alterations in the efficiency and fidelity of protein synthesis with aging are less well understood. Age-associated changes occur in both the protein synthetic machinery (ribosomal proteins and rRNA) and within regulatory factors controlling translation. At the same time, many of the interventions that prolong lifespan do so in part by pre-emptively decreasing protein synthesis rates to allow better harmonization to age-related declines in protein catabolism. Here we review the roles of translation regulation in aging, with a specific focus on factors implicated in age-related neurodegeneration. We discuss how emerging technologies such as ribosome profiling and superior mass spectrometric approaches are illuminating age-dependent mRNA-specific changes in translation rates across tissues to reveal a critical interplay between catabolic and anabolic pathways that likely contribute to functional decline. These new findings point to nodes in posttranscriptional gene regulation that both contribute to aging and offer targets for therapy. This article is categorized under: Translation > Translation Regulation Translation > Ribosome Biogenesis Translation > Translation Mechanisms.

Keywords:  aging; neurodegeneration; translation; translation regulation

DOI:  https://doi.org/10.1002/wrna.1628
Evid Based Complement Alternat Med. 2020 ;2020 7486041

Electroacupuncture Pretreatment against Cerebral Ischemia/Reperfusion Injury through Mitophagy.

Chenlu Mao, Cheng Hu, Yudi Zhou, Rong Zou, Sha Li, Yaomei Cui, Weiqian Tian.

Cerebral ischemia/reperfusion (I/R) injury can induce the mitophagy of neurons in the ischemic brain. Electroacupuncture (EA) pretreatment has a protective effect on cerebral ischemia/reperfusion injury. However, its internal mechanism still needs to be further studied. The present study's purpose is to investigate whether mitophagy is involved in neuroprotection elicited by EA pretreatment in a rat model of cerebral ischemia/reperfusion injury. The rats were pretreated with vehicle, EA at the Baihui (GV20) and Shuigou (GV26) acupoints 30 min daily, for 5 days consecutively prior to the focal cerebral ischemia injury induced by the middle cerebral artery occlusion (MCAO) model. Compared to the sham group, the neurological scores, infarction volume, number of autophagosomes, FUNDC1, p62, and the ratio of LC3-II/I were significantly increased but mitochondrial membrane potential and autophagy-related protein p-mTORC1 significantly decreased in the I/R group. However, EA pretreatment significantly reversed these trends. Overall, the results of this study demonstrated that EA pretreatment protected the cerebral ischemia/reperfusion injury which maybe correlated with mitophagy.

DOI: https://doi.org/10.1155/2020/7486041