bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒05‒17
28 papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2020 May 13. 1-17
      Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases.ABBREVIATIONS: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.
    Keywords:  Brown adipocyte; inflammasome; pink1; transcriptional activation; white adipocyte
  2. Cells. 2020 May 09. pii: E1184. [Epub ahead of print]9(5):
      In all eukaryotic cells, intracellular organization and spatial separation of incompatible biochemical processes is established by individual cellular subcompartments in form of membrane-bound organelles. Virtually all of these organelles are physically connected via membrane contact sites (MCS), allowing interorganellar communication and a functional integration of cellular processes. These MCS coordinate the exchange of diverse metabolites and serve as hubs for lipid synthesis and trafficking. While this of course indirectly impacts on a plethora of biological functions, including autophagy, accumulating evidence shows that MCS can also directly regulate autophagic processes. Here, we focus on the nexus between interorganellar contacts and autophagy in yeast and mammalian cells, highlighting similarities and differences. We discuss MCS connecting the ER to mitochondria or the plasma membrane, crucial for early steps of both selective and non-selective autophagy, the yeast-specific nuclear-vacuolar tethering system and its role in microautophagy, the emerging function of distinct autophagy-related proteins in organellar tethering as well as novel MCS transiently emanating from the growing phagophore and mature autophagosome.
    Keywords:  ERMES; ER–mitochondria encounter structure; MAMs; autophagy; lipophagy; membrane contact sites; mitochondria-associated membranes; mitophagy; nucleus–vacuole junction; pexophagy; piecemeal microautophagy of the nucleus
  3. Cells. 2020 May 10. pii: E1186. [Epub ahead of print]9(5):
      The MiT/TFE family of transcription factors (MITF, TFE3, and TFEB), which control transcriptional programs for autophagy and lysosome biogenesis have emerged as regulators of energy metabolism in cancer. Thus, their activation increases lysosomal catabolic function to sustain cancer cell growth and survival in stress conditions. Here, we found that TFEB depletion dramatically reduces basal expression levels of the cyclin-dependent kinase (CDK) inhibitor p21/WAF1 in various cell types. Conversely, TFEB overexpression increases p21 in a p53-dependent manner. Furthermore, induction of DNA damage using doxorubicin induces TFEB-mediated activation of p21, delays G2/M phase arrest, and promotes cell survival. Pharmacological inhibition of p21, instead, abrogates TFEB-mediated protection during the DNA damage response. Together, our findings uncover a novel and direct role of TFEB in the regulation of p21 expression in both steady-state conditions and during the induction of DNA-damage response (DDR). Our observations might open novel therapeutic strategies to promote cancer cell death by targeting the TFEB-p21 pathway in the presence of genotoxic agents.
    Keywords:  DNA-damage response; TFEB; autophagy; cancer; cell cycle; doxorubicin; genotoxic stress; p21
  4. Autophagy. 2020 May 13. 1-3
      Classical macroautophagy/autophagy functions to maintain cell health during stressful conditions by targeting cytosolic components for degradation and recycling through the lysosomal pathway. In contrast, nondegradative secretory autophagy functions as an alternative autophagy mechanism to mediate extracellular secretion. A recent study published in Nature Cell Biology from the laboratory of Jayanta Debnath has identified components of the LC3-conjugation machinery as mediators in the selection of cargo for nonclassical secretion. Termed LC3-dependent extracellular vesicle loading and secretion (LDELS), this mechanism provides an additional link between secretory autophagy and the release of extracellular vesicles.ABBREVIATIONS: ATG, autophagy-related; BioID, proximity-dependent biotinylation; CM, conditioned medium; EV, extracellular vesicle; HNRNPK, heterogeneous nuclear ribonuclear protein K; ILVs, intralumenal vesicles; LDELS, LC3-dependent EV loading and secretion; LIR, LC3-interacting region; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MS, mass spectrometry; MVBs, multivesicular bodies; ncRNA, non-coding RNA; NSMAF/FAN, neutral sphingomyelinase activation associated factor; P-bodies, processing bodies; PE, phosphatidylethanolamine; RB1CC1/FIP200, RB1 inducible coiled-coil 1; RBP, RNA-binding protein; RNA-seq, RNA sequencing; SAFB, scaffold-attachment factor B; SILAC, stable isotope labeling of amino acids in cell culture; SMPD3/nSMase2, sphingomyelin phosphodiesterase 3; TEM, transmission electron microscopy; TMT, tandem mass tagging.
    Keywords:  Autophagy; LC3-II; macroautophagy; multivesicular bodies; secretory autophagy
  5. Cells. 2020 May 07. pii: E1152. [Epub ahead of print]9(5):
      We recently identified elevated annexin A6 (AnxA6) protein levels in Niemann-Pick-type C1 (NPC1) mutant cells. In these cells, AnxA6 depletion rescued the cholesterol accumulation associated with NPC1 deficiency. Here, we demonstrate that elevated AnxA6 protein levels in NPC1 mutants or upon pharmacological NPC1 inhibition, using U18666A, were not due to upregulated AnxA6 mRNA expression, but caused by defects in AnxA6 protein degradation. Two KFERQ-motifs are believed to target AnxA6 to lysosomes for chaperone-mediated autophagy (CMA), and we hypothesized that the cholesterol accumulation in endolysosomes (LE/Lys) triggered by the NPC1 inhibition could interfere with the CMA pathway. Therefore, AnxA6 protein amounts and cholesterol levels in the LE/Lys (LE-Chol) compartment were analyzed in NPC1 mutant cells ectopically expressing lysosome-associated membrane protein 2A (Lamp2A), which is well known to induce the CMA pathway. Strikingly, AnxA6 protein amounts were strongly decreased and coincided with significantly reduced LE-Chol levels in NPC1 mutant cells upon Lamp2A overexpression. Therefore, these findings suggest Lamp2A-mediated restoration of CMA in NPC1 mutant cells to lower LE-Chol levels with concomitant lysosomal AnxA6 degradation. Collectively, we propose CMA to permit a feedback loop between AnxA6 and cholesterol levels in LE/Lys, encompassing a novel mechanism for regulating cholesterol homeostasis in NPC1 disease.
    Keywords:  AnxA6; Lamp2A.; NPC1; chaperone-mediated autophagy; cholesterol; endolysosomes
  6. Transl Neurodegener. 2020 May 11. 9(1): 17
      BACKGROUND: Lysosomes digest extracellular material from the endocytic pathway and intracellular material from the autophagic pathway. This process is performed by the resident hydrolytic enzymes activated by the highly acidic pH within the lysosomal lumen. Lysosome pH gradients are mainly maintained by the vacuolar (H+) ATPase (or V-ATPase), which pumps protons into lysosomal lumen by consuming ATP. Dysfunction of V-ATPase affects lysosomal acidification, which disrupts the clearance of substrates and leads to many disorders, including neurodegenerative diseases.MAIN BODY: As a large multi-subunit complex, the V-ATPase is composed of an integral membrane V0 domain involved in proton translocation and a peripheral V1 domain catalyzing ATP hydrolysis. The canonical functions of V-ATPase rely on its H+-pumping ability in multiple vesicle organelles to regulate endocytic traffic, protein processing and degradation, synaptic vesicle loading, and coupled transport. The other non-canonical effects of the V-ATPase that are not readily attributable to its proton-pumping activity include membrane fusion, pH sensing, amino-acid-induced activation of mTORC1, and scaffolding for protein-protein interaction. In response to various stimuli, V-ATPase complex can reversibly dissociate into V1 and V0 domains and thus close ATP-dependent proton transport. Dysregulation of pH and lysosomal dysfunction have been linked to many human diseases, including neurodegenerative disorders such as Alzheimer disease, Parkinson's disease, amyotrophic lateral sclerosis as well as neurodegenerative lysosomal storage disorders.
    CONCLUSION: V-ATPase complex is a universal proton pump and plays an important role in lysosome acidification in all types of cells. Since V-ATPase dysfunction contributes to the pathogenesis of multiple neurodegenerative diseases, further understanding the mechanisms that regulate the canonical and non-canonical functions of V-ATPase will reveal molecular details of disease process and help assess V-ATPase or molecules related to its regulation as therapeutic targets.
    Keywords:  Autophagy; Lysosomal acidification; Neurodegeneration; Vacuolar-type ATPase
  7. Autophagy. 2020 May 13. 1-19
      Autophagy is a highly conserved catabolic process and a major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles. An increasing body of evidence has unveiled autophagy as an indispensable biological function that helps to maintain normal tissue homeostasis and metabolic fitness that can also lead to severe consequences for the normal cellular functioning when altered. Recent accumulating data point to autophagy as a key player in a wide variety of physiological and pathophysiological conditions in the human endometrium, one of the most proficient self-regenerating tissues in the human body and an instrumental player in placental species reproductive function. The current review highlights the most recent findings regarding the process of autophagy in the normal and cancerous endometrial tissue. Current research efforts aiming to therapeutically exploit autophagy and the methodological approaches used are discussed.ABBREVIATIONS: 3-MA: 3-methyladenine; ACACA (acetyl-CoA carboxylase alpha); AICAR: 5-aminoimidazole-4-carboximide riboside; AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ATG: autophagy related; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; ATG3: autophagy related 3; ATG4C: autophagy related 4C cysteine peptidase; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9: autophagy related 9; Baf A1: bafilomycin A1; BAX: BCL2 associated X, apoptosis regulator; BCL2: BCL2 apoptosis regulator; BECN1: beclin 1; CACNA1D: calcium voltage-gated channel subunit alpha1 D; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CASP9: caspase 9; CD44: CD44 molecule (Indian blood group); CDH1: cadherin 1; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; CMA: chaperone-mediated autophagy; CQ: chloroquine; CTNNB1: catenin beta 1; DDIT3: DNA damage inducible transcript 3; EC: endometrial cancer; EGFR: epidermal growth factor receptor; EH: endometrial hyperplasia; EIF4E: eukaryotic translation initiation factor 4E; EPHB2/ERK: EPH receptor B2; ER: endoplasmic reticulum; ERBB2: er-b2 receptor tyrosine kinase 2; ERVW-1: endogenous retrovirus group W member 1, envelope; ESR1: estrogen receptor 1; FSH: follicle-stimulating hormone; GCG/GLP1: glucagon; GFP: green fluorescent protein; GIP: gastric inhibitory polypeptide; GLP1R: glucagon-like peptide-1 receptor; GLS: glutaminase; H2AX: H2A.X variant histone; HIF1A: hypoxia inducible factor 1 alpha; HMGB1: high mobility group box 1; HOTAIR: HOX transcript antisense RNA; HSPA5: heat shock protein family A (HSP70) member 5; HSPA8: heat shock protein family A (HSP70) member 8; IGF1: insulin like growth factor 1; IL27: interleukin 27; INS: insulin; ISL: isoliquiritigenin; KRAS: KRAS proto-oncogene, GTPase; LAMP2: lysosomal-associated membrane protein 2; lncRNA: long-non-coding RNA; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK8: mitogen-activated protein kinase 8; MAPK9: mitogen-activated protein kinase 9; MPA: medroxyprogesterone acetate; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; MYCBP: MYC-binding protein; NFE2L2: nuclear factor, erythroid 2 like 2; NFKB: nuclear factor kappa B; NFKBIA: NFKB inhibitor alpha; NK: natural killer; NR5A1: nuclear receptor subfamily 5 group A member 1; PARP1: poly(ADP-ribose) polymerase 1; PAX2: paired box 2; PDK1: pyruvate dehydrogenase kinase 1; PDX: patient-derived xenograft; PIK3C3/Vps34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3CA: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PIK3R1: phosphoinositide-3-kinase regulatory subunit 1; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PPD: protopanaxadiol; PRKCD: protein kinase C delta; PROM1/CD133: prominin 1; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PTEN: phosphatase and tensin homolog; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RFP: red fluorescent protein; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; RSV: resveratrol; SGK1: serum/glucocorticoid regulated kinase 1; SGK3: serum/glucocorticoid regulated kinase family member 3; SIRT: sirtuin; SLS: stone-like structures; SMAD2: SMAD family member 2; SMAD3: SMAD family member 3; SQSTM1: sequestosome 1; TALEN: transcription activator-like effector nuclease; TGFBR2: transforming growth factor beta receptor 2; TP53: tumor protein p53; TRIB3: tribbles pseudokinase 3; ULK1: unc-51 like autophagy activating kinase 1; ULK4: unc-51 like kinase 4; VEGFA: vascular endothelial growth factor A; WIPI2: WD repeat domain, phosphoinositide interacting 2; XBP1: X-box binding protein 1; ZFYVE1: zinc finger FYVE domain containing 1.
    Keywords:  Autophagy; endometrial cancer; endometrial hyperplasia; endometrium; menstrual cycle; obesity; reproduction
  8. Front Pharmacol. 2020 ;11 547
      Autophagy is a highly conserved multistep process and functions as passage for degrading and recycling protein aggregates and defective organelles in eukaryotic cells. Based on the nature of these materials, their size and degradation rate, four types of autophagy have been described, i.e. chaperone mediated autophagy, microautophagy, macroautophagy, and selective autophagy. One of the major regulators of this process is mTOR, which inhibits the downstream pathway of autophagy following the activation of its complex 1 (mTORC1). Alkylphosphocholine (APC) derivatives represent a novel class of antineoplastic agents that inhibit the serine-threonine kinase Akt (i.e. protein kinase B), which mediates cell survival and cause cell cycle arrest. They induce autophagy through inhibition of the Akt/mTOR cascade. They interfere with phospholipid turnover and thus modify signaling chains, which start from the cell membrane and modulate PI3K/Akt/mTOR, Ras-Raf-MAPK/ERK and SAPK/JNK pathways. APCs include miltefosine, perifosine, and erufosine, which represent the first-, second- and third generation of this class, respectively. In a high fraction of human cancers, constitutively active oncoprotein Akt1 suppresses autophagy in vitro and in vivo. mTOR is a down-stream target for Akt, the activation of which suppresses autophagy. However, treatment with APC derivatives will lead to dephosphorylation (hence deactivation) of mTOR and thus induces autophagy. Autophagy is a double-edged sword and may result in chemotherapeutic resistance as well as cancer cell death when apoptotic pathways are inactive. APCs display differential autophagy induction capabilities in different cancer cell types. Therefore, autophagy-dependent cellular responses need to be well understood in order to improve the chemotherapeutic outcome.
    Keywords:  Akt/mTOR pathway; alkylphosphocholines; autophagy as drug target; miltefosine/perifosine/erufosine; types of autophagy
  9. Trends Cell Biol. 2020 Jun;pii: S0962-8924(20)30052-0. [Epub ahead of print]30(6): 452-466
      Lysosomes are of major importance for the regulation of cellular cholesterol homeostasis. Food-derived cholesterol and cholesterol esters contained within lipoproteins are delivered to lysosomes by endocytosis. From the lysosomal lumen, cholesterol is transported to the inner surface of the lysosomal membrane through the glycocalyx; this shuttling requires Niemann-Pick C (NPC) 1 and NPC2 proteins. The lysosomal membrane proteins lysosomal-associated membrane protein (LAMP)-2 and lysosomal integral membrane protein (LIMP)-2/SCARB2 also bind cholesterol. LAMP-2 may serve as a cholesterol reservoir, whereas LIMP-2, like NPC1, is able to transport cholesterol through a transglycocalyx tunnel. Contact sites and fusion events between lysosomes and other organelles mediate the distribution of cholesterol. Lysosomal cholesterol content is sensed thereby regulating mammalian target of rapamycin complex (mTORC)-dependent signaling. This review summarizes our understanding of the major steps in cholesterol handling from the moment it enters the lysosome until it leaves this compartment.
    Keywords:  Niemann–Pick type C; cholesterol; contact sites; lysosomal integral membrane protein-2; lysosomes
  10. Front Cell Dev Biol. 2020 ;8 276
      During their development and overall life, mesenchymal stem cells (MSCs) encounter a plethora of internal and external stress signals and therefore, they need to put in action homeostatic changes in order to face these stresses. To this aim, similar to other mammalian cells, MSCs are endowed with two crucial biological responses, autophagy and senescence. Sharing of a number of stimuli like shrinkage of telomeres, oncogenic and oxidative stress, and DNA damage, suggest an intriguingly close relationship between autophagy and senescence. Autophagy is at first reported to suppress MSC senescence by clearing injured cytoplasmic organelles and impaired macromolecules, yet recent investigations also showed that autophagy can promote MSC senescence by inducing the production of senescence-associated secretory proteins (SASP). These apparently contrary contributions of autophagy may mirror an intricate image of autophagic regulation on MSC senescence. We here tackle the pro-senescence and anti-senescence roles of autophagy in MSCs while concentrating on some possible mechanistic explanations of such an intricate liaison. Clarifying the autophagy/senescence relationship in MSCs will help the development of more effective and safer therapeutic strategies.
    Keywords:  SASP; general autophagy; mesenchymal stem cell; selective autophagy; senescence
  11. J Biol Chem. 2020 May 14. pii: jbc.RA120.013223. [Epub ahead of print]
      The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that is essential for cellular homeostasis. V-ATPase activity is controlled by regulated assembly of the enzyme from its component V1 and V0 domains. We previously reported that amino acid starvation rapidly increases V-ATPase assembly and activity in mammalian lysosomes, but the signaling pathways controlling this effect are unknown. In testing inhibitors of pathways important for controlling cellular metabolism, we found here that the cAMP-dependent protein kinase (PKA) inhibitor H89 increases lysosomal V-ATPase activity and blocks any further change upon starvation. The AMP-activated protein kinase (AMPK) inhibitor dorsomorphin decreased lysosomal V-ATPase activity and also blocked any increase upon starvation. However, CRISPR-mediated gene editing revealed that PKA and AMPK are not required for the starvation-dependent increase in lysosomal V-ATPase activity, indicating that H89 and dorsomorphin modify V-ATPase activity through other cellular targets. We next found that the AKT Ser/Thr kinase (AKT) inhibitor MK2206 blocks the starvation-dependent increase in lysosomal V-ATPase activity without altering basal activity. Expression of AKT1 or AKT3, but not AKT2, was required for increased lysosomal V-ATPase activity in response to amino acid starvation in mouse fibroblasts. Finally, HEK293T cells expressing only AKT1 responded normally to starvation, whereas cells expressing only AKT2 displayed a significantly reduced increase in V-ATPase activity and assembly upon starvation. These results show that AKT is required for controlling the rapid response of lysosomal V-ATPase activity to changes in amino acid availability and that this response depends on specific AKT isoforms.
    Keywords:  Akt PKB; CRISPR/Cas; MK2206; amino acid homeostasis; kinase signaling; lysosomal acidification; nutient sensing; pH regulation; proton transport; vacuolar ATPase
  12. Mol Cell. 2020 May 07. pii: S1097-2765(20)30257-4. [Epub ahead of print]78(3): 379-381
      Fujioka et al. (2020) uncovered liquid-liquid phase separation of the PAS, a key driver of autophagosome formation in yeast. Moreover, the authors demonstrated that liquid-like PAS controls autophagic kinase activation and is itself regulated by the phosphorylation status of its constituents.
  13. Hepatology. 2020 May 11.
      Primary liver cancers, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), are highly lethal and frequent tumors worldwide, with few effective treatment options. The mammalian target of Rapamycin (mTOR) complex is a central regulator of cell growth and metabolism by its ability to integrate inputs from amino acids, nutrients and extracellular signals. The mTOR protein is incorporated into two distinct complexes: mammalian target of Rapamycin complex 1 (mTORC1) and mammalian target of Rapamycin complex 2 (mTORC2). Specifically, mTORC1 regulates protein synthesis, glucose and lipid metabolism, and autophagy, whereas mTORC2 promotes liver tumorigenesis via modulating the AGC family of serine/threonine kinases, especially the AKT (protein kinase B) proteins. In human HCC and iCCA samples, genomics analyses have unraveled the frequent de-regulation of the mTOR complexes. Both in vitro and in vivo studies have demonstrated the key role of mTORC1 and mTORC2 in liver tumor development and progression. The first generation mTOR inhibitors have been evaluated for effectiveness in liver tumor treatment and provided unsatisfactory results. Current research efforts are devoted to generating more efficacious mTOR inhibitors and identify biomarkers for patient selection as well as for novel combination therapies. Here, we provide a comprehensive review of the mechanisms leading to deregulated mTOR signaling cascade in liver cancers, the mechanisms whereby the mTOR pathway contributes to HCC and iCCA molecular pathogenesis, the therapeutic strategies, and the challenges that we need to overcome to effectively inhibit mTOR in liver cancer treatment. In conclusion: Deregulated mTOR signaling significantly contributes to HCC and iCCA molecular pathogenesis. mTOR inhibitors, presumably administered in association with other drugs, might be effective against subsets of human liver tumors.
    Keywords:  Cholangiocarcinoma; Hepatocellular carcinoma; mTOR inhibitor; mTORC1; mTORC2
  14. Int J Mol Sci. 2020 May 10. pii: E3369. [Epub ahead of print]21(9):
      Cells have developed elaborate quality-control mechanisms for proteins and organelles to maintain cellular homeostasis. Such quality-control mechanisms are maintained by conformational folding via molecular chaperones and by degradation through the ubiquitin-proteasome or autophagy-lysosome system. Accumulating evidence suggests that impaired autophagy contributes to the accumulation of intracellular inclusion bodies consisting of misfolded proteins, which is a hallmark of most neurodegenerative diseases. In addition, genetic mutations in core autophagy-related genes have been reported to be linked to neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Conversely, the pathogenic proteins, such as amyloid β and α-synuclein, are detrimental to the autophagy pathway. Here, we review the recent advances in understanding the relationship between autophagic defects and the pathogenesis of neurodegenerative diseases and suggest autophagy induction as a promising strategy for the treatment of these conditions.
    Keywords:  autophagy; neurodegenerative disease; protein aggregates
  15. Front Immunol. 2020 ;11 752
      Interaction between eosinophils and dermal fibroblasts is essential for provoking allergic inflammation in atopic dermatitis (AD). In vitro co-culture of human eosinophils and dermal fibroblasts upon AD-related IL-31 and IL-33 stimulation, and in vivo MC903-induced AD murine model were employed to investigate the anti-inflammatory mechanism of IL-1 family cytokine IL-37 in AD. Results showed that IL-37b could inhibit the in vitro induction of AD-related pro-inflammatory cytokines IL-6 and TNF-α, and chemokines CXCL8, CCL2 and CCL5, increase autophagosome biogenesis-related LC3B, and decrease autophagy-associated ubiquitinated protein p62 by regulating intracellular AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling pathway. In CRISPR/Cas9 human IL-37b knock-in mice, IL-37b could significantly alleviate MC903-stimulated ear tissue swelling, itching sensation and the level of circulating cytokine IL-6 and ear in situ expression of AD-related TNF-α, CCL5 and transforming growth factor-β. Moreover, IL-37b could significantly upregulate Foxp3+ regulatory T cells (Treg) in spleen and ear together with significantly increased serum Treg cytokine IL-10, and decrease eosinophil infiltration in ear lesion. IL-37b knock-in mice showed a distinct intestinal microbiota metabolic pattern upon MC903 stimulation. Furthermore, IL-37b restored the disordered gut microbiota diversity, through regulating the in vivo autophagy mechanism mediated by intestinal metabolite 3-methyladenine, adenosine monophosphate, 2-hydroxyglutarate, purine and melatonin. In summary, IL-37b could significantly ameliorate eosinophils-mediated allergic inflammation via the regulation of autophagy mechanism, intestinal bacterial diversity and their metabolites in AD. Results therefore suggest that IL-37 is a potential anti-inflammatory cytokine for AD treatment.
    Keywords:  IL-37; allergic inflammation; atopic dermatitis; autophagy; eosinophils; microbiota
  16. Cells. 2020 May 09. pii: E1179. [Epub ahead of print]9(5):
      Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric ATG12~5/16 complex, which is part of the essential ubiquitin-like conjugation systems in autophagy. Dictyostelium discoideum atg5 single, atg5/12 double, and atg5/12/16 triple gene knock-out mutant strains displayed similar defects in the conjugation of ATG8 to phosphatidylethanolamine, development, and cell viability upon nitrogen starvation. This implies that ATG5, 12 and 16 act as a functional unit in canonical autophagy. Macropinocytosis of TRITC dextran and phagocytosis of yeast were significantly decreased in ATG5¯ and ATG5¯/12¯ and even further in ATG5¯/12¯/16¯ cells. In contrast, plaque growth on Klebsiella aerogenes was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of Escherichia coli was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of E. coli, was seen for ATG5¯ and ATG5¯/12¯/16¯ cells. Proteasomal activity was also disturbed in a complex fashion, consistent with an inhibitory activity of ATG16 in the absence of ATG5 and/or ATG12. Our results confirm the essential function of the ATG12~5/16 complex in canonical autophagy, and furthermore are consistent with autophagy-independent functions of the complex and its individual components. They also strongly support the placement of autophagy upstream of the ubiquitin-proteasome system (UPS), as a fully functional UPS depends on autophagy.
    Keywords:  ATG12~5/16 complex; Dictyostelium; UPS; autophagy; phagocytosis; pinocytosis; proteasome; ubiquitin-like protein
  17. Elife. 2020 May 15. pii: e57089. [Epub ahead of print]9
      Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we found that NPC1's N-terminal domain need not release from the rest of the protein for efficient cholesterol export. Either introducing single disulfide bonds to constrain lumenal/extracellular domains or shortening a cytoplasmic loop abolishes transport activity by both NPC1 and NPC1L1. The widely prescribed cholesterol uptake inhibitor, ezetimibe, blocks NPC1L1; we show that residues that lie at the interface between NPC1L1's three extracellular domains comprise the drug's binding site. These data support a model in which cholesterol passes through the cores of NPC1/NPC1L1 proteins; concerted movement of various domains is needed for transfer and ezetimibe blocks transport by binding to multiple domains simultaneously.
    Keywords:  biochemistry; cell biology; chemical biology; cholesterol transport; ezetimibe; human; ldl cholesterol; lysosome; niemann pick c disease
  18. Spectrochim Acta A Mol Biomol Spectrosc. 2020 Apr 28. pii: S1386-1425(20)30406-6. [Epub ahead of print]237 118428
      In situ and real-time visualization of autophagy process is of vital importance for fundamental researches in biology. However, fluorescent probes for autophagy were rarely reported, which greatly hindered the study on autophagy. In this work, a fluorescent probe was rationally designed and fabricated for the ratiometric visualization of autophagy. The probe targeted non-lysosome organelles in healthy cells, and gave blue emission. During autophagy, the organelles together with the probe were delivered into acid lysosomes, and gave red fluorescence. In this way, the autophagy process could be monitored in ratiometric manner. The starvation induced autophagy was successfully visualized by means of the probe, and the inhibition effect of chloroquine to autophagy was also observed. We expect that the probe can serve as a powerful tool for the investigation of autophagy and relative areas.
    Keywords:  Autophagy; Fluorescent probe; Ratiometric; pH sensitive
  19. Autophagy. 2020 Jun;16(6): 973-974
      AMPK is one of the main regulators of energy homeostasis in the cell, achieving this role in part by upregulating autophagy in times of nutrient deprivation. Previous reports have described several AMPK substrates involved in autophagy regulation; however, there are still undiscovered AMPK downstream effectors that could play an important role in autophagy. In a new article, Dohmen et al. discovered that the CCNY-CDK16 complex is a novel AMPK substrate involved in autophagy activation.
    Keywords:  AICAR; AMPK; CDK16; cyclin Y; phosphorylation
  20. Front Cell Dev Biol. 2020 ;8 270
      Mitochondria are essential organelles important for energy production, proliferation, and cell death. Biogenesis, homeostasis, and degradation of this organelle are tightly controlled to match cellular needs and counteract chronic stress conditions. Despite providing their own DNA, the vast majority of mitochondrial proteins are encoded in the nucleus, synthesized by cytosolic ribosomes, and subsequently imported into different mitochondrial compartments. The integrity of the mitochondrial proteome is permanently challenged by defects in folding, transport, and turnover of mitochondrial proteins. Therefore, damaged proteins are constantly sequestered from the outer mitochondrial membrane and targeted for proteasomal degradation in the cytosol via mitochondrial-associated degradation (MAD). Recent studies identified specialized quality control mechanisms important to decrease mislocalized proteins, which affect the mitochondrial import machinery. Interestingly, central factors of these ubiquitin-dependent pathways are shared with the ER-associated degradation (ERAD) machinery, indicating close collaboration between both tubular organelles. Here, we summarize recently described cellular stress response mechanisms, which are triggered by defects in mitochondrial protein import and quality control. Moreover, we discuss how ubiquitin-dependent degradation is integrated with cytosolic stress responses, particularly focused on the crosstalk between MAD and ERAD.
    Keywords:  C. elegans; Cdc48; Msp1; mitochondria; mitochondria-associated degradation (MAD); p97; proteostasis; ubiquitin
  21. J Cell Sci. 2020 May 11. pii: jcs242040. [Epub ahead of print]133(9):
      Target of rapamycin (TOR) is a serine/threonine protein kinase conserved in most eukaryote organisms. TOR assembles into two multiprotein complexes (TORC1 and TORC2), which function as regulators of cellular growth and homeostasis by serving as direct transducers of extracellular biotic and abiotic signals, and, through their participation in intrinsic feedback loops, respectively. TORC1, the better-studied complex, is mainly involved in cell volume homeostasis through regulating accumulation of proteins and other macromolecules, while the functions of the lesser-studied TORC2 are only now starting to emerge. In this Cell Science at a Glance article and accompanying poster, we aim to highlight recent advances in our understanding of TORC2 signalling, particularly those derived from studies in yeast wherein TORC2 has emerged as a major regulator of cell surface homeostasis.
    Keywords:  Membrane tension homeostasis; Protein kinase; Signalling; TORC2
  22. Microb Cell. 2020 May 04. 7(5): 119-128
      Autophagy is a catabolic pathway with multifaceted roles in cellular homeostasis. This process is also involved in the antiviral response at multiple levels, including the direct elimination of intruding viruses (virophagy), the presentation of viral antigens, the fitness of immune cells, and the inhibition of excessive inflammatory reactions. In line with its central role in immunity, viruses have evolved mechanisms to interfere with or to evade the autophagic process, and in some cases, even to harness autophagy or constituents of the autophagic machinery for their replication. Given the devastating consequences of the current COVID-19 pandemic, the question arises whether manipulating autophagy might be an expedient approach to fight the novel coronavirus SARS-CoV-2. In this piece, we provide a short overview of the evidence linking autophagy to coronaviruses and discuss whether such links may provide actionable targets for therapeutic interventions.
    Keywords:  COVID-19; MERS; SARS; SARS-CoV-2; coronavirus; immunity; inflammation; virophagy
  23. iScience. 2020 Apr 27. pii: S2589-0042(20)30290-X. [Epub ahead of print]23(5): 101105
      Excessive levels of saturated fatty acids are toxic to vascular smooth muscle cells (VSMCs). We previously reported that mice lacking VSMC-stearoyl-CoA desaturase (SCD), a major enzyme catalyzing the detoxification of saturated fatty acids, develop severe vascular calcification from the massive accumulation of lipid metabolites containing saturated fatty acids. However, the mechanism by which SCD deficiency causes vascular calcification is not completely understood. Here, we demonstrate that saturated fatty acids significantly inhibit autophagic flux in VSMCs, contributing to vascular calcification and apoptosis. Mechanistically, saturated fatty acids are accumulated as saturated lysophosphatidic acids (LPAs) (i.e. 1-stearoyl-LPA) possibly synthesized through the reaction of GPAT4 at the contact site between omegasomes and the MAM. The accumulation of saturated LPAs at the contact site causes abnormal formation of omegasomes, resulting in accumulation of autophagosomal precursor isolation membranes, leading to inhibition of autophagic flux. Thus, saturated LPAs are major metabolites mediating autophagy inhibition and vascular calcification.
    Keywords:  Cell Biology; Lipidomics; Molecular Biology
  24. J Clin Med. 2020 May 12. pii: E1440. [Epub ahead of print]9(5):
      Mitochondria are intracellular organelles involved in a myriad of activities. To safeguard their vital functions, mitochondrial quality control (MQC) systems are in place to support organelle plasticity as well as physical and functional connections with other cellular compartments. In particular, mitochondrial interactions with the endosomal compartment support the shuttle of ions and metabolites across organelles, while those with lysosomes ensure the recycling of obsolete materials. The extrusion of mitochondrial components via the generation and release of mitochondrial-derived vesicles (MDVs) has recently been described. MDV trafficking is now included among MQC pathways, possibly operating via mitochondrial-lysosomal contacts. Since mitochondrial dysfunction is acknowledged as a hallmark of aging and a major pathogenic factor of multiple age-associated conditions, the analysis of MDVs and, more generally, of extracellular vesicles (EVs) is recognized as a valuable research tool. The dissection of EV trafficking may help unravel new pathophysiological pathways of aging and diseases as well as novel biomarkers to be used in research and clinical settings. Here, we discuss (1) MQC pathways with a focus on mitophagy and MDV generation; (2) changes of MQC pathways during aging and their contribution to inflamm-aging and progeroid conditions; and (3) the relevance of MQC failure to several disorders, including neurodegenerative conditions (i.e., Parkinson's disease, Alzheimer's disease) and cardiovascular disease.
    Keywords:  biomarkers; exosomes; extracellular vesicles; geroprotective interventions; mitochondrial damage; mitochondrial dynamics; mitochondrial-derived vesicles (MDVs); mitochondrial-lysosomal axis; mitophagy; neurodegeneration
  25. Biochim Biophys Acta Mol Cell Biol Lipids. 2020 May 10. pii: S1388-1981(20)30129-3. [Epub ahead of print] 158737
      Aberrant fatty acid (FA) metabolism is a hallmark of proliferating cells, including untransformed fibroblasts or cancer cells. Lipolysis of intracellular triglyceride (TG) stores by adipose triglyceride lipase (ATGL) provides an important source of FAs serving as energy substrates, signaling molecules, and precursors for membrane lipids. To investigate if ATGL-mediated lipolysis impacts cell proliferation, we modified ATGL activity in murine embryonic fibroblasts (MEFs) and in five different cancer cell lines to determine the consequences on cell growth and metabolism. Genetic or pharmacological inhibition of ATGL in MEFs causes impaired FA oxidation, decreased ROS production, and a substrate switch from FA to glucose leading to decreased AMPK-mTOR signaling and higher cell proliferation rates. ATGL expression in these cancer cells is low when compared to MEFs. Additional ATGL knockdown in cancer cells did not significantly affect cellular lipid metabolism or cell proliferation whereas the ectopic overexpression of ATGL increased lipolysis and reduced proliferation. In contrast to ATGL silencing, pharmacological inhibition of ATGL by Atglistatin© impeded the proliferation of diverse cancer cell lines, which points at an ATGL-independent effect. Our data indicate a crucial role of ATGL-mediated lipolysis in the regulation of cell proliferation. The observed low ATGL activity in cancer cells may represent an evolutionary selection process and mechanism to sustain high cell proliferation rates. As the increasing ATGL activity decelerates proliferation of five different cancer cell lines this may represent a novel therapeutic strategy to counteract uncontrolled cell growth.
    Keywords:  ATGL; Cancer; Lipid; Lipolysis; Proliferation
  26. Curr Opin Cell Biol. 2020 May 11. pii: S0955-0674(20)30048-X. [Epub ahead of print]65 96-102
      Misfolded and mistargeted proteins in the early secretory pathway present significant risks to the cell. A diverse and integrated network of quality control pathways protects the cell from these threats. We focus on the discovery of new mechanisms that contribute to this protective network. Biochemical and structural advances in endoplasmic reticulum targeting fidelity, and in the redistribution of mistargeted substrates are discussed. We further review new discoveries in quality control at the inner nuclear membrane in the context of orphaned subunits. We consider developments in our understanding of cargo selection for endoplasmic reticulum export. Conflicting data on quality control by cargo receptor proteins are discussed and we look to important future questions for the field.
    Keywords:  Endoplasmic reticulum; Protein quality control
  27. Glia. 2020 May 16.
      Mitophagy is essential for the health of dopaminergic neurons because mitochondrial damage is a keystone of Parkinson's disease. The aim of the present work was to study the degradation of mitochondria in the degenerating dopaminergic synapse. Adult Sprague-Dawley rats and YFP-Mito-DAn mice with fluorescent mitochondria in dopaminergic neurons were injected in the lateral ventricles with 6-hydroxydopamine, a toxic that inhibits the mitochondrial chain of dopaminergic neurons and blockades the axonal transport. Dopaminergic terminals closest to the lateral ventricle showed an axonal fragmentation and an accumulation of damaged mitochondria in 2-9 μ saccular structures (spheroids). Damaged mitochondria accumulated in spheroids initiated (showing high Pink1, parkin, ubiquitin, p-S65-Ubi, AMBRA1, and BCL2L13 immunoreactivity and developing autophagosomes) but did not complete (mitochondria were not polyubiquitinated, autophagosomes had no STX17, and no lysosomes were found in spheroids) the mitophagy process. Then, spheroids were penetrated by astrocytic processes and DAergic mitochondria were transferred to astrocytes where they were polyubiquitinated (UbiK63+) and linked to mature autophagosomes (STX17+) which became autophagolysosomes (Lamp1/Lamp2 which co-localized with LC3). Present data provide evidence that the mitophagy of degenerating dopaminergic terminals starts in the dopaminergic spheroids and finishes in the surrounding astrocytes (spheroid-mediated transmitophagy). The neuron-astrocyte transmitophagy could be critical for preventing the release of damaged mitochondria to the extracellular medium and the neuro-inflammatory activity which characterizes Parkinson's disease.
    Keywords:  Parkinson's disease; astrocyte; dopaminergic neurons; mitochondria; transmitophagy
  28. Cell Metab. 2020 May 05. pii: S1550-4131(20)30197-2. [Epub ahead of print]
      Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4+ T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes.
    Keywords:  T cells; autophagy; inflammaging; metformin; mitochondria