bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2022‒04‒03
33 papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2022 Mar 29. 1-3
      Neurons depend on macroautophagy/autophagy to maintain cellular homeostasis, and loss of autophagy leads to neurodegeneration. To better understand the role of basal autophagy in neurons, we enriched autophagic vesicles from healthy adult mouse brain and performed mass spectrometry to identify cargos cleared by autophagy. We found that synaptic and mitochondrial proteins comprise nearly half of the unique AV cargos identified in brain. Similarly, synaptic and mitochondrial proteins are major cargos for basal autophagy in neurons. Strikingly, we noted a specific enrichment of mitochondrial nucleoids within neuronal autophagosomes, which occurs through a mechanism distinct from damage-associated mitophagy. Here, we discuss the implications of these findings for our understanding of homeostatic mechanisms in neurons and how the age-dependent decline of autophagy in neurons may contribute to the onset or progression of neurodegenerative disease.
    Keywords:  DNM1L; SYN1; TFAM; macroautophagy; mitochondria; mitochondrial division; mitochondrial nucleoids; mitophagy; neurodegeneration; neuronal homeostasis
  2. Nat Commun. 2022 Apr 01. 13(1): 1760
      The evolutionarily conserved serine/threonine kinase mTORC1 is a central regulator of cell growth and proliferation. mTORC1 is activated on the lysosome surface. However, once mTORC1 is activated, it is unclear whether mTORC1 phosphorylates local lysosomal proteins to regulate specific aspects of lysosomal biology. Through cross-reference analyses of the lysosome proteome with the mTORC1-regulated phosphoproteome, we identify STK11IP as a lysosome-specific substrate of mTORC1. mTORC1 phosphorylates STK11IP at Ser404. Knockout of STK11IP leads to a robust increase of autophagy flux. Dephosphorylation of STK11IP at Ser404 represses the role of STK11IP as an autophagy inhibitor. Mechanistically, STK11IP binds to V-ATPase, and regulates the activity of V-ATPase. Knockout of STK11IP protects mice from fasting or Methionine/Choline-Deficient Diet (MCD)-induced fatty liver. Thus, our study demonstrates that STK11IP phosphorylation represents a mechanism for mTORC1 to regulate lysosomal acidification and autophagy, and points to STK11IP as a promising therapeutic target for the amelioration of diseases with aberrant autophagy signaling.
  3. PLoS Biol. 2022 Mar 31. 20(3): e3001594
      Mechanistic target of rapamycin complex I (mTORC1) is central to cellular metabolic regulation. mTORC1 phosphorylates a myriad of substrates, but how different substrate specificity is conferred on mTORC1 by different conditions remains poorly defined. Here, we show how loss of the mTORC1 regulator folliculin (FLCN) renders mTORC1 specifically incompetent to phosphorylate TFE3, a master regulator of lysosome biogenesis, without affecting phosphorylation of other canonical mTORC1 substrates, such as S6 kinase. FLCN is a GTPase-activating protein (GAP) for RagC, a component of the mTORC1 amino acid (AA) sensing pathway, and we show that active RagC is necessary and sufficient to recruit TFE3 onto the lysosomal surface, allowing subsequent phosphorylation of TFE3 by mTORC1. Active mutants of RagC, but not of RagA, rescue both phosphorylation and lysosomal recruitment of TFE3 in the absence of FLCN. These data thus advance the paradigm that mTORC1 substrate specificity is in part conferred by direct recruitment of substrates to the subcellular compartments where mTORC1 resides and identify potential targets for specific modulation of specific branches of the mTOR pathway.
  4. Mol Cell. 2022 Mar 29. pii: S1097-2765(22)00221-0. [Epub ahead of print]
      Selective autophagy specifically eliminates damaged or superfluous organelles, maintaining cellular health. In this process, a double membrane structure termed an autophagosome captures target organelles or proteins and delivers this cargo to the lysosome for degradation. The attachment of the small protein ubiquitin to cargo has emerged as a common mechanism for initiating organelle or protein capture by the autophagy machinery. In this process, a suite of ubiquitin-binding cargo receptors function to initiate autophagosome assembly in situ on the target cargo, thereby providing selectivity in cargo capture. Here, we review recent efforts to understand the biochemical mechanisms and principles by which cargo are marked with ubiquitin and how ubiquitin-binding cargo receptors use conserved structural modules to recruit the autophagosome initiation machinery, with a particular focus on mitochondria and intracellular bacteria as cargo. These emerging mechanisms provide answers to long-standing questions in the field concerning how selectivity in cargo degradation is achieved.
    Keywords:  cargo receptor; mitophagy; selective autophagy; ubiquitin; xenophagy
  5. Autophagy. 2022 Mar 29. 1-2
      Macroautophagy/autophagy occurs preferentially at synapses and responds to increased neuronal activity states. How synaptic autophagy is coupled to the neuronal activity state is largely unknown. Through genetic approaches we find that ATG-9, the only transmembrane protein in the core autophagy pathway, is transported from the trans-Golgi network to synapses in C. elegans via the AP-3 complex. At synapses ATG-9 undergoes exo-endocytosis in an activity-dependent manner. Mutations that disrupt the endocytosis pathway, including a mutation associated with early onset Parkinsonism (EOP), lead to abnormal ATG-9 accumulation into subsynaptic clathrin-rich foci, and defects in activity-induced synaptic autophagy. We propose that ATG-9 exo-endocytosis links the activity-dependent synaptic vesicle cycle with autophagosome formation at synapses.
    Keywords:  AP-3; ATG-9; Golgi apparatus; Parkinson disease; autophagy; clathrin; endocytosis; neuronal activity state; synaptic vesicle cycle; synaptojanin 1/UNC-26
  6. Life Sci Alliance. 2022 Jul;pii: e202101239. [Epub ahead of print]5(7):
      Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Here, we show that mammalian ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. The altered lysosome morphology upon ESCRT-I depletion coincided with elevated expression of genes annotated to biogenesis of lysosomes due to prolonged activation of TFEB/TFE3 transcription factors. Lack of ESCRT-I also induced transcription of cholesterol biosynthesis genes, in response to inefficient delivery of cholesterol from endolysosomal compartments. Among factors that could possibly activate TFEB/TFE3 signaling upon ESCRT-I deficiency, we excluded lysosomal cholesterol accumulation and Ca2+-mediated dephosphorylation of TFEB/TFE3. However, we discovered that this activation occurs due to the inhibition of Rag GTPase-dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at S112. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S112 phosphorylation and prevented TFEB/TFE3 activation. Our results indicate that ESCRT-I deficiency evokes a homeostatic response to counteract lysosomal nutrient starvation, that is, improper supply of nutrients derived from lysosomal degradation.
  7. Autophagy. 2022 Apr 01.
      Macroautophagy/autophagy, a highly conserved catabolic pathway that maintains proper cellular homeostasis is stringently regulated by numerous autophagy-related (Atg) proteins. Many studies have investigated autophagy regulation at the transcriptional level; however, relatively little is known about translational control. Here, we report the upstream open reading frames (uORFs)-mediated translational control of multiple Atg proteins in Saccharomyces cerevisiae and in human cells. The translation of several essential autophagy regulators in yeast, including Atg13, is suppressed by canonical uORFs under nutrient-rich conditions, and is activated during nitrogen-starvation conditions. We also found that the predicted human ATG4B and ATG12 non-canonical uORFs suppress downstream coding sequence translation. These results demonstrate that uORF-mediated translational control is a widely used mechanism among ATG genes from yeast to human and suggest a model for how some ATG genes bypass the general translational suppression that occurs under stress conditions to maintain a proper level of autophagy.
    Keywords:  Autophagy; human; lysosome; stress; translational regulation; vacuole; yeast
  8. Mol Cell. 2022 Mar 21. pii: S1097-2765(22)00211-8. [Epub ahead of print]
      mTORC1 controls cellular metabolic processes in response to nutrient availability. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which are localized on the lysosomal surface by the Ragulator complex. The Rag GTPases receive amino acid signals from multiple upstream regulators. One negative regulator, GATOR1, is a GTPase activating protein (GAP) for RagA. GATOR1 binds to the Rag GTPases via two modes: an inhibitory mode and a GAP mode. How these two binding interactions coordinate to process amino acid signals is unknown. Here, we resolved three cryo-EM structural models of the GATOR1-Rag-Ragulator complex, with the Rag-Ragulator subcomplex occupying the inhibitory site, the GAP site, and both binding sites simultaneously. When the Rag GTPases bind to GATOR1 at the GAP site, both Rag subunits contact GATOR1 to coordinate their nucleotide loading states. These results reveal a potential GAP mechanism of GATOR1 during the mTORC1 inactivation process.
    Keywords:  GAP; GATOR1; Rag GTPase; enzyme mechanism; mTOR complex 1; mTORC1; nutrient sensing
  9. J Cell Sci. 2022 Mar 28. pii: jcs.259421. [Epub ahead of print]
      Lysosomes mediate degradation of macromolecules to their precursors for their cellular recycling. Additionally, lysosome-related organelles mediate cell type-specific functions. The Chédiak-Higashi syndrome is an autosomal, recessive disease, in which loss of the protein LYST causes defects in lysosomes and lysosome-related organelles. The molecular function of LYST, however, is largely unknown. Here, we dissected the function of the yeast LYST homolog, Bph1. We show that Bph1 is an endosomal protein, and an effector of the minor Rab5 isoform Ypt52. Strikingly, the bph1▵ mutant has lipidated Atg8 on their endosomes, which is sorted via late endosomes into the vacuole lumen under non-autophagy inducing conditions. In agreement, proteomics of bph1▵ vacuoles reveal an accumulation of Atg8, reduced flux via selective autophagy, and defective endocytosis. Additionally, bph1▵ cells have reduced autophagic flux under starvation conditions. Our observations suggest that Bph1 is a novel Rab5 effector that maintains endosomal functioning. When lost, Atg8 is lipidated at endosomes even during normal growth and ends up in the vacuole lumen. Thus, our results contribute to the understanding of the role of LYST-related proteins and associated diseases.
    Keywords:  Atg8; Autophagy; Endosome; LYST; Rab5; Ypt52
  10. Biochim Biophys Acta Mol Basis Dis. 2022 Mar 25. pii: S0925-4439(22)00070-9. [Epub ahead of print]1868(7): 166400
      Autophagy is an intracellular self-degradative mechanism which responds to cellular conditions like stress or starvation and plays a key role in regulating cell metabolism, energy homeostasis, starvation adaptation, development and cell death. Numerous studies have stipulated the participation of autophagy in cancer, but the role of autophagy either as tumor suppressor or tumor promoter is not clearly understood. However, mechanisms by which autophagy promotes cancer involves a diverse range of modifications of autophagy associated proteins such as ATGs, Beclin-1, mTOR, p53, KRAS etc. and autophagy pathways like mTOR, PI3K, MAPK, EGFR, HIF and NFκB. Furthermore, several researches have highlighted a context-dependent, cell type and stage-dependent regulation of autophagy in cancer. Alongside this, the interaction between tumor cells and their microenvironment including hypoxia has a great potential in modulating autophagy response in favour to substantiate cancer cell metabolism, self-proliferation and metastasis. In this review article, we highlight the mechanism of autophagy and their contribution to cancer cell proliferation and development. In addition, we discuss about tumor microenvironment interaction and their consequence on selective autophagy pathways and the involvement of autophagy in various tumor types and their therapeutic interventions concentrated on exploiting autophagy as a potential target to improve cancer therapy.
    Keywords:  Autophagy; Cancer; Homeostasis; Hypoxia
  11. Aging Cell. 2022 Mar 29. e13603
      Protein quality control ensures the degradation of damaged and misfolded proteins. Derangement of proteostasis is a primary cause of aging and age-associated diseases. The ubiquitin-proteasome and autophagy-lysosome play key roles in proteostasis but, in addition to these systems, the human genome encodes for ~600 proteases, also known as peptidases. Here, we examine the role of proteases in aging and age-related neurodegeneration. Proteases are present across cell compartments, including the extracellular space, and their substrates encompass cellular constituents, proteins with signaling functions, and misfolded proteins. Proteolytic processing by proteases can lead to changes in the activity and localization of substrates or to their degradation. Proteases cooperate with the autophagy-lysosome and ubiquitin-proteasome systems but also have independent proteolytic roles that impact all hallmarks of cellular aging. Specifically, proteases regulate mitochondrial function, DNA damage repair, cellular senescence, nutrient sensing, stem cell properties and regeneration, protein quality control and stress responses, and intercellular signaling. The capacity of proteases to regulate cellular functions translates into important roles in preserving tissue homeostasis during aging. Consequently, proteases influence the onset and progression of age-related pathologies and are important determinants of health span. Specifically, we examine how certain proteases promote the progression of Alzheimer's, Huntington's, and/or Parkinson's disease whereas other proteases protect from neurodegeneration. Mechanistically, cleavage by proteases can lead to the degradation of a pathogenic protein and hence impede disease pathogenesis. Alternatively, proteases can generate substrate byproducts with increased toxicity, which promote disease progression. Altogether, these studies indicate the importance of proteases in aging and age-related neurodegeneration.
    Keywords:  aging; extracellular proteostasis; neurodegeneration; peptidase; protease; proteolysis
  12. Biol Reprod. 2022 Mar 25. pii: ioac060. [Epub ahead of print]
      Autophagy, an intracellular recycling system, is essential for the meiotic maturation of porcine oocytes. Multiple studies, sought to reveal the precise mechanism employed, commonly used autophagy inducers, such as rapamycin, which is a mammalian target of rapamycin (mTOR) inhibitor. However, it has a limitation as mTOR plays various roles in cell growth and metabolism beyond autophagy. Trehalose has been reported as a novel mTOR-independent autophagy inducer in many cells. Furthermore, our previous study demonstrated that trehalose supplementation during in vitro maturation of porcine oocytes improves the developmental competence of parthenogenetic embryos possibly via autophagic activation, whereas the underlying mechanisms remain unclear. Therefore, the aim of this study was to address this issue. In this study, we found that trehalose plays a role as an autophagy activator by autophagic flux assay and determined that it promotes PI3K/Akt inhibition and VPS34/mTOR activation by immunoblotting, both in cumulus cells (CCs) and oocytes. However, it is interesting to note that these effects caused by trehalose were worked totally varying between CCs and oocytes. In CCs, the autophagy was activated through the improvement of lysosomal function/autophagic clearance viability by upregulation of coordinated lysosomal expression and regulation genes via PI3K/Akt inhibition. Whereas in oocytes, autophagy was activated via VPS34 induction which directly influences autophagosome formation, and the precise meiotic process was ensured via Akt inhibition and mTOR activation. Taken together, this study provided evidence that trehalose could be used as an autophagy inductor during porcine oocyte maturation based on the revealed mechanism.
    Keywords:  Autophagy; Oocyte maturation; PI3K/Akt; Trehalose; VPS34/mTOR
  13. Redox Biol. 2022 Mar 24. pii: S2213-2317(22)00056-8. [Epub ahead of print]52 102284
      Autophagy is an evolutionarily conserved self-protecting mechanism implicated in cellular homeostasis. ATG4B plays a vital role in autophagy process via undertaking priming and delipidation of LC3. Chemical inhibitors and regulative modifications such as oxidation of ATG4B have been demonstrated to modulate autophagy function. Whether and how ATG4B could be regulated by metal ions is largely unknown. Copper is an essential trace metal served as static co-factors in redox reactions in physiology process. Excessive accumulation of copper in ATP7B mutant cells leads to pathology progression such as insoluble Mallory body (MB) in Wilson disease (WD). The clearance of MB via autophagy pathway was thought as a promising strategy for WD. Here, we discovered that copper ion instead of other ions could inhibit the activity of ATG4B followed by autophagy suppression. In addition, copper could induce ATG4B oligomers depending on cysteine oxidation which could be abolished in reduced condition. Copper also promotes the formation of insoluble ATG4B aggregates, as well as p62-and ubiquitin-positive aggregates, which is consistent with the components of MB caused by copper overload in WD cell model. Importantly, overexpression of ATG4B could partially reduce the formation of MB and rescue impaired autophagy. Taken together, our results uncovered for the first time a new damage mechanism mediated by copper and implied new insights of the crosstalk between the toxicity of copper and autophagy in the pathogenesis of WD.
    Keywords:  ATG4B; Aggregates; Autophagy; Copper ion; Mallory body
  14. Autophagy. 2022 Mar 28. 1-18
      FYCO1 (FYVE and coiled-coil domain containing 1) is an adaptor protein, expressed ubiquitously and required for microtubule-dependent, plus-end-directed transport of macroautophagic/autophagic vesicles. We have previously shown that loss-of-function mutations in FYCO1 cause cataracts with no other ocular and/or extra-ocular phenotype. Here, we show fyco1 homozygous knockout (fyco1-/-) mice recapitulate the cataract phenotype consistent with a critical role of FYCO1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many autophagy-associated genes, proteins, and lipids respectively perturbed in fyco1-/- mice lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial cells revealed a decrease in autophagic flux and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed cellular organelles accumulated in FYCO1 (c.2206C>T) KI lens-like organoid structures and in fyco1-/- mice lenses. In summary, our data confirm the loss of FYCO1 function results in a diminished autophagic flux, impaired organelle removal, and cataractogenesis.Abbreviations: CC: congenital cataracts; DE: differentially expressed; ER: endoplasmic reticulum; FYCO1: FYVE and coiled-coil domain containing 1; hESC: human embryonic stem cell; KI: knock-in; OFZ: organelle-free zone; qRT-PCR: quantitative real-time PCR; PE: phosphatidylethanolamine; RNA-Seq: RNA sequencing; SD: standard deviation; sgRNA: single guide RNA; shRNA: shorthairpin RNA; TEM: transmission electron microscopy; WT: wild type.
    Keywords:  Autophagy; cataracts; lens fiber cells; organelle removal; organelle-free zone
  15. Cell Commun Signal. 2022 Mar 31. 20(1): 43
      As an important mechanism to maintain cellular homeostasis, autophagy exerts critical functions via degrading misfolded proteins and damaged organelles. Recent years, alternative autophagy, a new type of autophagy has been revealed, which shares similar morphology with canonical autophagy but is independent of Atg5/Atg7. Investigations on different diseases showed the pivotal role of alternative autophagy during their physio-pathological processes, including heart diseases, neurodegenerative diseases, oncogenesis, inflammatory bowel disease (IBD), and bacterial infection. However, the studies are limited and the precise roles and mechanisms of alternative autophagy are far from clear. It is necessary to review current research on alternative autophagy and get some hint in order to provide new insight for further study. Video Abstract.
    Keywords:  Alternative autophagy; Canonical autophagy; Diseases; Mechanism; Non-canonical autophagy
  16. Ultrastruct Pathol. 2022 Mar 29. 1-8
      Acute alcohol feeding can activate autophagy and promotes the selection of autophagic vacuoles in the mitochondria, which is a key process regulating the occurrence and progression of alcohol steatohepatitis (ASH). In this study, ASH mice expressed more autophagy-associated proteins than healthy controls, as revealed by immunohistochemistry. In addition, transmission electron microscopy (TEM) detected a unique autophagy ultrastructure in ASH mouse liver cells, consisting of a large vesicle fused directly with mitochondria, which differed from the classical pattern. This novel type of mitophagy may provide a new avenue for a protective mechanism targeting mitophagy, which would benefit patients with ASH.Abbreviations: ASH: alcoholic steatohepatitis; ALD: Alcoholic liver disease; ALT: alanine aminotransferase; AST: aspartate aminotransferase; HE: hematoxylin and eosin; TEM: transmission electron microscope; LC3: microtubule-associated protein 1 light chain 3; SQSTM1/p62: sequestosome 1; UQCRC2: ubiquinol-cytochrome c reductase core protein 2; PINK1: PTEN induced kinase 1; AMPK: AMP-activated protein kinase.
    Keywords:  Alcoholic steatohepatitis; mitophagy; selective autophagy; transmission electron microscope
  17. J Integr Neurosci. 2022 Mar 18. 21(2): 46
      Alzheimer's disease (AD) is a neurodegeneration csharacterized by amyloid-β (Aβ) deposition and abnormally phosphorylated Tau protein aggregation. Autophagy, as an important cellular metabolic activity, is closely related to the production, secretion and clearance of Aβ peptide and Tau phosphorylation level. Therefore, autophagy may become a potential target for AD treatment. A large number of molecules are involved in the mammalian target of rapamycin (mTOR)-dependent or mTOR-independent pathway of autophagy. More and more evidences show that statins can intervene autophagy by regulating the activity or expression level of autophagy-related proteins and genes. On the one hand, statins can induce autophagy through Sirtuin1 (SIRT1), P21, nuclear P53 and adenylate activated protein kinase (AMPK). On the other hand, statins inhibit the mevalonate metabolism pathway, thereby interfering with the prenylation of small GTPases, leading to autophagy dysfunction. Statins can also reduce the levels of LAMP2 and dynein, destroying autophagy. In this review, we focused on the role of autophagy in AD and the autophagy mechanism of statins in the potential treatment of AD.
    Keywords:  Alzheimer's disease; Amyloid-β; Autophagy; Autophagy flux; Mevalonate pathway; Statin; Tau protein
  18. Dev Comp Immunol. 2022 Mar 23. pii: S0145-305X(22)00063-5. [Epub ahead of print]132 104401
      MyD88 is a typical street protein of the TLRs signaling pathway and is a central player in innate immune signaling, which can regulate the NF-κB signaling pathway and promote downstream inflammatory factors. However, studies on the molecular mechanisms of the MyD88-mediated NF-κB signaling pathway in teleosts have been poorly reported. In this study, we report that Zw10 targets MyD88 to inhibit NF-κB activation. Zw10 inhibits cell proliferation and MyD88-mediated innate immunity in fish. Zw10 interacts with MyD88, and its Δ2 domain is very critical for MyD88 degradation. In addition, we found that Zw10 degrade MyD88 by autophagy, thereby negatively regulating the MyD88-mediated NF-κB signaling pathway. This study not only enriches the research on the innate immunity of teleost fish, but also provides insights for the regulating mechanism for mammals.
    Keywords:  Autophagy; MyD88; NF-κB; Teleost fish; Zw10
  19. Front Oncol. 2022 ;12 852859
      The tuberous sclerosis complex (TSC) is a rare genetic syndrome and multisystem disease resulting in tumor formation in major organs. A molecular hallmark of TSC is a dysregulation of the mammalian target of rapamycin (mTOR) through loss-of-function mutations in either tumor suppressor TSC1 or TSC2. Here, we sought to identify drug vulnerabilities conferred by TSC2 tumor-suppressor loss through cell-based chemical biology screening. Our small-molecule chemical screens reveal a sensitivity to inhibitors of checkpoint kinase 1/2 (CHK1/2), regulators of cell cycle, and DNA damage response, in both in vitro and in vivo models of TSC2-deficient renal angiomyolipoma (RA) tumors. Further, we performed transcriptional profiling on TSC2-deficient RA cell models and discovered that these recapitulate some of the features from TSC patient kidney tumors compared to normal kidneys. Taken together, our study provides a connection between mTOR-dependent tumor growth and CHK1/2, highlighting the importance of CHK1/2 inhibition as a potential antitumor strategy in TSC2-deficient tumors.
    Keywords:  AZD7762; CHEK1/2; Chk1/2; TSC2; checkpoint kinase inhibitors; mTOR; tuberous sclerosis complex; tumor xenografts
  20. Autophagy. 2022 Mar 28. 1-14
      The notion that macroautophagy/autophagy is a potentially attractive therapeutic target for a variety of diseases, including cancer, largely stems from pre-clinical mouse studies. Most of these examine the effects of irreversible and organ confined autophagy deletion using site specific Cre-loxP recombination of the essential autophagy regulating genes Atg7 or Atg5. Model systems with the ability to impair autophagy systemically and reversibly at all disease stages would allow a more realistic approach to evaluate the consequences of authophagy inhibition as a therapeutic concept and its potential side effects. Here, we present shRNA transgenic mice that via doxycycline (DOX) regulable expression of a highly efficient miR30-E-based shRNA enabled knockdown of Atg7 simultaneously in the majority of organs, with the brain and spleen being noteable exceptions. Induced animals deteriorated rapidly and experienced profound destruction of the exocrine pancreas, severe hypoglycemia and depletion of hepatic glycogen storages. Cessation of DOX application restored apparent health, glucose homeostasis and pancreatic integrity. In a similar Atg5 knockdown model we neither observed loss of pancreatic integrity nor diminished survival after DOX treatment, but identified histological changes consistent with steatohepatitis and hepatic fibrosis in the recovery period after termination of DOX. Regulable Atg7-shRNA mice are valuable tools that will enable further studies on the role of autophagy impairment at various disease stages and thereby help to evaluate the consequences of acute autophagy inhibition as a therapeutic concept.
    Keywords:  Atg5; Atg7; autophagy; liver; pancreas; shRNA transgenic mice
  21. Mol Omics. 2022 Mar 28.
      The major function of the lysosome is to degrade unwanted materials such as lipids, proteins, and nucleic acids; therefore, deficits of the lysosomal system can result in improper degradation and trafficking of these biomolecules. Diseases associated with lysosomal failure can be lethal and are termed lysosomal storage disorders (LSDs), which affect 1 in 5000 live births collectively. LSDs are inherited metabolic diseases caused by mutations in single lysosomal and non-lysosomal proteins and resulting in the subsequent accumulation of macromolecules within. Most LSD patients present with neurodegenerative clinical symptoms, as well as damage in other organs. The discovery of new biomarkers is necessary to understand and monitor these diseases and to track therapeutic progress. Over the past ten years, mass spectrometry (MS)-based proteomics has flourished in the biomarker studies in many diseases, including neurodegenerative, and more specifically, LSDs. In this review, biomarkers of disease pathophysiology and monitoring of LSDs revealed by MS-based proteomics are discussed, including examples from Niemann-Pick disease type C, Fabry disease, neuronal ceroid-lipofuscinoses, mucopolysaccharidosis, Krabbe disease, mucolipidosis, and Gaucher disease.
  22. Proc Natl Acad Sci U S A. 2022 Apr 05. 119(14): e2121133119
      Significance Cardiovascular diseases remain the leading cause of death worldwide, with atherosclerosis being the most common source of clinical events. Metabolic changes with aging associate with concurrent increased risk of both type 2 diabetes and cardiovascular disease, with the former further raising the risk of the latter. The activity of a selective type of autophagy, chaperone-mediated autophagy (CMA), decreases with age or upon dietary excesses. Here we study whether reduced CMA activity increases risk of atherosclerosis in mouse models. We have identified that CMA is up-regulated early in response to proatherogenic challenges and demonstrate that reduced systemic CMA aggravates vascular pathology in these conditions. We also provide proof-of-concept support that CMA up-regulation is an effective intervention to reduce atherosclerosis severity and progression.
    Keywords:  atherosclerotic plaques; lipid challenge; lysosomes; proteolysis; vascular disease
  23. EMBO Rep. 2022 Mar 28. e54453
      The NLRP3-directed inflammasome complex is crucial for the host to resist microbial infection and monitor cellular damage. However, the hyperactivation of NLRP3 inflammasome is implicated in pathogenesis of inflammatory diseases, including inflammatory bowel disease (IBD). Autophagy and autophagy-related genes are closely linked to NLRP3-mediated inflammation in these inflammatory disorders. Here, we report that CCDC50, a novel autophagy cargo receptor, negatively regulates NLRP3 inflammasome assembly and suppresses the cleavage of pro-caspase-1 and interleukin 1β (IL-1β) release by delivering NLRP3 for autophagic degradation. Transcriptome analysis showed that knockdown of CCDC50 results in upregulation of signaling pathways associated with autoinflammatory diseases. CCDC50 deficiency leads to enhanced proinflammatory cytokine response triggered by a wide range of endogenous and exogenous NLRP3 stimuli. Ccdc50-deficient mice are more susceptible to dextran sulfate (DSS)-induced colitis and exhibit more severe gut inflammation with elevated NLRP3 inflammasome activity. These results illustrate the physiological significance of CCDC50 in the pathogenicity of inflammatory diseases, suggesting protective roles of CCDC50 in keeping gut inflammation under control.
    Keywords:  CCDC50; IL-1β; NLRP3 inflammasome; autophagy receptor; inflammatory diseases
  24. Curr Biol. 2022 Mar 28. pii: S0960-9822(22)00265-2. [Epub ahead of print]32(6): R292-R294
      Endosomal sorting complex required for transport (ESCRT) proteins can promote extreme membrane deformations, including scission and sealing. New work uncovers a link between these proteins and the early secretory pathway that is functionally important for programmed autophagy during Drosophila development.
  25. J Nutr Biochem. 2022 Mar 25. pii: S0955-2863(22)00069-9. [Epub ahead of print] 108998
      Overly elevated circulating non-esterified fatty acids (NEFAs) is an emerging health concern of obesity-associated energy disorders. However, methods to reduce circulating NEFAs remain elusive. The present study determined the effect of piceatannol, a naturally occurring stilbene, on adipocyte lipolysis and its underlying mechanism. Differentiated 3T3-L1 adipocytes and brown adipocytes and isolated white adipose tissue were treated with various concentrations of piceatannol for 1.5-hr both in the basal and stimulated lipolysis conditions. Piceatannol significantly inhibited NEFAs and glycerol release with a concomitant reduction of ATGL, CGI-58 and PLIN1 expression in adipocytes. Using a series of inhibitor assays, piceatannol-induced degradation of these proteins was found to be mediated by upregulation of the autophagy-lysosome pathway. Moreover, we demonstrated that piceatannol is capable of stimulating autophagy in vitro. Importantly, piceatannol administration tended to lower fasting-induced serum glycerol levels in healthy mice. Furthermore, piceatannol administration lowered lipolysis, central adiposity and hyperinsulinemia in diet-induced obese mice. Our study provides profound evidence of a novel inhibitory role of piceatannol in lipolysis through autophagy-lysosome-dependent degradation of the key lipolytic proteins in adipocytes. This study offers a mechanistic foundation for investigating the potential of piceatannol-containing foods in reducing lipolysis and its associated metabolic disorders.
    Keywords:  ATGL; adipocytes; autophagy; degradation; lipolysis; piceatannol
  26. Int J Biol Macromol. 2022 Mar 28. pii: S0141-8130(22)00627-4. [Epub ahead of print]
      TANK-binding kinase 1 (TBK1) plays a fundamental role in regulating the cellular response to infecting pathogens and controlling several signaling cascades. It regulates inflammatory, interferon, NF-κB, autophagy, and Akt pathways. Post-translational modifications (PTM) of TBK1 strongly control its action and consequent cellular signaling. The dysregulation of the TBK1 pathway is correlated to many pathophysiological conditions, including cancer, that implicates the promising therapeutic advantage for targeting TBK1. The present study summarizes current information on the molecular mechanisms and cancer-inducing roles of TBK1. Designed inhibitors of TBK1 are considered a potential therapeutic agent for several diseases, including cancer. Data from pre-clinical tumor models recommend that the target of TBK1 can be a productive strategy and plan for anti-tumor therapy in particular settings. This review further highlighted the therapeutic potential of potent and selective TBK1 inhibitors, including Amlexanox, Compound II, BX795, MRT67307, SR8185 AZ13102909, CYT387, GSK8612, BAY985, and Domainex. These inhibitors may be implicated to facilitate therapeutic management of cancer and TBK1-associated diseases in the future.
    Keywords:  Anti-inflammatory; Cellular signaling; Kinase inhibitors; TANK-binding kinase 1; TBK1 inhibitors; Targeted therapy
  27. Mol Metab. 2022 Mar 25. pii: S2212-8778(22)00050-3. [Epub ahead of print] 101481
      Spatial compartmentalization of metabolic pathways within membrane-separated organelles is key to the ability of eukaryotic cells to precisely regulate their biochemical functions. Membrane-bound organelles such as mitochondria, endoplasmic reticulum (ER) and lysosomes enable the concentration of metabolic precursors within optimized chemical environments, greatly accelerating the efficiency of both anabolic and catabolic reactions, enabling division of labor and optimal utilization of resources. However, metabolic compartmentalization also poses a challenge to cells because it creates spatial discontinuities that must be bridged for reaction cascades to be connected and completed. To do so, cells employ different methods to coordinate metabolic fluxes occurring in different organelles, such as membrane-localized transporters to facilitate regulated metabolite exchange between mitochondria and lysosomes, non-vesicular transport pathways via physical contact sites connecting the ER with both mitochondria and lysosomes, as well as localized regulatory signaling processes that coordinately regulate the activity of all these organelles. Effective communication among these systems is essential to cellular health and function, whereas disruption of inter-organelle communication is an emerging driver in a multitude of diseases, from cancer to neurodegeneration.
    Keywords:  Contact sites; Lysosome; Metabolism; Mitochondria; Transporters; mTORC1
  28. Sci Adv. 2022 Apr;8(13): eabj1604
      Altered nucleolar and ribosomal dynamics are key hallmarks of aging, but their regulation remains unclear. Building on the knowledge that the conserved nuclear export receptor Exportin 1 (XPO-1/XPO1) modulates proteostasis and life span, we systematically analyzed the impact of nuclear export on protein metabolism. Using transcriptomic and subcellular proteomic analyses in nematodes, we demonstrate that XPO-1 modulates the nucleocytoplasmic distribution of key proteins involved in nucleolar dynamics and ribosome function, including fibrillarin (FIB-1/FBL) and RPL-11 (RPL11). Silencing xpo-1 led to marked reduction in global translation, which was accompanied by decreased nucleolar size and lower fibrillarin levels. A targeted screen of known proteostatic mediators revealed that the autophagy protein LGG-1/GABARAP modulates nucleolar size by regulating RPL-11 levels, linking specific protein degradation to ribosome metabolism. Together, our study reveals that nucleolar size and life span are regulated by LGG-1/GABARAP via ribosome protein surveillance.
  29. FASEB J. 2022 May;36(5): e22282
      Inflammatory bowel disease (IBD) represents a set of idiopathic and chronic inflammatory diseases of the gastrointestinal tract. Central to the pathogenesis of IBD is a dysregulation of normal intestinal epithelial homeostasis. cGAS is a DNA-sensing receptor demonstrated to promote autophagy, a mechanism that removes dysfunctional cellular components. Beclin-1 is a crucial protein involved in the initiation of autophagy. We hypothesized that cGAS plays a key role in intestinal homeostasis by upregulating Beclin-1-mediated autophagy. We evaluated intestinal cGAS levels in humans with IBD and in murine colonic tissue after performing a 2% dextran sulfate sodium (DSS) colitis model. Autophagy and cell death mechanisms were studied in cGAS KO and WT mice via qPCR, WB analysis, H&E, IF, and TUNEL staining. Autophagy was measured in stimulated intestinal epithelial cells (IECs) via WB analysis. Our data demonstrates cGAS to be upregulated during human and murine colitis. Furthermore, cGAS deficiency leads to worsened colitis and decreased levels of autophagy proteins including Beclin-1 and LC3-II. Co-IP demonstrates a direct binding between cGAS and Beclin-1 in IECs. Transfection of cGAS in stimulated HCT-116 cells leads to increased autophagy. IECs isolated from cGAS KO have diminished autophagic flux. cGAS KO mice subjected to DSS have increased cell death and cleaved caspase-3. Lastly, treatment of cGAS KO mice with rapamycin decreased the severity of colitis. Our data suggest that cGAS maintains intestinal epithelial homeostasis during human IBD and murine colitis by upregulating Beclin-1-mediated autophagy and preventing IEC death. Rescue of autophagy can attenuate the severity of colitis associated with cGAS deficiency.
    Keywords:  DSS; cGAS; colitis; intestinal epithelium
  30. Pharmacol Ther. 2022 Mar 26. pii: S0163-7258(22)00072-9. [Epub ahead of print] 108178
      Defects in cellular functions related to altered protein homeostasis and associated progressive accumulation of pathological intracellular material is a critical process involved in the pathogenesis of many neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Autophagy is an essential mechanism that ensures neuronal health by removing long-lived proteins or defective organelles and by doing so prevents cell toxicity and death within the central nervous system. Abundant evidence has shown that neuronal autophagy pathways are altered in Alzheimer's disease, Parkinson's disease and traumas of the central nervous system including Spinal Cord Injury and Traumatic Brain Injury. In this review, we aimed to summarize the latest studies on the role that altered neuronal autophagy plays in brain health and these pathological conditions, and how this knowledge can be leveraged for the development of novel therapeutics against them.
    Keywords:  Alzheimer's disease; Autophagy; Central nervous system; Neurodegeneration; Neurons; Parkinson's disease; Spinal cord injury; Traumatic brain injury
  31. Front Oncol. 2022 ;12 852424
      Cancer formation is a highly regulated and complex process, largely dependent on its microenvironment. This complexity highlights the need for developing novel target-based therapies depending on cancer phenotype and genotype. Autophagy, a catabolic process, removes damaged and defective cellular materials through lysosomes. It is activated in response to stress conditions such as nutrient deprivation, hypoxia, and oxidative stress. Oxidative stress is induced by excess reactive oxygen species (ROS) that are multifaceted molecules that drive several pathophysiological conditions, including cancer. Moreover, autophagy also plays a dual role, initially inhibiting tumor formation but promoting tumor progression during advanced stages. Mounting evidence has suggested an intricate crosstalk between autophagy and ROS where they can either suppress cancer formation or promote disease etiology. This review highlights the regulatory roles of autophagy and ROS from tumor induction to metastasis. We also discuss the therapeutic strategies that have been devised so far to combat cancer. Based on the review, we finally present some gap areas that could be targeted and may provide a basis for cancer suppression.
    Keywords:  ROS; anticancer therapy resistance; autophagy; epithelial–mesenchymal transition; metastasis; tumor microenvironment
  32. Mitochondrion. 2022 Mar 25. pii: S1567-7249(22)00025-3. [Epub ahead of print]64 73-81
      The correlation between mitochondrial function and oncogenesis is complex and is not fully understood. Here we determine the importance of mitochondrial-linked pyrimidine synthesis for the aggressiveness of cancer cells. The enzyme dihydroorotate dehydrogenase (DHODH) links oxidative phosphorylation to de novo synthesis of pyrimidines. We demonstrate that an inhibition of DHODH results in a respiration-independent significant increase of anchorage-independent growth but does not affect DNA repair ability. Instead, we show an autophagy-independent increase of lysosomes. The results of this study suggest that inhibition of mitochondrial-linked pyrimidine synthesis in cancer cells results in a more aggressive tumor phenotype.
    Keywords:  DNA repair; Lysosome increase; Mitochondria; Mitochondrial-linked pyrimidine synthesis; Tumorigenesis
  33. Traffic. 2022 Mar 28.
      Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has revealed that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling, and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyse lysosome morphology, positioning, motility, and function. We highlight the principles behind these methods, the methodological strategies, and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.
    Keywords:  Endolysosomes; Lysosomal storage diseases; Lysosome biogenesis; Lysosome exocytosis; Lysosome-related organelles; Lysosomes; Membrane contact sites; TFEB; mTOR