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  • 1
    Language: English
    In: Nature biotechnology, 2013-07, Vol.31 (7), p.653-658
    Description: Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.
    Subject(s): Biological transport ; Carrier Proteins ; Endocytosis ; Endocytosis - genetics ; Gene Silencing ; Gene Transfer Techniques ; Humans ; Lipids ; Lipids - administration & dosage ; Lipids - chemistry ; Lipids - genetics ; Membrane Glycoproteins ; Metal Nanoparticles - administration & dosage ; Metal Nanoparticles - chemistry ; Microscopy, Confocal ; Nanoparticles ; Pharmaceutical sciences ; Research ; Ribonucleic acid ; RNA ; RNA, Small Interfering - administration & dosage ; RNA, Small Interfering - chemistry ; RNA, Small Interfering - genetics ; Signal Transduction - genetics ; TOR Serine-Threonine Kinases - genetics ; TOR Serine-Threonine Kinases - metabolism
    ISSN: 1087-0156
    E-ISSN: 1546-1696
    Source: Academic Search Ultimate
    Source: Get It Now
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  • 2
    Language: English
    In: Frontiers in cell and developmental biology, 2018, Vol.6, p.147-147
    Description: Autophagy, a cellular homeostatic process, which ensures cellular survival under various stress conditions, has catapulted to the forefront of innate defense mechanisms during intracellular infections. The ability of autophagy to tag and target intracellular pathogens toward lysosomal degradation is central to this key defense function. However, studies involving the role and regulation of autophagy during intracellular infections largely tend to ignore the housekeeping function of autophagy. A growing number of evidences now suggest that the housekeeping function of autophagy, rather than the direct pathogen degradation function, may play a decisive role to determine the outcome of infection and immunological balance. We discuss herein the studies that establish the homeostatic and anti-inflammatory function of autophagy, as well as role of bacterial effectors in modulating and coopting these functions. Given that the core autophagy machinery remains largely the same across diverse cargos, how selectivity plays out during intracellular infection remains intriguing. We explore here, the contrasting role of autophagy adaptors being both selective as well as pleotropic in functions and discuss whether E3 ligases could bring in the specificity to cargo selectivity.
    Subject(s): Autophagy (Cytology) ; Cell membranes ; DUBs ; inflammation ; NDP52 ; OPTN ; p62 ; Physiology ; Pleiotropy ; Research ; TAX1BP1 ; ubiquitination ; xenophagy
    ISSN: 2296-634X
    E-ISSN: 2296-634X
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 3
    Language: English
    In: Nature cell biology, 2011-04, Vol.13 (4), p.453-460
    Description: mTOR (mammalian target of rapamycin) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes, including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates. Here we report that lysosomal positioning coordinates anabolic and catabolic responses with changes in nutrient availability by orchestrating early plasma-membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, whereas starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH. Lysosomal positioning regulates mTORC1 signalling, which in turn influences autophagosome formation. Lysosome positioning also influences autophagosome-lysosome fusion rates, and thus controls autophagic flux by acting at both the initiation and termination stages of the process. Our findings provide a physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux.
    Subject(s): Autophagy (Cytology) ; Autophagy - physiology ; Food ; HeLa Cells ; Humans ; Lysosomes ; Lysosomes - metabolism ; Lysosomes - ultrastructure ; Mechanistic Target of Rapamycin Complex 1 ; Multiprotein Complexes ; Physiological aspects ; Proteins - genetics ; Proteins - metabolism ; Research ; Signal Transduction - physiology ; TOR Serine-Threonine Kinases - genetics ; TOR Serine-Threonine Kinases - metabolism
    ISSN: 1465-7392
    E-ISSN: 1476-4679
    Source: Academic Search Ultimate
    Source: Get It Now
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  • 4
    Language: English
    In: eLife, 2016-01-07, Vol.5
    Description: The mammalian target of rapamycin complex 1 (mTORC1) is the key signaling hub that regulates cellular protein homeostasis, growth, and proliferation in health and disease. As a prerequisite for activation of mTORC1 by hormones and mitogens, there first has to be an available pool of intracellular amino acids. Arginine, an amino acid essential during mammalian embryogenesis and early development is one of the key activators of mTORC1. Herein, we demonstrate that arginine acts independently of its metabolism to allow maximal activation of mTORC1 by growth factors via a mechanism that does not involve regulation of mTORC1 localization to lysosomes. Instead, arginine specifically suppresses lysosomal localization of the TSC complex and interaction with its target small GTPase protein, Rheb. By interfering with TSC-Rheb complex, arginine relieves allosteric inhibition of Rheb by TSC. Arginine cooperates with growth factor signaling which further promotes dissociation of TSC2 from lysosomes and activation of mTORC1. Arginine is the main amino acid sensed by the mTORC1 pathway in several cell types including human embryonic stem cells (hESCs). Dependence on arginine is maintained once hESCs are differentiated to fibroblasts, neurons, and hepatocytes, highlighting the fundamental importance of arginine-sensing to mTORC1 signaling. Together, our data provide evidence that different growth promoting cues cooperate to a greater extent than previously recognized to achieve tight spatial and temporal regulation of mTORC1 signaling.
    Subject(s): Allosteric properties ; Amino acids ; Arginine ; Arginine - metabolism ; autophagy ; Biomedical research ; Cancer ; Cell Biology ; Cell Differentiation ; Cellular signal transduction ; Embryo cells ; Embryogenesis ; Embryonic Stem Cells - physiology ; Experiments ; Fibroblasts ; Growth factors ; Guanosine triphosphatases ; Health aspects ; Hepatocytes ; Homeostasis ; Human ; Humans ; Kinases ; Localization ; Lysosomes ; Mechanistic Target of Rapamycin Complex 1 ; Medical research ; Metabolism ; Mitogens ; Monomeric GTP-Binding Proteins - metabolism ; mTOR ; Multiprotein Complexes - metabolism ; Neuropeptides - metabolism ; Rapamycin ; Ras Homolog Enriched in Brain Protein ; Regulation ; Signal Transduction ; Stem cells ; TOR protein ; TOR Serine-Threonine Kinases - metabolism ; Tuberous Sclerosis Complex 2 ; Tuberous Sclerosis Complex 2 Protein ; Tumor Suppressor Proteins - metabolism
    ISSN: 2050-084X
    E-ISSN: 2050-084X
    Source: PubMed Central
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 5
    Language: English
    In: Nature chemical biology, 2008-05, Vol.4 (5), p.295-305
    Description: Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.
    Subject(s): Animals ; Autophagy - drug effects ; Calcium Channels, L-Type - drug effects ; Clonidine - pharmacology ; Cyclic AMP - metabolism ; Danio rerio ; Humans ; Huntington Disease - immunology ; Huntington Disease - physiopathology ; Imidazoline Receptors - antagonists & inhibitors ; Minoxidil - pharmacology ; Protein Kinases - physiology ; Signal Transduction ; TOR Serine-Threonine Kinases ; Type C Phospholipases - metabolism ; Verapamil - pharmacology
    ISSN: 1552-4450
    E-ISSN: 1552-4469
    Source: Academic Search Ultimate
    Source: Nature Journals Online
    Source: Get It Now
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  • 6
    Language: English
    In: The Journal of cell biology, 2005-09-26, Vol.170 (7), p.1101-1111
    Description: Macroautophagy is a key pathway for the clearance of aggregate-prone cytosolic proteins. Currently, the only suitable pharmacologic strategy for up-regulating autophagy in mammalian cells is to use rapamycin, which inhibits the mammalian target of rapamycin (mTOR), a negative regulator of autophagy. Here we describe a novel mTOR-independent pathway that regulates autophagy. We show that lithium induces autophagy, and thereby, enhances the clearance of autophagy substrates, like mutant huntingtin and . This effect is not mediated by glycogen synthase kinase 3β inhibition. The autophagy-enhancing properties of lithium were mediated by inhibition of inositol monophosphatase and led to free inositol depletion. This, in turn, decreased myo-inositol-1,4,5-triphosphate (IP ) levels. Our data suggest that the autophagy effect is mediated at the level of (or downstream of) lowered IP , because it was abrogated by pharmacologic treatments that increased IP . This novel pharmacologic strategy for autophagy induction is independent of mTOR, and may help treatment of neurodegenerative diseases, like Huntington's disease, where the toxic protein is an autophagy substrate.
    Subject(s): Actins ; alpha-Synuclein - genetics ; alpha-Synuclein - metabolism ; Animals ; Antibodies ; Autophagy - drug effects ; Cell aggregates ; Cell death ; Cell Line, Tumor ; Cell lines ; Chlorocebus aethiops ; COS Cells ; Enzyme Inhibitors - pharmacology ; Humans ; Huntingtin Protein ; Immunoblotting ; Inositol - metabolism ; Inositol phosphates ; Inositols ; Lithium ; Lithium - pharmacology ; Lithium compounds ; Mutation ; Nerve Tissue Proteins - genetics ; Nerve Tissue Proteins - metabolism ; Neurological disorders ; Nuclear Proteins - genetics ; Nuclear Proteins - metabolism ; PC12 cells ; Pharmacology ; Phosphoric Monoester Hydrolases - antagonists & inhibitors ; Phosphoric Monoester Hydrolases - metabolism ; Protein Kinases - metabolism ; Proteins ; Research ; Sirolimus - pharmacology ; TOR Serine-Threonine Kinases ; Toxicity ; Usage
    ISSN: 0021-9525
    E-ISSN: 1540-8140
    Source: Rockefeller University Press
    Source: PubMed Central
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  • 7
    Language: English
    In: The Journal of biological chemistry, 2010-04-09, Vol.285 (15), p.11061-11067
    Description: Many of the neurodegenerative diseases that afflict people are caused by intracytoplasmic aggregate-prone proteins. These include Parkinson disease, tauopathies, and polyglutamine expansion diseases such as Huntington disease. In Mendelian forms of these diseases, the mutations generally confer toxic novel functions on the relevant proteins. Thus, one potential strategy for dealing with these mutant proteins is to enhance their degradation. This can be achieved by up-regulating macroautophagy, which we will henceforth call autophagy. In this minireview, we will consider the reasons why autophagy up-regulation may be a powerful strategy for these diseases. In addition, we will consider some of the drugs and associated signaling pathways that can be used to induce autophagy with these therapeutic aims in mind.
    Subject(s): Amyloid ; Animals ; Autophagy ; Biochemistry - methods ; Brain ; Calmodulin ; Cell Biology ; Cytoplasm - metabolism ; Diseases ; Diseases/Amyloid ; Drug Action ; Drug Screen ; Drugs ; Gene Expression Regulation ; Humans ; Lysosomes ; Macrophages - metabolism ; Metabolism ; Minireviews ; Models, Biological ; Mutation ; Neurodegenerative Diseases - metabolism ; Neurodegenerative Diseases - pathology ; Organ Systems ; Protein Binding ; Protein Denaturation ; Protein Folding ; Protein Kinases ; Proteins - chemistry ; Signal Transduction ; Signal Transduction/Protein Kinases/Calmodulin ; Subcellular Organelles ; Subcellular Organelles/Lysosomes ; Tissue ; Tissue/Organ Systems/Brain ; Toxins ; Toxins/Drugs/Xenobiotics/Drug Action ; Xenobiotics
    ISSN: 0021-9258
    E-ISSN: 1083-351X
    Source: HighWire Press (Free Journals)
    Source: PubMed Central
    Source: Alma/SFX Local Collection
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 8
    Language: English
    In: The FEBS journal, 2008-09, Vol.275 (17), p.4263-4270
    Description: Autophagy is a nonspecific bulk degradation pathway for long‐lived cytoplasmic proteins, protein complexes, or damaged organelles. This process is also a major degradation pathway for many aggregate‐prone, disease‐causing proteins associated with neurodegenerative disorders, such as mutant huntingtin in Huntington’s disease. In this review, we discuss factors regulating the degradation of mutant huntingtin by autophagy. We also report the growing list of new drugs/pathways that upregulate autophagy to enhance the clearance of this mutant protein, as autophagy upregulation may be a tractable strategy for the treatment of Huntington’s disease.
    Subject(s): autophagy ; Autophagy - drug effects ; Biochemistry ; Cellular biology ; Genetic disorders ; Humans ; Huntingtin Protein ; Huntington Disease - genetics ; Huntington Disease - immunology ; Huntington Disease - metabolism ; Huntington's chorea ; Huntington's disease ; Inositol - antagonists & inhibitors ; lithium ; Lithium Compounds - pharmacology ; Medical genetics ; mTOR ; Mutation ; Nerve Tissue Proteins - genetics ; Nerve Tissue Proteins - metabolism ; Neurological disorders ; Nuclear Proteins - genetics ; Nuclear Proteins - metabolism ; Pharmacology ; polyglutamine ; Protein Kinases - metabolism ; rapamycin ; Sirolimus - pharmacology ; TOR Serine-Threonine Kinases ; Trehalose - pharmacology
    ISSN: 1742-464X
    E-ISSN: 1742-4658
    Source: Academic Search Ultimate
    Source: Alma/SFX Local Collection
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  • 9
    Language: English
    In: Cell reports (Cambridge), 2013-12-12, Vol.5 (5), p.1302-1315
    Description: Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid-storage disorders is not clear. Here, we show defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-β-cyclodextrin, which is used for cholesterol-depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. Our data suggest that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease. [Display omitted] •Defective autophagy in NPC1 disease is caused by failure of SNARE machinery•Loss of NPC1 protein impairs amphisome formation and autophagosome maturation•Cholesterol depletion with HP-β-cyclodextrin blocks autophagic flux•Induced autophagy rescues defects and is a rational therapeutic strategy for NPC1 Autophagy dysfunction is implicated in several human diseases. Now, Jaenisch and colleagues show that autophagic flux is impaired in Niemann-Pick type C1 disease, possibly contributing to disease pathology. Stimulating autophagy rescues the block in basal autophagy without the formation of amphisomes. Cholesterol-depletion treatment with HP-β-cyclodextrin also impedes autophagy, whereas using a lower dose that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy by removing both cholesterol and autophagic cargo.
    Subject(s): Animals ; Autophagy ; Autophagy enhancer ; beta-Cyclodextrins - pharmacology ; Cells, Cultured ; Cholesterol ; Cholesterol - deficiency ; Cholesterol - metabolism ; HEK293 Cells ; Humans ; Lipid storage disorder ; Lysosomal storage disorder ; Lysosomes - metabolism ; Membrane Glycoproteins - genetics ; Membrane Glycoproteins - metabolism ; Mice ; Neurodegeneration ; Neurons - drug effects ; Neurons - metabolism ; Niemann-Pick Disease, Type C - genetics ; Niemann-Pick Disease, Type C - metabolism ; Niemann-Pick type C1 disease ; Rats ; SNARE Proteins - metabolism
    ISSN: 2211-1247
    E-ISSN: 2211-1247
    Source: Alma/SFX Local Collection
    Source: DOAJ Directory of Open Access Journals - Not for CDI Discovery
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  • 10
    Language: English
    In: Amino acids, 2014-06-26, Vol.47 (10), p.2065-2088
    Description: Maintenance of amino acid homeostasis is important for healthy cellular function, metabolism and growth. Intracellular amino acid concentrations are dynamic; the high demand for protein synthesis must be met with constant dietary intake, followed by cellular influx, utilization and recycling of nutrients. Autophagy is a catabolic process via which superfluous or damaged proteins and organelles are delivered to the lysosome and degraded to release free amino acids into the cytoplasm. Furthermore, autophagy is specifically activated in response to amino acid starvation via two key signaling cascades: the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and the general control nonderepressible 2 (GCN2) pathways. These pathways are key regulators of the integration between anabolic (amino acid depleting) and catabolic (such as autophagy which is amino acid replenishing) processes to ensure intracellular amino acid homeostasis. Here, we discuss the key roles that amino acids, along with energy (ATP, glucose) and oxygen, are playing in cellular growth and proliferation. We further explore how sophisticated methods are employed by cells to sense intracellular amino acid concentrations, how amino acids can act as a switch to dictate the temporal and spatial activation of anabolic and catabolic processes and how autophagy contributes to the replenishment of free amino acids, all to ensure cell survival. Relevance of these molecular processes to cellular and organismal physiology and pathology is also discussed.
    Subject(s): Activation ; Amino acids ; Amino Acids - metabolism ; Analytical Chemistry ; Animals ; Autophagy ; Biochemical Engineering ; Biochemistry ; Biomedical and Life Sciences ; Cascade control ; Cell Physiological Phenomena ; Cellular ; Constants ; Dextrose ; general ; Glucose ; Glutamine ; Homeostasis ; Humans ; Life Sciences ; Neurobiology ; Pathways ; Physiology ; Protein biosynthesis ; Proteins ; Proteomics ; Replenishment ; Review Article ; Signal Transduction
    ISSN: 0939-4451
    E-ISSN: 1438-2199
    Source: Alma/SFX Local Collection
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