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Nature Cell Biology Mar 2023Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING...
Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs.
Topics: Animals; Humans; Adenosine Triphosphatases; Endosomal Sorting Complexes Required for Transport; Endosomes; Interferon Type I; Microautophagy; Protein Transport; Signal Transduction; Membrane Proteins
PubMed: 36918692
DOI: 10.1038/s41556-023-01098-9 -
Autophagy Jul 2021SCAP (SREBF chaperone) regulates SREBFs (sterol regulatory element binding transcription factors) processing and stability, and, thus, becomes an emerging drug target to...
SCAP (SREBF chaperone) regulates SREBFs (sterol regulatory element binding transcription factors) processing and stability, and, thus, becomes an emerging drug target to treat dyslipidemia and fatty liver disease. However, the current known SCAP inhibitors, such as oxysterols, induce endoplasmic reticulum (ER) stress and NR1H3/LXRα (nuclear receptor subfamily 1 group H member 3)-SREBF1/SREBP-1 c-mediated hepatic steatosis, which severely limited the clinical application of this inhibitor. In this study, we identified a small molecule, lycorine, which binds to SCAP, which suppressed the SREBF pathway without inducing ER stress or activating NR1H3. Mechanistically, lycorine promotes SCAP lysosomal degradation in a macroautophagy/autophagy-independent pathway, a mechanism completely distinct from current SCAP inhibitors. Furthermore, we determined that SQSTM1 captured SCAP after its exit from the ER. The interaction of SCAP and SQSTM1 requires the WD40 domain of SCAP and the TB domain of SQSTM1. Interestingly, lycorine triggers the lysosome translocation of SCAP independent of autophagy. We termed this novel protein degradation pathway as the SQSTM1-mediated autophagy-independent lysosomal degradation (SMAILD) pathway. , lycorine ameliorates high-fat diet-induced hyperlipidemia, hepatic steatosis, and insulin resistance in mice. Our study demonstrated that the inhibition of SCAP through the SMAILD pathway could be employed as a useful therapeutic strategy for treating metabolic diseases. 25-OHD: 25-hydroxyvitamin D; 3-MA: 3-methyladenine; ABCG5: ATP binding cassette subfamily G member 5; ABCG8: ATP binding cassette subfamily G member 8; ACACA: acetyl-CoA carboxylase alpha; AEBSF: 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; AHI: anhydroicaritin; AKT/protein kinase B: AKT serine/threonine kinase; APOE: apolipoprotein E; ATF6: activating transcription factor 6; ATG: autophagy-related; BAT: brown adipose tissue; CD274/PD-L1: CD274 molecule; CETSA: cellular thermal shift assay; CMA: chaperone-mediated autophagy; COPII: cytoplasmic coat protein complex-II; CQ: chloroquine; DDIT3/CHOP: DNA damage inducible transcript 3; DNL: lipogenesis; EE: energy expenditure; EGFR: epithelial growth factor receptor; eMI: endosomal microautophagy; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FADS2: fatty acid desaturase 2; FASN: fatty acid synthase; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvate transaminase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; HMGCS1: 3-hydroxy-3-methylglutaryl-CoA synthase 1; HSP90B1/GRP94: heat shock protein 90 beta family member 1; HSPA5/GRP78: heat hock protein family A (Hsp70) member 5; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; INSIG1: insulin induced gene 1; LAMP2A: lysosomal associated membrane protein 2A; LDLR: low density lipoprotein receptor; LyTACs: lysosome targeting chimeras; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MBTPS1: membrane bound transcription factor peptidase, site 1; MEF: mouse embryonic fibroblast; MST: microscale thermophoresis; MTOR: mechanistic target of rapamycin kinase; MVK: mevalonate kinase; PROTAC: proteolysis targeting chimera; RQ: respiratory quotient; SCAP: SREBF chaperone; SCD1: stearoyl-coenzemy A desaturase 1; SMAILD: sequestosome 1 mediated autophagy-independent lysosomal degradation; SQSTM1: sequestosome 1; SREBF: sterol regulatory element binding transcription factor; TNFRSF10B/DR5: TNF receptor superfamily member 10b; TRAF6: TNF receptor associated factor 6; UPR: unfolded protein response; WAT: white adipose tissue; XBP1: X-box binding protein 1.
Topics: Amaryllidaceae Alkaloids; Animals; Diet, High-Fat; Down-Regulation; HEK293 Cells; Hep G2 Cells; Humans; Hyperlipidemias; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Lysosomes; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Obesity; Phenanthridines; Sequestosome-1 Protein; Signal Transduction
PubMed: 32432943
DOI: 10.1080/15548627.2020.1757955 -
The Journal of Cell Biology Oct 2022A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to...
A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, it is unclear whether NCOA4 only acts as an autophagy receptor in ferritin degradation. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).
Topics: Autophagy; Endosomes; Ferritins; Iron; Lysosomes; Nuclear Receptor Coactivators; Phagosomes; Transcription Factors
PubMed: 36066504
DOI: 10.1083/jcb.202203102 -
Autophagy Jan 2023Impaired degradation of the transcriptional coactivator YAP1 and IL6ST (interleukin 6 cytokine family signal transducer), two proteins deregulated in liver cancer, has...
Impaired degradation of the transcriptional coactivator YAP1 and IL6ST (interleukin 6 cytokine family signal transducer), two proteins deregulated in liver cancer, has been shown to promote tumor growth. Here, we demonstrate that YAP1 and IL6ST are novel substrates of chaperone-mediated autophagy (CMA) in human hepatocellular carcinoma (HCC) and hepatocyte cell lines. Knockdown of the lysosomal CMA receptor LAMP2A increases protein levels of YAP1 and IL6ST, without changes in mRNA expression. Additionally, both proteins show KFERQ-dependent binding to the CMA chaperone HSPA8 and accumulate into isolated lysosomes after stimulation of CMA by prolonged starvation. We further show that LAMP2A downregulation promotes the proliferation and migration in HCC cells and a human hepatocyte cell line, and that it does so in a YAP1- and IL6ST-dependent manner. Finally, LAMP2A expression is downregulated, and YAP1 and IL6ST expression is upregulated, in human HCC biopsies. Taken together, our work reveals a novel mechanism that controls the turnover of two cancer-relevant proteins and suggests a tumor suppressor function of CMA in the liver, advocating for the exploitation of CMA activity for diagnostic and therapeutic purposes. ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; CMA: chaperone-mediated autophagy; eMI: endosomal microautophagy; HCC: hepatocellular carcinoma; HSPA8: heat shock protein family A (Hsp70) member 8; IL6ST: interleukin 6 cytokine family signal transducer; JAK: Janus kinase; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MAPK8: mitogen-activated protein kinase 8; P6: pyridine 6; SQSTM1: sequestosome 1; TUBA: tubulin alpha; VDAC1: voltage dependent anion channel 1; VP: verteporfin; YAP1: Yes1 associated transcriptional regulator.
Topics: Humans; Chaperone-Mediated Autophagy; Carcinoma, Hepatocellular; Autophagy; Liver Neoplasms; Interleukin-6; Cell Line; Adaptor Proteins, Signal Transducing; Cell Proliferation; Lysosomes; Lysosomal-Associated Membrane Protein 2; Cytokine Receptor gp130
PubMed: 35435804
DOI: 10.1080/15548627.2022.2063004 -
Journal of Biochemistry Nov 2023The cGAS-STING innate immune pathway has recently emerged as a critical driver of inflammation in a variety of settings, such as virus infection, cellular stress and... (Review)
Review
The cGAS-STING innate immune pathway has recently emerged as a critical driver of inflammation in a variety of settings, such as virus infection, cellular stress and tissue damage. The pathway detects microbial and host-derived double-stranded DNA (dsDNA) in the cytosol, and triggers the production of the type I interferons through the activation of IRF3. The detailed mechanistic and biochemical understanding of the pathway has enabled the development of pharmacological agents for the treatment of chronic inflammation and cancer. STING is an endoplasmic reticulum (ER)-localized transmembrane protein. Upon emergence of cytosolic dsDNA, STING exits the ER and migrates sequentially to the Golgi, recycling endosomes and lysosomes. Importantly, the intracellular translocation of STING is essential for the activation and inactivation of the STING signalling. In this review, I summarize the recent insights into the regulators of the membrane traffic of STING and STING-associated autoinflammatory diseases.
Topics: Humans; Signal Transduction; Membrane Proteins; Protein Transport; Nucleotidyltransferases; DNA; Inflammation; Immunity, Innate
PubMed: 37562849
DOI: 10.1093/jb/mvad064 -
Autophagy 2018The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerative conditions....
UNLABELLED
The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerative conditions. Alterations in autophagy processes, including macroautophagy and chaperone-mediated autophagy (CMA), have been described in Parkinson disease (PD). CMA is a selective autophagic process that depends on LAMP2A (lysosomal-associated membrane protein 2A), a mammal and bird-specific membrane glycoprotein that translocates cytosolic proteins containing a KFERQ-like peptide motif across the lysosomal membrane. Drosophila reportedly lack CMA and use endosomal microautophagy (eMI) as an alternative selective autophagic process. Here we report that neuronal expression of human LAMP2A protected Drosophila against starvation and oxidative stress, and delayed locomotor decline in aging flies without extending their lifespan. LAMP2A also prevented the progressive locomotor and oxidative defects induced by neuronal expression of PD-associated human SNCA (synuclein alpha) with alanine-to-proline mutation at position 30 (SNCA). Using KFERQ-tagged fluorescent biosensors, we observed that LAMP2A expression stimulated selective autophagy in the adult brain and not in the larval fat body, but did not increase this process under starvation conditions. Noteworthy, we found that neurally expressed LAMP2A markedly upregulated levels of Drosophila Atg5, a key macroautophagy initiation protein, and that it increased the density of Atg8a/LC3-positive puncta, which reflects the formation of autophagosomes. Furthermore, LAMP2A efficiently prevented accumulation of the autophagy defect marker Ref(2)P/p62 in the adult brain under acute oxidative stress. These results indicate that LAMP2A can potentiate autophagic flux in the Drosophila brain, leading to enhanced stress resistance and neuroprotection.
ABBREVIATIONS
Act5C: actin 5C; a.E.: after eclosion; Atg5: autophagy-related 5; Atg8a/LC3: autophagy-related 8a; CMA: chaperone-mediated autophagy; DHE: dihydroethidium; elav: embryonic lethal abnormal vision; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; GABARAP: GABA typeA receptor-associated protein; Hsc70-4: heat shock protein cognate 4; HSPA8/Hsc70: heat shock protein family A (Hsp70) member 8; LAMP2: lysosomal associated membrane protein 2; MDA: malondialdehyde; PA-mCherry: photoactivable mCherry; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PD: Parkinson disease; Ref(2)P/p62: refractory to sigma P; ROS: reactive oxygen species; RpL32/rp49: ribosomal protein L32; RT-PCR: reverse transcription polymerase chain reaction; SING: startle-induced negative geotaxis; SNCA/α-synuclein: synuclein alpha; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; UAS: upstream activating sequence.
Topics: Animals; Animals, Genetically Modified; Autophagy; Brain; Disease Models, Animal; Drosophila; Humans; Locomotion; Lysosomal-Associated Membrane Protein 2; Neuroprotection; Oxidative Stress; Parkinson Disease; Phenotype; Signal Transduction; alpha-Synuclein
PubMed: 29989488
DOI: 10.1080/15548627.2018.1491489 -
Progress in Molecular Biology and... 2020Nuclear recycling is essential for cell and organismal homeostasis. Nuclear architecture perturbations, such as nuclear loss or nuclear enlargement, have been observed... (Review)
Review
Nuclear recycling is essential for cell and organismal homeostasis. Nuclear architecture perturbations, such as nuclear loss or nuclear enlargement, have been observed in several pathological conditions. Apart from proteasomal components which reside in the nucleus, specific autophagic proteins also shuttle between the nucleus and the cytoplasm. Until recently, only the microautophagic degradation of nuclear components had been described. Recent studies, dissecting nuclear material recycling in organisms ranging from yeast to mammals, provide insight relevant to other forms of nucleophagy and the mediators involved. Nucleophagy has also been implicated in pathology. Lamins are degraded in cancer through direct interaction with LC3 in the nucleus. Similarly, in neurodegeneration, Golgi-associated nucleophagy is exacerbated. The physiological role of nucleophagy and its contribution to other pathologies remain to be elucidated. Here we discus recent findings that shed light into the molecular mechanisms and pathways that mediate the autophagic recycling of nuclear material.
Topics: Animals; Autophagy; Autophagy-Related Proteins; Caenorhabditis elegans; Cell Nucleolus; Cell Nucleus; Golgi Apparatus; Humans; Intranuclear Inclusion Bodies; Laminopathies; Lamins; Mammals; Mice; Microtubule-Associated Proteins; Neoplasms; Neurodegenerative Diseases; Nuclear Envelope; Proteolysis; RNA Stability; Saccharomyces cerevisiae
PubMed: 32620238
DOI: 10.1016/bs.pmbts.2020.01.003 -
The Biochemical Journal Sep 2021The ubiquitin-proteasome pathway (UPP) and autophagy play integral roles in cellular homeostasis. As part of their normal life cycle, most proteins undergo... (Review)
Review
The ubiquitin-proteasome pathway (UPP) and autophagy play integral roles in cellular homeostasis. As part of their normal life cycle, most proteins undergo ubiquitination for some form of redistribution, localization and/or functional modulation. However, ubiquitination is also important to the UPP and several autophagic processes. The UPP is initiated after specific lysine residues of short-lived, damaged or misfolded proteins are conjugated to ubiquitin, which targets these proteins to proteasomes. Autophagy is the endosomal/lysosomal-dependent degradation of organelles, invading microbes, zymogen granules and macromolecules such as protein, carbohydrates and lipids. Autophagy can be broadly separated into three distinct subtypes termed microautophagy, chaperone-mediated autophagy and macroautophagy. Although autophagy was once thought of as non-selective bulk degradation, advancements in the field have led to the discovery of several selective forms of autophagy. Here, we focus on the mechanisms of primary and selective mammalian autophagy pathways and highlight the current knowledge gaps in these molecular pathways.
Topics: Animals; Autophagosomes; Autophagy; Autophagy-Related Proteins; Endosomal Sorting Complexes Required for Transport; Endosomes; Eukaryotic Cells; Homeostasis; Humans; Lysosomes; Molecular Chaperones; Proteasome Endopeptidase Complex; Protein Folding; Protein Processing, Post-Translational; Proteolysis; Ubiquitin; Ubiquitination
PubMed: 34554214
DOI: 10.1042/BCJ20210314 -
Journal of Trace Elements in Medicine... Dec 2020Autophagy is a conserved catabolic process that plays an important role in cellular homeostasis. The study of the interplay between autophagy and zinc has gained... (Review)
Review
Autophagy is a conserved catabolic process that plays an important role in cellular homeostasis. The study of the interplay between autophagy and zinc has gained interest over the last years. Multiple studies have indicated that zinc stimulates autophagy and is critical for basal and induced autophagy in mammalian cells. Conversely, autophagy is induced by zinc starvation in yeast. There are no studies analyzing the role of zinc in either Microautophagy or Chaperone-Mediated-Autophagy. The mechanisms by which zinc modulates autophagy are still poorly understood. Studies examining loss of function of genes involved in cellular zinc homeostasis have provided novel insights into the role of zinc in autophagy. Autophagy may help cells adapt to changes in zinc availability in medium by controlling zinc mobilization, recycling, and secretion. Zinc is a key player in toxic and protective autophagy.
Topics: Animals; Autophagy; Humans; Lysosomes; Metallothionein; Mitophagy; Zinc
PubMed: 32957075
DOI: 10.1016/j.jtemb.2020.126636 -
Molecular Cell Apr 2022Organelles are continuously turned over as part of cellular homeostasis and adaptation. Most organelles, even including the nucleus, are degraded by lysosomes via...
Organelles are continuously turned over as part of cellular homeostasis and adaptation. Most organelles, even including the nucleus, are degraded by lysosomes via different pathways, such as macroautophagy, microautophagy, organelle-derived vesicle degradation, and crinophagy. In some specific cases-for example, in lens fiber cells-organelles are degraded by cytosolic phospholipases. To view this SnapShot, open or download the PDF.
Topics: Autophagy; Cytosol; Lens, Crystalline; Lysosomes; Organelles
PubMed: 35452619
DOI: 10.1016/j.molcel.2022.03.015