26 results on '"Jefferies, Harold B. J."'
Search Results
2. A switch from canonical to noncanonical autophagy shapes B cell responses
- Author
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Martinez-Martin, Nuria, Maldonado, Paula, Gasparrini, Francesca, Frederico, Bruno, Aggarwal, Shweta, Gaya, Mauro, Tsui, Carlson, Burbage, Marianne, Keppler, Selina Jessica, Montaner, Beatriz, Jefferies, Harold B. J., Nair, Usha, Zhao, Yan G., Domart, Marie-Charlotte, Collinson, Lucy, Bruckbauer, Andreas, Tooze, Sharon A., and Batista, Facundo D.
- Published
- 2017
3. Autophagosome membrane expansion is mediated by the N-terminus and cismembrane association of human ATG8s.
- Author
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Wenxin Zhang, Taki Nishimura, Gahlot, Deepanshi, Chieko Saito, Davis, Colin, Jefferies, Harold B. J., Schreiber, Anne, Thukral, Lipi, and Tooze, Sharon A.
- Published
- 2023
- Full Text
- View/download PDF
4. AUTOPHAGY: A switch from canonical to noncanonical autophagy shapes B cell responses
- Author
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Martinez-Martin, Nuria, Maldonado, Paula, Gasparrini, Francesca, Frederico, Bruno, Aggarwal, Shweta, Gaya, Mauro, Tsui, Carlson, Burbage, Marianne, Keppler, Selina Jessica, Montaner, Beatriz, Jefferies, Harold B. J., Nair, Usha, Zhao, Yan G., Domart, Marie-Charlotte, Collinson, Lucy, Bruckbauer, Andreas, Tooze, Sharon A., and Batista, Facundo D.
- Published
- 2017
- Full Text
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5. Phosphoproteomic identification of ULK substrates reveals VPS15‐dependent ULK/VPS34 interplay in the regulation of autophagy
- Author
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Mercer, Thomas John, primary, Ohashi, Yohei, additional, Boeing, Stefan, additional, Jefferies, Harold B J, additional, De Tito, Stefano, additional, Flynn, Helen, additional, Tremel, Shirley, additional, Zhang, Wenxin, additional, Wirth, Martina, additional, Frith, David, additional, Snijders, Ambrosius P, additional, Williams, Roger Lee, additional, and Tooze, Sharon A, additional
- Published
- 2021
- Full Text
- View/download PDF
6. A switch from canonical to noncanonical autophagy shapes B cell responses
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Francis Crick Institute, Cancer Research UK, Medical Research Council (UK), Wellcome Trust, National Institutes of Health (US), Ragon Institute of MGH, MIT and Harvard, European Commission, Comisión Nacional de Investigación Científica y Tecnológica (Chile), Institut Pasteur, Fondazione Cenci Bolognetti, EMBO, German Research Foundation, Martínez-Martín, Nuria, Maldonado, Paula, Gasparrini, Francesca, Frederico, Bruno, Aggarwal, Shweta, Gaya, Mauro, Tsui, Carlson, Burbage, Marianne, Keppler, Selina Jessica, Montaner, Beatriz, Jefferies, Harold B. J., Nair, Usha, Zhao, Yan G., Domart, Marie-Charlotte, Collinson, Lucy, Bruckbauer, Andreas, Tooze, Sharon A., Batista, Facundo D., Francis Crick Institute, Cancer Research UK, Medical Research Council (UK), Wellcome Trust, National Institutes of Health (US), Ragon Institute of MGH, MIT and Harvard, European Commission, Comisión Nacional de Investigación Científica y Tecnológica (Chile), Institut Pasteur, Fondazione Cenci Bolognetti, EMBO, German Research Foundation, Martínez-Martín, Nuria, Maldonado, Paula, Gasparrini, Francesca, Frederico, Bruno, Aggarwal, Shweta, Gaya, Mauro, Tsui, Carlson, Burbage, Marianne, Keppler, Selina Jessica, Montaner, Beatriz, Jefferies, Harold B. J., Nair, Usha, Zhao, Yan G., Domart, Marie-Charlotte, Collinson, Lucy, Bruckbauer, Andreas, Tooze, Sharon A., and Batista, Facundo D.
- Abstract
Autophagy is important in a variety of cellular and pathophysiological situations; however, its role in immune responses remains elusive. Here, we show that among B cells, germinal center (GC) cells exhibited the highest rate of autophagy during viral infection. In contrast to mechanistic target of rapamycin complex 1–dependent canonical autophagy, GC B cell autophagy occurred predominantly through a noncanonical pathway. B cell stimulation was sufficient to down-regulate canonical autophagy transiently while triggering noncanonical autophagy. Genetic ablation of WD repeat domain, phosphoinositide–interacting protein 2 in B cells alone enhanced this noncanonical autophagy, resulting in changes of mitochondrial homeostasis and alterations in GC and antibody-secreting cells. Thus, B cell activation prompts a temporal switch from canonical to noncanonical autophagy that is important in controlling B cell differentiation and fate.
- Published
- 2017
7. ATG9A shapes the forming autophagosome through Arfaptin 2 and phosphatidylinositol 4-kinase IIIβ.
- Author
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Judith, Delphine, Jefferies, Harold B. J., Tooze, Sharon A., Boeing, Stefan, Frith, David, and Snijders, Ambrosius P.
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AUTOPHAGY , *PHOSPHATIDYLINOSITOL 3-kinases , *GOLGI apparatus - Abstract
ATG9A is a multispanning membrane protein essential for autophagy. Normally resident in Golgi membranes and endosomes, during amino acid starvation, ATG9A traffics to sites of autophagosome formation. ATG9A is not incorporated into autophagosomes but is proposed to supply so-far-unidentified proteins and lipids to the autophagosome. To address this function of ATG9A, a quantitative analysis of ATG9A-positive compartments immunoisolated from amino acid-starved cells was performed. These ATG9A vesicles are depleted of Golgi proteins and enriched in BAR-domain containing proteins, Arfaptins, and phosphoinositide-metabolizing enzymes. Arfaptin2 regulates the starvation-dependent distribution of ATG9A vesicles, and these ATG9A vesicles deliver the PI4-kinase, PI4KIIIβ, to the autophagosome initiation site. PI4KIIIβ interacts with ATG9A and ATG13 to control PI4P production at the initiation membrane site and the autophagic response. PI4KIIIβ and PI4P likely function by recruiting the ULK1/2 initiation kinase complex subunit ATG13 to nascent autophagosomes. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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8. Assessing Mammalian Autophagy.
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Tooze, Sharon A., Dooley, Hannah C., Jefferies, Harold B. J., Joachim, Justin, Judith, Delphine, Lamb, Christopher A., Razi, Minoo, and Wirth, Martina
- Published
- 2015
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9. Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC
- Author
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McKnight, Nicole C, primary, Jefferies, Harold B J, additional, Alemu, Endalkachew A, additional, Saunders, Rebecca E, additional, Howell, Michael, additional, Johansen, Terje, additional, and Tooze, Sharon A, additional
- Published
- 2012
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10. Trafficking and signaling in mammalian autophagy
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Tooze, Sharon A., primary, Jefferies, Harold B. J., additional, Kalie, Eyal, additional, Longatti, Andrea, additional, Mcalpine, Fiona E., additional, Mcknight, Nicole C., additional, Orsi, Andrea, additional, Polson, Hannah E. J., additional, Razi, Minoo, additional, Robinson, Deborah J., additional, and Webber, Jemma L., additional
- Published
- 2010
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11. A selective PIKfyve inhibitor blocks PtdIns(3,5)P2production and disrupts endomembrane transport and retroviral budding
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Jefferies, Harold B J, primary, Cooke, Frank T, additional, Jat, Parmjit, additional, Boucheron, Christine, additional, Koizumi, Tomonobu, additional, Hayakawa, Masahiko, additional, Kaizawa, Hiroyuki, additional, Ohishi, Takahide, additional, Workman, Paul, additional, Waterfield, Michael D, additional, and Parker, Peter J, additional
- Published
- 2008
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12. A selective PIKfyve inhibitor blocks PtdIns(3,5)P2 production and disrupts endomembrane transport and retroviral budding.
- Author
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Jefferies, Harold B J, Cooke, Frank T, Jat, Parmjit, Boucheron, Christine, Koizumi, Tomonobu, Hayakawa, Masahiko, Kaizawa, Hiroyuki, Ohishi, Takahide, Workman, Paul, Waterfield, Michael D, and Parker, Peter J
- Abstract
Phosphoinositides have crucial roles in cellular controls, many of which have been established through the use of small-molecule inhibitors. Here, we describe YM201636, a potent inhibitor of the mammalian class III phosphatidylinositol phosphate kinase PIKfyve, which synthesizes phosphatidylinositol 3,5-bisphosphate. Acute treatment of cells with YM201636 shows that the PIKfyve pathway is involved in the sorting of endosomal transport, with inhibition leading to the accumulation of a late endosomal compartment and blockade of retroviral exit. Inhibitor specificity is shown by the use of short interfering RNA against the target, as well as by rescue with the drug-resistant yeast orthologue Fab1. We concluded that the phosphatidylinositol 3,5-bisphosphate pathway is integral to endosome formation, determining morphology and cargo flux. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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13. C16ORF70/MYTHO promotes healthy aging in C.elegans and prevents cellular senescence in mammals.
- Author
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Franco-Romero, Anais, Morbidoni, Valeria, Milan, Giulia, Sartori, Roberta, Wulff, Jesper, Romanello, Vanina, Armani, Andrea, Salviati, Leonardo, Conte, Maria, Salvioli, Stefano, Franceschi, Claudio, Buonomo, Viviana, Swoboda, Casey O., Grumati, Paolo, Pannone, Luca, Martinelli, Simone, Jefferies, Harold B. J., Dikic, Ivan, van der Laan Filipe Cabreiro, Jennifer, and Millay, Douglas P.
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CELLULAR aging , *ANIMAL life spans , *HUMAN DNA , *NUCLEOTIDE sequence , *CAENORHABDITIS elegans , *AGING , *LIFE spans - Abstract
The identification of genes that confer either extension of life span or accelerate age-related decline was a step forward in understanding the mechanisms of aging and revealed that it is partially controlled by genetics and transcriptional programs. Here, we discovered that the human DNA sequence C16ORF70 encodes a protein, named MYTHO (macroautophagy and youth optimizer), which controls life span and health span. MYTHO protein is conserved from Caenorhabditis elegans to humans and its mRNA was upregulated in aged mice and elderly people. Deletion of the orthologous myt-1 gene in C. elegans dramatically shortened life span and decreased animal survival upon exposure to oxidative stress. Mechanistically, MYTHO is required for autophagy likely because it acts as a scaffold that binds WIPI2 and BCAS3 to recruit and assemble the conjugation system at the phagophore, the nascent autophagosome. We conclude that MYTHO is a transcriptionally regulated initiator of autophagy that is central in promoting stress resistance and healthy aging. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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14. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k.
- Author
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Jefferies, Harold B. J., Fumagali, Stefano, Dennis, Patrick B., Reinhard, Christoph, Pearson, Richard B., and Thomas, George
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RAPAMYCIN , *IMMUNOSUPPRESSIVE agents , *DRUGS , *MACROLIDE antibiotics , *PROTEIN kinases , *PHOSPHOTRANSFERASES , *PHOSPHORYLATION , *CHEMICAL reactions - Abstract
Treatment of mammalian cells with the immunosuppressant rapamycin, a bacterial macrolide, selectively suppresses mitogeninduced translation of an essential class of mRNAs which contain an oligopyrimidine tract at their transcriptional start (5′TOP), most notably mRNAs encoding ribosomal proteins and elongation factors. In parallel, rapamycin blocks mitogen-induced p70 ribosomal protein S6 kinase (p70s6k) phosphorylation and activation. Utilizing chimeric mRNA constructs containing either a wild-type or disrupted 5′TOP, we demonstrate that an intact polypyrimidine tract is required for rapamycin to elicit an inhibitory effect on the translation of these transcripts. In turn, a dominant-interfering p70s6k, which selectively prevents p70s6k activation by blocking phosphorylation of the rapamycinsensitive sites, suppresses the translation of the chimeric mRNA containing the wild-type but not the disrupted 59TOP. Conversion of the principal rapamycinsensitive p70s6k phosphorylation site, T389, to an acidic residue confers rapamycin resistance on the kinase and negates the inhibitory effects of the macrolide on 5′TOP mRNA translation in cells expressing this mutant. The results demonstrate that the rapamycin block of mitogeninduced 59TOP mRNA translation is mediated through inhibition of p70s6k activation. [ABSTRACT FROM AUTHOR]
- Published
- 1997
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15. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k.
- Author
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Jefferies, Harold B. J., Fumagali, Stefano, Dennis, Patrick B., Reinhard, Christoph, Pearson, Richard B., and Thomas, George
- Subjects
RAPAMYCIN ,IMMUNOSUPPRESSIVE agents ,DRUGS ,MACROLIDE antibiotics ,PROTEIN kinases ,PHOSPHOTRANSFERASES ,PHOSPHORYLATION ,CHEMICAL reactions - Abstract
Treatment of mammalian cells with the immunosuppressant rapamycin, a bacterial macrolide, selectively suppresses mitogeninduced translation of an essential class of mRNAs which contain an oligopyrimidine tract at their transcriptional start (5′TOP), most notably mRNAs encoding ribosomal proteins and elongation factors. In parallel, rapamycin blocks mitogen-induced p70 ribosomal protein S6 kinase (p70
s6k ) phosphorylation and activation. Utilizing chimeric mRNA constructs containing either a wild-type or disrupted 5′TOP, we demonstrate that an intact polypyrimidine tract is required for rapamycin to elicit an inhibitory effect on the translation of these transcripts. In turn, a dominant-interfering p70s6k , which selectively prevents p70s6k activation by blocking phosphorylation of the rapamycinsensitive sites, suppresses the translation of the chimeric mRNA containing the wild-type but not the disrupted 59TOP. Conversion of the principal rapamycinsensitive p70s6k phosphorylation site, T389, to an acidic residue confers rapamycin resistance on the kinase and negates the inhibitory effects of the macrolide on 5′TOP mRNA translation in cells expressing this mutant. The results demonstrate that the rapamycin block of mitogeninduced 59TOP mRNA translation is mediated through inhibition of p70s6k activation. [ABSTRACT FROM AUTHOR]- Published
- 1997
- Full Text
- View/download PDF
16. Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves.
- Author
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Gomez-Sanchez, Jose A., Carty, Lucy, Iruarrizaga-Lejarreta, Marta, Palomo-Irigoyen, Marta, Varela-Rey, Marta, Griffith, Megan, Hantke, Janina, Macias-Camara, Nuria, Azkargorta, Mikel, Aurrekoetxea, Igor, De Juan, Virginia Gutiérrez, Jefferies, Harold B. J., Aspichueta, Patricia, Elortza, Félix, Aransay, Ana M., Martinez-Chantar, María L., Baas, Frank, Mato, José M., Mirsky, Rhona, and Woodhoo, Ashwin
- Subjects
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SCHWANN cells , *AUTOPHAGY , *MYELIN , *PERIPHERAL nervous system , *CYTOLOGICAL research - Abstract
Although Schwann cell myelin breakdown is the universal outcome of a remarkably wide range of conditions that cause disease or injury to peripheral nerves, the cellular and molecular mechanisms that make Schwann cell-mediated myelin digestion possible have not been established. We report that Schwann cells degrade myelin after injury by a novel form of selective autophagy, myelinophagy. Autophagy was up-regulated by myelinating Schwann cells after nerve injury, myelin debris was present in autophagosomes, and pharmacological and genetic inhibition of autophagy impaired myelin clearance. Myelinophagy was positively regulated by the Schwann cell JNK/c-Jun pathway, a central regulator of the Schwann cell reprogramming induced by nerve injury. We also present evidence that myelinophagy is defective in the injured central nervous system. These results reveal an important role for inductive autophagy during Wallerian degeneration, and point to potential mechanistic targets for accelerating myelin clearance and improving demyelinating disease. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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17. Autophagosome membrane expansion is mediated by the N-terminus and cis -membrane association of human ATG8s.
- Author
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Zhang W, Nishimura T, Gahlot D, Saito C, Davis C, Jefferies HBJ, Schreiber A, Thukral L, and Tooze SA
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- Humans, Autophagy-Related Protein 8 Family genetics, Autophagy-Related Protein 8 Family metabolism, Autophagy physiology, Autophagy-Related Proteins metabolism, Autophagosomes metabolism, Microtubule-Associated Proteins metabolism
- Abstract
Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. By using non-tagged GABARAPs, we show that GABARAP N-terminus and its cis -membrane insertion are crucial to regulate the size of autophagosomes in cells irrespectively of p62 degradation. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8., Competing Interests: WZ, TN, DG, CS, CD, HJ, AS, LT, ST No competing interests declared, (© 2023, Zhang, Nishimura et al.)
- Published
- 2023
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18. ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation.
- Author
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van Vliet AR, Chiduza GN, Maslen SL, Pye VE, Joshi D, De Tito S, Jefferies HBJ, Christodoulou E, Roustan C, Punch E, Hervás JH, O'Reilly N, Skehel JM, Cherepanov P, and Tooze SA
- Subjects
- Cryoelectron Microscopy, Biological Assay, Lipids, Autophagosomes, Autophagy
- Abstract
ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy., Competing Interests: Declaration of interests S.A.T. serves on the scientific advisory board of Casma Therapeutics., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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19. Corrigendum to Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity.
- Author
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Wirth M, Mouilleron S, Zhang W, Sjøttem E, Abudu YP, Jain A, Olsvik HL, Bruun JA, Razi M, Jefferies HBJ, Lee R, Joshi D, O'Reilly N, Johansen T, and Tooze SA
- Published
- 2021
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20. Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity.
- Author
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Wirth M, Mouilleron S, Zhang W, Sjøttem E, Princely Abudu Y, Jain A, Lauritz Olsvik H, Bruun JA, Razi M, Jefferies HBJ, Lee R, Joshi D, O'Reilly N, Johansen T, and Tooze SA
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Apoptosis Regulatory Proteins metabolism, HEK293 Cells, HeLa Cells, Humans, Mammals metabolism, Microtubule-Associated Proteins metabolism, Phosphorylation, Protein Binding, Protein Domains, Protein Serine-Threonine Kinases metabolism, Autophagy-Related Protein 8 Family metabolism, Carrier Proteins chemistry, Carrier Proteins metabolism, Membrane Proteins chemistry, Membrane Proteins metabolism
- Abstract
Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2021
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21. MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma.
- Author
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New M, Van Acker T, Sakamaki JI, Jiang M, Saunders RE, Long J, Wang VM, Behrens A, Cerveira J, Sudhakar P, Korcsmaros T, Jefferies HBJ, Ryan KM, Howell M, and Tooze SA
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Apoptosis, Autophagy-Related Protein-1 Homolog genetics, Autophagy-Related Protein-1 Homolog metabolism, Carcinoma, Pancreatic Ductal genetics, Carcinoma, Pancreatic Ductal metabolism, Cell Proliferation, Humans, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Malate Dehydrogenase antagonists & inhibitors, Malate Dehydrogenase genetics, Membrane Proteins genetics, Pancreatic Neoplasms genetics, Pancreatic Neoplasms metabolism, RNA, Small Interfering genetics, Signal Transduction, Transcription Factors genetics, Transcription Factors metabolism, Tumor Cells, Cultured, YAP-Signaling Proteins, Autophagy, Carcinoma, Pancreatic Ductal pathology, Gene Expression Regulation, Neoplastic, Malate Dehydrogenase metabolism, Membrane Proteins metabolism, Pancreatic Neoplasms pathology
- Abstract
Pancreatic ductal adenocarcinoma (PDAC) is driven by metabolic changes in pancreatic cells caused by oncogenic mutations and dysregulation of p53. PDAC cell lines and PDAC-derived xenografts grow as a result of altered metabolic pathways, changes in stroma, and autophagy. Selective targeting and inhibition of one of these may open avenues for the development of new therapeutic strategies. In this study, we performed a genome-wide siRNA screen in a PDAC cell line using endogenous autophagy as a readout and identified several regulators of autophagy that were required for autophagy-dependent PDAC cell survival. Validation of two promising candidates, MPP7 (MAGUK p55 subfamily member 7, a scaffolding protein involved in cell-cell contacts) and MDH1 (cytosolic Malate dehydrogenase 1), revealed their role in early stages of autophagy during autophagosome formation. MPP7 was involved in the activation of YAP1 (a transcriptional coactivator in the Hippo pathway), which in turn promoted autophagy, whereas MDH1 was required for maintenance of the levels of the essential autophagy initiator serine-threonine kinase ULK1, and increased in the activity upon induction of autophagy. Our results provide a possible explanation for how autophagy is regulated by MPP7 and MDH1, which adds to our understanding of autophagy regulation in PDAC. SIGNIFICANCE: This study identifies and characterizes MPP7 and MDH1 as novel regulators of autophagy, which is thought to be responsible for pancreatic cancer cell survival., (©2019 American Association for Cancer Research.)
- Published
- 2019
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22. Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy.
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Joachim J, Razi M, Judith D, Wirth M, Calamita E, Encheva V, Dynlacht BD, Snijders AP, O'Reilly N, Jefferies HBJ, and Tooze SA
- Subjects
- Apoptosis Regulatory Proteins, HEK293 Cells, Humans, Adaptor Proteins, Signal Transducing metabolism, Autophagy, Centrioles metabolism, Microtubule-Associated Proteins metabolism, Ubiquitination
- Abstract
Autophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2017
- Full Text
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23. Activation of ULK Kinase and Autophagy by GABARAP Trafficking from the Centrosome Is Regulated by WAC and GM130.
- Author
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Joachim J, Jefferies HB, Razi M, Frith D, Snijders AP, Chakravarty P, Judith D, and Tooze SA
- Subjects
- Animals, Apoptosis Regulatory Proteins, Autophagy, Cell Line, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, HCT116 Cells, HEK293 Cells, HeLa Cells, Humans, Mice, Protein Transport, Adaptor Proteins, Signal Transducing metabolism, Autoantigens metabolism, Centrosome metabolism, Membrane Proteins metabolism, Microtubule-Associated Proteins metabolism, Protein Serine-Threonine Kinases metabolism
- Abstract
Starvation-induced autophagy requires activation of the ULK complex at the phagophore. Two Golgi proteins, WAC and GM130, regulate autophagy, however their mechanism of regulation is unknown. In search of novel interaction partners of WAC, we found that GM130 directly interacts with WAC, and this interaction is required for autophagy. WAC is bound to the Golgi by GM130. WAC and GM130 interact with the Atg8 homolog GABARAP and regulate its subcellular localization. GABARAP is on the pericentriolar matrix, and this dynamic pool contributes to autophagosome formation. Tethering of GABARAP to the Golgi by GM130 inhibits autophagy, demonstrating an unexpected role for a golgin. WAC suppresses GM130 binding to GABARAP, regulating starvation-induced centrosomal GABARAP delivery to the phagophore. GABARAP, unlipidated and lipidated, but not LC3B, GABARAPL1, and GATE-16, specifically promotes ULK kinase activation dependent on the ULK1 LIR motif, elucidating a unique non-hierarchical role for GABARAP in starvation-induced activation of autophagy., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
24. Assessing mammalian autophagy.
- Author
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Tooze SA, Dooley HC, Jefferies HB, Joachim J, Judith D, Lamb CA, Razi M, and Wirth M
- Subjects
- Animals, Blotting, Western, Humans, Lysosomes metabolism, Microscopy, Fluorescence, Microtubule-Associated Proteins metabolism, Phagosomes metabolism, Autophagy physiology
- Abstract
Autophagy (self-eating) is a highly conserved, vesicular pathway that cells use to eat pieces of themselves, including damaged organelles, protein aggregates or invading pathogens, for self-preservation and survival (Choi et al., N Engl J Med 368:651-662, 2013; Lamb et al., Nat Rev Mol Cell Biol 14:759-774, 2013). Autophagy can be delineated into three major vesicular compartments (the phagophore, autophagosome, autolysosome, see Fig. 1). The initial stages of the pathway involve the formation of phagophores (also called isolation membranes), which are open, cup-shaped membranes that expand and sequester the cytosolic components, including organelles and aggregated proteins or intracellular pathogens. Closure of the phagophore creates an autophagosome, which is a double-membrane vesicle. Fusion of the autophagosome with the lysosome, to form an autolysosome, delivers the content of the autophagosome into the lysosomal lumen and allows degradation to occur.Autophagy is a dynamic process that is initiated within 15 min of amino acid starvation in cell culture systems (Köchl et al., Traffic 7:129-145, 2006) and is likely to occur as rapidly in vivo (Mizushima et al., J Cell Biol 152:657-668, 2001). To initiate studies on the formation of the autophagosomes, and trafficking to and from the autophagic pathway, an ideal starting approach is to do a morphological analysis in fixed cells. Additional validation of the morphological data can be obtained using simple Western blot analysis. Here we describe the most commonly used morphological technique to study autophagy, in particular, using the most reliable marker, microtubule-associated protein 1A/1B-light chain 3 (LC3). In addition, we describe a second immunofluorescence assay to determine if autophagy is being induced, using an antibody to WD repeat domain, phosphoinositide interacting 2 (WIPI2), an effector of the phosphatidylinositol (3)-phosphate (PI3P) produced during autophagosome formation.
- Published
- 2015
- Full Text
- View/download PDF
25. In vitro reconstitution of fusion between immature autophagosomes and endosomes.
- Author
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Morvan J, Köchl R, Watson R, Collinson LM, Jefferies HB, and Tooze SA
- Subjects
- Animals, Autophagy drug effects, Biological Assay, Cytosol ultrastructure, Endocytosis drug effects, Endosomes drug effects, Endosomes ultrastructure, Ethylmaleimide pharmacology, Humans, Immunoprecipitation, Microtubule-Associated Proteins metabolism, Nucleotides pharmacology, PC12 Cells, Phagosomes drug effects, Phagosomes ultrastructure, Protein Transport drug effects, Rats, Temperature, Vacuoles drug effects, Vacuoles metabolism, Vacuoles ultrastructure, Endosomes metabolism, Membrane Fusion drug effects, Phagosomes metabolism
- Abstract
Autophagy is a highly conserved degradative pathway whereby a double membrane engulfs cytoplasmic constituents to form an autophagic vacuole or autophagosome. An essential requirement for efficient autophagy is the acquisition of an adequate degradative capacity by the autophagosomes. To acquire this capacity the immature autophagic vacuoles (AVis) obtain lysosomal hydrolases by fusion with endosomes. The current models suggest that at least two types of endosomes, early and late, fuse with AVis to form mature, degradative AVds. This fusion and maturation requires proteins also involved in endosome maturation such as Rab7. However, it is not known if there are molecular requirements unique to AVi-endosome fusion. To identify and investigate the molecular requirements of this fusion we developed a cell-free fusion assay based on content mixing, which occurs after fusion of isolated AVis and different endosomal fractions. Our assay shows that isolated AVis can fuse to a similar extent in vitro with both early and late endosomes. Furthermore, fusion between autophagosomes and endosomes requires cytosolic and endosomal proteins, but does not show a nucleotide-dependence, and is partially N-ethylmaleimide sensitive. We also demonstrate that the lipidated form of the autophagosomal protein LC3 is dispensable for this fusion event.
- Published
- 2009
- Full Text
- View/download PDF
26. A selective PIKfyve inhibitor blocks PtdIns(3,5)P(2) production and disrupts endomembrane transport and retroviral budding.
- Author
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Jefferies HB, Cooke FT, Jat P, Boucheron C, Koizumi T, Hayakawa M, Kaizawa H, Ohishi T, Workman P, Waterfield MD, and Parker PJ
- Subjects
- Aminopyridines chemistry, Animals, Biological Transport drug effects, Biomarkers metabolism, Endosomes drug effects, Endosomes metabolism, Enzyme Inhibitors chemistry, Heterocyclic Compounds, 3-Ring chemistry, Lysosomes drug effects, Lysosomes metabolism, Mice, NIH 3T3 Cells, Aminopyridines pharmacology, Cell Membrane drug effects, Cell Membrane metabolism, Enzyme Inhibitors pharmacology, Heterocyclic Compounds, 3-Ring pharmacology, Phosphatidylinositol Phosphates biosynthesis, Phosphotransferases (Alcohol Group Acceptor) antagonists & inhibitors, Retroviridae drug effects, Retroviridae metabolism
- Abstract
Phosphoinositides have crucial roles in cellular controls, many of which have been established through the use of small-molecule inhibitors. Here, we describe YM201636, a potent inhibitor of the mammalian class III phosphatidylinositol phosphate kinase PIKfyve, which synthesizes phosphatidylinositol 3,5-bisphosphate. Acute treatment of cells with YM201636 shows that the PIKfyve pathway is involved in the sorting of endosomal transport, with inhibition leading to the accumulation of a late endosomal compartment and blockade of retroviral exit. Inhibitor specificity is shown by the use of short interfering RNA against the target, as well as by rescue with the drug-resistant yeast orthologue Fab1. We concluded that the phosphatidylinositol 3,5-bisphosphate pathway is integral to endosome formation, determining morphology and cargo flux.
- Published
- 2008
- Full Text
- View/download PDF
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