| 1. |
- Aftab, Obaid, 1984-, et al.
(author)
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Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter
- 2014
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In: Autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 10:1, s. 57-69
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Journal article (peer-reviewed)abstract
- Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.
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| 2. |
- Agostinis, P, et al.
(author)
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Autophagy researchers
- 2014
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 10:9, s. 1483-1486
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Journal article (other academic/artistic)
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| 6. |
- Brattås, Per Ludvik, et al.
(author)
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Impact of differential and time-dependent autophagy activation on therapeutic efficacy in a model of Huntington disease
- 2021
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In: Autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 17:6, s. 1316-1329
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Journal article (peer-reviewed)abstract
- Activation of macroautophagy/autophagy, a key mechanism involved in the degradation and removal of aggregated proteins, can successfully reverse Huntington disease phenotypes in various model systems. How neuronal autophagy impairments need to be considered in Huntington disease progression to achieve a therapeutic effect is currently not known. In this study, we used a mouse model of HTT (huntingtin) protein aggregation to investigate how different methods and timing of autophagy activation influence the efficacy of autophagy-activating treatment in vivo. We found that overexpression of human TFEB, a master regulator of autophagy, did not decrease mutant HTT aggregation. On the other hand, Becn1 overexpression, an autophagic regulator that plays a key role in autophagosome formation, partially cleared mutant HTT aggregates and restored neuronal pathology, but only when administered early in the disease progression. When Becn1 was administered at a later stage, when prominent mutant HTT accumulation and autophagy impairments have occurred, Becn1 overexpression did not rescue the mutant HTT-associated phenotypes. Together, these results demonstrate that the targets used to activate autophagy, as well as the timing of autophagy activation, are crucial for achieving efficient therapeutic effects.
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| 8. |
- Camougrand, N, et al.
(author)
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Autophagy researchers
- 2013
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 9:5, s. 635-638
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Journal article (peer-reviewed)
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| 10. |
- Corkery, Dale P., et al.
(author)
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ATG12–ATG5-TECPR1 : an alternative E3-like complex utilized during the cellular response to lysosomal membrane damage
- 2024
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In: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 20:2, s. 443-444
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Journal article (peer-reviewed)abstract
- ATG16L1 is an essential component of the Atg8-family protein conjugation machinery, providing membrane targeting for the ATG12–ATG5 conjugate. Recently, we identified an alternative E3-like complex that functions independently of ATG16L1. This complex utilizes the autophagosome-lysosome tethering factor TECPR1 for membrane targeting. TECPR1 is recruited to damaged lysosomal membranes via a direct interaction with sphingomyelin. At the damaged membrane, TECPR1 assembles into an E3-like complex with ATG12–ATG5 to regulate unconventional LC3 lipidation and promote efficient lysosomal repair.
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| 11. |
- Corkery, Dale P., et al.
(author)
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Eating while intoxicated : characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy
- 2023
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In: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 19:6, s. 1885-1886
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Journal article (peer-reviewed)abstract
- Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.
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| 13. |
- Cuervo, Ana Maria, et al.
(author)
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Autophagy and aging - The importance of maintaining "clean" cells
- 2005
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In: autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 1:3, s. 131-140
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Research review (peer-reviewed)abstract
- A decrease in the turnover of cellular components and the intracellular accumulation of altered macromolecules and organelles are features common to all aged cells. Diminished autophagic activity plays a major role in these age-related manifestations. In this work we review the molecular defects responsible for the malfunctioning of two forms of autophagy, macroautophagy and chaperone-mediated outophagy, in old mammals, and highlight general and cell-type specific consequences of dysfunction of the autophagic system with age. Dietary caloric restriction and antilipolytic agents have been proven to efficiently stimulate autophagy in old rodents. These and other possible experimental restorative efforts are discussed.
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| 14. |
- Dahmane, Selma, et al.
(author)
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A 3D view of how enteroviruses hijack autophagy
- 2023
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In: Autophagy. - : Taylor & Francis Group. - 1554-8627 .- 1554-8635. ; 19:7, s. 2156-2158
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Journal article (peer-reviewed)abstract
- Viruses are masters at using cellular pathways to aid their replication. Cryo-electron tomography of poliovirus-infected cells revealed how it utilizes macroautophagy to its advantage. Assembly of these non-enveloped virions takes place directly on membranes and requires PIK3C3/VPS34 activity to be completed, whereas the canonical autophagy inducer ULK1 restricts virus assembly. The tomograms further revealed that enterovirus-induced autophagy is selective for RNA-loaded virions, which may help ensure maximum infectivity of the virus-laden vesicles released through secretory autophagy.
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| 15. |
- Decressac, Mickael, et al.
(author)
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TFEB: Pathogenic role and therapeutic target in Parkinson disease.
- 2013
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 9:8, s. 1244-1246
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Journal article (peer-reviewed)abstract
- Parkinson disease (PD) is characterized by the progressive loss of nigral dopamine neurons and the presence of accumulations containing the disease-causing protein SNCA/α-synuclein. Here we review our recent findings describing how SNCA impairs the function of the master regulator of the autophagy-lysosomal pathway (ALP), the transcription factor EB (TFEB), and that genetic or pharmacological stimulation of its activity promotes protection of dopamine neurons. These findings suggest that strategies aimed at enhancing autophagy-mediated degradation of SNCA may hold great promise for disease intervention in PD.
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| 21. |
- Fiesel, Fabienne C., et al.
(author)
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Substitution of PINK1 Gly411 modulates substrate receptivity and turnover
- 2023
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In: Autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 19:6, s. 1711-1732
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Journal article (peer-reviewed)abstract
- The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development. Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.
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| 25. |
- Giovannucci, Tatiana A., et al.
(author)
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Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy
- 2022
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In: Autophagy. - : Taylor & Francis. - 1554-8627 .- 1554-8635. ; 18:7, s. 1486-1502
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Journal article (peer-reviewed)abstract
- The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.
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