A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B

Pengo, Niccolò; Agrotis, Alexander; Prak, Krisna; Jones, Joe M.; Ketteler, Robin

doi:10.1038/s41467-017-00303-2

Cited by 126 publications

(121 citation statements)

References 40 publications

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“…Spatial and temporal regulation of recruitment and dissociation of LC3 family proteins to and from autophagosomes is achieved through regulation of ATG4 activity [13][14][15][16][17]. Spatial and temporal regulation of recruitment and dissociation of LC3 family proteins to and from autophagosomes is achieved through regulation of ATG4 activity [13][14][15][16][17].…”

Section: Discussionmentioning

confidence: 99%

“…Spatial and temporal regulation of recruitment and dissociation of LC3 family proteins to and from autophagosomes is achieved through regulation of ATG4 activity [13][14][15][16][17]. This regulation is achieved through the phosphorylation and dephosphorylation of ATG4 itself [15,16] and the phosphorylation of LC3s by TBK1. This suggests that LC3-II conjugated to autophagosomes is protected from premature de-lipidation by a timely regulatory mechanism.…”

Section: Discussionmentioning

confidence: 99%

“…LC3 proteins undergo two processing steps, (i) an initial proteolytic cleavage of the peptide bond responsible for the conversion of pro-LC3 to active LC3 (LC3-I) and (ii) the subsequent cleavage of the amide bond for de-lipidation of LC3-II from autophagosomes to regenerate a free cytosolic LC3 pool. Phosphorylation of ATG4B at S383/392 increases its protease activity [14], especially during the LC3 de-lipidation phase, whereas ULK1-mediated phosphorylation of ATG4B at S316 (in humans) [15] or at S307 (in yeast) [16] reduces pro-LC3 binding and C-terminal tail cleavage. Among the four mammalian paralogs of ATG4, ATG4B is the most active protease followed by ATG4A and ATG4C/D, which exhibit minimal protease activity [10,11].…”

Section: Introductionmentioning

confidence: 99%

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TBK1‐mediated phosphorylation of LC3C and GABARAP‐L2 controls autophagosome shedding by ATG4 protease

et al. 2019

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Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surfaceexposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autophagosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation.

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Section: Discussionmentioning

confidence: 99%

“…Spatial and temporal regulation of recruitment and dissociation of LC3 family proteins to and from autophagosomes is achieved through regulation of ATG4 activity [13][14][15][16][17]. This regulation is achieved through the phosphorylation and dephosphorylation of ATG4 itself [15,16] and the phosphorylation of LC3s by TBK1. This suggests that LC3-II conjugated to autophagosomes is protected from premature de-lipidation by a timely regulatory mechanism.…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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TBK1‐mediated phosphorylation of LC3C and GABARAP‐L2 controls autophagosome shedding by ATG4 protease

et al. 2019

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“…Under nutrient sufficiency, mTOR prevents ULK1 activation; whereas, when mTOR is inhibited, ULK1 is activated and phosphorylates Atg13 and FIP200 to continue autophagy processes [42,43]. Additional evidence has shown ULK1 regulates autophagy protease ATG4B affecting downstream LC3B [44,45]. Further studies are necessary to determine how ULK1 affects the mTOR pathway in skeletal muscle turnover and the direct effects of n-3 and n-6 intermediates on the regulation of autophagy.…”

Section: Discussionmentioning

confidence: 99%

Delta-6-desaturase (FADS2) inhibition and omega-3 fatty acids in skeletal muscle protein turnover

Brown

Sharma

Baker

et al. 2019

Biochemistry and Biophysics Reports

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Polyunsaturated fatty acids (PUFAs) are essential dietary components. They are not only used for energy, but also act as signaling molecules. The delta-6 desaturase (D6D) enzyme, encoded by the FADS2 gene, is one of two rate limiting enzymes that convert the PUFA precursors – α-linolenic (n-3) and linoleic acid (n-6) to their respective metabolites. Alterations in the D6D enzyme activity alters fatty acid profiles and are associated with metabolic and inflammatory diseases including cardiovascular disease and type 2 diabetes. Omega-3 PUFAs, specifically its constituent fatty acids DHA and EPA, are known for their anti-inflammatory ability and are also beneficial in the prevention of skeletal muscle wasting, however the mechanism for muscle preservation is not well understood. Moreover, little is known of the effects of altering the n-6/n-3 ratio in the context of a high-fat diet, which is known to downregulate protein synthesis. Twenty C57BL6 male mice were fed a high-fat lard (HFL, 45% fat (mostly lard), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 13:1) diet for 6 weeks. Mice were then divided into 4 groups (n = 5 per group): HFL– , high-fat oil– (HFO, 45% fat (mostly Menhaden oil), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 1:3), HFL+ (HFL diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day), and HFO+ (HFO diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day). After 2 weeks on their respective diets and treatments, animals were sacrificed and gastrocnemius muscle harvested. Protein turnover signaling were analyzed via Western Blot. 4-EBP1 and ribosomal protein S6 expression were measured. A two-way ANOVA revealed no significant change in the phosphorylation of both 4EBP-1 and ribosomal protein S6 with diet or inhibitor. There was a significant reduction in STAT3 phosphorylation with the inhibition of FADS2 (p = 0.03). Additionally, we measured markers of protein degradation through levels of FOXO phosphorylation, ubiquitin, and LC3B expression; there was a trend towards increased phosphorylation of FOXO (p = 0.08) and ubiquitinated proteins (p = 0.05) with FADS2 inhibition. LC3B expression, a marker of autophagy, was significantly higher in the HFL plus FADS2 inhibition group from all other comparisons. Lastly, we analyzed activation of mitochondrial biogenesis which is closely linked with protein synthesis through PGC1-α and Cytochrome-C expression, however no significant differences were associated with either marker across all groups. Collectively, these data suggest that the protective effects of muscle mass by omega-3 fatty acids are from inhibition of protein degradation. Our aim was to determine the role of PUFA metabolites, DHA and EPA, in skeletal muscle protein turnover and assess the effects of n-3s independently. We observed that by inhibiting the FADS2 enzyme, the protective effect of n-3s on protein synthesis and proliferation was lost; concomitantly, protein degradation was increased with FADS2 inhibition regardless of diet.

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“…It is emerging that the activity of ATG4B is tightly controlled by post-translational modifications, including phosphorylation mediated by ATG1/ULK1 [12,13], AKT1 [14], and serine/threonine protein kinase MST4 [15]. For instance, phosphorylation of ATG4B by ULK1 on serine 316 results in a reduction of LC3-II hydrolysis [12], allowing the autophagosome to close before premature deconjugation of LC-II from the autophagosome membrane. Similarly, members of the LC3/GABARAP family can be phosphorylated by Tank-binding kinase 1 (TBK1), which renders them resistant to ATG4-mediated deconjugation [16].…”

Section: Introductionmentioning

confidence: 99%

On ATG4B as Drug Target for Treatment of Solid Tumours—The Knowns and the Unknowns

Agrotis

Ketteler

2019

Cells

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Autophagy is an evolutionary conserved stress survival pathway that has been shown to play an important role in the initiation, progression, and metastasis of multiple cancers; however, little progress has been made to date in translation of basic research to clinical application. This is partially due to an incomplete understanding of the role of autophagy in the different stages of cancer, and also to an incomplete assessment of potential drug targets in the autophagy pathway. While drug discovery efforts are on-going to target enzymes involved in the initiation phase of the autophagosome, e.g., unc51-like autophagy activating kinase (ULK)1/2, vacuolar protein sorting 34 (Vps34), and autophagy-related (ATG)7, we propose that the cysteine protease ATG4B is a bona fide drug target for the development of anti-cancer treatments. In this review, we highlight some of the recent advances in our understanding of the role of ATG4B in autophagy and its relevance to cancer, and perform a critical evaluation of ATG4B as a druggable cancer target.

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A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B

Cited by 126 publications

References 40 publications

TBK1‐mediated phosphorylation of LC3C and GABARAP‐L2 controls autophagosome shedding by ATG4 protease

TBK1‐mediated phosphorylation of LC3C and GABARAP‐L2 controls autophagosome shedding by ATG4 protease

Delta-6-desaturase (FADS2) inhibition and omega-3 fatty acids in skeletal muscle protein turnover

On ATG4B as Drug Target for Treatment of Solid Tumours—The Knowns and the Unknowns

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