Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3

Farnung, Jakob; Muhar, Matthias; Liang, Jin Rui; Tolmachova, Kateryna A.; Benoit, Roger; Corn, Jacob E.; Bode, Jeffrey W.

doi:10.1021/acscentsci.3c00009

Cited by 7 publications

(4 citation statements)

References 54 publications

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“…S1C). The AlphaFold-predicted intermolecular β sheet between ATG3 residues 95 to 110 and β2 of LC3 in our structural model is consistent with the presence of a noncanonical LIR motif in the flexible region of ATG3, which was recently shown to be required for LC3 lipidation in cellulo ( 37 ). Combined with crystallographic structures ( 21 , 38 ) of ATG12-ATG5 in quaternary complex with a bound fragment of ATG3 and the N-terminal ATG5-binding domain of ATG16L1, we present an atomistic model of the full LC3 lipidation machinery consisting of the E3-like ATG12–ATG5-ATG16L1 complex bound to the E2-substrate conjugate, ATG3-LC3 ( Fig.…”

Section: Resultssupporting

confidence: 87%

Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore

Rao,

Skulsuppaisarn,

Strong

et al. 2024

Sci. Adv.

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The covalent attachment of ubiquitin-like LC3 proteins (microtubule-associated proteins 1A/1B light chain 3) prepares the autophagic membrane for cargo recruitment. We resolve key steps in LC3 lipidation by combining molecular dynamics simulations and experiments in vitro and in cellulo. We show how the E3-like ligaseautophagy-related 12 (ATG12)–ATG5-ATG16L1 in complex with the E2-like conjugase ATG3 docks LC3 onto the membrane in three steps by (i) the phosphatidylinositol 3-phosphate effector protein WD repeat domain phosphoinositide-interacting protein 2 (WIPI2), (ii) helix α2 of ATG16L1, and (iii) a membrane-interacting surface of ATG3. Phosphatidylethanolamine (PE) lipids concentrate in a region around the thioester bond between ATG3 and LC3, highlighting residues with a possible role in the catalytic transfer of LC3 to PE, including two conserved histidines. In a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12–ATG5-ATG16L1 machinery establishes a high level of regulatory control.

show abstract

Section: Resultssupporting

confidence: 87%

Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore

Rao,

Skulsuppaisarn,

Strong

et al. 2024

Sci. Adv.

View full text Add to dashboard Cite

show abstract

“…S1C). The AlphaFoldpredicted intermolecular β sheet between ATG3 residues 95 to 110 and β2 of LC3 in our structural model is consistent with the presence of a noncanonical LIR motif in the flexible region of ATG3, which was recently shown to be required for LC3 lipidation in cellulo (37). Combined with crystallographic structures (21,38) of ATG12-ATG5 in quaternary complex with a bound fragment of ATG3 and the N-terminal ATG5-binding domain of ATG16L1, we present an atomistic model of the full LC3 lipidation machinery consisting of the E3-like ATG12-ATG5-ATG16L1 complex bound to the E2-substrate conjugate, ATG3-LC3 (Fig.…”

Section: Docking Step 1: Wipi2 Recruits Atg12-atg5-atg16l1 Loaded Wit...supporting

confidence: 88%

Three-step docking by WIPI2, ATG16L1 and ATG3 delivers LC3 to the phagophore

Rao

Strong

Ren

et al. 2023

Preprint

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The covalent attachment of ubiquitin-like LC3 proteins prepares the autophagic membrane for cargo recruitment. We resolve key steps in LC3 lipidation by combining molecular dynamics simulations and experimentsin vitroandin cellulo. We show how the E3-like ligase ATG12--ATG5-ATG16L1 in complex with the E2-like conjugase ATG3 docks LC3 onto the membrane in three steps by (1) the PI(3)P effector protein WIPI2, (2) helix α2 of ATG16L1, and (3) a membrane-interacting surface of ATG3. Phosphatidylethanolamine (PE) lipids concentrate in a region around the thioester bond between ATG3 and LC3, highlighting residues with a possible role in the catalytic transfer of LC3 to PE, including two conserved histidines. In a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12--ATG5-ATG16L1 machinery establishes a high level of regulatory control.

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“…Taken together, our results establish a conceptually new family of chemical tools for time-resolved studies on protein ubiquitination in live cells and further exemplify the utility of chemically synthesized customized probes that are more readily synthesizable and are of different structural designs for the study of Ub modifications. − It should be mentioned that although the current photocaging group, Nbg, requires 365 nm of UV light and may cause cytotoxicity in some experiments, our probe can be easily extended to use more benign photosensitive groups that would meet the requirements for specific biological conditions.…”

Section: Discussionmentioning

confidence: 62%

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells

Liang,

Wang,

Hua

et al. 2023

JACS Au

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Dynamic monitoring of intracellular ubiquitin (Ub) conjugates is instrumental to understanding the Ub regulatory machinery. Although many biochemical approaches have been developed to characterize protein ubiquitination, chemical tools capable of temporal resolution probing of ubiquitination events remain to be developed. Here, we report the development of the first cell-permeable and stimuli-responsive Ub probe and its application for the temporal resolution profiling of ubiquitinated substrates in live cells. The probe carrying the photolabile group N-(2-nitrobenzyl)-Gly (Nbg) on the amide bond between Ub Gly75 and Gly76 is readily prepared through chemical synthesis and can be delivered to live cells by conjugation via a disulfide bond with the cyclic cell-penetrating peptide cR10D (i.e., 4-((4-(dimethylamino)phenyl)-azo)-benzoic acid-modified cyclic deca-arginine). Both in vitro and in vivo experiments showed that Ub-modifying enzymes (E1, E2s, and E3s) could not install the Ub probe onto substrate proteins prior to removal of the nitrobenzyl group, which was easily accomplished via photoirradiation. The utility and practicality of this probe were exemplified by the timeresolved biochemical and proteomic investigation of ubiquitination events in live cells during a H 2 O 2 -mediated oxidative stress response. This work shows a conceptually new family of chemical Ub tools for the time-resolved studies on dynamic protein ubiquitination in different biological processes and highlights the utility of modern chemical protein synthesis in obtaining customdesigned tools for biological studies.

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Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3

Cited by 7 publications

References 54 publications

Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore

Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore

Three-step docking by WIPI2, ATG16L1 and ATG3 delivers LC3 to the phagophore

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells

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