Autophagy-regulating protease Atg4: structure, function, regulation and inhibition.
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ABSTRACT:
SUBMITTER: Maruyama T
PROVIDER: S-EPMC5799747 | biostudies-literature | 2017 Sep
REPOSITORIES: biostudies-literature
Publications
Autophagy-regulating protease Atg4: structure, function, regulation and inhibition.
Maruyama Tatsuro T Noda Nobuo N NN
The Journal of antibiotics 20170913
Autophagy is an intracellular degradation system that contributes to cellular homeostasis through degradation of various targets such as proteins, organelles and microbes. Since autophagy is related to various diseases such as infection, neurodegenerative diseases and cancer, it is attracting attention as a new therapeutic target. Autophagy is mediated by dozens of autophagy-related (Atg) proteins, among which Atg4 is the sole protease that regulates autophagy through the processing and deconjug ...[more]
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Project description:Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets. The papain-like protease of coronaviruses has been less explored than other viral proteins; however, its substantive role in viral replication and impact on the host immune response make it a suitable target to study. This review article focuses on the structure and function of the papain-like protease (PLpro ) of SARS-CoV-2, including variants of concern, and compares it to those of other coronaviruses, such as SARS-CoV-1 and MERS-CoV. The protease's recognition motif is mirrored in ubiquitin and ISG15, which are involved in the antiviral immune response. Inhibitors, including GRL0617 derivatives, and their prospects as potential future antiviral agents are also discussed.
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Project description:Hydrogen sulfide is a signaling molecule that regulates essential processes for plant performance, such as autophagy. In Arabidopsis thaliana, hydrogen sulfide negatively regulates autophagy independently of reactive oxygen species, but the underlying mechanism remains to be understood. To determine whether persulfidation, an emergent posttranslational modification, has a main role in the control of autophagy by sulfide, we have used a proteomic approach targeting ATG4, a cysteine protease that plays a crucial role in autophagy progression. We showed that AtATG4a from A. thaliana, which is the predominant ATG4 protease in this plant species, contains a specific site for persulfidation, the residue Cys170, included in the characteristic catalytic triad Cys-His-Asp of cysteine proteases. We tested whether persulfidation regulates the activity of ATG4 by setting up a heterologous assay using the Chlamydomonas reinhardtii CrATG8 protein as a substrate. Our findings demonstrate that sulfide significantly inactivates AtATG4a cleavage activity in a reversible manner. The biological significance of the reversible inhibition of the ATG4 protease by sulfide is supported by our findings in Arabidopsis leaves under both basal and autophagy-inducing conditions. We also observed a significant increase in the overall ATG4 activity in Arabidopsis under nitrogen starvation and osmotic stress, which is inhibited by sulfide. Therefore, our data strongly suggest that the negative regulation of autophagy by sulfide is mediated, at least, by the specific persulfidation of the protease ATG4.
2020-12-14 | PXD019802 | Pride
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Project description:Autophagy is a mechanism responsible for intracellular degradation and recycling of macromolecules and organelles, essential for cell survival in adverse conditions. More than 40 autophagy-related (ATG) genes have been identified and characterized in fungi, among them ATG4 and ATG8. ATG4 encodes a cysteine protease (Atg4) that plays an important role in autophagy by initially processing Atg8 at its C-terminus region. Atg8 is a ubiquitin-like protein essential for the synthesis of the double-layer membrane that constitutes the autophagosome vesicle, responsible for delivering the cargo from the cytoplasm to the vacuole lumen. The contributions of Atg-related proteins in the pathogenic yeast in the genus Cryptococcus remain to be explored, to elucidate the molecular basis of the autophagy pathway. In this context, we aimed to investigate the role of autophagy-related proteins 4 and 8 (Atg4 and Atg8) during autophagy induction and their contribution with non-autophagic events in C. neoformans. We found that Atg4 and Atg8 are conserved proteins and that they interact physically with each other. ATG gene deletions resulted in cells sensitive to nitrogen starvation. ATG4 gene disruption affects Atg8 degradation and its translocation to the vacuole lumen, after autophagy induction. Both atg4 and atg8 mutants are more resistant to oxidative stress, have an impaired growth in the presence of the cell wall-perturbing agent Congo Red, and are sensitive to the proteasome inhibitor bortezomib (BTZ). By that, we conclude that in C. neoformans the autophagy-related proteins Atg4 and Atg8 play an important role in the autophagy pathway; which are required for autophagy regulation, maintenance of amino acid levels and cell adaptation to stressful conditions.
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Project description:Autophagy is a highly conserved biological process during which double membrane bound autophagosomes carry intracellular cargo material to the vacuole or lysosome for degradation and/or recycling. Autophagosome biogenesis requires Autophagy 4 (Atg4) cysteine protease-mediated processing of ubiquitin-like Atg8 proteins. Unlike single Atg4 and Atg8 genes in yeast, the Arabidopsis genome contains two Atg4 (AtAtg4a and AtAtg4b) and nine Atg8 (AtAtg8a-AtAtg8i) genes. However, we know very little about specificity of different AtAtg4s for processing of different AtAtg8s. Here, we describe a unique bioluminescence resonance energy transfer-based AtAtg8 synthetic substrate to assess AtAtg4 activity in vitro and in vivo. In addition, we developed a unique native gel assay of superhRLUC catalytic activity assay to monitor cleavage of AtAtg8s in vitro. Our results indicate that AtAtg4a is the predominant protease and that it processes AtAtg8a, AtAtg8c, AtAtg8d, and AtAtg8i better than AtAtg4b in vitro. In addition, kinetic analyses indicate that although both AtAtg4s have similar substrate affinity, AtAtg4a is more active than AtAtg4b in vitro. Activity of AtAtg4s is reversibly inhibited in vitro by reactive oxygen species such as H2O2. Our in vivo bioluminescence resonance energy transfer analyses in Arabidopsis transgenic plants indicate that the AtAtg8 synthetic substrate is efficiently processed and this is AtAtg4 dependent. These results indicate that the synthetic AtAtg8 substrate is used efficiently in the biogenesis of autophagosomes in vivo. Transgenic Arabidopsis plants expressing the AtAtg8 synthetic substrate will be a valuable tool to dissect autophagy processes and the role of autophagy during different biological processes in plants.
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