Atg8-family Proteins Research Articles

TP53INP2 mediates autophagic degradation of ubiquitinated proteins through its ubiquitin-interacting motif.

The tumor protein p53-inducible nuclear protein 2 (TP53INP2) has been reported to participate in autophagy by interacting with autophagosome-localized autophagy-related protein 8 (Atg8) family proteins, including LC3. Here, we uncover a novel function for TP53INP2 in the autophagic degradation of proteins. We identify the ubiquitin-interacting motif (UIM) of TP53INP2 that mediates its binding to ubiquitin and ubiquitinated proteins. TP53INP2 lacking the UIM is able to displace autophagic adaptor p62 from LC3, which leads to accumulation of ubiquitinated proteins in cells. Furthermore, overexpression of TP53INP2 lacking the UIM sensitizes cells to chloroquine treatment. Our findings indicate that TP53INP2 may act as a novel autophagic adaptor through recruiting ubquitinated substrates to autophagosomes for degradation.

An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5

ABSTRACTShort linear motifs, known as LC3-interacting regions (LIRs), interact with mactoautophagy/autophagy modifiers (Atg8/LC3/GABARAP proteins) via a conserved universal mechanism. Typically, this includes the occupancy of 2 hydrophobic pockets on the surface of Atg8-family proteins by 2 specific aromatic and hydrophobic residues within the LIR motifs. Here, we describe an alternative mechanism of Atg8-family protein interaction with the non-canonical UBA5 LIR, an E1-like enzyme of the ufmylation pathway that preferentially interacts with GABARAP but not LC3 proteins. By solving the structures of both GABARAP and GABARAPL2 in complex with the UBA5 LIR, we show that in addition to the binding to the 2 canonical hydrophobic pockets (HP1 and HP2), a conserved tryptophan residue N-terminal of the LIR core sequence binds into a novel hydrophobic pocket on the surface of GABARAP proteins, which we term HP0. This mode of action is unique for UBA5 and accompanied by large rearrangements of key residues including the side chains of the gate-keeping K46 and the adjacent K/R47 in GABARAP proteins. Swapping mutations in LC3B and GABARAPL2 revealed that K/R47 is the key residue in the specific binding of GABARAP proteins to UBA5, with synergetic contributions of the composition and dynamics of the loop L3. Finally, we elucidate the physiological relevance of the interaction and show that GABARAP proteins regulate the localization and function of UBA5 on the endoplasmic reticulum membrane in a lipidation-independent manner.Abbreviations: ATG: AuTophaGy-related; EGFP: enhanced green fluorescent protein; GABARAP: GABA-type A receptor-associated protein; ITC: isothermal titration calorimetry; KO: knockout; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NMR: nuclear magnetic resonance; RMSD: root-mean-square deviation of atomic positions; TKO: triple knockout; UBA5: ubiquitin like modifier activating enzyme 5

Members of the autophagy class III phosphatidylinositol 3-kinase complex I interact with GABARAP and GABARAPL1 via LIR motifs

ABSTRACT Autophagosome formation depends on a carefully orchestrated interplay between membrane-associated protein complexes. Initiation of macroautophagy/autophagy is mediated by the ULK1 (unc-51 like autophagy activating kinase 1) protein kinase complex and the autophagy-specific class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1). The latter contains PIK3C3/VPS34, PIK3R4/VPS15, BECN1/Beclin 1 and ATG14 and phosphorylates phosphatidylinositol to generate phosphatidylinositol 3-phosphate (PtdIns3P). Here, we show that PIK3C3, BECN1 and ATG14 contain functional LIR motifs and interact with the Atg8-family proteins with a preference for GABARAP and GABARAPL1. High resolution crystal structures of the functional LIR motifs of these core components of PtdIns3K-C1were obtained. Variation in hydrophobic pocket 2 (HP2) may explain the specificity for the GABARAP family. Mutation of the LIR motif in ATG14 did not prevent formation of the PtdIns3K-C1 complex, but blocked colocalization with MAP1LC3B/LC3B and impaired mitophagy. The ULK-mediated phosphorylation of S29 in ATG14 was strongly dependent on a functional LIR motif in ATG14. GABARAP-preferring LIR motifs in PIK3C3, BECN1 and ATG14 may, via coincidence detection, contribute to scaffolding of PtdIns3K-C1 on membranes for efficient autophagosome formation. Abbreviations ATG: autophagy-related; BafA1: bafilomycin A1; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GFP: enhanced green fluorescent protein; KO: knockout; LDS: LIR docking site; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1/p62: sequestosome 1; VPS: Vacuolar protein sorting; ULK: unc-51 like autophagy activating kinase

NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy.

Parkin-mediated mitophagy is a quality control pathway that selectively removes damaged mitochondria via the autophagic machinery. Autophagic receptors, which interact with ubiquitin and Atg8 family proteins, contribute to the recognition of damaged mitochondria by autophagosomes. NDP52, an autophagy receptor, is required for autophagic engulfment of damaged mitochondria during mitochondrial uncoupler treatment. The N-terminal SKICH domain and C-terminal zinc finger motif of NDP52 are both required for its function in mitophagy. While the zinc finger motif contributes to poly-ubiquitin binding, the function of the SKICH domain remains unclear. Here, we show that NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP) via the SKICH domain. During mitophagy, NDP52 invades depolarized mitochondria and interacts with MTPAP dependent on the proteasome but independent of ubiquitin binding. Loss of MTPAP reduces NDP52-mediated mitophagy, and the NDP52-MTPAP complex attracts more LC3 than NDP52 alone. These results indicate that NDP52 and MTPAP form an autophagy receptor complex, which enhances autophagic elimination of damaged mitochondria.

Human ubiquitin-like proteins as central coordinators in autophagy.

Autophagy is one of the most versatile recycling systems of eukaryotic cells. It degrades diverse cytoplasmic components such as organelles, protein aggregates, ribosomes and multi-enzyme complexes. Not surprisingly, any failure of autophagy or reduced activity of the pathway contributes to the onset of various pathologies, including neurodegeneration, cancer and metabolic disorders such as diabetes or immune diseases. Furthermore, autophagy contributes to the innate immune response and combats bacterial or viral pathogens. The hallmark of macroautophagy is the formation of a membrane sack that sequesters cytoplasmic cargo and delivers it to lysosomes for degradation. More than 40 autophagy-related (ATG) proteins have so far been identified. A unique protein-conjugation system represents one of the core components of this highly elaborate machinery. It conjugates six homologous ATG8 family proteins to the autophagic membrane. In this review, we summarize the current knowledge regarding the various functions of ATG8 proteins in autophagy and briefly discuss how physical approaches and in vitro reconstitution contributed in deciphering their function.

Potent and specific Atg8-targeting autophagy inhibitory peptides from giant ankyrins.

The mammalian Atg8 family proteins are central drivers of autophagy and contain six members, classified into the LC3 and GABARAP subfamilies. Due to their high sequence similarity and consequent functional overlaps, it is difficult to delineate specific functions of Atg8 proteins in autophagy. Here we discover a super-strong GABARAP-selective inhibitory peptide harbored in 270/480 kDa ankyrin-G and a super-potent pan-Atg8 inhibitory peptide from 440 kDa ankyrin-B. Structural studies elucidate the mechanism governing the Atg8 binding potency and selectivity of the peptides, reveal a general Atg8-binding sequence motif, and allow development of a more GABARAP-selective inhibitory peptide. These peptides effectively blocked autophagy when expressed in cultured cells. Expression of these ankyrin-derived peptides in Caenorhabditis elegans also inhibited autophagy, causing accumulation of the p62 homolog SQST-1, delayed development and shortened life span. Thus, these genetically encodable autophagy inhibitory peptides can be used to occlude autophagy spatiotemporally in living animals.

Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases

ABSTRACTDuring macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.

ATG4B inhibitor FMK-9a induces autophagy independent on its enzyme inhibition

Atg4 is essential for autophagosome formation and Atg8 recycle with the function of processing the precursor and the lipidated Atg8-family proteins. Abnormal autophagic activity is involved in a variety of pathophysiological diseases and ATG4B is of interest as a potential therapeutic target due to its key roles in autophagy process. So ATG4B inhibitors are highly needed. FMK-9a is the most potent inhibitor reported so far. In this study, we confirmed FMK-9a could suppress ATG4B activity in vitro and in cells, with an IC50 of 260 nM. Besides, FMK-9a could also attenuate the process of cleavage of pro-LC3 and the delipidation of LC3-PE. Importantly, FMK-9a could induce autophagy both in HeLa and MEF cells regardless of its inhibition on ATG4B activity. Moreover, FMK-9a induced autophagy required FIP200 and ATG5. In conclusion, we demonstrated that ATG4B inhibitor FMK-9a induces autophagy independent on its enzyme inhibition. Thus, FMK-9a may plays multiple roles in autophagy process and cannot simply take it as an ATG4B inhibitor.

Regulation of selective autophagy: the p62/SQSTM1 paradigm.

In selective autophagy, cytoplasmic components are selected and tagged before being sequestered into an autophagosome by means of selective autophagy receptors such as p62/SQSTM1. In this review, we discuss how selective autophagy is regulated. An important level of regulation is the selection of proteins or organelles for degradation. Components selected for degradation are tagged, often with ubiquitin, to facilitate recognition by autophagy receptors. Another level of regulation is represented by the autophagy receptors themselves. For p62, its ability to co-aggregate with ubiquitinated substrates is strongly induced by post-translational modifications (PTMs). The transcription of p62 is also markedly increased during conditions in which selective autophagy substrates accumulate. For other autophagy receptors, the LC3-interacting region (LIR) motif is regulated by PTMs, inhibiting or stimulating the interaction with ATG8 family proteins. ATG8 proteins are also regulated by PTMs. Regulation of the capacity of the core autophagy machinery also affects selective autophagy. Importantly, autophagy receptors can induce local recruitment and activation of ULK1/2 and PI3KC3 complexes at the site of cargo sequestration.

Kenny mediates selective autophagic degradation of the IKK complex to control innate immune responses

Selective autophagy is a catabolic process with which cellular material is specifically targeted for degradation by lysosomes. The function of selective autophagic degradation of self-components in the regulation of innate immunity is still unclear. Here we show that Drosophila Kenny, the homolog of mammalian IKKγ, is a selective autophagy receptor that mediates the degradation of the IκB kinase complex. Selective autophagic degradation of the IκB kinase complex prevents constitutive activation of the immune deficiency pathway in response to commensal microbiota. We show that autophagy-deficient flies have a systemic innate immune response that promotes a hyperplasia phenotype in the midgut. Remarkably, human IKKγ does not interact with mammalian Atg8-family proteins. Using a mathematical model, we suggest mechanisms by which pathogen selection might have driven the loss of LIR motif functionality during evolution. Our results suggest that there may have been an autophagy-related switch during the evolution of the IKKγ proteins in metazoans.

ILIR@viral: A web resource for LIR motif-containing proteins in viruses

ABSTRACTMacroautophagy/autophagy has been shown to mediate the selective lysosomal degradation of pathogenic bacteria and viruses (xenophagy), and to contribute to the activation of innate and adaptative immune responses. Autophagy can serve as an antiviral defense mechanism but also as a proviral process during infection. Atg8-family proteins play a central role in the autophagy process due to their ability to interact with components of the autophagy machinery as well as selective autophagy receptors and adaptor proteins. Such interactions are usually mediated through LC3-interacting region (LIR) motifs. So far, only one viral protein has been experimentally shown to have a functional LIR motif, leaving open a vast field for investigation. Here, we have developed the iLIR@viral database (http://ilir.uk/virus/) as a freely accessible web resource listing all the putative canonical LIR motifs identified in viral proteins. Additionally, we used a curated text-mining analysis of the literature to identify novel putative LIR motif-containing proteins (LIRCPs) in viruses. We anticipate that iLIR@viral will assist with elucidating the full complement of LIRCPs in viruses.

Autophagy impairment mediated by S-nitrosation of ATG4B leads to neurotoxicity in response to hyperglycemia

ABSTRACTThe majority of diabetic patients develop neuropathy and there is an increasing prevalence of neurodegeneration in the central nervous system (CNS). However, the mechanism behind this is poorly understood. Here we first observed that macroautophagy/autophagy was suppressed in the hippocampus of diabetic GK rats with hyperglycemia, whereas it was unchanged in ob/ob mice without hyperglycemia. Autophagy could be directly inhibited by high glucose in mouse primary hippocampal neurons. Moreover, autophagy was protective in high-glucose-induced neurotoxicity. Further studies revealed that autophagic flux was suppressed by high glucose due to impaired autophagosome synthesis illustrated by mRFP-GFP-LC3 puncta analysis. We showed that decreased autophagy was dependent on NO produced under high glucose conditions. Therefore, (LC-MS/MS)-based quantitative proteomic analysis of protein S-nitrosation was performed and a core autophagy protein, ATG4B was found to be S-nitrosated in the hippocampus of GK rats. ATG4B was also verified to be S-nitrosated in neuronal cells cultured with high glucose. The activities of ATG4B in the processing of unmodified, precursor Atg8-family proteins and in the deconjugation of PE from lipidated Atg8-family proteins, which are essential for efficient autophagosome biogenesis were both compromised by S-nitrosation at Cys189 and Cys292 sites. In addition, ATG4B processing of the GABARAPL1 precursor was affected the least by S-nitrosation compared with other substrates. Finally, ATG4B S-nitrosation was verified to be responsible for decreased autophagy and neurotoxicity in response to high glucose. In conclusion, autophagy impairment mediated by S-nitrosation of ATG4B leads to neurotoxicity in response to hyperglycemia. Our research reveals a novel mechanism linking hyperglycemia with CNS neurotoxicity and shows that S-nitrosation is a novel post-transcriptional modification of the core autophagy machinery.

Identification of New ATG4B Inhibitors Based on a Novel High-Throughput Screening Platform

Autophagy is an evolutionarily conserved homeostasis process through which aggregated proteins or damaged organelles are enveloped in a double-membrane structure called an autophagosome and then digested in a lysosome-dependent manner. Growing evidence suggests that malfunction of autophagy contributes to the pathogenesis of a variety of diseases, including cancer, viral infection, and neurodegeneration. However, autophagy is a complicated process, and understanding of the relevance of autophagy to disease is limited by lack of specific and potent autophagy modulators. ATG4B, a Cys-protease that cleaves ATG8 family proteins, such as LC3B, is a key protein in autophagosome formation and maturation process. A novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay measuring protease activity of ATG4B was developed, validated, and adapted into a high-throughput screening (HTS) format. HTS was then conducted with a Roche focus library of 57,000 compounds. After hit confirmation and a counterscreen to filter out fluorescence interference compounds, 267 hits were confirmed, constituting a hit rate of 0.49%. Furthermore, among 65 hits with an IC50 < 50 µM, one compound mimics the LC3 peptide substrate (-TFG-). Chemistry modification based on this particular hit gave preliminary structure activity relationship (SAR) resulting in a compound with a 10-fold increase in potency. This compound forms a stable covalent bond with Cys74 of ATG4B in a 1:1 ratio as demonstrated by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Furthermore, this compound displayed cellular ATG4B inhibition activity. Overall, the novel TR-FRET ATG4B protease assay plus counterscreen assay provides a robust platform to identify ATG4B inhibitors, which would help to elucidate the mechanism of the autophagy pathway and offer opportunities for drug discovery.

Measurement of the Activity of the Atg4 Cysteine Proteases.

While only one Atg4 is present in yeast, there are four Atg4 homologues in human and in mouse with different substrate specificities and catalytic efficiencies. The molecule Atg4 is a type of cysteine protease, and is known for its crucial roles in cleavage of the Atg8 family proteins before they can be conjugated to phospholipids, and also in cleavage of the conjugated Atg8 molecules from the membrane, a process known as deconjugation. Both processes are required for the maximal efficiency in autophagosome biogenesis. Atg4 could thus be a target for intervention of the autophagy process. It is thus important to measure Atg4 activity to determine and to modulate the autophagy function. Here, we review the catalytic functions and regulatory mechanisms of human Atg4 proteases and discuss the methodology for analyzing Atg4 activity in details.

Investigating Structure and Dynamics of Atg8 Family Proteins.

Atg8 family members were the first autophagy-related proteins to be investigated in structural detail and continue to be among the best-understood molecules of the pathway. In this review, we will first provide a concise outline of the major methods that are being applied for structural characterization of these proteins and the complexes they are involved in. This includes a discussion of the strengths and limitations associated with each method, along with guidelines for successful adoption to a specific problem. Subsequently, we will present examples illustrating the application of these techniques, with a particular focus on the complementarity of information they provide.

ILIR database: A web resource for LIR motif-containing proteins in eukaryotes

ABSTRACTAtg8-family proteins are the best-studied proteins of the core autophagic machinery. They are essential for the elongation and closure of the phagophore into a proper autophagosome. Moreover, Atg8-family proteins are associated with the phagophore from the initiation of the autophagic process to, or just prior to, the fusion between autophagosomes with lysosomes. In addition to their implication in autophagosome biogenesis, they are crucial for selective autophagy through their ability to interact with selective autophagy receptor proteins necessary for the specific targeting of substrates for autophagic degradation. In the past few years it has been revealed that Atg8-interacting proteins include not only receptors but also components of the core autophagic machinery, proteins associated with vesicles and their transport, and specific proteins that are selectively degraded by autophagy. Atg8-interacting proteins contain a short linear LC3-interacting region/LC3 recognition sequence/Atg8-interacting motif (LIR/LRS/AIM) motif which is responsible for their interaction with Atg8-family proteins. These proteins are referred to as LIR-containing proteins (LIRCPs). So far, many experimental efforts have been carried out to identify new LIRCPs, leading to the characterization of some of them in the past 10 years. Given the need for the identification of LIRCPs in various organisms, we developed the iLIR database (https://ilir.warwick.ac.uk) as a freely available web resource, listing all the putative canonical LIRCPs identified in silico in the proteomes of 8 model organisms using the iLIR server, combined with a Gene Ontology (GO) term analysis. Additionally, a curated text-mining analysis of the literature permitted us to identify novel putative LICRPs in mammals that have not previously been associated with autophagy.

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors.

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed.

Structural basis of FYCO1 and MAP1LC3A interaction reveals a novel binding mode for Atg8-family proteins

ABSTRACTFYCO1 (FYVE and coiled-coil domain containing 1) functions as an autophagy adaptor in directly linking autophagosomes with the microtubule-based kinesin motor, and plays an essential role in the microtubule plus end-directed transport of autophagic vesicles. The specific association of FYCO1 with autophagosomes is mediated by its interaction with Atg8-family proteins decorated on the outer surface of autophagosome. However, the mechanistic basis governing the interaction between FYCO1 and Atg8-family proteins is largely unknown. Here, using biochemical and structural analyses, we demonstrated that FYCO1 contains a unique LC3-interacting region (LIR), which discriminately binds to mammalian Atg8 orthologs and preferentially binds to the MAP1LC3A and MAP1LC3B. In addition to uncovering the detailed molecular mechanism underlying the FYCO1 LIR and MAP1LC3A interaction, the determined FYCO1-LIR-MAP1LC3A complex structure also reveals a unique LIR binding mode for Atg8-family proteins, and demonstrates, first, the functional relevance of adjacent sequences C-terminal to the LIR core motif for binding to Atg8-family proteins. Taken together, our findings not only provide new mechanistic insight into FYCO1-mediated transport of autophagosomes, but also expand our understanding of the interaction modes between LIR motifs and Atg8-family proteins in general.

SLC27A4 regulate ATG4B activity and control reactions to chemotherapeutics-induced autophagy in human lung cancer cells.

Autophagy is a highly conserved self-digestion process to promote cell survival in response to nutrient starvation and other metabolic stresses in eukaryotic cells. Dysregulation of this system is linked with numerous human diseases, including cancers. ATG4B, a cysteine protease required for autophagy, cleaves the C-terminal amino acid of ATG8 family proteins to reveal a C-terminal glycine which is necessary for ATG8 proteins conjugation to phosphatidylethanolamine (PE) and insertion to autophagosome precursor membranes. However, the mechanism governing the protein stability of ATG4B in human cancer cells is not fully understood. In this study, tandem affinity purification/mass spectrometry (TAP/MS) were applied to the investigation of the interaction between ATG4B and potential candidate proteins. Then, co-immunoprecipitation (Co-IP) and GST-pull down assays indicated that the candidate protein-SLC27A4 directly interacts with ATG4B in lung cancer cell lines. Intriguingly, we also found that ATG4B protein expression was increased in parallel with SLC27A4 in lung cancer cell lines as well as lung tumor tissues. However, relevant functional research of SLC27A4 in autophagy or oncotherapy has not been investigated before. In this study, we hypothesized that SLC27A4 might act as a mediator of ATG4B, in some respects, through the protein binding directly. Further, we found that the high expression level of SLC7A4 increased the ATG4B stability and was conducive to rapid reaction to everolimus (RAD001)-induced autophagy in human lung cancer cells. As expected, the results showed that SLC27A4 could help to maintain the protein stability and intracellular concentration of ATG4B, thereby triggering rapid autophagy through releasing ATG4B to cytoplasm under conditions of reduced nutrient availability or during stress of chemotherapy in lung cancer cells. Reduced SLC27A4 by si-RNA also showed the enhanced therapeutic efficiency of everolimus, doxorubicin, and cisplatin in human lung cancer cell lines. Collectively, this study may help researchers better understand the mechanism of autophagy vitality in human cancers and SLC27A4/ATG4B complex might act as a new potential therapeutic target of lung tumor chemotherapy.

Reticulophagy and nucleophagy: New findings and unsolved issues

Autophagy targets various intracellular components ranging from proteins and nucleic acids to organelles for their degradation in lysosomes or vacuoles. In selective types of autophagy, receptor proteins play central roles in target selection. These proteins bind or localize to specific targets, and also interact with Atg8 family proteins on forming autophagosomal membranes, leading to the efficient sequestration of the targets by the membranes. Our recent study revealed that yeast cells actively degrade the endoplasmic reticulum (ER) and even part of the nucleus via selective autophagy under nitrogen-deprived conditions. We identified novel receptors, Atg39 and Atg40, specific to these pathways. Here, we summarize our findings on ‘reticulophagy’ (or ‘ER-phagy’) and ‘nucleophagy’, and discuss key issues that remain to be solved in future studies.