Drug repositioning identifies potential autophagy inhibitors for the LIR motif p62/SQSTM1 protein.

Autophagy is a critical cellular process for degrading damaged organelles and proteins under stressful conditions and has casually been shown to contribute to tumor survival and drug resistance. Sequestosome-1 (SQSTM1/p62) is an autophagy receptor that interacts with its binding partners via the LC3-interacting region (LIR). The p62 protein has been a highly researched target for its critical role in selective autophagy. In this study, we aimed to identify FDA-approved drugs that bind to the LIR motif of p62 and inhibit its LIR function, which could be useful targets for modulating autophagy. To this, the homology model of the p62 protein was predicted using biological data, and docking analysis was performed using Molegro Virtual Docker and PyRx softwares. We further assessed the toxicity profile of the drugs using the ProTox-II server and performed dynamics simulations on the effective candidate drugs identified. The results revealed that the kanamycin, velpatasvir, verteporfin, and temoporfin significantly decreased the binding of LIR to the p62 protein. Finally, we experimentally confirmed that Kanamycin can inhibit autophagy-associated acidic vesicular formation in breast cancer MCF-7 and MDA-MB 231 cells. These repositioned drugs may represent novel autophagy modulators in clinical management, warranting further investigation.

Identification of the GABARAP binding determinant in PI4K2A.

GABARAP is a member of the ATG8 family of ubiquitin-like autophagy related proteins. It was initially discovered as a facilitator of GABA-A receptor translocation to the plasma membrane and has since been shown to promote the intracellular transport of a variety of other proteins under non-autophagic conditions. We and others have shown that GABARAP interacts with the Type II phosphatidylinositol 4-kinase, PI4K2A, and that this interaction is important for autophagosome-lysosome fusion. Here, we identify a 7-amino acid segment within the PI4K2A catalytic domain that contains the GABARAP interaction motif (GIM). This segment resides in an exposed loop that is not conserved in the other mammalian Type II PI 4-kinase, PI4K2B, explaining the specificity of GABARAP binding to the PI4K2A isoform. Mutation of the PI4K2A GIM inhibits GABARAP binding and PI4K2A-mediated recruitment of cytosolic GABARAP to subcellular organelles. We further show that GABARAP binds to mono-phosphorylated phosphoinositides, PI3P, PI4P, and PI5P, raising the possibility that these lipids contribute to the binding energies that drive GABARAP-protein interactions on membranes.

High-Throughput Screening for LC3/GABARAP Binders Utilizing the Fluorescence Polarization Assay.

The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of Ki values for the measured compounds.

In Silico Assessment of LIR Motif-Containing Proteins Binding to ATG8-Family Proteins.

Selective autophagic degradation of cellular components has been shown to be mediated by the interaction of LIR motif-containing proteins with ATG8-family proteins. Here, we present a detailed methodology for the in silico evaluation of potential binding between LIR motif-containing proteins and ATG8-family proteins. We visualize AlphaFold-predicted protein complexes using PyMOL to assess potential interactions, providing an effective computational tool for this purpose.

Prohibitins, Phb1 and Phb2, function as Atg8 receptors to support yeast mitophagy and also play a negative regulatory role in Atg32 processing.

The prohibitins Phb1 and Phb2 assemble at the mitochondrial inner membrane to form a multi-dimeric complex. These scaffold proteins are highly conserved in eukaryotic cells, from yeast to mammals, and have been implicated in a variety of mitochondrial functions including aging, proliferation, and degenerative and metabolic diseases. In mammals, PHB2 regulates PINK1-PRKN mediated mitophagy by interacting with lipidated MAP1LC3B/LC3B. Despite their high conservation, prohibitins have not been linked to mitophagy in budding yeasts. In this study, we demonstrate that both Phb1 and Phb2 are required to sustain mitophagy in Saccharomyces cerevisiae. Prohibitin-dependent mitophagy requires formation of the Phb1-Phb2 complex and a conserved AIM/LIR-like motif identified in both yeast prohibitins. Furthermore, both Phb1 and Phb2 interact and exhibit mitochondrial colocalization with Atg8. Interestingly, we detected a basal C terminus processing of the mitophagy receptor Atg32 that depends on the presence of the i-AAA Yme1. In the absence of prohibitins this processing is highly enhanced but reverted by the inactivation of the rhomboid protease Pcp1. Together our results revealed a novel role of yeast prohibitins in mitophagy through its interaction with Atg8 and regulating an Atg32 proteolytic event. Abbreviation: AIM/LIR: Atg8-family interacting motif/LC3-interacting region; ANOVA: analysis of variance; ATG/Atg: autophagy related; C terminus/C-terminal: carboxyl terminus/carboxyl-terminal; GFP: green fluorescent protein; HA: human influenza hemagglutinin; Idh1: isocitrate dehydrogenase 1; MAP1C3B/LC3B: microtubule associated protein 1 light chain 3 beta; mCh: mCherry; MIM: mitochondrial inner membrane; MOM: mitochondrial outer membrane; N starvation: nitrogen starvation; N terminus: amino terminus; PARL: presenilin associated rhomboid like; Pcp1: processing of cytochrome c peroxidase 1; PCR: polymerase chain reaction; PGAM5: PGAM family member 5 mitochondrial serine/threonine protein phosphatase; PHBs/Phb: prohibitins; PINK1: PTEN induced kinase 1; PMSF: phenylmethylsulfonyl fluoride; PRKN: parkin RBR E3 ubiquitin protein ligase; SD: synthetic defined medium; SDS: sodium dodecyl sulfate; SMD-N: synthetic defined medium lacking nitrogen; WB: western blot; WT: wild type; Yme1: yeast mitochondrial escape 1; YPD: yeast extract-peptone-dextrose medium; YPLac: yeast extract-peptone-lactate medium.

Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α. Mitochondrial damage triggers TRIM5α's auto-ubiquitination and its interaction with ubiquitin-binding autophagy adaptors including NDP52, optineurin, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1. TRIM5α with intact ubiquitination function is required for the proper accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Additionally, we show that TRIM5α can directly recruit autophagy initiation machinery to damaged mitochondria. Our data support a model in which TRIM5α provides a self-amplifying, mitochondria-localized, ubiquitin-based, assembly platform for TBK1 and mitophagy adaptors that is ultimately required to recruit the core autophagy machinery.

Interaction of L1CAM with LC3 Is Required for L1-Dependent Neurite Outgrowth and Neuronal Survival.

The neural cell adhesion molecule L1 (also called L1CAM or CD171) functions not only in cell migration, but also in cell survival, differentiation, myelination, neurite outgrowth, and signaling during nervous system development and in adults. The proteolytic cleavage of L1 in its extracellular domain generates soluble fragments which are shed into the extracellular space and transmembrane fragments that are internalized into the cell and transported to various organelles to regulate cellular functions. To identify novel intracellular interaction partners of L1, we searched for protein-protein interaction motifs and found two potential microtubule-associated protein 1 light-chain 3 (LC3)-interacting region (LIR) motifs within L1, one in its extracellular domain and one in its intracellular domain. By ELISA, immunoprecipitation, and proximity ligation assay using L1 mutant mice lacking the 70 kDa L1 fragment (L1-70), we showed that L1-70 interacts with LC3 via the extracellular LIR motif in the fourth fibronectin type III domain, but not by the motif in the intracellular domain. The disruption of the L1-LC3 interaction reduces L1-mediated neurite outgrowth and neuronal survival.

Development of new tools to study membrane-anchored mammalian Atg8 proteins.

ABBREVIATIONS: A:C autophagic membrane:cytosol; ALS amyotrophic lateral sclerosis; ATG4 autophagy related 4; Atg8 autophagy related 8; BafA1 bafilomycin A1; BNIP3L/Nix BCL2 interacting protein 3 like; CALCOCO2/NDP52 calcium binding and coiled-coil domain 2; EBSS Earle's balanced salt solution; GABARAP GABA type A receptor-associated protein; GST glutathione S transferase; HKO hexa knockout; Kd dissociation constant; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NLS nuclear localization signal/sequence; PE phosphatidylethanolamine; SpHfl1 Schizosaccharomyces pombeorganic solute transmembrane transporter; SQSTM1/p62 SQSTM1/p62; TARDBP/TDP-43 TAR DNA binding protein; TKO triple knockout.

Selective autophagy and Golgi quality control in <i>Drosophila</i>.

The LIR motif-docking site (LDS) of Atg8/LC3 proteins is essential for the binding of LC3-interacting region (LIR)-containing proteins and their subsequent degradation by macroautophagy/autophagy. In our recent study, we created a mutated LDS site in Atg8a, the <i>Drosophila</i> homolog of Atg8/LC3 and found that LDS mutants accumulate known autophagy substrates and have reduced lifespan. We also conducted quantitative proteomics analyses and identified several proteins that are enriched in the LDS mutants, including Gmap (Golgi microtubule-associated protein). Gmap contains a LIR motif and accumulates in LDS mutants. We showed that Gmap and Atg8a interact in a LIR-LDS dependent manner and that the Golgi size and morphology are altered in Atg8a-LDS and Gmap-LIR motif mutants. Our findings highlight a role for Gmap in the regulation of Golgiphagy.

GMAP is an Atg8a-interacting protein that regulates Golgi turnover in Drosophila.

Selective autophagy receptors and adapters contain short linear motifs called LIR motifs (LC3-interacting region), which are required for the interaction with the Atg8-family proteins. LIR motifs bind to the hydrophobic pockets of the LIR motif docking site (LDS) of the respective Atg8-family proteins. The physiological significance of LDS docking sites has not been clarified in vivo. Here, we show that Atg8a-LDS mutant Drosophila flies accumulate autophagy substrates and have reduced lifespan. Using quantitative proteomics to identify the proteins that accumulate in Atg8a-LDS mutants, we identify the cis-Golgi protein GMAP (Golgi microtubule-associated protein) as a LIR motif-containing protein that interacts with Atg8a. GMAP LIR mutant flies exhibit accumulation of Golgi markers and elongated Golgi morphology. Our data suggest that GMAP mediates the turnover of Golgi by selective autophagy to regulate its morphology and size via its LIR motif-mediated interaction with Atg8a.

Focused Design of Novel Cyclic Peptides Endowed with GABARAP-Inhibiting Activity.

(1) Background: Disfunctions in autophagy machinery have been identified in various conditions, including neurodegenerative diseases, cancer, and inflammation. Among mammalian autophagy proteins, the Atg8 family member GABARAP has been shown to be greatly involved in the autophagy process of prostate cancer cells, supporting the idea that GABARAP inhibitors could be valuable tools to fight the progression of tumors. (2) Methods: In this paper, starting from the X-ray crystal structure of GABARAP in a complex with an AnkirinB-LIR domain, we identify two new peptides by applying in silico drug design techniques. The two ligands are synthesized, biophysically assayed, and biologically evaluated to ascertain their potential anticancer profile. (3) Results: Two cyclic peptides (WC8 and WC10) displayed promising biological activity, high conformational stability (due to the presence of disulfide bridges), and Kd values in the low micromolar range. The anticancer assays, performed on PC-3 cells, proved that both peptides exhibit antiproliferative effects comparable to those of peptide K1, a known GABARAP inhibitor. (4) Conclusions: WC8 and WC10 can be considered new GABARAP inhibitors to be employed as pharmacological tools or even templates for the rational design of new small molecules.

A yeast two-hybrid screening identifies novel Atg8a interactors in Drosophila.

Macroautophagy/autophagy-related protein Atg8/LC3 is important for autophagosome biogenesis and required for selective degradation of various substrates. In our recent study, we performed a yeast two-hybrid screening to identify proteins that interact with Atg8a, the Drosophila homolog of Atg8/LC3. The screening identified several Atg8a-interacting proteins. These proteins include: i) proteins which have already been experimentally verified to bind Atg8a, such as Atg1, DOR, ref(2)P and key (Kenny); ii) proteins for which their mammalian homologs interact with Atg8-family members, like Ank2, Atg4, and Nedd4; and iii) several novel Atg8a-interacting proteins, such as trc/STK38 and Tak1. We showed that Tak1, as well as its co-activator, Tab2, both interact with Atg8a and are substrates for selective autophagic clearance. We also determined that SH3PX1 interacts with Tab2 and is necessary for the effective regulation of the immune-deficiency (IMD) pathway. Our findings suggest a mechanism for the regulatory interactions between Tak1-Tab2-SH3PX1 and Atg8a, which contribute to the fine-tuning of the IMD pathway.

The RB1CC1 Claw-binding motif: a new piece in the puzzle of autophagy regulation.

RB1CC1/FIP200 is a subunit of the ULK1 complex in more complex eukaryotes. This large polypeptide was proposed to be a functional homolog of the Atg17 and Atg11 scaffolding proteins in yeast. Previous studies showed that RB1CC1 can bind to various proteins of the macroautophagy/autophagy machinery, where the RB1CC1 Claw domain directly interacts with a short linear segment of its interactors. A mechanistic insight into how the small globular RB1CC1 Claw domain can interact with such an array of structurally variable proteins has been elusive. The recent study by Zhou et al., discussed here, yields structural data that not only provide a unifying mechanistic explanation of these interactions, but also reveals previously unknown RB1CC1 interactors and opens a new field for exploration of autophagy regulation.Abbreviations: FIR: FIP200-interacting region; LIR: LC3-interacting region; pS/p-S: phosphorylated serine.

The interplay between pathogens and Atg8 family proteins: thousand-faced interactions.

Autophagy is an intracellular degradation and recycling process that can also remove pathogenic intracellular bacteria and viruses from within cells (referred to as xenophagy) and activate the adaptive immune responses. But autophagy-especially Atg proteins including Atg8 family members-can also have proviral and probacterial effects. In this review, we summarize known interactions of bacterial, parasitic, and viral proteins with Atg8 family proteins and the outcome of these interactions on pathogen replication, autophagy, or mitophagy. We discuss the value of prediction software and the research methodology in the study of pathogen protein-Atg8 family protein interactions, with selected examples of potential LC3-interacting region motif-containing SARS-CoV-2 proteins.

Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity.

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.

The BAX-binding protein MOAP1 associates with LC3 and promotes closure of the phagophore.

MOAP1 (modulator of apoptosis 1) is a BAX-binding protein tightly regulated by the ubiquitin-proteasome system. Apoptotic stimuli stabilize MOAP1 protein and facilitate its interaction with BAX to promote apoptosis. Here we show that in contrast to being resistant to apoptotic stimuli, MOAP1-deficient cells are hypersensitive to cell death mediated by starvation rendered by EBSS treatment. MOAP1-deficient cells exhibited impairment in macroautophagy/autophagy signaling induced by EBSS. Mechanistic analysis revealed that MOAP1-deficient cells had no notable defect in the recruitment of the pre-autophagosomal phosphatidylinositol-3-phosphate (PtdIns3P)-binding proteins, ZFYVE1/DFCP1 and WIPI2, nor in the LC3 lipidation mechanism regulated by the ATG12-ATG5-ATG16L1 complex upon EBSS treatment. Interestingly, MOAP1 is required for facilitating efficient closure of phagophore in the EBSS-treated cells. Analysis of LC3-positive membrane structures using Halo-tagged LC3 autophagosome completion assay showed that predominantly unclosed phagophore rather than closed autophagosome was present in the EBSS-treated MOAP1-deficient cells. The autophagy substrate SQSTM1/p62, which is normally contained within the enclosed autophagosome under EBSS condition, was also highly sensitive to degradation by proteinase K in the absence of MOAP1. MOAP1 binds LC3 and the binding is critically dependent on a LC3-interacting region (LIR) motif detected at its N-terminal region. Re-expression of MOAP1, but not its LC3-binding defective mutant, MOAP1-LIR, in the MOAP1-deficient cells, restored EBSS-induced autophagy. Together, these observations suggest that MOAP1 serves a distinct role in facilitating autophagy through interacting with LC3 to promote efficient phagophore closure during starvation.Abbreviations: CQ: Chloroquine; EBSS: Earle's Balanced Salt Solution; GABARAP: Gamma-Amino Butyric Acid Receptor Associated Protein; IF: Immunofluorescence; IP: Immunoprecipitation; LAMP1: Lysosomal-Associated Membrane Protein 1; LIR: LC3-Interacting Region; MAP1LC3/LC3: Microtubule Associated Protein 1 Light Chain 3; MEF: Mouse Embryonic Fibroblast; MOAP1: Modulator of Apoptosis 1; PE: Phosphatidylethanolamine; PtdIns3K: class III PtdIns3K complex I; PtdIns3P: Phosphatidylinositol-3-phosphate; STX17: Syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1.

Atg8-Family Proteins-Structural Features and Molecular Interactions in Autophagy and Beyond.

Autophagy is a common name for a number of catabolic processes, which keep the cellular homeostasis by removing damaged and dysfunctional intracellular components. Impairment or misbalance of autophagy can lead to various diseases, such as neurodegeneration, infection diseases, and cancer. A central axis of autophagy is formed along the interactions of autophagy modifiers (Atg8-family proteins) with a variety of their cellular counter partners. Besides autophagy, Atg8-proteins participate in many other pathways, among which membrane trafficking and neuronal signaling are the most known. Despite the fact that autophagy modifiers are well-studied, as the small globular proteins show similarity to ubiquitin on a structural level, the mechanism of their interactions are still not completely understood. A thorough analysis and classification of all known mechanisms of Atg8-protein interactions could shed light on their functioning and connect the pathways involving Atg8-proteins. In this review, we present our views of the key features of the Atg8-proteins and describe the basic principles of their recognition and binding by interaction partners. We discuss affinity and selectivity of their interactions as well as provide perspectives for discovery of new Atg8-interacting proteins and therapeutic approaches to tackle major human diseases.

A nuclear role for Atg8-family proteins.

Despite the growing evidence that the macroautophagy/autophagy-related protein LC3 is localized in the nucleus, why and how it is targeted to the nucleus are poorly understood. In our recent study, we found that transcription factor seq (sequoia) interacts via its LIR motif with Atg8a, the Drosophila homolog of LC3, to negatively regulate the transcription of autophagy genes. Atg8a was found to also interact with the nuclear acetyltransferase complex subunit YL-1 and deacetylase Sirt2. Modulation of the acetylation status of Atg8a by YL-1 and Sirt2 affects the interaction between seq and Atg8a, and controls the induction of autophagy. Our work revealed a novel nuclear role for Atg8a, which is linked with the transcriptional regulation of autophagy genes.

Structure and Dynamics in the ATG8 Family From Experimental to Computational Techniques.

Autophagy is a conserved and essential intracellular mechanism for the removal of damaged components. Since autophagy deregulation is linked to different kinds of pathologies, it is fundamental to gain knowledge on the fine molecular and structural details related to the core proteins of the autophagy machinery. Among these, the family of human ATG8 proteins plays a central role in recruiting other proteins to the different membrane structures involved in the autophagic pathway. Several experimental structures are available for the members of the ATG8 family alone or in complex with their different biological partners, including disordered regions of proteins containing a short linear motif called LC3 interacting motif. Recently, the first structural details of the interaction of ATG8 proteins with biological membranes came into light. The availability of structural data for human ATG8 proteins has been paving the way for studies on their structure-function-dynamic relationship using biomolecular simulations. Experimental and computational structural biology can help to address several outstanding questions on the mechanism of human ATG8 proteins, including their specificity toward different interactors, their association with membranes, the heterogeneity of their conformational ensemble, and their regulation by post-translational modifications. We here summarize the main results collected so far and discuss the future perspectives within the field and the knowledge gaps. Our review can serve as a roadmap for future structural and dynamics studies of the ATG8 family members in health and disease.