Decoding the molecular mechanism of selective autophagy of glycogen mediated by autophagy receptor STBD1.

Autophagy of glycogen (glycophagy) is crucial for the maintenance of cellular glucose homeostasis and physiology in mammals. STBD1 can serve as an autophagy receptor to mediate glycophagy by specifically recognizing glycogen and relevant key autophagic factors, but with poorly understood mechanisms. Here, we systematically characterize the interactions of STBD1 with glycogen and related saccharides, and determine the crystal structure of the STBD1 CBM20 domain with maltotetraose, uncovering a unique binding mode involving two different oligosaccharide-binding sites adopted by STBD1 CBM20 for recognizing glycogen. In addition, we demonstrate that the LC3-interacting region (LIR) motif of STBD1 can selectively bind to six mammalian ATG8 family members. We elucidate the detailed molecular mechanism underlying the selective interactions of STBD1 with ATG8 family proteins by solving the STBD1 LIR/GABARAPL1 complex structure. Importantly, our cell-based assays reveal that both the STBD1 LIR/GABARAPL1 interaction and the intact two oligosaccharide binding sites of STBD1 CBM20 are essential for the effective association of STBD1, GABARAPL1, and glycogen in cells. Finally, through mass spectrometry, biochemical, and structural modeling analyses, we unveil that STBD1 can directly bind to the Claw domain of RB1CC1 through its LIR, thereby recruiting the key autophagy initiation factor RB1CC1. In all, our findings provide mechanistic insights into the recognitions of glycogen, ATG8 family proteins, and RB1CC1 by STBD1 and shed light on the potential working mechanism of STBD1-mediated glycophagy.

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.

Determination of hATG8 Binding Selectivity of AIM (Autophagy-Interacting Motif) Peptides Using Native Electrospray Ionization Mass Spectrometry.

Establishing the hATG8 binding selectivity of AIM (autophagy-interacting motif) sequences found within autophagy system proteins provides insights into their biological roles, and in the case of disease-associated AIM mutations, potential pathophysiological mechanisms. Given the sometimes small differences in affinity for an individual AIM amongst the six hATG8 proteins, establishing AIM preferences can be experimentally challenging. We describe a native mass spectrometry method that is suitable for detecting such differences, using synthetic AIM peptides and recombinant hATG8 proteins, to probe hATG8-AIM interactions in the gas phase. Binding preferences of a single AIM peptide against multiple hATG8s, or two AIM peptides against a single hATG8 (e.g., wild-type versus mutant AIM), may be determined.

Computational and Experimental Approaches Towards Understanding the Role of ATG8 in Autophagy: A Therapeutic Paradigm in Leishmaniasis.

In the realm of parasitology, autophagy has emerged as a critical focal point, particularly in combating Leishmaniasis. Central to this endeavour is the recognition of the protein ATG8 as pivotal for the survival and infectivity of the parasitic organism Leishmania major, thereby making it a potential target for therapeutic intervention. Consequently, there is a pressing need to delve into the structural characteristics of ATG8 to facilitate the design of effective drugs. In this study, our efforts centered on the purification of ATG8 from Leishmania major, which enabled novel insights into its structural features through meticulous spectroscopic analysis. We aimed to comprehensively assess the stability and behaviour of ATG8 in the presence of various denaturants, including urea, guanidinium chloride, and SDS-based chemicals. Methodically, our approach included secondary structural analysis utilizing CD spectroscopy, which not only validated but also augmented computationally predicted structures of ATG8 reported in previous investigations. Remarkably, our findings unveiled that the purified ATG8 protein retained its folded conformation, exhibiting the anticipated secondary structure. Moreover, our exploration extended to the influence of lipids on ATG8 stability, yielding intriguing revelations. We uncovered a nuanced perspective suggesting that targeting both the lipid composition of Leishmania major and ATG8 could offer a promising strategy for future therapeutic approaches in combating leishmaniasis. Collectively, our study underscores the importance of understanding the structural intricacies of ATG8 in driving advancements towards the development of targeted therapies against Leishmaniasis, thereby providing a foundation for future investigations in this field.

Beyond the C-terminal Glycine of ATG8 Proteins - The Story of Some Neglected Amino Acids.

ATG8 proteins form a family of small ubiquitin-like modifiers, well-known for their importance in both macroautophagy and autophagy-independent processes. A unique feature of this protein family is their conjugation to membrane lipids through the covalent attachment of a glycine residue at the C-terminus of ATG8 proteins. Notably, most ATG8 proteins are expressed with additional amino acids at their C-terminus, shielding the key glycine residue. Consequently, lipidation requires the activation of the ATG8 precursors through proteolytic cleavage, known as priming. ATG4 proteases catalyze this priming process, and under physiological conditions, unprimed forms of ATG8 are not detected. This raises the question about the purpose of the C-terminal extension of ATG8 proteins. While the roles of lipidated and free, primed ATG8 proteins have been extensively studied, the potential function of their precursor form or the priming process itself remains largely unexplored. Here, we summarize information from existing literature and our own experiments to contribute to the understanding of these neglected amino acids.

AlphaFold2-multimer guided high-accuracy prediction of typical and atypical ATG8-binding motifs.

Macroautophagy/autophagy is an intracellular degradation process central to cellular homeostasis and defense against pathogens in eukaryotic cells. Regulation of autophagy relies on hierarchical binding of autophagy cargo receptors and adaptors to ATG8/LC3 protein family members. Interactions with ATG8/LC3 are typically facilitated by a conserved, short linear sequence, referred to as the ATG8/LC3 interacting motif/region (AIM/LIR), present in autophagy adaptors and receptors as well as pathogen virulence factors targeting host autophagy machinery. Since the canonical AIM/LIR sequence can be found in many proteins, identifying functional AIM/LIR motifs has proven challenging. Here, we show that protein modelling using Alphafold-Multimer (AF2-multimer) identifies both canonical and atypical AIM/LIR motifs with a high level of accuracy. AF2-multimer can be modified to detect additional functional AIM/LIR motifs by using protein sequences with mutations in primary AIM/LIR residues. By combining protein modelling data from AF2-multimer with phylogenetic analysis of protein sequences and protein-protein interaction assays, we demonstrate that AF2-multimer predicts the physiologically relevant AIM motif in the ATG8-interacting protein 2 (ATI-2) as well as the previously uncharacterized noncanonical AIM motif in ATG3 from potato (Solanum tuberosum). AF2-multimer also identified the AIM/LIR motifs in pathogen-encoded virulence factors that target ATG8 members in their plant and human hosts, revealing that cross-kingdom ATG8-LIR/AIM associations can also be predicted by AF2-multimer. We conclude that the AF2-guided discovery of autophagy adaptors/receptors will substantially accelerate our understanding of the molecular basis of autophagy in all biological kingdoms.

A Multifaceted Hit-Finding Approach Reveals Novel LC3 Family Ligands.

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.

Phosphorylation by casein kinase 2 enhances the interaction between ER-phagy receptor TEX264 and ATG8 proteins.

Selective autophagy cargos are recruited to autophagosomes primarily by interacting with autophagosomal ATG8 family proteins via the LC3-interacting region (LIR). The upstream sequence of most LIRs contains negatively charged residues such as Asp, Glu, and phosphorylated Ser and Thr. However, the significance of LIR phosphorylation (compared with having acidic amino acids) and the structural basis of phosphorylated LIR-ATG8 binding are not entirely understood. Here, we show that the serine residues upstream of the core LIR of the endoplasmic reticulum (ER)-phagy receptor TEX264 are phosphorylated by casein kinase 2, which is critical for its interaction with ATG8s, autophagosomal localization, and ER-phagy. Structural analysis shows that phosphorylation of these serine residues increases binding affinity by producing multiple hydrogen bonds with ATG8s that cannot be mimicked by acidic residues. This binding mode is different from those of other ER-phagy receptors that utilize a downstream helix, which is absent from TEX264, to increase affinity. These results suggest that phosphorylation of the LIR is critically important for strong LIR-ATG8 interactions, even in the absence of auxiliary interactions.

Exploration of potential inhibitors for autophagy-related protein 8 as antileishmanial agents.

Leishmaniasis is considered a tropical neglected disease, which is caused by an intramacrophagic parasite, Leishmania. It is endemic in 89 different countries. Autophagy-related protein 8 (Ldatg8) is responsible for the transformation of parasites from promastigote to amastigote differentiation. Ldatg8 is one of the key drug targets of Leishmania donovani (L. donovani) responsible for the defense of parasites during stress conditions. Virtual screening of natural ligand library had been performed against Ldatg8 to identify novel and potent inhibitors. Molecular docking and molecular dynamics simulation studies showed that urolithin A stably blocked Ldatg8. Urolithins are combinations of coumarin and isocoumarin. Further, we evaluated the antileishmanial effects of urolithin A by antileishmanial assays. Urolithin A inhibited the growth and proliferation of L. donovani promastigotes with an IC50  value of 90.3 ± 6.014 µM. It also inhibited the intramacrophagic parasite significantly with an IC50  value of 78.67 ± 4.62 µM. It showed limited cytotoxicity to the human THP-1 differentiated macrophages with a CC50  value of 190.80 ± 16.89 µM. Further, we assayed reactive oxygen species (ROS) generation and annexin V/PI staining upon urolithin A treatment of parasites to have an insight into the mechanism of its action. It induced ROS significantly in a dose-dependent manner, which caused apoptosis partially in parasites. The potential inhibitors for Ldatg8, identified in this study, would provide the platform for the development of an effective and affordable antileishmanial drug.

The crystal structure of the FAM134B-GABARAP complex provides mechanistic insights into the selective binding of FAM134 to the GABARAP subfamily.

The mammalian Atg8 family (Atg8s proteins) consists of two subfamilies: GABARAP and LC3. All members can bind to the LC3-interacting region (LIR) or Atg8-interacting motif and participate in multiple steps of autophagy. The endoplasmic reticulum (ER) autophagy receptor FAM134B contains an LIR motif that can bind to Atg8s, but whether it can differentially bind to the two subfamilies and, if so, the structural basis for this preference remains unknown. Here, we found that FAM134B bound to the GABARAP subfamily more strongly than to the LC3 subfamily. We then solved the crystal structure of the FAM134B-GABARAP complex and demonstrated that FAM134B used both its LIR core and the C-terminal helix to bind to GABARAP. We further showed that these properties might be conserved in FAM134A or FAM134C. The structure also allowed us to identify the structural determinants for the binding selectivity. Our work may be valuable for studying the differential functions of GABARAP and LC3 subfamilies in ER phagy in future.

The expanding role of Atg8.

It would be quite convenient if every protein had one distinct function, one distinct role in just a single cellular process. In the field of macroautophagy/autophagy, however, we are increasingly finding that this is not the case; several autophagy proteins have two or more roles within the process of autophagy and many even "moonlight" as functional members of entirely different cellular processes. This is perhaps best exemplified by the Atg8-family proteins. These dynamic proteins have already been reported to serve several functions both within autophagy (membrane tethering, membrane fusion, binding to cargo receptors, binding to autophagy machinery) and beyond (LC3-associated phagocytosis, formation of EDEMosomes, immune signaling) but as Maruyama and colleagues suggest in their recent report, this list of functions may not yet be complete.

Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis.

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.

A new crystal form of GABARAPL2.

The Atg8 protein family comprises the GABA type A receptor-associated proteins (GABARAPs) and microtubule-associated protein 1 light chains 3 (MAP1LC3s) that are essential mediators of autophagy. The LC3-interacting region (LIR) motifs of autophagy receptors and adaptors bind Atg8 proteins to promote autophagosome formation, cargo recruitment, and autophagosome closure and fusion to lysosomes. A crystal structure of human GABARAPL2 has been published [PDB entry 4co7; Ma et al. (2015), Biochemistry, 54, 5469-5479]. This was crystallized in space group P21 with a monoclinic angle of 90° and shows a pseudomerohedral twinning pathology. This article reports a new, untwinned GABARAPL2 crystal form, also in space group P21, but with a 98° monoclinic angle. No major conformational differences were observed between the structures. In the structure described here, the C-terminal Phe117 binds into the LIR docking site (LDS) of a neighbouring molecule within the asymmetric unit, as observed in the previously reported structure. This crystal contact blocks the LDS for co-crystallization with ligands. Phe117 of GABARAPL2 is normally removed during biological processing by Atg4 family proteases. These data indicate that to establish interactions with the LIR, Phe117 should be removed to eliminate the crystal contact and liberate the LDS for co-crystallization with LIR peptides.

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast.

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12-Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12-Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113-131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64-99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

A Selective Autophagy Pathway for Phase-Separated Endocytic Protein Deposits.

Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.

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.

Super-assembly of ER-phagy receptor Atg40 induces local ER remodeling at contacts with forming autophagosomal membranes.

The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles.