LC3-interacting Region Research Articles

Autophagy captures the retromer-TBC1D5 complex to inhibit receptor recycling

ABSTRACT Retromer prevents the destruction of numerous receptors by recycling them from endosomes to the trans-Golgi network or plasma membrane. This enables retromer to fine-tune the activity of many signaling pathways in parallel. However, the mechanism(s) by which retromer function adapts to environmental fluctuations such as nutrient withdrawal and how this affects the fate of its cargoes remains incompletely understood. Here, we reveal that macroautophagy/autophagy inhibition by MTORC1 controls the abundance of retromer+ endosomes under nutrient-replete conditions. Autophagy activation by chemical inhibition of MTOR or nutrient withdrawal does not affect retromer assembly or its interaction with the RAB7 GAP protein TBC1D5, but rather targets these endosomes for bulk destruction following their capture by phagophores. This process appears to be distinct from amphisome formation. TBC1D5 and its ability to bind to retromer, but not its C-terminal LC3-interacting region (LIR) or nutrient-regulated dephosphorylation, is critical for retromer to be captured by autophagosomes following MTOR inhibition. Consequently, endosomal recycling of its cargoes to the plasma membrane and trans-Golgi network is impaired, leading to their lysosomal turnover. These findings demonstrate a mechanistic link connecting nutrient abundance to receptor homeostasis. Abbreviations: AMPK, 5’-AMP-activated protein kinase; APP, amyloid beta precursor protein; ATG, autophagy related; BafA, bafilomycin A1; CQ, chloroquine; DMEM, Dulbecco’s minimum essential medium; DPBS, Dulbecco’s phosphate-buffered saline; EBSS, Earle’s balanced salt solution; FBS, fetal bovine serum; GAP, GTPase-activating protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; LANDO, LC3-associated endocytosis; LP, leupeptin and pepstatin; MTOR, mechanistic target of rapamycin kinase; MTORC1, MTOR complex 1; nutrient stress, withdrawal of amino acids and serum; PDZ, DLG4/PSD95, DLG1, and TJP1/zo-1; RPS6, ribosomal protein S6; RPS6KB1/S6K1, ribosomal protein S6 kinase B1; SLC2A1/GLUT1, solute carrier family 2 member 1; SORL1, sortillin related receptor 1; SORT1, sortillin 1; SNX, sorting nexin; TBC1D5, TBC1 domain family member 5; ULK1, unc-51 like autophagy activating kinase 1; WASH, WASH complex subunit.

Seneca Valley virus 3C protease cleaves OPTN (optineurin) to Impair selective autophagy and type I interferon signaling

ABSTRACT Seneca Valley virus (SVV) causes vesicular disease in pigs, posing a threat to global pork production. OPTN (optineurin) is a macroautophagy/autophagy receptor that restricts microbial propagation by targeting specific viral or bacterial proteins for degradation. OPTN is degraded and cleaved at glutamine 513 following SVV infection via the activity of viral 3C protease (3C[pro]), resulting in N-terminal and a C-terminal OPTN fragments. Moreover, OPTN interacts with VP1 and targets VP1 for degradation to inhibit viral replication. The N-terminal cleaved OPTN sustained its interaction with VP1, whereas the degradation capacity targeting VP1 decreased. The inhibitory effect of N-terminal OPTN against SVV infection was significantly reduced, C-terminal OPTN failed to inhibit viral replication, and degradation of VP1 was blocked. The knockdown of OPTN resulted in reduced TBK1 activation and phosphorylation of IRF3, whereas overexpression of OPTN led to increased TBK1-IRF3 signaling. Additionally, the N-terminal OPTN diminished the activation of the type I IFN (interferon) pathway. These results show that SVV 3C[pro] targets OPTN because its cleavage impairs its function in selective autophagy and type I IFN production, revealing a novel model in which the virus develops diverse strategies for evading host autophagic machinery and type I IFN response for survival. Abbreviations: Co-IP: co-immunoprecipitation; GFP-green fluorescent protein; hpi: hours post-infection; HRP: horseradish peroxidase; IFN: interferon; IFNB/IFN-β: interferon beta; IRF3: interferon regulatory factor 3; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; OPTN: optineurin; PBS: phosphate-buffered saline; SVV: Seneca Valley virus; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; UBAN: ubiquitin binding in TNIP/ABIN (TNFAIP3/A20 and inhibitor of NFKB/NF-kB) and IKBKG/NEMO; UBD: ubiquitin-binding domain; ZnF: zinc finger.

Ksp1 is an autophagic receptor protein for the Snx4-assisted autophagy of Ssn2/Med13

ABSTRACT Ksp1 is a casein II-like kinase whose activity prevents aberrant macroautophagy/autophagy induction in nutrient-rich conditions in yeast. Here, we describe a kinase-independent role of Ksp1 as a novel autophagic receptor protein for Ssn2/Med13, a known cargo of Snx4-assisted autophagy of transcription factors. In this pathway, a subset of conserved transcriptional regulators, Ssn2/Med13, Rim15, and Msn2, are selectively targeted for vacuolar proteolysis following nitrogen starvation, assisted by the sorting nexin heterodimer Snx4-Atg20. Here we show that phagophores also engulf Ksp1 alongside its cargo for vacuolar proteolysis. Ksp1 directly associates with Atg8 following nitrogen starvation at the interface of an Atg8-family interacting motif (AIM)/LC3-interacting region (LIR) in Ksp1 and the LIR/AIM docking site (LDS) in Atg8. Mutating the LDS site prevents the autophagic degradation of Ksp1. However, deletion of the C terminal canonical AIM still permitted Ssn2/Med13 proteolysis, suggesting that additional non-canonical AIMs may mediate the Ksp1-Atg8 interaction. Ksp1 is recruited to the perivacuolar phagophore assembly site by Atg29, a member of the trimeric scaffold complex. This interaction is independent of Atg8 and Snx4, suggesting that Ksp1 is recruited early to phagophores, with Snx4 delivering Ssn2/Med13 thereafter. Finally, normal cell survival following prolonged nitrogen starvation requires Ksp1. Together, these studies define a kinase-independent role for Ksp1 as an autophagic receptor protein mediating Ssn2/Med13 degradation. They also suggest that phagophores built by the trimeric scaffold complex are capable of receptor-mediated autophagy. These results demonstrate the dual functionality of Ksp1, whose kinase activity prevents autophagy while it plays a scaffolding role supporting autophagic degradation. Abbreviations: 3-AT: 3-aminotriazole; 17C: Atg17-Atg31-Atg29 trimeric scaffold complex; AIM: Atg8-family interacting motif; ATG: autophagy related; CKM: CDK8 kinase module; Cvt: cytoplasm-to-vacuole targeting; IDR: intrinsically disordered region; LIR: LC3-interacting region; LDS: LIR/AIM docking site; MoRF: molecular recognition feature; NPC: nuclear pore complex; PAS: phagophore assembly site; PKA: protein kinase A; RBP: RNA-binding protein; UPS: ubiquitin-proteasome system. SAA-TF: Snx4-assisted autophagy of transcription factors; Y2H: yeast two-hybrid.

Co-chaperone BAG3 enters autophagic pathway via its interaction with microtubule associated protein 1 light chain 3 beta.

The co-chaperone BAG3 is a hub for a variety of cellular pathways via its multiple domains and its interaction with chaperones of the HSP70 family or small HSPs. During aging and under cellular stress conditions in particular, BAG3, together with molecular chaperones, ensures the sequestration of aggregated or aggregation-prone ubiquitinated proteins to the autophagic-lysosomal system via ubiquitin receptors. Accumulating evidence for BAG3-mediated selective autophagy independent of cargo ubiquitination led to analyses predicting a direct interaction of BAG3 with LC3 proteins. Phylogenetically, BAG3 comprises several highly conserved potential LIRs, LC3-interacting regions, which might allow for the direct targeting of BAG3 including its cargo to autophagosomes and drive their autophagic degradation. Based on pull-down experiments, peptide arrays and proximity ligation assays, our results provide evidence of an interaction of BAG3 with LC3B. In addition, we could demonstrate that disabling all predicted LIRs abolished the inducibility of a colocalization of BAG3 with LC3B-positive structures and resulted in a substantial decrease of BAG3 levels within purified native autophagic vesicles compared with wild-type BAG3. These results suggest an autophagic targeting of BAG3 via interaction with LC3B. Therefore, we conclude that, in addition to being a key co-chaperone to HSP70, BAG3 may also act as a cargo receptor for client proteins, which would significantly extend the role of BAG3 in selective macroautophagy and protein quality control.

Targeted Degradation of Alpha-Synuclein by Autophagosome-Anchoring Chimera Peptides.

Targeted protein degradation (TPD) confers knockdown of "undruggable" targets such as alpha-synuclein (αSyn), a pathogenic protein in multiple neurodegenerative diseases. Though many of these proteins were mainly degraded through the autophagy-lysosome pathway (ALP), few TPD tools harnessing the ALP were reported. Herein, we developed a strategy termed autophagosome-anchoring chimera (ATACC), in which the protein of interest (POI) can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of an LC3-interacting region (LIR)-containing bifunctional peptide, and the selective autophagy of the POI is thus facilitated. A series of αSyn-targeting ATACC peptides were designed and synthesized. Biological evaluations demonstrated that these compounds could degrade αSyn specifically and effectively through a "chemical-induced cargo recognition-ALP degradation" mechanism. The neuroprotective effects of ATACC peptide P1 were further validated in vitro and in vivo. Collectively, our results provided a new TPD tool and revealed a potential therapeutic strategy against synucleinopathies.

P62 and NBR1 functions are dispensable for aggrephagy in mouse ESCs and ESC-derived neurons.

Accumulation of protein aggregates is a hallmark of various neurodegenerative diseases. Selective autophagy mediates the delivery of specific cytoplasmic cargo material into lysosomes for degradation. In aggrephagy, which is the selective autophagy of protein aggregates, the cargo receptors p62 and NBR1 were shown to play important roles in cargo selection. They bind ubiquitinated cargo material via their ubiquitin-associated domains and tether it to autophagic membranes via their LC3-interacting regions. We used mouse embryonic stem cells (ESCs) in combination with genome editing to obtain further insights into the roles of p62 and NBR1 in aggrephagy. Unexpectedly, our data reveal that both ESCs and ESC-derived neurons do not show strong defects in the clearance of protein aggregates upon knockout of p62 or NBR1 and upon mutation of the p62 ubiquitin-associated domain and the LC3-interacting region motif. Taken together, our results show a robust aggregate clearance in ESCs and ESC-derived neurons. Thus, redundancy between the cargo receptors, other factors, and pathways, such as the ubiquitin-proteasome system, may compensate for the loss of function of p62 and NBR1.

AutophagyNet: high-resolution data source for the analysis of autophagy and its regulation

ABSTRACT Macroautophagy/autophagy is a highly-conserved catabolic procss eliminating dysfunctional cellular components and invading pathogens. Autophagy malfunction contributes to disorders such as cancer, neurodegenerative and inflammatory diseases. Understanding autophagy regulation in health and disease has been the focus of the last decades. We previously provided an integrated database for autophagy research, the Autophagy Regulatory Network (ARN). For the last eight years, this resource has been used by thousands of users. Here, we present a new and upgraded resource, AutophagyNet. It builds on the previous database but contains major improvements to address user feedback and novel needs due to the advancement in omics data availability. AutophagyNet contains updated interaction curation and integration of over 280,000 experimentally verified interactions between core autophagy proteins and their protein, transcriptional and post-transcriptional regulators as well as their potential upstream pathway connections. AutophagyNet provides annotations for each core protein about their role: 1) in different types of autophagy (mitophagy, xenophagy, etc.); 2) in distinct stages of autophagy (initiation, expansion, termination, etc.); 3) with subcellular and tissue-specific localization. These annotations can be used to filter the dataset, providing customizable download options tailored to the user’s needs. The resource is available in various file formats (e.g. CSV, BioPAX and PSI-MI), and data can be analyzed and visualized directly in Cytoscape. The multi-layered regulation of autophagy can be analyzed by combining AutophagyNet with tissue- or cell type-specific (multi-)omics datasets (e.g. transcriptomic or proteomic data). The resource is publicly accessible at http://autophagynet.org. Abbreviations: ARN: Autophagy Regulatory Network; ATG: autophagy related; BCR: B cell receptor pathway; BECN1: beclin 1; GABARAP: GABA type A receptor-associated protein; IIP: innate immune pathway; LIR: LC3-interacting region; lncRNA: long non-coding RNA; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; miRNA: microRNA; NHR: nuclear hormone receptor; PTM: post-translational modification; RTK: receptor tyrosine kinase; TCR: T cell receptor; TLR: toll like receptor.

MCM8-mediated mitophagy protects vascular health in response to nitric oxide signaling in a mouse model of Kawasaki disease.

Mitophagy is a major quality control pathway that removes unwanted or dysfunctional mitochondria and plays an essential role in vascular health. Here we show that MCM8 expression is significantly decreased in children with Kawasaki disease (KD) who developed coronary artery aneurysms. Mechanistically, we discovered that nitric oxide signaling promotes TRIM21-mediated MCM8 ubiquitination, which disrupts its interaction with MCM9 and promotes its cytosolic export. In the cytosol, MCM8 relocates to the mitochondria pore-forming proteins and promotes their ubiquitination by TRIM21. In addition, MCM8 directly recruits LC3 via its LC3-interacting region (LIR) motif and initiates mitophagy. This suppresses mitochondrial DNA-mediated activation of type I interferon via cGAS and STING. Mice that are deficient in Mcm8, Trim21 and Nos2 or reconstituted with the East-Asian-specific MCM8-P276 variant develop more severe coronary artery vasculopathy in the Lactobacillus casei extract-induced KD model. Collectively, the data suggest that MCM8 protects vascular health in the KD setting.

LIRcentral: a manually curated online database of experimentally validated functional LIR motifs

ABSTRACT Several selective macroautophagy receptor and adaptor proteins bind members of the Atg8 (autophagy related 8) family using short linear motifs (SLiMs), most often referred to as Atg8-family interacting motifs (AIMs) or LC3-interacting regions (LIRs). AIM/LIR motifs have been extensively studied during the last fifteen years, since they can uncover the underlying biological mechanisms and possible substrates for this key catabolic process of eukaryotic cells. Prompted by the fact that experimental information regarding LIR motifs can be found scattered across heterogeneous literature resources, we have developed LIRcentral (https://lircentral.eu), a freely available online repository for user-friendly access to comprehensive, high-quality information regarding LIR motifs from manually curated publications. Herein, we describe the development of LIRcentral and showcase currently available data and features, along with our plans for the expansion of this resource. Information incorporated in LIRcentral is useful for accomplishing a variety of research tasks, including: (i) guiding wet biology researchers for the characterization of novel instances of LIR motifs, (ii) giving bioinformaticians/computational biologists access to high-quality LIR motifs for building novel prediction methods for LIR motifs and LIR containing proteins (LIRCPs) and (iii) performing analyses to better understand the biological importance/features of functional LIR motifs. We welcome feedback on the LIRcentral content and functionality by all interested researchers and anticipate this work to spearhead a community effort for sustaining this resource which will further promote progress in studying LIR motifs/LIRCPs. Abbreviations: AIM, Atg8-family interacting motif; Atg8, autophagy related 8; GABARAP, GABA type A receptor-associated protein; LIR, LC3-interacting region; LIRCP, LIR-containing protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; PMID, PubMed identifier; PPI, protein-protein interaction; SLiM, short linear motif

The mitophagy receptor NIX induces vIRF-1 oligomerization and interaction with GABARAPL1 for the promotion of HHV-8 reactivation-induced mitophagy.

Recently, viruses have been shown to regulate selective autophagy for productive infections. For instance, human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), activates selective autophagy of mitochondria, termed mitophagy, thereby inhibiting antiviral innate immune responses during lytic infection in host cells. We previously demonstrated that HHV-8 viral interferon regulatory factor 1 (vIRF-1) plays a crucial role in lytic replication-activated mitophagy by interacting with cellular mitophagic proteins, including NIX and TUFM. However, the precise molecular mechanisms by which these interactions lead to mitophagy activation remain to be determined. Here, we show that vIRF-1 binds directly to mammalian autophagy-related gene 8 (ATG8) proteins, preferentially GABARAPL1 in infected cells, in an LC3-interacting region (LIR)-independent manner. Accordingly, we identified key residues in vIRF-1 and GABARAPL1 required for mutual interaction and demonstrated that the interaction is essential for mitophagy activation and HHV-8 productive replication. Interestingly, the mitophagy receptor NIX promotes vIRF-1-GABARAPL1 interaction, and NIX/vIRF-1-induced mitophagy is significantly inhibited in GABARAPL1-deficient cells. Moreover, a vIRF-1 variant defective in GABARAPL1 binding substantially loses the ability to induce vIRF-1/NIX-induced mitophagy. These results suggest that NIX supports vIRF-1 activity as a mitophagy mediator. In addition, we found that NIX promotes vIRF-1 aggregation and stabilizes aggregated vIRF-1. Together, these findings indicate that vIRF-1 plays a role as a viral mitophagy mediator that can be activated by a cellular mitophagy receptor.

ATG4B antagonizes antiviral immunity by GABARAP-directed autophagic degradation of TBK1

ABSTRACT TBK1 (TANK binding kinase 1) is an essential kinase of antiviral immunity, yet the regulatory mechanisms responsible for its stringent control via autophagy are not fully understood. Here, we identify the macroautophagy/autophagy-related cysteine protease ATG4B as a negative regulator of human antiviral immune responses by targeting TBK1 for autophagic degradation at the advanced stage of viral infection. Mechanistically, ATG4B serves as an adaptor for recruiting TBK1 to GABARAP (GABA type A receptor-associated protein), which subsequently leads to the TBK1-GABARAP interaction through the LC3-interacting region (LIR) motif of TBK1 ULD domain. Moreover, pharmacological ATG4B inhibitor, a small molecule named S130, contributes to host defense against viral infection and blocks ATG4B-dependent autophagic degradation of TBK1. Accordingly, S130 increases antiviral response and inhibits the VSV infection both in vitro and in vivo. Altogether, our study reveals the regulatory role of ATG4B in modulating TBK1-centered type I interferon (IFN) signaling, and indicates that ATG4B suppression can provide a potential therapy target for viral infection. Abbreviations Baf A1: bafilomycin A1; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IFN: interferon; IKBKE/IKKi: inhibitor of nuclear factor kappa B kinase subunit epsilon; IRF3: interferon regulatory factor 3; ISG: interferon-stimulated gene; ISRE: IFN-stimulated response element; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMPs: pathogen-associated molecule patterns; RIGI/DDX58: RNA sensor RIG-I; SeV: Sendai virus; siRNA: small interfering RNA; TBK1: TANK binding kinase 1; WT: wild-type; VSV: vesicular stomatitis virus.

Tethering ATG16L1 or LC3 induces targeted autophagic degradation of protein aggregates and mitochondria

ABSTRACT Proteolysis-targeting chimeras (PROTACs) based on the ubiquitin-proteasome system have made great progress in the field of drug discovery. There is mounting evidence that the accumulation of aggregation-prone proteins or malfunctioning organelles is associated with the occurrence of various age-related neurodegenerative disorders and cancers. However, PROTACs are inefficient for the degradation of such large targets due to the narrow entrance channel of the proteasome. Macroautophagy (hereafter referred to as autophagy) is known as a self-degradative process involved in the degradation of bulk cytoplasmic components or specific cargoes that are sequestered into autophagosomes. In the present study, we report the development of a generalizable strategy for the targeted degradation of large targets. Our results suggested that tethering large target models to phagophore-associated ATG16L1 or LC3 induced targeted autophagic degradation of the large target models. Furthermore, we successfully applied this autophagy-targeting degradation strategy to the targeted degradation of HTT65Q aggregates and mitochondria. Specifically, chimeras consisting of polyQ-binding peptide 1 (QBP) and ATG16L1-binding peptide (ABP) or LC3-interacting region (LIR) induced targeted autophagic degradation of pathogenic HTT65Q aggregates; and the chimeras consisting of mitochondria-targeting sequence (MTS) and ABP or LIR promoted targeted autophagic degradation of dysfunctional mitochondria, hence ameliorating mitochondrial dysfunction in a Parkinson disease cell model and protecting cells from apoptosis induced by the mitochondrial stress agent FCCP. Therefore, this study provides a new strategy for the selective proteolysis of large targets and enrich the toolkit for autophagy-targeting degradation.

The inner mitochondrial membrane fission protein MTP18 serves as a mitophagy receptor to prevent apoptosis in oral cancer.

MTP18 (also known as MTFP1), an inner mitochondrial membrane protein, plays a vital role in maintaining mitochondrial morphology by regulating mitochondrial fission. Here, we found that MTP18 functions as a mitophagy receptor that targets dysfunctional mitochondria into autophagosomes for elimination. Interestingly, MTP18 interacts with members of the LC3 (also known as MAP1LC3) family through its LC3-interacting region (LIR) to induce mitochondrial autophagy. Mutation in the LIR motif (mLIR) inhibited that interaction, thus suppressing mitophagy. Moreover, Parkin or PINK1 deficiency abrogated mitophagy in MTP18-overexpressing human oral cancer-derived FaDu cells. Upon exposure to the mitochondrial oxidative phosphorylation uncoupler CCCP, MTP18[mLIR]-FaDu cells showed decreased TOM20 levels without affecting COX IV levels. Conversely, loss of Parkin or PINK1 resulted in inhibition of TOM20 and COX IV degradation in MTP18[mLIR]-FaDu cells exposed to CCCP, establishing Parkin-mediated proteasomal degradation of outer mitochondrial membrane as essential for effective mitophagy. We also found that MTP18 provides a survival advantage to oral cancer cells exposed to cellular stress and that inhibition of MTP18-dependent mitophagy induced cell death in oral cancer cells. These findings demonstrate that MTP18 is a novel mitophagy receptor and that MTP18-dependent mitophagy has pathophysiologic implications for oral cancer progression, indicating inhibition of MTP18-mitophagy could thus be a promising cancer therapy strategy.

Molecular characterization of wild-type and HSAN2B-linked FAM134B.

Family with sequence similarity 134, member B (FAM134B), also known as Reticulophagy regulator 1 (RETREG1), is an ER-phagy receptor involved in ER homeostasis. Congenital mutations in the FAM134B gene have been reported to be associated with hereditary sensory and autonomic neuropathy type 2B (HSAN2B); however, the molecular differences between wild-type and HSAN2B-linked FAM134B are not fully understood. We prepared several human FAM134B constructs, such as the HSAN2B-linked mutant, and compared their features with those of wild-type FAM134B by transfecting these constructs into FAM134B-deficient Neuro2a cells. Although intrinsic FAM134B protein expression in wild-type Neuro2a cells was affected by the supply of amino acids in the culture medium, the expression of each HSAN2B-linked mutant FAM134B protein was hardly affected by serum and amino acid deprivation. On the other hand, the intracellular localization of GFP-tagged HSAN2B-linked mutants, except for P7Gfs133X, overlapped well with ER-localized SP-RFPKDEL and did not differ from that of GFP-tagged wild-type FAM134B. However, analysis of protein‒protein interactions using the NanoBiT reporter assay revealed the difference between wild-type and C-terminal truncated mutant FAM134B. Furthermore, this NanoBiT assay demonstrated that both wild-type and G216R FAM134B interacted with LC3/GABARAPL1 to the same extent, but the FAM134B construct with mutations near the LC3-interacting region (LIR) did not. Similar to the NanoBiT assay, the C-terminal-truncated FAM134B showed lower ER-phagy activities, as assessed by the cotransfection of GFP-tagged reporters. We showed that wild-type and HSAN2B-linked FAM134B have different molecular characteristics by transfecting cells with various types of constructs. Thus, this study provides new insights into the molecular mechanisms underlying HSAN2B as well as the regulation of ER-phagy.

Targeting of biomolecular condensates to the autophagy pathway.

Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.

Mitophagy associated self-degradation of phosphorylated MAP4 guarantees the migration and proliferation responses of keratinocytes to hypoxia

Our previous study has announced that phosphorylated microtubule-associated protein 4 (p-MAP4) accelerated keratinocytes migration and proliferation under hypoxia through depolymerizing microtubules. However, p-MAP4 should exhibit inhibitory effects on wound healing, for it also impaired mitochondria. Thus, figuring out the outcome of p-MAP4 after it impaired mitochondria and how the outcome influenced wound healing were far-reaching significance. Herein, the results revealed that p-MAP4 might undergo self-degradation through autophagy in hypoxic keratinocytes. Next, p-MAP4 activated mitophagy which was unobstructed and was also the principal pathway of its self-degradation triggered by hypoxia. Moreover, both Bcl-2 homology 3 (BH3) and LC3 interacting region (LIR) domains had been verified in MAP4, and they endowed MAP4 with the capability to synchronously function as a mitophagy initiator and a mitophagy substrate receptor. And, mutating any one of them ruined hypoxia-induced self-degradation of p-MAP4, resulting in destroyed proliferation and migration responses of keratinocytes to hypoxia. Our findings unviewed that p-MAP4 experienced mitophagy-associated self-degradation through utilizing its BH3 and LIR domains under hypoxia. As a result, the mitophagy-associated self-degradation of p-MAP4 guaranteed the migration and proliferation responses of keratinocytes to hypoxia. Together, this research provided a bran-new pattern of proteins in regulating wound healing, and offered a new direction for intervening wound healing.

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

Macroautophagy is one of two major degradation systems in eukaryotic cells. Regulation and control of autophagy are often achieved through the presence of short peptide sequences called LC3 interacting regions (LIR) in autophagy-involved proteins. Using a combination of new protein-derived activity-based probes prepared from recombinant LC3 proteins, along with protein modeling and X-ray crystallography of the ATG3-LIR peptide complex, we identified a noncanonical LIR motif in the human E2 enzyme responsible for LC3 lipidation, ATG3. The LIR motif is present in the flexible region of ATG3 and adopts an uncommon β-sheet structure binding to the backside of LC3. We show that the β-sheet conformation is crucial for its interaction with LC3 and used this insight to design synthetic macrocyclic peptide-binders to ATG3. CRISPR-enabled in cellulo studies provide evidence that LIRATG3 is required for LC3 lipidation and ATG3∼LC3 thioester formation. Removal of LIRATG3 negatively impacts the rate of thioester transfer from ATG7 to ATG3.

Identification of potential selective autophagy receptors from protein‐content profiling of autophagosomes

Selective autophagy receptors (SARs) are central to cellular homeostatic and organellar recycling pathways. Over the last two decades, more than 30 SARs have been discovered and validated using a variety of experimental approaches ranging from cell biology to biochemistry, including high-throughput imaging and screening methods. Yet, the extent of selective autophagy pathways operating under various cellular contexts, for example, under basal and starvation conditions, remains unresolved. Currently, our knowledge of all known SARs and their associated cargo components is fragmentary and limited by experimental data with varying degrees of resolution. Here, we use classical predictive and modeling approaches to integrate high-quality autophagosome content profiling data with disparate datasets. We identify a global set of potential SARs and their associated cargo components active under basal autophagy, starvation-induced, and proteasome-inhibition conditions. We provide a detailed account of cellular components, biochemical pathways, and molecular processes that are degraded via autophagy. Our analysis yields a catalog of new potential SARs that satisfy the characteristics of bonafide, well-characterized SARs. We categorize them by the subcellular compartments they emerge from and classify them based on their likely mode of action. Our structural modeling validates a large subset of predicted interactions with the human ATG8 family of proteins and shows characteristic, conserved LC3-interacting region (LIR)-LIR docking site (LDS) and ubiquitin-interacting motif (UIM)-UIM docking site (UDS) binding modes. Our analysis also revealed the most abundant cargo molecules targeted by these new SARs. Our findings expand the repertoire of SARs and provide unprecedented details into the global autophagic state of HeLa cells. Taken together, our findings provide motivation for the design of new experiments, testing the role of these novel factors in selective autophagy.

LC3-independent autophagy is vital to prevent TNF cytotoxicity

ABSTRACT The (macro)autophagy field is facing a paradigm shift after the recent discovery that cytosolic cargoes can still be selectively targeted to phagophores (the precursors to autophagosomes) even in the absence of LC3 or other Atg8-protein family members. Several in vitro studies have indeed reported on the existence of an unconventional selective autophagic pathway that involves the in-situ formation of an autophagosome around the cargo through the direct selective autophagy receptor-mediated recruitment of RB1CC1/FIP200, thereby bypassing the requirement of LC3. In an article recently published in Science, we demonstrate the physiological importance of this unconventional autophagic pathway in the context of TNF (tumor necrosis factor) signaling. We show that it promotes the degradation of the cytotoxic TNFRSF1A/TNFR1 (TNF receptor superfamily member 1A) complex II that assembles upon TNF sensing and thereby protects mice from TNFRSF1A-driven embryonic lethality and skin inflammation. Abbreviations: ATG: autophagy related; CASP: caspase; FIR: RB1CC1/FIP200-interacting region; LIR: LC3-interacting region; M1: linear; PAS: phagophore assembly site; PtdIns3K: phosphatidylinositol 3-kinase; TNF: tumor necrosis factor; TNFRSF1A: TNF receptor superfamily member 1A

Atg8 family proteins, LIR/AIM motifs and other interaction modes.

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like "half-LIRs" and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.