Non-canonical Autophagy Pathway Research Articles

Genome-wide and targeted CRISPR screens identify RNF213 as a mediator of interferon gamma-dependent pathogen restriction in human cells.

To define cellular immunity to the intracellular pathogen Toxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to identify genes important for (interferon gamma) IFN-γ-dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-γ-mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial (Mycobacterium tuberculosis) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213-mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-γ. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii, indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells.

Construction of endothelial cell-related and autophagy-related prognostic models for hepatocellular carcinoma based on single-cell data.

We aimed to develop an autophagy-related prognostic model with single-cell RNA sequencing (ScRNA-Seq) data for hepatocellular carcinoma (HCC) patients. ScRNA-Seq datasets of HCC patients were analyzed by Seurat. The expression of genes involved in canonical and noncanonical autophagy pathways in scRNA-seq data was also compared. Cox regression was applied to construct an AutRG risk prediction model. Subsequently, we examined the characteristics of AutRG high-risk and low-risk group patients. Six major cell types (hepatocytes, myeloid cells, T/NK cells, B cells, fibroblast cells, and endothelial cells) were identified in the scRNA-Seq dataset. The results showed that most of the canonical and noncanonical autophagy genes were highly expressed in hepatocytes, with the exception of MAP 1LC3B, SQSTM1, MAP 1LC3A, CYBB, and ATG3. Six AutRG risk prediction models originating from different cell types were constructed and compared. The AutRG prognostic signature (GAPDH, HSP90AA1, and TUBA1C) in endothelial cells had the best overall performance for predicting the overall survival of HCC patients, with 1-year, 3-year, and 5-year AUCs equal to 0.758, 0.68, and 0.651 in the training cohort and 0.760, 0.796, and 0.840 in the validation cohort, respectively. The different tumor mutation burden, immune infiltration, and gene set enrichment characteristics of the AutRG high-risk and low-risk group patients were identified. We constructed an endothelial cell-related and autophagy-related prognostic model of HCC patients using the ScRNA-Seq dataset for the first time. This model demonstrated the good calibration ability of HCC patients and provided a new understanding of the evaluation of prognosis.

Non-canonical autophagy in aging and age-related diseases.

Autophagy, one of the arms of proteostasis, influences aging and age-related diseases. Recently, the discovery of additional roles of autophagy-related proteins in non-canonical degradation and secretion has revealed alternative fates of autophagic cargo. Some of these non-canonical pathways have been linked to neurodegenerative diseases and improving the understanding of this link is crucial for their potential targetability in aging and age-related diseases. This review discusses recent investigations of the involvement of non-canonical autophagy players and pathways in age-related diseases that are now beginning to be discovered. Unraveling these pathways and their relation to classical autophagy could unearth a fascinating new layer of proteostasis regulation during normal aging and in longevity.

Analysis of LC3-Associated Phagocytosis and Antigen Presentation in Macrophages and B Cells.

Canonical autophagy and the non-canonical autophagy pathway LC3-associated phagocytosis (LAP) play crucial roles in the immune system by processing antigens for major histocompatibility complex (MHC) class II restricted presentation to CD4+ T cells. Recent studies offer insight into the relationship between LAP, autophagy, and antigen processing in macrophages and dendritic cells; however their involvement during antigen processing in B cells is less well understood.In this chapter, we describe how to monitor, manipulate, and understand the role of LAP and classical autophagy during MHC-restricted antigen presentation by human monocyte-derived macrophages as well as in B cell lymphoblastoid cell lines (LCLs). It includes an explanation on how to generate LCLs and monocyte-derived macrophages from primary human cells. Then we describe two different approaches to manipulate the autophagy pathways: silencing of the atg4b gene using CRISPR/Cas9 technology and a lentivirus delivery system for specific ATG4B overexpression. We also propose a method for triggering LAP and measuring different ATG proteins using Western blot and immunofluorescence. Finally, we show an approach to investigate MHC class II antigen presentation by an in vitro co-culture assay that uses the measurement of secreted cytokines, released by activated CD4+ T cells, as readout.

Beyond autophagy: LC3-associated phagocytosis and endocytosis.

Noncanonical functions of the autophagy machinery in pathways including LC3-associated phagocytosis and LC3-associated endocytosis have garnered increasing interest in both normal physiology and pathobiology. New discoveries over the past decade of noncanonical uses of the autophagy machinery in these distinct molecular mechanisms have led to robust investigation into the roles of single-membrane LC3 lipidation. Noncanonical autophagy pathways have now been implicated in the regulation of multiple processes ranging from debris clearance, cellular signaling, and immune regulation and inflammation. Accumulating evidence is demonstrating roles in a variety of disease states including host-pathogen responses, autoimmunity, cancer, and neurological and neurodegenerative pathologies. Here, we broadly summarize the differences in the mechanistic regulation between autophagy and LAP and LANDO and highlight some of the key roles of LAP and LANDO in innate immune function, inflammation, and disease pathology.

Many roads lead to CASM: Diverse stimuli of noncanonical autophagy share a unifying molecular mechanism.

Autophagy is a fundamental catabolic process coordinated by a network of autophagy-related (ATG) proteins. These ATG proteins also perform an important parallel role in "noncanonical" autophagy, a lysosome-associated signaling pathway with key functions in immunity, inflammation, cancer, and neurodegeneration. While the noncanonical autophagy pathway shares the common ATG machinery, it bears key mechanistic and functional distinctions, and is characterized by conjugation of ATG8 to single membranes (CASM). Here, we review the diverse, and still expanding, collection of stimuli and processes now known to harness the noncanonical autophagy pathway, including engulfment processes, drug treatments, TRPML1 and STING signaling, viral infection, and other pathogenic factors. We discuss the multiple associated routes to CASM and assess their shared and distinctive molecular features. By integrating these findings, we propose an updated and unifying mechanism for noncanonical autophagy, centered on ATG16L1 and V-ATPase.

Insights on attenuating autophagy cellular and molecular pathways versus methotrexate-induced toxicity via liposomal turmeric therapy

BackgroundMethotrexate (MX), a competitive inhibitor of dihydrofolate reductase, can inhibit DNA and RNA production and is a powerful anticancer agent widely utilized in clinical practice for treating nonneoplastic maladies, as psoriasis and rheumatoid arthritis; meanwhile, its probable prescription dose and interval of administration are strictly limited due to dose-related organ damage. Former studies verified that kidney, brain, liver, and lung harms are prospective obstacles of methotrexate administration. To understand the machinery of methotrexate-prompt toxicity, various mechanisms were investigated. The former is an autophagy defense mechanism; autophagy is a self-digesting mechanism responsible for the removal of damaged organelles and malformed proteins by lysosome. The contemporary article hypothesized that turmeric or its liposomal analog could defeat autophagy of MX-induced acute toxicity. Methotrexate, in a dose of 1.5 mg/kg, was administered intravenously followed by turmeric and liposomal turmeric treatment in a dose of 5 mg/kg for 30 days in rats. ResultsIncrement in autophagy (AUTP) consent by MX administration was attenuated by concurrent treatment via turmeric and liposomal turmeric that was reliable on the alteration in apoptotic markers. The assembly of FOXO-3 in serum post methotrexate administration was suppressed by concurrent treatment via liposomal turmeric. Apoptosis/autophagic marker investigation was evaluated through the gene expression of Bax (BCL2-associated X protein)/Bcl2 (B-cell lymphoma 2)/P53 (tumor protein P53)/SiRT-1 (sirtuin silent mating-type information regulation 2 homolog 1) and FOXO-3 (forkhead box transcription factor-3)/ERDJ-4 (endoplasmic reticulum localized DnaJ homologs)/BNP (brain natriuretic peptide B) signaling. The cell death of all cells was categorized to achieve autophagy. Interestingly, Bax/Bcl2/P53/SiRT-1 signaling pathways were downregulated, contributing to inhibiting the initiation of autophagy. Meanwhile, FOXO-3/BNP/ERDJ-4 reduction-implicated noncanonical autophagy pathways were involved in methotrexate-induced autophagy, whereas this change was suppressed when turmeric was administered in liposomal form. ConclusionThese outcomes recommended that liposomal turmeric prevents MX-induced acute toxicity through its autophagy, antioxidant, and antiapoptotic properties.

DJ-1 binds to Rubicon to Impair LC-3 Associated Phagocytosis.

The ability to effectively clear infection is fundamental to host survival. Sepsis, defined as dysregulated host response to infection, is a heterogenous clinical syndrome that does not uniformly clear intact bacterial or sterile infection (i.e., lipopolysaccharide). These findings were further associated with increased survival in DJ-1 deficient animals exposed to intact bacteria relative to DJ-1 deficient challenged with lipopolysaccharide. We analyzed bacterial and lipopolysaccharide clearance in bone marrow macrophages (BMM) cultured ex vivo from wild-type and DJ-1 deficient mice. Importantly, we demonstrated that DJ-1 deficiency in BMM promotes Rubicon-dependent increase in L3C-associated phagocytosis, non-canonical autophagy pathway used for xenophagy, during bacterial but not lipopolysaccharide infection. In contrast to DJ-1 deficient BMM challenged with lipopolysaccharide, DJ-1 deficient BMM exposed to intact bacteria showed enhanced Rubicon complexing with Beclin-1 and UVRAG and consistently facilitated the assembly of complete autophagolysosomes that were decorated with LC3 molecules. Our data shows DJ-1 impairs or/and delays bacterial clearance and late autophagolysosome formation by binding to Rubicon resulting in Rubicon degradation, decreased L3C-associated phagocytosis, and decreased bacterial clearance in vitro and in vivo - implicating Rubicon and DJ-1 as critical regulators of bacterial clearance in experimental sepsis.

The importance of non-canonical autophagy pathways in Alzheimer’s disease pathology and potential therapies

The importance of non-canonical autophagy pathways in Alzheimer’s disease pathology and potential therapies

CCPG1 involved in corneal Aspergillus fumigatus infection

To investigate whether non-canonical autophagy transport receptor cell cycle progression 1 (CCPG1) is involved in the corneal antifungal immune response. Human corneal epithelial cells (HCECs) and human myeloid leukemia mononuclear cells (THP-1) macrophages stimulated by Aspergillus fumigatus (A. fumigatus) were used as cell models. The expression of CCPG1 mRNA was detected by qRT-PCR. Western blot was used to determine the protein expression of CCPG1 and interleukin-1β (IL-1β). The dectin-1 neutralizing antibody was used to detect the association between dectin-1 and CCPG1. Immunofluorescence was used to observe the colocalization of CCPG1 and C-type lectin-like receptor-1 (CLEC-1) in THP-1 macrophages. The expression of CCPG1 started to increase at 4h after infection and increased in a time-dependent manner in HCECs and THP-1 macrophages. With dectin-1 neutralizing antibody pretreatment, the expression of IL-1β was down-regulated. CCPG1 up-regulation in response to A. fumigatus infection was independent of dectin-1. Immunofluorescence showed the colocalization of CCPG1 and CLEC-1 in THP-1 macrophages. As a specific autophagy protein of non-canonical autophagy pathway, CCPG1 is involved in corneal infection with A. fumigatus.

Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis.

Iron is not only essential but also a toxic trace element. Under iron repletion, ferritin maintains cellular iron homeostasis by storing iron to avoid iron toxicity. Under iron depletion, the ferritin-specific autophagy adaptor NCOA4 delivers ferritin to lysosomes via macroautophagy to enable cells to use stored iron. Here, we show that NCOA4 also plays crucial roles in the regulation of ferritin fate under iron repletion. NCOA4 forms insoluble condensates via multivalent interactions generated by the binding of iron to its intrinsically disordered region. This sequesters NCOA4 away from ferritin and allows ferritin accumulation in the early phase of iron repletion. Under prolonged iron repletion, NCOA4 condensates can deliver ferritin to lysosomes via a TAX1BP1-dependent non-canonical autophagy pathway, thereby preventing relative iron deficiency due to excessive iron storage and reduced iron uptake. Together, these observations suggest that the NCOA4-ferritin axis modulates intracellular iron homeostasis in accordance with cellular iron availability.

When the Phagosome Gets Leaky: Pore-Forming Toxin-Induced Non-Canonical Autophagy (PINCA).

Macrophages remove bacteria from the extracellular milieu via phagocytosis. While most of the engulfed bacteria are degraded in the antimicrobial environment of the phagolysosome, several bacterial pathogens have evolved virulence factors, which evade degradation or allow escape into the cytosol. To counter this situation, macrophages activate LC3-associated phagocytosis (LAP), a highly bactericidal non-canonical autophagy pathway, which destroys the bacterial pathogens in so called LAPosomes. Moreover, macrophages can also target intracellular bacteria by pore-forming toxin-induced non-canonical autophagy (PINCA), a recently described non-canonical autophagy pathway, which is activated by phagosomal damage induced by bacteria-derived pore-forming toxins. Similar to LAP, PINCA involves LC3 recruitment to the bacteria-containing phagosome independently of the ULK complex, but in contrast to LAP, this process does not require ROS production by Nox2. As last resort of autophagic targeting, macrophages activate xenophagy, a selective form of macroautophagy, to recapture bacteria, which evaded successful targeting by LAP or PINCA through rupture of the phagosome. However, xenophagy can also be hijacked by bacterial pathogens for their benefit or can be completely inhibited resulting in intracellular growth of the bacterial pathogen. In this perspective, we discuss the molecular differences and similarities between LAP, PINCA and xenophagy in macrophages during bacterial infections.

Autophagy-Lysosomal Pathway as Potential Therapeutic Target in Parkinson's Disease.

Cellular quality control systems have gained much attention in recent decades. Among these, autophagy is a natural self-preservation mechanism that continuously eliminates toxic cellular components and acts as an anti-ageing process. It is vital for cell survival and to preserve homeostasis. Several cell-type-dependent canonical or non-canonical autophagy pathways have been reported showing varying degrees of selectivity with regard to the substrates targeted. Here, we provide an updated review of the autophagy machinery and discuss the role of various forms of autophagy in neurodegenerative diseases, with a particular focus on Parkinson’s disease. We describe recent findings that have led to the proposal of therapeutic strategies targeting autophagy to alter the course of Parkinson’s disease progression.

Noncanonical autophagy in systemic lupus erythematosus

AbstractBackgroundNoncanonical autophagy is generally described as a lysosomal degradation process that requires only a subset of the core autophagy‐related proteins to form functional autophagosomes.Review: Accumulating evidence implicates noncanonical autophagy pathways in expanding the versatility of the immune system via regulation of functions that include antigen presentation, dead cell clearance, inflammatory cytokine production, and immune cell homeostasis. In this review, we use microtubule‐associated protein 1 light chain 3‐associated phagocytosis (LAP) as an example of noncanonical autophagy, describing its distinctive molecular machinery and highlighting recent advances in its functioning in immunity. We also discuss the direct and indirect evidence supporting the pathogenic significance of abnormal levels of LAP in systemic lupus erythematosus (SLE).Future Perspectives: A better understanding of the role of noncanonical autophagy in SLE may reveal crucial information about the disease pathology, providing direction for therapeutic developments and improved prognosis.

Macrophages target Listeria monocytogenes by two discrete non-canonical autophagy pathways

ABSTRACT Non-canonical autophagy pathways decorate single-membrane vesicles with Atg8-family proteins such as MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). Phagosomes containing the bacterial pathogen Listeria monocytogenes (L.m.) can be targeted by a non-canonical autophagy pathway called LC3-associated phagocytosis (LAP), which substantially contributes to the anti-listerial activity of macrophages and immunity. We here characterized a second non-canonical autophagy pathway targeting L.m.-containing phagosomes, which is induced by damage caused to the phagosomal membrane by the pore-forming toxin of L.m., listeriolysin O. This pore-forming toxin-induced non-canonical autophagy pathway (PINCA) was the only autophagic pathway evoked in tissue macrophages deficient for the NADPH oxidase CYBB/NOX2 that produces the reactive oxygen species (ROS) that are required for LAP induction. Similarly, also bone marrow-derived macrophages (BMDM) exclusively targeted L.m. by PINCA as they completely failed to induce LAP because of insufficient production of ROS through CYBB, in part, due to low expression of some CYBB complex subunits. Priming of BMDM with proinflammatory cytokines such as TNF and IFNG/IFNγ increased ROS production by CYBB and endowed them with the ability to target L.m. by LAP. Targeting of L.m. by LAP remained relatively rare, though, preventing LAP from substantially contributing to the anti-listerial activity of BMDM. Similar to LAP, the targeting of L.m.-containing phagosomes by PINCA promoted their fusion with lysosomes. Surprisingly, however, this did not substantially contribute to anti-listerial activity of BMDM. Thus, in contrast to LAP, PINCA does not have clear anti-listerial function suggesting that the two different non-canonical autophagy pathways targeting L.m. may have discrete functions. Abbreviations: actA/ActA: actin assembly-inducing protein A; ATG: autophagy-related; BMDM: Bone marrow-derived macrophages; CALCOCO2/NDP52: calcium-binding and coiled-coil domain-containing protein 2; CYBA/p22phox: cytochrome b-245 light chain; CYBB/NOX2: cytochrome b(558) subunit beta; E. coli: Escherichia coli; IFNG/IFNγ: interferon gamma; L.m.: Listeria monocytogenes; LAP: LC3-associated phagocytosis; LGALS: galectin; LLO: listeriolysin O; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NCF1/p47phox: neutrophil cytosol factor 1; NCF2/p67phox: neutrophil cytosol factor 2; NCF4/p67phox: neutrophil cytosol factor 4; Peritoneal macrophages: PM; PINCA: pore-forming toxin-induced non-canonical autophagy; plc/PLC: 1-phosphatidylinositol phosphodiesterase; PMA: phorbol 12-myristate 13-acetate; RB1CC1/FIP200: RB1-inducible coiled-coil protein 1; ROS: reactive oxygen species; S. aureus: Staphylococcus aureus; S. flexneri: Shigella flexneri; SQSTM1/p62: sequestosome 1; S. typhimurium: Salmonella typhimurium; T3SS: type III secretion system; TNF: tumor necrosis factor; ULK: unc-51 like autophagy activating kinase; PM: peritoneal macrophages; WT: wild type.

Endothelial surface translocation of mitochondrial PDCE2 involves the non-canonical secretory autophagy pathway: Putative molecular target for radiation-guided drug delivery

Radiation has been proposed as a priming agent to induce discriminatory luminal biomarkers for vascular targeting and drug delivery in disorders such as brain arteriovenous malformations and cancers. We previously observed ectopic expression of intracellular proteins such as mitochondrial PDCE2 on irradiated endothelium in animal models. In this study we examined the mechanism of PDCE2 trafficking in human endothelial cells to better understand its suitability as a vascular target. Ionizing radiation induced PDCE2 surface localization in association with accumulation of autophagosome markers (L3CB and p62) indicative of late-stage inhibition of autophagic flux. This effect was abolished in the presence of Rapamycin, an autophagy-inducer, but replicated in the presence of Bafilomycin A, an autophagy blocker. PDCE2 co-localized with lysosomal markers of the canonical degradative autophagy pathway in response to radiation but also with recycling endosomes and SNARE proteins responsible for autophagosome-plasma membrane fusion. These findings demonstrate that radiation-induced blockade of autophagic flux stimulates redirection of intracellular molecules such as PDCE2 to the cell surface via a non-canonical secretory autophagy pathway. Intracellular membrane proteins trafficked in this way could provide a unique pool of radiation biomarkers for therapeutic drug delivery.

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

SummaryAutophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific “molecular signature” for the non-canonical autophagy pathway.

Autophagy Activation by Resveratrol Reduces Severity of Experimental Rheumatoid Arthritis.

Previous work reported that dietary supplementation with resveratrol lowers synovial hyperplasia, inflammatory and oxidative damage in an antigen-induced arthritis (AIA) model. Here, it is investigated whether resveratrol can regulate the abnormal synovial proliferation by inducing autophagy and controlling the associated inflammatory response. Animals treated with resveratrol 8 weeks before AIA induction show the highest significant signal for microtubule-associated protein 1 light chain 3 by confocal microscopy. Besides, resveratrol significantly reduces p62 expression, but it does not increase the signal of beclin-1. Also, active caspase-3 expression, as well as poly(ADP-ribose) polymerase, is upregulated in the AIA group, and is significantly reduced in resveratrol-treated AIA group. Resveratrol also mitigates angiopoietin-1 and vascular endothelial growth factor signals. Finally, resveratrol significantly reduces the serum levels of IL-1β, C reactive protein, and prostaglandin E2, as well as nuclear factor κB synovial tissue expression, which shows a significant correlation with p62 expression. Dietary supplementation with resveratrol induces the noncanonical autophagy pathway and limits the cross-talk with inflammation, which in consequence modulates the synovial hyperplasia. Preventive strategies that incorporate dietary intervention with resveratrol may offer a potential therapeutic alternative to drugs to influence the risk of rheumatoid arthritis and influence its course.

ISG15 Connects Autophagy and IFN-γ-Dependent Control of Toxoplasma gondii Infection in Human Cells.

The intracellular protozoan parasite Toxoplasma gondii is capable of infecting most nucleated cells, where it survives in a specially modified compartment called the parasitophorous vacuole (PV). Interferon gamma (IFN-γ) is the major cytokine involved in activating cell-autonomous immune responses to inhibit parasite growth within this intracellular niche. In HeLa cells, IFN-γ treatment leads to ubiquitination of susceptible parasite strains, recruitment of the adaptors p62 and NDP52, and engulfment in microtubule-associated protein 1 light chain 3 (LC3)-positive membranes that restrict parasite growth. IFN-γ-mediated growth restriction depends on core members of the autophagy (ATG) pathway but not the initiation or degradative steps in the process. To explore the connection between these different pathways, we used permissive biotin ligation to identify proteins that interact with ATG5 in an IFN-γ-dependent fashion. Network analysis of the ATG5 interactome identified interferon-stimulated gene 15 (ISG15), which is highly upregulated by IFN treatment, as a hub connecting the ATG complex with other IFN-γ-induced genes, suggesting that it forms a functional link between the pathways. Deletion of ISG15 resulted in impaired recruitment of p62, NDP52, and LC3 to the PV and loss of IFN-γ-restricted parasite growth. The function of ISG15 required conjugation, and a number of ISGylated targets overlapped with the IFN-γ-dependent ATG5 interactome, including the adapter p62. Collectively, our findings establish a role for ISG15 in connecting the ATG pathway with IFN-γ-dependent restriction of T. gondii in human cells.IMPORTANCE Interferon(s) provide the primary defense against intracellular pathogens, a property ascribed to their ability to upregulate interferon-stimulated genes. Due to the sequestered niche occupied by Toxoplasma gondii, the host has elaborated intricate ways to target the parasite within its vacuole. One such mechanism is the recognition by a noncanonical autophagy pathway that envelops the parasite-containing vacuole and stunts growth in human cells. Remarkably, autophagy-dependent growth restriction requires interferon-γ, yet none of the classical components of autophagy are induced by interferon. Our studies draw a connection between these pathways by demonstrating that the antiviral protein ISG15, which is normally upregulated by interferons, links the autophagy-mediated control to ubiquitination of the vacuole. These findings suggest a similar link between interferon-γ signaling and autophagy that may underlie defense against other intracellular pathogens.

Canonical and Noncanonical Autophagy as Potential Targets for COVID-19.

The SARS-CoV-2 pandemic necessitates a review of the molecular mechanisms underlying cellular infection by coronaviruses, in order to identify potential therapeutic targets against the associated new disease (COVID-19). Previous studies on its counterparts prove a complex and concomitant interaction between coronaviruses and autophagy. The precise manipulation of this pathway allows these viruses to exploit the autophagy molecular machinery while avoiding its protective apoptotic drift and cellular innate immune responses. In turn, the maneuverability margins of such hijacking appear to be so narrow that the modulation of the autophagy, regardless of whether using inducers or inhibitors (many of which are FDA-approved for the treatment of other diseases), is usually detrimental to viral replication, including SARS-CoV-2. Recent discoveries indicate that these interactions stretch into the still poorly explored noncanonical autophagy pathway, which might play a substantial role in coronavirus replication. Still, some potential therapeutic targets within this pathway, such as RAB9 and its interacting proteins, look promising considering current knowledge. Thus, the combinatory treatment of COVID-19 with drugs affecting both canonical and noncanonical autophagy pathways may be a turning point in the fight against this and other viral infections, which may also imply beneficial prospects of long-term protection.