Autophagy

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,700 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. We provided in a previous manuscript a list of chaperones and folding enzymes as well as the components that make up the machineries for protein synthesis, protein trafficking into and out of organelles, and organelle-specific degradation pathways. Here, we provide a curated list of 838 unique high-confidence components of the autophagy-lysosome pathway, one of the two major protein degradation systems in human cells.

Disruption of macroautophagy/autophagy has emerged as a common feature in many neurodegenerative diseases. Autophagy is a membrane-dependent pathway that requires many key regulators to quickly localize on and off membranes during induction promoting membrane fusion. Previously, our bioinformatic approaches have shown that autophagy and Huntington disease (HD) are enriched in S-acylated proteins. S-acylation involves the reversible addition of long chain fatty acids to promote membrane binding. Herein, we show that inhibition of S-acylation regulates the abundance of several key regulators of autophagy and leads to a partial block of autophagic flux. We show that the autophagy receptor SQSTM1/p62 (sequestosome 1) is S-acylated and directed to the lysosome. Importantly, we see that SQSTM1 S-acylation is significantly reduced in HD patient and mouse model brains, thus providing a novel mechanism for the generation of empty autophagosomes previously seen in HD models and patient cells.

Autophagy is a fundamental intracellular degradation pathway with vital physiological functions. Although it is well known that autophagy is activated during starvation, the extent of basal autophagy remains unclear owing to challenges in measuring autophagic flux in vivo. In this study, we developed autophagy reporter (GFP-LC3-RFP) mice and quantified basal autophagic flux across tissues by comparing normal and autophagy-deficient conditions. Comparative analyses revealed uniformly low basal autophagic flux during embryogenesis, but significant tissue-specific variation in adult mice. In contrast to previous assumptions that basal autophagy in the brain is low, the brain, along with the liver and kidney, exhibited higher basal autophagic flux than the heart, skeletal muscle, and intestine. These data serve as foundational information on basal autophagic flux in mammals and provide a plausible explanation for the severe neurological phenotypes linked to autophagy gene mutations in mice and humans.

Identifying novel genes associated with autophagy (ATG) in man remains an important task for gaining complete understanding on this fundamental physiological process. A machine-learning guided approach can highlight potentially \"missing pieces\" linking core autophagy genes with understudied, \"dark\" genes that can help us gain deeper insight into these processes. In this study, we used a set of 103 (out of 288 genes from the Autophagy Database, ATGdb), based on the presence of ATG-associated terms annotated from 3 secondary sources: GO (gene ontology), KEGG pathway and UniProt keywords, respectively. We regarded these as additional confirmation for their importance in ATG. As negative labels, we used the OMIM list of genes associated with monogenic diseases (after excluding the 288 ATG-associated genes). Data associated with these genes from 17 different public sources were compiled and used to derive a Meta Path/XGBoost (MPxgb) machine learning model trained to distinguish ATG and non-ATG genes (10-fold cross-validated, 100-times randomized models, median AUC = 0.994 +/- 0.0084). Sixteen ATG-relevant variables explain 64% of the total model gain, and 23% of the top 251 predicted genes are annotated in ATGdb. Another 15 genes have potential ATG associations, whereas 193 do not. We suggest that some of these 193 genes may represent \"autophagy dark genes\", and argue that machine learning can be used to guide autophagy research in order to gain a more complete functional and pathway annotation of this complex process.

Intracellular recycling via autophagy is governed by post-translational modifications of the autophagy-related (ATG) proteins. One notable example is ATG4-dependent delipidation of ATG8, a process that plays critical but distinct roles in autophagosome formation in yeast and mammals. Here, we aimed to elucidate the specific contribution of this process to autophagosome formation in species representative of evolutionary distant green plant lineages: unicellular green alga Chlamydomonas reinhardtii, with a relatively simple set of ATG genes, and a vascular plant Arabidopsis thaliana, harboring expanded ATG gene families. Remarkably, the more complex autophagy machinery of Arabidopsis rendered ATG8 delipidation entirely dispensable for the maturation of autophagosomes, autophagic flux and related stress tolerance; whereas autophagy in Chlamydomonas strictly depended on the ATG4-mediated delipidation of ATG8. Importantly, we uncovered the distinct impact of different Arabidopsis ATG8 orthologs on autophagosome formation, especially prevalent under nitrogen depletion, providing a new insight into potential drivers behind the expansion of the ATG8 family in higher plants. Our findings underscore the evolutionary diversification of the molecular mechanism governing the maturation of autophagosomes in eukaryotic lineages and highlight how this conserved pathway is tailored to diverse organisms.

Atg8 proteins play a crucial role in autophagy. There is a single Atg8 isoform in yeast, while mammals have up to seven homologs categorized into LC3s and GABARAPs. The GABARAP subfamily consists of GABARAP, GABARAPL1, and GABARAPL2/GATE16, implicated in various stages along the pathway. However, the intricacies among GABARAP proteins are complex and require a more precise delineation. Here, we introduce a new cellular platform to study autophagy using CRISPR/Cas9-mediated tagging of endogenous genes of the GABARAP subfamily with different fluorescent proteins. This platform allows robust examination of autophagy by flow cytometry of cell populations and monitoring of GABARAP homologs at single-cell resolution using fluorescence microscopy. Strikingly, the simultaneous labeling of the different endogenous GABARAPs allows the identification and isolation of autophagosomes differentially marked by these proteins. Using this system, we found that the different GABARAPs are associated with different autophagosomes. We argue that this new cellular platform will be crucial in studying the unique roles of individual GABARAP proteins in autophagy and other putative cellular processes.

Autophagy is a conserved degradative process that promotes cellular homeostasis under stress conditions. Under nutrient starvation autophagy is largely non-selective, promoting the indiscriminate breakdown of cytosolic components. Conversely, selective autophagy is responsible for the specific turnover of damaged organelles including endoplasmic reticula, lysosomes, mitochondria, and peroxisomes. The mechanisms of selective autophagy are best understood through the activity of cargo-specific receptors called autophagy receptors, which facilitate the engulfment of the targeted cargo within autophagosomes, leading to subsequent degradation. We hypothesized that selective autophagy may be regulated by distinct upstream signaling from starvation induced autophagy, providing an additional layer of regulatory control to targeted autophagic degradation. To comprehensively address this question we conducted kinome-wide CRISPR screens to identify distinct signaling pathways responsible for the regulation of basal autophagy, starvation-induced autophagy, and two types of selective autophagy, ER-phagy and pexophagy. These parallel screens identified established and novel autophagy shared regulators under these conditions, as well as kinases specifically required for ER-phagy or pexophagy. More specifically, CDK11A and NME3 were further characterized to be selective ER-phagy regulators. Meanwhile, PAN3 and CDC42BPG were identified as activator or inhibitor of pexophagy, respectively. Collectively, these datasets provide the first comparative description of the kinase signaling specificity, separating regulation of selective autophagy and bulk autophagy. HighlightsO_LIParallel pooled kinome genetic knockout screens reveal known and novel regulators of autophagy under basal conditions, nutrient starvation, ER stress, and peroxisomal stress C_LIO_LISelective ER and peroxisomal autophagy both have unique activators and inhibitors that distinguish them from bulk autophagy C_LIO_LICDK11A and NME3 specifically induce and inhibit ER-phagy, respectively C_LIO_LIPAN3 and CDC42BPG specifically induce and inhibit pexophagy, respectively C_LI

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

Naked mole-rats (NMRs, Heterocephalus glaber) display unusual longevity and resistance to age-related decline, and accumulating evidence suggests that their autophagy-lysosome pathway (ALP) is regulated differently from that of conventional mammalian models. However, most studies in NMR cells have relied on static biochemical or ultrastructural readouts, leaving the dynamic organisation of autophagy in living cells poorly defined. Here, we establish a stable tandem fluorescent autophagy reporter in NMR skin fibroblasts using an mCherry-EGFP-LC3NMR construct to enable live-cell, single-cell resolution analysis of ALP dynamics. Under basal conditions, NMR skin fibroblasts exhibit a greater abundance of LC3-positive structures than HeLa cells, together with a mixed population of autophagosomes and autolysosomes, indicating a distinct steady-state organisation of the ALP. Chloroquine (CQ)-induced lysosomal stress caused the expected accumulation of LC3-positive structures but also triggered the formation of large cytoplasmic vacuoles in NMR skin fibroblasts. Importantly, this vacuolation was not associated with acute cytotoxicity and progressively resolved following CQ removal, accompanied by reorganisation of LC3-positive compartments and recovery of lysosomal acidity. Electron microscopy showed that CQ-induced vacuoles are membrane-bound, containing internal material and co-existing with multiple ALP-related vesicular compartments. Primary NMR skin fibroblasts display a similar vacuolation phenotype, indicating that this response is not an artefact of immortalisation or reporter expression. Together, these findings establish a live-cell platform for analysing autophagy in NMR cells and identify a distinctive, reversible vacuolation response to lysosomal stress, consistent with dynamic remodelling of the lysosomal system within NMR skin fibroblasts.

Autophagy is a conserved pro-survival pathway for delivering misfolded proteins and damaged organelles to lysosomes for degradation and protein homeostasis. Anomaly in autophagy leads to aberrant protein aggregation in neuronal cells, which is a common etiology of neurodegenerative disorders. Endo-lysosomal cation channel TRPML3 (Transient Receptor Potential Mucolipin-3) has been shown to induce autophagy in cell line models. However, the mechanism of TRPML3 mediated autophagy induction and the underlying gene expression changes are not clearly understood. Here, by using Ca2+-and electrical-current measurements, RNA sequencing and RT PCR studies, we explored the cellular function of TRPML3 and the global transcriptomic profile in a cell-based serum starvation model of autophagy. We report that serum starvation leads to downregulation of neuronal developmental genes during autophagy induction. TRPML3 overexpression further amplifies the effect of starvation in downregulating neuronal gene expression. But, when nutrition is not a limiting condition, TRPML3 overexpression upregulated neuronal genes including those responsible for axon guidance, synaptogenesis, and dendritic arborization. TRPML3 mediated neuronal gene expression changes were, presumably, due to transcription factors (TF) TFEB, FOXO1 and neuron-specific TFs such as SOX2, and ETV5. To further validate the role of TRPML3 in neuronal gene regulation, we performed meta-analysis of publicly available RNAseq datasets on neurodegenerative disorders which provided insight into the heterogeneity in the molecular mechanisms of autophagy and corroborated the TFEB-mediated autophagy induction and neuronal gene expression in TRPML3 overexpression condition. Based on our results, we propose that TRPML3 may act as a potential genetic marker for familial neurodegenerative disorders.

Synapses are specialized neuronal compartments essential for brain communication. Neuronal communication mostly relies on the adequate supply and renovation of synaptic vesicles that fuse with the plasma membrane and release neurotransmitters in response to action potentials. Autophagy is an evolutionary conserved cellular mechanism essential for homeostasis that can be locally regulated in the neuronal synapse. However, the precise mechanisms controlling synaptic autophagy, especially during neuronal communication and pathological scenarios, remain elusive. Here, we report that neuronal activity and amino-acid deprivation regulate synaptic autophagy via distinct molecular mechanisms. We show that Synaptogyrin, a highly abundant presynaptic protein found in synaptic vesicles, is a novel negative regulator of synaptic autophagy in response to neuronal activity without affecting autophagy induction via amino-acid deprivation. We demonstrate that loss of Synaptogyrin modifies the localization of the autophagy protein Atg9 and boosts autophagosome formation at the synapse. Furthermore, activation of synaptic autophagy by loss of Synaptogyrin, but not by amino acid deprivation, leads to the degradation of synaptic vesicle components via autophagy. Reducing the levels of Synaptogyrin results in the degradation of synaptic TAU via autophagy and restores autophagy dysfunction observed in a Drosophila Tau model of Frontotemporal Dementia (FTD). Our data provide novel and valuable information to understand how autophagy is regulated at the synapse in response to neuronal activity and how this process participates in neuronal (dys)function.

Reduced autophagy is associated with the aberrant humoral response observed in lipopolysaccharide-responsive beige-like anchor protein (LRBA) deficiency; however, the exact molecular mechanism and its impact on T-cell responses remain unknown. We identified two novel LRBA interactors, phosphoinositide 3-kinase regulatory subunit 4 (PIK3R4) and FYVE And Coiled-Coil Domain Autophagy Adaptor 1 (FYCO1). Both proteins play essential roles in different stages of autophagy. PIK3R4 facilitates the production of phosphatidylinositol-3 phosphate (PI(3)P) required for autophagosome formation and autophagosome-lysosome fusion, whereas FYCO1 allows autophagosome movement. LRBA-KO cells showed an impaired PI(3)P production, a delayed autophagosome-lysosome fusion, an accumulation of enlarged autophagosomes, and an atypical lysosomal positioning. These abnormalities led to decreased cargo material degradation and prolonged antigen presentation to T-cells via autophagy, resulting in increased production of proinflammatory cytokines, as autophagy is a major intracellular degradation system for major histocompatibility class II complex (MHCII) loading. Aberrant autophagosome formation, cargo degradation and antigen presentation were rescued by ectopic expression of WT-LRBA. In summary, we identified a novel function of LRBA that is crucial for T-cell-driven response through the interaction with two proteins of the autophagy machinery. These observations may contribute to the exacerbated T-cell dysregulation observed in LRBA-deficient patients.

Autophagosome formation involves the sequential actions of conserved ATG family proteins that regulate the lipidation of the ubiquitin-like modifier ATG8 at the nascent isolation membrane. Although the molecular steps driving this process are well understood, the source of membranes supplied for the expanding autophagosome and their mode of delivery remain uncertain. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12[~]ATG5 conjugate that is crucial for ATG8 lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins caused defects in autophagic flux in cell-based starvation assays. To determine whether the ATG12[~]ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. Proteins whose surface expression increased contingent on autophagic capability included EPHB2, SLC12A4, and JAG1. Those whose surface expression was decreased included CASK, SLC27A4 and LAMP1. These data provide evidence for direct regulatory coupling between the ATG12[~]ATG5 conjugate and the clathrin membrane trafficking system, and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery.

The pathogenic tropical flagellates Leishmania belong to an early-branching eukaryotic lineage (Kinetoplastida) with several unique features. Here, we explore three ancient protein targeting linear motif systems and their receptors and demonstrate how they resemble or differ from other eukaryotic organisms, including their hosts. Secretory signal peptides, endoplasmic reticulum (ER) retention motifs (KDEL motifs), and autophagy signals (motifs interacting with ATG8 family members) are essential components of cellular life. Although expected to be conserved, we observe that all three systems show a varying degree of divergence from the eukaryotic version observed in animals, plants, or fungi. We not only describe their behavior but also build predictive models that allow the prediction of localization or function for several proteins in Leishmania species for the first time. Several of these critical protein-protein interactions could serve as targets of selective antimicrobial agents against Leishmaniasis due to their divergence from the host.

Autophagy is a highly conserved, intracellular recycling process by which cytoplasmic contents are degraded in the lysosome. This process occurs at a low level constitutively; however, it is induced robustly in response to stressors, in particular, starvation of critical nutrients such as amino acids and glucose. That said, the relative contribution of these inputs is ambiguous and many starvation medias are poorly defined or devoid of multiple nutrients. Here, we sought to generate a quantitative catalog of autophagy across multiple stages and in single, living cells under normal growth conditions as well as in media starved specifically of amino acids or glucose. We found that autophagy is induced by starvation of amino acids, but not glucose, in U2OS cells, and that MTORC1-mediated ULK1 regulation and autophagy are tightly linked to amino acid levels. While autophagy is engaged immediately during amino acid starvation, a heightened response occurs during a period marked by transcriptional upregulation of autophagy genes during sustained starvation. Finally, we demonstrated that cells immediately return to their initial, low-autophagy state when nutrients are restored, highlighting the dynamic relationship between autophagy and environmental conditions. In addition to sharing our findings here, we provide our data as a high-quality resource for others interested in mathematical modeling or otherwise exploring autophagy in individual cells across a population.

SARS-CoV-2 is the causative agent for the COVID-19 pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin-1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 28 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate Beclin-1-Vps34-Atg14 complex but selectively inhibit Beclin-1-Vps34-UVRAG complex. Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raises the possibility of targeting the autophagic pathway for the treatment of COVID-19.

Autophagy is a critical cellular process, yet its genomic definition remains inconsistent across digital repositories. This lack of standardisation hinders reproducibility in high-throughput studies and clinical research. Here, we present Au_Sus, a high-confidence human autophagy census established through a frequency-based majority consensus of seven primary databases and literature sources. After rigorous manual curation and nomenclature standardisation, we defined a tiered framework: Maxim_Au (2,581 genes), Au_Sus (the 201-gene core consensus), and Minim_Au (77 universal genes). Functional enrichment and protein-protein interaction analysis confirm that Au_Sus captures a highly integrated and purified autophagic machinery, with significant associations to neurodegeneration and oncology. Furthermore, an analysis of nearly 100 published cancer gene signatures revealed profound functional dilution, with 60% of signature genes absent from our consensus. These findings suggest that many of these models incorporate peripheral stress markers rather than core autophagic effectors. Hence, Au_Sus (freely accessible at ausis.uniovi.es) provides a reliable, ready-to-use benchmark to standardise the study of autophagy in health and disease.

Autophagy is an intracellular catabolic and recycling process critical for plant stress tolerance. Upon autophagy induction, a unique and very specialized membrane named phagophore nucleates, shapes, expands and fuses to yield a double membrane vesicle sequestering and trafficking cargo for degradation. To better understand how this intense series of membrane remodeling events is orchestrated and identify the underlying actors, we aimed at establishing the molecular footprint of the phagophore. In this study, we first report on a method to purify autophagic compartments in Arabidopsis, by combining cell fractionation and immuno-isolation in native conditions. Second, proteomic analyses of ATG-isolated membranes allowed us to establish a list of candidate proteins involved in autophagy, membrane remodeling, vesicular trafficking and lipid metabolism and colocalizing with autophagic compartments after transient expression in Nicotiana benthamiana. Third, lipidomic analyses revealed that ATG-isolated membranes are mostly composed of glycerophospholipids-with a minor contribution of sterols and sphingolipids-, including a large proportion of phosphatidylcholine and phosphatidylglycerol. Together, our study unravels the singular composition of the plant phagophore and provides key protein and lipid candidates to explore how lipid and membrane dynamics instruct the autophagy pathway.

Plant growth is governed by the integration of environmental cues and nutritional status. Under stress conditions, growth is usually attenuated in favor of stress response, creating a trade-off between growth and stress. Autophagy is a vital process in eukaryotes, maintaining cellular balance by degrading and recycling cellular components. It is triggered by various nutrient-deprivation conditions and both biotic and abiotic stresses in plants. Surprisingly, over-expressing autophagy-related genes across multiple plant species resulted in increased plant size, yield, and stress resistance, posing autophagy as a regulator of the stress-growth balance. Yet, the molecular mechanisms governing its induction remain partially understood. In the current work, we identified raffinose-a plant-derived sugar known for its role in stress responses-as a novel plant autophagy inducer. Raffinose treatment resulted in increased biomass and yield in an autophagy-dependent manner in several plant species. We also show that raffinose activates autophagy through the SnRK1 kinase complex, independent of TOR signaling, and that raffinose treatment results in increased expression of ATG5 and ATG7. We also point to possible downstream candidates operating autophagy-related biomass accumulation. Our findings offer new perspectives on the role of autophagy in maintaining a balance between plant growth and stress responses, underscoring the significance of raffinose in its regulation. SIGNIFICANCE STATEMENTThe intricate balance between plant growth and stress responses is crucial for agricultural productivity, particularly as climate change intensifies environmental stressors such as drought and extreme temperatures. Usually, there is a trade-off between growth and stress response. Autophagy--a cellular recycling process essential for maintaining cellular homeostasis--plays a pivotal role in this balance. Yet, the molecular mechanisms modulating it are partially understood. Raffinose treatment enhances biomass and yields in various plant species by inducing autophagy. By elucidating the molecular mechanisms of raffinose-mediated autophagy induction, our findings provide valuable insights into potential strategies for enhancing plant resilience against climate-induced stress.

Autophagy is an intracellular catabolic process conserved across eukaryotes and critical for plant stress tolerance. Upon their delivery in the vacuole, how autophagic bodies containing cargo are hydrolyzed to warrant autophagy degradation remains poorly characterized. Here, we identify two Arabidopsis phospholipases as core components of the autophagy machinery. We find that LCAT3 and LCAT4 traffic to the vacuolar lumen and converge on autophagic bodies using differential pathways, placing them on the outer and inner side of the vesicle, respectively. Double knockouts lcat3,4 accumulate autophagic bodies and show reduced autophagy activity. In vivo reconstitution demonstrates that LCAT3 can hydrolyze the membrane of autophagic bodies, enabling the activity of LCAT4 to enhance this process. In sum, our work reveals that the concerted action of a multi-component system is required for the efficient and specific disruption of autophagic bodies as an obligatory step for the completion of the autophagy pathway.