Pho81 is a novel regulator of pexophagy induced by phosphate starvation.

In the budding yeast Saccharomyces cerevisiae, macroautophagy/autophagy can be induced by various types of starvation. It is thought that potential autophagic substrates vary to meet specific nutritional demands under different starvation conditions. In a recent study, Gross et al. found that autophagy induced by phosphate starvation includes many selective aspects. For example, this work identified Pho81 as a regulator of pexophagy under conditions of phosphate starvation. Pho81 senses phosphate metabolites and directly interacts with Atg11 to promote Atg1-mediated Atg11 phosphorylation. This finding provides an example of how modulation of the Atg1/ULK kinase complex can convey specific metabolic information to regulate autophagic substrates.Abbreviation: AKC: Atg1/ULK kinase complex.

Unlocking a New Path: An Autophagometer that Measures Flux Using a Non-Fluorescent Immunohistochemistry Method.

Macroautophagy/autophagy, a crucial cellular process, is typically measured using fluorescence-based techniques, which can be costly, complex, and impractical for clinical settings. In this paper, we introduce a novel, cost-effective, non-fluorescent immunohistochemistry (IHC) method for evaluating autophagy flux. This technique, based on antigen-antibody reactions and chromogenic detection, provides clear, quantifiable results under standard light microscopy, eliminating the need for expensive equipment and specialized reagents. Our method simplifies technical requirements, making it accessible to routine clinical laboratories and research settings with limited resources. By comparing our approach with traditional fluorescence methods, we demonstrate its superior effectiveness, cost-efficiency, and applicability to patient samples. This innovative technique has the potential to significantly advance autophagy research and improve clinical diagnostics, offering a practical and robust tool for studying autophagy mechanisms in diseases such as cancer and neurodegenerative disorders. Our non-fluorescent IHC method represents a significant step forward in evaluating autophagy flux, making it more accessible and reliable, with the promise of enhancing our understanding and treatment of autophagy-related diseases.

The emerging significance of Vac8, a multi-purpose armadillo-repeat protein in yeast.

Vac8 is the sole armadillo-repeat (ARM) protein in yeast. The function of Vac8 in the cytoplasm-to-vacuole targeting pathway has been known for a long time but its role in the phagophore assembly site localization and recruitment of autophagy-related protein complexes is slowly coming to light. Because Vac8 is also involved in formation of the nuclear-vacuole junction and vacuole inheritance, the protein needs to be a competent and wide-ranging mediator of cellular processes. In this article, we discuss two recent studies reporting on Vac8 and its binding partners. We describe Vac8 in the context of crystallized protein complexes as well as predicted models to reveal the versatility of Vac8 and its potential to become a subject of future autophagy research.Abbreviation: ARM, armadillo repeat; Cvt, cytoplasm-to-vacuole targeting; IDPR, intrinsically disordered protein region NVJ, nucleus-vacuole junction; SEC, size-exclusion chromatography.

Autophagy in aging-related diseases and cancer: Principles, regulatory mechanisms and therapeutic potential.

Macroautophagy/autophagy is primarily accountable for the degradation of damaged organelles and toxic macromolecules in the cells. Regarding the essential function of autophagy for preserving cellular homeostasis, changes in, or dysfunction of, autophagy flux can lead to disease development. In the current paper, the complicated function of autophagy in aging-associated pathologies and cancer is evaluated, highlighting the underlying molecular mechanisms that can affect longevity and disease pathogenesis. As a natural biological process, a reduction in autophagy is observed with aging, resulting in an accumulation of cell damage and the development of different diseases, including neurological disorders, cardiovascular diseases, and cancer. The MTOR, AMPK, and ATG proteins demonstrate changes during aging, and they are promising therapeutic targets. Insulin/IGF1, TOR, PKA, AKT/PKB, caloric restriction and mitochondrial respiration are vital for lifespan regulation and can modulate or have an interaction with autophagy. The specific types of autophagy, such as mitophagy that degrades mitochondria, can regulate aging by affecting these organelles and eliminating those mitochondria with genomic mutations. Autophagy and its specific types contribute to the regulation of carcinogenesis and they are able to dually enhance or decrease cancer progression. Cancer hallmarks, including proliferation, metastasis, therapy resistance and immune reactions, are tightly regulated by autophagy, supporting the conclusion that autophagy is a promising target in cancer therapy.

TgATG9 is required for autophagosome biogenesis and maintenance of chronic infection in Toxoplasma gondii.

Toxoplasma gondii is a ubiquitous protozoan parasite that can reside long-term within hosts as intracellular tissue cysts comprised of chronic stage bradyzoites. To perturb chronic infection requires a better understanding of the cellular processes that mediate parasite persistence. Macroautophagy/autophagy is a catabolic and homeostatic pathway that is required for T. gondii chronic infection, although the molecular details of this process remain poorly understood. A key step in autophagy is the initial formation of the phagophore that sequesters cytoplasmic components and matures into a double-membraned autophagosome for delivery of the cargo to a cell's digestive organelle for degradative recycling. While T. gondii appears to have a reduced repertoire of autophagy proteins, it possesses a putative phospholipid scramblase, TgATG9. Through structural modeling and complementation assays, we show herein that TgATG9 can partially rescue bulk autophagy in atg9Δ yeast. We demonstrated the importance of TgATG9 for proper autophagosome dynamics at the subcellular level using three-dimensional live cell lattice light sheet microscopy. Conditional knockdown of TgATG9 in T. gondii after bradyzoite differentiation resulted in markedly reduced parasite viability. Together, our findings provide insights into the molecular dynamics of autophagosome biogenesis within an early-branching eukaryote and pinpoint the indispensable role of autophagy in maintaining T. gondii chronic infection.

Ferroptosis contributes to the progression of female-specific neoplasms, from breast cancer to gynecological malignancies in a manner regulated by non-coding RNAs: Mechanistic implications.

Ferroptosis, a recently identified type of non-apoptotic cell death, triggers the elimination of cells in the presence of lipid peroxidation and in an iron-dependent manner. Indeed, ferroptosis-stimulating factors have the ability of suppressing antioxidant capacity, leading to the accumulation of reactive oxygen species (ROS) and the subsequent oxidative death of the cells. Ferroptosis is involved in the pathophysiological basis of different maladies, such as multiple cancers, among which female-oriented malignancies have attracted much attention in recent years. In this context, it has also been unveiled that non-coding RNA transcripts, including microRNAs, long non-coding RNAs, and circular RNAs have regulatory interconnections with the ferroptotic flux, which controls the pathogenic development of diseases. Furthermore, the potential of employing these RNA transcripts as therapeutic targets during the onset of female-specific neoplasms to modulate ferroptosis has become a research hotspot; however, the molecular mechanisms and functional alterations of ferroptosis still require further investigation. The current review comprehensively highlights ferroptosis and its association with non-coding RNAs with a focus on how this crosstalk affects the pathogenesis of female-oriented malignancies, from breast cancer to ovarian, cervical, and endometrial neoplasms, suggesting novel therapeutic targets to decelerate and even block the expansion and development of these tumors.

Integrating bioengineering, super-resolution microscopy and mechanobiology in autophagy research: addendum to the guidelines (4th edition).

Recent key technological developments, such as super-resolution microscopy and microfabrication, enabled investigation of biological processes, including macroautophagy/autophagy, with unprecedented spatiotemporal resolution and control over experimental conditions. Such disruptive innovations deepened our capability to provide mechanistic understandings of the autophagic process and its causes. This addendum aims to expand the guidelines on autophagy in three key directions: optical methods enabling visualization of autophagic machinery beyond the diffraction-limited resolution; bioengineering enabling accurate designs and control over experimental conditions; and theoretical advances in mechanobiology connecting autophagy and mechanical processes of the cell. Abbreviation: 3D: three-dimensional; SIM: structured illumination microscopy; STORM: stochastic optical reconstruction microscopy.

Dysregulation of pancreatic ß-cell autophagy and the risk of type 2 diabetes.

Macroautophagy/autophagy is an essential degradation process that removes abnormal cellular components, maintains homeostasis within cells, and provides nutrition during starvation. Activated autophagy enhances cell survival during stressful conditions, although overactivation of autophagy triggers induction of autophagic cell death. Therefore, early-onset autophagy promotes cell survival whereas late-onset autophagy provokes programmed cell death, which can prevent disease progression. Moreover, autophagy regulates pancreatic ß-cell functions by different mechanisms, although the precise role of autophagy in type 2 diabetes (T2D) is not completely understood. Consequently, this mini-review discusses the protective and harmful roles of autophagy in the pancreatic ß cell and in the pathophysiology of T2D.

Extracellular NCOA4 is a mediator of septic death by activating the AGER-NFKB pathway.

Sepsis, a life-threatening condition resulting from a dysregulated response to pathogen infection, poses a significant challenge in clinical management. Here, we report a novel role for the autophagy receptor NCOA4 in the pathogenesis of sepsis. Activated macrophages and monocytes secrete NCOA4, which acts as a mediator of septic death in mice. Mechanistically, lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria, induces NCOA4 secretion through autophagy-dependent lysosomal exocytosis mediated by ATG5 and MCOLN1. Moreover, bacterial infection with E. coli or S. enterica leads to passive release of NCOA4 during GSDMD-mediated pyroptosis. Upon release, extracellular NCOA4 triggers the activation of the proinflammatory transcription factor NFKB/NF-κB by promoting the degradation of NFKBIA/IκB molecules. This process is dependent on the pattern recognition receptor AGER, rather than TLR4. In vivo studies employing endotoxemia and polymicrobial sepsis mouse models reveal that a monoclonal neutralizing antibody targeting NCOA4 or AGER delays animal death, protects against organ damage, and attenuates systemic inflammation. Furthermore, elevated plasma NCOA4 levels in septic patients, particularly in non-survivors, correlate positively with the sequential organ failure assessment score and concentrations of lactate and proinflammatory mediators, such as TNF, IL1B, IL6, and HMGB1. These findings demonstrate a previously unrecognized role of extracellular NCOA4 in inflammation, suggesting it as a potential therapeutic target for severe infectious diseases. Abbreviation: BMDMs: bone marrow-derived macrophages; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; ELISA: enzyme-linked immunosorbent assay; LPS: lipopolysaccharide; NO: nitric oxide; SOFA: sequential organ failure assessment.

The DNA damage response and autophagy during cancer development: an antagonistic pleiotropy entanglement.

The DNA damage response (DDR) pathway is a cardinal cellular stress response mechanism that during cancer development follows an antagonistic pleiotropy mode of action. Given that DDR activation is an energy demanding process, interplay with macroautophagy/autophagy, a stress response and energy providing mechanism, is likely to take place. While molecular connections between both mechanisms have been reported, an open question regards whether autophagy activation follows solely or is entangled with DDR in a similar antagonistic pleiotropy pattern during cancer development. Combing evidence on the spatiotemporal relationship of DDR and autophagy in the entire spectrum of carcinogenesis from our previous studies, we discuss these issues in the current addendum.Abbreviation: AMPK: AMP-dependent protein kinase; DDR: DNA damage response.

A delicate decision between repair and degradation of damaged lysosomes.

The destination of a damaged lysosome is either being repaired if the damage is small or degraded through a lysosome-specific macroautophagy/autophagy pathway named lysophagy when the damage is too extensive to repair. Even though previous studies report lumenal glycan exposure during lysosome damage as a signal to trigger lysophagy, it is possibly beneficial for cells to initiate lysophagy earlier than membrane rupture. In a recently published article, Gahlot et al. determined that SPART/SPG20 senses lipid-packing defects and recruits and activates the ubiquitin ligase ITCH, which labels damaged lysosomes with ubiquitin chains to initiate lysophagy.

When an underdog becomes a major player: the role of protein structural disorder in the Atg8 conjugation system.

The noncanonical ubiquitin-like conjugation cascade involving the E1 (Atg7), E2 (Atg3, Atg10), and E3 (Atg12-Atg5-Atg16 complex) enzymes is essential for incorporation of Atg8 into the growing phagophore via covalent linkage to PE. This process is an indispensable step in autophagy. Atg8 and E1-E3 enzymes are the first subset from the core autophagy protein machinery structures that were investigated in earlier studies by crystallographic analyses of globular domains. However, research over the past decade shows that many important functions in the conjugation machinery are mediated by intrinsically disordered protein regions (IDPRs) - parts of the protein that do not adopt a stable secondary or tertiary structure, which are inherently dynamic and well suited for protein-membrane interactions but are invisible in protein crystals. Here, we summarize earlier and recent findings on the autophagy conjugation machinery by focusing on the IDPRs. This summary reveals that IDPRs, originally considered dispensable, are in fact major players and a driving force in the function of the autophagy conjugation system. Abbreviation: AD, activation domain of Atg7; AH, amphipathic helix; AIM, Atg8-family interacting motif; CL, catalytic loop (of Atg7); CTD, C-terminal domain; FR, flexible region (of Atg3 or Atg10); GUV, giant unilammelar vesicles; HR, handle region (of Atg3); IDPR, intrinsically disordered protein region; IDPs: intrinsically disordered proteins; LIR, LC3-interacting region; NHD: N-terminal helical domain; NMR, nuclear magnetic resonance; PE, phosphatidylethanolamine; UBL, ubiquitin like.

ATG14 and STX18: gatekeepers of lipid droplet degradation and the implications for disease modulation.

Lipophagy, a form of autophagy specific to the degradation of lipid droplets (LDs), plays an important role in the maintenance of cellular homeostasis and metabolic processes. A recent study has identified ATG14 (autophagy related 14) as a molecule that targets LDs and marks them for degradation via lipophagy; a process that is inhibited by the binding of STX18 (syntaxin 18) to ATG14 in mammalian cells. The exact mechanism of regulation of lipophagy, and subsequently of cellular LD levels, is still under investigation; however, dysregulation of this process has been linked to a number of disease phenotypes. An imbalance of lipid levels can result in a wide variety of conditions depending on the cell/tissue type in which they occur. In cells of the retinal pigment epithelium, lipid accumulation can result in dry age-related macular degeneration, in hepatocytes it can result in nonalcoholic fatty liver diseases and in neural cells it can result in the pathogenesis of neurodegenerative conditions such as Alzheimer and Parkinson diseases. Based upon its wide range of implications in diseases, modulation of lipophagy is currently being further investigated for its potential as a treatment for a variety of conditions ranging from viral infection to developmental illnesses.

Autophagy and machine learning: Unanswered questions.

Autophagy is a critical conserved cellular process in maintaining cellular homeostasis by clearing and recycling damaged organelles and intracellular components in lysosomes and vacuoles. Autophagy plays a vital role in cell survival, bioenergetic homeostasis, organism development, and cell death regulation. Malfunctions in autophagy are associated with various human diseases and health disorders, such as cancers and neurodegenerative diseases. Significant effort has been devoted to autophagy-related research in the context of genes, proteins, diagnosis, etc. In recent years, there has been a surge of studies utilizing state of the art machine learning (ML) tools to analyze and understand the roles of autophagy in various biological processes. We taxonomize ML techniques that are applicable in an autophagy context, comprehensively review existing efforts being taken in this direction, and outline principles to consider in a biomedical context. In recognition of recent groundbreaking advances in the deep-learning community, we discuss new opportunities in interdisciplinary collaborations and seek to engage autophagy and computer science researchers to promote autophagy research with joint efforts.

CLU (clusterin) and PPARGC1A/PGC1α coordinately control mitophagy and mitochondrial biogenesis for oral cancer cell survival.

Mitophagy involves the selective elimination of defective mitochondria during chemotherapeutic stress to maintain mitochondrial homeostasis and sustain cancer growth. Here, we showed that CLU (clusterin) is localized to mitochondria to induce mitophagy controlling mitochondrial damage in oral cancer cells. Moreover, overexpression and knockdown of CLU establish its mitophagy-specific role, where CLU acts as an adaptor protein that coordinately interacts with BAX and LC3 recruiting autophagic machinery around damaged mitochondria in response to cisplatin treatment. Interestingly, CLU triggers class III phosphatidylinositol 3-kinase (PtdIns3K) activity around damaged mitochondria, and inhibition of mitophagic flux causes the accumulation of excessive mitophagosomes resulting in reactive oxygen species (ROS)-dependent apoptosis during cisplatin treatment in oral cancer cells. In parallel, we determined that PPARGC1A/PGC1α (PPARG coactivator 1 alpha) activates mitochondrial biogenesis during CLU-induced mitophagy to maintain the mitochondrial pool. Intriguingly, PPARGC1A inhibition through small interfering RNA (siPPARGC1A) and pharmacological inhibitor (SR-18292) treatment counteracts CLU-dependent cytoprotection leading to mitophagy-associated cell death. Furthermore, co-treatment of SR-18292 with cisplatin synergistically suppresses tumor growth in oral cancer xenograft models. In conclusion, CLU and PPARGC1A are essential for sustained cancer cell growth by activating mitophagy and mitochondrial biogenesis, respectively, and their inhibition could provide better therapeutic benefits against oral cancer.

Identification of the YIPF3-YIPF4 heterodimer as a novel Golgiphagy receptor.

Golgiphagy is a selective form of macroautophagy, characterized by the targeted degradation of Golgi compartments through specific receptors. In two recent studies, the YIPF3-YIPF4 heterodimer has been independently identified as the first Golgiphagy receptor within mammalian cells. This heterodimeric complex exhibits a direct affinity for mammalian Atg8-family proteins (ATG8s), thereby facilitating the expansion of phagophores in proximity to Golgi regions. Notably, the interaction between YIPF3-YIPF4 heterodimers and ATG8s undergoes regulatory modulation through phosphorylation. Furthermore, cells lacking either YIPF3 or YIPF4 display defects in Golgiphagy. To elucidate the physiological relevance of these proteins, the necessity of YIPF3-YIPF4 in orchestrating Golgi proteome remodeling was substantiated through experimentation in an in vitro neuronal differentiation model.Abbreviation: ATG: autophagy related; ATG8s: mammalian Atg8-family proteins; LIR, LC3-interacting region.

Emerging dimensions of autophagy in melanoma.

Macroautophagy/autophagy has previously been regarded as simply a way for cells to deal with nutrient emergency. But explosive work in the last 15 years has given increasingly new knowledge to our understanding of this process. Many of the functions of autophagy that are unveiled from recent studies, however, cannot be reconciled with this conventional view of cell survival but, instead, point to autophagy being integrally involved at a deeper level of cell biology, playing a critical role in maintaining homeostasis and promoting an integrated stress/immune response. The new appreciation of the role of autophagy in the evolutionary trajectory of cancer and cancer interaction with the immune system provides a mechanistic framework for understanding the clinical benefits of autophagy-based therapies. Here, we examine current knowledge of the mechanisms and functions of autophagy in highly plastic and aggressive melanoma as a model disease of human malignancy, while highlighting emerging dimensions indicating that autophagy is at play beyond its classical face.Abbreviation: AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; ATG: autophagy related; BRAF: B-Raf proto-oncogene, serine/threonine kinase; CAFs: cancer-associated fibroblasts; CCL5: C-C motif chemokine ligand 5; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; CTLA4: cytotoxic T-lymphocyte associated protein 4; CTL: cytotoxic T lymphocyte; DAMPs: danger/damage-associated molecular patterns; EGFR: epidermal growth factor receptor; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; FITM2: fat storage inducing transmembrane protein 2; HCQ: hydroxychloroquine; ICB: immune checkpoint blockade; ICD: immunogenic cell death; LDH: lactate dehydrogenase; MAPK: mitogen-activated protein kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NDP52: nuclear dot protein 52; NFKB/NF-κ B: nuclear factor kappa B; NBR1: the neighbor of BRCA1; NK: natural killer; NRF1: nuclear respiratory factor 1; NSCLC: non-small-cell lung cancer; OPTN: optineurin; PDAC: pancreatic ductal adenocarcinoma; PDCD1/PD-1: programmed cell death 1; PPT1: palmitoyl-protein thioesterase 1; PTEN: phosphatase and tensin homolog; PTK2/FAK1: protein tyrosine kinase 2; RAS: rat sarcoma; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TGFB/TGF-ß: transforming growth factor beta; TMB: tumor mutational burden; TME: tumor microenvironment; TSC1: TSC complex subunit 1; TSC2: TSC complex subunit 2; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated.

Biosensors; a novel concept in real-time detection of autophagy.

Autophagy is an early-stage response with self-degradation properties against several insulting conditions. To date, the critical role of autophagy has been well-documented in physiological and pathological conditions. This process involves various signaling and functional biomolecules, which are involved in different steps of the autophagic response. During recent decades, a range of biochemical analyses, chemical assays, and varied imaging techniques have been used for monitoring this pathway. Due to the complexity and dynamic aspects of autophagy, the application of the conventional methodology for following autophagic progression is frequently associated with a mistake in discrimination between a complete and incomplete autophagic response. Biosensors provide a de novo platform for precise and accurate analysis of target molecules in different biological settings. It has been suggested that these devices are applicable for real-time monitoring and highly sensitive detection of autophagy effectors. In this review article, we focus on cutting-edge biosensing technologies associated with autophagy detection.

Pink1 balances reticulophagy and mitophagy by regulating distinct E3 ubiquitin ligases.

The selective clearance of unwanted, damaged or dangerous components by macroautophagy/autophagy is critical for maintaining cellular homeostasis in organisms from yeast to humans. In recent years, significant progress has been made in understanding how phagophores selectively sequester specific cargo. Nevertheless, a fundamental question remains: Can distinct selective autophagy programs simultaneously operate within the same cell? A recent study from the Baehrecke lab has unveiled a developmentally programmed Pink1-dependent reticulophagy process in the Drosophila intestine. Furthermore, this study demonstrated that autophagy differentially clears mitochondria and ER in the same cell under the regulation of Pink1 through different E3 ubiquitin ligases, highlighting the need for further exploration in understanding the complexity of autophagic substrate selection and crosstalk between diverse autophagy programs.

Defective autophagy and autophagy activators in myasthenia gravis: a rare entity and unusual scenario.

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ) that results from autoantibodies against nicotinic acetylcholine receptors (nAchRs) at NMJs. These autoantibodies are mainly originated from autoreactive B cells that bind and destroy nAchRs at NMJs preventing nerve impulses from activating the end-plates of skeletal muscle. Indeed, immune dysregulation plays a crucial role in the pathogenesis of MG. Autoreactive B cells are increased in MG due to the defect in the central and peripheral tolerance mechanisms. As well, autoreactive T cells are augmented in MG due to the diversion of regulatory T (Treg) cells or a defect in thymic anergy leading to T cell-mediated autoimmunity. Furthermore, macroautophagy/autophagy, which is a conserved cellular catabolic process, plays a critical role in autoimmune diseases by regulating antigen presentation, survival of immune cells and cytokine-mediated inflammation. Abnormal autophagic flux is associated with different autoimmune disorders. Autophagy regulates the connection between innate and adaptive immune responses by controlling the production of cytokines and survival of Tregs. As autophagy is involved in autoimmune disorders, it may play a major role in the pathogenesis of MG. Therefore, this mini-review demonstrates the potential role of autophagy and autophagy activators in MG.Abbreviations: Ach, acetylcholine; Breg, regulatory B; IgG, immunoglobulin G; MG, myasthenia gravis; NMJ, neuromuscular junction; ROS, reactive oxygen species; Treg, regulatory T; Ubl, ubiquitin-like.