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Molecular Cell Nov 2022ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas...
ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.
Topics: Autophagosomes; Cryoelectron Microscopy; Autophagy; Biological Assay; Lipids
PubMed: 36347259
DOI: 10.1016/j.molcel.2022.10.017 -
The Journal of Cell Biology Sep 2023Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key...
Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2.
Topics: Animals; Dictyostelium; Endosomes; Mammals; Phagosomes; Phosphatidylinositols; Phosphatidylinositol 3-Kinases
PubMed: 37382666
DOI: 10.1083/jcb.202209077 -
Autophagy Aug 2019SIRT3 (sirtuin 3), a mitochondrial protein deacetylase, maintains respiratory function, but its role in the regulation of innate immune defense is largely unknown....
SIRT3 (sirtuin 3), a mitochondrial protein deacetylase, maintains respiratory function, but its role in the regulation of innate immune defense is largely unknown. Herein, we show that SIRT3 coordinates mitochondrial function and macroautophagy/autophagy activation to promote anti-mycobacterial responses through PPARA (peroxisome proliferator activated receptor alpha). SIRT3 deficiency enhanced inflammatory responses and mitochondrial dysfunction, leading to defective host defense and pathological inflammation during mycobacterial infection. Antibody-mediated depletion of polymorphonuclear neutrophils significantly increased protection against mycobacterial infection in mice. In addition, mitochondrial oxidative stress promoted excessive inflammation induced by infection in macrophages. Notably, SIRT3 was essential for the enhancement of PPARA, a key regulator of mitochondrial homeostasis and autophagy activation in the context of infection. Importantly, overexpression of either PPARA or TFEB (transcription factor EB) in macrophages recovered antimicrobial activity through autophagy activation. Furthermore, pharmacological activation of SIRT3 enhanced antibacterial autophagy and functional mitochondrial pools during mycobacterial infection. Finally, the levels of and were downregulated and inversely correlated with (tumor necrosis factor) levels in peripheral blood mononuclear cells from tuberculosis patients. Collectively, these data demonstrate a previously unappreciated function of SIRT3 in orchestrating mitochondrial and autophagic functions to promote antimycobacterial responses. Ab: antibody; BCG: Bacillus Calmette-Guérin; Baf-A: bafilomycin A; BMDMs: bone marrow-derived macrophages; CFU: colony forming unit; CXCL5: C-X-C motif chemokine ligand 5; EGFP: enhanced green fluorescent protein; ERFP: enhanced red fluorescent protein; FOXO3: forkhead box O3; HC: healthy controls; H&E: haematoxylin and eosin; HKL: honokiol; IHC: immunohistochemistry; IL1B: interleukin 1 beta; IL6: interleukin 6; IL12B: interleukin 12B; MDMs: monocyte-derived macrophages; MMP: mitochondrial membrane potential; Mtb: ; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffered saline; PMN: polymorphonuclear neutrophil; PPARA: peroxisome proliferator activated receptor alpha; ROS: reactive oxygen species; SIRT3: sirtuin 3; TB: tuberculosis; TEM: transmission electron microscopy; TFEB: transcription factor EB; TNF: tumor necrosis factor.
Topics: Anti-Bacterial Agents; Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Female; Homeostasis; Humans; Inflammation; Lung; Lysosomes; Macrophages; Male; Middle Aged; Mitochondria; Mycobacterium; Neutrophils; Oxidative Stress; PPAR alpha; Phagosomes; Sirtuin 3; Tuberculosis; Tumor Necrosis Factor-alpha
PubMed: 30774023
DOI: 10.1080/15548627.2019.1582743 -
The Biochemical Journal May 2021Amphisomes are intermediate/hybrid organelles produced through the fusion of endosomes with autophagosomes within cells. Amphisome formation is an essential step during... (Review)
Review
Amphisomes are intermediate/hybrid organelles produced through the fusion of endosomes with autophagosomes within cells. Amphisome formation is an essential step during a sequential maturation process of autophagosomes before their ultimate fusion with lysosomes for cargo degradation. This process is highly regulated with multiple protein machineries, such as SNAREs, Rab GTPases, tethering complexes, and ESCRTs, are involved to facilitate autophagic flux to proceed. In neurons, autophagosomes are robustly generated in axonal terminals and then rapidly fuse with late endosomes to form amphisomes. This fusion event allows newly generated autophagosomes to gain retrograde transport motility and move toward the soma, where proteolytically active lysosomes are predominantly located. Amphisomes are not only the products of autophagosome maturation but also the intersection of the autophagy and endo-lysosomal pathways. Importantly, amphisomes can also participate in non-canonical functions, such as retrograde neurotrophic signaling or autophagy-based unconventional secretion by fusion with the plasma membrane. In this review, we provide an updated overview of the recent discoveries and advancements on the molecular and cellular mechanisms underlying amphisome biogenesis and the emerging roles of amphisomes. We discuss recent developments towards the understanding of amphisome regulation as well as the implications in the context of major neurodegenerative diseases, with a comparative focus on Alzheimer's disease and Parkinson's disease.
Topics: Animals; Autophagosomes; Autophagy; Endosomes; Humans; Neurodegenerative Diseases; Neurons
PubMed: 34047789
DOI: 10.1042/BCJ20200917 -
Autophagy May 2021NRBF2, a regulatory subunit of the ATG14-BECN1/Beclin 1-PIK3C3/VPS34 complex, positively regulates macroautophagy/autophagy. In this study, we report that NRBF2 is...
NRBF2, a regulatory subunit of the ATG14-BECN1/Beclin 1-PIK3C3/VPS34 complex, positively regulates macroautophagy/autophagy. In this study, we report that NRBF2 is required for the clearance of apoptotic cells and alleviation of inflammation during colitis in mice. NRBF2-deficient mice displayed much more severe colitis symptoms after the administration of ulcerative colitis inducer, dextran sulfate sodium salt (DSS), accompanied by prominent intestinal inflammation and apoptotic cell accumulation. Interestingly, we found that mice and macrophages displayed impaired apoptotic cell clearance capability, while adoptive transfer of macrophages to mice alleviated DSS-induced colitis lesions. Mechanistically, NRBF2 is required for the generation of the active form of RAB7 to promote the fusion between phagosomes containing engulfed apoptotic cells and lysosomes via interacting with the MON1-CCZ1 complex and regulating the guanine nucleotide exchange factor (GEF) activity of the complex. Evidence from clinical samples further reveals the physiological role of NRBF2 in maintaining intestinal homeostasis. In biopsies of UC patient colon, we observed upregulated NRBF2 in the colon macrophages and the engulfment of apoptotic cells by NRBF2-positive cells, suggesting a potential protective role for NRBF2 in UC. To confirm the relationship between apoptotic cell clearance and IBD development, we compared TUNEL-stained cell counts in the UC with UC severity (Mayo Score) and observed a strong correlation between the two indexes, indicating that apoptotic cell population in colon tissue correlates with UC severity. The findings of our study reveal a novel role for NRBF2 in regulating apoptotic cell clearance to restrict intestinal inflammation. ANOVA: analysis of variance; ATG14: autophagy related 14; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BMDM: bone marrow-derived macrophage; BSA: bovine serum albumin; CD: Crohn disease; CD68: CD68 molecule; CFP: cyan fluorescent protein; CMFDA: 5-chloromethylfluorescein diacetate; Co-IP, co-immunoprecipitation; CPR: C-reactive protein; Cy7: cyanine 7 maleimide; DAB: diaminobezidine 3; DAI: disease activity indexes; DAPI: 4'6-diamidino-2-phenylindole; DMEM: dulbecco's modified eagle's medium; DMSO: dimethyl sulfoxide; DOC: sodium deoxycholate; DSS: dextran sulfate sodium; EDTA: ethylenediaminetetraacetic acid; EGTA: ethylenebis (oxyethylenenitrilo) tetraacetic acid; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; FRET: Förster resonance energy transfer; GDP: guanine dinucleotide phosphate; GEF: guanine nucleotide exchange factor; GFP: green fluorescent protein; GTP: guanine trinucleotide phosphate; GWAS: genome-wide association studies; HEK293: human embryonic kidney 293 cells; HRP: horseradish peroxidase; IBD: inflammatory bowel disease; IgG: immunoglobin G; IL1B/IL-1β: interleukin 1 beta; IL6: interleukin 6; IRGM: immunity related GTPase M; ITGAM/CD11b: integrin subunit alpha M; KO: knockout; LRRK2: leucine rich repeat kinase 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MPO: myeloperoxidase; NaCl: sodium chloride; NEU: neutrophil; NOD2: nucleotide binding oligomerization domain containing 2; NP40: nonidet-P40; NRBF2: nuclear receptor binding factor 2; PBS: phosphate buffer saline; PCR: polymerase chain reaction; PE: P-phycoerythrin; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PTPRC/CD45: protein tyrosine phosphatase receptor type C; SDS-PAGE: sodium dodecylsulphate-polyacrylamide gel electrophoresis; TBST: tris-buffered saline Tween-20; Tris-HCl: trihydroxymethyl aminomethane hydrochloride; TUNEL: TdT-mediated dUTP nick-end labeling; UC: ulcerative colitis; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WT: wild type; YFP: yellow fluorescent protein.
Topics: Animals; Humans; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Class III Phosphatidylinositol 3-Kinases; Inflammation; Lysosomes; Phagosomes; Trans-Activators; Mice
PubMed: 32160108
DOI: 10.1080/15548627.2020.1741332 -
The Journal of Cell Biology Oct 2022A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to...
A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, it is unclear whether NCOA4 only acts as an autophagy receptor in ferritin degradation. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).
Topics: Autophagy; Endosomes; Ferritins; Iron; Lysosomes; Nuclear Receptor Coactivators; Phagosomes; Transcription Factors
PubMed: 36066504
DOI: 10.1083/jcb.202203102 -
Cells Aug 2021Autophagy is a conserved self-degradation process that is activated under a wide variety of stresses and physiological conditions [...].
Autophagy is a conserved self-degradation process that is activated under a wide variety of stresses and physiological conditions [...].
Topics: Animals; Apoptosis; Autophagosomes; Autophagy; Autophagy-Related Proteins; Humans; Signal Transduction
PubMed: 34440756
DOI: 10.3390/cells10081987 -
Neuron Mar 2022Neurons depend on autophagy to maintain cellular homeostasis, and defects in autophagy are pathological hallmarks of neurodegenerative disease. To probe the role of...
Neurons depend on autophagy to maintain cellular homeostasis, and defects in autophagy are pathological hallmarks of neurodegenerative disease. To probe the role of basal autophagy in the maintenance of neuronal health, we isolated autophagic vesicles from mouse brain tissue and used proteomics to identify the major cargos engulfed within autophagosomes, validating our findings in rodent primary and human iPSC-derived neurons. Mitochondrial proteins were identified as a major cargo in the absence of mitophagy adaptors such as OPTN. We found that nucleoid-associated proteins are enriched compared with other mitochondrial components. In the axon, autophagic engulfment of nucleoid-enriched mitochondrial fragments requires the mitochondrial fission machinery Drp1. We proposed that localized Drp1-dependent fission of nucleoid-enriched fragments in proximity to the sites of autophagosome biogenesis enhances their capture. The resulting efficient autophagic turnover of nucleoids may prevent accumulation of mitochondrial DNA in the neuron, thus mitigating activation of proinflammatory pathways that contribute to neurodegeneration.
Topics: Animals; Autophagosomes; Autophagy; Brain; Mice; Neurodegenerative Diseases; Neurons
PubMed: 35051374
DOI: 10.1016/j.neuron.2021.12.029 -
Cells Nov 2021Trogocytosis is a mode of internalization of a part of a live cell by nibbling and is mechanistically distinct from phagocytosis, which implies internalization of a... (Review)
Review
Trogocytosis is a mode of internalization of a part of a live cell by nibbling and is mechanistically distinct from phagocytosis, which implies internalization of a whole cell or a particle. Trogocytosis has been demonstrated in a broad range of cell types in multicellular organisms and is also known to be involved in a plethora of functions. In immune cells, trogocytosis is involved in the "cross-dressing" between antigen presenting cells and T cells, and is thus considered to mediate intercellular communication. On the other hand, trogocytosis has also been reported in a variety of unicellular organisms including the protistan (protozoan) parasite . ingests human T cell line by trogocytosis and acquires complement resistance and cross-dresses major histocompatibility complex (MHC) class I on the cell surface. Furthermore, trogocytosis and trogocytosis-like phenomena (nibbling of a live cell, not previously described as trogocytosis) have also been reported in other parasitic protists such as , , , and free-living amoebae. Thus, trogocytosis is conserved in diverse eukaryotic supergroups as a means of intercellular communication. It is depicting the universality of trogocytosis among eukaryotes. In this review, we summarize our current understanding of trogocytosis in unicellular organisms, including the history of its discovery, taxonomical distribution, roles, and molecular mechanisms.
Topics: Animals; Entamoeba histolytica; Eukaryota; Models, Biological; Parasites; Phagosomes; Trogocytosis
PubMed: 34831198
DOI: 10.3390/cells10112975 -
Virulence Dec 2023Tularaemia is a zoonotic disease caused by the Gram-negative bacterium, . Depending on its entry route into the organism, causes different diseases, ranging from... (Review)
Review
Tularaemia is a zoonotic disease caused by the Gram-negative bacterium, . Depending on its entry route into the organism, causes different diseases, ranging from life-threatening pneumonia to less severe ulceroglandular tularaemia. Various strains with different geographical distributions exhibit different levels of virulence. is an intracellular bacterium that replicates primarily in the cytosol of the phagocytes. The main virulence attribute of is the type 6 secretion system (T6SS) and its effectors that promote escape from the phagosome. In addition, has evolved a peculiar envelope that allows it to escape detection by the immune system. In this review, we cover tularaemia, different strains, and their pathogenicity. We particularly emphasize the intracellular life cycle, associated virulence factors, and metabolic adaptations. Finally, we present how largely escapes immune detection to be one of the most infectious and lethal bacterial pathogens.
Topics: Humans; Francisella tularensis; Virulence; Tularemia; Virulence Factors; Phagosomes
PubMed: 37941380
DOI: 10.1080/21505594.2023.2274638