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The Journal of Biological Chemistry Nov 2023The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral...
The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway.
Topics: Humans; Autophagy; DNA-Binding Proteins; HeLa Cells; Intercellular Signaling Peptides and Proteins; Lysosomes; Progranulins; Microtubule-Associated Proteins; Gene Expression Regulation; Extracellular Vesicles; Enzyme Inhibitors; Autophagosomes; Autophagy-Related Proteins
PubMed: 37739033
DOI: 10.1016/j.jbc.2023.105272 -
Communications Biology Oct 2023Phagosome maturation is critical for immune defense, defining whether ingested material is destroyed or converted into antigens. Sec22b regulates phagosome maturation,...
Phagosome maturation is critical for immune defense, defining whether ingested material is destroyed or converted into antigens. Sec22b regulates phagosome maturation, yet how has remained unclear. Here we show Sec22b tethers endoplasmic reticulum-phagosome membrane contact sites (MCS) independently of the known tether STIM1. Sec22b knockdown increases calcium signaling, phagolysosome fusion and antigen degradation and alters phagosomal phospholipids PI(3)P, PS and PI(4)P. Levels of PI(4)P, a lysosome docking lipid, are rescued by Sec22b re-expression and by expression of the artificial tether MAPPER but not the MCS-disrupting mutant Sec22b-P33. Moreover, Sec22b co-precipitates with the PS/PI(4)P exchange protein ORP8. Wild-type, but not mutant ORP8 rescues phagosomal PI(4)P and reduces antigen degradation. Sec22b, MAPPER and ORP8 but not P33 or mutant-ORP8 restores phagolysosome fusion in knockdown cells. These findings clarify an alternative mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.
Topics: Phagosomes; Phagocytosis; Endoplasmic Reticulum; Phosphatidylinositol Phosphates
PubMed: 37794132
DOI: 10.1038/s42003-023-05382-0 -
Proceedings of the National Academy of... Jan 2024Lipid droplets (LDs) are organelles critical for energy storage and membrane lipid homeostasis, whose number and size are carefully regulated in response to cellular...
Lipid droplets (LDs) are organelles critical for energy storage and membrane lipid homeostasis, whose number and size are carefully regulated in response to cellular conditions. The molecular mechanisms underlying lipid droplet biogenesis and degradation, however, are not well understood. The Troyer syndrome protein spartin (SPG20) supports LD delivery to autophagosomes for turnover via lipophagy. Here, we characterize spartin as a lipid transfer protein whose transfer ability is required for LD degradation. Spartin copurifies with phospholipids and neutral lipids from cells and transfers phospholipids in vitro via its senescence domain. A senescence domain truncation that impairs lipid transfer in vitro also impairs LD turnover in cells while not affecting spartin association with either LDs or autophagosomes, supporting that spartin's lipid transfer ability is physiologically relevant. Our data indicate a role for spartin-mediated lipid transfer in LD turnover.
Topics: Lipid Droplets; Autophagosomes; Autophagy; Membrane Lipids
PubMed: 38190532
DOI: 10.1073/pnas.2314093121 -
Journal of Thoracic Disease Oct 2023Cystic fibrosis (CF) is a disorder that affects the cystic fibrosis transmembrane conductance regulator (CFTR). Without properly functioning CFTR channels, chloride does... (Review)
Review
BACKGROUND AND OBJECTIVE
Cystic fibrosis (CF) is a disorder that affects the cystic fibrosis transmembrane conductance regulator (CFTR). Without properly functioning CFTR channels, chloride does not exit respiratory epithelial cells, and consequently the mucus lining the surface of the cells becomes thick. This viscous mucus accumulates and causes abnormal function of the mucociliary apparatus, which can lead to bacterial colonization, infections with () and (), and eventually lung damage. Recent studies have shown that the increased susceptibility to respiratory infections in CF patients may also be due to defects in neutrophil function, but the exact mechanism is uncertain.
METHODS
The PubMed database was searched on February 10, 2023 and again on July 23, 2023 to compile a comprehensive list of clinical and experimental studies to evaluate neutrophil function in CF. The first search included a combination of MeSH terms: "cystic fibrosis" and "neutrophils/physiology". A separate second search included a combination of the MeSH terms: "neutrophils" and "cystic fibrosis transmembrane conductance regulator".
KEY CONTENT AND FINDINGS
Neutrophils from patients with CF have decreased transfer of chloride into phagolysosomes after bacterial ingestion and have dysregulated degranulation. This reduces the production of toxic oxidative radicals, especially hypochlorous acid (HOCl), and reduces bactericidal activity. CFTR potentiators correct the dysregulated degranulation in patients with CF and increased neutrophil killing activity. A reduced concentration of chloride in assays also reduces neutrophil killing activity; these observations are relevant to the reduced chloride concentrations in respiratory secretions in patients with CF.
CONCLUSIONS
This literature review summarizes studies that demonstrate that an important defect in CF neutrophils lies in the oxygen-dependent pathway in phagolysosomes and studies with ivacaftor demonstrate that this drug corrects CF neutrophil function. These studies demonstrate the potential utility of using easily available neutrophils to study drug effects in CF patients.
PubMed: 37969285
DOI: 10.21037/jtd-23-846 -
ELife Jul 2023The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and...
The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and their association with the autophagosome membrane through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosome membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in , but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function. Our data question the use of lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.
Topics: Humans; Animals; Caenorhabditis elegans; Microtubule-Associated Proteins; Autophagy; Autophagosomes; Phagocytosis; Mammals; Apoptosis Regulatory Proteins; Caenorhabditis elegans Proteins
PubMed: 37395461
DOI: 10.7554/eLife.85748 -
Cell Death & Disease Aug 2023Accumulating evidence has shown that the quality of proteins must be tightly monitored and controlled to maintain cellular proteostasis. Misfolded proteins and protein...
Accumulating evidence has shown that the quality of proteins must be tightly monitored and controlled to maintain cellular proteostasis. Misfolded proteins and protein aggregates are targeted for degradation through the ubiquitin proteasome (UPS) and autophagy-lysosome systems. The ubiquitination and deubiquitinating enzymes (DUBs) have been reported to play pivotal roles in the regulation of the UPS system. However, the function of DUBs in the regulation of autophagy remain to be elucidated. In this study, we found that knockdown of Leon/USP5 caused a marked increase in the formation of autophagosomes and autophagic flux under well-fed conditions. Genetic analysis revealed that overexpression of Leon suppressed Atg1-induced cell death in Drosophila. Immunoblotting assays further showed a strong interaction between Leon/USP5 and the autophagy initiating kinase Atg1/ULK1. Depletion of Leon/USP5 led to increased levels of Atg1/ULK1. Our findings indicate that Leon/USP5 is an autophagic DUB that interacts with Atg1/ULK1, negatively regulating the autophagic process.
Topics: Animals; Autophagy; Autophagosomes; Cell Death; Drosophila; Lysosomes; Proteasome Endopeptidase Complex; Ubiquitin; Deubiquitinating Enzymes; Autophagy-Related Protein-1 Homolog; Drosophila Proteins; Ubiquitin-Specific Proteases
PubMed: 37607937
DOI: 10.1038/s41419-023-06062-x -
The Journal of Cell Biology Mar 2024In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane...
In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR.
Topics: Humans; Actin Cytoskeleton; Autophagosomes; Dynactin Complex; Dyneins; GTPase-Activating Proteins; Lysosomes; rab GTP-Binding Proteins; Sorting Nexins
PubMed: 38323995
DOI: 10.1083/jcb.202306040 -
Function (Oxford, England) 2024The skeletal system is crucial for supporting bodily functions, protecting vital organs, facilitating hematopoiesis, and storing essential minerals. Skeletal... (Review)
Review
The skeletal system is crucial for supporting bodily functions, protecting vital organs, facilitating hematopoiesis, and storing essential minerals. Skeletal homeostasis, which includes aspects such as bone density, structural integrity, and regenerative processes, is essential for normal skeletal function. Autophagy, an intricate intracellular mechanism for degrading and recycling cellular components, plays a multifaceted role in bone metabolism. It involves sequestering cellular waste, damaged proteins, and organelles within autophagosomes, which are then degraded and recycled. Autophagy's impact on bone health varies depending on factors such as regulation, cell type, environmental cues, and physiological context. Despite being traditionally considered a cytoplasmic process, autophagy is subject to transcriptional and epigenetic regulation within the nucleus. However, the precise influence of epigenetic regulation, including DNA methylation, histone modifications, and non-coding RNA expression, on cellular fate remains incompletely understood. The interplay between autophagy and epigenetic modifications adds complexity to bone cell regulation. This article provides an in-depth exploration of the intricate interplay between these two regulatory paradigms, with a focus on the epigenetic control of autophagy in bone metabolism. Such an understanding enhances our knowledge of bone metabolism-related disorders and offers insights for the development of targeted therapeutic strategies.
Topics: Humans; Epigenesis, Genetic; Autophagy; Homeostasis; Autophagosomes; Bone Density; Bone Diseases, Metabolic
PubMed: 38486976
DOI: 10.1093/function/zqae004 -
Disease Models & Mechanisms Oct 2023Invasive fungal infections represent a significant global health problem, and present several clinical challenges, including limited treatment options, increasing rates... (Review)
Review
Invasive fungal infections represent a significant global health problem, and present several clinical challenges, including limited treatment options, increasing rates of antifungal drug resistance and compounding comorbidities in affected patients. Metals, such as copper, iron and zinc, are critical for various biological and cellular processes across phyla. In mammals, these metals are important determinants of immune responses, but pathogenic microbes, including fungi, also require access to these metals to fuel their own growth and drive expression of major virulence traits. Therefore, host immune cells have developed strategies to either restrict access to metals to induce starvation of invading pathogens or deploy toxic concentrations within phagosomes to cause metal poisoning. In this Review, we describe the mechanisms regulating fungal scavenging and detoxification of copper, iron and zinc and the importance of these mechanisms for virulence and infection. We also outline how these metals are involved in host immune responses and the consequences of metal deficiencies or overloads on how the host controls invasive fungal infections.
Topics: Animals; Humans; Copper; Virulence; Metals; Iron; Zinc; Invasive Fungal Infections; Mammals
PubMed: 37905492
DOI: 10.1242/dmm.050393 -
The Journal of Cell Biology Mar 2024Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson's disease and several inflammatory diseases, has been shown to localize to stressed...
Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson's disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here, we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.
Topics: Adenosine Triphosphatases; Autophagosomes; Autophagy; Cell Cycle Proteins; Lysosomes; Humans; Animals; Mice; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Autophagy-Related Proteins
PubMed: 38227290
DOI: 10.1083/jcb.202302067