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The Journal of Cell Biology Aug 2024Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged...
Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example, by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK-mediated inhibition of phagophore tethering to donor membrane. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.
Topics: Humans; AMP-Activated Protein Kinases; Autophagosomes; Autophagy; Glucose; Cell Line
PubMed: 38775785
DOI: 10.1083/jcb.202309145 -
Molecular Cell May 2024Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases,...
Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.
Topics: Peptides; Autophagy; Humans; Huntingtin Protein; Autophagosomes; Sequestosome-1 Protein; Amyloid; Huntington Disease; Protein Aggregates; Cryoelectron Microscopy; Animals; Protein Aggregation, Pathological
PubMed: 38759629
DOI: 10.1016/j.molcel.2024.04.018 -
Cellular and Molecular Life Sciences :... May 2024The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along...
The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2β and p150. We found that the mTORC1 inhibitor rapamycin enhanced p150-AP-2β complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150-AP-2β interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150-AP-2β interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150-AP-2β interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150-AP-2β interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.
Topics: Animals; Humans; Mice; Adaptor Protein Complex 2; Autophagosomes; Autophagy; Dynactin Complex; Lysosomal-Associated Membrane Protein 1; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Neurons; Protein Binding; Sirolimus
PubMed: 38758395
DOI: 10.1007/s00018-024-05256-6 -
PLoS Pathogens May 2024Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of...
Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.
Topics: Legionella pneumophila; Animals; rab GTP-Binding Proteins; Mice; Phagosomes; rab7 GTP-Binding Proteins; Lysosomes; Macrophages; Legionnaires' Disease; Sumoylation; Mice, Inbred C57BL; Endosomes
PubMed: 38739652
DOI: 10.1371/journal.ppat.1011783 -
Virulence Dec 2024() is the causative agent of Q fever, a zoonotic disease. Intracellular replication of requires the maturation of a phagolysosome-like compartment known as the...
() is the causative agent of Q fever, a zoonotic disease. Intracellular replication of requires the maturation of a phagolysosome-like compartment known as the replication permissive -containing vacuole (CCV). Effector proteins secreted by the Dot/Icm secretion system are indispensable for maturation of a single large CCV by facilitating the fusion of promiscuous vesicles. However, the mechanisms of CCV maintenance and evasion of host cell clearance remain to be defined. Here, we show that secreted vacuolar protein E (CvpE) contributes to CCV biogenesis by inducing lysosome-like vacuole (LLV) enlargement. LLV fission by tubulation and autolysosome degradation is impaired in CvpE-expressing cells. Subsequently, we found that CvpE suppresses lysosomal Ca channel transient receptor potential channel mucolipin 1 (TRPML1) activity in an indirect manner, in which CvpE binds phosphatidylinositol 3-phosphate [PI(3)P] and perturbs PIKfyve activity in lysosomes. Finally, the agonist of TRPML1, ML-SA5, inhibits CCV biogenesis and replication. These results provide insight into the mechanisms of CCV maintenance by CvpE and suggest that the agonist of TRPML1 can be a novel potential treatment that does not rely on antibiotics for Q fever by enhancing Coxiella-containing vacuoles (CCVs) fission.
Topics: Animals; Humans; Bacterial Proteins; Coxiella burnetii; HeLa Cells; Host-Pathogen Interactions; Lysosomes; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Q Fever; Transient Receptor Potential Channels; Vacuoles
PubMed: 38725096
DOI: 10.1080/21505594.2024.2350893 -
Nature Communications May 2024Patients with decreased levels of CD18 (β2 integrins) suffer from life-threatening bacterial and fungal infections. CD11b, the α subunit of integrin CR3 (CD11b/CD18,...
Patients with decreased levels of CD18 (β2 integrins) suffer from life-threatening bacterial and fungal infections. CD11b, the α subunit of integrin CR3 (CD11b/CD18, αβ), is essential for mice to fight against systemic Candida albicans infections. Live elongating C. albicans activates CR3 in immune cells. However, the hyphal ligands that activate CR3 are not well defined. Here, we discovered that the C. albicans Als family proteins are recognized by the I domain of CD11b in macrophages. This recognition synergizes with the β-glucan-bound lectin-like domain to activate CR3, thereby promoting Syk signaling and inflammasome activation. Dectin-2 activation serves as the "outside-in signaling" for CR3 activation at the entry site of incompletely sealed phagosomes, where a thick cuff of F-actin forms to strengthen the local interaction. In vitro, CD18 partially contributes to IL-1β release from dendritic cells induced by purified hyphal Als3. In vivo, Als3 is vital for C. albicans clearance in mouse kidneys. These findings uncover a novel family of ligands for the CR3 I domain that promotes fungal clearance.
Topics: Animals; Mice; beta-Glucans; Candida albicans; Candidiasis; CD11b Antigen; CD18 Antigens; Dendritic Cells; Fungal Proteins; Lectins, C-Type; Macrophages; Signal Transduction
PubMed: 38724513
DOI: 10.1038/s41467-024-48093-8 -
BioEssays : News and Reviews in... Jun 2024Autophagy, an essential cellular process for maintaining cellular homeostasis and eliminating harmful cytoplasmic objects, involves the de novo formation of... (Review)
Review
Autophagy, an essential cellular process for maintaining cellular homeostasis and eliminating harmful cytoplasmic objects, involves the de novo formation of double-membraned autophagosomes that engulf and degrade cellular debris, protein aggregates, damaged organelles, and pathogens. Central to this process is the phagophore, which forms from donor membranes rich in lipids synthesized at various cellular sites, including the endoplasmic reticulum (ER), which has emerged as a primary source. The ER-associated omegasomes, characterized by their distinctive omega-shaped structure and accumulation of phosphatidylinositol 3-phosphate (PI3P), play a pivotal role in autophagosome formation. Omegasomes are thought to serve as platforms for phagophore assembly by recruiting essential proteins such as DFCP1/ZFYVE1 and facilitating lipid transfer to expand the phagophore. Despite the critical importance of phagophore biogenesis, many aspects remain poorly understood, particularly the complete range of proteins involved in omegasome dynamics, and the detailed mechanisms of lipid transfer and membrane contact site formation.
Topics: Autophagosomes; Endoplasmic Reticulum; Autophagy; Humans; Animals; Phosphatidylinositol Phosphates
PubMed: 38724256
DOI: 10.1002/bies.202400038 -
Infection and Immunity Jun 2024The human-specific bacterial pathogen group A (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous...
The human-specific bacterial pathogen group A (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.
Topics: Streptococcus pyogenes; Humans; Macrophages; Lysosomes; Streptolysins; Bacterial Proteins; Phagosomes; THP-1 Cells; Phagocytosis; Streptococcal Infections; Cathepsin B; Hydrogen-Ion Concentration
PubMed: 38722166
DOI: 10.1128/iai.00141-24 -
Biochemical and Biophysical Research... Jul 2024Macro-autophagy (autophagy hereafter) is an evolutionarily conserved cellular process that has long been recognized as an intracellular mechanism for maintaining... (Review)
Review
Macro-autophagy (autophagy hereafter) is an evolutionarily conserved cellular process that has long been recognized as an intracellular mechanism for maintaining cellular homeostasis. It involves the formation of a membraned structure called the autophagosome, which carries cargo that includes toxic protein aggregates and dysfunctional organelles to the lysosome for degradation and recycling. Autophagy is primarily considered and studied as a cell-autonomous mechanism. However, recent studies have illuminated an underappreciated facet of autophagy, i.e., non-autonomously regulated autophagy. Non-autonomously regulated autophagy involves the degradation of autophagic components, including organelles, cargo, and signaling molecules, and is induced in neighboring cells by signals from primary adjacent or distant cells/tissues/organs. This review provides insight into the complex molecular mechanisms governing non-autonomously regulated autophagy, highlighting the dynamic interplay between cells within tissue/organ or distinct cell types in different tissues/organs. Emphasis is placed on modes of intercellular communication that include secreted molecules, including microRNAs, and their regulatory roles in orchestrating this phenomenon. Furthermore, we explore the multidimensional roles of non-autonomously regulated autophagy in various physiological contexts, spanning tissue development and aging, as well as its importance in diverse pathological conditions, including cancer and neurodegeneration. By studying the complexities of non-autonomously regulated autophagy, we hope to gain insights into the sophisticated intercellular dynamics within multicellular organisms, including mammals. These studies will uncover novel avenues for therapeutic intervention to modulate intercellular autophagic pathways in altered human physiology.
Topics: Humans; Autophagy; Animals; Cell Communication; MicroRNAs; Neoplasms; Signal Transduction; Autophagosomes
PubMed: 38701555
DOI: 10.1016/j.bbrc.2024.150024 -
Medicine May 2024Hepatocellular carcinoma (HCC) is one of the most common malignant tumors globally and often develops on the foundation of chronic liver disease or cirrhosis. Cirrhosis... (Observational Study)
Observational Study
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors globally and often develops on the foundation of chronic liver disease or cirrhosis. Cirrhosis is a clinically prevalent chronic progressive liver disease characterized by diffuse liver damage resulting from long-term or repeated actions of 1 or more etiological factors. However, the impact of CENPF and nuclear division cycle 80 (NDC80) genes on rehabilitation nursing of HCC and cirrhosis remains unclear. HCC and cirrhosis datasets GSE63898 and GSE89377 profile files were downloaded from the gene expression omnibus database generated on platforms GPL13667 and GPL6947, respectively. Differentially expressed genes (DEGs) screening, weighted gene co-expression network analysis (WGCNA), construction and analysis of protein-protein interaction (PPI) networks, functional enrichment analysis, gene set enrichment analysis (GSEA), survival analysis, immune infiltration analysis, and comparative toxicogenomics database (CTD) analysis were conducted. Gene expression heatmaps were plotted. miRNAs regulating central DEGs were selected through TargetScan. A total of 626 DEGs were identified. According to gene ontology (GO) analysis, they were primarily enriched in small molecule metabolic processes, drug metabolic processes, binding of identical proteins, and lipid metabolic processes. Kyoto Encyclopedia of Gene and Genome (KEGG) analysis results indicated that the target genes were mainly enriched in metabolic pathways, phagosomes, glycine, serine, and threonine metabolism. The construction and analysis of the PPI network revealed 3 core genes (NDC80, CENPF, RRM2). Gene expression heatmaps showed that core genes (CENPF, NDC80) were highly expressed in HCC and cirrhosis samples. CTD analysis found that 2 genes (CENPF and NDC80) were associated with liver, jaundice, ascites, fever, dyspepsia, and hepatic encephalopathy. CENPF and NDC80 are highly expressed in HCC and cirrhosis, and CENPF and NDC80 might be the biomarkers of rehabilitation nursing of HCC and cirrhosis.
Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Cirrhosis; Nuclear Proteins; Protein Interaction Maps; Gene Expression Profiling; Cytoskeletal Proteins
PubMed: 38701255
DOI: 10.1097/MD.0000000000037984