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European Journal of Cell Biology Jun 2024Efficient degradation of phagocytic cargo in lysosomes is crucial to maintain cellular homeostasis and defending cells against pathogens. However, the mechanisms...
Efficient degradation of phagocytic cargo in lysosomes is crucial to maintain cellular homeostasis and defending cells against pathogens. However, the mechanisms underlying the degradation and recycling of macromolecular cargo within the phagolysosome remain incompletely understood. We previously reported that the phagolysosome containing the corpse of the polar body in C. elegans tubulates into small vesicles to facilitate corpse clearance, a process that requires cargo protein degradation and amino acid export. Here we show that degradation of hexosylceramides by the prosaposin ortholog SPP-10 and glucosylceramidases is required for timely corpse clearance. We observed accumulation of membranous structures inside endolysosomes of spp-10-deficient worms, which are likely caused by increased hexosylceramide species. spp-10 deficiency also caused alteration of additional sphingolipid subclasses, like dihydroceramides, 2-OH-ceramides, and dihydrosphingomyelins. While corpse engulfment, initial breakdown of corpse membrane inside the phagolysosome and lumen acidification proceeded normally in spp-10-deficient worms, formation of the cargo-containing vesicles from the corpse phagolysosome was reduced, resulting in delayed cargo degradation and phagolysosome resolution. Thus, by combining ultrastructural studies and sphingolipidomic analysis with observing single phagolysosomes over time, we identified a role of prosaposin/SPP-10 in maintaining phagolysosomal structure, which promotes efficient resolution of phagocytic cargos.
Topics: Animals; Caenorhabditis elegans; Phagosomes; Caenorhabditis elegans Proteins; Saposins; Lysosomes; Phagocytosis; Ceramides
PubMed: 38582051
DOI: 10.1016/j.ejcb.2024.151411 -
Microbial Pathogenesis May 2024Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious...
Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious Bronchitis Virus (IBV) and autophagy. This study aims to investigate the impact of IBV on autophagy and the role of autophagy in viral replication. We observed that IBV infection increased the expression of microtubule-associated protein 1 light chain 3, a marker of autophagy, decreased the expression of sequestosome 1, and led to elevated intracellular LC3 puncta levels. These findings suggest that IBV infection activates the autophagic process in cells. To investigate the impact of autophagy on the replication of IBV, we utilized rapamycin as an autophagy activator and 3-methyladenine as an autophagy inhibitor. Our results indicate that IBV promotes viral replication by inducing autophagy. Further investigation revealed that IBV induces autophagosome formation by inhibiting the mTOR-ULK1 pathway and activating the activity of vacuolar protein sorting 34 (VPS34), autophagy-related gene 14, and the Beclin-1 complex. VPS34 plays a crucial role in this process, as inhibiting VPS34 protein activity enhances cell proliferation after IBV infection. Additionally, inhibiting VPS34 significantly improves the survival rate of IBV-infected chicks, suppresses IBV replication in the kidney, and alleviates tracheal, lung, and kidney damage caused by IBV infection. In summary, IBV infection can induce autophagy by modulating the mTOR/ULK1 signaling pathway and activating the VPS34 complex, while autophagy serves to promote virus replication.
Topics: Infectious bronchitis virus; Virus Replication; Autophagy; Animals; Class III Phosphatidylinositol 3-Kinases; Chickens; Coronavirus Infections; Sirolimus; Beclin-1; TOR Serine-Threonine Kinases; Signal Transduction; Cell Line; Poultry Diseases; Autophagosomes; Chlorocebus aethiops; Microtubule-Associated Proteins
PubMed: 38574829
DOI: 10.1016/j.micpath.2024.106638 -
Bioscience Reports May 2024Chloride is a key anion involved in cellular physiology by regulating its homeostasis and rheostatic processes. Changes in cellular Cl- concentration result in... (Review)
Review
Chloride is a key anion involved in cellular physiology by regulating its homeostasis and rheostatic processes. Changes in cellular Cl- concentration result in differential regulation of cellular functions such as transcription and translation, post-translation modifications, cell cycle and proliferation, cell volume, and pH levels. In intracellular compartments, Cl- modulates the function of lysosomes, mitochondria, endosomes, phagosomes, the nucleus, and the endoplasmic reticulum. In extracellular fluid (ECF), Cl- is present in blood/plasma and interstitial fluid compartments. A reduction in Cl- levels in ECF can result in cell volume contraction. Cl- is the key physiological anion and is a principal compensatory ion for the movement of the major cations such as Na+, K+, and Ca2+. Over the past 25 years, we have increased our understanding of cellular signaling mediated by Cl-, which has helped in understanding the molecular and metabolic changes observed in pathologies with altered Cl- levels. Here, we review the concentration of Cl- in various organs and cellular compartments, ion channels responsible for its transportation, and recent information on its physiological roles.
Topics: Humans; Chlorides; Animals; Homeostasis; Chloride Channels; Signal Transduction; Extracellular Fluid; Ion Transport
PubMed: 38573803
DOI: 10.1042/BSR20240029 -
The Journal of Cell Biology Jul 2024Autophagy serves as a stress response pathway by mediating the degradation of cellular material within lysosomes. In autophagy, this material is encapsulated in...
Autophagy serves as a stress response pathway by mediating the degradation of cellular material within lysosomes. In autophagy, this material is encapsulated in double-membrane vesicles termed autophagosomes, which form from precursors referred to as phagophores. Phagophores grow by lipid influx from the endoplasmic reticulum into Atg9-positive compartments and local lipid synthesis provides lipids for their expansion. How phagophore nucleation and expansion are coordinated with lipid synthesis is unclear. Here, we show that Faa1, an enzyme activating fatty acids, is recruited to Atg9 vesicles by directly binding to negatively charged membranes with a preference for phosphoinositides such as PI3P and PI4P. We define the membrane-binding surface of Faa1 and show that its direct interaction with the membrane is required for its recruitment to phagophores. Furthermore, the physiological localization of Faa1 is key for its efficient catalysis and promotes phagophore expansion. Our results suggest a positive feedback loop coupling phagophore nucleation and expansion to lipid synthesis.
Topics: Autophagosomes; Autophagy; Fatty Acids; Feedback; Macroautophagy; Saccharomyces cerevisiae
PubMed: 38573225
DOI: 10.1083/jcb.202309057 -
Acta Neuropathologica Mar 2024Prader-Willi Syndrome (PWS) is a rare neurodevelopmental disorder of genetic etiology, characterized by paternal deletion of genes located at chromosome 15 in 70% of...
Prader-Willi Syndrome (PWS) is a rare neurodevelopmental disorder of genetic etiology, characterized by paternal deletion of genes located at chromosome 15 in 70% of cases. Two distinct genetic subtypes of PWS deletions are characterized, where type I (PWS T1) carries four extra haploinsufficient genes compared to type II (PWS T2). PWS T1 individuals display more pronounced physiological and cognitive abnormalities than PWS T2, yet the exact neuropathological mechanisms behind these differences remain unclear. Our study employed postmortem hypothalamic tissues from PWS T1 and T2 individuals, conducting transcriptomic analyses and cell-specific protein profiling in white matter, neurons, and glial cells to unravel the cellular and molecular basis of phenotypic severity in PWS sub-genotypes. In PWS T1, key pathways for cell structure, integrity, and neuronal communication are notably diminished, while glymphatic system activity is heightened compared to PWS T2. The microglial defect in PWS T1 appears to stem from gene haploinsufficiency, as global and myeloid-specific Cyfip1 haploinsufficiency in murine models demonstrated. Our findings emphasize microglial phagolysosome dysfunction and altered neural communication as crucial contributors to the severity of PWS T1's phenotype.
Topics: Humans; Mice; Animals; Prader-Willi Syndrome; Microglia; Carrier Proteins; Phenotype; Phagosomes; Adaptor Proteins, Signal Transducing
PubMed: 38556574
DOI: 10.1007/s00401-024-02714-0 -
Nature Communications Mar 2024Hospital-acquired pneumonia (HAP) is associated with high mortality and costs, and frequently caused by multidrug-resistant (MDR) bacteria. Although prior antimicrobial...
Hospital-acquired pneumonia (HAP) is associated with high mortality and costs, and frequently caused by multidrug-resistant (MDR) bacteria. Although prior antimicrobial therapy is a major risk factor for HAP, the underlying mechanism remains incompletely understood. Here, we demonstrate that antibiotic therapy in hospitalized patients is associated with decreased diversity of the gut microbiome and depletion of short-chain fatty acid (SCFA) producers. Infection experiments with mice transplanted with patient fecal material reveal that these antibiotic-induced microbiota perturbations impair pulmonary defense against MDR Klebsiella pneumoniae. This is dependent on inflammatory monocytes (IMs), whose fatty acid receptor (FFAR)2/3-controlled and phagolysosome-dependent antibacterial activity is compromized in mice transplanted with antibiotic-associated patient microbiota. Collectively, we characterize how clinically relevant antibiotics affect antimicrobial defense in the context of human microbiota, and reveal a critical impairment of IM´s antimicrobial activity. Our study provides additional arguments for the rational use of antibiotics and offers mechanistic insights for the development of novel prophylactic strategies to protect high-risk patients from HAP.
Topics: Humans; Mice; Animals; Anti-Bacterial Agents; Monocytes; Anti-Infective Agents; Klebsiella pneumoniae; Lung
PubMed: 38555356
DOI: 10.1038/s41467-024-47149-z -
Communications Biology Mar 2024Autophagy is a dynamic self-renovation biological process that maintains cell homeostasis and is responsible for the quality control of proteins, organelles, and energy... (Review)
Review
Autophagy is a dynamic self-renovation biological process that maintains cell homeostasis and is responsible for the quality control of proteins, organelles, and energy metabolism. The E1-like ubiquitin-activating enzyme autophagy-related gene 7 (ATG7) is a critical factor that initiates classic autophagy reactions by promoting the formation and extension of autophagosome membranes. Recent studies have identified the key functions of ATG7 in regulating the cell cycle, apoptosis, and metabolism associated with the occurrence and development of multiple diseases. This review summarizes how ATG7 is precisely programmed by genetic, transcriptional, and epigenetic modifications in cells and the relationship between ATG7 and aging-related diseases.
Topics: Autophagy-Related Protein 7; Autophagosomes; Autophagy; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes
PubMed: 38553562
DOI: 10.1038/s42003-024-06080-1 -
PLoS Pathogens Mar 2024Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the...
Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote differentiation into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastidae. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of pkar3 or pkac3 null mutants lose their elongated shape and become rounded but remain flagellated. Truncation of an N-terminal formin homology (FH)-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARIβ and PKARIIβ binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 binds nucleoside analogs, but not cAMP, with a high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early-diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape.
Topics: Animals; Humans; Female; Leishmania; Cyclic AMP-Dependent Protein Kinases; Macrophages; Cell Differentiation; Morphogenesis; Mammals
PubMed: 38551993
DOI: 10.1371/journal.ppat.1012073 -
Cells Mar 2024In eukaryotes, targeting intracellular components for lysosomal degradation by autophagy represents a catabolic process that evolutionarily regulates cellular... (Review)
Review
In eukaryotes, targeting intracellular components for lysosomal degradation by autophagy represents a catabolic process that evolutionarily regulates cellular homeostasis. The successful completion of autophagy initiates the engulfment of cytoplasmic materials within double-membrane autophagosomes and subsequent delivery to autolysosomes for degradation by acidic proteases. The formation of autolysosomes relies on the precise fusion of autophagosomes with lysosomes. In recent decades, numerous studies have provided insights into the molecular regulation of autophagosome-lysosome fusion. In this review, an overview of the molecules that function in the fusion of autophagosomes with lysosomes is provided. Moreover, the molecular mechanism underlying how these functional molecules regulate autophagosome-lysosome fusion is summarized.
Topics: Animals; Autophagosomes; Autophagy; Macroautophagy; Homeostasis; Lysosomes; Mammals
PubMed: 38534345
DOI: 10.3390/cells13060500 -
Microbiology Spectrum May 2024To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This...
To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This cocktail is delivered to macrophages, through a targeted liposome presented here as ENTX_001. Endolytix Cocktail 1 (EC1) leverages mycobacteriophage lysin enzymes LysA and LysB, while also including α-amylase and isoamylase for degradation of the mycobacterial envelope from outside of the cell. The LysA family of proteins from mycobacteriophages has been shown to cleave the peptidoglycan layer, whereas LysB is an esterase that hydrolyzes the linkage between arabinogalactan and mycolic acids of the mycomembrane. The challenge of gaining access to the substrates of LysA and LysB provided exogenously was addressed by adding amylase enzymes that degrade the extracellular capsule shown to be present in . This enzybiotic approach avoids antimicrobial resistance, specific receptor-mediated binding, and intracellular DNA surveillance pathways that limit many bacteriophage applications. We show this cocktail of enzymes is bactericidal against both rapid- and slow-growing nontuberculous mycobacteria (NTM) as well as strains. The EC1 cocktail shows superior killing activity when compared to previously characterized LysB alone. EC1 is also powerfully synergistic with standard-of-care antibiotics. In addition to killing of NTM, ENTX_001 demonstrates the rescue of infected macrophages from necrotic death by and . Here, we demonstrate shredding of mycobacterial cells by EC1 into cellular debris as a mechanism of bactericide.IMPORTANCEThe world needs entirely new forms of antibiotics as resistance to chemical antibiotics is a critical problem facing society. We addressed this need by developing a targeted enzyme therapy for a broad range of species and strains within mycobacteria and highly related genera including nontuberculous mycobacteria such as , , as well as . One advantage of this approach is the ability to drive our lytic enzymes through encapsulation into macrophage-targeted liposomes resulting in attack of mycobacteria in the cells that harbor them where they hide from the adaptive immune system and grow. Furthermore, this approach shreds mycobacteria independent of cell physiology as the drug targets the mycobacterial envelope while sidestepping the host range limitations observed with phage therapy and resistance to chemical antibiotics.
Topics: Mycobacterium tuberculosis; Mycobacteriophages; Macrophages; Humans; Nontuberculous Mycobacteria; Liposomes; Anti-Bacterial Agents; Peptidoglycan; Microbial Sensitivity Tests; Endopeptidases; Galactans
PubMed: 38534149
DOI: 10.1128/spectrum.03534-23