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Autophagy Apr 2019Helicobacter pylori (H. pylori) is a common human pathogenic bacterium. Once infected, it is difficult for the host to clear this organism using the innate immune...
Helicobacter pylori (H. pylori) is a common human pathogenic bacterium. Once infected, it is difficult for the host to clear this organism using the innate immune system. Increased antibiotic resistance further makes it challenging for effective eradication. However, the mechanisms of immune evasion still remain obscure, and novel strategies should be developed to efficiently eliminate H. pylori infection in stomachs. Here we uncovered desirable anti-H. pylori effect of vitamin D3 both in vitro and in vivo, even against antibiotic-resistant strains. We showed that H. pylori can invade into the gastric epithelium where they became sequestered and survived in autophagosomes with impaired lysosomal acidification. Vitamin D3 treatment caused a restored lysosomal degradation function by activating the PDIA3 receptor, thereby promoting the nuclear translocation of PDIA3-STAT3 protein complex and the subsequent upregulation of MCOLN3 channels, resulting in an enhanced Ca release from lysosomes and normalized lysosomal acidification. The recovered lysosomal degradation function drives H. pylori to be eliminated through the autolysosomal pathway. These findings provide a novel pathogenic mechanism on how H. pylori can survive in the gastric epithelium, and a unique pathway for vitamin D3 to reactivate the autolysosomal degradation function, which is critical for the antibacterial action of vitamin D3 both in cells and in animals, and perhaps further in humans. Abbreviations: 1,25D3: 1α, 25-dihydroxyvitamin D3; ATG5: autophagy related 5; Baf A1: bafilomycin A; BECN1: beclin 1; CagA: cytotoxin-associated gene A; CFU: colony-forming unit; ChIP-PCR: chromatin immunoprecipitation-polymerase chain reaction; Con A: concanamycin A; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; CTSD: cathepsin D; GPN: Gly-Phe-β-naphthylamide; H. pylori: Helicobacter pylori; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; MCOLN3: mucolipin 3; MCU: mitochondrial calcium uniporter; MOI: multiplicity of infection; NAGLU: N-acetyl-alpha-glucosaminidase; PDIA3: protein disulfide isomerase family A member 3; PMA: phorbol 12-myristate 13-acetate; PRKC: protein kinase C; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; SS1: Sydney Strain 1; TRP: transient receptor potential; VacA: vacuolating cytotoxin; VD3: vitamin D3; VDR: vitamin D receptor.
Topics: Acetylglucosaminidase; Acid Phosphatase; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Autophagosomes; Autophagy; Autophagy-Related Protein 5; Calcium; Carrier Proteins; Cell Line; Cholecalciferol; Epithelial Cells; Helicobacter Infections; Helicobacter pylori; Humans; Lysosomes; Male; Mice, Inbred C57BL; Protein Disulfide-Isomerases; Receptors, Calcitriol; STAT3 Transcription Factor; Stomach; Transient Receptor Potential Channels; Cathelicidins
PubMed: 30612517
DOI: 10.1080/15548627.2018.1557835 -
Nature Reviews. Molecular Cell Biology Jun 2021
Topics: Biological Transport; Lysosomes
PubMed: 33911233
DOI: 10.1038/s41580-021-00376-4 -
Biochemical and Biophysical Research... Jul 2022Macroautophagy (hereafter autophagy) is a conserved cellular degradation system, impairments in which have been implicated in the development of a wide range of...
Macroautophagy (hereafter autophagy) is a conserved cellular degradation system, impairments in which have been implicated in the development of a wide range of diseases, including cancer and neurodegenerative diseases. Autophagy is mainly comprised of two processes: the formation of autophagosomes and autolysosomes. A detailed understanding of the formation of autophagosomes has been obtained in the past several decades. However, limited information is currently available on the formation of autolysosomes, which may partially be attributed to fewer methods to study the formation of autolysosomes than that of autophagosomes. Abemaciclib (Abe) and vacuolin-1 (Vac) are drugs that suppress the progression of breast cancer and induce characteristic vacuole formation in cells. Since Abe-induced vacuoles have the appearance of autolysosomes, they may be used to examine the formation of autolysosomes. However, it remains unknown whether Abe-/Vac-induced vacuoles are regulated by autophagosome-lysosome fusion. Markers for endosomes, lysosomes, and autophagosomes (Rab7, LAMP1, and mRFP-GFP-LC3, respectively) indicated that Abe-/Vac-induced vacuoles were autolysosomes. Abe and Vac failed to induce vacuolation in ATG16L1-deficient autophagy-null cells. Furthermore, Abe-/Vac-induced vacuolation was suppressed by bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, whereas it was facilitated by rapamycin and the overexpression of Beclin-1, inducers of autophagosome-lysosome fusion. Moreover, vacuole formation was inhibited by the knockdown of progranulin (PGRN), a regulator of autophagosome-lysosome fusion, and promoted by its overexpression. The present results suggest the potential of Abe-/Vac-induced vacuole-like autolysosomes as a tool for evaluating autophagosome-lysosome fusion and examining the effects of PGRN in autophagy.
Topics: Aminopyridines; Autophagosomes; Autophagy; Benzimidazoles; Heterocyclic Compounds, 4 or More Rings; Lysosomes; Macroautophagy; Progranulins; Vacuoles
PubMed: 35598430
DOI: 10.1016/j.bbrc.2022.05.027 -
The Journal of Biological Chemistry Apr 2018Ubiquitination is a widespread post-translational modification that controls multiple steps in autophagy, a major lysosome-mediated intracellular degradation pathway. A... (Review)
Review
Ubiquitination is a widespread post-translational modification that controls multiple steps in autophagy, a major lysosome-mediated intracellular degradation pathway. A variety of ubiquitin chains are attached as selective labels on protein aggregates and dysfunctional organelles, thus promoting their autophagy-dependent degradation. Moreover, ubiquitin modification of autophagy regulatory components is essential to positively or negatively regulate autophagy flux in both non-selective and selective pathways. We review the current findings that elucidate the components, timing, and kinetics of the multivalent role of ubiquitin signals in control of amplitude and selectivity of autophagy pathways as well as their impact on the development of human diseases.
Topics: Animals; Humans; Lysosomes; Protein Aggregates; Proteolysis; Signal Transduction; Ubiquitin; Ubiquitination
PubMed: 29187595
DOI: 10.1074/jbc.TM117.000117 -
Cells Mar 2019The small GTPase, Rab7a, and the regulators of its GDP/GTP-binding status were shown to have roles in both endocytic membrane traffic and autophagy. Classically known to... (Review)
Review
The small GTPase, Rab7a, and the regulators of its GDP/GTP-binding status were shown to have roles in both endocytic membrane traffic and autophagy. Classically known to regulate endosomal retrograde transport and late endosome-lysosome fusion, earlier work has indicated a role for Rab7a in autophagosome-lysosome fusion as well as autolysosome maturation. However, as suggested by recent findings on PTEN-induced kinase 1 (PINK1)-Parkin-mediated mitophagy, Rab7a and its regulators are critical for the correct targeting of Atg9a-bearing vesicles to effect autophagosome formation around damaged mitochondria. This mitophagosome formation role for Rab7a is dependent on an intact Rab cycling process mediated by the Rab7a-specific guanine nucleotide exchange factor (GEF) and GTPase activating proteins (GAPs). Rab7a activity in this regard is also dependent on the retromer complex, as well as phosphorylation by the TRAF family-associated NF-κB activator binding kinase 1 (TBK1). Here, we discuss these recent findings and broadened perspectives on the role of the Rab7a network in PINK1-Parkin mediated mitophagy.
Topics: Animals; Humans; Lysosomes; Mitophagy; Models, Biological; Phagosomes; Phosphorylation; rab GTP-Binding Proteins
PubMed: 30857122
DOI: 10.3390/cells8030224 -
Acta Tropica Jun 2017Autophagy has emerged as an essential component of the defense system against intracellular pathogens. We demonstrated that Trypanosoma cruzi, an intracellular protozoan...
Autophagy has emerged as an essential component of the defense system against intracellular pathogens. We demonstrated that Trypanosoma cruzi, an intracellular protozoan parasite, was not eliminated by the host's autophagic machinery despite exposure to the host cell cytoplasm. Puncta of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and LC3-II, a lipidated form of LC3, were significantly increased after infection with T. cruzi, indicating that the parasite activated the early steps of host autophagy and induced autophagosome formation. However, autolysosomes were not observed in the infected cells. In addition, T. cruzi was not enwrapped by autophagosomes, suggesting that the parasite has mechanisms to allow it to evade autophagic capture. The results of this study indicate that host autophagy is incomplete following T. cruzi infection.
Topics: Animals; Autophagy; Biomarkers; Chagas Disease; Humans; Lysosomes; Microtubule-Associated Proteins; Phagosomes; Trypanosoma cruzi
PubMed: 28232068
DOI: 10.1016/j.actatropica.2017.02.021 -
Journal of Trace Elements in Medicine... Dec 2020Autophagy is a conserved catabolic process that plays an important role in cellular homeostasis. The study of the interplay between autophagy and zinc has gained... (Review)
Review
Autophagy is a conserved catabolic process that plays an important role in cellular homeostasis. The study of the interplay between autophagy and zinc has gained interest over the last years. Multiple studies have indicated that zinc stimulates autophagy and is critical for basal and induced autophagy in mammalian cells. Conversely, autophagy is induced by zinc starvation in yeast. There are no studies analyzing the role of zinc in either Microautophagy or Chaperone-Mediated-Autophagy. The mechanisms by which zinc modulates autophagy are still poorly understood. Studies examining loss of function of genes involved in cellular zinc homeostasis have provided novel insights into the role of zinc in autophagy. Autophagy may help cells adapt to changes in zinc availability in medium by controlling zinc mobilization, recycling, and secretion. Zinc is a key player in toxic and protective autophagy.
Topics: Animals; Autophagy; Humans; Lysosomes; Metallothionein; Mitophagy; Zinc
PubMed: 32957075
DOI: 10.1016/j.jtemb.2020.126636 -
Autophagy Aug 2012Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux...
Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP(+)RFP(+) puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.
Topics: Animals; Autophagy; Cisplatin; Dose-Response Relationship, Drug; Embryo, Mammalian; Fibroblasts; Fluorescence; Green Fluorescent Proteins; HeLa Cells; Humans; Luminescent Proteins; Lysosomes; Mice; Microscopy, Confocal; Microtubule-Associated Proteins; Neoplasms; Recombinant Fusion Proteins; Sirolimus; Staurosporine; Red Fluorescent Protein
PubMed: 22647982
DOI: 10.4161/auto.20284 -
Frontiers in Immunology 2021Autophagy is a vital conserved degradative process that maintains cellular homeostasis by recycling or eliminating dysfunctional cellular organelles and proteins. More... (Review)
Review
Autophagy is a vital conserved degradative process that maintains cellular homeostasis by recycling or eliminating dysfunctional cellular organelles and proteins. More recently, autophagy has become a well-recognized host defense mechanism against intracellular pathogens through a process known as xenophagy. On the host-microbe battlefield many intracellular bacterial pathogens have developed the ability to subvert xenophagy to establish infection. Obligately intracellular bacterial pathogens of the family, including , and have developed a dichotomous strategy to exploit the host autophagic pathway to obtain nutrients while escaping lysosomal destruction for intracellular survival within the host cell. In this review, the recent findings regarding how these master manipulators engage and inhibit autophagy for infection are explored. Future investigation to understand mechanisms used by to exploit autophagy may advance novel antimicrobial therapies and provide new insights into how intracellular microbes exploit autophagy to survive.
Topics: Anaplasmataceae; Anaplasmataceae Infections; Animals; Autophagy; Host Microbial Interactions; Humans; Immunity, Innate; Lysosomes; Signal Transduction; Wnt Signaling Pathway
PubMed: 33912170
DOI: 10.3389/fimmu.2021.642771 -
Talanta Aug 2020Understanding lysosome-related physiology needs specific lysosome probes to track the biological processes of lysosome in living cells. Here, we report an...
Understanding lysosome-related physiology needs specific lysosome probes to track the biological processes of lysosome in living cells. Here, we report an azacyclo-modified fluorescent probe that has a large Stokes shift, good photostability and negligible cytotoxicity for highly specific labeling of lysosome and autolysosome in living cells. The probes with different kinds of azacyclo groups on parent dye dansyl are screened to show that dansyl-cycleanine (DNS-C) with four nitrogen atoms possesses the best lysosome-localized ability. And DNS-C as a universal tracker exhibits excellent ability for lysosome labeling in different cell lines with high overlap coefficients (≥0.90). Different from a commercially available LysoTracker, the Stokes shift of DNS-C up to 240 nm (λ = 330/570 nm), is much larger than that of LysoTracker ~20 nm (λ = 573/595 nm). More importantly, the fluorescence of DNS-C keeps still high brightness after a time-lapsed imaging for 40 min in living cells, implying its remarkable photostability for long-term tracking. In addition, DNS-C can also clearly image the autolysosome, a critical subcellular compartment, forming by the fusion of lysosome with autophagosome in autophagy. These results suggest the promising utility of our probe as a powerful tool to real-time trace physiological processes of lysosomes.
Topics: Aza Compounds; Fluorescent Dyes; HeLa Cells; Humans; Lysosomes; Optical Imaging; Tumor Cells, Cultured
PubMed: 32456907
DOI: 10.1016/j.talanta.2020.120941