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Autophagy Jul 2023Autophagosomes are crucial components of the cellular recycling machinery that form at endoplasmic reticulum (ER)-associated sites. As the autophagosome membrane is...
Autophagosomes are crucial components of the cellular recycling machinery that form at endoplasmic reticulum (ER)-associated sites. As the autophagosome membrane is largely devoid of transmembrane proteins, autophagosome biogenesis is thought to be largely regulated by lipid transfer and lipid modifications, as well as membrane-associated proteins. While the membrane origin of autophagosomes and their lipid composition are still incompletely understood, previous studies have found the autophagosome membrane to be enriched in unsaturated fatty acids and have little cholesterol, suggesting that cholesterol removal is an integral step during autophagosome biogenesis. In our study, we demonstrate that short term cholesterol depletion leads to a rapid induction of autophagy and identify the ER-localized cholesterol transport protein GRAMD1C as a negative regulator of starvation-induced macroautophagy/autophagy. ATG: autophagy related; ccRCC: clear cell renal cell carcinoma; ER: endoplasmic reticulum; GRAM: glucosyltransferases, RAB-like GTPase activators and myotubularins; GRAMD: GRAM domain containing; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCBD: methyl-cyclodextrin; MTOR: mechanistic target of rapamycin kinase; VASt: VAD1 analog of StAR-related lipid transfer.
Topics: Autophagosomes; Autophagy; Carrier Proteins; Macroautophagy; Membrane Proteins; Cholesterol; Lipids
PubMed: 36469687
DOI: 10.1080/15548627.2022.2155020 -
Phytomedicine : International Journal... Mar 2024Autophagy, a cellular process involving lysosomal self-digestion, plays a crucial role in recycling biomolecules and degrading dysfunctional proteins and damaged...
BACKGROUND
Autophagy, a cellular process involving lysosomal self-digestion, plays a crucial role in recycling biomolecules and degrading dysfunctional proteins and damaged organelles. However, in non-small cell lung cancer (NSCLC), cancer cells can exploit autophagy to survive metabolic stress and develop resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), which reduce treatment efficacies. Currently, most studies have found that late-stage autophagy inhibitors can hinder EGFR-TKIs resistance, while research on early-stage autophagy inhibitors is still limited.
PURPOSE
This study investigates the mechanism via which the Xie-Bai-San (XBS) formula enhances NSCLC cell sensitivity to gefitinib, revealing the relationship between XBS-induced cell death and the inhibition of autophagosome formation.
METHODS
Cell viability was assessed using CCK-8 and EdU assays, lentivirus transfection was utilized to generate PC9 cells harboring the PIK3CA E545K mutation (referred to as PC9-M), autophagic flux was monitored using mCherry-GFP-LC3 adenovirus. Protein expression and colocalization were observed through immunofluorescence staining. The interaction between Bcl-2 and Beclin-1 in PC9-GR and PC9-M cells was determined via co-immunoprecipitation (Co-IP) assay, cell apoptosis was assessed by flow cytometry and PI staining, and overall survival analysis of lung adenocarcinoma patients was conducted using the TCGA database. In vivo experiments included a patient-derived xenograft (PDX) model with EGFR and PIK3CA mutations and subcutaneous mice xenografts of NSCLC cell lines (PC9 and PC9-GR). In addition, autophagic vesicles in mouse tumor tissues were observed via transmission electron microscopy analysis.
RESULTS
XBS effectively inhibits the proliferation of gefitinib-resistant NSCLC cells and induces apoptosis both in vitro and in vivo. Mechanistically, XBS suppresses gefitinib-induced autophagic flux by inhibiting autophagy through the upregulation of p-mTOR and Bcl-2 and downregulation of Beclin-1. Additionally, XBS enhances the interaction between Bcl-2 and Beclin-1, and the overexpression of Beclin-1 promotes NSCLC cell proliferation and counteracts XBS-induced cell death, while XBS demonstrates minimal impact on autophagosome-lysosome fusion or lysosome function.
CONCLUSION
This study reveals a novel role for the XBS formula in impeding autophagy initiation and demonstrates its potential as a candidate drug to counteract autophagy-induced treatment resistance in NSCLC.
Topics: Humans; Animals; Mice; Carcinoma, Non-Small-Cell Lung; Gefitinib; Beclin-1; Lung Neoplasms; Antineoplastic Agents; Autophagosomes; ErbB Receptors; Quinazolines; Protein Kinase Inhibitors; Drug Resistance, Neoplasm; Apoptosis; Proto-Oncogene Proteins c-bcl-2; Cell Line, Tumor
PubMed: 38232540
DOI: 10.1016/j.phymed.2024.155351 -
Journal of Neuroinflammation Nov 2023Traumatic spinal cord injury can cause immediate physical damage to the spinal cord and result in severe neurological deficits. The primary, mechanical tissue damage...
Traumatic spinal cord injury can cause immediate physical damage to the spinal cord and result in severe neurological deficits. The primary, mechanical tissue damage triggers a variety of secondary damage mechanisms at the injury site which significantly contribute to a larger lesion size and increased functional damage. Inflammatory mechanisms which directly involve both microglia (MG) and monocyte-derived macrophages (MDM) play important roles in the post-injury processes, including inflammation and debris clearing. In the current study, we investigated changes in the structure and function of MG/MDM in the injured spinal cord of adult female mice, 7 days after a thoracic contusion SCI. With the use of chip mapping scanning electron microscopy, which allows to image large samples at the nanoscale, we performed an ultrastructural comparison of MG/MDM located near the lesion vs adjacent regions to provide novel insights into the mechanisms at play post-injury. We found that MG/MDM located near the lesion had more mitochondria overall, including mitochondria with and without morphological alterations, and had a higher proportion of altered mitochondria. MG/MDM near the lesion also showed an increased number of phagosomes, including phagosomes containing myelin and partiallydigested materials. MG/MDM near the injury interacted differently with the spinal cord parenchyma, as shown by their reduced number of direct contacts with synaptic elements, axon terminals and dendritic spines. In this study, we characterized the ultrastructural changes of MG/MDM in response to spinal cord tissue damage in mice, uncovering changes in phagocytic activity, mitochondrial ultrastructure, and inter-cellular interactions within the spinal cord parenchyma.
Topics: Mice; Female; Animals; Microglia; Macrophages; Spinal Cord Injuries; Phagocytes; Spinal Cord
PubMed: 37990235
DOI: 10.1186/s12974-023-02953-0 -
Cell Communication and Signaling : CCS Oct 2023The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it...
BACKGROUND
The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified.
METHODS
We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy.
RESULTS
We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound.
CONCLUSION
Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract.
Topics: Humans; Prodigiosin; Golgi Apparatus; Lysosomes; Autophagosomes; Autophagy
PubMed: 37798768
DOI: 10.1186/s12964-023-01275-1 -
Journal of Cell Science Feb 2024ATG9A, a transmembrane protein of the core autophagy pathway, cycles between the Golgi, endosomes and a vesicular compartment. ATG9A was recently shown to act as a lipid...
ATG9A, a transmembrane protein of the core autophagy pathway, cycles between the Golgi, endosomes and a vesicular compartment. ATG9A was recently shown to act as a lipid scramblase, and this function is thought to require its interaction with another core autophagy protein, ATG2A, which acts as a lipid transfer protein. Together, ATG9A and ATG2A are proposed to function to expand the growing autophagosome. However, ATG9A is implicated in other pathways including membrane repair and lipid droplet homeostasis. To elucidate other ATG9A interactors within the autophagy pathway, or interactors beyond autophagy, we performed an interactome analysis through mass spectrometry. This analysis revealed a host of proteins involved in lipid synthesis and trafficking, including ACSL3, VPS13A and VPS13C. Furthermore, we show that ATG9A directly interacts with VPS13A and forms a complex that is distinct from the ATG9A-ATG2A complex.
Topics: Vesicular Transport Proteins; Membrane Proteins; Autophagosomes; Autophagy; Lipids; Autophagy-Related Proteins
PubMed: 38294121
DOI: 10.1242/jcs.261081 -
Emerging Microbes & Infections Dec 2024The discovery of promising cytokines and clarification of their immunological mechanisms in controlling the intracellular fate of (Mtb) are necessary to identify...
The discovery of promising cytokines and clarification of their immunological mechanisms in controlling the intracellular fate of (Mtb) are necessary to identify effective diagnostic biomarkers and therapeutic targets. To escape immune clearance, Mtb can manipulate and inhibit the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. In this study, we found that interleukin 16 (IL-16) is elevated in the serum samples of Tuberculosis (TB) patients and can serve as a specific target for treatment TB. There was a significant difference in IL-16 levels among active TB, latent TB infection (LTBI), and non-TB patients. This study first revealed that macrophages are the major source of IL-16 production in response to Mtb infection, and elucidated that IL-16 can promote Mtb intracellular survival by inhibiting phagosome maturation and suppressing the expression of Rev-erbα which can inhibit IL-10 secretion. The experiments using zebrafish larvae infected with and mice challenged with H37Rv demonstrated that reducing IL-16 levels resulted in less severe pathology and improved survival, respectively. In conclusion, this study provided direct evidence that Mtb hijacks the host macrophages-derived interleukin 16 to enhance intracellular growth. It is suggesting the immunosuppressive role of IL-16 during Mtb infection, supporting IL-16 as a promising therapeutic target.
Topics: Animals; Humans; Mice; Interleukin-16; Macrophages; Mycobacterium tuberculosis; Phagosomes; Tuberculosis; Zebrafish
PubMed: 38380651
DOI: 10.1080/22221751.2024.2322663 -
Autophagy May 2024Macroautophagy/autophagy is a tightly regulated cellular process integral to homeostasis and innate immunity. As such, dysregulation of autophagy is associated with...
Macroautophagy/autophagy is a tightly regulated cellular process integral to homeostasis and innate immunity. As such, dysregulation of autophagy is associated with cancer, neurodegenerative disorders, and infectious diseases. While numerous factors that promote autophagy have been characterized, the key mechanisms that prevent excessive autophagy are less well understood. Here, we identify CSNK2/CK2 (casein kinase 2) as a negative regulator of autophagy. Pharmacological inhibition of CSNK2 activity or siRNA-mediated depletion of CSNK2 increased basal autophagic flux in cell lines and primary human lung cells. , ectopic expression of CSNK2 reduced autophagic flux. Mechanistically, CSNK2 interacted with the FLN (filamin)-NHL domain-containing tripartite motif (TRIM) family members TRIM2, TRIM3 and TRIM71. Our data show that recruitment of CSNK2 to the C-terminal NHL domain of TRIM3 lead to its robust phosphorylation at serine 661 by CSNK2. A phosphorylation-defective mutant of TRIM3 was unable to reduce autophagosome numbers indicating that phosphorylation by CSNK2 is required for TRIM-mediated autophagy inhibition. All three TRIMs facilitated inactivation of the ULK1-BECN1 autophagy initiation complex by facilitating ULK1 serine 757 phosphorylation. Inhibition of CSNK2 promoted autophagy upon influenza A virus (IAV) and measles virus (MeV) infection. In line with this, targeting of CSNK2 or depletion of TRIM2, TRIM3 or TRIM71 enhanced autophagy-dependent restriction of IAV, MeV and human immunodeficiency virus 1 (HIV-1). Thus, our results identify the CSNK2-TRIM2, -TRIM3, -TRIM71 axis as a key regulatory pathway that limits autophagy. Targeting this axis may allow for therapeutic induction of autophagy against viral infections and in diseases associated with dysregulated autophagy. ATG5: autophagy related 5; BafA1: bafilomycin A; BECN1: beclin 1; CCD: coiled-coil domain; CSNK2/CK2: casein kinase 2; CSNK2A1: casein kinase 2 alpha 1; CSNK2A2: casein kinase 2 alpha 2; CSNK2B: casein kinase 2 beta; FLN: filamin; HeLa GL: HeLa cells stably expressing eGFP-LC3B; HIV-1: human immunodeficiency virus 1; IAV: influenza A virus; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3; MeV: measles virus; MTOR: mechanistic target of rapamycin kinase; RING: really interesting new gene; SQSTM1/p62: sequestosome 1; TRIM: tripartite motif; ULK1: unc-51 like autophagy activating kinase 1.
Topics: Humans; Autophagy; Casein Kinase II; Tripartite Motif Proteins; Autophagy-Related Protein-1 Homolog; Phosphorylation; HEK293 Cells; Beclin-1; Intracellular Signaling Peptides and Proteins; Autophagosomes; HeLa Cells; Ubiquitin-Protein Ligases; Carrier Proteins
PubMed: 37938186
DOI: 10.1080/15548627.2023.2281128 -
The Journal of Cell Biology Sep 2023Formation and fission of tubules from autolysosomes, endolysosomes, or phagolysosomes are required for lysosome reformation. However, the mechanisms governing these...
Formation and fission of tubules from autolysosomes, endolysosomes, or phagolysosomes are required for lysosome reformation. However, the mechanisms governing these processes in these different lysosomal organelles are poorly understood. Thus, the role of phosphatidylinositol-4-phosphate (PI(4)P) is unclear as it was shown to promote the formation of tubules from phagolysosomes but was proposed to inhibit tubule formation on autolysosomes because the loss of PI4KIIIβ causes extensive lysosomal tubulation. Using super-resolution live-cell imaging, we show that Arf1-PI4KIIIβ positive vesicles are recruited to tubule fission sites from autolysosomes, endolysosomes, and phagolysosomes. Moreover, we show that PI(4)P is required to form autolysosomal tubules and that increased lysosomal tubulation caused by loss of PI4KIIIβ represents impaired tubule fission. At the site of fission, we propose that Arf1-PI4KIIIβ positive vesicles mediate a PI(3)P signal on lysosomes in a process requiring the lipid transfer protein SEC14L2. Our findings indicate that Arf1-PI4KIIIβ positive vesicles and their regulation of PI(3)P are critical components of the lysosomal tubule fission machinery.
Topics: Lysosomes; Signal Transduction; ADP-Ribosylation Factor 1; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 37289133
DOI: 10.1083/jcb.202205128 -
Molecules (Basel, Switzerland) Dec 2023The SH2-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) enzyme opposes the activity of PI3K and therefore is of interest in the treatment of inflammatory...
The SH2-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) enzyme opposes the activity of PI3K and therefore is of interest in the treatment of inflammatory disorders. Recent results also indicate that SHIP1 promotes phagolysosomal degradation of lipids by microglia, suggesting that the enzyme may be a target for the treatment of Alzheimer's disease. Therefore, small molecules that increase SHIP1 activity may have benefits in these areas. Recently we discovered a bis-sulfonamide that increases the enzymatic activity of SHIP1. A series of similar SHIP1 activators have been synthesized and evaluated to determine structure-activity relationships and improve in vivo stability. Some new analogs have now been found with improved potency. In addition, both the thiophene and the thiomorpholine in the parent structure can be replaced by groups without a low valent sulfur atom, which provides a way to access activators that are less prone to oxidative degradation.
Topics: Phosphoric Monoester Hydrolases
PubMed: 38138538
DOI: 10.3390/molecules28248048 -
Microorganisms Dec 2023Through the promotion of phagolysosome formation, autophagy has emerged as a crucial mechanism to eradicate intracellular (Mtb). A cell-autonomous host defense... (Review)
Review
Through the promotion of phagolysosome formation, autophagy has emerged as a crucial mechanism to eradicate intracellular (Mtb). A cell-autonomous host defense mechanism called lysosome biogenesis and autophagy transports cytoplasmic cargos and bacterial phagosomes to lysosomes for destruction during infection. Similar occurrences occurred in stressful or starvation circumstances and led to autophagy, which is harmful to the cell. It is interesting to note that under both hunger and infection states, the transcription factor EB (TFEB) acts as a master regulator of lysosomal activities and autophagy. This review highlighted recent research on the multitier regulation of TFEB-induced autophagy by a variety of host effectors and Mtb sulfolipid during Mtb infection and starvation. In general, the research presented here sheds light on how lysosome biogenesis and autophagy are differentially regulated by the TFEB during Mtb infection and starvation.
PubMed: 38138088
DOI: 10.3390/microorganisms11122944