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Biochemical and Biophysical Research... Sep 2024Autophagy is a critical catabolic pathway that enables cells to survive and adapt to stressful conditions, especially nutrient deprivation. The fusion of autophagic...
Autophagy is a critical catabolic pathway that enables cells to survive and adapt to stressful conditions, especially nutrient deprivation. The fusion of autophagic vacuoles with lysosomes is the final step of autophagy, which degrades the engulfed contents into metabolic precursors for re-use by the cell. O-GlcNAc transferase (OGT) plays a crucial role in regulating autophagy flux in response to nutrient stress, particularly by targeting key proteins involved in autophagosome-lysosome fusion. However, the role of OGT in basal autophagy, which occurs at a low and constitutive levels under growth conditions, remains poorly understood. Silencing or inhibition of OGT was used to compare the effect of OGT downregulation on autophagy flux in the non-cancerous CCD841CoN and cancerous HCT116 human colon cell lines under nutrient-rich conditions. We provide evidence that the reduction of OGT activity impairs the maturation of autophagosomes, thereby blocking the completion of basal autophagy in both cell lines. Additionally, OGT inhibition results in the accumulation of lysosomes and enlarged late endosomes in the perinuclear region, as demonstrated by confocal imaging. This is associated with a defect in the localization of the small GTPase Rab7 to these organelles. The regulation of transport and fusion events between the endosomal and lysosomal compartments is crucial for maintaining the autophagic flux. These findings suggest an interplay between OGT and the homeostasis of the endolysosomal network in human cells.
Topics: Humans; N-Acetylglucosaminyltransferases; Autophagy; Endosomes; Down-Regulation; Lysosomes; rab7 GTP-Binding Proteins; Nutrients; rab GTP-Binding Proteins; Colon; HCT116 Cells; Autophagosomes
PubMed: 38852504
DOI: 10.1016/j.bbrc.2024.150198 -
International Journal of Biological... Jun 2024Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease with an unclear pathogenesis and no available specific drug treatment. The principal...
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease with an unclear pathogenesis and no available specific drug treatment. The principal etiological factors are lung inflammation caused by environmental factors, damage to alveolar epithelial cells, leading to epithelial-mesenchymal transition (EMT), and the abnormal proliferation of fibroblasts. Here, we have demonstrated that fibroblast growth factor 21 (FGF21) ameliorates IPF via the autophagy pathway. We administered FGF21 to bleomycin (BLM)-treated mice, which ameliorated their defects in lung function, reduced the accumulation of collagen, restored tissue structure, reduced the deposition of hydroxyproline, reduced the expression of collagen I and α-SMA and increased the expression of E-cadherin. The expression of LC3BII and the number of autophagosomes were significantly higher in the lungs. The expression of AKT and mTOR was significantly reduced by FGF21 treatment. We also determined the effects of FGF21 in A549 cells treated with TGF-β, and found that FGF21 significantly inhibits activation of the AKT signaling pathway, thereby reducing TGF-β-induced EMT and preventing the uncontrolled proliferation of fibroblasts. We conclude that FGF21 ameliorates IPF by inhibiting the PI3K-AKT-mTOR signaling pathway and activating autophagy, which provides a theoretical basis for FGF21 to be used for the treatment of IPF.
PubMed: 38851619
DOI: 10.1016/j.ijbiomac.2024.132896 -
Nature Structural & Molecular Biology Jun 2024The hallmark of non-selective autophagy is the formation of cup-shaped phagophores that capture bulk cytoplasm. The process is accompanied by the conjugation of LC3B to...
The hallmark of non-selective autophagy is the formation of cup-shaped phagophores that capture bulk cytoplasm. The process is accompanied by the conjugation of LC3B to phagophores by an E3 ligase complex comprising ATG12-ATG5 and ATG16L1. Here we combined two complementary reconstitution approaches to reveal the function of LC3B and its ligase complex during phagophore expansion. We found that LC3B forms together with ATG12-ATG5-ATG16L1 a membrane coat that remodels flat membranes into cups that closely resemble phagophores. Mechanistically, we revealed that cup formation strictly depends on a close collaboration between LC3B and ATG16L1. Moreover, only LC3B, but no other member of the ATG8 protein family, promotes cup formation. ATG16L1 truncates that lacked the C-terminal membrane binding domain catalyzed LC3B lipidation but failed to assemble coats, did not promote cup formation and inhibited the biogenesis of non-selective autophagosomes. Our results thus demonstrate that ATG16L1 and LC3B induce and stabilize the characteristic cup-like shape of phagophores.
PubMed: 38834913
DOI: 10.1038/s41594-024-01300-y -
Proceedings of the National Academy of... Jun 2024Synapse maintenance is essential for generating functional circuitry, and decrement in this process is a hallmark of neurodegenerative disease. Yet, little is known...
Synapse maintenance is essential for generating functional circuitry, and decrement in this process is a hallmark of neurodegenerative disease. Yet, little is known about synapse maintenance in vivo. Cysteine string protein α (CSPα), encoded by the gene, is a synaptic vesicle chaperone that is necessary for synapse maintenance and linked to neurodegeneration. To investigate the transcriptional changes associated with synapse maintenance, we performed single-nucleus transcriptomics on the cortex of young CSPα knockout (KO) mice and littermate controls. Through differential expression and gene ontology analysis, we observed that both neurons and glial cells exhibit unique signatures in the CSPα KO brain. Significantly, all neuronal classes in CSPα KO brains show strong signatures of repression in synaptic pathways, while up-regulating autophagy-related genes. Through visualization of synapses and autophagosomes by electron microscopy, we confirmed these alterations especially in inhibitory synapses. Glial responses varied by cell type, with microglia exhibiting activation. By imputing cell-cell interactions, we found that neuron-glia interactions were specifically increased in CSPα KO mice. This was mediated by synaptogenic adhesion molecules, with the classical Neurexin1-Neuroligin 1 pair being the most prominent, suggesting that communication of glial cells with neurons is strengthened in CSPα KO mice to preserve synapse maintenance. Together, this study provides a rich dataset of transcriptional changes in the CSPα KO cortex and reveals insights into synapse maintenance and neurodegeneration.
Topics: Animals; Synapses; Mice; Mice, Knockout; HSP40 Heat-Shock Proteins; Transcriptome; Neurons; Membrane Proteins; Neuroglia
PubMed: 38833477
DOI: 10.1073/pnas.2320064121 -
IScience Jun 2024The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded...
The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded remains largely unknown. In this study, we examine autophagy of glycogen, a storage form of glucose. We find that cells accumulate glycogen in the cytoplasm during nitrogen starvation and that this carbohydrate is rarely observed within autophagosomes and autophagic bodies. However, sequestration of glycogen by autophagy is observed following prolonged nitrogen starvation. We identify a yet-uncharacterized open reading frame, Yil024c (herein Atg45), as encoding a cytosolic receptor protein that mediates autophagy of glycogen (glycophagy). Furthermore, we show that, during sporulation, Atg45 is highly expressed and is associated with an increase in glycophagy. Our results suggest that cells regulate glycophagic activity by controlling the expression level of Atg45.
PubMed: 38832010
DOI: 10.1016/j.isci.2024.109810 -
ELife Jun 2024During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures....
During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.
Topics: Qa-SNARE Proteins; Autophagosomes; Phosphatidylinositol Phosphates; Humans; Molecular Dynamics Simulation; Autophagy
PubMed: 38831696
DOI: 10.7554/eLife.92189 -
ELife Jun 2024A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during...
A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during autophagy.
Topics: Autophagy; Autophagosomes; Membrane Fusion; SNARE Proteins; Humans; Animals
PubMed: 38831693
DOI: 10.7554/eLife.99181 -
Heliyon Jun 2024The aim of this study was to investigate the protective effect of HLJDD on septic rats and the underlying mechanisms.
AIMS
The aim of this study was to investigate the protective effect of HLJDD on septic rats and the underlying mechanisms.
MATERIALS AND METHODS
Adult male Sprague-Dawley (SD) adult rats (150-180 g) were randomly divided into the following 5 groups (n = 7 per group): the Sham group, caecal ligation and puncture (CLP) group, HLJDD + CLP (Huang Lian Jie Du Decoction, HLJDD) group (1 g/mL/100 g), HLJDD + Rap + CLP (H. Rap) group (Rap: 3 mg/kg), and HLJDD+3-MA + CLP (H. 3-MA) group (3-MA: 30 mg/kg). Rapamycin (Rap) and 3-methyladenosine (3-MA) were used to activate and inhibit autophagy, respectively. HLJDD was purchased from Beijing Tong Ren Tang Guiyang Branch and verified by experts as a genuine product. We used CLP to establish an animal model of sepsis in the last four groups. Survival was analysed by the Kaplan‒Meier method. Then, we examined autophagy-related genes () and proteins using real-time PCR and Western blotting, respectively. The microstructure of the ileum and the number of autophagosomes were observed by transmission electron microscopy (TEM). Analyses of HE-stained pathological ileum and inflammatory factor levels were examined to assess the extent of septic injury. The effect of HLJDD on the gut microbiota was analysed by 16S rRNA gene sequencing of faeces.
RESULTS
In this study, we identified the protective effects of HLJDD on mortality and inflammation in septic rats. Several key proteins, including LC3-II, Beclin-1 and p62, were examined and showed that HLJDD could effectively reverse the sepsis-induced decrease in autophagy. TEM was performed and the expression of s was assessed to evaluate fluctuations in autophagy. Then, we examined the intestinal tight junction protein zona occludens (ZO-1), lipopolysaccharide (LPS) and inflammatory factors, and found that HLJDD effectively alleviated the increase in gene expression, the level of LPS and serum level of inflammatory factors caused by sepsis. These results were consistent with those obtained from pathological sectioning and TEM analysis. Moreover, autophagy activation effectively ameliorated sepsis, and autophagy inhibition exacerbated the systemic symptoms caused by infection. By examining the expression of key proteins upstream of the autophagy pathway, we found that HLJDD inhibited mTOR via the MAPK/PI3K signalling pathway to promote autophagy in septic rats. 16S rRNA sequencing revealed that HLJDD significantly affected the diversity and physiological function of the gut microbiota in septic rats.
CONCLUSIONS
The results of this study indicate that autophagy activation is a potential mechanism underlying the protective effect of HLJDD on the intestine in septic rats.
PubMed: 38828290
DOI: 10.1016/j.heliyon.2024.e31607 -
Bioactive Materials Sep 2024While mild hyperthermia holds great potential in the treatment of solid tumors, the thermal stress-triggered self-repairing autophagy significantly compromises its...
While mild hyperthermia holds great potential in the treatment of solid tumors, the thermal stress-triggered self-repairing autophagy significantly compromises its efficacy. To circumvent this obstacle, an injectable hydrogel (NO-Gel) composed of thermosensitive poly(ethylene glycol)-polypeptide copolymers modified with abundant NO donors on their side chains is developed. Meanwhile, ferrimagnetic ZnFeO magnetic nanoparticles (MNPs) with high magnetic-heat conversion efficiency are synthesized and loaded into NO-Gel to obtain MNPs@NO-Gel. The MNPs@NO-Gel system exhibits a sol-gel transition upon heating, and has the ability to perform multiple magnetic hyperthermia therapy (MHT) after only one administration due to the even distribution and strong immobilization of MNPs in NO-Gel. NO can be continuously liberated from NO-Gel and this process is markedly accelerated by MHT. Additionally, MNPs@NO-Gel maintains its integrity for over one month and the released MNPs are metabolized by the spleen. After a single administration of MNPs@NO-Gel at the tumor site, three mild MHT treatments with similar effects are fulfilled, and the sufficient supply of NO effectively inhibits MHT-induced autophagic flux blocking the formation of autophagosomes and synchronously destroying lysosomes, thereby substantially boosting the efficacy of mild MHT. As a consequence, CT-26 colon tumors are completely eliminated without causing severe side-effects.
PubMed: 38827171
DOI: 10.1016/j.bioactmat.2024.05.032 -
International Journal of Biological... May 2024One of the major diseases affecting people globally is colorectal cancer (CRC), which is primarily caused by a lack of effective medical treatment and a limited... (Review)
Review
One of the major diseases affecting people globally is colorectal cancer (CRC), which is primarily caused by a lack of effective medical treatment and a limited understanding of its underlying mechanisms. Cellular autophagy functions to break down and eliminate superfluous proteins and substances, thereby facilitating the continual replacement of cellular elements and generating vital energy for cell processes. Non-coding RNAs and exosomal ncRNAs have a crucial impact on regulating gene expression and essential cellular functions such as autophagy, metastasis, and treatment resistance. The latest research has indicated that specific ncRNAs and exosomal ncRNA to influence the process of autophagy in CRC cells, which could have significant consequences for the advancement and treatment of this disease. It has been determined that a variety of ncRNAs have a vital function in regulating the genes essential for the formation and maturation of autophagosomes. Furthermore, it has been confirmed that ncRNAs have a considerable influence on the signaling pathways associated with autophagy, such as those involving AMPK, AKT, and mTOR. Additionally, numerous ncRNAs have the potential to affect specific genes involved in autophagy. This study delves into the control mechanisms of ncRNAs and exosomal ncRNAs and examines how they simultaneously influence autophagy in CRC.
PubMed: 38823748
DOI: 10.1016/j.ijbiomac.2024.132732