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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 -
Molecular Biology Reports Jun 2024Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality worldwide, and new diagnostic markers are urgently needed. We aimed to...
BACKGROUND
Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality worldwide, and new diagnostic markers are urgently needed. We aimed to investigate the mechanism by which hsa_circ_0096157 regulates autophagy and cisplatin (DDP) resistance in NSCLC.
METHODS
A549 cells were treated with DDP (0 μg/mL or 3 μg/mL). Then, the autophagy activator rapamycin (200 nm) was applied to the A549/DDP cells. Moreover, hsa_circ_0096157 and Nrf2 were knocked down, and Nrf2 was overexpressed in A549/DDP cells. The expression of Hsa_circ_0096157, the Nrf2/ARE pathway-related factors Nrf2, HO-1, and NQO1, and the autophagy-related factors LC3, Beclin-1, and p62 was evaluated by qRT‒PCR or western blotting. Autophagosomes were detected through TEM. An MTS assay was utilized to measure cell proliferation. The associated miRNA levels were also tested by qRT‒PCR.
RESULTS
DDP (3 μg/mL) promoted hsa_circ_0096157, LC3 II/I, and Beclin-1 expression and decreased p62 expression. Knocking down hsa_circ_0096157 resulted in the downregulation of LC3 II/I and Beclin-1 expression, upregulation of p62 expression, and decreased proliferation. Rapamycin reversed the effect of interfering with hsa_circ_0096157. Keap1 expression was lower, and Nrf2, HO-1, and NQO1 expression was greater in the A549/DDP group than in the A549 group. HO-1 expression was repressed after Nrf2 interference. In addition, activation of the Nrf2/ARE pathway promoted autophagy in A549/DDP cells. Moreover, hsa_circ_0096157 activated the Nrf2/ARE pathway. The silencing of hsa_circ_0096157 reduced Nrf2 expression by releasing miR-142-5p or miR-548n. Finally, we found that hsa_circ_0096157 promoted A549/DDP cell autophagy by activating the Nrf2/ARE pathway.
CONCLUSION
Knockdown of hsa_circ_0096157 inhibits autophagy and DDP resistance in NSCLC cells by downregulating the Nrf2/ARE signaling pathway.
Topics: Humans; Cisplatin; NF-E2-Related Factor 2; Carcinoma, Non-Small-Cell Lung; Drug Resistance, Neoplasm; Autophagy; Signal Transduction; Lung Neoplasms; A549 Cells; Gene Expression Regulation, Neoplastic; MicroRNAs; Cell Proliferation; Cell Line, Tumor; Antioxidant Response Elements; Antineoplastic Agents; Heme Oxygenase-1
PubMed: 38822881
DOI: 10.1007/s11033-024-09552-z -
Cellular & Molecular Biology Letters May 2024Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression...
BACKGROUND
Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis.
METHODS
To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs.
RESULTS
Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs.
CONCLUSION
This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.
Topics: Animals; Dogs; 14-3-3 Proteins; Female; Actinin; Mammary Neoplasms, Animal; Cell Line, Tumor; Chemotaxis; Autophagy; Endoplasmic Reticulum Stress; Mucoproteins; Oncogene Proteins
PubMed: 38822246
DOI: 10.1186/s11658-024-00601-w -
Molecular Immunology Jul 2024Chlamydia trachomatis (CT) is the leading cause of bacterial sexually transmitted diseases worldwide, which can cause diseases such as pelvic inflammatory disease, and...
Chlamydia trachomatis (CT) is the leading cause of bacterial sexually transmitted diseases worldwide, which can cause diseases such as pelvic inflammatory disease, and cervical and fallopian tube inflammation, and poses a threat to human health. Rosmarinic acid (RosA) is an active ingredient of natural products with anti-inflammatory and immunomodulatory effects. This study aimed to investigate the role of RosA in inhibiting autophagy-regulated immune cells-CD8+ T cells via the Ras/Raf/MEK/ERK signaling pathway in a CT-infected mouse model. Mice were inoculated with CT infection solution vaginally, and the mechanistic basis of RosA treatment was established using H&E staining, flow cytometry, immunofluorescence, transmission electron microscopy, and western blot. The key factors involved in RosA treatment were further validated using the MEK inhibitor cobimetinib. Experimental results showed that both RosA and the reference drug azithromycin could attenuate the pathological damage to the endometrium caused by CT infection; flow cytometry showed that peripheral blood CD8+ T cells increased after CT infection and decreased after treatment with RosA and the positive drug azithromycin (positive control); immunofluorescence showed that endometrial CD8 and LC3 increased after CT infection and decreased after RosA and positive drug treatment; the results of transmission electron microscopy showed that RosA and the positive drug azithromycin inhibited the accumulation of autophagosomes; western bolt experiments confirmed the activation of autophagy proteins LC3Ⅱ/Ⅰ, ATG5, Beclin-1, and p62 after CT infection, as well as the inhibition of Ras/Raf/MEK/ERK signaling. RosA and azithromycin inhibition of autophagy proteins activates Ras/Raf/MEK/ERK signaling. In addition, the MEK inhibitor cobimetinib attenuated RosA's protective effect on endometrium by further activating CD8+ T cells on a CT-induced basis, while transmission electron microscopy, immunofluorescence, and western blots showed that cobimetinib blocked ERK signals activation and further induced phagocytosis on a CT-induced basis. These data indicated that RosA can activate the Ras/Raf/MEK/ERK signaling pathway to inhibit autophagy, and RosA could also regulate the activation of immune cells-CD8+T cells to protect the reproductive tract of CT-infected mice.
Topics: Animals; CD8-Positive T-Lymphocytes; Autophagy; Female; Chlamydia Infections; Chlamydia trachomatis; Mice; Depsides; MAP Kinase Signaling System; Rosmarinic Acid; Cinnamates; ras Proteins; raf Kinases; Disease Models, Animal; Mice, Inbred C57BL
PubMed: 38820902
DOI: 10.1016/j.molimm.2024.05.007 -
Hearing Research Aug 2024The study focuses on the underlying regulatory mechanism of age-related hearing loss (ARHL), which results from autophagy dysregulation mediated by miR-130b-3p targeting...
The study focuses on the underlying regulatory mechanism of age-related hearing loss (ARHL), which results from autophagy dysregulation mediated by miR-130b-3p targeting PPARγ. We constructed miR-130b-3p knockout (antagomir) and PPARγ over-expression (OE-PPARγ) mice model by injecting mmu-miR-130b-3p antagomir and HBAAV2/Anc80-m-Pparg-T2A-mCHerry into the right ear' round window of each mouse, respectively. In vitro, we introduced oxidative stress within HEI-OC1 cells by HO and exogenously changed the miR-130b-3p and PPARγ levels. MiRNA level was detected by RT-qPCR, proteins by western blotting and immunohistochemistry. Morphology of autophagosomes was observed by electron microscopy. In vivo, the cochlea of aged mice showed higher miR-130b-3p expression and lower PPARγ expression, while exogenous inhibition of miR-130b-3p up-regulated PPARγ expression. Autophagy-related biomarkers expression (ATG5, Beclin-1 and LC3B II/I) decreased in aged mice, which reversely increased after the inhibition of miR-130b-3p. The elevation of PPARγ demonstrated similar effects. Contrarily, exogenous overexpression of miR-130b-3p resulted in the decrease of ATG5, Beclin-1 and LC3B II/I. We created oxidative stress within HEI-OC1 by HO, subsequently observed the formation of autophagosomes under electron microscope, so as the elevated cell apoptosis rate and weakened cell viability. MiR-130b-3p/PPARγ contributed to the premature senescence of these HO-induced HEI-OC1 cells. MiR-130b-3p regulated HEI-OC1 cell growth by targeting PPARγ, thus leading to ARHL.
Topics: Animals; PPAR gamma; Autophagy; MicroRNAs; Oxidative Stress; Mice, Knockout; Disease Models, Animal; Mice; Presbycusis; Cell Line; Aging; Mice, Inbred C57BL; Age Factors; Signal Transduction; Hearing; Cochlea; Apoptosis; Gene Expression Regulation
PubMed: 38820739
DOI: 10.1016/j.heares.2024.109029