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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 -
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 -
Frontiers in Cellular and Infection... 2024Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. is a parasitic tapeworm that causes cystic echinococcosis in animals...
Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. is a parasitic tapeworm that causes cystic echinococcosis in animals and humans. The hydatid fluid released by the parasite is known to contain various immunomodulatory components that manipulate host´s defense mechanism. In this study, we focused on understanding the effect of hydatid fluid on dendritic cells and its impact on autophagy induction and subsequent T cell responses. Initially, we observed a marked downregulation of two C-type lectin receptors in the cell membrane, CLEC9A and CD205 and an increase in lysosomal activity, suggesting an active cellular response to hydatid fluid. Subsequently, we visualized ultrastructural changes in stimulated dendritic cells, revealing the presence of macroautophagy, characterized by the formation of autophagosomes, phagophores, and phagolysosomes in the cell cytoplasm. To further elucidate the underlying molecular mechanisms involved in hydatid fluid-induced autophagy, we analyzed the expression of autophagy-related genes in stimulated dendritic cells. Our results demonstrated a significant upregulation of and , indicating the induction of autophagy machinery in response to hydatid fluid exposure. Additionally, using confocal microscopy, we observed an accumulation of LC3 in dendritic cell autophagosomes, confirming the activation of this catabolic pathway associated with antigen presentation. Finally, to evaluate the functional consequences of hydatid fluid-induced autophagy in DCs, we evaluated cytokine transcription in the splenocytes. Remarkably, a robust polyfunctional T cell response, with inhibition of Th2 profile, is characterized by an increase in the expression of and genes. These findings suggest that hydatid fluid-induced autophagy in dendritic cells plays a crucial role in shaping the subsequent T cell responses, which is important for a better understanding of host-parasite interactions in cystic echinococcosis.
Topics: Dendritic Cells; Animals; Echinococcus granulosus; Autophagy; Echinococcosis; T-Lymphocytes; Mice; Lectins, C-Type; Cytokines; Female; Autophagosomes
PubMed: 38817444
DOI: 10.3389/fcimb.2024.1334211 -
The Journal of Biological Chemistry May 2024Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion,...
Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1 mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.
PubMed: 38815862
DOI: 10.1016/j.jbc.2024.107419 -
Frontiers in Bioscience (Landmark... May 2024Ibrutinib could increase the risk of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) patients. However, the precise mechanism underlying ibrutinib-induced...
BACKGROUND
Ibrutinib could increase the risk of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) patients. However, the precise mechanism underlying ibrutinib-induced AF remains incompletely elucidated.
METHODS
We investigated the proportion of ibrutinib-treated CLL patients with new-onset AF. Optical mapping was conducted to reveal the proarrhythmic effect of ibrutinib on HL-1 cells. Fluorescence staining and western blot were used to compare connexins 43 and 40 expression in ibrutinib-treated and control groups. To identify autophagy phenotypes, we used western blot to detect autophagy-related proteins, transmission electron microscopy to picture autophagosomes, and transfected mCherry-GFP-LC3 virus to label autophagosomes and lysosomes. Hydroxychloroquine as an autophagy inhibitor was administered to rescue ibrutinib-induced Cx43 and Cx40 degradation.
RESULTS
About 2.67% of patients developed atrial arrhythmias after ibrutinib administration. HL-1 cells treated with ibrutinib exhibited diminished conduction velocity and a higher incidence of reentry-like arrhythmias compared to controls. Cx43 and Cx40 expression reduced along with autophagy markers increased in HL-1 cells treated with ibrutinib. Inhibiting autophagy upregulated Cx43 and Cx40.
CONCLUSIONS
The off-target effect of ibrutinib on the PI3K-AKT-mTOR signaling pathway caused connexin degradation and atrial arrhythmia via promoting autophagy.
CLINICAL TRIAL REGISTRATION
ChiCTR2100046062, https://clin.larvol.com/trial-detail/ChiCTR2100046062.
Topics: Humans; Adenine; TOR Serine-Threonine Kinases; Autophagy; Proto-Oncogene Proteins c-akt; Piperidines; Signal Transduction; Phosphatidylinositol 3-Kinases; Connexin 43; Female; Atrial Fibrillation; Connexins; Male; Aged; Middle Aged; Gap Junction alpha-5 Protein; Arrhythmias, Cardiac
PubMed: 38812314
DOI: 10.31083/j.fbl2905201 -
Brazilian Journal of Medical and... 2024Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4...
Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4 loss causes early arrest of meiosis and increased apoptosis of spermatogenic cells in male mice, its other potential functions in male fertility remain unexplored. Here, we utilized Ant4 knockout mice to delineate the effects of Ant4-deficiency on male reproduction. Our observations demonstrated that Ant4-deficiency led to infertility and impaired testicular development, which was further investigated by evaluating testicular oxidative stress, autophagy, and inflammation. Specifically, the loss of Ant4 led to an imbalance of oxidation and antioxidants. Significant ultrastructural alterations were identified in the testicular tissues of Ant4-deficient mice, including swelling of mitochondria, loss of cristae, and accumulation of autophagosomes. Our results also showed that autophagic flux and AKT-AMPK-mTOR signaling pathway were affected in Ant4-deficient mice. Moreover, Ant4 loss increased the expression of pro-inflammatory factors. Overall, our findings underscored the importance of Ant4 in regulating oxidative stress, autophagy, and inflammation in testicular tissues. Taken together, these insights provided a nuanced understanding of the significance of Ant4 in testicular development.
Topics: Animals; Male; Mice, Knockout; Testis; Oxidative Stress; Mitochondrial ADP, ATP Translocases; Mice; Autophagy; Infertility, Male; Spermatogenesis; Apoptosis; Signal Transduction
PubMed: 38808891
DOI: 10.1590/1414-431X2024e13590