Choline dehydrogenase interacts with SQSTM1 to activate mitophagy and promote coelomocyte survival in Apostichopus japonicus following Vibrio splendidus infection.

NEDD4 activates mitophagy by interacting with LC3 to restrain reactive oxygen species and apoptosis in Apostichopus japonicus challenged with Vibrio splendidus

Organisms produce high levels of reactive oxygen species (ROS) to kill pathogens or act as signaling molecules to induce immune responses; however, excessive ROS can result in cell death. To maintain ROS balance and cell survival, mitophagy selectively eliminates damaged mitochondria via mitophagy receptors in vertebrates. In marine invertebrates, however, mitophagy and its functions remain largely unknown. In the current study, Vibrio splendidus infection damaged mitochondrial morphology in coelomocytes and reduced mitochondrial membrane potential (ΔΨm) and mitophagosome formation. The colocalization of mitochondria and lysosomes further confirmed that lipopolysaccharide (LPS) treatment increased mitophagy flux. To explore the regulatory mechanism of mitophagy, we cloned Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a common mitophagy receptor, from sea cucumber Apostichopus japonicus (AjBNIP3) and confirmed that AjBNIP3 was significantly induced and accumulated in mitochondria after V. splendidus infection and LPS exposure. At the mitochondrial membrane, AjBNIP3 interacts with microtubule-associated protein 1 light chain 3 (LC3) on phagophore membranes to mediate mitophagy. After AjBNIP3 interference, mitophagy flux decreased significantly. Furthermore, AjBNIP3-mediated mitophagy was activated by ROS following the addition of exogenous hydrogen peroxide (H2O2), ROS scavengers, and ROS inhibitors. Finally, inhibition of BNIP3-mediated mitophagy by AjBNIP3 small interfering RNA (siRNA) or high concentrations of lactate increased apoptosis and decreased coelomocyte survival. These findings highlight the essential role of AjBNIP3 in damaged mitochondrial degradation during mitophagy. This mitophagy activity is required for coelomocyte survival in A. japonicus against V. splendidus infection.

Comparative analysis of midgut bacterial community under Vibrio splendidus infection in Apostichopus japonicus with hindgut as a reference

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related gamma-aminobutyrate receptor-associated protein and gamma-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 microm diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.

Purpose: To investigate the relationship between autophagy and radiation damage of human hepatoma cells and to explore the role of reactive oxygen species (ROS).Materials and methods: HepG2 cells were exposed to X-rays, then the protein expressions of microtubule-associated protein 1 light chain 3 (LC3) and poly ADP-ribose polymerase-1 (PARP-1) were measured by Western blot assay, the formation of autophagosomes was detected by an autophagy detection kit, the intracellular ROS level was measured by flow cytometer, and DNA damage was evaluated by the incidence of micronuclei (MN). A CCK-8 kit was used to measure the proliferation ability of irradiated cells with or without N-acetyl-l-cysteine (NAC) treatment. In some experiments, the hepatoma cells were transferred with LC3 siRNA or PARP-1 siRNA before irradiation.Results: The protein expressions of LC3 and PARP-1 and the inductions of autophagosomes and intracellular ROS were increased in the irradiated HepG2 cells. Pretreatment of cells with NAC relieved the irradiation-induced inhibition of cell proliferation. When HepG2 cells were transfected with the LC3 siRNA, the over-expression of PARP-1 was diminished in the irradiated cells. Compared with the control group, the inhibitions of LC3 and PARP-1 increased ROS level in the irradiated HepG2 cells and hence sensitized radiation responses of both proliferation inhibition and MN induction.Conclusion: Autophagy upregulates the expression of PARP-1 and relieves radiation damage by reducing the generation of ROS.

Impairment of autophagic degradation of the ubiquitin- and LC3-binding protein "p62" leads to the formation of cytoplasmic inclusion bodies. However, little is known about the sorting mechanism of p62 to autophagic degradation. Here we identified a motif of murine p62 consisting of 11 amino acids (Ser334-Ser344) containing conserved acidic and hydrophobic residues across species, as an LC3 recognition sequence (LRS). The crystal structure of the LC3-LRS complex at 1.56 angstroms resolution revealed interaction of Trp340 and Leu343 of p62 with different hydrophobic pockets on the ubiquitin fold of LC3. In vivo analyses demonstrated that p62 mutants lacking LC3 binding ability accumulated without entrapping into autophagosomes in the cytoplasm and subsequently formed ubiquitin-positive inclusion bodies as in autophagy-deficient cells. These results demonstrate that the intracellular level of p62 is tightly regulated by autophagy through the direct interaction of LC3 with p62 and reveal that selective turnover of p62 via autophagy controls inclusion body formation.

Effects of dietary β-glucan on the growth, immune responses and resistance of sea cucumber, Apostichopus japonicus against Vibrio splendidus infection

The sea cucumber (Apostichopus japonicus) is highly susceptible to environmental stress during aquaculture, storage, and transportation, often resulting in autolysis and considerable economic losses. UVA irradiation and Vibrio splendidus infection were used to induce skin ulceration in A. japonicus. In this study, UVA irradiation and V. splendidus infection were used to induce skin ulceration, and the effectiveness of a compound inhibitor in delaying its onset was evaluated. The degree of skin ulceration in A. japonicus was evaluated. Body wall tissues were collected to measure the activities of self-digesting enzymes, AchE, cathepsin L, SOD, and CAT. Caspase-3 expression was also analyzed to assess apoptosis and tissue damage. The results indicated that soaking A. japonicus in the inhibitor composition significantly delayed the onset of skin ulceration. After 72 h of UVA irradiation, the skin ulceration in group Eg was 0.55%, which was significantly lower than that in groups Cg and Wg. In the V. splendidus infection model, group Eg showed a 4-day delay in the onset of skin ulceration, compared to group Cg. Enzyme activity and gene expression analysis revealed that the inhibitor composition significantly reduced self-digesting enzyme expression in the A. japonicus body wall, increased SOD and CAT activities, and inhibited Caspase-3 expression. This study provides valuable theoretical insights into controlling skin ulceration in A. japonicus during aquaculture, preservation, and transportation.

Global N6-methyladenosine methylation analysis reveals the positive correlation between m6A modification and mRNA abundance during Apostichopus japonicus disease development

Patients with tuberculosis, who are treated with long-term high-dose combined use of anti-tuberculosis drugs, can cause many adverse reactions such as liver damage, but the mechanism is still unclear. Although phosphatase and tensin homolog induced kinase 1 (PINK1)/Parkin axis might participate in the process of liver damage through regulating mitochondrial autophagy and oxidative stress in liver cells. However, the association between the mitochondrial autophagy regulated by the PINK1/Parkin axis and the liver injury induced by anti-tuberculosis drugs is unknown. This study aims to explore the mechanism of PINK1/Parkin signal axis in regulating hepatocellular injury induced by anti-tuberculosis drugs through mitochondrial autophagy, and to provide new therapeutic targets for the patients with liver damage caused by anti-tuberculosis drugs. Mouse hepatocytes AML-12 were treated with isoniazide (INH) to induce liver injury. The mRNA and protein levels of PINK1, Parkin and autophagy associated factors were detected by real-time PCR and Western blotting in the normal AML-12 cells (control group), the AML-12 cells treated by INH (model group) and the AML-12 cells treated with INH and salidroside together (intervention group). Meantime, ELISA kit was used to detect the level of reactive oxygen species (ROS) in AML-12 cells, and the cell morphology and injury was observed by HE staining and transmission electron microscope (TEM). Compared with the control group, the mRNA (all P <0.01) and protein levels (all P <0.05) of PINK1, Parkin and microtubule associated protein 1 light chain 3 (LC3) were significantly decreased, the ubiquitination level was significantly decreased ( P <0.05), and the ROS level was increased ( P <0.05), and the hepatocellular cell showed obvious damage in the model group. Compared with the model group, the expression of PINK1, Parkin and LC3 were significantly increased in the intervention group (all P <0.05), as well as the ubiquitination level was also increased ( P <0.05). The ROS level mediated by mitochondria was decreased ( P <0.05). The results of HE staining and TEM showed that the cell status was improved and the damage of hepatocytes was significantly attenuated. In this study, the mechanism of mitochondrial autophagy mediated by PINK1/Parkin signal axis may be related to INH-induced injury in liver cells and it can regulate the damage state in vitro, indicating that Parkin is a potential molecular target for the prediction, diagnosis and treatment for liver injury induced by anti-tuberculosis drugs.

LRPPRC regulates metastasis and glycolysis by modulating autophagy and the ROS/HIF1-α pathway in retinoblastoma

Circular RNAs (circRNAs) are involved in various physiological and pathological processes in both vertebrates and invertebrates. However, most studies on circRNAs have focused on their roles as endogenous competitive RNAs. Here, we report a novel function of circRNA derived from the Fibrinogen-like protein 1 gene (circ-FGL1) that inhibits coelomocyte apoptosis via competing with the deubiquitinase AjOTUB1 to bind AjMyc in Apostichopus japonicus during Vibrio splendidus infection. The results showed that circ-FGL1 is significantly downregulated in coelomocytes of V. splendidus-induced A. japonicus and negatively regulates coelomocyte apoptosis through the AjBax-AjCyt c pathway. Mechanistically, the deubiquitinase AjOTUB1 and circ-FGL1 could interact with the transcription factor protein AjMyc in the same region with circ-FGL1/AjMyc having greater affinity. Under normal conditions, high levels of circ-FGL1 bind directly to AjMyc, inhibiting the deubiquitylation of AjMyc by AjOTUB1 and leading to the degradation of AjMyc. After V. splendidus infection, AjMyc disassociates from the depressed expression of circ-FGL1, promoting its deubiquitylation by binding to the induced deubiquitinase AjOTUB1 to inhibit its degradation. AjMyc is then transferred to the nucleus and promotes the transcription of AjCyt c and AjBax to induce coelomocyte apoptosis. The new finding will expand our present outstanding on the functional role of circRNAs and suggest new therapeutic targets for the treatment of echinoderms during bacterial invasion.

Characterization of c-Jun N-terminal kinase (JNK) gene reveals involvement of immune defense against Vibrio splendidus infection in Apostichopus japonicus

LC3 promotes the nuclear translocation of the vitamin D receptor and decreases fibrogenic gene expression in proximal renal tubules

Objective To evaluate the relationship between the hippocampal neuron-protective mechanism of hydrogen in a rat model of oxygen-glucose deprivation and restoration (OGD/R) and mitochondrial autophagy. Methods Hippocampal neurons isolated from healthy Sprague-Dawley rats (24 h after birth) were cultured in vitro, seeded in polylysine-coated 6-well plates at a density of 7×105 cells/well and then divided into 5 groups (n=30 each) using a random number table method: control group (C group), OGD/R group, OGD/R+ H2 group, OGD/R plus 3-methyladenine (3-MA) group (OGD/R+ 3-MA group), and OGD/R plus H2 plus 3-MA group (OGD/R+ H2+ 3-MA group). The cells were cultured for 24 h in normal culture atmosphere (75%N2-20%O2-5%CO2) in group C, and cells were subjected to oxygen-glucose deprivation for 2 h followed by O2-glucose supply for 24 h to establish the model of OGD/R injury in OGD/R, OGD/R+ H2, OGD/R+ 3-MA and OGD/R+ H2+ 3-MA groups.The cells were cultured for 24 h in a hydrogen-rich incubator (60% H2-10% O2-5% CO2-25% N2) after establishing the model in group OGD/R+ H2.Autophagy inhibitor 3-MA 10 mmol/L was added, and then cultured for 24 h in normal culture atmosphere after establishing the model in group OGD/R+ 3-MA.Autophagy inhibitor 3-MA 10 mmol/L was added, and then cultured for 24 h in hydrogen-rich incubator after establishing the model in group OGD/R+ H2+ 3-MA.The cell survival rate was measured using MTT assay.DCFH-DA fluorescent probe was applied for determination of reactive oxygen species (ROS) activity.The mitochondrial membrane potential was measured using a JC-10 assay kit.The neuronal apoptosis was detected by flow cytometry, and apoptosis rate was calculated.The expression of mitophagy-related protein microtubule-associated protein 1 light chain 3 (LC3), PINK1 and Parkin was determined by Western blot, and LC3Ⅱ/LC3Ⅰ ratio was calculated. Results Compared with group C, the cell survival rate and MMP were significantly decreased, the apoptosis rate and ROS activity were increased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were increased in OGD/R and OGD/R+ H2 groups (P<0.05). Compared with group OGD/R, the cell survival rate and MMP were significantly increased, the apoptosis rate and ROS activity were decreased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were increased in group OGD/R+ H2(P<0.05), and the cell survival rate and MMP were significantly decreased, the apoptosis rate and ROS activity were increased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were decreased in group OGD/R+ 3-MA (P<0.05). Compared with group OGD/R+ H2, the cell survival rate and MMP were significantly decreased, the apoptosis rate and ROS activity were increased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were decreased in OGD/R+ 3-MA and OGD/R+ H2+ 3-MA groups (P<0.05). Conclusion Hippocampal neuron-protective mechanism of hydrogen against OGDR injury is related to promoting mitochondrial autophagy in rats. Key words: Hydrogen; Hypoxia-ischemia, brain; Reperfusion injury; Hippocampus; Neurons; Mitochondria; Autophagy