Ulk1/FUNDC1 Prevents Nerve Cells from Hypoxia-Induced Apoptosis by Promoting Cell Autophagy.

Cell autophagy and cell apoptosis are both observed in the process of hypoxia-induced ischemic cerebral infarction (ICI). Unc-51 like autophagy activating kinase 1 (Ulk1) and FUN14 Domain-containing Protein 1 (FUNDC1) are both involved in the regulation of cell autophagy. This study aimed to investigate the regulatory effects of Ulk1 and FUNDC1 on hypoxia-induced nerve cell autophagy and apoptosis. Cell viability was measured using cell counting kit-8 (CCK-8) assay. Cell apoptosis was detected using Annexin V-PE/7-ADD staining assay. qRT-PCR was used to quantify the mRNA levels of Ulk1 and FUNDC1 in PC-12 cells. Cell transfection was performed to up-regulate the expression of Ulk1. 3-Methyladenine (3-MA) was used as autophagy inhibitor and rapamycin was used as autophagy activator in our experiments. SP600125 was used as c-Jun N-terminal kinase (JNK) inhibitor. Western blotting was performed to analyze the expression levels of key factors that are related to cell autophagy, apoptosis and JNK pathway. We found that hypoxia simultaneously induced apoptosis and autophagy of PC-12 cells. The activation of Ulk1 and FUNDC1 were also found in PC-12 cells after hypoxia induction. Overexpression of Ulk1 promoted the activation of FUNDC1 and prevented PC-12 cells from hypoxia-induced apoptosis. Suppression of Ulk1 had opposite effects. Furthermore, we also found that JNK pathway participated in the effects of Ulk1 overexpression on PC-12 cell apoptosis reduction. To conclude, Ulk1/FUNDC1 played critical regulatory roles in hypoxia-induced nerve cell autophagy and apoptosis. Overexpression of Ulk1 prevented nerve cells from hypoxia-induced apoptosis by promoting cell autophagy.

The structure and function of FUN14 domain-containing protein 1 and its contribution to cardioprotection by mediating mitophagy.

Cardiovascular disease (CVD) is a serious public health risk, and prevention and treatment efforts are urgently needed. Effective preventive and therapeutic programs for cardiovascular disease are still lacking, as the causes of CVD are varied and may be the result of a multifactorial combination. Mitophagy is a form of cell-selective autophagy, and there is increasing evidence that mitophagy is involved in cardioprotective processes. Recently, many studies have shown that FUN14 domain-containing protein 1 (FUNDC1) levels and phosphorylation status are highly associated with many diseases, including heart disease. Here, we review the structure and functions of FUNDC1 and the path-ways of its mediated mitophagy, and show that mitophagy can be effectively activated by dephosphorylation of Ser13 and Tyr18 sites, phosphorylation of Ser17 site and ubiquitination of Lys119 site in FUNDC1. By effectively activating or inhibiting excessive mitophagy, the quality of mitochondria can be effectively controlled. The main reason is that, on the one hand, improper clearance of mitochondria and accumulation of damaged mitochondria are avoided, and on the other hand, excessive mitophagy causing apoptosis is avoided, both serving to protect the heart. In addition, we explore the possible mechanisms by which FUNDC1-mediated mitophagy is involved in exercise preconditioning (EP) for cardioprotection. Finally, we also point out unresolved issues in FUNDC1 and its mediated mitophagy and give directions where further research may be needed.

Transcription Factor Forkhead Box P (Foxp) 1 Reduces Brain Damage During Cerebral Ischemia-Reperfusion Injury in Mice Through FUN14 Domain-containing Protein 1.

Mitophagy plays a significant role in modulating the activation of pyrin domain-containing protein 3 (NLRP3) inflammasome, which is a major contributor to the inflammatory response that exacerbates cerebral ischemia-reperfusion (I/R) injury. Despite this, the transcriptional regulation mechanism that governs mitophagy remains unclear. This study sought to explore the potential mechanism of Forkhead Box P1 (Foxp1) and its impact on cerebral I/R injury. We investigated the potential neuroprotective role of Foxp1 in cerebral I/R injury by the middle cerebral artery occlusion (MCAO) mouse model. Additionally, we assessed whether FUN14 domain-containing protein 1 (FUNDC1) could rescue the protective effect of Foxp1. Our results showed that overexpression of Foxp1 prevented brain damage during cerebral I/R injury and promoted NLRP3 inflammasome activation, whereas knockdown of Foxp1 had the opposite effect. Notably, Foxp1 overexpression directly promotes FUNDC1 expression, enhanced mitophagy activation, and inhibited the inflammatory response mediated by the NLRP3 inflammasome. Furthermore, we confirmed through chromatin immunoprecipitation (ChIP) and luciferase reporter assays that FUNDC1 is a direct target gene of Foxp1 downstream. Furthermore, the knockdown of FUNDC1 reversed the increased activation of mitophagy and suppressed NLRP3 inflammasome activation induced by Foxp1 overexpression. Collectively, our findings suggest that Foxp1 inhibits NLRP3 inflammasome activation through FUNDC1 to reduce cerebral I/R injury.

Role of Mitophagy in Myocardial Ischemia/Reperfusion Injury and Chinese Medicine Treatment.

Mitophagy is one of the important targets for the prevention and treatment of myocardial ischemia/reperfusion injury (MIRI). Moderate mitophagy can remove damaged mitochondria, inhibit excessive reactive oxygen species accumulation, and protect mitochondria from damage. However, excessive enhancement of mitophagy greatly reduces adenosine triphosphate production and energy supply for cell survival, and aggravates cell death. How dysfunctional mitochondria are selectively recognized and engulfed is related to the interaction of adaptors on the mitochondrial membrane, which mainly include phosphatase and tensin homolog deleted on chromosome ten (PTEN)-induced kinase 1/Parkin, hypoxia-inducible factor-1 α/Bcl-2 and adenovirus e1b19k Da interacting protein 3, FUN-14 domain containing protein 1 receptor-mediated mitophagy pathway and so on. In this review, the authors briefly summarize the main pathways currently studied on mitophagy and the relationship between mitophagy and MIRI, and incorporate and analyze research data on prevention and treatment of MIRI with Chinese medicine, thereby provide relevant theoretical basis and treatment ideas for clinical prevention of MIRI.

[Tanshinone IIA alleviates lipopolysaccharide-induced renal tubular epithelial cell apoptosis by inhibiting RIP3/FUNDC1 signaling pathway].

OBJECTIVE: To investigate the effect of tanshinone IIA pretreatment on acute renal injury in lipopolysaccharide (LPS)-induced septic mice and explore the possible mechanism. METHODS: Thirty C57BL/6 mice were randomized for treatment with saline (control), 10 mg/kg LPS for 24 h, or 10 mg/kg tanshinone IIA 15 min before LPS treatment. After the treatments, serum creatinine and blood urea nitrogen levels of the mice were detected, renal pathologies were observed with PAS staining, and renal expressions of RIP3, cleaved caspase-3 and p18-FUNDC1 were detected with Western blotting. In the cell experiment, cultured normal human renal tubular epithelial cells (HK-2) were treated with LPS (10 mg/mL), LPS+ siNC, LPS+ siRIP3, or LPS+tanshinone IIA (10 mg/L), and the changes in cell apoptosis were examined with TUNEL staining; Western blotting was performed to detect the expression levels of RIP3, cleaved caspase-3 and p18-FUNDC1, and qRT-PCR was used to detect the expression of RIP3 mRNA. RESULTS: LPS challenge for 24 h significantly increased serum creatinine and blood urea nitrogen levels in the mice, caused obviously damages in the proximal renal tubules, and increased renal expressions of RIP3, cleaved caspase-3 and p18-FUNDC1 proteins. Tanshinone IIA pretreatment significantly improved LPS-induced renal injury in the mice, alleviated apoptosis of the renal cells, and inhibited the expressions of RIP3, cleaved caspase-3 and p18-FUNDC1 proteins. In HK-2 cells, LPS stimulation significantly increased the protein expressions of RIP3, cleaved caspase-3 and p18-FUNDC1 and induced obvious cell apoptosis. Pretreatment with tanshinone IIA strongly inhibited the expression of RIP3 and p18-FUNDC1 and reduced LPS-induced apoptosis of HK-2 cells. CONCLUSION: Tanshinone IIA can reduce LPS-induced apoptosis of renal tubular epithelial cells by inhibiting RIP3/FUNDC1 signal pathway.

Mitophagy Disequilibrium, a Prominent Pathological Mechanism in Metabolic Heart Diseases.

With overall food intake among the general population as high as ever, metabolic syndrome (MetS) has become a global epidemic and is responsible for many serious life-threatening diseases, especially heart failure. In multiple metabolic disorders, maintaining a dynamic balance of mitochondrial number and function is necessary to prevent the overproduction of reactive oxygen species (ROS), which has been proved to be one of the important mechanisms of cardiomyocyte injury due to the mismatching of oxygen consumption and mitochondrial population and finally to heart failure. Mitophagy is a process that eliminates damaged or redundant mitochondria. It is mediated by a series of signaling molecules, including PINK, parkin, BINP3, FUNDC1, CTSD, Drp1, Rab9 and mTOR. Meanwhile, increasing evidence also showed that the interaction between ferroptosis and mitophagy interfered with mitochondrial homeostasis. This review will focus on these essential molecules and pathways of mitophagy and cell homeostasis affected by hypoxia and other stimuli in metabolic heart diseases.

Role of Mitophagy in Myocardial Ischemia/Reperfusion Injury and Chinese Medicine Treatment / 中国结合医学杂志

Mitophagy is one of the important targets for the prevention and treatment of myocardial ischemia/reperfusion injury (MIRI). Moderate mitophagy can remove damaged mitochondria, inhibit excessive reactive oxygen species accumulation, and protect mitochondria from damage. However, excessive enhancement of mitophagy greatly reduces adenosine triphosphate production and energy supply for cell survival, and aggravates cell death. How dysfunctional mitochondria are selectively recognized and engulfed is related to the interaction of adaptors on the mitochondrial membrane, which mainly include phosphatase and tensin homolog deleted on chromosome ten (PTEN)-induced kinase 1/Parkin, hypoxia-inducible factor-1 α/Bcl-2 and adenovirus e1b19k Da interacting protein 3, FUN-14 domain containing protein 1 receptor-mediated mitophagy pathway and so on. In this review, the authors briefly summarize the main pathways currently studied on mitophagy and the relationship between mitophagy and MIRI, and incorporate and analyze research data on prevention and treatment of MIRI with Chinese medicine, thereby provide relevant theoretical basis and treatment ideas for clinical prevention of MIRI.

Evaluation of Expression and Prognostic Significance of FUNDC1 Protein in Non-small Cell Lung Cancer Based on TCGA Database and Clinicopathology / 肿瘤防治研究

Objective To evaluate the expression of FUNDC1 and its clinical significance in non-small cell lung cancer. Methods We used TCGA database to analyze the difference of mitochondrial receptors (DRP1, BNIP3, FUNDC1, NIX, RHEB, LC3, OPA1 and MFN1) expression between normal and NSCLC tissues, as well as its effect on the prognosis of NSCLC patients. Immunohistochemistry was used to detect FUNDC1 expression. The correlations between FUNDC1 expression and clinicopathological characteristics, prognosis were evaluated by SPSS 22.0 statistical software. Results FUNDC1 expression was increased in NSCLC tissues, compared with normal tissues. FUNDC1 expression was related to the degree of differentiation and lymph node metastasis, but not to gender, age, pathological type, distant metastasis or TNM classification. The Cox regression analysis showed that FUNDC1 protein expression, lymph node metastasis, differentiation degree were independent prognostic factors of NSCLC. Increased FUNDC1 expression was related to decreased OS and PFS (P < 0.01). Conclusion The up-regulation of FUNDC1 expression can affect the prognosis of patients with NSCLC. It may be a new potential target for treating with NSCLC.

Structural insights into the recognition of phosphorylated FUNDC1 by LC3B in mitophagy.

Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria and maintains cellular homeostasis. Mitophagy is regulated by the post-translational modification of mitophagy receptors. Fun14 domain-containing protein 1 (FUNDC1) was reported to be a new receptor for hypoxia-induced mitophagy in mammalian cells and interact with microtubule-associated protein light chain 3 beta (LC3B) through its LC3 interaction region (LIR). Moreover, the phosphorylation modification of FUNDC1 affects its binding affinity for LC3B and regulates selective mitophagy. However, the structural basis of this regulation mechanism remains unclear. Here, we present the crystal structure of LC3B in complex with a FUNDC1 LIR peptide phosphorylated at Ser17 (pS17), demonstrating the key residues of LC3B for the specific recognition of the phosphorylated or dephosphorylated FUNDC1. Intriguingly, the side chain of LC3B Lys49 shifts remarkably and forms a hydrogen bond and electrostatic interaction with the phosphate group of FUNDC1 pS17. Alternatively, phosphorylated Tyr18 (pY18) and Ser13 (pS13) in FUNDC1 significantly obstruct their interaction with the hydrophobic pocket and Arg10 of LC3B, respectively. Structural observations are further validated by mutation and isothermal titration calorimetry (ITC) assays. Therefore, our structural and biochemical results reveal a working model for the specific recognition of FUNDC1 by LC3B and imply that the reversible phosphorylation modification of mitophagy receptors may be a switch for selective mitophagy.

Structural insights into the recognition of phosphorylated FUNDC1 by LC3B in mitophagy

Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria and maintains cellular homeostasis. Mitophagy is regulated by the post-translational modification of mitophagy receptors. Fun14 domain-containing protein 1 (FUNDC1) was reported to be a new receptor for hypoxia-induced mitophagy in mammalian cells and interact with microtubule-associated protein light chain 3 beta (LC3B) through its LC3 interaction region (LIR). Moreover, the phosphorylation modification of FUNDC1 affects its binding affinity for LC3B and regulates selective mitophagy. However, the structural basis of this regulation mechanism remains unclear. Here, we present the crystal structure of LC3B in complex with a FUNDC1 LIR peptide phosphorylated at Ser17 (pS), demonstrating the key residues of LC3B for the specific recognition of the phosphorylated or dephosphorylated FUNDC1. Intriguingly, the side chain of LC3B Lys49 shifts remarkably and forms a hydrogen bond and electrostatic interaction with the phosphate group of FUNDC1 pS. Alternatively, phosphorylated Tyr18 (pY) and Ser13 (pS) in FUNDC1 significantly obstruct their interaction with the hydrophobic pocket and Arg10 of LC3B, respectively. Structural observations are further validated by mutation and isothermal titration calorimetry (ITC) assays. Therefore, our structural and biochemical results reveal a working model for the specific recognition of FUNDC1 by LC3B and imply that the reversible phosphorylation modification of mitophagy receptors may be a switch for selective mitophagy.