Research progress in mitochondrial autophagy mediated by BNIP3

HUANG Jing-zhu, CHENG Qiu-chen, LI Fu-jian, Liu Ze-feng, Zhang Guo✉

1. Graduate School of Youjiang Medical College for Nationalities, Baise 533000, China

2. Department of Gastroenterology, Guangxi Academy of Medical Sciences, Guangxi Zhuang Autonomous Hospital, Nanning 530000, China

3. Department of Gastroenterology, Guangxi Hospital, The First Affiliated Hospital of Sun Yat-sen University, Nanning 530000, China

Keywords:

ABSTRACT Mitochondrial autophagy is widely found in mammals, and plays an important role in maintaining mitochondrial balance and mitochondrial quality control in cells.In this review,we reviewed the research progress of BNIP3-mediated mitochondrial autophagy and diseases in recent 5 years, providing new ideas for clinical diagnosis and treatment.

Autophagy is a biological process through which cells use the lysosome to degrade damaged organelles and proteins.Autophagy functions to maintain the stability of the intracellular environment when they are stimulated by external stimuli and nutrient deficiency.Autophagy can be divided into selective and non-selective autophagy according to its substrate.Generalized autophagy is usually manifested as nonselective autophagy.When autophagy is directed to clear damaged organelles, it manifests as corresponding selective autophagy,Such as mitochondrial autophagy, peroxisome autophagy,endoplasmic reticulum autophagy, etc.As a key component of mitochondrial quality control, mitophagy targets the timely elimination of damaged or dysfunctional mitochondria, thereby preventing excess reactive oxygen species (ROS) production, as well as the release of mitochondrial apoptotic factors and damageassociated molecular patterns (DAMPs)[1].These factors and damage-related molecular patterns (DAMPs) may be closely related to disease occurrence and progression.Mitochondrial autophagy requires induction of autophagy and mitochondrial initiation for autophagy recognition, and there are two initiation mechanisms for mitochondrial autophagy, one that relies on the PTENinduced kinase 1 (PINK1)-Parkin RBR E3 ubiquitin-protein ligase(PARK2) pathway, and the other that relies on the mitochondrial autophagy receptor BCL2 interacting protein 3 (BNIP3) or NIX(BNIP3 analog, BNIP3L) pathway[2].The latest research shows that mitophagy is closely related to human diseases[3].

Originally found on human chromosome 10q26.3 and known as“NIP3” BNIP3 is thought to belong to the Bcl-2 family of cell death regulators.Biochemically, BNIP3 is a protein that can interact with adenovirus E1B19kD protein, which is mainly composed of four functional domains, namely the PEST domain involved in protein degradation, the BH3 domain that mediates Caspase-dependent mitochondrial apoptosis, the NH2 domain, the transmembrane domain and the carboxyl-terminal domain, which are the most important domains for BNIP3 to exercise its Caspase-independent apoptosis function[4].In recent years, with the deepening of research,the role of BNIP3 in mitochondrial autophagy has attracted much attention, and the related research fields have made rapid progress.This article reviews the mechanism of BNIP3-mediated mitophagy in recent years and its research progress in diseases.

1.Bnip3-mediated mitochondrial autophagy mechanism

Mitophagy is a selective form of autophagy in which mitochondria are specifically targeted for degradation in autolysosomes, which are not limited to the turnover of dysfunctional mitochondria but also reduce the overall mass of mitochondria in response to stress,such as hypoxia and nutrient starvation[5].BNIP3 is a protein that dimerizes on the outer mitochondrial membrane via its C-terminal transmembrane domain (TMD).It contains a WXXL mode sequence structure (i.e., LIR) next to the N-terminal facing the cytoplasmic side, through which it can directly bind LC3 or GABARAP (LC3 homologous) to induce mitophagy[6].In addition, BNIP3 is also a mitochondrial autophagy receptor while acting as an induction molecular receptor, interacting directly through the conserved LC3 interaction region (LIR) motif and selectively targeting mitochondria for autophagosome degradation[7].At the same time, it was found that BNIP3 must first undergo dimerization to form homodimers,and its mitochondrial autophagy activity is regulated by its homodimerization, which promotes the interaction between BNIP3 and LC3, resulting in autophagosome recruitment into mitochondria for mitochondrial autophagy, which is related to its TMD structure[7].In addition to binding to LC3 to induce mitochondrial autophagy,BNIP3 can also indirectly induce mitochondrial autophagy[3], (1)The promoter region of the BNIP3 gene also contains a binding site of hypoxia-inducible factor 1 (HIF-1), which upregulates its expression under hypoxic conditions to induce mitochondrial autophagy.(2) BNIP3 may participate in ubiquitin-dependent mitophagy by promoting mitochondrial translocation of PARKIN and promoting PARKIN-mediated mitochondrial autophagy.(3)BNIP3 can activate autophagy by isolating RHEB to block the activation of mTOR.Fission-induced mitochondrial depolarization is an important factor triggering mitochondrial autophagy, and studies have confirmed a number of mechanisms to clear mitochondrial depolarization, and depolarization in physiological states may be the result of mitochondrial membrane permeability conversion(MPT) activation or indirect effect of mitochondrial outer membrane permeabilization (MOMP).Studies have shown that BNIP3 sensitizes BAX (BCL2-associated X, apoptosis regulator) and BAK (BCL2 antagonist/killer 1) insertion and activation in mitochondria, which is a key step in the permeabilization of the outer mitochondrial membrane, resulting in the release of pro-apoptotic factors in the intermembrane mitochondrial space into the cytoplasm to initiate the apoptosis cascade[1].

2.BNIP3-mediated study of mitochondrial autophagy in disease

2.1 Tumour

The mechanism of action of mitophagy in tumors is still not clear,but there is evidence that mitophagy is closely related to tumor occurrence and development[8].As one of the important mediators of mitochondrial autophagy, BNIP3 plays a very important role in regulating mitochondrial autophagy, plays a dual role in the development of tumors, and can promote cell survival or inhibit tumor cell growth.

In some tumors, BNIP3-mediated mitochondrial autophagy promotes tumorigenesis and development.Hu T et al[9]used bioinformatics methods to find that the expression of four genes(BCL2,LEF1,PLK1,and BNIP3) contained in BRCA could be used as prognostic biomarkers for BRCA, and comprehensive analysis showed that BRCA patients with high expression of BNIP3 had poor OS, DSS,and DFS,which showed that BNIP3 could promote the occurrence and development of invasive breast cancer.In breast cancer cells, downregulation of BNIP3 inhibits mitochondrial autophagy, thereby promoting apoptosis in breast cancer cells[10].In cervical cancer cells, upregulation of BNIP3 can promote the proliferation of HeLa cells and promote the occurrence and development of cervical cancer[11].In ovarian cancer cells,downregulation of BNIP3 expression inhibits mitophagy and reduces cell migration and invasion[12].In the cutaneous melanoma (SKCM)cohort, SKCM patients with “high” BNIP3 levels and “low”BNIP3 levels were compared to SKCM patients with “low” BNIP3 levels and found that SKCM patients with higher BNIP3 expression showed significantly reduced overall survival (OS), and further in vivo and in vitro experiments showed that removal of BNIP3 from melanoma cells delayed tumor cell growth[13].In non-small cell lung cancer cell lines A549 and SK-1 cells, the growth of non-small cell lung cancer cells is induced by downregulating BNIP3 and NIX by inhibiting mitochondrial autophagy and thereby inducing apoptosis[14].In hepatocellular carcinoma, Yao J et al[15] found that inhibiting the transcription of BNIP3 blocked the initiation of PINK1-PRKN-mediated mitochondrial autophagy, induced the instability of PINK1 protein and reduced the recruitment of PRKN in mitochondria, inhibiting tumor cell genesis and development;Dai X et al[16]found that upregulation of BNIP3-mediated mitophagy induced apoptosis in HepG2 cells.In colorectal cancer cells, silencing BNIP3 reduces tumor initiation cell (TIC) colony formation under hypoxic conditions[17].

In addition to promoting the occurrence and development of tumors, BNIP3-mediated mitochondrial autophagy has also been shown to inhibit the growth of tumor cells.In gastric cancer cells,BNIP3 expression is upregulated and mitophagy is activated, thereby inhibiting gastric cancer cell proliferation[18].In lung cancer cells,Zeng C et al[19]found that the treatment of ciclovirobusin D (CVB-D)in lung cancer cells can significantly inhibit the expression of BNIP3 transcription inhibitor p65, p65 downregulation can significantly alleviate its inhibition of BNIP3 transcription after CVB-D treatment;and can cause BNIP3 expression enhancement, thereby enhancing its interaction with LC3, mediating mitophagy activation,this mitochondrial autophagy-mediated mitochondrial dysfunction by targeting p65/BNIP3 The LC3 axis is the main mechanism of CVB-D-induced apoptosis, and further in vivo experiments revealed that subcutaneous transplanted tumors of tumor-bearing mice showed growth retardation after CVB-D treatment.

Tab 1 Mechanism of action of BNIP3 in tumors

2.2 Cardiovascular

BNIP3 is strongly associated with cardiac disease, with increased expression of the BNIP3 protein during hypoxia, cardiac hypertrophy, or ischemia[20].It has also been shown that when hypoxia is associated with cardiomyocyte acidosis, BNIP3 induces apoptosis rather than autophagy[21].The role of BNIP3 in heart disease remains to be further studied.

Overexpression of BNIP3 impairs the myocardial ischemiareperfusion (I/R) phenotype with apoptosis, ROS production,mitochondrial fragmentation, and increased dysfunction, while inducing BNIP3-mediated mitophagy in I/R helps remove damaged mitochondria and protect cardiomyocytes from apoptosis[20]; In the treatment of reoxygenated (HR) damage, it is observed that HR damage promotes Bnip3 phosphorylation at the Ser17 site, and phosphorylated Bnip3 has an increased ability for Bnip3 to interact with LC3, activating BNIP3-mediated mitochondrial autophagy[22].In recent years, many studies have shown to downregulate the expression of BNIP3 protein and inhibit mitochondrial autophagy[22-25], thereby preventing myocardial ischemia-reperfusion injury(IR).In contrast, Zhang YN et al[26]found that the Jumonji domain containing 5 (JMJD5) enhances BNIP3 expression by mediating the HIF-1α-BNIP3 pathway, thereby protecting OGD/R-induced cardiomyocyte damage.In recent years, there are many people who mistakenly eat aconitine, and a series of symptoms will occur after ingestion of aconitine, the more serious of which is cardiotoxicity,which can cause myocardial damage, Peng F et al[27]Studies found that aconitine inhibits cardiomyocyte proliferation and induces inflammation and apoptosis in a dose- and time-dependent manner, further through RNA sequencing, gene set enrichment analysis (GSEA) results show that cardiomyocyte inflammation,apoptosis and autophagy after aconitine intervention are related,the team further showed that Aconitine-induced cardiomyocyte damage may induce TNFαactivation, and then inhibit BNIP3-dependent mitochondrial autophagy, in vivo experiments, BNIP3 overexpression adenovirus by intracardiac injection, the results show that BNIP3 overexpression significantly inhibits aconitineinduced myocardial injury, and the mechanism may be by enhancing mitochondrial autophagy to effectively reverse aconitine-induced cardiotoxicity, which provides new ideas for clinical treatment.

In the dilated cardiomyopathy (DCM) model, it was found that the expression of BNIP3 protein decreased and mitochondrial autophagy was impaired, which in turn led to the impaired macroscopic phenotype of cardiomyocytes and dilated cardiomyopathy (DCM),which indicates that BNIP3-mediated mitochondrial autophagy plays an important role in DCM, enhances BNIP3 protein expression levels, and may provide a new therapeutic strategy for the treatment of DCM[28].

The expression of Bnip3, which plays a key role in the receptordependent pathway of mitochondrial autophagy, can be increased after IL-6 stimulation, Huo S et al[29]found that myocardial inflammation is induced by transverse aortic contraction (TAC), and after raloxifene intervention, it can be observed that raloxifene can block overactivated IL-6/STAT3 signaling and alleviate inflammation by reducing oxidative stress and regulating mitophagy levels, further experimental studies found that Bnip3 protein expression decreased in this experiment, The team showed that decreased expression of Bnip3 protein regulates mitochondrial autophagy to alleviate myocardial inflammation, cardiac remodeling and dysfunction induced by transverse aortic contraction (TAC), which has certain clinical significance for heart failure diseases.

2.3 Diseases of the Liver

BNIP3-mediated mitochondrial autophagy is associated with nonalcoholic fatty liver disease, alcoholic fatty liver disease,liver fibrosis,and other diseases.In the study of nonalcoholic fatty liver disease, Li R et al[30]found that upregulation of Bnip3-mediated mitochondrial autophagy can reduce mitochondrial damage and inhibit mitochondria-dependent apoptosis, thereby improving dietinduced nonalcoholic fatty liver disease.Gong LL et al[31]found that upregulating BNIP3 protein enhanced mitochondrial autophagy and alleviated the degree of hepatic steatosis.In the study of alcoholic fatty liver disease, Wu MF et al[32]found that there may be a negative correlation between Hif-2α levels and mitochondrial autophagy,and in vivo and in vitro experiments showed that the deletion of Hif-2α initiated mitochondrial autophagy, which can reduce the accumulation of liver lipids, and the PPAR- /PGC-1α signaling pathway involved in the β oxidation of liver fatty acids is negatively regulated by BNIP3-mediated mitochondrial autophagy.The team showed that BNIP3-mediated mitochondrial autophagy regulates lipid metabolism in alcoholic fatty liver disease.In the study of liver fibrosis, Wang Q et al[33]found that XBP1 activation significantly inhibited BNIP3-mediated mitochondrial autophagy and exacerbated liver fibrosis, and further experiments revealed that Xbp1 knockout reduces ROS production by promoting BNIP3-mediated mitophagy autophagy activation in macrophages, helping to improve liver fibrosis.

2.4 Renal disease

In renal proximal tubular epithelial cells cultured after oxygenglucose deprivation reperfusion (OGD-R) and in mouse injury models induced by renal ischemia-reperfusion (IR),it was found that BNIP3 expression in renal tubules increased,and further by silencing Bnip3 in renal tubular cells, OGD-R-induced mitochondrial autophagy and enhanced OGD-R-induced cell death was reduced.At the same time,in vivo,experiments have also confirmed that Bnip3 knockout aggravates renal ischemia-reperfusion (IR) damage,reduces mitochondrial autophagy, leads to the accumulation of damaged mitochondria, increases the production of reactive oxygen species,and enhances the cell death and inflammatory response of the kidney after renal IR[1].In addition,studies have shown that BNIP3-mediated mitochondrial autophagy is closely related to the progression of diabetic nephropathy, and by regulating BNIP3-mediated mitophagy,abnormal mitochondrial dysfunction,and mitochondrial quality control is restored,which can reverse the progression of diabetic nephropathy[34].

2.5 Diseases of the Nervous System

Autophagy is closely related to the process of neuronal differentiation, and in some cases,autophagy supports cellular homeostasis by eliminating the degradation of protein aggregates and senescent organelles,both of which are critical for neuronal cell survival and differentiation during development and adulthood.Studies have shown that the BNIP3 gene in neurons is downregulated in the ischemic CA3 region of the hippocampus, but still plays a key role in activating unique neuronal protectors or death without removing altered proteins or mitochondria[35].

Chronic hypoxia, intermittent hypoxia (IH),chronic cerebral hypoperfusion (CCH),neuronal ischemia-reperfusion injury, etc.can cause neuronal cell damage.In chronic hypoxia, impaired autophagy degradation and BNIP3-mediated inefficient mitochondrial autophagy may be mechanisms of neuronal cell damage[36].Yu D et al[37]found that knockdown of the expression of the BNIP3 gene protected spinal cord neurons from hypoxia-induced cell death.BNIP3 plays different roles in the mechanism of hypoxia-induced neuronal damage, and its mechanism needs to be confirmed by more studies.Studies have shown that activating BNIP3-mediated mitophagy can significantly inhibit the formation of NLRP3 inflammasome and microglia infiltration in the hippocampus of IH mice,reduce neuronal apoptosis and hippocampal inflammation[38];Neuronal ischemia-reperfusion injury studies have also been found to protect neurons from ischemia-reperfusion injury by activating BNIP3-mediated mitophagy[39].In CCH studies,it has been found that unlike chronic hypoxia, IH, and neuronal ischemiareperfusion injury, CCH cuts off mitochondrial autophagy required for BNIP3, ultimately preventing abnormal hyperphagia and mitochondrial autophagy,and slowing CCH-induced apoptosis of neurons[40].Founded in acute model mice of Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),pramipexole (PPX) weakens MPTP-induced neuronal damage in mice by modulating BNIP3-mediated mitochondrial autophagy[41].

2.6 Other

Recently, studies have shown that BNIP3 expression is reduced in preeclampsia (PE) patients, and further studies have shown that dysregulated BNIP3 is associated with impaired mitochondrial autophagy, oxidative stress, and apoptosis in PE[42].BNIP3-mediated mitochondrial autophagy shows different effects in chondrocytes,and Kim D et al[43]found that the overexpression of BNIP3 stimulates mitochondrial autophagy and cartilage degradation by upregulating chondrodegrading enzymes and chondrocytes death, which is of great significance for maintaining cartilage homeostasis.Lin J et al[44]found that HIF-1α inhibits the expression of BNIP3, thereby mediating the mitochondrial autophagy process in chondrocytes,partially weakening the protective effect of deferoxamine (DFO)on iron overload in chondrocytes, and preventing the development of hemophilic arthropathy (HA) through mitochondrial autophagy.BNIP3 plays a truly important role in reducing asthma symptoms and improving airway smooth muscle (ASM) remodeling, regulating adhesion, migration, and proliferation of ASM through Bnip3 regulating mitochondrial function and the expression of adhesion proteins[45].

3.Summary and outlook

In summary, BNIP3 is a key factor in regulating mitochondrial autophagy,and has attracted much attention in the field of mitochondrial autophagy research.This article reviews the latest research progress of BNIP3-mediated mitophagy in the past five years, and summarizes the relationship between BNIP3-mediated mitophagy and tumors, cardiovascular diseases, liver diseases,kidney diseases, neurological diseases and other diseases.Because autophagy plays different roles in disease, its role also varies.BNIP3 has shown dual effects in tumors, myocardial ischemia, perfusion injury, hypoxia-induced neuronal cell damage and other diseases,which can promote the occurrence and development of the disease and play a protective role in the disease, which may be related to the type of disease or the stage of the disease.Compared with the classic PINK1-parkin pathway, BNIP3-mediated mitophagy research is still insufficient and in-depth in the study of various diseases, and the specific mechanism of action is still not clear.

Authors’ Contribution

Huang Jingzhu conceived and designed the article,and wrote the paper: Cheng Qiuchen, Li Fujian, Liu Zefeng collected and sorted out literature/data; Zhang Guo conducts the feasibility analysis of the article and the revision of the paper, is responsible for the quality control and review of the article, and is responsible for the overall supervision and management of the article.

All authors declare that there is no conflict of interest.