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Asa B Gustafsson - One of the best experts on this subject based on the ideXlab platform.
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BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation
2019Co-Authors: Mark A. Lampert, Amabel M Orogo, Babette C Hammerling, Rita H. Najor, Leonardo J. Leon, Bingyan J. Wang, Taeyong Kim, Mark A. Sussman, Asa B GustafssonAbstract:Cell-based therapies represent a very promising strategy to repair and regenerate the injured heart to prevent progression to heart failure. To date, these therapies have had limited success due to a lack of survival and retention of the infused cells. Therefore, it is important to increase our understanding of the biology of these cells and utilize this information to enhance their survival and function in the injured heart. Mitochondria are critical for progenitor cell function and survival. Here, we demonstrate the importance of mitochondrial autophagy, or mitophagy, in the differentiation process in adult cardiac progenitor cells (CPCs). We found that mitophagy was rapidly induced upon initiation of differentiation in CPCs. We also found that mitophagy was mediated by mitophagy receptors, rather than the PINK1-PRKN/PARKIN pathway. Mitophagy mediated by BNIP3L/NIX and FUNDC1 was not involved in regulating progenitor cell fate determination, mitochondrial biogenesis, or reprogramming. Instead, mitophagy facilitated the CPCs to undergo proper mitochondrial network reorganization during differentiation. Abrogating BNIP3L- and FUNDC1-mediated mitophagy during differentiation led to sustained mitochondrial fission and formation of donut-shaped impaired mitochondria. It also resulted in increased susceptibility to cell death and failure to survive the infarcted heart. Finally, aging is associated with accumulation of mitochondrial DNA (mtDNA) damage in cells and we found that acquiring mtDNA mutations selectively disrupted the differentiation-activated mitophagy program in CPCs. These findings demonstrate the importance of BNIP3L- and FUNDC1-mediated mitophagy as a critical regulator of mitochondrial network formation during differentiation, as well as the consequences of accumulating mtDNA mutations. Abbreviations: Baf: bafilomycin A1; BCL2L13: BCL2 like 13; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CPCs: cardiac progenitor cells; DM: differentiation media; DNM1L: dynamin 1 like; EPCs: endothelial progenitor cells; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FUNDC1: FUN14 domain containing 1; HSCs: hematopoietic stem cells; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MFN1/2: mitofusin 1/2; MSCs: mesenchymal stem cells; mtDNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; PPARGC1A: PPARG coactivator 1 alpha; PHB2: prohibitin 2; POLG: DNA polymerase gamma, catalytic subunit; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester
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Abstract 174: The BH3-Only Protein BNIP3 Induces Mitochondrial Clearance via Multiple Pathways
Circulation Research, 2015Co-Authors: Eileen R Gonzalez, Dieter A. Kubli, Rita A. Hanna, Babette C Hammerling, Asa B GustafssonAbstract:Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. BNIP3 is an atypical BH3-only protein which is known to cause mitochondrial dysfunction and cell death in the myocardium. Interestingly, BNIP3 can also protect against cell death by promoting removal of dysfunctional mitochondria via autophagy (mitophagy). We have previously reported that BNIP3 is a potent inducer of mitophagy in cardiac myocytes and that BNIP3 contains an LC3 Interacting Region (LIR) that binds to LC3 on the autophagosome, tethering the mitochondrion to the autophagosome for engulfment. However, the molecular mechanism(s) underlying BNIP3-mediated mitophagy are still unclear. In this study, we discovered that BNIP3 can mediate mitochondrial clearance in cells even in the absence of a functional autophagy pathway. We found that overexpression of BNIP3 led to significant clearance of mitochondria in both wild type (WT) and autophagy deficient Atg5-/- MEFs. BNIP3 caused an increase in LC3II levels in WT MEFs, indicating increased formation of autophagosomes. In contrast, LC3II was undetectable in Atg5-/- MEFs. Furthermore, we found that BNIP3-mediated clearance in WT and Atg5-/- MEFs did not require the presence of Parkin, an E3 ubiquitin ligase which plays a critical role in clearing dysfunctional mitochondria in cells. Also, overexpression of Parkin did not enhance BNIP3-mediated mitochondrial clearance. When investigating activation of alternative cellular degradation pathways, we found that BNIP3 induced activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. Mutating the LC3 binding site in BNIP3 did not interfere with the activation of the endosomal pathway and clearance of mitochondria in Atg5-/- MEFs. Thus, these findings suggest that BNIP3 can promote clearance of mitochondria via multiple pathways in cells. The role of autophagy in removing mitochondria is already well established and we are currently exploring the roles of the endosomal and alternative autophagy pathways in BNIP3-mediated mitochondrial clearance in myocytes.
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microtubule associated protein 1 light chain 3 lc3 interacts with BNIP3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy
Journal of Biological Chemistry, 2012Co-Authors: Rita A. Hanna, Melissa N Quinsay, Amabel M Orogo, Kayla Giang, Shivaji Rikka, Asa B GustafssonAbstract:Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. BNIP3 is an atypical BH3-only protein that is known to cause mitochondrial dysfunction and cell death. Interestingly, BNIP3 can also protect against cell death by inducing mitochondrial autophagy. The mechanism for this process, however, remains poorly understood. BNIP3 contains a C-terminal transmembrane domain that is essential for homodimerization and proapoptotic function. In this study, we show that homodimerization of BNIP3 is also a requirement for induction of autophagy. Several BNIP3 mutants that do not interfere with its mitochondrial localization but disrupt homodimerization failed to induce autophagy in cells. In addition, we discovered that endogenous BNIP3 is localized to both mitochondria and the endoplasmic reticulum (ER). To investigate the effects of BNIP3 at mitochondria or the ER on autophagy, BNIP3 was targeted specifically to each organelle by substituting the BNIP3 transmembrane domain with that of Acta or cytochrome b5. We found that BNIP3 enhanced autophagy in cells from both sites. We also discovered that BNIP3 induced removal of both ER (ERphagy) and mitochondria (mitophagy) via autophagy. The clearance of these organelles was mediated in part via binding of BNIP3 to LC3 on the autophagosome. Although ablation of the BNIP3-LC3 interaction by mutating the LC3 binding site did not impair the prodeath activity of BNIP3, it significantly reduced both mitophagy and ERphagy. Our data indicate that BNIP3 regulates the apoptotic balance as an autophagy receptor that induces removal of both mitochondria and ER.
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mitochondrial autophagy by BNIP3 involves drp1 mediated mitochondrial fission and recruitment of parkin in cardiac myocytes
American Journal of Physiology-heart and Circulatory Physiology, 2011Co-Authors: Rita A. Hanna, Asa B GustafssonAbstract:The Bcl2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) is an atypical BH3-only protein that is associated with mitochondrial dysfunction and cell death. BNIP3 is also a potent inducer of mitochondrial autophagy, and in this study we have investigated the mechanisms by which BNIP3 induces autophagy in cardiac myocytes. We found that BNIP3 induced mitochondrial translocation of dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission in adult myocytes. Drp1-mediated mitochondrial fission correlated with increased autophagy, and inhibition of Drp1 reduced BNIP3-mediated autophagy. Overexpression of Drp1K38E, a dominant negative of Drp1, or mitofusin 1 prevented mitochondrial fission and autophagy by BNIP3. Also, inhibition of mitochondrial fission or autophagy resulted in increased death of myocytes overexpressing BNIP3. Moreover, BNIP3 promoted translocation of the E3 ubiquitin ligase Parkin to mitochondria, which was prevented in the presence of a Drp1 inhibitor. Interestingly, induction of autophagy by BNIP3 was reduced in Parkin-deficient myocytes. Thus our data suggest that induction of autophagy in response to BNIP3 is a protective response activated by the cell that involves Drp1-mediated mitochondrial fission and recruitment of Parkin.
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BNIP3 as a Dual Regulator of Mitochondrial Turnover and Cell Death in the Myocardium
Pediatric cardiology, 2011Co-Authors: Asa B GustafssonAbstract:The Bcl-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a pro-apoptotic BH3-only protein associated with the pathogenesis of many diseases, including cancer and cardiovascular disease. Studies over the past decade have provided insight into how BNIP3 induces mitochondrial dysfunction and subsequent cell death in cells. More recently, BNIP3 was identified as a potent inducer of autophagy in cells. However, the functional role of BNIP3-mediated autophagy has been difficult to define and remains controversial. New evidence has emerged suggesting that BNIP3 is an important regulator of mitochondrial turnover via autophagy in the myocardium. Also, studies suggest that the induction of BNIP3-dependent mitochondrial autophagy is a separately activated process independent of Bax/Bak and the mitochondrial permeability transition pore (mPTP). This review discusses the current understanding of the functional role that BNIP3 plays in the myocardium. Recent studies suggest that BNIP3 might have a dual function in the myocardium, where it regulates both mitochondrial turnover via autophagy and cell death and that these are two separate processes activated by BNIP3.
Lorrie A Kirshenbaum - One of the best experts on this subject based on the ideXlab platform.
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antagonism of e2f 1 regulated BNIP3 transcription by nf κb is essential for basal cell survival
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: James Shaw, Natalia Yurkova, Danielle Weidman, Tong Zhang, Hongying Gang, Floribeth Aguilar, Carly Scramstad, Harvey Weisman, Lorrie A KirshenbaumAbstract:The transcription factor E2F-1 drives proliferation and death, but the mechanisms that differentially regulate these divergent actions are poorly understood. The hypoxia-inducible death factor BNIP3 is an E2F-1 target gene and integral component of the intrinsic mitochondrial death pathway. The mechanisms that govern BNIP3 gene activity remain cryptic. Herein we show that the transcription factor NF-κB provides a molecular switch that determines whether E2F-1 signals proliferation or death under physiological conditions. We show under basal nonapoptotic conditions that NF-κB constitutively occupies and transcriptionally silences BNIP3 gene transcription by competing with E2F-1 for BNIP3 promoter binding. Conversely, in the absence of NF-κB, or during hypoxia when NF-κB abundance is reduced, basal BNIP3 gene transcription is activated by the unrestricted binding of E2F-1 to the BNIP3 promoter. Genetic knock-down of E2F-1 or retinoblastoma gene product over-expression in cardiac and human pancreatic cancer cells deficient for NF-κB signaling abrogated basal and hypoxia-inducible BNIP3 transcription. The survival kinase PI3K/Akt inhibited BNIP3 expression levels in cells in a manner dependent upon NF-κB activation. Hence, by way of example, we show that the transcriptional inhibition of E2F-1-dependent BNIP3 expression by NF-κB highlights a survival pathway that overrides the E2F-1 tumor suppressor program. Our data may explain more fundamentally how cells, by selectively inhibiting E2F-1-dependent death gene transcription, avert apoptosis down-stream of the retinoblastoma/E2F-1 cell cycle pathway.
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cardiac reanimation targeting cardiomyocyte death by BNIP3 and nix BNIP3l
Oncogene, 2008Co-Authors: G W Dorn, Lorrie A KirshenbaumAbstract:Programmed cardiac myocyte death contributes to pathological ventricular remodeling and the progression of myocardial infarction or pressure overload hypertrophy to dilated cardiomyopathy. Recent work has identified importance of stress-mediated transcriptional induction of BNIP3 (BCL2 and 19-kDa interacting protein-3) and NIX/BNIP3L in cardiac remodeling. Here, the regulatory mechanisms for these two factors in the heart and their effects on programmed cardiomyocyte death are reviewed, with a focus on information derived from studies using mouse models of cardiac BNIP3 and NIX/BNIP3L overexpression and gene ablation.
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the cell cycle factor e2f 1 activates BNIP3 and the intrinsic death pathway in ventricular myocytes
Circulation Research, 2008Co-Authors: Natalia Yurkova, James Shaw, Karen Blackie, Danielle Weidman, Ravi Jayas, Bryan Flynn, Lorrie A KirshenbaumAbstract:The cell cycle factor E2F-1 is known to regulate a variety of cellular processes including apoptosis. Previously we showed that disruption of Rb-E2F-1 complexes provoked apoptosis of postmitotic adult and neonatal ventricular myocytes; however, the underlying mechanism was undetermined. In this report, we show that E2F-1 provokes cell death of ventricular myocytes through a mechanism that directly impinges on the intrinsic death pathway. Furthermore, we show mechanistically that the hypoxia-inducible death factor BNIP3 is a direct transcriptional target of E2F-1 that is necessary and sufficient for E2F-1-induced cell death. Expression of E2F-1 resulted in a 4.9-fold increase (P<0.001) in nucleosomal DNA fragmentation and cell death by Hoechst 33258 dye and vital staining. E2F-1 provoked mitochondrial perturbations that were consistent with permeability transition pore opening. As determined by quantitative real-time PCR analysis, a 6.2-fold increase (P<0.001) in endogenous BNIP3 gene transcription was observed in cells expressing wild-type E2F-1 but not in cells expressing a mutation of E2F-1 defective for DNA binding. Rb, the principle regulator of cellular E2F-1 activity, was proteolytically cleaved and inactivated in ventricular myocytes during hypoxia. Consistent with the proteolytic cleavage of Rb, chromatin immunoprecipitation analysis revealed increased binding of E2F-1 to the BNIP3 promoter during hypoxia, a finding concordant with the induction of BNIP3 gene transcription. The BNIP3 homolog Nix/BNIP3L was unaffected in ventricular myocytes by either E2F-1 or hypoxia. Genetic knockdown of E2F-1 or expression of a caspase-resistant form of Rb suppressed basal and hypoxia-inducible BNIP3 gene transcription. Loss-of-function mutations of BNIP3 defective for mitochondrial membrane insertion or small interference RNA directed against BNIP3 suppressed cell death signals elicited by E2F-1. To our knowledge, the data provide the first direct evidence that activation of the intrinsic mitochondrial death pathway by E2F-1 is mutually dependent on and obligatorily linked to the transcriptional activation of BNIP3.
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inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of BNIP3 restrains postinfarction remodeling in mice
Journal of Clinical Investigation, 2007Co-Authors: Abhinav Diwan, Lorrie A Kirshenbaum, Maike Krenz, Faisal M Syed, Janaka Wansapura, Xiaoping Ren, Andrew G Koesters, Harvey S Hahn, Jeffrey Robbins, Keith W JonesAbstract:Following myocardial infarction, nonischemic myocyte death results in infarct expansion, myocardial loss, and ventricular dysfunction. Here, we demonstrate that a specific proapoptotic gene, BNIP3, minimizes ventricular remodeling in the mouse, despite having no effect on early or late infarct size. We evaluated the effects of ablating BNIP3 on cardiomyocyte death, infarct size, and ventricular remodeling after surgical ischemia/ reperfusion (IR) injury in mice. Immediately following IR, no significant differences were observed between BNIP3 -/- and WT mice. However, at 2 days after IR, apoptosis was diminished in BNIP3 -/- periinfarct and remote myocardium, and at 3 weeks after IR, BNIP3 -/- mice exhibited preserved LV systolic performance, diminished LV dilation, and decreased ventricular sphericalization. These results suggest myocardial salvage by inhibition of apoptosis. Forced cardiac expression of BNIP3 increased cardiomyocyte apoptosis in unstressed mice, causing progressive LV dilation and diminished systolic function. Conditional BNIP3 overexpression prior to coronary ligation increased apoptosis and infarct size. These studies identify postischemic apoptosis by myocardial BNIP3 as a major determinant of ventricular remodeling in the infarcted heart, suggesting that BNIP3 may be an attractive therapeutic target.
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response to myocardial ischemia reperfusion injury involves BNIP3 and autophagy
Cell Death & Differentiation, 2007Co-Authors: Anne Hamacherbrady, Nathan R. Brady, Roberta A. Gottlieb, Lorrie A Kirshenbaum, Susan E Logue, M R Sayen, M Jinno, Asa B GustafssonAbstract:Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. BNIP3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if BNIP3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of BNIP3 (TAT-BNIP3ΔTM) which has been shown to act as a dominant negative to block BNIP3-induced cell death. Perfusion with TAT-BNIP3ΔTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, BNIP3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against BNIP3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that BNIP3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.
Keith A. Webster - One of the best experts on this subject based on the ideXlab platform.
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BNIP3 Binds and Activates p300: Possible Role in Cardiac Transcription and Myocyte Morphology.
PloS one, 2015Co-Authors: John W. Thompson, Jianqin Wei, Kweku Appau, Huilan Wang, Maria Grazia Spiga, Regina M. Graham, Keith A. WebsterAbstract:BNIP3 is a hypoxia-regulated member of the Bcl-2 family of proteins that is implicated in apoptosis, programmed necrosis, autophagy and mitophagy. Mitochondria are thought to be the primary targets of BNIP3 although its activities may extend to the ER, cytoplasm, and nucleus. BNIP3 is induced in the heart by ischemia and pressure-overload, and may contribute to cardiomyopathy and heart failure. Only mitochondrial-dependent programmed death actions have been described for BNIP3 in the heart. Here we describe a novel activity of BNIP3 in cultured cardiac myocytes and transgenic mice overexpressing BNIP3 in the heart (BNIP3-TG). In cultured myocytes BNIP3 bound and activated the acetyltransferase p300, increased acetylation of histones and the transcription factor GATA4, and conferred p300 and GATA4-sensitive cellular morphological changes. In intact BNIP3-TG hearts BNIP3 also bound p300 and GATA4 and conferred enhanced GATA4 acetylation. BNIP3-TG mice underwent age-dependent ventricular dilation and heart failure that was partially prevented by p300 inhibition with curcumin. The results suggest that BNIP3 regulates cardiac gene expression and perhaps myocyte morphology by activating nuclear p300 acetyltransferase activity and hyperacetylating histones and p300-selective transcription factors.
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Regulation of BNIP3 death pathways by calcium, phosphorylation, and hypoxia-reoxygenation.
Antioxidants & redox signaling, 2007Co-Authors: Regina M. Graham, John W. Thompson, Jianqin Wei, Nanette H. Bishopric, Keith A. WebsterAbstract:BNIP3 is a proapoptotic member of the Bcl-2 family of death-regulating proteins that promote the intrinsic pathway of programmed cell death. The BNIP3 death program requires membrane insertion through an N-terminal transmembrane domain that directs the protein to mitochondrial and endoplasmic reticular (ER) membranes. We have reported that simulated ischemia induces transcription of the BNIP3 gene, and BNIP3 protein is stabilized by acidosis. BNIP3 programmed death is atypical, with features of both apoptosis and necrosis. Here we demonstrate that hypoxia–reoxygenation and agents that activate protein kinase C, including calcium ionophore, phorbol 12-myristate 13-acetate, and okadaic acid, also induce BNIP3. The molecular size of BNIP3 predicted from the amino acid sequence is 21.5 kDa, but the protein typically migrates in SDS-PAGE as a 31-kDa monomer and 60-kDa dimer. Treatment of cell extracts containing BNIP3 with phosphatase yielded a series of rapidly migrating species, the smallest of which corresp...
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Focused Issue onApoptosis and the Heart BNIP3 and signal-specific programmed death in the heart
2005Co-Authors: Keith A. Webster, Regina M. Graham, Nanette H. BishopricAbstract:The BH3-only proteins, including BNIP3, are members of the Bcl-2 family of cell death-regulating factors. Whereas proteins such as Bax and Bak play a central role in most forms of apoptosis, the BH3-only proteins appear to modulate apoptosis through cell type- and signalspecific pathways. This review will focus on our studies of the specific role of BNIP3 in cardiac myocyte apoptotic signaling during ischemia. We recently showed that hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the [pH]o drops below 6.5.A micro-array analysis of 20,000 genes identified the pro-apoptotic Bcl-2 family member BNIP3 as one of the most strongly upregulated of >100 hypoxia-inducible genes in cardiac myocytes. BNIP3 mRNA increased by 12-fold, and BNIP3 protein by sixfold, during 24 h of hypoxia; BNIP3 protein accumulation was further enhanced by acidosis. BNIP3 was loosely bound to mitochondria under conditions of neutral hypoxia but became more tightly associated at acid pH, coincident with opening of the mitochondrial permeability transition pore (MPTP). Subsequent DNA fragmentation and cell death were not blocked by caspase inhibitors, but were inhibited by antisense BNIP3 oligonucleotides and MPTP inhibitors, indicating that BNIP3 activates an atypical programmed death pathway with features of both apoptosis and necrosis.The sequential induction and activation of BNIP3 by hypoxia and acidosis provide a molecular basis for the observation that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes. © 2004 Elsevier Ltd. All rights reserved.
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BNIP3 and signal-specific programmed death in the heart.
Journal of molecular and cellular cardiology, 2004Co-Authors: Keith A. Webster, Regina M. Graham, Nanette H. BishopricAbstract:Abstract The BH3-only proteins, including BNIP3, are members of the Bcl-2 family of cell death-regulating factors. Whereas proteins such as Bax and Bak play a central role in most forms of apoptosis, the BH3-only proteins appear to modulate apoptosis through cell type- and signal-specific pathways. This review will focus on our studies of the specific role of BNIP3 in cardiac myocyte apoptotic signaling during ischemia. We recently showed that hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the [pH] o drops below 6.5. A micro-array analysis of 20,000 genes identified the pro-apoptotic Bcl-2 family member BNIP3 as one of the most strongly upregulated of >100 hypoxia-inducible genes in cardiac myocytes. BNIP3 mRNA increased by 12-fold, and BNIP3 protein by sixfold, during 24 h of hypoxia; BNIP3 protein accumulation was further enhanced by acidosis. BNIP3 was loosely bound to mitochondria under conditions of neutral hypoxia but became more tightly associated at acid pH, coincident with opening of the mitochondrial permeability transition pore (MPTP). Subsequent DNA fragmentation and cell death were not blocked by caspase inhibitors, but were inhibited by antisense BNIP3 oligonucleotides and MPTP inhibitors, indicating that BNIP3 activates an atypical programmed death pathway with features of both apoptosis and necrosis. The sequential induction and activation of BNIP3 by hypoxia and acidosis provide a molecular basis for the observation that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes.
Jiming Kong - One of the best experts on this subject based on the ideXlab platform.
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The proapoptotic protein BNIP3 interacts with VDAC to induce mitochondrial release of endonuclease G.
PloS one, 2014Co-Authors: Xiaosha Zhang, Xiu-wu Bian, Jiming KongAbstract:BNIP3 is a proapoptotic protein that induces cell death through a mitochondria-mediated pathway. We reported previously that mitochondrial localization of BNIP3 and translocation of EndoG from mitochondria to the nucleus are critical steps of the BNIP3 pathway. It is not clear, however, that how BNIP3 interacts with mitochondria. Here we show that expression of BNIP3 resulted in mitochondrial release and nuclear translocation of EndoG. Incubation of a recombinant GST-BNIP3 protein with freshly isolated mitochondria led to the integration of BNIP3 into mitochondria, reduction in the levels of EndoG in mitochondria and the presence of EndoG in the supernatant that was able to cleave chromatin DNA. Co-immunoprecipitation and mass spectrometry analysis reveals that BNIP3 interacted with the voltage-dependent anion channel (VDAC) to increase opening probabilities of mitochondrial permeability transition (PT) pores and induce mitochondrial release of EndoG. Blocking VDAC with a VDAC antibody largely abolished mitochondrial localization of BNIP3 and prevented EndoG release. Together, the data identify VDAC as an interacting partner of BNIP3 and support endonuclease G as a mediator of the BNIP3 pathway.
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BNIP3 interacting with LC3 triggers excessive mitophagy in delayed neuronal death in stroke.
CNS neuroscience & therapeutics, 2014Co-Authors: Ruoyang Shi, Shenghua Zhu, Spencer B. Gibson, Jiming KongAbstract:SUMMARY Introduction: A basal level of mitophagy is essential in mitochondrial quality control in physiological conditions, while excessive mitophagy contributes to cell death in a number of diseases including ischemic stroke. Signals regulating this process remain unknown. BNIP3, a pro-apoptotic BH3-only protein, has been implicated as a regulator of mitophagy. Aims: Both in vivo and in vitro models of stroke, as well as BNIP3 wild-type and knock out mice were used in this study. Results: We show that BNIP3 and its homologue BNIP3L (NIX) are highly expressed in a "delayed" manner and contribute to delayed neuronal loss following stroke. Deficiency in BNIP3 significantly decreases both neuronal mitophagy and apoptosis but increases nonselective autophagy following ischemic/hypoxic insults. The mitochondria-localized BNIP3 interacts with the autophagosome-localized LC3, suggesting that BNIP3, similar to NIX, functions as a LC3-binding receptor on mitochondria. Although NIX expression is upregulated when BNIP3 is silenced, up-regulation of NIX cannot func- tionally compensate for the loss of BNIP3 in activating excessive mitophagy. Conclusions: NIX primarily regulates basal level of mitophagy in physiological conditions, whereas BNIP3 exclusively activates excessive mitophagy leading to cell death.
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BNIP3 mediates pre‐myelinating oligodendrocyte cell death in hypoxia and ischemia
Journal of neurochemistry, 2013Co-Authors: Teng Guan, Xueping Chen, Qiyan Cai, Jianqin Niu, Lan Xiao, Jiming KongAbstract:Developing oligodendrocytes, collectively termed ‘pre-myelinating oligodendrocytes’ (preOLs), are vulnerable to hypoxic or ischemic insults. The underlying mechanism of this vulnerability remains unclear. Previously, we showed that Bcl-2⁄E1B-19K-interacting protein 3 (BNIP3), a proapoptotic member of the Bcl-2 family proteins, induced neuronal death in a caspase-independent manner in stroke. In this study, we investigated the role of BNIP3 in preOL cell death induced by hypoxia or ischemia. In primary oligodendrocyte progenitor cell (OPC) cultures exposed to oxygen–glucose deprivation, we found that BNIP3 was upregulated and levels of BNIP3 expression correlated with the death of OPCs. Up-regulation of BNIP3 was observed in preOLs in the white matter in a neonatal rat model of stroke. Knockout of BNIP3 significantly reduced death of preOLs in the middle cerebral artery occlusion model in mice. Our results demonstrate a role of BNIP3 in mediating preOLs cell death induced by hypoxia or ischemia, and suggest that BNIP3 may be a new target for protecting oligodendrocytes from death after stroke. Pre-myelinating oligodendrocytes (preOLs) are known to be highly vulnerable to ischemic insults. It remains unclear, however, how preOLs die. This study shows that BNIP3, a proapoptotic member of the Bcl-2 family proteins, is a mediator of hypoxia/ischemia-induced preOLs death. The BNIP3 cell death pathway may therefore be a new target for protecting oligodendrocytes from death after stroke.
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BNIP3 Upregulation and EndoG Translocation in Delayed Neuronal Death in Stroke and in Hypoxia
Stroke, 2007Co-Authors: Zhengfeng Zhang, Surong Zhang, Xuefen Yang, Jiming KongAbstract:Background and Purpose— Delayed neuronal death is a hallmark feature of stroke and the primary target of neuroprotective strategies. Caspase-independent apoptosis pathways are suggested as a mechanism for the delayed neuronal injury. Here we test the hypothesis that one of the caspase-independent apoptosis pathways is activated by BNIP3 and mediated by EndoG. Methods— We performed immunohistochemistry, Western blotting, cell transfection, subcellular fractionation, and RNA interfering to analyze the expression and localization of BNIP3 and EndoG in degenerating neurons in models of stroke and hypoxia. Results— BNIP3 was upregulated in brain neurons in a rat model of stroke and in cultured primary neurons exposed to hypoxia. The expressed BNIP3 was localized to mitochondria. Both forced expression of BNIP3 by plasmid transfection and induced expression of BNIP3 by hypoxia in neurons resulted in mitochondrial release and nuclear translocation of EndoG and neuronal cell death. Knockdown of BNIP3 by RNAi inhi...
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Evidence of oxidative stress-induced BNIP3 expression in amyloid beta neurotoxicity.
Brain research, 2007Co-Authors: Surong Zhang, Zhengfeng Zhang, Garry Sandhu, Xuefen Yang, Jonathan D. Geiger, Jiming KongAbstract:The formation of Aβ and its subsequent deposition in senile plaques are considered to be initial events that lead to a cascade of pathological changes in AD. Mediators of Aβ-induced oxidative stress are known to cause oxidative damage to macromolecules. However, the molecular mechanisms by which Aβ-induced oxidative stress leads to neuronal cell death are not fully understood. Here we show that Aβ-induced oxidative stress activates the pro-death gene BNIP3. Aβ treatment results in mitochondrial dysfunction, accumulation of reactive oxygen species, and subsequent expression of BNIP3 in rat primary cortical neurons. Pretreatment with antioxidants abolished Aβ-induced BNIP3 expression and attenuated cell death, demonstrating the role of oxidative stress in BNIP3 induction. Aβ-induced BNIP3 expression may be mediated by hypoxia-inducible factor-1 (HIF-1) because Aβ-treatment induced accumulation and nuclear translocation of HIF-1 and knock-down of HIF-1 by RNAi inhibited BNIP3 expression. Finally, knockdown of BNIP3 reduced Aβ-induced neuronal death. Together, these results suggest a potential pathological role of BNIP3 in the etiology of AD.
G Chinnadurai - One of the best experts on this subject based on the ideXlab platform.
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Overexpression of BH3-Only Protein BNIP3 Leads to Enhanced Tumor Growth.
Genes & cancer, 2010Co-Authors: S Vijayalingam, T Subramanian, Sreeraj G. Pillai, Ramachandran Rashmi, John E. Sagartz, G ChinnaduraiAbstract:BCL-2/E1B-19 kDa–interacting protein 3 (BNIP3) is a BH3-only mitochondrial protein. Expression of BNIP3 is strongly stimulated by hypoxia. Up-regulation of BNIP3 has been detected in several human carcinomas including carcinomas of the lung and breast. The significance of BNIP3 overexpression in these cancers is not known. To determine whether BNIP3 plays a role in tumor growth, we generated A549 lung carcinoma cells that overexpressed BNIP3 and examined their ability to form tumors in the mouse xenograft model. All cell lines that overexpressed BNIP3 formed larger tumors compared to the parental or vector-transformed A549 cells. Breast carcinoma cell lines that overexpressed BNIP3 also induced tumors in athymic mice in the absence of hormone administration, while the parental cell line did not. Stable shRNA-mediated knockdown of endogenous BNIP3 severely impaired the tumorigenic activity of A549 cells. The tumor growth-enhancing activity was reduced by deletion of the BH3 domain of BNIP3. Expression of a dominant-negative mutant of BNIP3 lacking the C-terminal transmembrane domain also inhibited the tumorigenic potential of A549 cells. These results suggest that BNIP3 plays a fundamental role in the development of certain solid tumors such as the lung and breast carcinomas.
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BNIP3 subfamily BH3-only proteins: mitochondrial stress sensors in normal and pathological functions.
Oncogene, 2008Co-Authors: G Chinnadurai, S Vijayalingam, S B GibsonAbstract:The BNIP3 subfamily of BH3-only proteins consists of BNIP3 and BNIP3-like (BNIP3L) proteins. These proteins form stable homodimerization complexes that localize to the outer membrane of the mitochondria after cellular stress. This promotes either apoptotic or non-apoptotic cell death such as autophagic cell death. Although the mammalian cells contain both members of this subfamily, the genome of Caenorhabditis elegans codes for a single BNIP3 ortholog, ceBNIP3, which shares homology in the transmembrane (TM) domain and in a conserved region close to the BH3 domain of mammalian BNIP3 protein. The cell death activities of BNIP3 and BNIP3L are determined by either the BH3 domain or the C-terminal TM domain. The TM domain of BNIP3 is unique, as it is capable of autonomous stable dimerization and contributes to mitochondrial localization of BNIP3. In knockout mouse models, BNIP3L was shown to be essential for normal erythrocyte differentiation and hematopoietic homeostasis, whereas BNIP3 plays a role in cellular responses to ischemia/reperfusion injury in the heart. Both BNIP3 and BNIP3L play a role in cellular responses to stress. Under hypoxia, both BNIP3 and BNIP3L expression levels are elevated and contribute to hypoxia-induced cell death. In addition, these proteins play critical roles in disease states. In heart disease, both BNIP3 and BNIP3L play a critical role in cardiomyocyte cell death following ischemic and non-ischemic injuries. In cancer, expression of BNIP3 and BNIP3L is downregulated by promoter hypermethylation or by homozygous deletion of the gene locus in certain cancers, whereas their expression was increased in other cancers. In addition, BNIP3 expression has been correlated with poor prognosis in some cancers. The results reviewed here suggest that BNIP3 and BNIP3L may be novel therapeutic targets for intervention because of their pathological roles in regulating cell death in disease states.
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BNIP3α A Human Homolog of Mitochondrial Proapoptotic Protein BNIP3
Cancer research, 1999Co-Authors: Motoaki Yasuda, Jia-wen Han, Cheryl A. Dionne, Janice M. Boyd, G ChinnaduraiAbstract:Apoptosis is regulated by interaction of viral and cellular BCL-2 family antiapoptotic proteins with various pro-apoptotic proteins, several of which are also members of the BCL-2 family. Cellular protein BNIP3 is a BCL-2 family proapoptotic protein that interacts with viral antiapoptosis proteins such as adenoviruses E1B-19K and EBV-BHRF1 and cellular antiapoptosis proteins such as BCL-2 and BCL-x L . Database searches indicate that the human genome encodes an open reading frame for a protein, BNIP3α, that shares substantial homology with BNIP3. The BNIP3α open reading frame encodes a protein of 219 amino acids that contains a conserved BH3 domain and a COOH-terminal trans- membrane domain, characteristic of several BCL-2 family proapoptotic proteins. BNIP3α interacts with viral antiapoptosis protein E1B-19K and cellular antiapoptosis proteins BCL-2 and BCL-x L . Overexpression of BNIP3α in transfected cells results in apoptosis and suppresses the antiapoptosis activity of E1B-19K and BCL-x L . Like BNIP3, BNIP3α seems to be predominantly localized in mitochondria. These results suggest that BNIP3α is a structural and functional homologue of BNIP3. BNIP3 and BNIP3α seem to be the first examples of homologues among the various human proapoptotic proteins. Northern blot analysis reveals that BNIP3α is expressed ubiquitously in most human tissues. In contrast, BNIP3 is expressed well in several human tissues and less abundantly in certain tissues such as placenta and lung. These results suggest that although BNIP3 and BNIP3α may promote apoptosis simultaneously in most human tissues, BNIP3α may play a more universal role.
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adenovirus e1b 19k bcl 2 interacting protein BNIP3 contains a bh3 domain and a mitochondrial targeting sequence
Journal of Biological Chemistry, 1998Co-Authors: Motoaki Yasuda, Paul Theodorakis, T Subramanian, G ChinnaduraiAbstract:Abstract Adenovirus E1B-19K and BCL-2 anti-apoptosis proteins interact with certain BCL-2 family pro-apoptotic proteins. A conserved domain, BH3, present in these proteins is essential for their pro-apoptotic activity and for heterodimerization with anti-apoptosis proteins. Cellular protein BNIP3 (previously NIP3) interacts with E1B-19K, BCL-2, BCL-xL, and EBV-BHRF1. BNIP3 contains a motif similar to the BH3 domain. Deletion of the BH3-like motif in BNIP3 abrogates its ability to heterodimerize with E1B-19K and BCL-xL. Substitution of the BH3 domain of BNIP3 for the corresponding sequences of BAX functionally restores the pro-apoptotic and protein heterodimerization activities of BAX. BNIP3 exhibits a delayed cell death activity that is partially relieved by deletion of the BH3 domain. BNIP3 suppresses the anti-apoptosis activity of BCL-xL in a BH3-dependent manner. BNIP3 contains a C-terminal trans-membrane (TM) domain similar to other BCL-2 family proteins and BNIP1 (previously NIP1). The TM domains of BNIP3 and BNIP1 can functionally substitute for the TM domain of a BCL-2 family member EBV-BHRF1. The BNIP3 TM domain exclusively targets the heterologous green fluorescent protein (GFP) to mitochondria. These results suggest that BNIP3 is a member of the BH3-contaning BCL-2 family of pro-apoptotic proteins and functions in mitochondria.