微管相关蛋白4在缺氧心肌细胞线粒体通透性转换孔开放和凋亡中的作用及机制研究
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摘要
细胞缺氧是严重烧伤最常见的病理生理现象之一。严重烧伤早期即存在器质性心肌损害,线粒体是心肌细胞缺氧损害的核心靶细胞器,线粒体通透性转换孔(MPTP)开放是启动线粒体途径凋亡的关键步骤,但其具体调节机制目前还不清楚。电压依赖的阴离子通道( VDAC )作为MPTP相关蛋白之一,可以调控MPTP的开放。有研究表明微管相关蛋白(microtubule associated proteins,MAPs)中的MAP2和Tau蛋白可以与线粒体外膜上的VDAC相互作用。
我们推测微管相关蛋白4(MAP4)在缺氧引起MPTP开放和细胞凋亡中发挥了重要作用。为验证这一设想,本研究利用体外培养的缺氧心肌细胞模型,通过构建MAP4野生型和磷酸化位点突变型重组腺病毒载体和MAP4 RNA干扰重组腺病毒载体,探讨MAP4在缺氧引起心肌细胞MPTP开放和凋亡中的作用和机制。
一、研究目的:
探讨微管相关蛋白4在缺氧心肌细胞线粒体通透性转换孔开放和凋亡中的作用和可能的机制。
二、材料方法:
1.将分离培养的乳鼠心肌细胞在94% N2,5% CO_2,1% O_2的混合气体中培养,制作缺氧模型。
2.提取大鼠心肌组织和心肌细胞线粒体蛋白,Western-blot检测缺氧前后MAP4表达变化。
3.免疫共沉淀和免疫荧光共定位检测MAP4和VDAC相互作用。
4.构建MAP4磷酸化位点突变(MAP4(Glu))和MAP4 SiRNA重组腺病毒载体,包装产生重组腺病毒。
5.提取心肌细胞胞质溶胶蛋白,Western-blot检测细胞色素C含量变化,用四甲基罗丹明乙酯(TMRE)检测线粒体膜电位,使用TUNEL染色检测心肌细胞凋亡情况。
三、主要结果
1.正常培养的大鼠乳鼠心肌细胞和成年大鼠心肌组织线粒体中均存在MAP4蛋白表达,而且在缺氧处理后MAP4蛋白表达显著增加。
2. MAP4可以和VDAC结合,同时MAP4和VDAC在心肌细胞的分布存在良好的共定位,而且在缺氧30min处理后二者结合明显增加。
3.常氧培养心肌细胞线粒体膜电位较高,其线粒体荧光强度较强。缺氧30min时线粒体膜电位显著降低,表现为线粒体荧光强度减弱。并且随着缺氧时间延长,线粒体膜电位进一步降低,线粒体荧光强度逐渐减弱。MAP4高表达可以减轻缺氧引起的心肌细胞线粒体膜电位降低,而MAP4 RNA干扰或高表达MAP4(Glu)均可引起心肌细胞线粒体膜电位降低。
4.常氧培养心肌细胞胞质溶胶蛋白细胞色素C含量较低,缺氧30min时细胞色素
C含量即较对照组显著增高,并且随着缺氧时间延长,细胞色素C含量进一步增高。MAP4高表达可以减轻缺氧引起的心肌细胞胞质溶胶细胞色素C含量增高,而MAP4 RNA干扰或高表达MAP4(Glu)均可引起心肌细胞胞质溶胶细胞色素C含量增高。
5.常氧培养心肌细胞只有极少数心肌细胞出现凋亡,缺氧处理后,心肌细胞凋亡比例显著升高,缺氧3小时后,心肌细胞凋亡比例较正常组显著升高,随着缺氧时间延长,心肌细胞凋亡比例逐渐升高。MAP4高表达可以减轻缺氧引起的心肌细胞凋亡,而MAP4 RNA干扰或高表达MAP4(Glu)可以引起心肌细胞凋亡增加。
四、讨论与结论
1.本研究首次发现心肌细胞线粒体存在MAP4蛋白的表达,缺氧处理后线粒体MAP4蛋白表达增加。
2. MAP4与心肌细胞线粒体外膜蛋白VDAC存在相互作用,缺氧时二者相互作用增强。
3.缺氧早期心肌细胞MAP4磷酸化增加,线粒体膜电位下降,胞质溶胶细胞色素C含量显著增加,心肌细胞凋亡显著增加。
4.高表达MAP4蛋白能够减轻缺氧引起的心肌细胞MPTP开放和心肌细胞凋亡,而抑制MAP4蛋白表达和高表达磷酸化位点突变MAP4蛋白(MAP4(Glu))均可以引起MPTP开放、心肌细胞凋亡增加。MAP4在缺氧所致的心肌细胞MPTP开放和凋亡中发挥了重要作用,而缺氧导致MAP4磷酸化增加是其重要机制。
5. MAP4在缺氧心肌细胞凋亡调控中发挥了重要作用,其机制可能是缺氧时MAP4磷酸化增加,MAP4和VDAC相互作用增强,引起MPTP开放,从而导致了心肌细胞凋亡增加。
Cellular hypoxia is one of the most common pathologic phenomenon in severe burns. Cardiac damage exists in the early stage of severe burns. Mitochondria dysfunction in hypoxia plays an important role in the cardiac damage. Mitochondrial permeability transition pore (MPTP) openning is the key process of apoptosis, but its mechanism is still unclear. Voltage-dependent anion channel(VDAC) is a component protein of MPTP in mitochondrial outer membrane, which can modulate MPTP. Studies showed that MAP2 and Tau, which are the member of microtubule associated protein,can interact with VDAC.
We presumed that MAP4 has an important role in modulating hypoxia-induced MPTP opening and apoptosis .. In this study, neonatal rat cadiocytes cultured in the hypoxic gas were employed as the hypoxic model. We construced wild-type and phosphorylation sites mutation as well as MAP4 RNA interference recombination adaenovirus to investigate the effects and mechanisms of MAP4 in hypoxia-induced MPTP openning and cardiocyte apoptosis.
Objectives
To investigate the mechanism of microtubule-associated protein 4 on the opening of mitochondrial permeability transition pore and apoptosis in hypoxic cardiomyocytes.
Methods
1. Neonatal rat cadiocytes were isolated, then, cultured in the mixed gas (94%N_2,5%CO_2,1% O_2) to employ the hypoxic model.
2. Rat myocard tissues and cadiocytes mitochondrial proteins were obtained after hypoxia treatment, and the expression of MAP4 protein was assayed using western- blot, normal myocard tissues and cadiocytes were used as control.
3. Interactions between MAP4 and VDAC were evaluated by co-immunoprecipitation and immunofluorence co-localization.
4. We construced recombination plasmids of MAP4(Glu) and MAP4 SiRNA, and packaged recombinant adenovirus.
5. Cadiocytes cytosol protein were obtained and cytochrome C content was evaluated by western-blot. The mitochondrial membrane potential was tested by TMRE and the apoptosis of cadiocytes were detected by TUNEL.
Results
1. MAP4 protein expresses in the mitochondria of both the neonatal rat cadiocytes and the adult rat myocardial tissues, and it increased after hypoxia treatement.
2. MAP4 can bind to VDAC and they have well co-localization in cardiocyte, the binding of MAP4 to VDAC enhanced after 30-min hypoxia.
3. Mitochondrial inner membrane potential loss was observed after 30-min hypoxia, and showed a dramatic reduction in TMRE fluorescence intensity. As the hypoxia time prolong, the decrease became more and more significant. MAP4 overexpression was founded to be effectively protective for mitochondrial membrane potential loss induced by hypoxia. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced mitochondrial inner membrane potential loss.
4. Normal cadiocytes cytosol cytochrome C content was very low. However, cytosol cytochrome C was increased significant after 30-min hypoxia. With the hypoxia time prolong,the increase became more and more significant. MAP4 Overexpressed cadiocytes were protected from hypoxia-induced cytochrome C release from mitochondria. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced cytochrome c release from mitochondria of cadiocytes.
5. Very little cadiocytes aptosis were observed under normal condition. After 3 h hypoxia treatment, cell death was increased significantly. As the hypoxia time prolong, the increase became more and more significant. MAP4 overexpressed cadiocytes were protected from hypoxia-induced cell death. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced incease of cadiocyte apoptosis.
Discussion and Conclusions
1. We first found that MAP4 protein expresses in the mitochondria of cadiocytes,and the expression of MAP4 increased after hypoxia.
2. MAP4 can bind to VDAC, furthermore 30-min hypoxia enhances the binding between them.
3. MAP4 phosphorylation, mitochondrial membrane potential lost, cytochrome C in cytosol increased and cardiocyte apoptosis were abserved in the early stage of hypoxia.
4. MAP4 overexpression cadiocytes were protected from hypoxia-induced MPTP opening and cadiocyte apoptosis. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced MPTP opening and cadiocyte apoptosis. These results demonstrate that MAP4 has an important role in modulating hypoxia-induced MPTP opening and apoptosis, and MAP4 phosphorylation is the key mechanism of this signal pathway.
5. The data presented in this study are consistent with a model in which MAP4 interacts with VDAC proteins to induce MPTP openning and ultimately, cardiocyte apoptosis. Hypoxia induced MAP4 phosphorylation which enhances interact between MAP4 and VDAC. These results demonstrate that MAP4 has an important role in modulating hypoxia-induced cardiocyte apoptosis.
我们推测微管相关蛋白4(MAP4)在缺氧引起MPTP开放和细胞凋亡中发挥了重要作用。为验证这一设想,本研究利用体外培养的缺氧心肌细胞模型,通过构建MAP4野生型和磷酸化位点突变型重组腺病毒载体和MAP4 RNA干扰重组腺病毒载体,探讨MAP4在缺氧引起心肌细胞MPTP开放和凋亡中的作用和机制。
一、研究目的:
探讨微管相关蛋白4在缺氧心肌细胞线粒体通透性转换孔开放和凋亡中的作用和可能的机制。
二、材料方法:
1.将分离培养的乳鼠心肌细胞在94% N2,5% CO_2,1% O_2的混合气体中培养,制作缺氧模型。
2.提取大鼠心肌组织和心肌细胞线粒体蛋白,Western-blot检测缺氧前后MAP4表达变化。
3.免疫共沉淀和免疫荧光共定位检测MAP4和VDAC相互作用。
4.构建MAP4磷酸化位点突变(MAP4(Glu))和MAP4 SiRNA重组腺病毒载体,包装产生重组腺病毒。
5.提取心肌细胞胞质溶胶蛋白,Western-blot检测细胞色素C含量变化,用四甲基罗丹明乙酯(TMRE)检测线粒体膜电位,使用TUNEL染色检测心肌细胞凋亡情况。
三、主要结果
1.正常培养的大鼠乳鼠心肌细胞和成年大鼠心肌组织线粒体中均存在MAP4蛋白表达,而且在缺氧处理后MAP4蛋白表达显著增加。
2. MAP4可以和VDAC结合,同时MAP4和VDAC在心肌细胞的分布存在良好的共定位,而且在缺氧30min处理后二者结合明显增加。
3.常氧培养心肌细胞线粒体膜电位较高,其线粒体荧光强度较强。缺氧30min时线粒体膜电位显著降低,表现为线粒体荧光强度减弱。并且随着缺氧时间延长,线粒体膜电位进一步降低,线粒体荧光强度逐渐减弱。MAP4高表达可以减轻缺氧引起的心肌细胞线粒体膜电位降低,而MAP4 RNA干扰或高表达MAP4(Glu)均可引起心肌细胞线粒体膜电位降低。
4.常氧培养心肌细胞胞质溶胶蛋白细胞色素C含量较低,缺氧30min时细胞色素
C含量即较对照组显著增高,并且随着缺氧时间延长,细胞色素C含量进一步增高。MAP4高表达可以减轻缺氧引起的心肌细胞胞质溶胶细胞色素C含量增高,而MAP4 RNA干扰或高表达MAP4(Glu)均可引起心肌细胞胞质溶胶细胞色素C含量增高。
5.常氧培养心肌细胞只有极少数心肌细胞出现凋亡,缺氧处理后,心肌细胞凋亡比例显著升高,缺氧3小时后,心肌细胞凋亡比例较正常组显著升高,随着缺氧时间延长,心肌细胞凋亡比例逐渐升高。MAP4高表达可以减轻缺氧引起的心肌细胞凋亡,而MAP4 RNA干扰或高表达MAP4(Glu)可以引起心肌细胞凋亡增加。
四、讨论与结论
1.本研究首次发现心肌细胞线粒体存在MAP4蛋白的表达,缺氧处理后线粒体MAP4蛋白表达增加。
2. MAP4与心肌细胞线粒体外膜蛋白VDAC存在相互作用,缺氧时二者相互作用增强。
3.缺氧早期心肌细胞MAP4磷酸化增加,线粒体膜电位下降,胞质溶胶细胞色素C含量显著增加,心肌细胞凋亡显著增加。
4.高表达MAP4蛋白能够减轻缺氧引起的心肌细胞MPTP开放和心肌细胞凋亡,而抑制MAP4蛋白表达和高表达磷酸化位点突变MAP4蛋白(MAP4(Glu))均可以引起MPTP开放、心肌细胞凋亡增加。MAP4在缺氧所致的心肌细胞MPTP开放和凋亡中发挥了重要作用,而缺氧导致MAP4磷酸化增加是其重要机制。
5. MAP4在缺氧心肌细胞凋亡调控中发挥了重要作用,其机制可能是缺氧时MAP4磷酸化增加,MAP4和VDAC相互作用增强,引起MPTP开放,从而导致了心肌细胞凋亡增加。
Cellular hypoxia is one of the most common pathologic phenomenon in severe burns. Cardiac damage exists in the early stage of severe burns. Mitochondria dysfunction in hypoxia plays an important role in the cardiac damage. Mitochondrial permeability transition pore (MPTP) openning is the key process of apoptosis, but its mechanism is still unclear. Voltage-dependent anion channel(VDAC) is a component protein of MPTP in mitochondrial outer membrane, which can modulate MPTP. Studies showed that MAP2 and Tau, which are the member of microtubule associated protein,can interact with VDAC.
We presumed that MAP4 has an important role in modulating hypoxia-induced MPTP opening and apoptosis .. In this study, neonatal rat cadiocytes cultured in the hypoxic gas were employed as the hypoxic model. We construced wild-type and phosphorylation sites mutation as well as MAP4 RNA interference recombination adaenovirus to investigate the effects and mechanisms of MAP4 in hypoxia-induced MPTP openning and cardiocyte apoptosis.
Objectives
To investigate the mechanism of microtubule-associated protein 4 on the opening of mitochondrial permeability transition pore and apoptosis in hypoxic cardiomyocytes.
Methods
1. Neonatal rat cadiocytes were isolated, then, cultured in the mixed gas (94%N_2,5%CO_2,1% O_2) to employ the hypoxic model.
2. Rat myocard tissues and cadiocytes mitochondrial proteins were obtained after hypoxia treatment, and the expression of MAP4 protein was assayed using western- blot, normal myocard tissues and cadiocytes were used as control.
3. Interactions between MAP4 and VDAC were evaluated by co-immunoprecipitation and immunofluorence co-localization.
4. We construced recombination plasmids of MAP4(Glu) and MAP4 SiRNA, and packaged recombinant adenovirus.
5. Cadiocytes cytosol protein were obtained and cytochrome C content was evaluated by western-blot. The mitochondrial membrane potential was tested by TMRE and the apoptosis of cadiocytes were detected by TUNEL.
Results
1. MAP4 protein expresses in the mitochondria of both the neonatal rat cadiocytes and the adult rat myocardial tissues, and it increased after hypoxia treatement.
2. MAP4 can bind to VDAC and they have well co-localization in cardiocyte, the binding of MAP4 to VDAC enhanced after 30-min hypoxia.
3. Mitochondrial inner membrane potential loss was observed after 30-min hypoxia, and showed a dramatic reduction in TMRE fluorescence intensity. As the hypoxia time prolong, the decrease became more and more significant. MAP4 overexpression was founded to be effectively protective for mitochondrial membrane potential loss induced by hypoxia. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced mitochondrial inner membrane potential loss.
4. Normal cadiocytes cytosol cytochrome C content was very low. However, cytosol cytochrome C was increased significant after 30-min hypoxia. With the hypoxia time prolong,the increase became more and more significant. MAP4 Overexpressed cadiocytes were protected from hypoxia-induced cytochrome C release from mitochondria. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced cytochrome c release from mitochondria of cadiocytes.
5. Very little cadiocytes aptosis were observed under normal condition. After 3 h hypoxia treatment, cell death was increased significantly. As the hypoxia time prolong, the increase became more and more significant. MAP4 overexpressed cadiocytes were protected from hypoxia-induced cell death. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced incease of cadiocyte apoptosis.
Discussion and Conclusions
1. We first found that MAP4 protein expresses in the mitochondria of cadiocytes,and the expression of MAP4 increased after hypoxia.
2. MAP4 can bind to VDAC, furthermore 30-min hypoxia enhances the binding between them.
3. MAP4 phosphorylation, mitochondrial membrane potential lost, cytochrome C in cytosol increased and cardiocyte apoptosis were abserved in the early stage of hypoxia.
4. MAP4 overexpression cadiocytes were protected from hypoxia-induced MPTP opening and cadiocyte apoptosis. However, knock down of MAP4 or overexpression of MAP4(Glu) both induced MPTP opening and cadiocyte apoptosis. These results demonstrate that MAP4 has an important role in modulating hypoxia-induced MPTP opening and apoptosis, and MAP4 phosphorylation is the key mechanism of this signal pathway.
5. The data presented in this study are consistent with a model in which MAP4 interacts with VDAC proteins to induce MPTP openning and ultimately, cardiocyte apoptosis. Hypoxia induced MAP4 phosphorylation which enhances interact between MAP4 and VDAC. These results demonstrate that MAP4 has an important role in modulating hypoxia-induced cardiocyte apoptosis.
引文
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36. Yoshida H. Apaf - 1 is required for mitochondrial pat hways of apoptosis and brain development. Cell ,2002 ,94 :739-750.
37. Li Jia ,Srinivasa M. Srinivasula ,Feng - Ting Liu ,et al . Apaf– 1 protein deficiency confers resistance to cytochrome c - depend2 ent apoptosis in human leukemia cells. Blood , 2001 , 98 :414-421.
38. Miho K, Kiyotaka T, Hiromu M, et al. Functional analysis of microtubule-binding domain of bovine MAP4. 1999 Cell Stru Func; 24: 337-344.
39. Chang W,Gruber D, Chari S, et al. Phosphorylation of MAP4 affects microtubule properties and cell cycle progression. J Cell Sci. 2001, 114:2879-2887.
40. Kitazawa H, Iida J, Uchida A, et al. Ser787 in the proline-rich region of human MAP4 is a critical phosphorylation site that reduces its activity to promote tubulin polymerization. Cell Struct Funct. 2000, 25:33-39.
41. Egelhoff TT, Lee RJ, Spudich JA. Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo. Cell. 1993, 75:363-371.
42. Fischer D, Mukrasch MD, Biernat J, et al. Conformational changes specific for pseudophosphorylation at serine 262 selectively impair binding of tau to microtubules. Biochemistry. 2009, 48:10047-10055.
43. Léger J, Kempf M, Lee G, et al. Conversion of serine to aspartate imitates phosphorylation-induced changes in the structure and function of microtubule-associated protein tau. J Biol Chem. 1997, 272:8441-8446.
1. Newmeyer DD, Farschon DM, Reed JC.Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell. 1994,21;79(2):353-364.
2. Green DR , Reed JC. Mitochondria and apoptosis . Science, 1998, 281: 1309- 1312.
3. Desagher S, Martinou JC. Mitochondria as the central control point of apoptosis. Trends Cell Biol . 2000, 10:369–377.
4. Tsujimoto Y. Cell death regulation by the Bcl-2 protein family in the mitochondria. J Cell Physiol. 2003, 195:158–167.
5. Kroemer G, Petit P, Zamzami N, et al. The biochemistry of programmed cell death. FASEB J. 1995, 9:1277–1287.
6. Yang J, Wang JH, Zhu SS, et al. C-reactive protein augments hypoxia-induced apoptosis through mitochondrion-dependent pathway in cardiac myocytes. Mol Cell Biochem. 2008, 310: 215-226.
7. Di Lisa F, Canton M, MenabòR, et al. Mitochondria and cardioprotection. Heart Fail Rev. 2007, 12:249–260.
8. Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion—a target for cardioprotection. Cardiovasc Res. 2004, 61:372–385.
9. Kroemer G. The mitochondrial permeability transition pore complex as a pharmacological target. An introduction. Curr Med Chem. 2003, 10:1469–1472.
10. Kroemer G, Galluzzi L, Brenner C. Mitochondrial permeabilization in cell death. PhysiolRev. 2007, 87:99–163.
11. Leung AW, Halestrap AP. Recent progress in elucidating the molecular mechanism of the mitochondrial permeability transition pore. Biochim Biophys Acta. 2008, 1777:946–952.
12. Weiss JN, Korge P, Honda HM, et al. Role of the mitochondrial permeability transition in myocardial disease. Circ Res. 2003, 93:292–301.
13. Crompton M, Costi A. A heart mitochondrial Ca2+-dependent pore of possible relevance to re-perfusion-induced injury—evidence that ADP facilitates pore interconversion between the closed and open states. Biochem J. 1990, 266:33-39.
14. Haworth RA, Hunter DR. The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch Biochem Biophys. 1979, 195:460-467.
15. Rostovtseva TK,Tan W, Colombini M. On the role of VDAC in apoptosis: fact and fiction. J Bioenerg Biomembr. 2005, 37:129–142.
16. Blachly-Dyson E, Forte M. VDAC channels. IUBMB Life. 2001, 52:113–118.
17. Szabo I, Zoratti M. The mitochondrial permeability transition pore may comprise VDAC molecules. I. Binary structure and voltage dependence of the pore. FEBS Lett. 1993, 330:201–205.
18. Szabo I, De Pinto V, Zoratti M. The mitochondrial permeability transition pore may comprise VDAC molecules. II. The electrophysiological properties of VDAC are compatible with those of the mitochondrial megachannel. FEBS Lett. 1993, 330: 206–210.
19. Zizi M, Forte M, Blachly-Dyson E, et al. NADH regulates the gating of VDAC, the mitochondrial outer membrane channel. J Biol Chem. 1994, 269:1614–1616.
20. Gincel D, Zaid H, Shoshan-Barmatz V. Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function. Biochem J. 2001, 358:147–155.
21. Gincel D, Shoshan-Barmatz V. Glutamate interacts with VDAC and modulates opening of the mitochondrial permeability transition pore. J Bioenerg Biomembr. 2004, 36:179–186.
22. Pastorino JG, Shulga N, Hoek JB. Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis. J Biol Chem. 2002, 277: 7610– 7618.
23. Pastorino JG, Hoek JB, Shulga N. Activation of glycogen synthase kinase 3beta disrupts the binding of hexokinase II to mitochondria by phosphorylating voltage-dependent anion channel and potentiates chemotherapy-induced cytotoxicity. Cancer Res. 2005, 65:10545–10554.
24. Costantini P, Chernyak BV, Petronilli V, et al. Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. J Biol Chem. 1996, 271:6746–6751.
25. Fontaine E, Eriksson O, Ichas F, et al. Regulation of the permeability transition pore in skeletal muscle mitochondria. ModulationBy electron flowthrough the respiratory chain complex i. J Biol Chem. 1998, 273:12662–12668.
26. Pastorino JG, Hoek JB. Hexokinase II: the integration of energy metabolism and control of apoptosis. Curr Med Chem. 2003, 10:1535–1551.
27. Shimizu S, Narita M , Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channelVDAC. Nature. 1999, 399: 483-487.
28. Crompton M, Virji S, Ward JM. Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore. Eur J Biochem. 1998, 258:729–735.
29. Cesura AM, Pinard E, Schubenel R, et al. The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore. J Biol Chem. 2003, 278:49812–49818.
30. Zheng Y, Shi Y, Tian C, et al. Essential role of the voltage-dependent anion channel (VDAC) in mitochondrial permeability transition pore opening and cytochrome c release induced by arsenic trioxide. Oncogene. 2004, 23:1239–1247.
31. Krauskopf A, Eriksson O, Craigen WJ, et al. Properties of the permeability transition in VDAC1?/? mitochondria. Biochim Biophys Acta. 2006, 1757: 590– 595.
32. Baines CP, Kaiser RA, Sheiko T, et al. Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. Nat Cell Biol. 2007, 9: 550– 555.
33. 24. Fiore C, Trézéguet V, Le Saux A, et al. The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects. Biochimie. 1998, 80:137–150.
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36. Yoshida H. Apaf - 1 is required for mitochondrial pat hways of apoptosis and brain development. Cell ,2002 ,94 :739-750.
37. Li Jia ,Srinivasa M. Srinivasula ,Feng - Ting Liu ,et al . Apaf– 1 protein deficiency confers resistance to cytochrome c - depend2 ent apoptosis in human leukemia cells. Blood , 2001 , 98 :414-421.
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39. Chang W,Gruber D, Chari S, et al. Phosphorylation of MAP4 affects microtubule properties and cell cycle progression. J Cell Sci. 2001, 114:2879-2887.
40. Kitazawa H, Iida J, Uchida A, et al. Ser787 in the proline-rich region of human MAP4 is a critical phosphorylation site that reduces its activity to promote tubulin polymerization. Cell Struct Funct. 2000, 25:33-39.
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42. Fischer D, Mukrasch MD, Biernat J, et al. Conformational changes specific for pseudophosphorylation at serine 262 selectively impair binding of tau to microtubules. Biochemistry. 2009, 48:10047-10055.
43. Léger J, Kempf M, Lee G, et al. Conversion of serine to aspartate imitates phosphorylation-induced changes in the structure and function of microtubule-associated protein tau. J Biol Chem. 1997, 272:8441-8446.
1. Newmeyer DD, Farschon DM, Reed JC.Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell. 1994,21;79(2):353-364.
2. Green DR , Reed JC. Mitochondria and apoptosis . Science, 1998, 281: 1309- 1312.
3. Desagher S, Martinou JC. Mitochondria as the central control point of apoptosis. Trends Cell Biol . 2000, 10:369–377.
4. Tsujimoto Y. Cell death regulation by the Bcl-2 protein family in the mitochondria. J Cell Physiol. 2003, 195:158–167.
5. Kroemer G, Petit P, Zamzami N, et al. The biochemistry of programmed cell death. FASEB J. 1995, 9:1277–1287.
6. Yang J, Wang JH, Zhu SS, et al. C-reactive protein augments hypoxia-induced apoptosis through mitochondrion-dependent pathway in cardiac myocytes. Mol Cell Biochem. 2008, 310: 215-226.
7. Di Lisa F, Canton M, MenabòR, et al. Mitochondria and cardioprotection. Heart Fail Rev. 2007, 12:249–260.
8. Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion—a target for cardioprotection. Cardiovasc Res. 2004, 61:372–385.
9. Kroemer G. The mitochondrial permeability transition pore complex as a pharmacological target. An introduction. Curr Med Chem. 2003, 10:1469–1472.
10. Kroemer G, Galluzzi L, Brenner C. Mitochondrial permeabilization in cell death. PhysiolRev. 2007, 87:99–163.
11. Leung AW, Halestrap AP. Recent progress in elucidating the molecular mechanism of the mitochondrial permeability transition pore. Biochim Biophys Acta. 2008, 1777:946–952.
12. Weiss JN, Korge P, Honda HM, et al. Role of the mitochondrial permeability transition in myocardial disease. Circ Res. 2003, 93:292–301.
13. Crompton M, Costi A. A heart mitochondrial Ca2+-dependent pore of possible relevance to re-perfusion-induced injury—evidence that ADP facilitates pore interconversion between the closed and open states. Biochem J. 1990, 266:33-39.
14. Haworth RA, Hunter DR. The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch Biochem Biophys. 1979, 195:460-467.
15. Rostovtseva TK,Tan W, Colombini M. On the role of VDAC in apoptosis: fact and fiction. J Bioenerg Biomembr. 2005, 37:129–142.
16. Blachly-Dyson E, Forte M. VDAC channels. IUBMB Life. 2001, 52:113–118.
17. Szabo I, Zoratti M. The mitochondrial permeability transition pore may comprise VDAC molecules. I. Binary structure and voltage dependence of the pore. FEBS Lett. 1993, 330:201–205.
18. Szabo I, De Pinto V, Zoratti M. The mitochondrial permeability transition pore may comprise VDAC molecules. II. The electrophysiological properties of VDAC are compatible with those of the mitochondrial megachannel. FEBS Lett. 1993, 330: 206–210.
19. Zizi M, Forte M, Blachly-Dyson E, et al. NADH regulates the gating of VDAC, the mitochondrial outer membrane channel. J Biol Chem. 1994, 269:1614–1616.
20. Gincel D, Zaid H, Shoshan-Barmatz V. Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function. Biochem J. 2001, 358:147–155.
21. Gincel D, Shoshan-Barmatz V. Glutamate interacts with VDAC and modulates opening of the mitochondrial permeability transition pore. J Bioenerg Biomembr. 2004, 36:179–186.
22. Pastorino JG, Shulga N, Hoek JB. Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis. J Biol Chem. 2002, 277: 7610– 7618.
23. Pastorino JG, Hoek JB, Shulga N. Activation of glycogen synthase kinase 3beta disrupts the binding of hexokinase II to mitochondria by phosphorylating voltage-dependent anion channel and potentiates chemotherapy-induced cytotoxicity. Cancer Res. 2005, 65:10545–10554.
24. Costantini P, Chernyak BV, Petronilli V, et al. Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. J Biol Chem. 1996, 271:6746–6751.
25. Fontaine E, Eriksson O, Ichas F, et al. Regulation of the permeability transition pore in skeletal muscle mitochondria. ModulationBy electron flowthrough the respiratory chain complex i. J Biol Chem. 1998, 273:12662–12668.
26. Pastorino JG, Hoek JB. Hexokinase II: the integration of energy metabolism and control of apoptosis. Curr Med Chem. 2003, 10:1535–1551.
27. Shimizu S, Narita M , Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channelVDAC. Nature. 1999, 399: 483-487.
28. Crompton M, Virji S, Ward JM. Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore. Eur J Biochem. 1998, 258:729–735.
29. Cesura AM, Pinard E, Schubenel R, et al. The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore. J Biol Chem. 2003, 278:49812–49818.
30. Zheng Y, Shi Y, Tian C, et al. Essential role of the voltage-dependent anion channel (VDAC) in mitochondrial permeability transition pore opening and cytochrome c release induced by arsenic trioxide. Oncogene. 2004, 23:1239–1247.
31. Krauskopf A, Eriksson O, Craigen WJ, et al. Properties of the permeability transition in VDAC1?/? mitochondria. Biochim Biophys Acta. 2006, 1757: 590– 595.
32. Baines CP, Kaiser RA, Sheiko T, et al. Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. Nat Cell Biol. 2007, 9: 550– 555.
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