细胞凋亡的线粒体途径

1、细胞凋亡的线粒体途径细胞凋亡的线粒体途径 董霞 程在全2002-12-17报告提纲报告提纲 n细胞凋亡的特征损伤性死亡损伤性死亡 Necrosis程序化死亡、细胞凋亡程序化死亡、细胞凋亡Programmed Cell DeathApoptosis 染色质的凝集染色质的凝集, 嗜碱性染色增强嗜碱性染色增强,细胞核崩解此时线粒体保持形态正常细胞核崩解此时线粒体保持形态正常. 细胞体积缩小细胞体积缩小,一部分细胞质和核碎片进入由膜一部分细胞质和核碎片进入由膜包被的程序死亡小体包被的程序死亡小体,他们从细胞表面出芽脱落他们从细胞表面出芽脱落,并被巨噬细胞并被巨噬细胞.上皮细胞吞噬上皮细胞吞噬. 染色质

2、降解染色质降解, 核小体间连接核小体间连接DNA部位被降解部位被降解,产产生寡聚核小体生寡聚核小体DNA片段片段,即即180-200DP 整数倍整数倍的不同长度的的不同长度的DNA片断片断. Fig.1. Schematic summary of biochemical mechanisms of apoptosis.Mitochondria and Commitment to Cell Death n线粒体是真核细胞的重要细胞器，是动物细胞生成ATP的主要地点。线粒体基质的三羧酸循环酶系通过底物脱氢氧化生成NADH。NADH通过线粒体内膜呼吸链氧化。与此同时，导致跨膜质子移位形成跨膜质子梯度

3、和/或跨膜电位。线粒体内膜上的ATP合成酶利用跨膜质子梯度能量合成ATP。合成的ATP通过线粒体内膜ADP/ATP载体与细胞质中ADP交换进入细胞质，参与细胞的各种需能过程。 Mitochondria and Commitment to Cell Death nthe effectors of apoptosis are represented by a family of intracellular cysteine proteases known as caspases. nInhibiting caspases, however, does not always inhibit cell

4、 death induced by proapoptotic stimuli. Although caspase inhibitors block some or all of the apoptotic morphology induced by growth factor withdrawal, etoposide, actinomycin D, ultraviolet (UV) radiation, staurosporine, enforced c-Myc expression, or glucocorticoids, they do not necessarily maintain

5、replicative or clonogenic potential; ultimately, the cells die despite inactivation of caspases by way of a slower, nonapoptotic cell death (6-9). nIn contrast, antiapoptotic proteins such as Bcl-2, Bcl-xL, and oncogenic Abl can maintain survival and clonogenicity in the face of these treatments. Co

6、nversely, some proapoptotic proteins such as Bax, a mammalian cell death protein that targets mitochondrial membranes, can induce mitochondrial damage and cell death even when caspases are inactivated (10). Such experimental observations argue that a caspase-independent mechanism for commitment to d

7、eath exists. This mechanism is likely to involve mitochondria, as we will see. Mitochondrial Pathways in physiological cell death the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reducti

8、on, participation of pro- and antiapoptotic Bcl-2 family proteins. Mitochondrial Pathways in physiological cell death If mitochondria are pivotal in controlling cell life and death, then how do these organelles kill? At least three general mechanisms are known, and their effects may be interrelated,

9、 including(i) disruption of electron transport, oxidative phosphorylation, and adenosine triphosphate (ATP) production;(ii) release of proteins that trigger activation of caspase family proteases; and(iii) alteration of cellular reduction-oxidation (redox) potential Disruption of electron transport

10、and energy metabolism ndisruption of electron transport has been recognized as an early feature of cell death.n -Irradiation induces apoptosis in thymocytes and a disruption in the electron transport chain, probably at the cytochrome b-c1/cytochrome c (cyto c) step.nCeramide (a second messenger impl

11、icated in apoptosis signaling) disrupts electron transport at the same step in cells as well as in isolated mitochondria.n Ligation of Fas also leads to a disruption in cyto c function in electron transport.Disruption of electron transport and energy metabolism nOne consequence of the loss of electr

12、on transport should be a drop in ATP production. Although such a drop has been observed during apoptosis, it often occurs relatively late in the process (14). nIndeed, ATP appears to be required for downstream events in apoptosis (15). nThus, although loss of mitochondrial ATP production can kill a

13、cell, it is unlikely that this is a mechanism for induction of apoptosis. Disruption of electron transport and energy metabolism n线粒体跨膜电位的耗散与细胞凋亡有密切关系线粒体跨膜电位的耗散与细胞凋亡有密切关系n近年来陆续有报道说明线粒体跨膜电位的耗散早于核酸酶的激活，也早于磷酯酰丝氨酸暴露于细胞表面。而一旦线粒体跨膜电位耗散，细胞就会进入不可逆的凋亡过程。线粒体解联的呼吸链会产生大量活性氧，氧化线粒体内膜上的心磷脂。实验证明，用解偶联剂mClCCP会导致淋巴细胞凋

14、亡。而如果能稳定线粒体跨膜电位就能防止细胞凋亡。Release of caspase-activating proteins nThe importance of mitochondria in apoptosis was suggested by studies with a cell-free system in which spontaneous, nBcl-2-inhibitable nuclear condensation and DNA fragmentation were found to be dependent on the presence of mitochondria

15、(16). nSubsequently, studies in another cell-free system showed that induction of caspase activation by addition of deoxyadenosine triphosphate depended on the presence of cyto c released from mitochondria during extract preparation (17). During apoptosis (in vitro and in vivo) cyto c is released fr

16、om mitochondria and this is inhibited by the presence of Bcl-2 on these organelles (18, 19). nCytosolic cyto c forms an essential part of the vertebrate apoptosome, which is composed of cyto c, Apaf-1, and procaspase-9 (20). The result is activation of caspase-9, which then processes and activates o

17、ther caspases to orchestrate the biochemical execution of cells.Release of caspase-activating proteins nSignificantly, caspase inhibitors do not prevent cyto c release induced by several apoptogenic agents, including UV irradiation, staurosporine, and overexpression of Bax (14, 21, 22). An exception

18、 is cyto c release from mitochondria induced by the tumor necrosis factor receptor family member Fas, in which cyto c release is prevented by inhibition of caspases (primarily caspase-8) recruited to the cytosolic domain of ligated Fas (21). Nevertheless, cyto c release can sometimes contribute to F

19、as-mediated apoptosis by amplifying the effects of caspase-8 on activation of downstream caspases (23).nThe emergent view is that once cyto c is released, this commits the cell to die by either a rapid apoptotic mechanism involving Apaf-1-mediated caspase activation or a slower necrotic process due

20、to collapse of electron transport, which occurs when cyto c is depleted from mitochondria, resulting in a variety of deleterious sequelae including generation of oxygen free radicals and decreased production of ATP Reactive oxygen species and cellular redox. nMitochondria are the major source of sup

21、eroxide anion production in cells.nDuring transfer of electrons to molecular oxygen, an estimated 1 to 5% of electrons in the respiratory chain lose their way, most participating in formation of O2. Anything that decreases the coupling efficiency of electron chain transport can therefore increase pr

22、oduction of superoxides. Reactive oxygen species and cellular redox. nSuperoxides and lipid peroxidation are increased during apoptosis induced by myriad stimuli (28). nHowever, generation of ROS may be a relatively late event, occurring after cells have embarked on a process of caspase activation.

23、nIn this regard, attempts to study apoptosis under conditions of anoxia have demonstrated that at least some proapoptotic stimuli function in the absence or near absence of oxygen, which implies that ROSs are not the sine qua non of apoptosis (29, 30).n However, ROSs can be generated under condition

24、s of virtual anaerobiosis (31), and thus their role in apoptosis cannot be excluded solely on this basis. Figure 2. Model for caspase activation by mitochondria.osmotic disequilibrium leading to an expansion of the matrix space, organellar swelling, and subsequent rupture of the outer membranethe ot

25、her envisions opening of channels in the outer membrane thus releasing cyto c from the intermembrane space of mitochondria into the cytosol.PT Pore nIn many apoptosis scenarios, the mitochondrial inner transmembrane potential (m) collapses (32), indicating the opening of a large conductance channel

26、known as the mitochondrial PT pore (33) (Fig. 2). nits constituents include both inner membrane proteins, such as the adenine nucleotide translocator (ANT), and outer membrane proteins, such as porin (voltage-dependent anion channel; VDAC), which operate in concert, presumably at inner and outer mem

27、brane contact sites, and create a channel through which molecules 1.5 kD pass (32, 34). PT Pore nOpening of this nonselective channel in the inner membrane allows for an equilibration of ions within the matrix and intermembrane space of mitochondria, thus dissipating the H+ gradient across the inner

28、 membrane and uncoupling the respiratory chain. nPerhaps more importantly, PT pore opening results in a volume dysregulation of mitochondria due to the hyperosmolality of the matrix, which causes the matrix space to expand. nBecause the inner membrane with its folded cristae possesses a larger surfa

29、ce area than the outer membrane, this matrix volume expansion can eventually cause outer membrane rupture, releasing caspase-activating proteins located within the intermembrane space into the cytosol (Fig. 1).Fig. 3. The mitochondrial permeability transition.PT Pore 的性质的性质 通过一些实验室的研究，以下诸点值得指出：n线粒体内

30、膜通透性转变既是细胞凋亡的必须条件，也是它的充足条件。nPT孔道打开后导致线粒体许多功能的致命性变化从而启动了死亡途径。nPT孔道作为许多生理效应的感受器（二价阳离子、ATP、ADP、NAD、m、pH、巯基与多肽)，整合了电生理、氧化还原与细胞代谢状态的信息。nPT孔道的组成成分ADP-ATP载体是能量代谢的重要分子，由于ADP-ATP载体是由一个基因家族的几个成员所编码，它的表达有严格的组织专一性。因此，PT孔道在不同细胞中的调节可能稍有不同。nPT孔道的作用有自放大的效应。PT诱导m耗散，而反过来 mClCCP使m去极化会导致PT。一些PT的结果例如m 耗散，活性氧的生成本身也会导致PT。

31、这就说明PT会有正反馈， 从而在细胞凋亡中有自摧毁的作用。反过来，如果能防止m的耗散，就能避免氧化还原不平衡、磷酯酰丝氨酸的暴露与蛋白酶和核酸酶的激活。 PT Pore 有开放与关闭二种构象有开放与关闭二种构象nPT孔道开放导致细胞凋亡。n在PT孔道开放时线粒体释放细胞凋亡诱导因子(AIF)。AIF可能是一种蛋白水解酶，位于线粒体膜间间隙，它能被蛋白酶抑制剂如N-苄氧羰基-缬氨酰-丙氨酰-门冬氨酰氟甲基酮(N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone)所抑制。n苍术苷促进PT通道开放。苍术苷只能与ADP-ATP载体的胞液侧结合。n此外从线

32、粒体释放的细胞色素C也是一种细胞凋亡诱导因子。n而PT孔道关闭能防止细胞凋亡。n当PT孔道与环孢菌素A(cyclosporin A)或SH，或米酵菌酸(bongkrek acid)结合时PT孔道被关闭。米酵菌酸可与ADP-ATP载体的胞液及基质二侧结合Figure 2. The mitochondrial permeability transition.nA speculative model showing some of the components of the permeability transition pore. The roles of porin and the benzod

33、iazepine receptor remain circumstantial. In the open configuration, water and solutes enter the matrix, causing matrix swelling and outer membrane disruption (see Fig. 1), leading to release of cyto c and other proteins. Fig. 4 The cytochrome c-induced caspase activation pathway.OligomerizationRecru

34、itCleaveActiviateFigure 1. The cytochrome c-induced caspase activation pathway. nApoptotic stimuli exert their effects on mitochondria to cause the release of cytochrome c. Cytochrome c in turn binds to Apaf-1, a cytosolic protein that normally exists as an inactive monomer. The binding of cytochrom

35、e c induces a conformational change in Apaf-1, allowing it to bind the nucleotide dATP or ATP. The nucleotide binding to the Apaf-1-cytochrome c complex triggers its oligomerization to form the apoptosome, which recruits procaspase-9. The binding of procaspase-9 to the apoptosome forms the caspase-9

36、 holoenzyme that cleaves and activates the downstream caspases, such as caspase-3. Fig. 5. Displacement of IAPs from caspases by Smac/Diablo.Figure 2. Displacement of IAPs from caspases by Smac/Diablo. nThe precursor of Smac/Diablo is synthesized in the cytosol and transported to the mitochondria. A

37、fter mitochondrial entry, the mitochondrial targeting sequence of Smac (dark purple rectangle) is cleaved, exposing the four amino acid residues Ala-Val-Pro-Ile through which Smac binds to the BIR domain of IAPs. The mature Smac (aqua rectangle) is normally located in the mitochondrial intermembrane space. During apoptosis, cytochrome c (light purple circles) and Smac are released from the mitochondria. Cytochrome c triggers the activation of caspase-9 (gre