細胞凋亡的線粒體途徑

1、報告提綱 細胞凋亡的特征細胞死亡損傷性死亡 Necrosis程序化死亡、細胞凋亡Programmed Cell DeathApoptosis細胞凋亡的特征形態(tài)學特征: 染色質(zhì)的凝集, 嗜堿性染色增強,細胞核崩解此時線粒體保持形態(tài)正常. 細胞體積縮小,一部分細胞質(zhì)和核碎片進入由膜包被的程序死亡小體,他們從細胞表面出芽脫落,并被巨噬細胞.上皮細胞吞噬. 生化特征: 染色質(zhì)降解, 核小體間連接DNA部位被降解,產(chǎn)生寡聚核小體DNA片段,即180-200DP 整數(shù)倍的不同長度的DNA片斷. Fig.1. Schematic summary of biochemical mechanisms of ap

2、optosis.Mitochondria and Commitment to Cell Death 線粒體是真核細胞的重要細胞器，是動物細胞生成ATP的主要地點。線粒體基質(zhì)的三羧酸循環(huán)酶系通過底物脫氫氧化生成NADH。NADH通過線粒體內(nèi)膜呼吸鏈氧化。與此同時，導致跨膜質(zhì)子移位形成跨膜質(zhì)子梯度和/或跨膜電位。線粒體內(nèi)膜上的ATP合成酶利用跨膜質(zhì)子梯度能量合成ATP。合成的ATP通過線粒體內(nèi)膜ADP/ATP載體與細胞質(zhì)中ADP交換進入細胞質(zhì)，參與細胞的各種需能過程。 Mitochondria and Commitment to Cell Death the effectors of apopto

3、sis are represented by a family of intracellular cysteine proteases known as caspases. Inhibiting caspases, however, does not always inhibit cell death induced by proapoptotic stimuli. Although caspase inhibitors block some or all of the apoptotic morphology induced by growth factor withdrawal, etop

4、oside, actinomycin D, ultraviolet (UV) radiation, staurosporine, enforced c-Myc expression, or glucocorticoids, they do not necessarily maintain replicative or clonogenic potential; ultimately, the cells die despite inactivation of caspases by way of a slower, nonapoptotic cell death (6-9). In contr

5、ast, antiapoptotic proteins such as Bcl-2, Bcl-xL, and oncogenic Abl can maintain survival and clonogenicity in the face of these treatments. Conversely, some proapoptotic proteins such as Bax, a mammalian cell death protein that targets mitochondrial membranes, can induce mitochondrial damage and c

6、ell death even when caspases are inactivated (10). Such experimental observations argue that a caspase-independent mechanism for commitment to death exists. This mechanism is likely to involve mitochondria, as we will see. Mitochondrial Pathways in physiological cell death the release of caspase act

7、ivators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, participation of pro- and antiapoptotic Bcl-2 family proteins. Mitochondrial Pathways in physiological cell death If mitochondria are pivotal in control

8、ling cell life and death, then how do these organelles kill? At least three general mechanisms are known, and their effects may be interrelated, including(i) disruption of electron transport, oxidative phosphorylation, and adenosine triphosphate (ATP) production;(ii) release of proteins that trigger

9、 activation of caspase family proteases; and(iii) alteration of cellular reduction-oxidation (redox) potential Disruption of electron transport and energy metabolism disruption of electron transport has been recognized as an early feature of cell death. -Irradiation induces apoptosis in thymocytes a

10、nd a disruption in the electron transport chain, probably at the cytochrome b-c1/cytochrome c (cyto c) step.Ceramide (a second messenger implicated in apoptosis signaling) disrupts electron transport at the same step in cells as well as in isolated mitochondria. Ligation of Fas also leads to a disru

11、ption in cyto c function in electron transport.Disruption of electron transport and energy metabolism One consequence of the loss of electron 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). In

12、deed, ATP appears to be required for downstream events in apoptosis (15). Thus, although loss of mitochondrial ATP production can kill a cell, it is unlikely that this is a mechanism for induction of apoptosis. Disruption of electron transport and energy metabolism 線粒體跨膜電位的耗散與細胞凋亡有密切關系近年來陸續(xù)有報道說明線粒體跨

13、膜電位的耗散早于核酸酶的激活，也早于磷酯酰絲氨酸暴露于細胞表面。而一旦線粒體跨膜電位耗散，細胞就會進入不可逆的凋亡過程。線粒體解聯(lián)的呼吸鏈會產(chǎn)生大量活性氧，氧化線粒體內(nèi)膜上的心磷脂。實驗證明，用解偶聯(lián)劑mClCCP會導致淋巴細胞凋亡。而如果能穩(wěn)定線粒體跨膜電位就能防止細胞凋亡。Release of caspase-activating proteins The importance of mitochondria in apoptosis was suggested by studies with a cell-free system in which spontaneous, Bcl-2-i

14、nhibitable nuclear condensation and DNA fragmentation were found to be dependent on the presence of mitochondria (16). Subsequently, 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

15、from mitochondria during extract preparation (17). During apoptosis (in vitro and in vivo) cyto c is released from mitochondria and this is inhibited by the presence of Bcl-2 on these organelles (18, 19). Cytosolic cyto c forms an essential part of the vertebrate apoptosome, which is composed of cyt

16、o c, Apaf-1, and procaspase-9 (20). The result is activation of caspase-9, which then processes and activates other caspases to orchestrate the biochemical execution of cells.Release of caspase-activating proteins Significantly, caspase inhibitors do not prevent cyto c release induced by several apo

17、ptogenic agents, including UV irradiation, staurosporine, and overexpression of Bax (14, 21, 22). An exception 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) rec

18、ruited to the cytosolic domain of ligated Fas (21). Nevertheless, cyto c release can sometimes contribute to Fas-mediated apoptosis by lifying the effects of caspase-8 on activation of downstream caspases (23).The emergent view is that once cyto c is released, this commits the cell to die by either

19、a rapid apoptotic mechanism involving Apaf-1-mediated caspase activation or a slower necrotic process due 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 decreas

20、ed production of ATP Reactive oxygen species and cellular redox. Mitochondria are the major source of superoxide anion production in cells.During transfer of electrons to molecular oxygen, an estimated 1to 5% of electrons in the respiratory chain lose their way, most participating in formation of O2

21、. Anything that decreases the coupling efficiency of electron chain transport can therefore increase production of superoxides. Reactive oxygen species and cellular redox. Superoxides and lipid peroxidation are increased during apoptosis induced by myriad stimuli (28). However, generation of ROS may

22、 be a relatively late event, occurring after cells have embarked on a process of caspase activation. In 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

23、ROSs are not the sine qua non of apoptosis (29, 30). However, ROSs can be generated under conditions 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 exp

24、ansion of the matrix space, organellar swelling, and subsequent rupture of the outer membranethe other envisions opening of channels in the outer membrane thus releasing cyto c from the intermembrane space of mitochondria into the cytosol.PT Pore In many apoptosis scenarios, the mitochondrial inner

25、transmembrane potential (m) collapses (32), indicating the opening of a large conductance channel known as the mitochondrial PT pore (33) (Fig. 2). its constituents include both inner membrane proteins, such as the adenine nucleotide translocator (ANT), and outer membrane proteins, such as porin (vo

26、ltage-dependent anion channel; VDAC), which operate in concert, presumably at inner and outer membrane contact sites, and create a channel through which molecules 1.5 kD pass (32, 34). PT Pore Opening of this nonselective channel in the inner membrane allows for an equilibration of ions within the m

27、atrix and intermembrane space of mitochondria, thus dissipating the H+ gradient across the inner membrane and uncoupling the respiratory chain. Perhaps more importantly, PT pore opening results in a volume dysregulation of mitochondria due to the hyperosmolality of the matrix, which causes the matri

28、x space to expand. Because the inner membrane with its folded cristae possesses a larger surface 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.

29、 1).Fig. 3. The mitochondrial permeability transition.PT Pore 的性質(zhì) 通過一些實驗室的研究，以下諸點值得指出：線粒體內(nèi)膜通透性轉(zhuǎn)變既是細胞凋亡的必須條件，也是它的充足條件。PT孔道打開后導致線粒體許多功能的致命性變化從而啟動了死亡途徑。PT孔道作為許多生理效應的感受器（二價陽離子、ATP、ADP、NAD、m、pH、巰基與多肽)，整合了電生理、氧化還原與細胞代謝狀態(tài)的信息。PT孔道的組成成分ADP-ATP載體是能量代謝的重要分子，由于ADP-ATP載體是由一個基因家族的幾個成員所編碼，它的表達有嚴格的組織專一性。因此，PT孔道在不同細

30、胞中的調(diào)節(jié)可能稍有不同。PT孔道的作用有自放大的效應。PT誘導m耗散，而反過來 mClCCP使m去極化會導致PT。一些PT的結(jié)果例如m 耗散，活性氧的生成本身也會導致PT。這就說明PT會有正反饋， 從而在細胞凋亡中有自摧毀的作用。反過來，如果能防止m的耗散，就能避免氧化還原不平衡、磷酯酰絲氨酸的暴露與蛋白酶和核酸酶的激活。 PT Pore 有開放與關閉二種構(gòu)象PT孔道開放導致細胞凋亡。在PT孔道開放時線粒體釋放細胞凋亡誘導因子(AIF)。AIF可能是一種蛋白水解酶，位于線粒體膜間間隙，它能被蛋白酶抑制劑如N-芐氧羰基-纈氨酰-丙氨酰-門冬氨酰氟甲基酮(N-benzyloxycarbonyl-V

31、al-Ala-Asp-fluoromethylketone)所抑制。蒼術苷促進PT通道開放。蒼術苷只能與ADP-ATP載體的胞液側(cè)結(jié)合。此外從線粒體釋放的細胞色素C也是一種細胞凋亡誘導因子。而PT孔道關閉能防止細胞凋亡。當PT孔道與環(huán)孢菌素A(cyclosporin A)或SH，或米酵菌酸(bongkrek acid)結(jié)合時PT孔道被關閉。米酵菌酸可與ADP-ATP載體的胞液及基質(zhì)二側(cè)結(jié)合Figure 2. The mitochondrial permeability transition.A speculative model showing some of the components o

32、f the permeability transition pore. The roles of porin and the benzodiazepine 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

33、 cytochrome c-induced caspase activation pathway.OligomerizationRecruitCleaveActiviateFigure 1. The cytochrome c-induced caspase activation pathway. Apoptotic stimuli exert their effects on mitochondria to cause the release of cytochrome c. Cytochrome c in turn binds to Apaf-1, a cytosolic protein t

34、hat normally exists as an inactive monomer. The binding of cytochrome 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-

35、9. The binding of procaspase-9 to the apoptosome forms the caspase-9 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. The precursor of Smac/Diablo i

36、s synthesized in the cytosol and transported to the mitochondria. After 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 (green rec