Indian Journal of Experimental Biology Vol. 43, January 2005, pp 25-34

Review Article

Apoptosis: A mitochondrial perspective on cell death

Neerad C Mishra l * & Shailendra Kuma?

IRespiratory Immunology and Asthma Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr. S E, Albuquerque, NM 87108, USA

2V A Boston Healthcare System, West Roxbury, MA 02132, USA; Department of Neurology, Harvard Medical School, Boston, MA 02115, USA; Department of Neurology, Brigham and Women's Hospital, Boston, MA 02115, USA

Mitochondria play an important role in both the life and death of cells. The past 7-8 years have seen an intense surge in research devoted toward understanding the critical role of mitochondria in the regulation of cell death. Mitochondria have, next to their function in respiration, an important role in apoptotic signaling pathway. Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Apoptosis can be initiated by a wide array of stimuli, including mUltiple signaling pathways that, for the most part, converge at the mitochondria. Although classically considered the powerhouses of the cell, it is now understood that mitochondria are also "gatekeepers" that ulti­mately determine the fate of the cell. Malfunctioning at any level of the cell is eventually translated in the release of apop­togenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell . These mitochon­drial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death. In addi­tion, several BcI-2 family members and other upstream proteins also contribute to and regulate the apoptosis. In this review, we attempt to summarize our current view of the mechanism that leads to the influx and efflux of many proteins fromlto mitochondria during apoptosis.

Keywords: c-Abl, AIF, Apafl, Apoptosis, BcI-2, Caspases, Cell death, Cytochrome c, DIABLO, JNK, Mitochondria, PKCo, p53, Smac, Translocation, TR3

Programmed cell death or apoptosis plays an integral role in a variety of biological events, including mor­phogenesis, tissue homeostasis, and removal of un­wanted or harmful cells 1. Apoptosis is a continuous physiologic process and is one of today's most active fields of biomedical research2

-6

. Failure to accurately undergo apoptosis can cause severe anomalies in hu­mans, either due to the accumulation or due to the deficiency of a particular cell type. Abnormal inhibi­tion of apoptosis is a hallmark of cancer and autoim­mune diseases, whereas excessive cell death has been implicated in a number of neurodegenerative disor­ders7

• Extensive studies performed over a decade have revealed a large part of the molecular basis of cell death. It is now apparent that mitochondria are the central regulators of cell death and survival.

Cell death via. apoptosis follows the activation of effector proteases called caspases, which participate in enzymic cascades that terminate in cellular disas­sembll·9• Effector caspases, such as caspase-3 and -7, are activated by initiator caspases, such as caspase-9,

*Corresponding author-Phone: (505) 348-9125 Fax: (505) 348-8567 E-mail: nmishra@Irri.org

through proteolytic cleavage. Once activated, effector caspases are responsible for the proteolytic cleavage of a diverse array of structural and regulatory pro­teins, re!,ulting in an apoptotic phenotype lO

• Death by the terminal pathways of apoptosis is frequently com­pared with death by necrosis, but it is distinguished from necrotic death in that apoptosis is a closely regulated process induced by a specific stimulus, and occurs without the release of inflammatory mediators. Death by necrosis occurs because of failure to control cellular homeostasis after undergoing damage. Al­though apoptosis can be initiated via. a plethora of stimuli - including ultraviolet light, oxidative stress, viruses, chemicals, drugs, cytokines, and ligands. Most of the pathways ultimately converge at the mi­tochondria, which then converts these signals into a pro-apoptotic response. The complex role of mito­chondria came into focus when biochemical studies identified several mitochondrial proteins that are able to activate cellular apoptotic programs directly 1

1-15. In response to apoptotic stimuli, they are released to the cytosol and/or nucleus. They promote apoptosis either by activating caspases and nucleases or by neutraliz­ing cytosolic inhibitors of this process. This review summarizes the current understanding as to how the

26 INDIAN J EXP BIOL, JANUARY 200S

mitochondrion deciphers pro-apoptotic signals and how it responds to these signals to initiate the final executioner stage of apoptosis.

Cell death cascades Mitochondria play an essential role as a power

plant of the cell, providing energy for specialized cell functions. In the context of cell death, they play a central role in apoptotic pathways. Apoptosis is often referred to as caspase-dependent process. Caspases are a family of cysteinyl proteases and are expressed as inactive zymogens (procaspases). Activation of the proenzyme requires autocleavage or cleavage by other caspases at specific aspartate residues. Two pathways leading to caspase activation and proapoptotic cell death have been well characterized. One is initiated from death receptors at the cell surface, and other is triggered by mitochondrial6

. Death receptors belong to the tumor necrosis factor a (TNF-a) superfamily and include, for example, the Fas/CD95 receptor. Binding of the death ligand to its receptor is followed by the recruitment of procaspase-8 to the plasma membrane and autoproteolytic activation. Once acti­vated, caspase-8 activates procaspase-3 and other downstream procaspases 17. Caspase-8 knockout cells are therefore resistant to TNF and Fas-mediated apoptosis ls.

DNA damage, genotoxic agents, oxidative stress, ultraviolet radiation are some of the apoptotic signals channelled through the mitochondria to activate caspases. This path is independent of the cell mem­brane receptor-mediated activation of cell death 19.

Recently published articles, however, contradicted this view of stress-induced mitochondrial-only apop­tosis. Lassus and coworkers2o demonstrated that stress-induced cytokines can directly activate caspase-2. Therefore, mitochondrial permeabilization may function as amplification of caspase activation. The final steps to cell death signal transduction to the nu­cleus and subsequent DNA fragmentation by specific nucleases, however, are shared by both conduits, the receptor-mediated and mitochondrial-initiated apopto­sis.

Mitochondrial release of apoptogenic factors Cytochrome c-Holo-cytochrome c (cytochrome c

with attached heme) remains sequestered in the mito­chondrial intermembrane space where it serves as an electron shuttle between complex III and IV of the mitochondrial respiratory chain. Xiodong Wang and

co-workers21 have reported the surprising observation that holo-cytochrome c (but not apo-cytochrome c) is required for the activation of caspase-3 in a cell free _ system II. Following cell exposure to apoptotic stim­uli, cytochrome c was shown to be released into the cytosoe2

•23

. Today it is an established fact that cyto­chrome c, once present in the cytosol, drives the as­sembly of a high molecular weight caspase activating complex termed "apoptosome". Cytochrome c binds to apoptotic protein activating factor-l (Apaf-I) in the presence of ATP/dATP, leading to the formation of apoptosome. The cytosolic protein containing a caspase-recruitment domain (CARD) of Apaf-l be­come exposed in the apoptosome, which subsequently recruit multiple procaspase-9 molecules to the com­plex and facilitate their autoactivation. Only the caspase-9 bound to the apoptosome is able to cleave and activate downstream caspases such as caspase-324

• The knockout experiments also verify the linearity of the cytochrome c-Apaf-l-caspase-9-caspase-3 pathwal5

•26

.

DIABLOISmac-DIABLO (direct lAP-binding protein with a low isoelectric point)/Smac (second mitochondrial-derived activator of caspase) is another protein released from mitochondria during apopto­SiS I3

•14

• DIABLO/Smac, a 25 kD protein normally lo­calizes to the mitochondrial intermembrane space and is released to the cytosol only during apoptosis. In response to various apoptotic stimuli, DIABLO/Smac is released into the cytosol where it inhibits the in­hibitor of apoptotic proteins (lAPs). By binding to lAP, Smac displaces active caspases or prevents lAPs binding active caspases and thus promote death of the cell. When Smac binds to X chromosome-encoded lAP (X lAP) it prevents it from binding caspase-9 and thus promotes death following UV irradiation27

•

However, caspase activation facilitated by Smac may involve additional mechanisms that are independent from its interaction with XIAp2s.

HtrA210mi-Mammalian mitochondria also re­leases a protein called high-temperature requirement A2 (HtrA2)/Omi, which like DIABLO/Smac binds to lAPs during apoptosis29

-32

• The Omi precursor protein possesses an amino-terminal mitochondrial transloca­tion sequence that directs the protein into mitochon­dria. Once in mitochondria, the translocation se- · quence is cleaved to generate a mature 36 kD protein. During apoptosis, mitochondria release Omi together with cytochrome c and Smac32

. Omi has an IAP­binding motif that allows it to bind lAPs and suppress

MISHRA & KUMAR: APOPTOSIS: A MITOCHONDRIAL PERSPECTIVE ON CELL DEATH 27

their caspase-inhibitory activity. Interestingly, dele­tion of the lAP-binding motif prevents its interaction with lAPs but does not abolish its apoptosis-inducing activity, suggesting that Omi has additional effects on apoptotic cascade. Omi also has a trypsin-like serine protease domain, indicating that it may induce apop­tosis in a caspase-independent manner through its protease activitl9.31 . Recently, it has been reported that the protease activity of HtrA2/0mi contributes to its ability to potentiate caspase activation and apopto­sis via at least two different mechanisms. It cleaves and/or degrades lAPs and an unidentified substrate, resulting in inactivation of lAPs and permeabilization of the outer mitochondrial membrane followed by cytochrome c-dependent caspase activation, respec­tively33.

Apoptosis-inducing Jactor- The apoptosis-inducing factor (AIF) is a 57 kD flavoprotein located in the mitochondrial intermembrane spacel2. AIF is one of the first evidenced mitochondrial apoptotic activity. Upon induction of apoptosis, AIF translo­cates to the nucleus where it induces condensation of chromatin. Unlike cytochrome c, AIF activation of apoptosis is independent of caspases34. However, cy­tochrome c usually accompanies AIF release from mitochondria, resulting in caspase activation and sub­sequent DNA fragmentation35. Because the molecular weight of AIF is much greater than cytochrome c, it still remain unclear whether these proteins are re­leased from mitochondria via the same mechanism.

Endonuclease G-Endonuclease G (En doG) has been shown to be required for early embryogenesis and normal apoptosis36. EndoG is a nuclear-encoded mitochondrial protein reported to be important for nuclear DNA fragmentation during apoptosis. Like AIF, EndoG in mammalian cells is released from mi­tochondria during apoptosis and translocate to the nucleus to cleve DNA into nucleosomal fragments independent of caspases l5.Mitochondria release En­doG together with other apoptogenic proteins, indi­cating that EndoG may be located in the mitochon­drial intermembrane space. The identification of AIF and EndoG indicates that apoptosis can proceed in the absence of caspase activity when the mitochondria are damaged. In this case, release of AIF and EndoG from mitochondria starts an apoptotic program parallel to caspase activation.

The release of cytochrome c and other apoptogenic proteins from mitochondria is known to be regulated by the Bcl-2 family of proteins. The pro-death mem-

bers of this group of protein promote the release of these apoptogenic factors whereas the anti-death members prevent ie7

,38. .

Regulation of mitochondrial apoptotic signals One striking feature of apoptosis signaling is pro­

tein translocation of signal and effector molecules between three major cellular compartments. This in­cludes translocation to and from mitochondria, the cytoplasm and the nucleus. Of particular interest are a growing list of pro-apoptotic proteins that undergo translocation to mitochondria, where they exert their pro-apoptotic functions by inducing organellar dys­function (Fig. 1).

Translocation of Bcl-2 family (BH3-domain con­taining) proteins

A wide variety of mitochondrial events have been reported to be modulated by Bcl-2 and its homologs. The release of cytochrome-c and other apoptogenic proteins from the mitochondria is known to be regu­lated by Bcl-2 family members. It is widely accepted ' that the pro-apoptotic members of Bcl-2 family pro­mote the release of the apoptogenic factors whereas the anti-apoptotic members prevent it. Anti-apoptotic members of Bcl-2 family (such as Bcl-2 and Bcl-xd reside mainly, but not exclusively, in mitochondrial membrane, where they locally inhibit mitochondrial membrane permeabilization (MMP)39.

Proapoptotic members of the Bcl-2 family such as Bax can translocate to the mitochondria while under­going a conformational change; they then oligomerize within mitochondrial membranes and facilitate MMP. This translocation and oligomerization reaction is in­hibited by anti-apoptotic members of Bcl-2 family and is stimulated by pro-apoptotic BH3-only mem­bers of Bcl-2 family (such as Bid). Bid is exclusively cytosolic in living cells and upon activation of cell surface receptor, it is cleaved by caspase-8. The trun­cated Bid translocates to the mitochondria and in­duces cytochrome-c release40.41 .

Bad, another BH3-only protein, is regulated by phosphorylation and dephoshorylation42. The BH3 domain of Bad binds to and inactivates the anti­apoptotic members of the Bcl-2 family at the outer mitochondrial membrane, thereby promoting cell death. Conversely, in the presence of tropic factors, Akt and mitochondrial-anchored PKA phosphorylate Bad, allowing it to bind 14-3-3 protein and to remain in the cytosoI43.44

. Phosphorylation of Bad also disso-

28 INDIAN J EXP BIOL, JANUARY 2005

Apoptotic Stimuli

! Bad, Bid, Bax etc.

! I--- Bel-2, Bel-xL

l Loss of Mitochondrial

AIF Cytochrome c EndoG

l Apaf-t

l lAP Caspase-9

l ~ !

Apoptosis

Fig. I-Multiple apOptotic pathways originate from the mitochondria. Apoptotic signals are transduced to mitochondria by the BH3-only proteins and possibly by additional pathways. These signals can be neutralized by the anti-apoptotic proteins, such as BcI-2 or BcI-xL, The mitochondrial damage, in apoptotic response, triggers the release of apoptogenic proteins including cytochrome c, Smac, AlF, and EndoG. Cytochrome c triggers caspase activation through Apaf-l, and Smac relieves lAP inhibition of caspases. AlP and EndoG cause! apoptosis through chromatin condensation and fragmentation, independent of caspases activation. The mitochondrial damage may also lead to cell death due to loss of mitochondrial functions.

ciates its interaction with anti-apoptotic Bcl-2 family of proteins, allowing these proteins to promote sur­vival.

Translocation of other proteins (non-BH3 contain­ing) to the mitochondria

In addition to Bcl-2 family members, several other proteins have been shown to migrate to the mitochon­dria in response to certain stimuli and regulate the apoptosis by causing mitochondrial damage. These proteins include p5345

,46, an orphan receptor TR347,

c-Jun N-terminal kinase48.49, c-Abl tyrosine kinase50

-52

and protein kinase C053-55

• How these proteins, and possibly others, translocate to the mitochondria and regulate its functions is now fully known. In this sec­tion, we summarize the recent reports on the follow­ing proteins, which translocate to mitochondria in re­sponse to specific stimuli.

p53-p53 is a nuclear phosphoprotein and a tran­scription factor56

• p53 is a multi-functional protein involved in the control of cell cycle progression, apoptosis and genomic integrity in cells exposed to

DNA-damaging agents. Evidence suggests that p53 induces cell death by a dual mode of action involving activation of target genes and transcriptionally­independent direct signaling. p53 is present predomi­nantly in the cytoplasm of primary cultured cells and various cancer cells. p53 translocates to the nucleus to initiate gene activation and DNA synthesis for cell proliferation 57.

Recent studies have indicated that p53 has a direct signalling role in the induction of apoptosis58

,59, al­though the mechanisms involved are not completely understood. Marchenko and coworkers45 have demon­strated that a fraction of stress-activated p53 translo­cates to mitochondria after an apoptotic stimulus, but not during p53-dependent growth arrest. The translo­cation of p53 to mitochondria is rapid (within 1 hr after p53 activation) and precedes changes in MMP, cytochrome-c release and procaspase-3 activation. In contrast, p53 does not translocate during p53-independent apoptosis or p53-mediated cell cycle ar­rest. They have further identified that p53 protein can directly induce permeabilization of the outer mito-

MISHRA & KUMAR: APOPTOSIS: A MITOCHONDRIAL PERSPECTIVE ON CELL DEATH 29

chondrial membrane by forming complexes with the protective BclXL and BcI-2 proteins, resulting in cyto­chrome c release46. Others have shown that p53 inter­acts with the pro-apoptotic mitochondrial membrane protein Bak. Interaction of p53 with Bak: causes oli­gomerization of Bak: and release of cytochrome-c · from mitochondria60. Further, the formation of the p53-Bak: complex coincides with loss of an interac­tion between Bak: and the anti-apoptotic BcI-2-family member MCll60' These studies support a direct mito­chondrial role for p53 in the induction of apoptosis, but the relative contribution of this activity to p53-mediated cell death remains to be assessed.

TR3-The nuclear orphan steroid receptor TR3 (also called Nur77 or NGFIB), a member of the ster­oid/thyroid receptor family is a bonafide transcription factor with a zipc finger DNA-binding domain flanked by transactivation domains and a binding do­main for an as yet unknown ligand. TR3 is induced and acts as a transcription factor in response to epi­dermal growth factor and all-trans-retinoic acid. TR3 mediates apoptosis in different cell types in vivo, e.g. in neurons, T-cells and human cancer cells47. Unex­pectedly, when TR3 works as an apoptotic factor, its transcriptional activation function is turned offl7. In­stead, in response to wide variety of apoptotic stimuli, TR3 translocates from the nucleus to mitochondria to induce cytochrome-c release and apoptosis. The TR3 DNA-binding domain, required for transcriptional activity, is not required for mitochondrial targeting. Its mitochondrial action is also blocked by BcI_247. Thus, both p53 and TR3 (nuclear transcription fac­tors) by virtue of subcellular targeting, appear to be capable of inducing apoptosis.

c-Jun N-tenninal kinase-The c-Jun N-terminal kinases (JNKs) are classic stress-activated protein ki­nases61 . JNKs are potently and preferentially activated following various cell stress applications including UV irradiation, heat and osmotic shock62, treatment with a protein synthesis inhibitor63, inflammatory cy­tOkines63, growth factor withdrawal64

, and treatment with chemotherapy drugs including paclitaxeI65.66, adriamycin, vinblastine, and etoposide67. JNK is also activated in response to cell stress induced by certain DNA-damaging agents including 1-D-arabinofur­anosy1cytosine68, cis-platinum, and mitomycin C68. Generally, JNK has been implicated in the regulation of apoptosis69.7o. Recently, we and others have re­ported that ionizing radiation (IR) and phorbol ester induce translocation of JNK to mitochondria, which

in turn causes phosphorylation, and inactivation of anti-apoptotic BcI_xL48.49. Deng and coworkers71 have reported that JNK also work as BcI-2 kinase. JNK has been found to phosphorylate and co-localize with BcI-2 in the mitochondria where it plays an important role in regulating apoptosis71 . On the other hand, report suggests that activated JNK promotes Bax transloca­tion to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax. Phosphorylation of 14-3-3 led to dissociation of Bax from this protein. Expression of phosphorylation-defective mutants of 14-3-3 blocked JNK-induced Bax translocation to mitochondria, cytochrome-c release and apoptosis72. These reports open up a new exciting field for the identification of JNK-mediated apoptotic signaling . .

c-Abl tyrosine kinase-The c-Abl protein tyrosine kinase, product of the cellular Abelson (c-abl) gene is ubiquitously expressed and localized mainly in the nucleus and cytoplasm. c-Abl has been shown to me­diate inhibition of cell cycle progression and apopto­sis when cells are exposed to genotoxic stress. Moreover, it has also been shown that c-Abl regulates the G 1 growth arrest in response to DNA damage by p53 and its analog p73 dependent mechanism. Re­cently, we have demonstrated that c-Abl exerts apop­totic effects not only in response to DNA damaging agents but also in response to other stimuli such as oxidative stress, ER and microtubule-stress73.52. In addition, we found that c-Abl in response to certain apoptosis-inducing agents translocates from cyto­plasm and/or other organelles to the mitochondria. These include oxidative5o.51 , ER-stress52 and microtu­bule-stress (unpublished observation). Its transloca­tion induces change in MMP, cytochrome-c release, caspase-3 activation and apoptosis5

0-52. ROS-induced

localization of c-Abl to mitochondria is dependent on activation of protein kinase c delta and the c-Abl ki- . nase function5o.51 . These reports further demonstrate that stress-induced cytochrome-c release is apparently dependent on the c-Abl tyrosine kinase, because c­Abl null mouse embryonic fibroblasts are resistant to ER and oxidative stess-induced cytochrome-c release and apoptosis52.73.5o. These studies enhance the under­standing of the mechanism by which c-Abl exerts apoptotic effects by regulating mitochondrial func­tions.

Protein kinase CO--Protein kinase Co (PKCO) be­longs to the novel PKC subfamily and is, therefore, activated by DAG/phorbol esters in caIcium­independent manner. Following its activation, PKCO

30' INDIAN J EXP BIOL, JANUARY 2005

undergoes proteolytic degradation or down regulation via. the ubiquitin-proteasome system74

• PKCo has been reported to play a critical role in the control of cell growth. Loss of PKCo leads to cell transforma­tion in fibroblases whereas, its overexpression results in G21M arrest of the cell cycle76

• PKCo has been re­ported to translocate to nearly all-subcellular organ­elles, including nucleus, mitochondria, Golgi com­plex, ER and plasma membrane77.78. At each subcel­lular organelle, PKCo phosphorylates different sub­strates inducing various responses that eventually lead

to cell death. It has been known for a long time that upon activation, full-length PKCo translocates from the cytoplasm to the plasma membrane79

. One of the main targets of PKCo is -the mitochondria. Indeed,

. PKCo has been reported to localize to the mitochon­dria in response to PMA in mouse keratinocytesS3 and U-937 myeloid leukemia cellss4. The translocation can also be induced by oxidative stressss. In addition, these studies have demonstrated that over-expression and activation of PKCo exert change in mitochondrial functions such as a decrease in MMP and release of

~ ~

Apoptotic Stimuli

Survival Factors

j [BidjS (Caspase 8) c:: '"

~'"

1 I Apoptosis I

Apoptotic Stimuli

Phosphatases

Radiation, PMA

(AKT, PKA etc.)

\ IpKca] 1

ROS, I Apoptosis I ROS, ER-Stress

PMA

Fig. 2-Regulation of mitochondrial apoptotic signals. Many death signals converge onto mitochondria and are mediated through mem­bers of the BcI-2 protein family (BH3-domain containing) and other proteins which are having role in specific apoptotic pathways. In some cells, binding of Fas ligand to its receptor Fas leads to the caspase-8 activation, which proteolytically cleaves Bid, whose C­terminal fragment (t-Bid) translocates to mitochondria, where it activates Bax or Bax-like proteins and results in cytochrome-c (cyt c) release. Once in the cytosol, cyt c activates caspase-9 by binding to Apaf-l and dATP. Caspase-8 can also initiate a direct signaling pathway that is independent of mitochondria by activating downstream caspases. Apoptotic stimuli (death receptor independent) and growth-factor deprivation can trigger apoptosis by inducing translocation of bax or Bad to mitochondria. In normal cells, Bad can be phosphorylated by AKT, PKA and other enzymes which results it sequestration in cytosol through its binding to 14-3-3. Bad is also dephosphorylated by the phosphatases and translocate to mitochondria, where it binds to Bcl-xL. This displaces BcI-xL from BcI-xL-Bax heterodimers, thereby inhibiting the death-repressor activity of BcI-xL. In certain cells, in response to apoptotic stimuli p53 trans locates to mitochondra and permeabilize the outer mitochondrial membrane by forming complexes with the protective BclxL and BcI-2 proteins, resulting in cyt c release. TR3, another transcription factor also translocates to mitochondria from the nucleus and releases cyt c. In re­sponse to ionizing radiation and phorbol ester, c-jun-N-terminal kinase (INK) translocates to mitochondria, where it causes phosphoryla­tion and inactivation of anti-apoptotic Bcl-xL and BcI-2. INK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3. Other kinases like c-Abl tyrosine kinase (in ROS and ER-stress response) and Protein kinase Co (in ROS and PMA response) translocates to mitochondria and facilitate apoptosis by causing loss of mitochondrial transmembrane potential and release of cyt c and caspase-3 activation.

MISHRA & KUMAR: APOPTOSIS: A MITOCHONDRIAL PERSPECTIVE ON CELL DEATH 31

cytochrome c. In contrast, cytosine arabinoside (Ara­C) and etoposide (VP-16) induced nuclear transloca­tion of PKC8 preceded its cleavage by caspase-380

,81.

However, the catalytic fragment of 'PKC8, generated following treatment with cisplatin or UV radiation has been found to be localized to the mitochondrial frac­tion82

,83. Recently, Kajimoto and coworkers84 have reported that activation and translocation of PKC8 to the Golgi complex is critical for the ceramide-induced apoptosis. These studies suggest that depending on the cell types and apopototic stimuli, the targets of PKC8 may resides in the plasma membrane, nucleus or mitochondria.

The evolving theme here is that these mitochondri­ally translocating proteins belong to unrelated bio­chemical classes of molecules, most of which are pre­viously not associated with mitochondrial functions. However, these studies suggest a new exciting direc­tion for future research that ultimately may reveal other unidentified apoptotic pathways.

Concluding remarks Existence of multiple programs of cell death is

strongly supported by the vast amount of information disclosed in recent years. It has now become apparent that mitochondria act as integrators of pro-apoptotic signals, transducing these to the final execution machinery of apoptosis. In summary, mitochondrial­initiated apoptosis has two important signaling events. First, as illustrated in Figure 1, multiple factors function work together to trigger apoptosis. The release of cytochrome c activates caspases, and the release of Smac removes lAP inhibition of caspases, and the release of EndoG and AIF induces DNA fragmentation and chromatin condensation. Second, as illustrated in Figure 2, the regulation of mitochondrial apoptotic signals. These signals are executed by translocation of cytosolic BH3-only (Bax, Bad, t-Bid) and other non-BH3 proteins (p53, TR3, INK, c-Abl and PKC8) to mitochondria, thereby releasing pro-apoptotic factors required for the cell death. Unraveling the precise mechanism of action of these proteins will undoubtedly help our understanding of how signals from and to mitochondria regulates cell death.

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