Role of Bcl-2 family proteins and caspases in the regulationof apoptosis

Mohammad Shamsul Ola • Mohd. Nawaz •

Haseeb Ahsan

Received: 29 September 2010 / Accepted: 13 December 2010 / Published online: 6 January 2011

� Springer Science+Business Media, LLC. 2011

Abstract Apoptosis, or programmed cell death, plays a

pivotal role in the elimination of unwanted, damaged, or

infected cells in multicellular organisms and also in diverse

biological processes, including development, cell differ-

entiation, and proliferation. Apoptosis is a highly regulated

form of cell death, and dysregulation of apoptosis results in

pathological conditions including cancer, autoimmune and

neurodegenerative diseases. The Bcl-2 family proteins are

key regulators of apoptosis, which include both anti- and

pro-apoptotic proteins, and a slight change in the dynamic

balance of these proteins may result either in inhibition or

promotion of cell death. Execution of apoptosis by various

stimuli is initiated by activating either intrinsic or extrinsic

pathways which lead to a series of downstream cascade of

events, releasing of various apoptotic mediators from

mitochondria and activation of caspases, important for the

cell fate. In view of recent research advances about

underlying mechanism of apoptosis, this review highlights

the basics concept of apoptosis and its regulation by Bcl-2

family of protein. Furthermore, this review discusses the

interplay of various apoptotic mediators and caspases to

decide the fate of the cell. We expect that this review will

add to the pool of basic information necessary to under-

stand the mechanism of apoptosis which may implicate in

designing better strategy to develop biomedical therapy to

control apoptosis.

Keywords Apoptosis � Bcl-2 � BH-3 only proteins �Caspases � Mitochondrial proteins � Programmed cell death

Abbreviations

PCD Programmed cell death

Bcl-2 B cell lymphoma-2 protein

Bax Bcl-2 associated X protein

Bid Bcl-2 interacting domain death agonist

Bad Bcl-2 antagonist of cell death

Bcl-xl Bcl-extra long

Bim Bcl-2 interacting mediator of cell death

Bik Bcl-2 interacting killer

Bmf Bcl-2 modifying factor

Boo Bcl-2 homolog of ovary

Bcl-xs Bcl-extra short

Bak Bcl-2 antagonistic killer

Bok Bcl-2 related ovarian killer

Apaf-1 Apoptosis protease-activating factor-1

Diablo Direct IAP binding Protein with low pI

FADD Fas-associated death domain protein

TNF-R Tumor necrosis factor receptor

Fas-L Fas ligand

HtrA High-temperature requirement

IAP Inhibitor of apoptosis protein

IMM Inner mitochondrial membrane

Omi/HtrA2 Mammalian serine protease

SMAC Second mitochondrial activator of caspase

TNF-a Tumor Necrosis Factor alpha

TRADD TNF-receptor-1 associated death domain

protein

VDAC Voltage-dependent anion channel

Cyt c Cytochrome c

PIDDosome p53-Inducible death domain containing

protein complex

DISC Death-inducing signaling complex

M. S. Ola � Mohd. Nawaz

Department of Ophthalmology, College of Medicine, King Saud

University, Riyadh 11411, KSA

H. Ahsan (&)

Department of Biochemistry, Faculty of Dentistry, Jamia Millia

Islamia (A Central University), New Delhi 110025, India

e-mail: hahsan@rediffmail.com

123

Mol Cell Biochem (2011) 351:41–58

DOI 10.1007/s11010-010-0709-x

Introduction

Apoptosis is an ancient Greek word which means ‘‘the

falling of leaves from a tree’’. John Kerr first introduced the

name which refers to the morphological feature of forma-

tion of ‘‘apoptotic bodies’’ from a cell [1]. Apoptosis, also

known as programmed cell death (PCD), plays an integral

role in a variety of biological events including morpho-

genesis, tissue homeostasis, aging, and removal of

unwanted harmful cells [2]. Changes typical for apoptosis

include condensation of the nuclei, DNA fragmentation,

chromatin condensation, generation of envoluted mem-

brane segments, cellular shrinkage, and disintegration of

mitochondria [3, 4]. Apoptosis can be induced by a variety

of physiological and pathophysiological stimuli, such as

specific receptor molecules-CD95 [5, 6], tumor necrosis

factor (TNFa) [7], growth factors [8], ultraviolet light [9,

10], irradiation [11], heat shock [12], cytotoxic drugs [13,

14], oxidative stress [15], ceramide treatment [16], and

bacteria [17]. Dysfunction of apoptotic pathways result in

various pathological conditions including cancer, autoim-

mune, and neurodegenerative diseases [18–21].

A significant progress has been made toward under-

standing the mechanism of apoptosis and other apoptosis

related dysfunction, but it needs more intensive research to

better understand the whole cascade of events involved in

apoptosis and also the mechanism of their regulatory

pathways [22–24]. The nematode, Caenorhabditis elegans

(C. elegans), has been widely used to elucidate the

molecular pathways implicated in the regulation of PCD

[25–27]. For elucidating the underlying genetics and bio-

chemical pathways in PCD, three scientists, namely,

S. Brenner, J. Sulston, and R. Horvitz were awarded the

Noble Prize in medicine (2002).

The Bcl-2 and apoptosis

The underlying mechanism and genetics of apoptosis

began a new approach of research after the landmark dis-

covery by Horvitz, 1999 [28]. The protein expression of

two C. elegans cell death genes (CED-3 and CED-4) is

necessary for PCD during development, and CED-9 is the

functional homolog to mammalian protein Bcl-2, which is

shown to correct the phenotype of C. elegans by its anti-

apoptotic function [29, 30]. CED-9 was found to act

upstream of aspartate-directed cysteine protease (CED-3

and CED-4) known as caspases [31]. Previously, it was

revealed that proteins that are encoded by the mutant genes

discovered in C. elegans shared homology with mamma-

lian proteins, including Bcl-2 [32].

The role of Bcl-2 (B cell lymphoma 2), a founder

member of the Bcl-2 family of apoptosis regulator proteins,

has been elucidated in tumor development by dysfunction

in apoptotic pathways [20, 30]. In mammals, there is an

extrinsic apoptotic pathway that involves death receptors

and an intrinsic apoptotic pathway that involves the mito-

chondria. Mammalian mitochondria control apoptosis by

releasing many apoptogenic mediators from intermem-

brane space [33] and that further leads to a cascade of

reaction with the assistance of caspases, proteases that

cleave key cellular proteins [34, 35]. A major difference

between apoptosis in worm and mammals is the greater

role of mitochondria in the mammals but not in the worm.

Bcl-2 expression specifically blocks the morphologic fea-

tures of apoptosis, including the plasma membrane bleb-

bing, DNA cleavage, and nuclear condensation. Bcl-2

plays a role in cell survival and also inhibits cell death

induced by various stimuli such as chemotherapeutic

agents, ethanol and heat shock, indicating Bcl-2 as a neg-

ative regulator of cell death. The anti-death role is also

demonstrated in vivo by generation of mice lacking the

bcl-2 gene, which shows a variety of abnormalities

including excessive cell death.

Members and structural classification of Bcl-2 proteins

On the basis of various structural and functional charac-

teristics, the Bcl-2 family is divided into anti-apoptotic and

pro-apoptotic proteins [36–38]. All of the members of Bcl-

2 family have one or more homology domains labeled as

Bcl-2 homology (BH1, -2, -3, and -4) which are important

for heterodimeric interaction among members of the Bcl-2

family [27] (Fig. 1). Anti-apoptotic Bcl-2 family multido-

main proteins include Bcl-2, Bcl-xL (Bcl-extra long), A1,

Bcl-w, and Boo (Bcl-2 homolog of ovary) contain BH-

(1-4) domains. The Bcl-xL and Bcl-2 have a carboxy-ter-

minal hydrophobic transmembrane tail domain which helps

in localization of protein in outer mitochondrial membrane

(OMM) with the exception that Bcl-2 also resides in the

nuclear and endoplasmic reticulum membrane and is

translocated to OMM upon an apoptotic signal. Myeloid

cell leukemia factor-1 (Mcl-1) is the only antiapoptotic

Bcl-2 protein with three BH domains (BH-1, -2, and -3).

Pro-apoptotic Bcl-2 family of proteins are divided into

two subgroups according to the number of BH domains

(e.g., Bax, Bak, and Box) or those proteins that have only

the BH3 domain (e.g., Bid, Bim, and Bad) with the

exception of Bcl-xS which has only BH3 and BH4 domains

(Fig. 1). There are eight members of BH3 only protein,

which include hara-kiri (Hrk), BH3 interacting domain

death agonist (Bid), Bcl-2 interacting mediator of cell

death (Bim), Bcl-2 modifying factor (Bmf), p53, promoter-

upregulated modulator of apoptosis (Puma), Noxa (named

for ‘‘damage’’), Bcl-2 antagonist of cell death (Bad), and

42 Mol Cell Biochem (2011) 351:41–58

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Bcl-2 interacting killer (Bik). Bid, Bad, and Bim have

cytosolic location with respect to mitochondria. Following

death signal, BH3 domain-only proteins also known as

minimal death domain can neutralize or depress antiapo-

ptotic Bcl-2 proteins allowing pro-apoptotic proteins such

as Bax/Bak-like proteins to induce apoptosis [39, 40].

A restricted subset of these BH3 proteins undergoes

post-translation modification such as cleavage, phosphor-

ylation, transcriptional upregulation, and dissociation. Such

modifications result in their activation and translocation to

outer mitochondrial membrane (OMM) which helps it to

interact with multidomain pro-apototic members, Bax/Bak-

like proteins, leading to their oligomerization and forma-

tion of pore. Such pore may further lead to release of other

pro-apoptotic protein on the intermembrane space (IMS)

that enhances the processes of apoptosis [41]. Thus the

regulation of expression and function of BH3 domain-only

proteins is critical in mediating cell death and under-

standing their expression and function necessary for cyto-

protective strategies. In general, the relative ratio of pro-

survival (Bcl-2) and pro-apoptotic (Bax and BH3-only)

proteins seems to determine the cell sensitivity or resis-

tance to the apoptotic stimuli.

Mechanism of action and role of Bcl-2 family members

in the regulation of mitochondrial apoptosis

Antiapoptotic or pro-survival proteins

It is now becoming evident that almost every nucleated cell

requires protection by at least one of the member Bcl-2

homolog, and that regulates homeostasis in mammalian cell

and tissues. Over-expression of Bcl-2 protein in hemato-

poietic lineages yields excess T, B, and myeloid cells and

enhance the survival of these progenitor cells that are hard to

avoid cytotoxic insults [29, 42]. Alternatively, inactivation

of the Bcl-2 gene increases apoptosis in specific cell types,

presumably because the concentrations of other Bcl-2

homolog are too low to compensate. Bcl-2 itself is required

for the survival of kidney, mature lymphocytes and mela-

nocyte stem cells [43], Bcl-w for sperm progenitors in adult

mice [44], an A1 gene for neutrophils [45], Mcl-1 for zygote

implantation [46], and Bcl-xL for erythroid and neuronal

cells [47]. The hydrophobic carboxy-terminal domain of

these antiapoptotic proteins help to anchor themselves to the

cytoplasmic face of three intracellular membranes: the outer

mitochondrial membrane, the nuclear envelope, and the

endoplasmic reticulum (ER). Bcl-2 is an integral membrane

protein, whereas Bcl-xL and Bcl-w only become tightly

associated with the membrane after cytotoxic signals which

induce conformational changes to protect cell. The antiap-

optotic member of Bcl-2 family, including Bcl-2 itself,

decreases apoptosis mainly by preventing mitochondrial

outer membrane permeabilization (MOMP) either by neu-

tralizing the activity of both pro-apoptotic members (BH3

only protein and multidomain members) [48]. If Bcl-2 or

Bcl-xl cannot exert their antiapoptotic property, Bax oligo-

merization takes place and apoptosis is promoted.

Pro-apoptotic or anti-survival protein

It includes both multi-domain and BH3-only protein

members.

Multi-domain pro-apoptotic member

Antiapoptotic members such as Bcl-2 interact with Bak and

Bax and inhibit their oligomerization and/or bind to BH3

only proteins to block apoptosis [48]. Bak interacts with

two mitochondrial fusion proteins (Mfn1 and Mfn2). After

receiving apoptotic signal, Bak dissociates from Mfn2 to

become associated with Mfn1. It has been shown that

reconstitution of Bak into Bax/Bak double knockout cells

Fig. 1 According to conserve

domain (BH) and its anti or pro-

apoptotic action, the Bcl-2

super-family can be divided into

three sub-families: Anti-

apoptotic or Pro-survival multi-

domain, pro-apoptotic multi-

domain and pro-apoptotic BH3

only. Few examples of protein

from each group of the family

are given in parenthesis. TM

(transmembrane domain)

Mol Cell Biochem (2011) 351:41–58 43

123

restores mitochondrial fragmentation and Bak in associa-

tion with Bax permeabilize the outer membrane of mito-

chondria rendering apoptotic cascade [49]. Bak may

contribute to early mitochondrial fragmentation while Bax

is probably more important for subsequent pores develop-

ment and degeneration in the outer membrane [49]. A

number of studies have shown that insertion of activated

oligomerized Bax and/or Bak into the OMM exerts stress

on the membrane, leading to supramolecular pores that

include lipids (lipidic pores) in the OMM [50–52].

In healthy cells, Bak is inactive in the OMM through its

association with Voltage-dependent Anion channel

(VDAC-2) [48], whereas Bax is dormant in the cytosol

through interactions with several proteins, including

Ku-70, 14-3-3, and the humanin peptide. Many apoptotic

signals can trigger BH3-only-protein-dependent transloca-

tion of Bax, followed by its insertion into the OMM and the

formation of Bak or Bax homo-oligomers. Several models

have been proposed for how Bcl-2 anti-apoptotic proteins

antagonize the functions of Bax, Bak, and BH3-only pro-

teins. Activation of Bax and Bak leads to a conformational

change that exposes the N-terminus of the proteins, which is

otherwise hidden in the inactive state [53]. After Bak and

Bax form homo-oligomers, they participate in forming pores

in OMM and cause permeabilization leading to the release of

the contents of the mitochondrial intermembrane space

(IMS) including Smac and cytochrome c into the cytosol

[54, 55]. The released mitochondrial protein then activates

caspases, which causes a series of cascade reactions that

cleave essential proteins complement throughout the cell.

Two models of Bax and Bak activation, exist the direct

and indirect models. According to the direct model various

BH3 pro-apoptotic proteins called as activator proteins (Bid,

Bim, Puma, and p53) directly interact and induce confor-

mational changes in Bak and Bax [56]. Members of anti-

apoptotic proteins bind and sequester such activator, and

also bind to activated Bak and Bax proteins that might be

present and thus prevent apoptosis. BH3-only proteins are

further divided into activator and sensitizer categories [57].

Sensitizers bind to anti-apoptotic proteins and cause the

release of activator BH3-only proteins leading to activation

of Bak and Bax. However, it is likely that additional factors

other than Bid and Bim can act as activators such as Puma,

p53, and heat as activators of Bax and Bak [58–60], and

possibly others. Gavathiotis and colleagues have demon-

strated that stabilized alpha-helix of Bcl-2 domains (SA-

HBs) directly initiates Bax-mediated mitochondrial

apoptosis [61].

According to the indirect model, anti-apoptotic proteins

always bind to Bak and Bax and prevent their activation,

whereas in response to death signal, BH3-only proteins

bind to antiapoptotic proteins causing its release and ini-

tiating death signal through activation of Bax and Bak [62].

The sequestered forms of Bak and Bax are those fractions

of the total Bak and Bax population that are already

activated either spontaneously, or by other unspecified

means [63]. Bax and Bak can also alternatively interact

with proteins that remove the requirement for sequestra-

tion by anti-apoptotic proteins [64]. Activated population

of Bak and Bax are needed to kill, and that should be

sequestered by anti-apoptotic proteins to maintain survival

of the cell. Activated Bak and Bax are responsible for the

permeabilization of membranes. The activation is

achieved, either by interacting with activator proteins or

through some agents, while the anti-apoptotic proteins

inhibit death by sequestering activated Bak and Bax or

activator proteins.

BH3-only pro-apoptotic members

BH3-only pro-apoptotic proteins are involved in main-

taining the functionality and cellular integrity of the cell in

which Bim acts as sensor for cytoskeleton integrity, Bid

acts as sensor for death domain receptor signaling, and Bad

acts as an inhibitor for growth factors withdrawal. Indi-

vidual BH3-only proteins are normally controlled by

diverse mechanisms.

Cleavage of Bid Activation of cell surface receptors such

as tumor necrosis factor receptor (TNF-R) activates cas-

pase-8, which leads to the cleavage of Bid, and then

truncated Bid (active Bid or tBid) translocates from cytosol

to OMM and induces cytochrome c release [65–67]. Oh

and his group have demonstrated that at nanomolar con-

centrations of a synthetic Bid activates Bax almost as

efficiently as tBid itself thus tBid engages Bax to trigger its

pro-apoptotic activity [68]. It is now well established that

tBid leads to oligomerization of Bax and Bak, which forms

pores in OMM which in turn permits the release of IMS

proteins into the cytosol [54, 69]. During apoptosis, Bid

induces the mobilization of cyt c by remodeling mito-

chondrial cristae by interacting Bid with cardiolipin [70,

71]. Addition of tBid to permeabilized cells leads to

hydrolysis of cardiolipin molecules thereby decreasing the

cardiolipin levels [72, 73]. But tBid alone does not cause

mitochondrial outer membrane permeabilization [74].

Phosphorylation of Bad In the absence of various sur-

vival factors, Bad is activated by dephosphorylation [75].

The BH3 domain of Bad binds and inactivates Bcl-2 and/or

Bcl-xL at the OMM, thereby promoting cell death. Con-

versely, in the presence of survival factors, Bad is phos-

phorylated, making it to dissociate from Bcl-2 and/or Bcl-

xl, permitting, and survival promotion. There are several

phosphatases that dephosphorylate Bad in vitro [75, 76].

44 Mol Cell Biochem (2011) 351:41–58

123

However, a report by Datta and his group showed that Akt

phosphorylates Bad, both in vitro and in vivo, and blocks

the Bad-induced death of primary neurons [77]. Further-

more, several studies elucidate the importance of Bad

phosphorylation and underlying mechanism of cell death

[78–80].

Dissociation of Bim and Bmf In response to several

apoptotic stimuli, such as detachment of adherent cells

from their substratum (anoikis) or ultraviolet irradiation,

Bmf is released from the myosin V motor complex,

translocates and binds to antiapoptotic family proteins,

such as Bcl-2, Bcl-xl, and Bcl-w, but does not interact with

the pro-apoptotic family protein, such as Bax, Bid, and Bad

[81]. However, c-Jun N-terminal protein kinase (JNK) that

causes phosphorylation of Bim is involved in ischemia-

induced neuronal apoptosis through activation and trans-

location of Bax [82, 83]. Bim is the main regulator of

hematopoietic homeostasis [84], essential for the elimina-

tion of autoreactive lymphocytes [85] and plays vital role

in neuronal death [86]. Recently, it has been shown that

Bim displaces Bcl-xl in the mitochondria and promotes

Bax translocation during intrinsic pathways assisted by

TNFa [87] or UV-induced [88] apoptosis. Histone deace-

tylase (HDAC) inhibitors increase ionizing radiation-

induced apoptosis in several cancer cells via activation of

Bmf transcription [89, 90]. In TGF beta-induced cell death,

there is upregulation of Bmf and Bim, and thus, inhibition

of the TGFb provides an important therapeutic and pro-

tection of cells from apoptosis [91].

Transcriptional regulation of BH3-only proteins BH3-

only proteins are also transcriptionally regulated as in DNA

damage-induced apoptosis which requires synthesis of new

protein. Transcription of Noxa and Puma, the BH3-only

group members, is induced by p53 [92–94]. Apoptosis in

fibroblast in response to DNA damage is decreased in mice

knocked out in Noxa/puma whereas mutations of the BH3

domain suppress the pro-apoptotic activity of Noxa [94],

and Noxa-induced apoptosis is inhibited by Bcl-xl and

Bcl-2 [95]. Recently, it has been reported that Puma

expressed independent of p53 regulation [96, 97] and ini-

tiates apoptosis by dissociating Bcl-xl and Bax, promoting

Bax multimerization and mitochondrial translocation

[9, 96]. Puma rapidly induces apoptosis in cells lacking not

only the BH3-only proteins but in the absence of Bid and

Bim [98]. Puma induced apoptosis is associated by

regeneration of superoxide and H2O2 which is Bax-

dependent and that can be confirmed by the presence of

antioxidants that prevent Puma-dependent apoptosis [99].

Whereas Bax inactivation confers a resistance to Puma-

dependent apoptosis [100].

Apoptotic pathways

Apoptosis or PCD has conserved genetic and biochemical

pathways [27, 101]. In vertebrates, caspase-dependent

apoptosis occurs through two main interconnected path-

ways which are intrinsic and extrinsic pathways [102]. The

intrinsic pathway or intracellular path is mediated by Bcl-2

family, whereas the death receptor or extrinsic pathway is

activated by signal from other cells [7, 103, 104].

Intrinsic pathway

The intrinsic pathways also known as mitochondrial path-

ways or stress pathways are activated by a diverse array of

death stress, genomic stress, metabolic stress, presence of

unfolded proteins, and other stimuli that lead to perme-

abilization of OMM and release of apoptotic proteins into

the cytosol (Fig. 2). Several of these proteins including

cytochrome c (cyt c) initiate or regulate caspase activation.

The cyt c plays the main role in this pathway which is

activated after its interaction with apoptotic protease-acti-

vating factor (Apaf1) and deoxyadenosine triphosphate

(dATP) to form apoptosome [102, 105]. The apoptosome

creates a platform to bring together molecules of the ini-

tiator caspase of the intrinsic pathway. Progression through

the pathway usually leads to activation of caspase-9,

enabling their auto-activation. Caspase-8 also has a major

role to play by activating the Bid leading to the formation

of activated truncated Bid (tBid), which translocates to

mitochondria and releases cyt c. The activated caspase-8

then cleaves procaspase-3, giving activated caspase-3,

which acts as an executioner, by cleaving multiple of other

substrate within the cells [34, 106].

Extrinsic pathway

The extrinsic pathway involves the association of receptor-

mediated transmembrane death receptor (FAS and TNF-a)

and its extracellular ligand (FAS-L and TNF-aL) [107–

109]. For association the receptor trimerizes and death

adapter molecules are recruited on the cytoplasmic side of

the mitochondrial membrane. Fas recruits Fas-associated

death domain protein (FADD) whereas TNF-R recruits

TNF-R1-associated death domain protein (TRADD) which

again recruits FADD [7]. Such association leads to for-

mation of death-inducing signaling complex (DISC) con-

sisting of a complex of receptor, its ligand, the initiator

procaspase-8 (or procaspase-10), and some other regulators

and co-factor [110]. The complex helps in recruiting more

of procaspase-8 (or procaspase-10) and enables their

autoactivation (Fig. 2).

Mol Cell Biochem (2011) 351:41–58 45

123

Fig. 2 The underlying mechanisms in the execution of apoptotic

pathways. The Apoptotic stimulus received are communicated to the

cell by extrinsic or intrinsic pathways. The extrinsic pathway involves

the engagement of various transmembrane death receptors such as

FAS and TNF-a and their corresponding extracellular ligands (FASL,

TNF-aL). Fas recruits, Fas-associated death domain protein (FADD)

whereas TNF-R recruits TNF-R1 associated death domain protein

(TRADD) which again recruits FADD. Such association leads to

formation of Death-inducing signaling complex (DISC) which acts as

platform for many co-factors and regulator for cleavage and

activation of procaspase-8 (or 10), an initiator caspase of the extrinsic

pathway. The activated caspase-8 (or 10) leads to their own

autoactivation which further activate the caspase-3 and caspase-7,

an effectors caspase. The intrinsic pathway involves the mitochondria

and after receiving the stimulus, the pro-apototic BH3-only members

(Bid, bad, bim, etc.) bind and neutralize the antiapoptotic proteins

(Bcl-2, Bcl-xL, etc.). This leads to oligomerization of multi-domain

pro-apoptotic member (Bax/Bak) present on mitochondrial membrane

surface whose activation leads to permeabilization and formation of

pores or VDAC in outer mitochondrial membrane (OMM), releasing

various apoptotic mediators (HtrA2/Omi, Smac/DIABLO, Cyt c, endo

G and AIF). The release of Cyt c in cytosol causes the association of

Apaf-1 and ATP/dATP to form apoptosome, the initiator caspase of

the intrinsic pathway, caspase-9, enabling their autoactivation. This in

turn activates the series of downstream of cascade to activate the

caspase-3 and caspase-9, an effectors caspase involved in cell death.

The role of IAP is to act a guardian inside a cell to defend against the

mediator of apoptosis (HtrA2/Omi, Smac/DIABLO) by binding to

Caspase 3/7/9 where as Endo G and AIF acts in a caspase independent

manner by causing chromatin condensation and fragmentation. The

pro-apototic BH3-only members, Bid, is also activated by caspase-8,

making it an important converging point of both pathways. The

activated truncated (t)Bid further activates downstream targets, such

as BAX and BAK

46 Mol Cell Biochem (2011) 351:41–58

123

Apoptotic mediators

Permeabilization of the outer mitochondrial membrane

allows the leakage of at least five apoptotic mediators

(apoptogenic proteins) from the mitochondrial intermem-

brane space, such as cyt c, second mitochondrial activator

of caspases/Direct IAP protein with low pI (Smac/DIA-

BLO), HtrA2/Omi, apoptosis-inducing factors (AIF), and

endonuclease G [32, 37]. These proteins induce apoptosis

in different ways. Smac/DIABLO and HtrA2/Omi suppress

the ability of IAPs (inhibitors of apoptosis proteins) to

inhibit caspases. Endonuclease G and AIF are involved in

DNA fragmentation, and AIF is also involved in chromatin

condensation (Fig. 2). The release of these apoptotic

mediators from mitochondria is known to be regulated by

Bcl-2 family of proteins [55, 111]. However, caspase-

independent mitochondrial cell death results from loss of

respiration and not from the release of cytotoxic apoptotic

mediators [112].

Release of cytochrome c

Cyt c is a water soluble 13 kDa protein encoded by nuclear

gene that is translated in cytosol to be finally imported into

mitochondria. It normally resides in the spaces within

cristae of the inner mitochondrial membrane (IMM) and at

narrow cristae junctions [113]. The role of cyt c in the

intrinsic pathways in mammalian cells is well known.

Addition of dATP to cytosolic extract induces caspase

activity [114] and depletion of cyt c in cell extract inhibits

its apoptotic potential and also microinjection of cyt c in

various cell types enhances the apoptotic pathways [115,

116]. Thus, cyt c is the main mediator of apoptosis [117,

118], and the release of cyt c occurs due to DNA damage

[119, 120]. Over-expression of Bcl-2 blocks the release of

cyt c from mitochondria and inhibits the initiation of

apoptosis [121].

Release of endonuclease G (EndoG)

EndoG is a 30 kDa nuclease protein located in mitochon-

drial intermembrane space [122, 123]. The release of

EndoG after apoptotic signal leads to DNA fragmentation

as found in inhibitor of caspase-activated DNase (ICAD)-

deficient cells after induction of apoptosis by TNF treat-

ment and UV-irradiation [123, 124]. Once released, EndoG

participates in DNA fragmentation but without assistance

of caspases [125–127].

Release of apoptosis-inducing factors (AIF)

Apoptosis-inducing factor (AIF), which resembles bacterial

oxidoreductase, is a 57 kDa flavoprotein present in the

mitochondrial intermembrane space [128]. Upon induction

of apoptosis, AIF translocates from the mitochondria to the

nucleus and causes DNA fragmentation and chromatin

condensation [128]. These effects are independent of its

oxidoreductase and caspases activity [129, 130]. Disruption

of AIF in mice prevents normal apoptosis necessary for the

activation of embryoid bodies in the embryo [131]. In

addition, AIF is required for specific cell death pathways

including lethal responses to excitotoxins such as gluta-

mate and N-methyl-D-aspartate (NMDA), DNA-alkylating

agents, hypoxia–ischemia, or growth factor deprivation

[132]. Recently, Schulthess and coworkers have shown a

protective role of AIF on b-cell turnover and the loss of

AIF increases b-cell apoptosis. AIF is essential for main-

taining b-cell mass and oxidative stress response [133].

The mechanism of AIF-induced large-scale chromatin

condensation and DNA fragmentation is still not clear

[128, 131, 134–136]. More recently, the role of AIF in

12/15-lipoxygenase (LOX)-dependent organelle damage

pathway has been reported showing that AIF and 12/15-

LOX are important mediators in a common cell death

pathway in stroke-induced brain damage [137].

Release of Smac/DIABLO

SMAC (Second Mitochondrial Activator of Caspases) or

DIABLO (Direct IAP Binding Protein with Low pI) are a

25 kDa, pro-apoptotic protein released from the inter-

membrane space that neutralizes the inhibitory activity of

IAP leading to activation of caspases and apoptosis [138–

140]. During reovirus-induced apoptosis, Smac/DIABLO

are released that decreases the level of IAP’s and thus

activates apoptosis [139]. Sphingosine 1-phosphate inhibits

the release of Smac/DIABLO from mitochondria and

antagonizes apoptosis of human leukemia cells. Smac/

DIABLO is involved in many cancer manifestation and

progression [141] such as cervical cancer [142], colon

cancer [143], and hepatocellular carcinoma [144].

Release of Omi/HtrA2

The mammalian serine protease Omi/HtrA2 (high-tem-

perature requirement) is a 49 kD protein, homologous to

the bacterial endoprotease also known as DegP [145].

Unlike Smac/DIABLO, the pro-apoptotic activity of Omi/

HtrA2 involves both IAP binding and serine protease

activity. Omi/HtrA2 has a dual function, when residing

inside the mitochondria it promotes cell survival, but when

released into the cytoplasm it participates in both caspase-

dependent and -independent cell death. It prevents the

IAP’s action via amino-terminal reaper-related motif which

induces caspase activity [99, 146–148]. It also mediates

caspase-independent cell death through its own protease

Mol Cell Biochem (2011) 351:41–58 47

123

activity, by the fact that simultaneous deletion of the other

IAP binding protein, Smac/DIABLO, does not alter the

phenotype of Omi/HtrA2 knockout mice or cells derived

from them [149]. The caspase-independent role of Omi/

HtrA2 in apoptosis is evident in human cardiac-specific

inhibitor of cell cycle protein, Thanatos-Associated Protein

5 (THAP5) that are cleaved by pro-apoptotic Omi/HtrA2

during cardiomyocytic cell death [150]. The role of Omi/

HtrA2 in colon cancer [151] and oxidative stress of pig-

ment epithelial cell [152] further supports its application of

mediating apoptosis in cells. The role of Omi/HtrA2 in

promoting cell death by binding and degrading ped/pea-15,

an anti-apoptotic protein, establishes the pro-apoptotic

effect of Omi/HtrA2 [153] and its role in the apoptosis of

prostate cancer cell, PC-3 [154]. However, a contradictory

role of mitochondrial Omi/HtrA2 has been reported. In

response to some extracellular inducers of mitochondrial

stress, Omi/HtrA2 stabilizes mitochondrial membrane

potential and inhibits mitochondrial superoxide generation

and hence controls apoptosis [155].

Regulation of apoptosis by IAPs

Inhibitor of apoptosis (IAPs) is a family of antiapoptotic

proteins that associate with caspases in response to diverse

stimuli. IAP has been discovered both in invertebrate and

vertebrates. So far, eight human IAP homologs have been

identified which includes X-chromosome-linked IAP

(XIAP, also known as hILP, MIHA, or BIRC4), survivin

(also known as TIAP or BIRC5), cellular IAP1 (c-IAP1, also

known as HIAP2, MIHB or BIRC2), c-IAP2 (also known as

HIAP1, MIHC, or BIRC3), neuronal apoptosis inhibitory

protein (also known as BIRC1), IAP-like protein 2 (also

known as BIRC8, or Ts-IAP), apollon (also known as Bruce

or BIRC6), melanoma IAP (ML-IAP, also known as KIAP,

livin, or BIRC7) [156, 157]. The best characterized IAPs

such as XIAP, c-IAP1, and c-IAP2, bind caspase-3, caspase-

7, and caspase-9, thereby inhibiting their activation and

preventing apoptosis (Table 1) [156, 158–160]. Also, cIAP1

and cIAP2 have been shown to bind caspases, although how

they inhibit apoptosis at the molecular level is not com-

pletely understood [161]. Direct inhibition of caspase

activity by c-IAPs is an important means of regulation in

order to protect cell death. Activity of XIAP is blocked by

binding to Omi/Htr2A and Smac/DIABLO proteins released

from mitochondria after pro-apoptotic stimuli. Thus, Smac/

DIABLO is a negative regulator of IAPs [140, 162]. Simi-

larly, Omi/HtrA2 also inhibits the XIAP through a reaper-

like motif [148] and has a prognostic significance in hepa-

tocellular [144] and renal cell carcinoma [163]. Inhibition of

apoptosis increases the survival rate of cancer cells and

facilitates their escape from cytotoxic therapies and immune

surveillance [144, 164, 165].

Caspases and underlying mechanism in controlling

cellular apoptosis

Caspases are not only involved in the process of apoptosis

but also needed for the development and maturation of

cytokines leading to cell growth and differentiation [166].

Apoptotic cell death is dependent on a family of aspartate-

specific cysteine proteases (caspases) that cleave certain

vital structural proteins (e.g., lamins, gelsolin) and pro-

teolytically activate latent enzymes (e.g., nucleases) that

contribute to cell death. These enzymes exist in most cells

as inactive precursors (procaspases) that are converted into

their active forms by proteolytic cleavage at internal

aspartic acid residues, which separates the caspase into

small and large subunits [167] and then they become

activated by autoproteolysis.

Through genetic analysis of cell-death defective (CED)

mutants, it was found that the product of the aspartate-

directed cysteine protease (CED-3) is required for all

developmental-related programmed cell deaths in the

worm [168]. In this multicellular model, various genes that

encode for proteins essential for the regulation and exe-

cution of apoptosis were identified and their mammalian

homologs were described [33, 76]. For example, ced-9 and

Bcl-2 encode proteins that inhibit apoptosis, Apaf-1 and

ced-4 encode an adaptor protein that permits the interaction

between initiator proteins ced-3 and caspase-9. Ced-3

encodes a protein homologous to caspase-9 which is

responsible for the apoptotic initiation process. Fifteen

mammalian caspases have been identified, with caspase-11

and 12 identified only in mouse as shown in Table 1.

All caspases are synthesized as zymogens sharing a com-

mon domain structure consisting of a large (p10) and a small

(p20) catalytic subunit. Among two fundamentally different

groups, the first group, termed as initiator caspases, is char-

acterized by a long prodomain which provides a protein–

protein interaction platform [167, 169]. Prodomains allow the

recruitment of procaspases into an activating protein com-

plex. Long prodomain caspases are caspase-1, -2, -4, -5, -9,

-11, and -12 with an N-terminal caspase-activating recruit-

ment domain (CARD), and caspase-8 and -10 with an

N-terminal death effector domain (DED) (Table 1). In con-

trast to the initiator caspases, the second group consisting of

executioner caspases-3, -6, and -7 lack the large N-terminal

non-enzymatic domain and they are responsible for the

majority of cellular destruction during apoptosis [170]. When

procaspase-8 or -10 recruited to ligate death receptors by Fas-

Associated Death Domain (FADD), they undergo autocatal-

ysis, releasing the p10 and p20 subunits that form the active

(tetrameric) enzyme. Caspase-9 is activated in the presence of

ATP and cyt c by an allosteric change on a heptameric scaf-

fold of apoptotic protease-activating factor 1 (Apaf1) proteins

termed as apoptosome. Caspase-9 processing occurs

48 Mol Cell Biochem (2011) 351:41–58

123

secondary to caspase-3 activation during Smac-induced

apoptosis. In a heat shock-induced death, Caspase-2 induces

apoptosis via cleavage of Bid [171].

Interdependence of caspases and Bcl-2 in the regulation

of apoptosis

Numerous studies suggest that many apoptotic signaling

pathways converge at the mitochondria, where signals are

processed through a series of molecular events culminating

in the release of potent death factors that trigger either

through the extrinsic or the intrinsic pathway. Basically,

the release of the pro-apoptotic proteins from the inter-

membrane space triggers apoptosis, in a caspase-dependent

(through cyt c, Omi/HtrA2, and SMAC) as well as in a

caspase-independent form (through AIF and Endo G).

In mammalian cells, caspases-9, -8, and -2 rely on the

formation of apoptosome (Apaf-1), death-inducing signal-

ing complex (DISC), and PIDDosome, respectively, for

activation of apoptotic signals. Apoptosome is composed of

seven molecules (heptamer) of Apaf-1 bound to cyt c in the

presence of ATP/dATP, Fig. 2. DISC is assembled follow-

ing binding of death ligand to its receptor and contains

FADD and caspase-8 (or –10) whereas PIDDosome contains

at least three components, PIDD, RAIDD, and caspase-2.

Apoptosome complex

Apoptosome is a multimeric protein complex that mediates

activation of an initiator caspase at the onset of apoptosis.

Biochemical and structural investigations revealed

insights into the assembly and function of the various

apoptosomes from fruit fly (Drosophilia melanogaster),

worm (C. elgans) and mammals [172, 173]. The assembly

of the mammalian apoptosome which is responsible for the

activation of caspase-9 requires the binding of Apaf-1, cyt

c, and ATP/dATP. The apoptosome is an oligomeric sig-

naling platform that has a core of seven apoptotic Apaf1.

Each Apaf-1 monomer contains an N-terminal caspase

recruitment domain (CARD), a nucleotide-binding and

oligomerization domain (NOD), and a string of WD40

(tryptophan-aspartic acid) repeats at the C terminus

(Fig. 2). The WD40 repeats are thought to be the site of cyt

c binding [102, 174].

Death-inducing signaling complex (DISC)

DISC consist of complex of the death receptor (FAS), the

adaptor FAS-associated death domain protein (FADD), the

initiator caspase procaspase-8 (or procaspase-10), and

possibly other co-factors and regulators [175]. Kischkel

and coworkers reported the formation of a protein complex

in the dying cell and named it as DISC. Upon receiving the

death signal the activated death ligands homo-trimerizes,

which in turn, induces oligomerization of the Fas death

receptors (Fig. 2) [176, 177]. Dimerization of caspase-8 is

a crucial factor for activation and suggests that DISC may

facilitate the activation of caspase-8 through dimerization

[178]. The DISC creates a platform that brings together

molecules of the initiator caspase-8 (or caspase-10) and

other co-factors and regulators for execution of extrinsic

Table 1 Subfamily of caspase showing their functional role, functional domain and corresponding specificity to member of Inhibitor of

Apoptosis (IAP’s)

Sub-family Caspase Mammals Funtional domain Specificity to IAP’s

I- Caspase-2 Human CARD –

Apoptosis activator –

Caspase-8 Human DED XIAP, c-IAP1, c-IAP2, Ts-IAP, Ts-IAP

Caspase-9 Human DED –

Caspase-10 Human CARD –

II- Caspase-3 Human NED NAIP, XIAP, c-IAP1, c-IAP2, Survivin, livin

Apoptosis executioner Caspase-6 Human NED –

Caspase-7 Human NED NAIP, XIAP, c-IAP1, c-IAP2, Survivin, livin

III- Caspase-1 Human CARD –

Inflammatory mediator Caspase-4 Human CARD –

Caspase-5 Human CARD –

Caspase-11 Mouse CARD –

Caspase-12 Mouse CARD –

Caspase-13 Human CARD –

Caspase-14 Human CARD –

Caspase-14 Human CARD –

Caspase-Activating Recruitment domain (CARD), Death Effector Domain (DED), and Non-Enzymatic Domain (NED)

Mol Cell Biochem (2011) 351:41–58 49

123

apoptotic pathway which finally leads to their auto-acti-

vation [179, 180].

p53-Inducible death domain containing protein

complex (PIDDosome)

The PIDDosome complex under physiological conditions

contains PIDD and associate with the activation of another

initiator caspase, caspase-2 [181]. Although, the role of the

PIDDosome in apoptosis remains controversial, its

expression is inducible upon DNA damage [173, 181, 182].

However, PIDD-deficient mice undergo apoptosis not only

in response to DNA damage, but also in response to various

p53-independent stress signals and to death receptor

engagement. In the absence of PIDD, both caspase-2 pro-

cessing and activation occur in response to DNA damage

indicating that PIDD does not play an essential role for all

p53-mediated or p53-independent apoptotic pathways

[182]. The role of caspase-2 in the mitochondrial pathway

is now widely accepted [183–185]. Thus, the initial stage

of DNA damage facilitated by p53-mediated apoptosis

occurs by a PIDD and caspase 2-dependent mechanism.

For events that are downstream of cyt c release, p53’s full

transcriptional regulatory functions are required [186].

Apoptosis and the caspase-independent pathway

There also exists a caspase-independent apoptotic pathway

that is associated to AIF [131, 134], endonuclease G [187],

as well as Omi/HtrA2 [150–152]. As already discussed

above in this review, cells after receiving the apoptotic

signals release the nuclear AIF molecule and endonuclease

G protein which are translocated to the nucleus causing a

large-scale chromatin condensation and DNA fragmenta-

tion independently of caspase activation. Similarly Omi/

HtrA2 mediates caspase-independent cell death through its

own protease activity as can be observed during apoptosis.

A specific Omi/HtrA2 inhibitor can stop degradation of

THAP5 protein (THAP family of proteins), which leads to

reduced cell death [150]. A few studies have focused the

role of Heat Shock Proteins (HSPs) that are either consti-

tutively expressed or expressed under variety of stresses

stimuli [188], have shown their role in apoptosis via cas-

pase-independent pathway [189]. Members of HSP protein

such as HSP27 and HSP70 participate in oncogenesis,

probably by interfering apoptotic pathways. First, they act

as chaperones and play a role in proteasome-mediated

degradation of apoptosis-regulatory proteins. Second, they

inhibit key effectors of the apoptotic machinery including

the apoptosome and apoptosis-inducing factor [190].

However recent work on caspase-independent apoptotic

pathway has lead to the discovery of various other

molecules which has a major role in the pathway. For

example, the work by Yuan and his group have shown that

Ste20-like protein kinase 3 respond to apoptosis of HeLa

cells to trigger the caspase-independent apoptotic pathway

[191]. Similarly activated analog of CY, 4-hydroperoxy-

cyclophosphamide (4-OOH-CY) is being used for the

therapy for hematological malignancies and autoimmune

disorders through caspase-independent T-cell apoptosis

[192].

Therapeutic implication in the control of apoptosis

Lack of the phenomena of apoptosis results in excessive

increase of cell number and has implications in autoim-

munity and tumorigenesis. On the other hand, excessive

apoptosis decreases the cell population, which is linked to

many neurodegenerative disorders such as Parkinson’s

disease, Alzheimer’s disease, Huntington’s disease, and

spinal muscular atrophy. Alzheimer’s disease is a complex

neurological disorder in which the beta-amyloid peptides

are formed in the brain. In this, the Bcl-2 is down-regulated

and Bax is up-regulated [193, 194]. With respect to the role

of Bcl-2 proteins and its role in Parkinson’s disease, it has

been anticipated that pro-apoptotic family members par-

ticipate in neuronal death in a variety of Parkinson’s dis-

ease models [195]. Numerous studies show that activation

of apoptosis has also been found to be involved in the

pathogenesis of other human diseases such as chronic heart

failure [196], diabetes [197, 198], and atherosclerosis

[199]. The increased expression of Bcl-2 in the vascular

endothelium inhibits the diabetes-induced degeneration of

retinal capillaries and superoxide generation [198, 200].

An imbalance among the Bcl-2 family of proteins, in

favor of the anti-apoptotic members, is a phenomenon that

naturally, and frequently occurs in cancer cells [201–203].

Over-expression of anti-apoptotic Bcl-2 or Bcl-xl probably

occurs in more than half of all cancers. Moreover, loss of

expression of Bax is also found in some colorectal cancers

and in hematopoietic malignancies, whereas the expression

of a highly apoptogenic variant of Bax (Baxw) is correlated

with an increased survival of patients with glioblastoma

multiforme, an aggressive form of brain tumors [204]. The

defects may arise from the fact that neoplastic cells are

under strong selective pressure to stabilize their mito-

chondrial permeability, even if they harbor alterations in

the p53 tumor suppressor pathway. c-Myc, for instance,

can induce mitochondrial damage independently from the

transcriptional activity of p53. Since p53 also activates the

mitochondrial death pathway, the mitochondrion appears to

integrate the diverse pro-apoptotic mechanisms induced by

oncogenes. Studies in transgenic mice have revealed that

Bcl-2 (and/or Bcl-xl) over-expression and p53 mutations

50 Mol Cell Biochem (2011) 351:41–58

123

(or ARF loss) are selected independently during Myc-

induced lymphomagenesis [205–207].

The emerging knowledge about proteins that are

involved in apoptosis, including their 3-D structures and

biochemical mechanisms, has provided therapeutic ave-

nues by discovering molecules or targets which may

modulate apoptosis. A number of therapeutic approaches

are undergoing using an antisense RNA [208], various

small molecules [209–218, 222, 223], and peptidic com-

pounds [219–221, 224, 225] classified as potential thera-

peutics to target the pathway of apoptosis, as briefly

summarized in Table 2. Most of these therapeutics involve

the targeting of structurally defined multidomain of the

member of Bcl-2 family of protein. Some of these mole-

cules/agents are in clinical trials.

The combination of IAP antagonists with drugs that

target ErbB receptors promotes apoptosis thereby reduces

the cell turnover of breast cancer cells [226]. The apoptotic

response to most chemotherapeutic drugs in mammalian

cells involves the induction of mitochondrial pathway in

which mitochondrial membrane permeabilization con-

trolled by the Bcl-2 protein family, is induced. Other

strategies include the IAP proteins as therapeutic targets

that are expressed in the majority of human tumor through

the inhibition of cellular death and participation in sig-

naling pathways associated with malignancies [157, 227].

Further more, Table 2 has summarized the list of antisense

[228], small molecules [229–235], and peptidic compounds

[234, 236, 237] that are under investigation to develop

potential therapeutics against IAPs protein which are

involved in cancer.

Acknowledgments MSO and MN thank Medical Research Chair in

Ophthalmology funded by Dr. Nasser Al-Rasheed, College of Med-

icine, Kind Saud University for support. HA would like to thank Dr.

Nihal Ahmad, School of Medicine and Public Health, University of

Wiscosnsin, Madison for a cancer cell biology research fellowship.

The authors would also like to thank Ms. Crisalis Longanilla-Bautista

and Mr. Miaraj Siddiquei in helping with figures and proof reading.

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