Bcl-2 Family Proteins

Walid Nabil Fouad

Microbiology Department
Medical Research Institute - Alexandria
University
:INTRODUCTION

“Bcl-2 family proteins are proteins

responsible for constituting a ‘life or death’
decision point for cells. Their main function
is to regulate the process of apoptosis.”
?What is Apoptosis. 1
 Apoptosis (also known as: Programmed Cell Death - PCD) is a
cellular self-destruction mechanism involved in a variety of bio-
logical events, such as developmental sculpturing, tissue homeo-
stasis, and selective removal of ‘unwanted’ cells (such as pre-
cancerous cells).

 Apoptosis is usually induced by events such as growth factor

withdrawal and toxins.

 It is controlled by regulators, which have either an inhibitory effect on

programmed cell death (anti-apoptotic) or block the protective
effect of inhibitors (pro-apoptotic).
:Significance of Apoptosis. 2
 Apoptosis is an essential physiological process that plays a
very important role in regulating a variety of diseases that
have enormous social impacts. For example: Disruption of the
regulation of apoptosis may result in various diseases,
including:

• Cancer associated with the inhibition of apoptosis, and

• Neurodegenerative disorders (e.g. Schizophrenia)
associated with the enhancement of apoptosis.
?How Apoptosis works. 3
 Apoptosis manifests in two major execution programs
downstream of the death signal:

(1) Caspase Pathway:

A family of cysteine proteases, called caspases, are activated
in a cascade manner, and represent the key component
responsible for the mechanism of apoptosis. These caspases
are the ones responsible for the apoptotic-specific changes,
such as cleavage of critical cellular proteins, and cell
disassembly, leading to apoptosis.
.)How Apoptosis works? (Cont

(2) Mitochondrial Dysfunction:

The organelle dysfunction, of which mitochondrial dysfunction
is the best characterized includes:

• A change in the mitochondrial membrane potential.

• Production of reactive oxygen species (ROS).
• Opening of the permeability transition pore (PTP), and
• Release of the intermembrane space protein,
cytochrome c (Cyt c).

Released cytochrome c activates Apaf-1, which in turn

activates a downstream caspase program.
:Apoptosis and Bcl-2 proteins. 4
 Although caspases represent a central point in apoptosis, their
activation is in turn regulated by the Bcl-2 family proteins.
 The main function of the Bcl-2 proteins is to act as "central
regulators of caspase activation", deciding whether a cell will
live or die.

 Bcl-2 proteins also play a key role in cell death by regulating

the integrity of the mitochondrial and endoplasmic
reticulum (ER) membranes.

 The following diagram illustrates how Bcl-2 family proteins

regulate apoptosis:
:BCL-2 FAMILY PROTEINS
 The Bcl-2 family is the best characterized protein family
involved in the regulation of apoptotic cell death. And thus,
responsible for constituting a "life or death" decision point for
cells.

 Bcl-2 derives its name from B-cell lymphoma 2, as it is the

second member of a range of proteins initially described as a
reciprocal gene translocation in chromosomes 14 and 18 in
follicular lymphomas.

 The Bcl-2 family of proteins has expanded significantly (at least

20 Bcl-2 proteins have been reported in mammals, and
several others have been identified in viruses).
?What are Bcl-2 proteins. 1
 The Bcl-2 family proteins include both pro-apoptotic as well as
anti-apoptotic molecules.

 Indeed, the ratio between these two subsets helps determine,

in part, the susceptibility of cells to a death signal. Thus, the
balance between antagonistic family members is believed to
play a role in determining cell fate.

 Considerable portions of the pro- vs. anti-apoptotic Bcl-2

members localize to separate sub-cellular compartments in the
absence of a death signal. For example:
:Anti-apoptotic vs. Pro-apoptotic. 2
 Anti-apoptotic members are initially integral membrane
proteins found in the mitochondria, endoplasmic reticulum
(ER), or nuclear membrane.

 In contrast, a substantial fraction of the pro-apoptotic

members localize to cytosol or cytoskeleton prior to a
death signal.

 Following a death signal, the pro-apoptotic members undergo

a conformational change that enables them to target and
integrate into membranes, especially the mitochondrial outer
membrane.
:Structure of Bcl-2 proteins. 3
 In other words, we can say that the anti-apoptotic Bcl-2
molecules are “guarding the mitochondrial gate” from the
pro-apoptotic Bcl-2 members that “gain access” following a
death signal.

 Bcl-2 family members possess up to four conserved Bcl-2

homology (BH) domains designated BH1, BH2, BH3, and BH4,
which correspond to α-helical segments.

 One of the striking features of Bcl-2 family proteins is their

ability to form homodimers and heterodimers.
:Classification of Bcl-2 family proteins. 4
 The Bcl-2 family is classified into the following three subfamilies
depending on the homology and functions of each protein:

(i) A subfamily including Bcl-2, Bcl-xL and Bcl-w, all of which

exert anti-cell death activity and share sequence homology
particularly within four regions, BH1 through BH4.

(ii) A subfamily represented by Bax and Bak, which share

sequence homology at BH1, BH2 and BH3 but not at BH4,
although significant homology at BH4 is also noticed in some
members. All these proteins exert pro-apoptotic activity.
.)Classification of Bcl-2 family proteins (Cont

(iii) A subfamily including Bik

and Bid, all of which are pro-
apoptotic and share sequence
homology only within BH3 (for
this reason, the members of this
subfamily are called BH3
proteins).
:Types of Bcl-2 proteins. 5
A. Anti-apoptotic Members
 The anti-apoptotic members of this family, include Bcl-2, Bcl-xl
and Bcl-w.

 These protein molecules prevent apoptosis either by:

• Preventing the release of mitochondrial apoptogenic factors

such as cytochrome c and AIF (apoptosis-inducing factor) into the
cytoplasm, or
• Sequestering caspase activation.
:Anti-apoptotic Members

 Over-expression of these "pro-survival" members is associated

with tumor progression. Abnormal expression, on the other
hand, may result in the development of some neuro-
degenerative disorders.

 The most common example of the anti-apoptotic proteins is the Bcl-2

(the first anti-cell death gene).

 Bcl-2 was originally identified as an oncogene involved in human

follicular lymphoma of B cell origin.

 Bcl-2 was shown to prevent apoptosis induced by various stimuli,

including serum deprivation, heat shock, and chemotherapeutic
reagents.
?How Bcl-2/Bcl-xL work. 6
 Bcl-2 prevents cell death by blocking a step which leads to the
activation of caspases.

 Concerning how Bcl-2 prevents the activation of caspases, basically

two independent mechanisms have been considered, which are
schematically summarized in the following figure.

A- Prevention of the release of mitochondrial apoptogenic

factors by Bcl-2/Bcl-xL:

 Bcl-2 and Bcl-xL are localized in the mitochondrial membrane as well

as in the endoplasmic reticulum membrane and the nuclear envelope.
.)How Bcl-2/Bcl-xL work? (Cont

 Bcl-2 and Bcl-xL in the mitochondria prevent the apoptosis-

associated release of apoptogenic factors, such as cytochrome
c and apoptosis-inducing factor (AIF) from the mitochondrial inter-
membrane space into the cytoplasm.

 Once cytochrome c and AIF reach the cytoplasm, they directly

activate caspases. The release of AIF is dependent upon the
occurrence of mitochondrial dysfunction, including membrane
potential loss and the membrane permeability transition (PT), and
Bcl-2/Bcl-xL prevents these types of mitochondrial
dysfunction by as yet unidentified mechanisms.
.)How Bcl-2/Bcl-xL work? (Cont

 It is thought that Bcl-2 and Bcl-

xL efficiently inhibit all of the
mitochondrial changes
induced by Bax and Bak
proteins in in vitro systems.
 Bcl-2 might prevent PT partly by
antagonizing Bax.
 It has also been suggested that
Bcl-2 enhances proton efflux
across the mitochondrial
inner membrane to prevent
potential loss and PT.
.)How Bcl-2/Bcl-xL work? (Cont

B- Sequestration of caspases via formation of

apoptosomes by Bcl-2/Bcl-xL:

 Another mechanism by which Bcl-2/Bcl-xL prevent the activation of

caspases is through their abilities to sequester pro-caspases.
 The mechanism by which caspases are activated after cytochrome c
release has been well established from the in vitro studies:

• Cytochrome c, which is released to the cytoplasm, binds to a

cytoplasmic protein Apaf-1 via the C-terminal WD-40 repeat
domain in the presence of ATP or dATP.
• The resulting complex recruits procaspase-9 (through homophilic
association of the N-terminal CARDs ‘caspase recruitment
domains’ of procaspase-9 and Apaf-1), inducing the self-
cleavage/activation of caspase-9.
.)How Bcl-2/Bcl-xL work? (Cont

 Based on the observations that Bcl-xL binds indirectly to

both pro-caspase-8 and to the Apaf-1/pro-caspase-9
complex (this complex is called an ‘apoptosome’), it has been
proposed that Bcl-xL sequesters caspases to prevent their
activation.

B. Pro-apoptotic Members
 In contrast to anti-apoptotic members, pro-apoptotic members
of this family, such as (Bax, BAD, Bak and Bok), trigger the
release of caspases from death antagonists via
heterodimerization.
:Pro-apoptotic Members

 Pro-apoptotic proteins also induce the release of mitochon-

drial apoptogenic factors (cytochrome c and AIF) into the
cytoplasm via acting on mitochondrial permeability transition
(PT) pore.

 After entering the cytoplasm, cytochrome c and AIF directly

activate caspases that cleave a set of cellular proteins to
cause apoptotic changes.

 Over-expression of these pro-apoptotic members may

promote sustained cell death during both acute injuries and
chronic degenerative disorders.
?How are pro-apoptotic members activated. 7
 Activation of the pro-apoptotic molecule Bax appears to involve
sub cellular translocation and dimerization.

(1) In viable cells a substantial portion of Bax is monomeric

and found either in the cytosol or loosely attached to
membranes.

(2) Following a death stimulus, cytosolic and monomeric Bax

translocates to the mitochondria where it becomes an integral
membrane protein and cross-linkable as a homodimer.
?How Bax works. 8
 Bax has been shown to interact with the mitochondrial PT pore.
Although Bax-mediated cytochrome c release is dependent
on PT pore opening, it is still unclear exactly how cytochrome
is released.

 It has been suggested that cytochrome c release occur through

outer membrane rupture resulting from mitochondrial
swelling caused by PT pore opening.

 It has also been suggested that Bax might form large pores
through which cytochrome c is able to pass.
THANK YOU