Bcl-2 Family Proteins

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Bcl-2 Family Proteins

Walid Nabil FouadMicrobiology Department

Medical Research Institute - AlexandriaUniversity


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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.


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1.What is Apoptosis? 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 onprogrammed cell death (anti-apoptotic) or block the protective

effect of inhibitors (pro-apoptotic).


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3.How Apoptosis works? Apoptosis manifests in two major execution programs

downstream of the death signal:

(1) Caspase Pathway:A family ofcysteine 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.


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How Apoptosis works? (Cont.)

(2) Mitochondrial Dysfunction:

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

A change in the mitochondrial membrane potential. Production ofreactive oxygen species (ROS). Opening of the permeability transition pore (PTP), and Release of the intermembrane space protein,

cytochrome c(Cyt c).

Released cytochrome cactivates Apaf-1, which in turnactivates a downstream caspase program.


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4.Apoptosis and Bcl-2 proteins: Although caspases represent a central point in apoptosis, their

activation is in turn regulated by theBcl-2family proteins.

The main function of the Bcl-2 proteins is to act as "central

regulators of caspase activation", deciding whether a cell willlive or die.

Bcl-2 proteins also play a key role in cell death by regulatingthe integrity of the mitochondrial and endoplasmicreticulum (ER) membranes.

The following diagram illustrates how Bcl-2 family proteinsregulate apoptosis:


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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 forcells.

Bcl-2 derives its name from B-cell lymphoma 2, as it is thesecond member of a range of proteins initially described as areciprocal gene translocation in chromosomes 14 and 18 infollicular lymphomas.

The Bcl-2 family of proteins has expanded significantly (at least20 Bcl-2 proteins have been reported in mammals, andseveral others have been identified in viruses).


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1.What are Bcl-2 proteins? 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 theabsence of a death signal. For example:


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2.Anti-apoptotic vs. Pro-apoptotic: Anti-apoptoticmembers are initially integral membrane

proteins found in the mitochondria, endoplasmic reticulum

(ER), or nuclear membrane.

In contrast, a substantial fraction of the pro-apoptoticmembers localize to cytosolorcytoskeleton prior to a

death signal.

Following a death signal, the pro-apoptotic members undergo

a conformational change that enables them to target andintegrate into membranes, especially the mitochondrial outer

membrane.


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3.Structure of Bcl-2 proteins: 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 -helical segments.

One of the striking features of Bcl-2 family proteins is theirability to form homodimers and heterodimers.


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4.Classification of Bcl-2 family proteins: 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 homologyparticularly 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 somemembers. All these proteins exert pro-apoptotic activity.


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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).


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5.Types of Bcl-2 proteins: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 releaseof mitochondrial apoptogenic factorssuch as cytochrome c and AIF (apoptosis-inducing factor) into the

cytoplasm, or

Sequestering caspase activation.


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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

(thefirst 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.


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6.How Bcl-2/Bcl-xL work?

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 areschematically 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 wellas in the endoplasmic reticulum membrane and the nuclear envelope.


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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 cytochromec and apoptosis-inducing factor (AIF) from the mitochondrial inter-membrane space into the cytoplasm.

Once cytochrome c and AIF reach the cytoplasm, they directlyactivate caspases. The release of AIF is dependent upon theoccurrence of mitochondrial dysfunction, including membranepotential loss and the membrane permeability transition (PT), andBcl-2/Bcl-xL prevents these types of mitochondrial

dysfunction by as yet unidentified mechanisms.


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It is thought that Bcl-2 and Bcl-xL efficiently inhibit all of themitochondrial changesinduced by Bax and Bak

proteins in in vitro systems.

Bcl-2 might prevent PT partly byantagonizing Bax.

It has also been suggested thatBcl-2 enhances proton efflux

across the mitochondrialinner membrane to preventpotential loss and PT.


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B- Sequestration of caspases via formation ofapoptosomes by Bcl-2/Bcl-xL:

Another mechanism by which Bcl-2/Bcl-xL prevent the activation ofcaspases is through their abilities to sequester pro-caspases.

The mechanism by which caspases are activated after cytochrome crelease has been well established from the in vitro studies:

Cytochrome c, which is released to the cytoplasm, binds to acytoplasmic protein Apaf-1 via the C-terminal WD-40 repeatdomain in the presence of ATP or dATP.

The resulting complex recruits procaspase-9 (through homophilicassociation of the N-terminal CARDs caspase recruitmentdomains of procaspase-9 and Apaf-1), inducing the self-cleavage/activation of caspase-9.


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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.


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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 maypromote sustained cell death during both acute injuries and

chronic degenerative disorders.


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7.How are pro-apoptotic members activated?

Activation of the pro-apoptotic molecule Bax appears to involvesub cellular translocation and dimerization.

(1) In viable cells a substantial portion of Bax is monomericand found either in the cytosol orloosely attached tomembranes.

(2) Following a death stimulus, cytosolic and monomeric Baxtranslocates to the mitochondria where it becomes an integralmembrane protein and cross-linkable as a homodimer.


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8.How Bax works?

Bax has been shown to interact with the mitochondrial PT pore.Although Bax-mediated cytochrome c release is dependenton PT pore opening, it is still unclear exactly how cytochromeis released.

It has been suggested that cytochrome c release occur throughouter membrane rupture resulting from mitochondrialswelling caused by PT pore opening.

It has also been suggested that Bax might form large pores

through which cytochrome c is able to pass.


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Bcl-2 Family Proteins