Protein Function

10 Major Functions of Bcl-2

Regulates Apoptosis (21)
Regulating Calcium Homeostasis (21)
Neuronal activity (2)
Autophagy (20)
Mitochondria dynamics and energetics (20)
Binding (20)
- Protease binding
- Ubiquitin protein ligase binding
- transcription factor binding
- sequence specific DNA binding
- Protein phosphatase 2A binding
- Idential protein binding
- BH3 domain binding
Channel Inhibition/Activation (20)
Protein Homodimerization (20)
Protein Heterodimerization (20)

Apoptotic Pathway

Apoptosis, or programmed cell death, is a crucial biological event that happens thousands upon thousands of times daily in each one of our bodies. Apoptosis pathways have been well studied over the latter of the last several decades as they play a critical role in many diseases and disorders, most noticeably cancer. Bcl-2 proteins are apoptotic regulatory proteins found in mammals with conserved machinary spanning to organisms such as C. elegans, which is where their function were first discovered. (22)

C. Elegans and BH3 Only Proteins

Detailed genetic studies on C. elegans have illustrated that two Bcl-2 related proteins, EGL-1 and CED-9, are critical for controlling the programmed cell death of somatic cells. EGL-1 is expressed as a death activator in response to damage signals by the cell. Binding of EGL-1 to CED-9, which is the Bcl-2 ortholog of the worm, causes a release of the adapter protein CED-4. The release of CED-4 binds to and activates caspase CED-3 to induce apoptosis. This relates to the Bcl-2 proteins containing BH3 only domains. The Bcl-2 protein acts as CED-9 does, forming a complex with the mammalian versions of EGL-1; Bik/Nbk/Blk, Bid, Bad, Hrk/DP5, Bim/Bod, Noxa, Puma/Bbc3, Bmf and Bcl-G . This breaches outer mitochondrial membranes and allows cytochrome c to complex with APAF-1 forming "apoptosome", the mammalian CED-4 ortholog. This activates caspase-9 and other downstream caspase's which degrade cellular organelles and induce cellular apoptosis. (22)

Bcl-2 Pro-Survival Proteins

BH3 only proteins are pro-apoptotic molecules that induce apoptosis via the eventual activation of caspase enzymes. However, Bcl-2 proteins can function as pro-survival molecules by their ability to inhibit the actions of BH3 only proteins which are potent death domain containing. Bcl-2 proteins actually bind to BH3 domains to inhibit their pro-apoptotic actions. This does not allow pro-apoptotic proteins to induce apoptosis to breach mitochondrial membranes. Without the breach of mitochindrial membranes, cytochrome c cannot be released to form to apoptosome with APAF-1, never activating the capase enzymes for degradation. (21,22)

Fig.9 Intrinsic apoptotic pathway most closely related to BCl-2 proteins. The red arrow indicates the inhibition of BH3 only proteins of Noxa, Puma, Bad, Bim, Bid by Bcl-2 to inhibit the permeabilization of mitochondria to inhibit the release of cytochrome c. 5) indicates the formation of of the apoptosome complex that is blocked by bcl-2 to prevent the activation of caspase 9 and eventually of caspase 3, the enzyme that induces organelle degradation. (21)

Calcium Homeostasis Regulation

Bcl-2 has been a main focus as an apoptotic regulator but serves many other functions (listed below). Within in the last 8 years, Bcl-2 has gained attention as a regulating factor in calcium homeostasis, specifically regarding the endoplasmic reticulum (ER) and mitochondria. Newer studies indicate that Bcl-2 actually regulates ER calcium levels both directly and indirectly, actually having the ability to inactivate the Sarcoplasmic/endoplasmic Reticulum calcium pump SERCA of muscle cells (23) and SERCA2b levels in the non muscle isoforms(24). The ability of Bcl-2 to regulate calcium homeostasis has also been cited to be related to it's pro-survival actions. Several studies have illustrated it's pro-survival actions at the ER relate to its ability to increase ER Ca²⁺ leak and causing low-basal ER Ca²⁺ concentrations. This has been cited as a pro-survival mechanism as it reduces stress-induced ER Ca²⁺ release which can occur when a cell is undergoing apoptosis (25,26). Bcl-2 has also been found to reduce this stress-induced ER Ca²⁺ release by inhibiting IP3R- a membrane glycoprotein complex that acts a calcium ion channel when activated by Inositol Triphosphate. Bcl-2 binds with IP3R due to the presence of its BH4 domain that has an affinity for the regulatory and coupling domain of the IP3R leading to a reduction in stress-induced IP3R Ca²⁺ release and an increase in cytosolic calcium levels (27).

Implications for Anti-Apoptotic Activity'

The ability of Bcl-2 to work on calcium homeostasis is tied to it's anti-apoptotic activity by relieving stress induced calcium release from the ER. Stress-induced calcium release from the ER is a step associated with apoptosis from the prolonged expression of a transcription factor called CHOP. Although how this machinery induces apoptosis is not exactly known, it's trigger is calcium release from the ER, allowing Bcl-2 to act as an inhibitor to this apoptotic process. (28)

Fig. 10 Diagram showing how calcium homeostasis regulation plays a role in apoptosis. Shown is how the presence of Bcl-2 reduces stress-induced calcium release by the ER and allows mitochondria to function normally avoiding apoptosis(28)

Major Biochemical Pathways

Sub-Cellular Localization

Bcl-2 has been found to be localized in(8,9):

Outer Mitochondrial Membrane
Smooth Endoplasmic Reticulum Membrane
Nuclear Envelope

Fig.11 Sub-cellular localization of Bcl-2 (8)

The subcellular localization of the Bcl-2 protein is related to its function as an apoptosis regulator. Its function on the outer mitochondrial membrane relates to its mechanism in forming heterodimer complexes with other Bcl-2 family proteins responsible for apoptosis. Since the intrinsic apoptotic pathway it acts on relies upon the permeabilization of the outer mitochondrial membrane to release cytochrome c, its location at the membrane is critical to its function. Here, is where the bcl-2 protein will exhibit the majority of its anti-apoptotic function(22). Localization in the ER is related to its anti-apoptotic properties that are related to the calcium release associated with apoptotic pathways. Referenced under calcium homeostasis, the localization at this organelle allows it to inhibit stress-induced calcium release. Bcl-2 is also contained in the nuclear envelope due to its association with the smooth ER(23).

Other Major Protein Interactions

Bcl-2 Homologous antagonist Killer (BAK1)

A pro-apoptotic Bcl-2 family protein that contains all four Bcl-2 homologous domains and complexes with Bcl-2 to form a heterodimer complex that inhibits it's anti-apoptotic activity. This is due to it's ability to induce apoptosis by not allowing it to permeabilize the outer mitochondrial matrix(20)

(30)

Bcl-2-associated death promoter (BAD)

A pro-apoptotic protein that contains only a BH3 binding domain without containing a a c-terminal transmembrane domain that can target outer mitochondrial membranes and nuclear envelopes. Once activated it can form a heterodimer with anti-apoptotic proteins, such as Bcl-2, to prevent them from inhibiting apoptosis (20).

(30)

Bcl-2-like protein 11 (Bcl2L11)

Commonly referred to as BIM, this protein is encoded by the Bcl2L11 gene and is part of the Bcl-2 family of proteins. It only contains a BH3 domain leading it to be a pro-apoptotic protein that complexes with Bcl-2 to have its anti-apoptotic function inhibited. Cancer cells suppress BIM activation (20).

(30)

Apoptosis Regulator BAX (Bcl-like-protein 4)

The BAX protein is an apoptotic activator when it forms a heterodimer with Bcl-2 inhibiting its anti-apoptotic activity and causing it to open the mitochondrial voltage-dependent anion channel (mvda). This causes a loss in membrane potential and the release of cytochrome c to induce apoptosis. The BAX gene is known to be regulated by tumor suppressor p53 (20)

(30)

Caspase 9

Caspase 9 is protein that acts a protease and is an initiator in the intrinsic apoptotic pathway. Apoptotic signals result in the release of cytochrome c from the mitochondria and result in the activation of the apoptosome when it activates APAF-1. This then cleaves the pro-enzyme of caspase-9 to result in it's active dimer form and activating caspase-3. This process is blocked by Bcl-2 anti-apoptotic proteins which never allow for the release of cytochrome c (29)

(30)

Tissues Where Protein Is Expressed(2):

thyroid gland
appendix
bone marrow
lymph node
tonsil
spleen
nasopharynx
bronchus
prostate
epididymis
seminal cesicles
fallopian tube
endometrium
ocary
placenta
skin
parathyroid gland
smooth muscle
colon
rectum

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