Sandbox Reserved 1122

Human Bcl-2 (B-Cell Lymphoma 2), isoform 1 is an oncoprotein of 239 residues regulating cell death (apoptosis), notably acting as an anti-apoptotic. It is encoded by the BCL-2 gene located on the 18th chromosome (63.12-63.32 Mb). There are 2 isoforms of this protein (1 and 2), produced by alternative splicing, and which differ by 2 amino acids (residues 96 (A→T) and 110 (R→G))^[1]. Alteration of this protein is the cause of many cancers, and is also likely to be involved in schyzophrenia and autoimmunity.

Structure

Human Bcl-2, isoform 1 is a 26kDa protein of 239 residues which is negatively charged at pH 7. The linear structure highlights 5 domains: (10-30), (93-107), (136-155), (187-202) and a transmembrane domain (212-233) (not present in the presented 3D structure because of the poor behaviour in solution of the protein containing the transmembrane region). It organizes as eight alpha-helices : from 11 to 25 (α1) , from 93 to 107 (α2), from 109 to 118 (α3), from 126 to 137 (α4), from 144-163 (α5), from 169 to 184 (α6), from 186 to 191 (α7) and from 194 to 202 (α8) and there are also 3 turns (32-34, 123-125, 138-140). The is a 3(10) helix. ^[2]

Helices 5 and 6 are mostly hydrophobic and they are surrounded by four other helices characterized by their amphipathic properties. This type of structure with alpha-helices is also encountered in some bacterial toxins such as diphteria toxin where it permits to form pores in membranes. This similarity of arrangement suggests that Bcl-2, as well as diphteria toxin, is able to interfere in permeability of organelles like endoplasmic reticulum or mitochondria. ^[3]

Position of the BH (Bcl-2 homology) and Transmembrane (TM) domains on the linear structure of Bcl-2.^[4]

The transmembrane domain of Bcl-2 is made of 21 amino acids and is located at the carboxy-terminal tail of the protein. It allows the docking of Bcl-2 in the membrane of cellular organelles especially mitochondria and endoplasmic reticulum. The localization at the membrane permits an efficient interaction with effectors.^[5]

The tertiary structure of Bcl-2 shows that this protein contains a hydrophobic groove made of the BH1, BH2 and BH3 domains on its surface that allows dimerization with other members of the Bcl-2 family. This region needs to be highly conserved to keep the ability of interacting with the BH3 domain of the proapoptotic protein of the family, in fact, it has been shown that a mutation in this structure leads to the silencing of the dimerization thus may inhibit the activity of Bcl-2. The isoform 1 and 2 differs from two amino acid in the hydrophobic groove but this difference doesn’t induce any change in the conformation. However as expected, it affects the affinity with Bad and Bak proteins (from the Bcl-2 family). Indeed Bcl-2 isoform 1 shows to have a weaker affinity for Bad and Bak compared to isoform 2.^[6]

Moreover, Gly145 in the BH1 domain and Trp188 in the BH2 domain of Bcl-2 are also clue residues for the interaction between the hydrophobic pocket and the BH3 domain of the partner. In fact it has been shown that a mutation on one of these two amino acids suppress such interaction.^[7]

Function

Bcl-2 is mainly found attached to the membranes through its C-terminal transmembrane domain. It can be anchored to the nucleus, the endoplasmic reticulum (ER) or the mitochondrion. It normally acts as an antiapoptotic protein and the way it works depends on its localization.

IP3R inhibition

Bcl-2 localized at the ER membrane participates in the control of Ca2+ content and release. The inositol 1,4,5-trisphosphate receptor (IP3R) is the Ca2+ release channel localized in the ER. Its pro-apoptotic activity can be directly inhibited by Bcl-2, its homology domain 4 (BH4) being essential for this effect. comprises 20 amino acids organized in alpha-helical structure which is required to inhibit IP3R. Residues K17, H20, Y21 and R26 participate in the inhibition of IP3R because they are very accessible and proximal in the secondary structure. ^[8]

Regulation of the mitochondrial pathway of apoptosis

BH3-only proteins which belong to the Bcl-2 family activate pro-apoptotic proteins such as Bcl-2-associated X protein (Bax) or Bcl-2 antagonist/killer-1 (Bak) at the mitochondrion. When Bax or Bak are activated, they homo-oligomerize and form pores in the outer mitochondrial membrane which are necessary for the pro-apoptotic molecules (including second mitochondria-derived activator of caspase and cytochrome c) to escape. Then cytochrome c leads to the activation of caspases which are actually proteases that degrade the key proteins of the cell.

On the other hand, Bcl-2 may prevent the activation and homo-oligomerization of Bax and Bak thus blocking the cell death. This is achieved by sequestering BH3-only proteins or activated and monomeric Bax and Bak. and are essential for Bcl-2/Bax heterodimer formation. The conservation of each amino acid seems to be very important to this interaction. ^{[9] [10]}

Therefore, the neutralization of Bcl-2 is required for efficient cell-death. BH3 proteins Bad, Bim and Puma bind Bcl-2 and disable its anti-apoptotic activity. 4 hydrophobic residues of BH3 peptides occupy the hydrophobic pocket of Bcl-2 and the sequestered pro-apoptotic proteins are released. This hydrophobic pocket is formed by F97 and Y101 and conserved residues Asp in BH3 and Arg in the BH1 of Bcl-2 form a salt bridge which strenghtens the interaction. ^{[11] [12] [13]}

Bcl-2 localized on external mitochondrial membrane can also inhibit the release of cytochrome c from mitochondria. ^{[14] [15]}

Regulation of proinflammatory caspase-1 activation

NALP1 is a member of a NLR-family proteins. Its function is to activate the members of the proinflammatory caspase family which participate in the cytokine activation pathway (especially caspase-1). The Bcl-2 loop regions between the and bind NALP1. This interaction is exclusively reserved to two members of Bcl-2 family: Bcl-XL and Bcl-2 itself because this interacting region is poorly conserved in Bcl-2 family. By binding to NALP1, Bcl-2 inhibits the inflammatory caspase activation. Hence, it protects cells from the stress. The posttranslational modifications found on the loops (rich in Ser and Thr residues) between and modify the anti-apoptotic activity of Bcl-2. Hence, the Bcl-2 binding to NALP1 can be affected by these modifications. ^[16]

Interaction with c-Myc

When Bcl-2 is anchored to nuclear membrane, it can interact with nuclear proteins, for example, transcription factors such as c-Myc. domain of Bcl-2 binds MBII domain of c-Myc. This interaction has several consequences: increase of half-life of c-Myc and its transcriptional activity and inhibition of DNA repair (through down-regulation of AP endonuclease (APE1) expression). Interestingly, c-Myc can also be found in cytoplasm where it is able to interact with mitochondrial Bcl-2.

Inhibition of autophagy

Autophagy is a very conserved event during which different cytoplasmic organelles and other structures are delivered to the lysosome where they are recycled. This mechanism may enhance cell survival because it is responsible for getting rid of intracellular pathogens, toxic molecules or damaged organelles. On the other hand, if autophagy is excessive, it may lead to the cell death. This process has many regulatory pathways including the interaction between the autophagy protein Beclin 1 and Bcl-2. Only the Bcl-2 which is localized in the ER is capable to bind Beclin 1 through its and domains. Normally Beclin 1 forms a complex with hVps34 (Class III PI3K). This complex is important for the localization of other autophagy proteins in the lysosome membrane. It is one of the first steps in the autophagosome formation and it can be inhibited by Bcl-2. At normal conditions this interaction is minor but it increases when the cell is starving. Thus, Bcl-2 prevents starvation-induced autophagy. ^[17]

Cell cycle control

Bcl-2 is able to exert an anti-proliferative activity which is completely independant from its anti-apoptotic activity. In quiescent cells, Bcl-2 is responsible for faster G0/G1 arrest and for slower G0-G1/S transition. Posttranslational phosphorylations are very important for Bcl-2 anti-proliferative activity, for example, phosphorylation at Thr 56 by CDK1 delays G2/M exit. and unstructured loop between and are crucial for this cell cycle control activity. Tyr28 is critical for anti-proliferative activity, a point mutation reduces the ability of Bcl-2 to block the re-entry of quiescent cells. Inhibition of G0-G1/S is a consequence of increased levels of p130 and p27 which are regulated by Bcl-2. Bcl-2 also inhibits Raf-1 which normally activates ERK. ERK then inhibits Rb and this allows cell to pass the restriction point which leads to G1. Bcl-2 anti-proliferatif activity together with prevention of starvation-induced autophagy allow cells to survive in poor conditions. ^[18]

Diseases

Cancer

Bcl-2 is involved in many cancers such as breast, melanoma, prostate, chronic lymphocytic leukemia and lung cancers. In fact, its overexpression combined with an overexpression of c-Myc (another oncoprotein) produce aggressive B-lymphocytes. ^[19]. It has also be shown that Bcl-2 overpexpression supresses DNA repair by enhancing Myc transcriptional activity. ^[20] In fact, Bcl-2 is encoded by the BCL-2 gene (0,20 Mb) located on the 18th chromosome. Nevertheless, translocation between chromosome 14 and 18 juxtaposes the BCL-2 gene and the immunoglobulin locus. This brings the BCL-2 gene under the regulation of the immunoglobulin heavy-chain enhancer, diregulating BCL-2 expression level (involved in non-Hodgkin's lymphomas). ^[21] Other mechanisms appear to regulate and enhance the level of expression of BCL-2 : loss of endogenous microRNAs which normally repress Bcl-2 expression, and also hypomethylation.^[22]

Bcl-2 domain mediates the interaction with MBII domain of Myc. This interaction enhance c-Myc half-life, resulting in an enhancing of its total activity. c-Myc is a well known oncoprotein (transcription factor) involved in many cancers as it regulates a lot of genes of the cell cycle.

Autoimmunity

Bcl-2 family members and notably Bcl-2 protein seem to play an important role in autoimmune diseases, along with the gene Bim. This gene encodes a protein Bcl-2L11 (Bcl-2 like) belonging to the Bcl-2 family. It controls the apoptosis of T and B lymphocytes as a proapoptotic. When mutated, the loss of apoptotic activity in the lymphocytes population can lead to an accumulation of aggressive lymphocytes developing autoantibodies resulting in autoimmune disorders in certain genetic background. ^[23]; In fact, a selection process of T-cells occur at several levels (for example when entering the thymus). Only 5% of these cells survive. When Bim is inactive, this proportion is much higer due to overexpression of Bcl-2 and lack of Bim proteins.^[24] Bcl-2 overexpression is also involved in the inhibition of the elimination of B-lymphocytes presenting B-Cell receptor targeting self-antigens.^[25]

Resistance to chemotherapy

Bcl-2 and other members of Bcl-2 family have been shown to inhibit cell death induced by cytotoxic anticancer therapy. In fact, they are responsible for an intrinsic chemoresistance, different from mechanisms of efflux pumps or drug metabolism.The level of action of many cancer drugs seems to depend on the level of expression of Bcl-2, and notably Bcl-2/Bax mechanism of apoptosis.^[26] Thus Bcl-2 is an important resistance factor as it can counteract apoptosis induced by cancer drugs. It inhibits the activation of mitochondria thus inhibiting apoptosis. ^[27] That’s why Bcl-2 is a prime target for new cancer therapies.

Therapies targeting Bcl-2

As a prime target for new cancer therapies, many new drugs target Bcl-2 in order to treat cancers. That’s the case of Genasense, which an antisens oligonucleotide drug. Its aim is to hybridises with Bcl-2 mRNA, thus avoiding its translation and the formation of the protein (even if further studies have shown limited results). Many other drugs are under development, each targeting Bcl-2 ( for example, Small molecule inhibitors, such as Fenretinide, down-regulating Bcl-2). Molecules directly interacting with the protein such as Gossypol are also studied, showing relatively good results. ^[28]