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 (𝛼 and 𝛽), produced by alternative splicing, and which differ by 2 aminoacids (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(93-107), (136-155), (187-202) and a transmembrane domain (212-233) (due to its poor behavior in solution, it has been replaced by a segment of Bcl-xl in the presented 3D structure). 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). Helices 5 and 6 are mostly hydrophobic and they are surrounded by four other helices characterized by their amphipathic properties. There are also 3 turns (32-34, 123-125, 138-140). The is a 3(10) helix. ^[2]

The transmembrane domain of Bcl-2 is made of 21 aminoacids 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.^[3]

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 of this protein. However as expected, it affect 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.^[4]

Function

Bcl-2 is mainly found attached to the membranes through its C-terminal transmembrane domain. It can be anchored to nucleus, endoplasmic reticulum (ER) or 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 membranes participates in the control of Ca2+ content and release. The inositol 1,4,5-trisphosphate receptor (IP3R) is the primary Ca2+ release channel localized in the ER. Its pro-apoptotic activity can be directly inhibited by the Bcl-2, homology domain 4 (BH4) being essential and sufficient for this effect. comprises 20 amino acids (10-30) 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. ^[5]

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. ^{[6] [7]}

Therefore, the neutralization of Bcl-2 is required for eficient 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 thus strenghtening the interaction. ^{[8] [9] [10]}

Bcl-2 localized on external mitochondrial membrane can also inhibit the release of cytochrome c from mitochondria. ^{[11] [12]}

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 cytokine activation pathway (especially caspase-1). The Bcl-2 loop regions between the BH3 and BH4 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 highly variable in Bcl-2 family. By binding to NALP1, Bcl-2 inhibits the inflammatory caspase activation. Hence, it protects cell from the stress. The posttranslational modifications found on the loops between BH3 and BH4 modify the anti-apoptotic activity of Bcl-2. Hence, the Bcl-2 binding to NALP1 can be affected by these modifications. ^[13]

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 BH1 and BH2 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. ^[14]

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. ^[15]. It has also be shown that Bcl-2 overpexpression supresses DNA repair by enhancing Myc transcriptional activity. ^[16] 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). ^[17] 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.^[18]

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.

Structural highlights