Protein Structure

Isoform Alpha

Isoform Beta

Fig.6 The BCL-2 protein shown in both isoforms of Alpha(PDB ID:5JSN.A) and Beta(PDB ID:5JSN.B)(16)

Fig.7 Shows the amino acid positions of Bcl-2 homologous domains (BH1-4)(14)

Fig.8 Shows conserved sequences that relate to the Bcl-2 Homology Domains. A distinct C-terminus , a free carboxyl group, is present in Isoform Beta. While locations are conserved between Isoform Alpha and Isoform Beta, domains towards the C-terminus vary as it was shown there are no active domains in Isoform Beta past the 155aa location(7).

BCL-2 apoptosis regulator

The Bcl-2 protein shown on the left is its crystal structure in complexed with novel orally active inhibitor S55746. Bcl-2 is a dimeric protein which is made up of 7 alpha helices and 4 functional sites found across the protein. The protein consists of hydrophobic side chains that cause the hydrophobic amino acid sides to face each other and turn the protein in on itself due to water interactions. The quantary structure of the BCL-2 gene is unknown currently, there may not be one. Bcl-2 proteins are characterized by the presence of one to four relatively short sequence motifs, which are approximately or less than 20 amino acid residues in length and are referred to as Bcl-2 homology domains (BH1, BH2, BH3, and BH4).(11)

Bcl-2 proteins are regulators of caspase activation, a major role player in cell apoptosis by regulating the integrity of both mitochondrial and endoplasmic reticulum (ER) membranes.(12) There have been more than 20 Bcl-2 family proteins reported in mammals and some viruses with the BCL-2 gene encoding for two isoforms found in Homo sapiens, alpha and beta. The Bcl-2 family of proteins located in Homo sapiens are either pro-apoptotic or anti-apoptotic proteins that activate or suppress apoptosis in an array of cell systems, most noticeably being that of the lymphohematopoietic and nervous system(2). Their apoptotic activity is based on the domains contained in structure; whether it has BH1-BH3(pro-apoptotic) or BH1-BH4 motifs (anti-apoptotic)(18). The Bcl-2 protein is able to regulate mitochondrial and endoplasmic reticulum membrane integrity by working in a feedback mechanism with caspase enzymes- this is a group of proteases part of our cells apoptotic pathway that degrade cellular organelles in a controlled manner. BCL-2 is able to inhibit the actions of the caspase enzymes by either preventing the release of cytochrom c from the mitochondria or being the APAF-1 (apoptosis protease-activating factor 1) thus not allowing it's protease to function. (13)

Functional Domains

There are 4 functional domains that determine the activity of the Bcl-2 protein. They contain Bcl-2 homology regions (BH) which allow it to complex to other proteins(Fig. 7):

BH1+BH2-required for the interaction with BAX and for anti-apoptotic activity. These two C-terminal regions have a head and tail interaction with the distant N-terminal region BH4 for homodimerization(18)
BH3- is found in both anti- and pro-apoptotic bcl-2 proteins and it's sole presence is a typical key feature of pro-apoptotic proteins, but others such as BAX, BAK (13)
BH4- The BH4 motif is required for anti-apoptotic activity and for interaction with RAF1 and EGLN3(19). It is the N-terminal region that interacts with BH1+BH2(18)
BH3-Homology Binding Domain: Where pro-apoptotic proteins bind to anti-apoptotic proteins by the BH3 domain(2,7)

The protein isoforms of the BCL-2 gene contain BH1+BH2 motifs with an additional N-terminus BH4 which is never found in pro-apoptotic proteins. Even though BCL-2 does contain a BH3 motif it does not exhibit pro-apoptotic properties. BH1,BH2, and BH3 conserved domains fold together to form a hydrophobic groove on pro-survival molecules where pro-apoptotic molecules can ligand too.

Anti-Apoptotic Bcl-2: contain all 4 homology motifs and are related to other sub-families including Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B (13)
Pro-Apoptotic Bcl-2 Family Proteins: contain domains BH1-3, in the cases of BH3-only proteins only BH3. Consist of Bcl-2 family proteins such as Bax, and Bak which form heterodimer complexes with Bcl-2 to prohibit their anti-apoptosis activity(13)

Bcl-2 Isoform Variations

The transcript variants of the BCL-2 gene lead to the synthesis of two variant proteins; Alpha (NP_000624.2)(2) and Beta (NP_000648.2)(7). While these isoforms are similar to each other there are some key differences. The shortened transcript of the beta isofrom leads it to code a 34 amino acid shorter polypeptide compared to the Alpha isoform. While there are many similarites between the two when it comes to BH1,BH3, BH4, and their cleave sites by Caspase-3, there are noticable differences in the structure of the polypeptides towards the c-terminus(Fig.8). According to NCBI(7) , Uniprot(15), and PDB(16), there are no indication of BH2 in the beta form; as well as an altered BH3-Homology binding domain that does not include the 201aa position(7). The transmembrane region occurs at 212aa-233aa(Fig.8) which is after the termination of the Beta isoform at 205aa. All found research relates to the "canonical sequence" which has a length of 239aa, meaning it references the Alpha isoform(12,16,17). Noticeably, the differences between the two isoforms occur around or after the 201aa position which is almost at the termination of isoform beta. Since BH2 domains are needed for anti-apoptotic action as they interact with BH4 for the homodimerization of Bcl-2(18) and the loss of its transmembrane function(7) it is inferred that isoform beta is less active as an apoptosis inhibitor.

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