BH3-interacting domain death agonist (BID), a highly conserved protein [1], is a proapoptotic member of the Bcl-2 family [2]. Its apoptotic function is exhibited through its ability to regulate the permeability of the outer mitochondrial membrane (OMM) [3].
BID is a cytosolic protein, which when cleaved by enzymes such as caspase-8 , granzyme B, calpains or cathepsins, activates other proapoptotic members of the Bcl-2 family, namely Bax and Bak [4]. Bax and Bak are nuclear-encoded proteins and found as monomers in healthy cells, having a globular α-helical structure. Following stress signals, they change their conformations and assemble as oligomeric complexes on the OMM; these changes enable them to become pore-forming proteins [5].
Cleavage of BID by caspase-8 generates two fragments: the C-terminal truncated fragment, p15 (tBID, the major proteolytic product) and the N-terminal fragment, p7 [6]. These two fragments are held together by hydrophobic interactions until they come into contact with the OMM [7]. Interaction with the OMM separates the two fragments rapidly [8]. tBID subsequently undergoes a conformational change, facilitated by Mtch2, to embed into the membrane [9]. Its BH3 domain then interacts with proapoptotic Bcl-2 family proteins such as Bax and Bak, inducing mitochondrial outer membrane permeabilization (MOMP) which ultimately results in apoptosis [10].
Lysosomal cathepsins cleave BID into 15kDa tBID and degrade the pro-survival members of BCL-2 family such as Bcl-2, Bcl-xL [11].
Granzyme B (grB) cleaves BID to generate grB-truncated Bid (14kDa) and similarly translocates to the mitochondria whilst causing a conformational change in Bax, allowing Bax to integrate into the OMM and initiate the release of cytochrome c (Cytc) from the mitochondria into the cytosol [12]. In the cytosol, Cytc binds apoptotic protease-activating factor 1 (Apaf-1). The Cytc-Apaf-1 complex has higher affinity for dATP, whose binding is essential for oligomerisation and formation of the apopotosome. Subsequently, the apoptosome recruits procaspase-9 and promotes its cleavage to an active form, caspase-9. Apoptosome-bound caspase-9 acts as the cleavage factor of caspase-3, resulting in induction of apoptosis. Cytc release also initiates apoptosis through binding inositol-trisphosphate (IP3) receptors, triggering calcium release into the cytosol followed by the calpain activation and release of apoptosis-inducing factor (AIF) [13]. These events are displayed in Fig. 1.
In humans, three novel BID isoforms are generated by alternative splicing as part of the necessary stringent regulation of cleaved Bid [14]. Bid(S) contains N-terminal regulatory domains without the BH3 domain, negates the apoptotic effects of tBID, and inhibits Fas-mediated apoptosis. Bid(EL) induces apoptosis and contains the full-length BID with additional N-terminal sequence. Bid(ES) contains the Bid sequence downstream of the BH3 domain, induces apoptosis, and partially inhibits tBID. BID contains eight alpha-helices, with the core comprising the hydrophobic α6 and α7 helices [15]. Bid(L) has identical C-terminus to Bid (EL) and contains the BH3 domain; therefore, both are likely to induce apoptosis in a similar fashion with their differing N-terminal sequences being related to differing subcellular localisations [16].
BID
BH3 interacting-domain death agonist
About BID
Fig. 1. Involvement of BID in apoptotic pathways (Partially adapted from Westphal et al.)
About techniques used
BLAST Program
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The Basic Local Alignment Search Tool (BLAST) finds the regions of similarity between sequences. The similarities between sequences can be used to infer functional and evolutionary relationships between sequences as well as helping to identify members of gene families. There are many different types of BLAST search such as BLASTn, BLASTp, etc [17].
In this project, we used BLASTp, which compares protein sequences to the NCBI protein sequence database by performing a local alignment and calculates the statistical significance of the matches. BLASTp generates a bit score and expected value (E-value) for each of the alignment pair. The bit score gives an indication of how good the alignment is. A higher bit score corresponds to a better alignment. The E-value gives the expected number of alignments due to chance. A lower E-value corresponds to a more significant alignment. Bit scores are normalised, which enables us to compare different alignments, but E-values differ from one BLAST to another and cannot be used for comparison [17].
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Multiple Sequence Alignment
MSAs allow us to compare sequences of proteins. MSAs enable us to analyse protein structure, predict protein function, and infer phylogenetic relationships. Protein sequences are arranged in an array such that each column contains similar homologous amino acids [18].
We used Clustal Omega to perform the MSAs displayed on our site. Clustal Omega was developed in 2011 and has the advantage of aligning a large number of protein sequences with high accuracy [19]. The first step in how Clustal Omega generates MSAs is the production of guide trees via a modified version of mBed [20]. The sequences are replaced by an element vector to compare them to the reference sequences and align accordingly. Once aligned to the guide, Clustal Omega uses the HHalign package which subjects the alignment to Hidden Markov Models (HMMs) to further align the sequences. HMMs exploit the linearity of the genome, based on statistical models to give a probability of a feature existing at certain positions.
The highlighted black-regions indicate highly conserved regions. The highlighted grey regions are poorly conserved regions. Finally, no highlight indicates lack of consensus.
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