An Integrated Bioinformatics and Computational Biology Approach Identifies New BH3-Only Protein Candidates

Abstract

In this study, we utilized an integrated bioinformatics and computational biology approach in search of new BH3-only proteins belonging to the BCL2 family of apoptotic regulators. The BH3 (BCL2 homology 3) domain mediates specific binding interactions among various BCL2 family members. It is composed of an amphipathic α-helical region of approximately 13 residues that has only a few amino acids that are highly conserved across all members. Using a generalized motif, we performed a genome-wide search for novel BH3-containing proteins in the NCBI Consensus Coding Sequence (CCDS) database. In addition to known pro-apoptotic BH3-only proteins, 197 proteins were recovered that satisfied the search criteria. These were categorized according to α-helical content and predictive binding to BCL-xL (encoded by BCL2L1) and MCL-1, two representative anti-apoptotic BCL2 family members, using position-specific scoring matrix models. Notably, the list is enriched for proteins associated with autophagy as well as a broad spectrum of cellular stress responses such as endoplasmic reticulum stress, oxidative stress, antiviral defense, and the DNA damage response. Several potential novel BH3-containing proteins are highlighted. In particular, the analysis strongly suggests that the apoptosis inhibitor and DNA damage response regulator, AVEN, which was originally isolated as a BCL-xL-interacting protein, is a functional BH3-only protein representing a distinct subclass of BCL2 family members.

Fig. (1)

Alignment of AVEN proteins of human (NP_065104.1), chimpanzee (XP_510277.3), dog (XP_535418.2), mouse (NP_083120.2), chicken (NP_001005791.1) and fruit fly (NP_572817.1). Conserved BH3-like motifs in the human, chimpanzee, dog, mouse and chicken proteins are highlighted in blue with conserved L and D residues in red. Numbering using the heptad convention of Dutta and colleagues [11] is shown at the top. Note that the Drosophila protein has diverged considerably from the other proteins and does not contain a putative BH3 motif, but some common residues have been retained within the boxed region (referred to as Region II by Zou and colleagues [34]). The 25-mer AVEN and dAven peptides used in the molecular dynamics simulations of putative BH3 interactions with BCL-xL (Fig. 3) are underlined.

Fig. (2)

ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) sequence alignment of AVEN versus BIK. “*”, identical; “:”, conserved substitutions; “.”, semi-conserved substitutions. The putative BH3-like motif in AVEN and the BIK BH3 domain are highlighted.

Fig. (3)

Results of molecular dynamics simulation of putative AVEN BH3 motif with BCL-xL. (A). Ribbon diagram of a peptide encompassing the putative BH3 motif of human AVEN (ESQRGTDFSVLLSSAGDSFSQFRFA; shown in red) complexed with BCL-xL. (B) Ribbon diagram of the corresponding peptide from Drosophila Aven (AQLRAGDFQQLAQFPSLGGGHFTFG; shown in red). See Fig. 1 and text for details. Simulations (20 ns) are based on an analogous 25-mer of the BAD BH3 motif and the BAD/BCL-xL structure (PDB code: 1G5J).

Fig. (4)

Known and hypothetical interactions between selected autophagy, DNA damage checkpoint, and apoptotic proteins. Known physical associations for ATG12, ATG16L1, AVEN and BECN1 were imported from BioGrid (http://thebiogrid.org/). Binding of ATG12 to BCL2 via a BH3-like motif was reported in Ref. [100]. A putative interaction between ATG16L1 and a representative anti-apoptotic BCL2 family member (BCL2) is indicated. Proteins highlighted in red were recovered in the screen for new BH3-containing protein candidates.