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The Evolution of Beta Amyloid Cleaving Enzyme (BACE1): an Alzheimer’s Disease Drug Target

Christopher Southan1 and John M. Hancock2

1) EMBL-EBI, Cambridge CB101SD and 2) MRC Harwell, Oxford, OX110RD

Methods

Dr Christopher SouthanELIXIR Database Survey Co-ordinatorsouthan@ebi.ac.ukhttp://www.cdsouthan.info/CDS_proff.htm

Plaque formation in Alzheimer's Disease (AD) is seeded by excision products of Amyloid precursor protein (APP) generated by the combined activities of beta and gamma secretases. To reduce the production of these neurotoxic peptides the inhibition of beta-secretase (Beta-site APP Cleaving Enzyme, BACE1, Swiss-Prot P56817) is being intensively pursued as a possible therapy for AD and all drug candidates are screened for selectivity against the paralogue (BACE2, Swiss-Prot Q9Y5Z0) which shares 50% sequence identity.

Introduction

Fig.1 Gene structure, protein features and a bound inhibitor for human BACE1

Literature data for BACE1 support pleiotropic neuronal roles which may involve the processing of not only APP but a also wide range of different substrates (reviewed in PMID: 18005427). This is supported by neurochemical deficits and behavioral changes in BACE1 knock-out mice. While BACE2 knock-out mice are normal the increased neonatal mortality observed in BACE1/2 double knockout mice suggests some functional overlap between the paralogues. Because a clear picture of their individual roles has not yet emerged we have utilised the expanding eukaryotic genome data to examine the evolution of BACE.

Conclusions

By searching UniProt, dbEST and Ensembl we were able to retrieve BACE-like protein sequences from a wide range of species. This facilitated multiple alignments, the identification of conserved sequence blocks, maximum likelihood phylogenetic tree generation and the calculation of non-synonymous (Ka) and synonymous (Ks) sequence differences from alignments of mammalian cDNAs. We also searched the nine reported BACE1 human substrates against representative organisms.

Results The most basal organisms for which we could detect high-

similarity scores were the sea urchin (Strongylocentrotus), amphioxus (Branchiostoma), cnidarians (Nematostella, Hydra), ascidians (Ciona, Halocynthia) and the lamprey (Petromyzontinae). The emergence of the single Ur-BACE is very clear with amino acid identities of 40%-50% to human BACE1 and the characteristic C-terminal transmembrane domain. The best hits in Drosophila and C.elegans are cathepsin D/E-like homologues with less than 30% identity. The tree of BACE-like sequences is in fig.1. The vertebrate paralogues show BACE2 branch lengths approximately twice those for BACE1 with the intriguing split of BACE2 in the tetraploid Xenopus laevis .

For the whole coding region, Ka/Ks ratios were all well below 1, showing a predominant role for purifying selection in mammals. Values for BACE2 were higher (more relaxed) than for BACE1. Analysis of individual exons (fig.3) showed weaker selection acting on the N-terminals.

We propose the following evolutionary history. As a consequence of genome duplications during the emergence of chordates, the single Ur-BACE was derived from a cathepsin D/E-like ancestor and evolved distinct sequence properties. After another duplication event the accelerated change in BACE2 suggests neofunctionalisation, perhaps related to its peripheral tissue distribution. This contrasts with the more conserved sequence for BACE1 and preservation of the ancestral, possibly neuronal, Ur-BACE function. In the mammalian lineage Ka/Ks ratios suggest continuing adaptation of the terminal exons but that purifying selection has been maintained in regions associated with catalysis. The branch lengths to the ascidian sequences are generally longer than those in the BACE1 and BACE2 families, which suggests different evolutionary constraints. Related to this is the observation that most of the reported human BACE1 substrates, including APP, seem to be absent in Ciona (results not shown) but have high-scoring matches in the other species.  

Our data will facilitate the choice of new functional genomics experiments to illuminate the roles of BACE. Comparisons between knock-outs or knock-downs for the single Ur-BACE, for example against Zebra fish for each and both of the two paralogues, could reveal important undiscovered ancestral functions that the mammalian orthologues may still have. The availability of BACE1-selective and BACE2 cross-reactive inhibitors offers additional experimental options. Given the pressing medical need it is to be hoped that drug candidates entering clinical development for AD will progress. However, advancing our understanding of the evolution of these proteases remains important not only to assess possible consequences of BACE1 inhibition but also what level of BACE1/BACE2 specificity is necessary.

Fig.2 Phylogenetic tree of BACE-like sequences

Fig.3 Exon Ka/Ks ratios for the cDNAs of human, mouse and rat BACE1 (blue) and BACE2 (magenta)

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