The presentation addresses the challenges in validating bace1 and bace2 as drug targets for Alzheimer's and diabetes, highlighting issues with incorrect protein sequences and unfinished representative genomes. It discusses the phylogenetic analysis of these proteases, the evolution of bace enzymes, and the prevalence of errors in genomic data. The document also proposes potential solutions and raises questions for future research in this field.

1. Will the correct ORFs please stand up?
tribulations of using genome data for
deep phylogeny of BACE1/2
Christopher Southan, Deanery of Biomedical Sciences
and TW2Informatics, Göteborg, Sweden
Presentation for the Barton Group, University of Dundee, Dec 2019

2. 2
Outline
• BACE1 and BACE2 are protease targets for Alzheimer's and diabetes,
respectively but their validation is now questioned
• Phylogenetic analysis can added functional insights
• This came up against two key problems
• A surprising prevalence of incorrect protein sequences predicted from genomes
• Many BACE1 and BACE2 orthologues had truncation and/or indel errors.
• Key phylogenetic representative genomes are languishing in an unfinished state
• Some options for amelioration of these problems will be described
• An update on the evolution of these enzymes will be shown

3. 3
Context

4. 4
BACE1 as a drug target

5. 5
BACE1 normal function
Suggested normal cleaved APP (P05067) ectodomain involvement nerve cells and
Aβ peptide dampening of neuronal hyperactivity
Voltage-gated sodium channel subunits (SCN4B, O60939) substrates for regulation
of Nav1 channel metabolism
Neuregulin (NRG1, Q022979) substrate for control of nerve cell myelination
Amyloid-like protein 2 (APP2, Q06481) substrate for ectodomain fragments
BACE2 paralogue has 50% sequence identity

6. 6
Alzheimer’s secretases as drug targets

7. 7
GtoPdb BACE1/2 inhibitor curation
BACE1
24 leads
BACE2
12 leads

8. 8
The bad news in 2018• There are 8741
compounds in ChEMBL
(compared to 8363 for
thrombin)
• 342 structures in PDB
• Most large pharma
companies had programs
• But three Phase III
candidates have failed,
some did even worse
than placebo
• De facto target de-
validation?
• Sheds doubt on causality
of amyloid

9. 9
Ensembl: looking at the gift horse

10. 10
Ensembl BACE2 paralogues ranked by % ID vs
human BACE2: ~ 50% erroneous ORFs

11. 11
Ensembl BACE2 paralogues (II)

12. 12
Poor old Lesser Hedgehog – lesser BACE2?

13. 13
Poor old Horse – too much BACE2?

14. 14
Danio rerio BACE2
• After 2.5 years of assembly curation
the 2017 zebrafish reference genome
(and UniProt) still has a clipped 372aa
BACE2
• NCBI has a 503aa version

15. 15
TreeFam: A different
way of looking at the
bad news for the
BACE2 ORFs

16. 16
BACE evolution

17. 17
BACE in
Chordates (I)

18. 18
BACE in Chordates (II)
• Amphioxus outgroup consistent with protochordate 2R whole-genome
duplication followed by paralog persistence
• Distinctly accelerated evolution of BACE2 (neofunctionalisation ?)
• Inferred neuronal role for BAC1 but no data outside human, mouse, fish
• Long branches are partial sequences
• Birds group with turtles
• Coelacanth groups with reptiles and tetrapods (not ray-finned fish)
• Xenopus laevis tetraploidisation has maintained “double” paralogs
• No evidence for pseudogenes or “dead” variants
• Implication of common origin between nerve and pancreatic cells

19. 19
Finding the UrBACE:
Human BACE1 vs Monosiga (Choanoflagellate)
• 33% identity over 432 residues,
9% gaps
• Divergence time ~0.8 billion years
• Two paralogues?

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But Monosiga still draft shotgun sequence from 2008
Has two paralogues we cant position on an assembly

21. 21
The placezoan: version 1 for 13 years
Three paralogues not complete and can not be positioned or nearest

22. 22
UrBACEs
and
cathepsin
paralogs

23. 23
Ur-BACE evolutionary trajectory
• After duplication from a cathepsin ancestor the emergence of the
UrBACE protein sequence is distinct
• Cathepsin paralogs form a clear outgroup
• “Shuffling-in” of the CTM is the defining post-duplication shift in cellular
location and function
• Multiple divergent paralogs in basal phyla (2 in Monosiga, 3 in
Trichoplax) predate the nervous system
• Found in cnidaria with only nerve nets
• Long branch lengths mainly due to partial sequences
• Major orders now represented by draft genome assemblies
• Basal relationships of Eumetazoans still unresolved
• Beyond limited EST coverage no tissue distribution or functional data

24. 24
Oily tails: key
to secretase
function

25. 25
Invertebrate
phyla without
BACE
homologues
Non-orthologous replacement of UrBACE for pre-
neuronal RIP-related secretase functions ?

26. 26
Evolution updates

27. 27
Fixing dogy ORFs
o BLASTP on both sides of the Atlantic
o TBLASTN against genome reads, ESTs, and the HTS division
o Run gene prediction on contigs (e.g. GeneMark etc)
o Stich the bits together (by hand)
o Iterative repeat searching with those bits
o Run InterPro scan
o If you are still left with partials, they can still slot into trees in informative
positions

28. 28
Latest NCBI results: new creatures

29. 29
New Ur-BACE

30. 30
Rounding off

31. 31
Summarising the ORF problem
o Protein analysis is difficult when ORFs are not correct
o Ensembl gene builds have significant levels of dodgy sequences
o These include many error types
o Searching BACE homologues at EBI or NCBI gives different results from
different pipelines (e.g. Ensembl, JGI and NCBI)
o Many eukaryotic genomes remain in the draft state for a decade or more
o Cordinating deep transcript coverage and a genome assembly seems rare
o Seems paradoxical now we can sequence a genome a day
o This is particularly problematic for species in key phylogentic positions

32. 32
Plans and questions
o Despite problems, make an update
o Crank up Jalview :)
o What is the Ur-BACE function in early metazoan?
o Could this be relevant to contempory mamallian function?
o Could we identify the non-homologous replacement in the Ecdyszoans?
o Can we encourage anyone to try BAC1/2 specific inhibitors for
functional proping in model organisms in lower phyla (e.g. Zebrafish)
o Do BACE1 inhibitors have any clinical future?

33. 33
Questions and endpiece
o https://sites.google.com/view/tw2informatics/home homepage
o https://europepmc.org/search?query=AUTH:%22Southan+C%22 publications