1. An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

2. Summary Introduction Goal The trypsin inhibition pathway Involved proteins Ortholog identification methods The pathway by ortholog analysis CCK analysis Trypsin analysis Conclusions Acknowledgements An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

3. Introduction (1) Orthology: describes “the evolutionary relationship between homologous genes whose independent evolution reflects a speciation event” (Fitch, 1970) An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

4. Introduction (2) Orthology is often used for the transfer of functional annotation from proteins in an a model organism to proteins in human Can even be used for complete pathways Wanted! Case study in which interspecial differences might be explained by using orthologies An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

5. Introduction (3) Organon: thrombin inhibitors Needed to stop thrombosis (blood clotting) Thrombin inhibitor on the market: (xi)melagatran, sold as Exanta (AstraZeneca) Proven to be better than warfarin, but … An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

6. Introduction (4) Side effect of thrombin inhibitors: inhibition of trypsin Trypsin inhibition -> rise in cholecystokinin (CCK) levels -> stimulation of pancreas -> pancreatic tumors Difficult to test in model organisms: –Rat: very strong CCK response –Mouse: weak CCK response –Human: almost no CCK response An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

7. Summary Introduction Goal The trypsin inhibition pathway Involved proteins Ortholog identification methods The pathway by ortholog analysis CCK analysis Trypsin analysis Conclusions Acknowledgements An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

8. Goal Explain the interspecial differences in CCK response, using –several ortholog identification methods –if needed, other information: regulatory data expression data structural data An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

9. Summary Introduction Goal The trypsin inhibition pathway Involved proteins Ortholog identification methods The pathway by ortholog analysis CCK analysis Trypsin analysis Conclusions Acknowledgements An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

10. The trypsin inhibition pathway An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

11. Summary Introduction Goal The trypsin inhibition pathway Involved proteins Ortholog identification methods The pathway by ortholog analysis CCK analysis Trypsin analysis Conclusions Acknowledgements An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

12. Involved proteins: Cholecystokinin (CCK) Identical to pancreozyme Stimulates secretion by exocrine acinary cells of the pancreas Stimulates secretion of bile Stimulates secretion of insulin Production of CCK is stimulated by the presence of digestion products in the duodenum CCK is chemically related to gastrin Human SPTrEMBL proteins: –CCKN_HUMAN –CCKR_HUMAN (CCKA receptor) –GASR_HUMAN (CCKB / gastrin receptor) An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

13. Involved proteins: Gastrin Hormone that stimulates the stomach Stimulates stomach glands to produce gastric acid Stimulates production of pancreatic juice Secreted in the last part of the stomach (antrum) Production of gastrin stimulated by the presence of meat products or alcohol in the stomach Production stops when enough acid is present in the stomach (pH 2.5) Human SPTrEMBL proteins: –GAST_HUMAN –GASR_HUMAN (CCKB / gastrin receptor) An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

14. Involved proteins: Trypsin Proteinase: degrades proteins Next to pepsin and chymotrypsin, the most important proteinase in the digestive system During digestion, trypsin interacts with the other proteinases to degrade proteins; it continues in the duodenum where it has a maximal enzymatic activity at pH 8 Very similar to chymotrypsin Human SPTrEMBL proteins: –TRY1_HUMAN –TRY2_HUMAN –TRY3_HUMAN –TRY4_HUMAN An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

15. Involved proteins: Trypsin inhibitors Alpha-1-antitrypsin: protein that can block the activity of trypsin and other enzymes, such as elastase Most alpha-1-antitrypsin is produced in the liver ‘Acute phase protein’: production increases during inflammatory processes Human SPTrEMBL proteins: –A1AT_HUMAN (precursor) –A1AU_HUMAN An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

16. Involved proteins: Elastase Pancreatic elastase (EC 3.4.21.36) is a serine protease with a broad substrate specificity Can degrade elastine, a protein that occurs often in fibres, tendons and ligaments Human SPTrEMBL proteins: –EL1_HUMAN –EL2A_HUMAN –EL2B_HUMAN –EL3A_HUMAN –EL3B_HUMAN –ELNE_HUMAN An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

17. Involved proteins: Thrombin Proteolytic enzyme Converts fibrinogen to fibrin, an insoluble protein that forms fibrils and causes blood clotting Human SPTrEMBL proteins: –PAR1_HUMAN (receptor) –PAR2_HUMAN (receptor) –PAR3_HUMAN (receptor) –PAR4_HUMAN (receptor) –PTI6_HUMAN (inhibitor) An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

18. Summary Introduction Goal The trypsin inhibition pathway Involved proteins Ortholog identification methods The pathway by ortholog analysis CCK analysis Trypsin analysis Conclusions Acknowledgements An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

19. Ortholog identification methods 1.Using functional annotation (SPTrEMBL): 2.Best Bidirectional Hit (BBH)  one-to-one relationships 3.PhyloGenetic Trees (PGT)  many-to-many relationships An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

20. Ortholog identification methods (2): BBH Very easy and quick Human protein (1)  SW  best hit in mouse/rat (2) Mouse/rat protein (2)  SW  best hit in human (3) If 3 equals 1, the human and mouse/rat protein are considered to be orthologs An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

21. Ortholog identification methods (3): PGT An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07) Human All eukaryotic proteomes Z>20, RH>0.5*QL ~25,000 groups PHYLOME SELECTION OF HOMOLOGS ALIGNMENTS AND TREES PROTEOME PROTEOMES TREE SCANNING LIST Hs-Mm pairs Hs-Rn pairs

22. The pathway by ortholog analysis An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07) Mm – Hs – Rn - by annotation - BBH - PGT

23. The pathway by ortholog analysis An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07) PGT method: in some cases too many orthologous relationships, especially for trypsin (73 in mouse and 62 in rat!) BBH method seems to be more usable for this study, but still not gives an explanation for the differences in CCK levels  Combine ortholog analysis with other data  Focus on the molecules that are most likely to be responsible for these differences: CCK and trypsin

24. Summary Introduction Goal The trypsin inhibition pathway Involved proteins Ortholog identification methods The pathway by ortholog analysis CCK analysis Trypsin analysis Conclusions Acknowledgements An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

25. CCK analysis Human: –CCKN_HUMAN Mouse: –CCKN_MOUSE –Q8R041 –Q9DCL5 Rat: –CCKN_RAT  Three orthologs in mouse? Maybe different regulation? An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

26. CCK analysis ID CCKN_MOUSE STANDARD; PRT; 115 AA. DE Cholecystokinins precursor (CCK) [Contains: Cholecystokinin 33 DE (CCK33); Cholecystokinin 12 (CCK12); Cholecystokinin 8 (CCK8)]. GN Name=Cck; OS Mus musculus (Mouse). SQ SEQUENCE 115 AA; 12866 MW; 0EEDABAB8F7D839A CRC64; MKSGVCLCVV MAVLAAGALA QPVVPAEATD PVEQRAEEAP RRQLRAVLRP DREPRARLGA LLARYIQQVR KAPSGRMSVL KNLQSLDPSH RISDRDYMGW MDFGRRSAED YEYPS ID Q8R041 PRELIMINARY; PRT; 134 AA. DE Cck protein. GN Name=Cck; OS Mus musculus (Mouse). SQ SEQUENCE 134 AA; 15163 MW; 9651DDD6C1D785E0 CRC64; MKSGVCLCVV MAVLAAGALA QPVVPAEATD PVEQRAQEAP RRQLRAVLRT DGEPRARLGA LLARYIQQVR KVAWMVTSGW VLTWTSRAGL KHRRWASFLW SSRTQFFLPA FEQPMACRPV CIWLDCSFWP HVRS ID Q9DCL5 PRELIMINARY; PRT; 115 AA. DE Mus musculus adult male kidney cDNA, RIKEN full-length enriched DE library, clone:0610025O15 product:PROCHOLECYSTOKININ, full insert DE sequence. GN Name=Cck; OS Mus musculus (Mouse). SQ SEQUENCE 115 AA; 12770 MW; 7841B11D39BB52DA CRC64; MKSGVCLCVV MAVLAAGALA QPVVPAEATD PVEQRAQEAP RRQLRAVLRT DGEPRARLGA LLARYIQQVR KAPSGRMSVL KNLQSLDPSH RISDRDYMGW MDFGRRSAED YEYPS An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

27. CCK analysis Ensembl Gene: ENSMUSG00000032532 Ensembl gene ENSMUSG00000032532 has 2 transcripts: ENSMUST00000060307, ENSMUST00000035120 Procholecystokinin precursor (CCK). [Source:Uniprot/SWISSPROT;Acc:P09240] The gene has the following external identifiers mapped to it: AFFY_MG_U74Av2: 96055_at AFFY_Mouse430A_2: 1419473_a_at AFFY_Mu11KsubB: Msa.512.0_f_at, x59520_f_at EMBL: X59520, BC028487, AK002677, M11739 EntrezGene: 12424 GO: GO:0005615, GO:0005179 MarkerSymbol: Cck, MGI:88297 protein_id: BAB22279.1, AAA37382.1, CAA42104.1, AAH28487.1 RefSeq_dna: NM_031161 Uniprot/SPTREMBL: Q8R041, Q9DCL5 Uniprot/SWISSPROT: CCKN_MOUSE, P09240 http://www.ensembl.org:80/Mus_musculus/geneview?gene=ENSMUSG00000032532ENSMUSG00000032532 http://www.ensembl.org:80/Mus_musculus/geneview?gene=ENSMUSG00000032532  Just alternative transcripts! An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

28. Summary Previous work: orthology benchmarking Introduction Goal The trypsin inhibition pathway Ortholog identification methods Involved proteins The pathway by ortholog analysis CCK analysis Trypsin analysis Conclusions Acknowledgements An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

29. Trypsin analysis (1) An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07) UniProtEnsemblChromosomeStartEnd TRY1_HUMAN / TRY2_HUMANENSG000001736367141675279141989078 TRY3_HUMANENSG0000001043893374051533789230 TRY4_HUMAN---- TRY2_MOUSEENSMUSG0000005716364144629141449583 TRY4_MOUSEENSMUSG0000005410664123812241241385 TRY1_RAT / TRY2_RATENSRNOG0000001410046928924969465210 TRY3_RATENSRNOG0000001338246894841368952121 TRY4_RATENSRNOG0000001324546887297968876725

30. Trypsin analysis (2): mouse An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

31. Trypsin analysis (3): rat An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

32. Summary Previous work: orthology benchmarking Introduction Goal The trypsin inhibition pathway Involved proteins The pathway by ortholog analysis CCK analysis Trypsin analysis Conclusions Acknowledgements An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

33. Conclusions (1) Different ortholog identification methods can give very different results Less inclusive methods (BBH) seem to be more useful in pathway prediction Our problem (different CCK responses in Human, Mouse and Rat) cannot be solved only by orthology identification Multiple orthologs are often caused by alternative splicing An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

34. Conclusions (2) Future plans: –Take a better look at regulation: promoter detection? –Use expression data? –Structural explanation? Modelling of interactions between the involved molecules An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

35. Summary Previous work: orthology benchmarking Introduction Goal The trypsin inhibition pathway Involved proteins The pathway by ortholog analysis CCK analysis Trypsin analysis Conclusions Acknowledgements An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)

36. Acknowledgements Peter Groenen (Organon MDI) Diels van den Dobbelsteen (Organon Tox.) Others at Organon MDI and CMBI YOU for listening! An orthology case study: the trypsin inhibition pathway Tim Hulsen (2005/03/07)