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An orthology case study: the trypsin inhibition pathway

Tim Hulsen (2005/03/07). An orthology case study: the trypsin inhibition pathway. Tim Hulsen (2005/03/07). An orthology case study: the trypsin inhibition pathway. Summary. Introduction Goal The trypsin inhibition pathway Involved proteins Ortholog identification methods

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An orthology case study: the trypsin inhibition pathway

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  27. Tim Hulsen (2005/03/07) An orthology case study: the trypsin inhibition pathway CCK analysis Ensembl Gene: ENSMUSG00000032532Ensembl gene ENSMUSG00000032532 has 2 transcripts: ENSMUST00000060307, ENSMUST00000035120Procholecystokinin precursor (CCK). [Source:Uniprot/SWISSPROT;Acc:P09240]The gene has the following external identifiers mapped to it:AFFY_MG_U74Av2: 96055_atAFFY_Mouse430A_2: 1419473_a_atAFFY_Mu11KsubB: Msa.512.0_f_at, x59520_f_atEMBL: X59520, BC028487, AK002677, M11739EntrezGene: 12424GO: GO:0005615, GO:0005179MarkerSymbol: Cck, MGI:88297protein_id: BAB22279.1, AAA37382.1, CAA42104.1, AAH28487.1RefSeq_dna: NM_031161Uniprot/SPTREMBL: Q8R041, Q9DCL5Uniprot/SWISSPROT: CCKN_MOUSE, P09240http://www.ensembl.org:80/Mus_musculus/geneview?gene=ENSMUSG00000032532  Just alternative transcripts!

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

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

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

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

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

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

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

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

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

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