an orthology case study: the trypsin inhibition pathway tim hulsen (2005/03/07)
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An orthology case study:the trypsin inhibition pathway
Tim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
The trypsin inhibition pathway
An orthology case study: the trypsin inhibition pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
Ortholog identification methods (3): PGT
An orthology case study: the trypsin inhibition pathwayTim 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 pairsHs-Rn pairs
The pathway by ortholog analysis
An orthology case study: the trypsin inhibition pathwayTim Hulsen (2005/03/07)
Mm – Hs – Rn - by annotation- BBH- PGT
The pathway by ortholog analysis
An orthology case study: the trypsin inhibition pathwayTim 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
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
CCK analysisID CCKN_MOUSE STANDARD; PRT; 115 AA.DE Cholecystokinins precursor (CCK) [Contains: Cholecystokinin 33DE (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 enrichedDE library, clone:0610025O15 product:PROCHOLECYSTOKININ, full insertDE 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 pathwayTim Hulsen (2005/03/07)
CCK analysisEnsembl Gene: ENSMUSG00000032532
Ensembl 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!
An orthology case study: the trypsin inhibition pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
Trypsin analysis (1)
An orthology case study: the trypsin inhibition pathwayTim Hulsen (2005/03/07)
UniProt Ensembl Chromosome Start End
TRY1_HUMAN / TRY2_HUMAN ENSG00000173636 7 141675279 141989078
TRY3_HUMAN ENSG00000010438 9 33740515 33789230
TRY4_HUMAN - - - -
TRY2_MOUSE ENSMUSG00000057163 6 41446291 41449583
TRY4_MOUSE ENSMUSG00000054106 6 41238122 41241385
TRY1_RAT / TRY2_RAT ENSRNOG00000014100 4 69289249 69465210
TRY3_RAT ENSRNOG00000013382 4 68948413 68952121
TRY4_RAT ENSRNOG00000013245 4 68872979 68876725
Trypsin analysis (2): mouse
An orthology case study: the trypsin inhibition pathwayTim Hulsen (2005/03/07)
Trypsin analysis (3): rat
An orthology case study: the trypsin inhibition pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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 pathwayTim Hulsen (2005/03/07)
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