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)

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)


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


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 …


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


Summary



Goal

• Explain the interspecial differences in CCK response, using– several ortholog identification methods– if needed, other information:

• regulatory data• expression data• structural data


Summary



The trypsin inhibition pathway


Summary



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)


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)


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


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


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


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)


Summary



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


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


Ortholog identification methods (3): PGT


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


Mm – Hs – Rn - by annotation- BBH- PGT

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

Summary



CCK analysis

• Human:– CCKN_HUMAN

• Mouse:– CCKN_MOUSE

– Q8R041

– Q9DCL5

• Rat:– CCKN_RAT

Three orthologs in mouse? Maybe different regulation?


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


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!


http://www.ensembl.org/Mus_musculus/geneview?gene=ENSMUSG00000032532

http://www.ensembl.org/Mus_musculus/geneview?gene=ENSMUSG00000032532

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


Trypsin analysis (1)


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


Trypsin analysis (3): rat


Summary

• Previous work: orthology benchmarking• Introduction• Goal• The trypsin inhibition pathway• Involved proteins• The pathway by ortholog analysis• CCK analysis• Trypsin analysis• Conclusions• Acknowledgements


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


Conclusions (2)

• Future plans:– Take a better look at regulation: promoter detection?– Use expression data?– Structural explanation? Modelling of interactions

between the involved molecules


Summary

• Previous work: orthology benchmarking• Introduction• Goal• The trypsin inhibition pathway• Involved proteins• The pathway by ortholog analysis• CCK analysis• Trypsin analysis• Conclusions• Acknowledgements


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)

Documents

strong cck responsemouse

weak cck responsehuman

gastrinhuman sptrembl

interspecial differences

cholecystokinin cckidentical

thrombin inhibitorsneeded

model organisms

human ccka receptorgasr