An orthology case study: the trypsin inhibition pathway

1
An orthology case studythe trypsin inhibition
pathway

• Tim Hulsen (2005/03/07)

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

• Introduction
• Goal
• The trypsin inhibition pathway
• Involved proteins
• Ortholog identification methods
• The pathway by ortholog analysis
• CCK analysis
• Trypsin analysis
• Conclusions
• Acknowledgements

3
Introduction (1)
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Orthology describes the evolutionary
  relationship between homologous genes whose
  independent evolution reflects a speciation
  event (Fitch, 1970)

4
Introduction (2)
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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

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Introduction (3)
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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

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

• Side effect of thrombin inhibitors inhibition of
  trypsin
• Trypsin inhibition -gt rise in cholecystokinin
  (CCK) levels -gt stimulation of pancreas -gt
  pancreatic tumors
• Difficult to test in model organisms
• Rat very strong CCK response
• Mouse weak CCK response
• Human almost no CCK response

7
Summary
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Introduction
• Goal
• The trypsin inhibition pathway
• Involved proteins
• Ortholog identification methods
• The pathway by ortholog analysis
• CCK analysis
• Trypsin analysis
• Conclusions
• Acknowledgements

8
Goal
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Explain the interspecial differences in CCK
  response, using
• several ortholog identification methods
• if needed, other information
• regulatory data
• expression data
• structural data

9
Summary
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Introduction
• Goal
• The trypsin inhibition pathway
• Involved proteins
• Ortholog identification methods
• The pathway by ortholog analysis
• CCK analysis
• Trypsin analysis
• Conclusions
• Acknowledgements

10
The trypsin inhibition pathway
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway
11
Summary
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Introduction
• Goal
• The trypsin inhibition pathway
• Involved proteins
• Ortholog identification methods
• The pathway by ortholog analysis
• CCK analysis
• Trypsin analysis
• Conclusions
• Acknowledgements

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Involved proteins Cholecystokinin (CCK)
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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)

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Involved proteins Gastrin
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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)

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Involved proteins Trypsin
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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

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Involved proteins Trypsin inhibitors
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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

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Involved proteins Elastase
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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

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Involved proteins Thrombin
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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)

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

• Introduction
• Goal
• The trypsin inhibition pathway
• Involved proteins
• Ortholog identification methods
• The pathway by ortholog analysis
• CCK analysis
• Trypsin analysis
• Conclusions
• Acknowledgements

19
Ortholog identification methods
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Using functional annotation (SPTrEMBL)
• Best Bidirectional Hit (BBH)
• ? one-to-one relationships
• PhyloGenetic Trees (PGT)
• ? many-to-many relationships

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Ortholog identification methods (2) BBH
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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

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Ortholog identification methods (3) PGT
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Human
• 
  All eukaryotic proteomes
• Zgt20,
  RHgt0.5QL
• 25,000 groups

PROTEOME
Hs-Mm pairs Hs-Rn pairs
TREE SCANNING
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The pathway by ortholog analysis
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Mm Hs Rn
• by annotation
• BBH
• PGT

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The pathway by ortholog analysis
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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

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

• Introduction
• Goal
• The trypsin inhibition pathway
• Involved proteins
• Ortholog identification methods
• The pathway by ortholog analysis
• CCK analysis
• Trypsin analysis
• Conclusions
• Acknowledgements

25
CCK analysis
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Human
• CCKN_HUMAN
• Mouse
• CCKN_MOUSE
• Q8R041
• Q9DCL5
• Rat
• CCKN_RAT
• ? Three orthologs in mouse? Maybe different
  regulation?

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CCK analysis
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• ID CCKN_MOUSE STANDARD PRT 115 AA.
• DE Cholecystokinins precursor (CCK) Contains
  Cholecystokinin 33
• DE (CCK33) Cholecystokinin 12 (CCK12)
  Cholecystokinin 8 (CCK8).
• GN NameCck
• 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 NameCck
• 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.

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CCK analysis
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Ensembl Gene ENSMUSG00000032532Ensembl gene
  ENSMUSG00000032532 has 2 transcripts
  ENSMUST00000060307, ENSMUST00000035120Procholecys
  tokinin precursor (CCK). SourceUniprot/SWISSPROT
  AccP09240The gene has the following external
  identifiers mapped to itAFFY_MG_U74Av2
  96055_atAFFY_Mouse430A_2 1419473_a_atAFFY_Mu11K
  subB Msa.512.0_f_at, x59520_f_atEMBL X59520,
  BC028487, AK002677, M11739EntrezGene 12424GO
  GO0005615, GO0005179MarkerSymbol Cck,
  MGI88297protein_id BAB22279.1, AAA37382.1,
  CAA42104.1, AAH28487.1RefSeq_dna
  NM_031161Uniprot/SPTREMBL Q8R041,
  Q9DCL5Uniprot/SWISSPROT CCKN_MOUSE,
  P09240http//www.ensembl.org80/Mus_musculus/gene
  view?geneENSMUSG00000032532
• ? Just alternative transcripts!

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

• 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

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Trypsin analysis (1)
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway
30
Trypsin analysis (2) mouse
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway
31
Trypsin analysis (3) rat
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway
32
Summary
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

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

33
Conclusions (1)
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• 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

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Conclusions (2)
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Future plans
• Take a better look at regulation promoter
  detection?
• Use expression data?
• Structural explanation? Modelling of interactions
  between the involved molecules

35
Summary
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

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

36
Acknowledgements
Tim Hulsen (2005/03/07)
An orthology case study the trypsin inhibition
pathway

• Peter Groenen (Organon MDI)
• Diels van den Dobbelsteen (Organon Tox.)
• Others at Organon MDI and CMBI
• YOU for listening!