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Class I pathway Prediction of proteasomal cleavage and TAP binding

Class I pathway Prediction of proteasomal cleavage and TAP binding. Outline. MHC class I epitopes Antigen processing Proteasome Specificity and Polymorphism Prediction methods TAP Binding motif Evolution Immune escape. Peptide generation in the class I pathway. Proteasomal cleavage.

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Class I pathway Prediction of proteasomal cleavage and TAP binding

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  1. Class I pathwayPrediction of proteasomal cleavage and TAP binding

  2. Outline • MHC class I epitopes • Antigen processing • Proteasome • Specificity and Polymorphism • Prediction methods • TAP • Binding motif • Evolution • Immune escape

  3. Peptide generation in the class I pathway

  4. Proteasomal cleavage • ~20% of all peptide bonds are cleaved • Average peptide length 6-8 amino acids • Not all peptide bonds are equally likely cleaved • Cleavage more likely after hydrophobic than after hydrophilic amino acids

  5. Proteasome specificity • Low polymorphism • Constitutive & Immuno-proteasome • Evolutionary conserved • Stochastic and low specificity • Only 70-80% of the cleavage sites are reproduced in repeated experiments

  6. Proteasome evolution (b1 unit) Human (Hs) - Human Drosophila (Dm) - Fly Bos Taurus (Bota) - Cow Oncorhynchus mykiss (Om) - Fish Arabidopsis thaliana (Didi)- Plant Trichomonas vaginalis (SP)- Bacteria … Constitutive Immuno

  7. Immuno- and Constitutive proteasome specificity Immuno Constitutive P1 P1’ ...LVGPTPVNIIGRNMLTQL..

  8. Immuno- and Constitutive proteasome specificity Immuno Constitutive P1 P1’ ...LVGPTPVNIIGRNMLTQL..

  9. Predicting proteasomal cleavage • NetChop • Neural network based method • PaProc • Partially non-linear method (a neural network without hidden neurons????) • SMM (stabilized matrix method) • FragPredict • Based on a statistical analysis of cleavage-determining amino acid motifs present around the scissile bond (i.e. also weight matrix like)

  10. NetChop20S-3.0In vitro digest data from the constitutive proteasome Toes et al., J.exp.med. 2001

  11. NetChop 3.0 Cterm (MHC ligands) • NetChop-3.0 C-term • Trained on class I epitopes • Most epitopes are generated by the immunoproteasome • Predicts the processing specificity LDFVRFMGVMSSCNNPA LVQEKYLEYRQVPDSDP RTQDENPVVHFFKNIVT TPLIPLTIFVGENTGVP LVPVEPDKVEEATEGEN YMLDLQPETTDLYCYEQ PVESMETTMRSPVFTDN ISEYRHYCYSLYGTTLE AAVDAGMAMAGQSPVLR QPKKVKRRLFETRELTD LGEFYNQMMVKAGLNDD GYGGRASDYKSAHKGLK KTKDIVNGLRSVQTFAD LVGFLLLKYRAREPVTK SVDPKNYPKKKMEKRFV SSSSTPLLYPSLALPAP FLYGALLLAEGFYTTGA

  12. Predicting proteasomal cleavage NetChop-3.0 NetChop20S--3.0 • Relative poor predictive performance • For MHC prediction CC~0.92 and AUC~0.95

  13. Proteasome specificity

  14. What does TAP do?

  15. TAP affinity prediction • Transporter Associated with antigen Processing • Binds peptides 9-18 long • Binding determined mostly by N1-3 and C terminal amino acids

  16. A low matrix entry corresponds to an amino acid well suited for TAP binding TAP binding motif matrix Peters et el., 2003. JI, 171: 1741.

  17. TAP affinity prediction

  18. Predicting TAP affinity 9 meric peptides >9 meric ILRGTSFVYV -0.11 + 0.09 - 0.42 - 0.3 = -0.74 Peters et el., 2003. JI, 171: 1741.

  19. Proteasome, TAP and MHC co-evolution • Antigen processing and presentation is highly ineffective • Only 1 in 200 peptides will bind a given MHC complex • If proteasome and TAP do not effectively produce MHC restricted peptides, antigen processing would be a severe bottleneck for antigen recognition

  20. Co-evolution of Proteasome, TAP and MHC • CP-P1: Constitutive proteasome specificity at P1 position • TAP-9: TAP motif at P9 position • MHC-9: Average MHC motif at P9

  21. Co-evolution of Proteasome, TAP and MHC • IP-P1: Immuno proteasome specificity at P1 position • CP-P1: Constitutive proteasome specificity at P1 position • TAP-9: TAP motif at P9 position • MHC-9: Average MHC motif at P9

  22. More evolution Constitutive proteasome!!!

  23. What is going on at the N terminal?

  24. 0.0101 0.6483 0.9955 0.9984 0.4299 0.2261 0.0103 0.0265 0.0099 0.0099 0.9590 0.4670 0.9989 Epitope identification • TAP precursor A2 Epitope FLDGNEMTL FLDGNEMTL 2.0100 KFLDGNEMTL -2.5300 RKFLDGNEMTL -3.7400 TRKFLDGNEMTL -2.4400 • Proteasomal cleavage S T R K F L D G N E M T L . . .

  25. >50% need 2-3 amino acids N terminal trimming N terminal trimming S T R K F L D G N E M T L . . . 0.0101 0.6483 0.9955 0.9984 0.4299 0.2261

  26. Immune escape • Pathogens evolve under strong selection pressure to avoid CTL recognition • Generate point mutations or insertions/deletions to disturb • Peptide binding to MHC • CTL recognition • Only involve the antigenic peptide region • Antigen processing • Can involve peptide flanking region

  27. Immune escape via antigen processing HIV-1 Nef epitope VPLRPMTY (Milicic et al. JI, 2005, 4618) IP IP CP

  28. Summary • The most important players (MHC, TAP and proteasome) in the MHC class I pathway have co evolved to a share a common C terminal pathway specificity • We can predict (up to a degree) proteasomal cleavage • TAP binding motif characterized in a weight matrix • Binding mostly determined by the N1-3 and C terminal amino acids • Proteasome produces and TAP transports precursor T cell epitopes of length 8-13 amino acids • Epitope trimming in the ER by several amino peptidases (ERAP) • We still do not understand everything • Many more important players are involved in the class I path way

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