1. Class I pathway Prediction of proteasomal cleavage and TAP binidng Can Keşmir, TBB, Utrecht University, NL & CBS, BioCentrum, DTU

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 8-9 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 (  1 unit) Constitutive Immuno Human (Hs) - Human Drosophila (Dm) - Fly Bos Taurus (Bota) - Cow Oncorhynchus mykiss (Om) - Fish …

7. Immuno- and Constitutive proteasome specificity... LVGPTPVNIIGRNMLTQL.. P1P1’ Immuno Constitutive

8. 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) Predicting proteasomal cleavage

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

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

11. Prediction performance TP FP AP AN A roc =0.5 A roc =0.8 1 - spec Sens

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

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

18. 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

19. 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

20. 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

21. Co-evolution (continued) Kesmir et al. Immunogenetics, 2003, 55:437

22. What is going on at the N terminal?

23. S T R K F L D G N E M T L... Epitope identification TAP precursor A2 Epitope FLDGNEMTL FLDGNEMTL 2.0100 KFLDGNEMTL -2.5300 RKFLDGNEMTL -3.7400 TRKFLDGNEMTL -2.4400 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 Proteasomal cleavage

24. N terminal trimming >50% need 2-3 amino acids N terminal trimming

25. TAP and proteasome independent presentation CTL epitopes are presented at the cell surface on TAP deficient cell lines Some CTL epitopes have very poor TAP binding affinity Dominant CTL epitopes can have very poor C terminal cleavage signal Many CTL epitope have strong internal cleavage sites Other important players in the class I pathway –Signal peptides –Sec61 –Diffusion –Proteases

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 antigentic 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 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