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Class I pathway Prediction of proteasomal cleavage and TAP binidng

Class I pathway Prediction of proteasomal cleavage and TAP binidng. Morten Nielsen, CBS, BioCentrum, DTU. Outline. MHC class I epitopes Antigen processing Proteasome Specificity and Polymorphism Prediction methods TAP Binding motif Evolution Immune escape.

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Class I pathway Prediction of proteasomal cleavage and TAP binidng

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  1. Class I pathwayPrediction of proteasomal cleavage and TAP binidng Morten Nielsen, 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 (b1 unit) Human (Hs) - Human Drosophila (Dm) - Fly Bos Taurus (Bota) - Cow Oncorhynchus mykiss (Om) - Fish … Constitutive Immuno

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

  8. Predicting proteasomal cleavage • NetChop • Neural network based method • PaProc • Weight matrix based method • FragPredict • Based on a statistical analysis of cleavage-determining amino acid motifs present around the scissile bond • i.e. also weight matrix like

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

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

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

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

  13. Proteasome specificity • NetChop is the best available cleavage method • www.cbs.dtu.dk/services/NetChop-3.0

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

  23. More evolution Constitutive proteasome!!!

  24. What is going on at the N terminal?

  25. 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 . . .

  26. >50% need 2-3 amino acids N terminal trimming N terminal trimming

  27. CTL epitopes are presented at the cell surface on TAP deficient cell lines Some CTL epitopes have very poor TAP binding affinity Dominate 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 Mette will tell you more TAP and proteasome independent presentation

  28. 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

  29. 189 S N 0.38 SSWDFITV 190 S S 0.59 SSWDFITV 191 W S 0.92 SSWDFITV 192 D N 0.23 SSWDFITV 193 F S 0.87 SSWDFITV 194 I S 0.84 SSWDFITV 195 T N 0.27 SSWDFITV 196 V S 0.96 SSWDFITV 197 N S 0.82 SSWDFITV 189 S N 0.38 SSWDFITV S S 0.59 SSWDFITV W S 0.92 SSWDFITV 192 D N 0.23 SSWDFITV 193 F S 0.87 SSWDFITV 194 I S 0.83 SSWDFITV 195 T N 0.13 SSWDFITV 196 V S 0.92 SSWDFITV 197 D S 0.97 SSWDFITV Processed as SSWDFITVD and has wrong C terminal for MHC binding, not recognized by CTL Immune escape via antigen processing Moloney murine leukemia virus (MuLV) epitope SSWDFITV Processed right and recognized by CTL

  30. Immune escape • Proteasome-mediated digestion analysis of a synthetic 26-mer peptide derived from the Friend sequence shows that cleavage takes place predominantly C-terminal of D, instead of V as is the case for the Moloney MuLV sequence. Therefore, the C terminus of the epitope is not properly generated. Epitope-containing peptide fragments extended with an additional C-terminal D are not efficiently translocated by TAP and do not show significant binding affinity to MHC class I-Kb molecules.. Beekmanet al., JI 2000

  31. 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 • Proteasomal cleavage prediction tools exist • NetChop3.0 and NetChop20S-3.0 are among the best • 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 9-13 amino acids • Epitope trimming in the ER by several amino peptidases (ERAP) • We still do not understand every thing • Many more important players are involved in the class I path way

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