PCPS 
Proteasome Cleavage Prediction Server

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BACKGROUND

       The proteasome is the main responsible of proteolytic degradation of cytosolic proteins, generating protein fragments between 7 and 15 amino acids. Some of these peptides can be transported into the endoplasmic reticulum by TAP. Whereas proteases of the endoplasmic reticulum trim amino acids from the amino-terminus of the peptides the carboxy-terminus of MHCI peptide ligands is generated by the proteasome. Therefore the proteasome plays a central role in the MHCI antigen processing pathway. There are two different proteasomes, the constitutive proteasome, presented in all types of cells, and the immunoproteasome, constitutively presented only in dendritic cells.
       PCPS is a server for the prediction of cleavage sites generated by both, the constitutive proteasome and the immunoproteasome


USER GUIDE

  • Input

    •        Input query for PCPS can be one or more protein sequences in FASTA, GenBank, EMBL or Phylip formats. Example of a protein sequence in FASTA Format. 

    >A56881  PIR2 release 71.00 
    MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEAT
    NITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQW
    KEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPG
    YENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKI
    VIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPG
    GGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYY
    DAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTN
    EVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVR
    SFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYI
    NADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKK
    SPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYP
    LYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDY
    AVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERL
    QDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKY
    AGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA
           Protein input sequence/s can be pasted or uploaded from a local file. Sequence uploading is achieved in two sequential steps, first browse/choose the local file with the sequences and second hit the upload bottom. This is done to facilitate preprocessing and error checking of input data prior to submission to the server.

  • Cleavage models


    •       The user can select a single cleavage prediction model, proteasome or immunoproteasome, or both models simultaneously. The different available models correspond to the constitutive proteasome (PCP-p) or the immunoproteasome (PCP-ip), models were trained on 382 MHCI-eluted peptide ligands and 553 CD8 T cell epitopes and their flanking regions, respectively.PCPs were obtained using N-GRAM-COUNT and tested using HIDDEN-NGRAM. Different models were obtained from different training sets consisting of peptide fragments containing the carboxy-terminus (P1 residue of cleavage site) of the MHCI-restricted peptides (MHCI-eluted peptides and CD8 T cell epitopes) and a variable number of flanking residues. The predictive cleavage models for proteasome (PCP-p) and immunoproteasome (PCP-ip) available in PCPS are the following:


          Frag. Size (N): This is the size of the peptide fragments in training and testing sets.
          SE: Sensitivity.
          SP:Specificity.
          ECS: Expected Cleavage Sites. Calculated using the equation 100*C/(N-1) where C is the average number of cutpoint per fragment yield by a given model LMPCP when tested in a file of fragments size N.
          MCC: Matthews Correlation Coefficient.
          BTR: Better Than Random. Calculated as the difference between SE and ECS (BTR = SE - ECS). The bigger the difference between SE and ECS the better the prediction capacity of the model.

  • Output

    • There are two posibles output acording the user detition:

           The server will compute cleavage prediction after each residue of the protein by the proteasome and/or the immunoproteasome and return a table with all the residues and their cleavage prediction score, marking with a tick when the score is higher than the threshold value. This threshold value can be selected by the user in the option "Threshold". When both, proteasome and immunoproteasome, are selected, the server return a table with both cleavage predictions. Here follows a representative output taking as theshole value 0.5.

       At the begining of the page, if the option "Graphics" is selected, a figure will be displayed indicating the position of each epitope in the sequence. As it is show here, the sequence is represented as a black fine line, while the diferent epitopes are represented in blue boxes unther the black line. Here follows a representative output.


       The server will also return a table showing a list of peptides, size selected by the users, with predicted cleavage after the C-terminal end: cleavage score above the the threshold value choosen by the user in the "Threshold"options.  For each peptide, it will also be indicated if there is NO internal cleavage sites (labelled with a green cross) within the peptide or if there IS internal cleavage sites (labelled with a red cross) site (with a red cross). A peptide with an internal cleavage site means that there is at least site within the peptide with a cleavage score above the threshold for internal cleavage sites selected by users. Users can select to discard peptides with internal cleavage sites from the input page. Here follows a representative output.





    CONTACT: For any questions: Pedro Reche



    Last change: March 2020