The 20S proteasome splicing activity discovered by SpliceMet.
The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.
Liepe
Juliane
J
Institut für Biochemie, Charité, Universitätsmedizin Berlin, Berlin, Germany.
Mishto
Michele
M
Textoris-Taube
Kathrin
K
Janek
Katharina
K
Keller
Christin
C
Henklein
Petra
P
Kloetzel
Peter Michael
PM
Zaikin
Alexey
A
D000465:Algorithms; D000595:Amino Acid Sequence; D034961:Antigens, Nuclear / Q000737:chemistry / Q000378:metabolism; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes; D019295:Computational Biology / Q000379:methods*; D003198:Computer Simulation; D030562:Databases, Protein; D018984:Epitopes, T-Lymphocyte; D051521:Fibroblast Growth Factor 5 / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D015203:Reproducibility of Results; D012984:Software; D053719:Tandem Mass Spectrometry; D058951:gp100 Melanoma Antigen
D034961:Antigens, Nuclear; D001324:Autoantigens; D018984:Epitopes, T-Lymphocyte; C496449:FGF5 protein, human; D008562:Membrane Glycoproteins; C548637:PMEL protein, human; D010446:Peptide Fragments; D058951:gp100 Melanoma Antigen; D051521:Fibroblast Growth Factor 5; C066642:SP100 protein, human; D046988:Proteasome Endopeptidase Complex