Abstract
Proteasome-catalyzed peptide splicing (PCPS) represents an additional activity of mammalian 20S proteasomes recently identified in connection with antigen presentation. We show here that PCPS is not restricted to mammalians but that it is also a feature of yeast 20S proteasomes catalyzed by all three active site β subunits. No major differences in splicing efficiency exist between human 20S standard- and immuno-proteasome or yeast 20S proteasome. Using H(2)(18)O to monitor the splicing reaction we also demonstrate that PCPS occurs via direct transpeptidation that slightly favors the generation of peptides spliced in cis over peptides spliced in trans. Splicing efficiency itself is shown to be controlled by proteasomal cleavage site preference as well as by the sequence characteristics of the spliced peptides. By use of kinetic data and quantitative analyses of PCPS obtained by mass spectrometry we developed a structural model with two PCPS binding sites in the neighborhood of the active Thr1.
Highlights
The 20S proteasome with its proteolytically active site -subunits (1, 2, and 5) is a N-terminal nucleophilic hydrolase, widely conserved during evolution from yeast to mammals
To determine proteasomal cleavage and splicing preferences, and to investigate the quantitative relevance of proteasome-catalyzed peptide splicing (PCPS) and its underlying biochemical mechanisms it was mandatory to compute the absolute amount of reactant peptides that are available for peptide splicing, i.e. the proteasome-generated cleaved peptides (PCPs) and the amount of proteasomegenerated spliced peptides (PSPs) produced during the PCPS reaction
By focusing on the production of the two major proteasomal cleavage products (PCPs), i.e. gp10040 – 46 (RTKAWNR) and gp10047–52 (QLYPEW) (Figs. 1D, 1E) we noted that applying the titration method, the values exceeded the limit of 1 nmol, indicating that this method could in some cases overestimate the real ⌺ PCP/PSP amount
Summary
Peptides and Peptide Synthesis—The sequence enumeration for the polypeptides gp10040–52 (RTKAWNRQLYPEW), gp10035–57 (VSRQLRTKAWNRQLYPEWTEAQR) and gp100201–229 (AHSSSAFTITDQVPFSVSVSQLRALDGGNK) is referred to the human protein gp100PMEL17, for the peptide pp8916–40 (RLMYDMYPHFMPTNLGPSEKRVWMS) to the murine cytomegalovirus pp protein and for the peptide LLO291–317 (AYISSVAYGRQVYLKLSTNSHSTKVKA) to the murine Listeria monocytogenes’s Listeriolysin O protein. Peptide sequences of the 14 previously described PSPs [10] as well as the 25 new PSPs identified in the proteasomal processing of the four synthetic substrates are reported in supplemental Table S1. The relative quantification of the ratio direct transpeptidation/(hydrolysis ϩ transpeptidation) has been based on the isotopic pattern of the PSPs [RTK][QLYPEW] (gp10040 – 42/47–52) and [VSRQL] [VSRQL] (gp10035–39/35–39) from the digestions, in H218O-TEAD buffer and by LcL and yeast wild type 20S proteasomes, of the polypeptides gp10040–52 and gp10035–57, respectively. The means and S.D. reported in Table IIA and Table IVB represent the means, for each 20S proteasome, obtained from the sum of the four substrates degradation and the S.D. over time This type of statistical analysis is supposed to better mimic the in vivo situation where proteasomes are processing different substrates at the same time producing a unique pool of peptides. A complete description of the methods can be found in supplemental material
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