Abstract
Some HLA class I molecules bind a significant fraction of their constitutive peptidomes in the presence of proteasome inhibitors. In this study, A*68:01-bound peptides, and their parental proteins, were characterized through massive mass spectrometry sequencing to refine its binding motif, including the nearly exclusive preference for C-terminal basic residues. Stable isotope tagging was used to distinguish proteasome-inhibitor sensitive and resistant ligands. The latter accounted for less than 20% of the peptidome and, like in HLA-B27, arose predominantly from small and basic proteins. Under the conditions used for proteasome inhibition in vivo, epoxomicin and MG-132 incompletely inhibited the hydrolysis of fluorogenic substrates specific for the tryptic or for both the tryptic and chymotryptic subspecificities, respectively. This incomplete inhibition was also reflected in the cleavage of synthetic peptide precursors of A*68:01 ligands. For these substrates, the inhibition of the proteasome resulted in altered cleavage patterns. However these alterations did not upset the balance between cleavage at peptide bonds resulting in epitope destruction and those leading to their generation. The results indicate that inhibitor-resistant HLA class I ligands are not necessarily produced by non-proteasomal pathways. However, their generation is not simply explained by decreased epitope destruction upon incomplete proteasomal inhibition and may require additional proteolytic steps acting on incompletely processed proteasomal products.
Highlights
Major Histocompatibility complex class I (MHC-I)1 molecules constitutively bind and present at the cell surface large
We previously reported that a significant fraction of the HLA-B27-bound peptidome was efficiently presented in cells treated with epoxomicin and MG-132 in a way essentially unaffected by the concentration of inhibitor
Incomplete inhibition of the proteasome may lead to altered cleavage, because impairment of a catalytic site may allosterically modulate the activity of other sites [25]
Summary
Manual inspection of the spectrum, which was facilitated by using a simple tool implemented as an Excel macro, usually allowed us to derive a tentative sequence This was used to screen the human proteome in the human protein entries of the Uniprot/Swiss-prot database (Release 14.0: 2008/07/22, with 19329 entries), using a window of 0.5 m/z units for both precursor and fragment ions, for a possible match using the Mascot server 2.2 software. For those sequences showing the highest scores in this preliminary search, the MS-product tool (version 5.1.8) at http://prospector.ucsf.edu (University of California, San Francisco), which generates a list of theoretical fragment ions from a given peptide sequence, was used to match the candidate sequences to our experimental MS/MS spectra. The relative yields of individual digestion products were calculated directly from the relative intensities of the ion peaks in the MALDI-TOF MS of the unfractionated digestion mixture
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