Kurzfassung

Peptide microarrays should be applicable in the mapping of proteasome specificity. The molecular dimensions of these protein complexes may require increased sterical availability of the surface bound peptides, but since incorporation of long water-soluble linker molecules is already a standard procedure in peptides synthesis this problem should be readily addressed. FRET-based assays are routinely used for detection of protease digestion of peptides immobilized on various surfaces, and since...Peptide microarrays should be applicable in the mapping of proteasome specificity. The molecular dimensions of these protein complexes may require increased sterical availability of the surface bound peptides, but since incorporation of long water-soluble linker molecules is already a standard procedure in peptides synthesis this problem should be readily addressed. FRET-based assays are routinely used for detection of protease digestion of peptides immobilized on various surfaces, and since standard synthesis reagents are used in the current approach, the synthesis of peptide-arrays suitable for FRET-assays is not expected to constitute a major problem. This workpackage aims at the elucidation of the cleavage preferences of different subtypes of proteasomes and other peptidases involved in antigen processing. In the last years, we have established the methodology and specific skills to analyze and quantify peptide fragments generated by proteasomes and other peptidases involved in antigen processing (e.g. aminipeptidases, TPPII). These enzymes are crucially involved in the generation of peptides which are recognized by cytotoxic T-lymphocytes including peptides derived from pathogen or tumor-associated or –specific proteins [37-45][46]. Until now, analyses of proteases specificities were performed using digestion data from only a few model peptides or sometimes full length proteins [47-49]. However, the amount of data that can be generated using the conventional approach of in-vitro digesting model substrates followed by analysis of the fragments yields only a very narrow perspective into the universe of possible protease substrates. The possibility to evaluate the digestion kinetics of more than 10.000 peptides at a time will give a far better representation of the proteome and will therefore allow the development of much more powerful prediction algorithms. In conclusion, we attempt to extend the current knowledge on the specificity of these proteases and their effect on the generation of pathogen or tumor-specific CTL epitopes. The final goal is to define the contribution of these proteases and their specificities in order to allow, in combination with predictions of MHC-peptide interactions, a rapid and reliable identification of CTL epitopes in any given protein.» weiterlesen» einklappen