Census of cytosolic aminopeptidase activity reveals two novel cytosolic aminopeptidases.

Activation of CD8(+) cytotoxic T cells is crucial for the adaptive immune response against viral infections and the control of malignant transformed cells. Together with activation of costimulatory molecules like CD3 and CD28, CD8(+) T cells need activation of their unique T cell receptor via recognition of foreign peptide epitopes in combination with major histocompatibility complexes class I on the cell surface of professional antigen-presenting cells. Presentation of pathogen-associated proteins is the result of a complex proteolytic process. It starts with the breakdown of proteins by a cytosolic endopeptidase, the proteasome, and is continued by subsequent N-terminal trimming events in the cytosol and/or the endoplasmic reticulum. Analysis of the proteolytic aminopeptidase activity in the former cellular compartment showed that the cytosol harbors a multitude of aminopeptidases that have singular specificities, but on the other hand also show redundancy in the trimming of N-terminal residues. The observed pattern of the overall trimming in the cytosol is reflected by the activity of the four identified aminopeptidases, and the administration of protease inhibitors made it possible to assign specificity of cleaving of proteinogenic amino acids to one or more identified aminopeptidase. The only exception was the cleavage of aspartic acid, which is performed by one yet unidentified enzyme.

Characterizing the N-terminal processing motif of MHC class I ligands.

Most peptide ligands presented by MHC class I molecules are the product of an intracellular pathway comprising protein breakdown in the cytosol, transport into the endoplasmic reticulum, and successive N-terminal trimming events. The efficiency of each of these processes depends on the amino acid sequence of the presented ligand and its precursors. Thus, relating the amino acid composition N-terminal of presented ligands to the sequence specificity of processes in the pathway gives insight into the usage of ligand precursors in vivo. Examining the amino acid composition upstream the true N terminus of MHC class I ligands, we demonstrate the existence of a distinct N-terminal processing motif comprising approximately seven residues and matching the known preferences of proteasome and TAP, two key players in ligand processing. Furthermore, we find that some residues, which are preferred by both TAP and the proteasome, are underrepresented at positions immediately preceding the N terminus of MHC class I ligands. Based on experimentally determined aminopeptidase activities, this pattern suggests trimming next to the final N terminus to take place predominantly in the endoplasmic reticulum.