The Cytosolic Endopeptidase, Thimet Oligopeptidase, Destroys Antigenic Peptides and Limits the Extent of MHC Class I Antigen Presentation
ABSTRACT Most antigenic peptides presented on MHC class I molecules are generated by proteasomes during protein breakdown. It is unknown whether these peptides are protected from destruction by cytosolic peptidases. In cytosolic extracts, most antigenic peptides are degraded by the metalloendopeptidase, thimet oligopeptidase (TOP). We therefore examined whether TOP destroys antigenic peptides in vivo. When TOP was overexpressed in cells, class I presentation of antigenic peptides was reduced. In contrast, TOP overexpression didn't reduce presentation of peptides generated in the endoplasmic reticulum or endosomes. Conversely, preventing TOP expression with siRNA enhanced presentation of antigenic peptides. TOP therefore plays an important role in vivo in degrading peptides released by proteasomes and is a significant factor limiting the extent of antigen presentation.
- SourceAvailable from: Dipankar Nandi[Show abstract] [Hide abstract]
ABSTRACT: PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH5alphaDeltapepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E. coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.Microbiology 01/2004; 149(Pt 12):3437-47. DOI:10.1099/mic.0.26518-0 · 2.84 Impact Factor
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ABSTRACT: Cytotoxic CD8+ T lymphocytes (CTLs) constitute one of the main effector mechanisms against tumors and viral infections. CTLs specifically recognize short peptides ( 8 - 10 residues long) displayed on the surface of 'target' cells, which result from the processing of foreign or abnormal proteins ( e. g. virus and tumor proteins) and are bound to major histocompatibility complex (MHC) class I molecules. Virtually all nucleated cells display on their surface fragments of intracellularly produced polypeptides. When there are signs of invasion or transformation, CTLs take control of the situation by destroying these 'labeled' target cells. This is an extremely efficient mechanism. However, the efficient differentiation of naive CD8+ T cells into CTLs is a limiting prerequisite. To achieve this differentiation, dendritic cells (DCs) are critical since only these professional antigen-presenting cells (APCs) can provide not only the peptide presented onto the MHC class I molecules but also the costimulatory signals required for this activation. To this end, DCs take up antigens and degrade them into peptides which are loaded on MHC class I and presented onto the surface to prime specific T lymphocytes. In this review, we summarize the current knowledge on the mechanisms used by professional APCs in the processing and presentation of endogenous and exogenous antigens in the context of MHC class I molecules (i.e. priming and cross-priming). We will also discuss new vaccination strategies that take advantage of these physiological mechanisms in order to improve the elicitation of cytotoxic responses to eliminate intracellular pathogens and tumors.Transfusion Medicine and Hemotherapy 01/2004; 31(6):398-411. DOI:10.1159/000082484 · 2.01 Impact Factor