Identification and characterization of a mammalian protein interacting with 20S proteasome precursors.

The assembly of individual mammalian proteasome subunits into catalytically active 20S proteasome is not well understood. Herein, we report the identification and characterization of human and mouse homologues of the yeast proteasome maturating factor Ump1p. We delineate the region of hUMP1 implicated in the specific interaction with proteasome precursors and show that hUMP1 protein is absent from the mature form of the 20S proteasome. We also show that the transcript level of mammalian UMP1 is increased after IFN-gamma treatment and that mammalian UMP1 is functionally related to but not interchangeable with its yeast homologue.

Naturally processed and concealed HLA-A2.1-restricted epitopes from tumor-associated antigen tyrosinase-related protein-2.

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8(+) cytotoxic T lymphocytes. HLA-A2.1(+) melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2(360-368) (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2(476-484) (VMGTLVALV) were unable to lyse HLA-matched TRP-2(+) cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini-genes encoding the TRP-2(476-484) peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2(476-484) peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminus-flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2(476-484) epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A(27-35) immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2(476-484) epitope in HLA-A2.1(+) and TRP-2(+) tumor lines, as witnessed by cytokine release by specific T-cell clones.

The open reading frame III product of cauliflower mosaic virus forms a tetramer through a N-terminal coiled-coil.

The open reading frame III product of cauliflower mosaic virus is a protein of 15 kDa (p15) that is essential for the virus life cycle. It was shown that the 34 N-terminal amino acids are sufficient to support protein-protein interaction with the full-length p15 in the yeast two-hybrid system. A corresponding peptide was synthesized and a recombinant p15 was expressed in Escherichia coli and purified. Circular dichroism spectroscopy showed that the peptide and the full-length protein can assume an alpha-helical conformation. Analytical centrifugation allowed to determine that p15 assembles as a rod-shaped tetramer. Oxidative cross-linking of N-terminal cysteines of the peptide generated specific covalent oligomers, indicating that the N terminus of p15 is a coiled-coil that assembles as a parallel tetramer. Mutation of Lys22 into Asp destabilized the tetramer and put forward the presence of a salt bridge between Lys22 and Asp24 in a model building of the stalk. These results suggest a model in which the stalk segment of p15 is located at its N terminus, followed by a hinge that provides the space for presenting the C terminus for interactions with nucleic acids and/or proteins.