ABSTRACT
Proteasomes have been implicated in the production of the majority of peptides that associate with MHC class I molecules. We used two different proteasome inhibitors, the peptide aldehyde N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL) and the highly specific inhibitor lactacystin, to examine the role of proteasomes in generating peptide epitopes associated with HLA-A*0201. Neither LLnL nor lactacystin was able to completely block the expression of the HLA-A*0201. Furthermore, the effects of LLnL and lactacystin on the expression of different categories of specific epitopes, TAP independent vs TAP dependent and derived from either cytosolic or membrane proteins, were assessed. As predicted, presentation of two TAP-dependent epitopes was blocked by LLnL and lactacystin, while a TAP-independent epitope that is processed in the endoplasmic reticulum was unaffected by either inhibitor. Surprisingly, both LLnL and lactacystin increased rather than inhibited the expression of a cytosolically transcribed and TAP-dependent peptide from the influenza A virus M1 protein. Mass spectrometric analyses of in vitro proteasome digests of a synthetic 24 mer containing this epitope revealed no digestion products of any length that included the intact epitope. Instead, the major species resulted from cleavage sites within the epitope. Although cleavage at these sites was inhibitable by LLnL and lactacystin, epitope-containing species were still not produced. We conclude that proteasomes may in some cases actually destroy epitopes that would otherwise be destined for presentation by class I molecules. These results suggest that some epitopes are generated by nonproteasomal proteases in the cytosol.
Subject(s)
Cysteine Endopeptidases/metabolism , Cysteine Endopeptidases/physiology , Cytosol/enzymology , Cytosol/immunology , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/metabolism , Histocompatibility Antigens Class I/metabolism , Multienzyme Complexes/metabolism , Multienzyme Complexes/physiology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/physiology , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Amino Acid Sequence , Animals , Cell Line , Cell-Free System/immunology , Cysteine Endopeptidases/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Cytotoxicity, Immunologic/drug effects , Epitopes, T-Lymphocyte/biosynthesis , Epitopes, T-Lymphocyte/drug effects , Glycine/pharmacology , HLA-A Antigens/biosynthesis , HLA-A Antigens/drug effects , Humans , Leupeptins/pharmacology , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Multienzyme Complexes/drug effects , Proteasome Endopeptidase Complex , T-Lymphocytes, Cytotoxic/enzymology , T-Lymphocytes, Cytotoxic/immunology , Time Factors , Viral Matrix Proteins/biosynthesis , Viral Matrix Proteins/immunologySubject(s)
Extraterrestrial Environment , Plant Development , Chloroplasts/cytology , Culture Media , Culture Techniques , Cytoplasm , Endoplasmic Reticulum , Golgi Apparatus , Microscopy, Electron , Mitochondria , Nitrogen/analysis , Oryza/growth & development , Oxygen Consumption , Phosphorus/analysis , Plant Cells , Plants/analysis , Plants/metabolism , Plants, Toxic , Nicotiana/growth & development , Trees/growth & development , Zea mays/growth & developmentABSTRACT
Tissue cultures of Nicotiana labacum consisting of green, albino and habituated (normal origin) and teratoma (tomorous origin) were grown under asceptic conditions for 6 to 8 weeks and their extracts were analyzed for phosphatase activity. Comparative enzyme analyses were also made on crude stem extracts of greenhouse-grown normal and tumor tissues of Nicotiana tabacum (var. Wisconsin) and a hybrid (N. glauca x N. langsdorffii).All the crude extracts showed acid phosphatase activity with a pH optimum at 5.8 to 6.0. The total protein content and enzyme acivity of teratoma tissue (tumor) was higher than that of green, albino or habituated tissue (normal). Similar increased levels were seen in tumor tissue grown in greenhouse in comparison with greenhouse-grown normal tissues. The crude extracts of each of the tissues did not exhibit any qualitative difference in specificity with the 5 different substrates tested; however, differences in the level of activity was observed.The effect of 4 different culture media was tested on the growth, protein content and acid phosphatase activity of habituated tobacco in tissue culture. Tissues growing in medium containing high salt concentrations showed higher activities than tissues grown in a basal control medium. From the results, it is suggested that although many factors like auxin and other growth factors can influence growth of habituated tobacco tissue, they need not necessarily affect this specific enzyme activity.
