Belich, Monica Polidoro; (1997) Proteasome subunits related to the MHC-encoded LMP proteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The objective of this work was to identify and characterize new 20S proteasome subunits that are homologous to the human subunits LMP2 and LMP7. The LMP genes are tightly linked to the TAP peptide transport loci in the class II region of the MHC implicating this multi-subunit protease in antigen processing for class I presentation. Nevertheless, studies with .174 mutant cell line which lacks the LMP genes show that they were able to process and present antigens at the cell surface at a similar level to wild type 721 cells. The results presented in chapter 3 suggest that MB1 and Delta gene subunits are most probably the LMP7 and LMP2 subunit homologues. MB1 gene was discovered by screening cDNA libraries under non-stringent conditions with a LMP7 probe. Delta was cloned by hybridise complete cDNA libraries with a Delta PCR probe. Protein analysis showed that MB1 is 68.1% identical to the LMP7 sequence and Delta 60.8% identical to LMP2. The data presented in chapter 4 shows the chromosomal localization of MB1 and Delta loci examined by using fluorescence in situ hybridization (FISH) method. MB1 and Delta genes localized to different chromosomes, 14q11.2 and 17p13 respectively. Antisera raised against the C-termini of MB1 and Delta protein were produced to further characterize these ? subunits. Results presented on chapter 5 shows that MB1 and Delta proteins are part of the 20S proteasome complex and that they have reciprocal expression to that of the LMP genes: MB1 and Delta are up- regulated in mutant cells lacking the LMPs genes and down-regulated in the presence of γ-IFN. The data presented in chapter 6 shows that proteasomes from 721 and .174 cell lines have similar quaternary structure and the immunolocalization of MB1 and Delta subunits into. 174 cells supports the proposed role of these subunits in directly substituting the genes LMP2 and LMP7. Chapter 7 describes the analysis of a large number of B cell and non B cell lines for LMP2, LMP7, MB1 and Delta expression. It shows that these subunits are expressed at different levels and compositions and that 20S proteasomes are formed from many different combinations of the available subunits.

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