Juvenile rheumatoid arthritis (JRA) is a heterogeneous autoimmune disease likely to result from multiple genetic and environmental factors. One genetic factor encoded in the class I region of the major histocompatibility complex (MHC), and associated with late onset particular JRA, is HLA-B27. In preliminary studies we have identified homozygosity for the B allele of LMP2, one of two MHC-encoded proteasome subunit genes (LMP2 and LMP7), as an additional genetic risk factor for susceptibility to arthritis in HLA-B27-positive children. Since proteasomes ar involved in the generation of peptides presented by MHC class 1 molecules like B27, we hypothesize that polymorphisms in LMP7 may also affect susceptibility to JRA. Furthermore, LMP2, LMP7, and their allelic variants may influence the production of peptide epitopes presented by HLA-B27, thus influencing disease susceptibility in these individuals. This proposal outlines a career development program designed to complete the training of a physician-scientist interested in the molecular mechanisms of HLA-B27-associated autoimmune disease pathogenesis. It relies on the strengths and potential of a Howard Hughes Medical Institute research laboratory headed by Dr. John Monaco to provide further training in modern techniques of molecular immunology, particularly with regard to proteasome function, and strong Division of Rheumatology headed by Dr. David Glass, with long-term interests and expertise in genetic associations in JRA. In addition to the basic research component, the training program will provide the candidate an expanded knowledge base in immunology through participation in lab meetings, journal clubs, and research seminars.
In Specific Aim 1 we propose to use molecular genotyping to confirm and extend our initial observations wit LMP2, and determine whether LMP7 polymorphisms are associated with subgroups of JRA.
In Specific Aim 2 we address the role of LMP subunits in influencing the cleavage site specificity of proteasomes by digesting oligopeptides containing known HLA class I-restricted peptide epitopes. Epitopes will be identified and their relative quantities assessed, using mass spectroscopy (MS) and cytotoxic T lymphocyte (CTL) assays.
In Specific Aim 3 we will test hypothesis that LMP2 and LMP7 polymorphisms result in functional differences in proteasome activity. In vitro mutagenesis will be used to mimic naturally-occurring polymorphisms in LMP2 and LMP7, and cleavage site specificity of proteasomes containing these allelic variants will be assessed using MS and CTL recognition assays. These studies will define the association between LMP alleles and JRA, and contribute to our understanding of the function of MHC-encoded proteasome subunits and the significance of their allelic variation. Furthermore, they will essential to our determining how polymorphic gene products involved at different sites in the class I antigen processing pathway might interact to predispose certain individuals to JRA.
