T LYMPHOCYTE DYSFUNCTION IN LUPUS ERYTHEMATOSUS

点击翻译按钮获取中文摘要

DESCRIPTION (Adapted from investigator's Abstract): An important defect in SLE is T cell dysfunction, manifested as reduced proliferation to mitogens or antigens, reduced release of IL-2 and impaired generation of suppressor T cells. In this proposal, the Principal Investigator builds on his carefully established observation that the activity of the adenyl cyclase/cyclic AMP/protein kinase A isozyme phosphotransferase signal transduction pathway (specifically of the R1 subunit of PKA-1), is defective in 88% of people with SLE. PKA is the only cytosolic pathway for cAMP-mediated phosphorylation of multiple membrane and cytosolic proteins. The Principal Investigator has isolated the abnormality to the R1 subunit of the holoenzyme, and has shown that quantities of R1 are normal in SLE patients, but activity is reduced. His hypotheses are 1) that the defect is due to point mutations in R1a and/or R1b isoforms comprising the regulatory subunits of PKA-1, and 2) that these defects are inherited. A large body of previous work and preliminary data are presented. Specific Aims are: 1. To study a cohort of unrelated people with SLE to estimate the prevalence of deficient PKA-1 activity in DLE, SCLE and SLE, determine whether activity of enzyme relates to activity of disease, whether it differs in different races and sexes, and if it is a heritable trait in the families of these patients; 2. To identify mutations of the R1a or R1b subunit by SSCP and sequencing of cDNA from cloned PCR products and genomic DNA. Preliminary data show 2 examples of such mutations - both are T to A with F replacing I - one in R1a and the other in R1b, both located in regions likely to impair folding of the A and B subunits which permits full cAMP binding. Screening for mutations will use gel shift in SSCP or competitive PCR; bands of interest will be sequenced. 3. To prepare mutant and wild-type recombinant R1a and/or R1b subunit proteins from lupus subjects to quantify isozyme kinetics and phosphotransferase activities to determine if and how the mutations affect function. 4. To examine transcriptional and posttranscriptional regulation of R1a and R1b subunit mRNA in T cells from active and inactive SLE vs controls by quantifying the mRNA content, cytoplasmic mRNA turnover and amounts of each isoform protein. Transcription will be inhibited at various stages by actinomycin D and dichloro-b-D-ribouranosylbenzimidazole). Amounts will be measured by immunoblotting of 35S-labeled material. 5. To screen lupus families to determine whether the mutation is passed through lupus families and is associated with a PKA-1 isozyme deficiency. The Principal Investigator will study 14 families with 3 generations available - beginning with a lupus proband with a mutation.