T LYMPHOCYTE DYSFUNCTION IN LUPUS ERYTHEMATOSUS

描述(改编自调查人员摘要)： SLE是T细胞功能障碍，表现为对有丝分裂原的增殖减少 或抗原，IL-2的释放减少，抑制T细胞的生成受损 细胞。在这项建议中，首席调查员建立在他谨慎的基础上 已建立的观察结果表明，腺苷环化酶/环化 AMP/蛋白激酶A同工酶磷酸转移酶信号转导途径 (特别是PKA-1的R1亚基)，88%的人有缺陷 系统性红斑狼疮。PKA是cAMP介导的唯一胞浆途径 多个膜和胞质蛋白的磷酸化。《校长》 研究人员已经将异常分离到了R1亚单位 全酶，并表明在SLE患者中，R1的数量是正常的， 但活动减少了。他的假设是：1)缺陷是由于 R1a和/或R1b亚型的点突变包括 PKA-1亚基，以及2)这些缺陷是遗传的。一大堆 给出了以前的工作和初步数据。具体目标是：1. 研究一组无血缘关系的系统性红斑狼疮患者以估计其患病率 DLE、SCLE和SLE中PKA-1活性缺乏，决定了PKA-1活性是否 酶与疾病的活动性有关，无论它在不同的种族中是不同的。 以及性别，如果这是这些患者家庭中的一种可遗传特征； 2.用SSCP和测序鉴定R1a或R1b亚基的突变 从克隆的聚合酶链式反应产物和基因组DNA中提取cDNA.初步数据显示2 这类突变的例子-都是T到A，在R1a中用F取代I-One 另一个在R1b，两者都位于可能损害折叠的区域 允许完全cAMP结合的A和B亚基。筛选 突变将在SSCP或竞争性PCR中使用凝胶移位；感兴趣的条带 将被测序。3.制备突变型和野生型重组R1a 狼疮患者的和/或R1b亚基蛋白定量同工酶动力学 和磷酸转移酶活性来确定突变是否以及如何 影响功能。4.检查转录和转录后 活化和非活化T细胞中R1a和R1b亚单位mRNA的调节 通过定量检测SLE与对照组的mRNA含量、胞浆mRNA周转率 以及每种异构体蛋白质的量。转录将被抑制在 不同阶段的放线菌素D和二氯-b-D-核糖基苯并咪唑)。 量将通过免疫印迹35S标记的材料来测量。5.至 筛查狼疮家系以确定突变是否传递 狼疮家族，并与PKA-1同工酶缺乏症相关。这个 首席调查员将研究14个家庭，包括3代人- 从一个带有突变的狼疮先证者开始。

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.