T LYMPHOCYTE DYSFUNCTION IN LUPUS ERYTHEMATOSUS

红斑狼疮 T 淋巴细胞功能障碍

• 批准号: 2006162
• 负责人: GARY M KAMMER
• 金额: $ 33.67万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2006162
• 关键词: African American; T lymphocyte; caucasian American; clinical research; discoid lupus erythematosus; enzyme activity; gender difference; gene mutation; human genetic material tag; human subject; isozymes; protein kinase A; racial /ethnic difference; recombinant proteins; systemic lupus erythematosus; tissue /cell culture

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.

描述（改编自研究者摘要）： 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，减少IL-2的释放和抑制T细胞生成受损 细胞 在这份建议书中，首席研究员仔细地建立在他的基础上， 已建立的观察表明，腺苷酸环化酶/环化酶的活性 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），在88%的人中有缺陷 关于SLE PKA是cAMP介导的唯一胞质途径。 多种膜和胞质蛋白的磷酸化。 校长 研究者已将异常分离至 全酶，并已表明R1的量在SLE患者中是正常的， 但活性降低。 他的假设是：（1）缺陷是由于 包含调节的R1 a和/或R1 b同种型中的点突变 PKA-1的亚基，和2）这些缺陷是遗传的。 大量的 介绍了以前的工作和初步数据。 具体目标是：1。 研究一组无关的SLE患者， DLE、SCLE和SLE中PKA-1活性缺陷，确定是否存在PKA-1活性缺陷。 酶与疾病的活动有关，它是否在不同的种族中存在差异 和性别，以及它是否是这些患者家庭中的遗传特征; 2. 通过SSCP和测序鉴定R1 a或R1 b亚基的突变 从克隆的PCR产物和基因组DNA的cDNA。 初步数据显示2 这类突变的实例-两者都是T到A，其中F替换I -一个在R1 a中 另一个在R1 b中，两者都位于可能损害折叠的区域， A和B亚基允许完全cAMP结合。 筛查 突变将使用SSCP或竞争性PCR中的凝胶移位;感兴趣的条带 将被排序。 3. 制备突变型和野生型重组R1 a 和/或R1 b亚单位蛋白质来定量同工酶动力学 和磷酸转移酶活性来确定突变是否以及如何 影响功能。 4. 为了检测转录和转录后 R1 a和R1 b亚基mRNA在活化和非活化T细胞中的调节 通过定量mRNA含量、细胞质mRNA周转， 和每种同种型蛋白质的量。 转录将被抑制， 通过放线菌素D和二氯-b-D-核糖基苯并咪唑的不同阶段）。 将通过35 S标记材料的免疫印迹法测定含量。 5. 到 筛查狼疮家族以确定突变是否通过 与PKA-1同工酶缺乏有关。 的 首席研究员将研究14个家庭，其中有3代人- 从一个突变的狼疮先证者开始