T LYMPHOCYTE DYSFUNCTION IN LUPUS ERYTHEMATOSUS

红斑狼疮 T 淋巴细胞功能障碍

• 批准号: 2079546
• 负责人: GARY M KAMMER
• 金额: $ 15.76万
• 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 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2079546
• 关键词: T lymphocyte; disease /disorder proneness /risk; enzyme activity; enzyme deficiency; family genetics; human subject; isozymes; molecular pathology; phosphotransferases; protein isoforms; protein kinase A; protein structure function; systemic lupus erythematosus

We have described a disorder of cAMP metabolism in T lymphocytes of subjects with systemic lupus erythematosus (SLE) Our recent identification of defective cAMP -dependent phosphorylation of proteins in intact SLE T lymphocytes suggests a disorder of cAMP -dependent protein kinase (Protein kinase A) The objective of this proposal is to identify and characterize the abnormal protein kinase A function in SLE T lymphocytes by quantifying cAMP -dependent protein kinase activities and the amounts of the enzyme's constituent subunits, the types I (RI) and II (RII) regulatory and catalytic subunits (C subunit). The specific aims of this proposal are (a) to compare protein kinase A activities in the nonparticulate and particulate fractions of T lymphocytes and T lymphocyte subsets from active and inactive SLE subjects with controls. cAMP -dependent protein kinase activity will be quantified by measuring the phosphorylation of histone and Kemptide in nonparticulate and particulate T cell extracts. (b) We will purify partially the cAMP -dependent protein kinases of SLE T lymphocytes by DEAE -cellulose chromatography in order to determine the presence of anomalous RI, RII and/or C subunits. Anomalous RI or RII subunits can exhibit altered cAMP binding while anomalous C subunits can possess reduced binding affinity for ATP. (c) The amounts of protein kinase A subunits will be quantified by ELISA and nitrocellulose immunolabeling to determine whether a deficiency of one or more subunits exists to explain altered protein kinase activities. Differences in the kinase activities and/or amounts of a kinase of a kinase subunit between T cell subsets will be quantified to determine whether a restricted kinase defect exists. Since the T lymphocyte plays an integral role in both cellular and humoral immunity, it is important to discover whether a defective cAMP pathway accounts, in part, for the aberrant immunoregulation in SLE. Thus, our longterm goal is to determine whether defective protein kinase A function results in abnormal suppressor cell activity. The identification of a protein kinase A defect should provide important insights into the pathogenesis of this autoimmune disorder.

我们已经描述了T细胞的cAMP代谢紊乱 系统性红斑狼疮(SLE)患者的淋巴细胞 我们最近发现的有缺陷的cAMP依赖 完整SLE T淋巴细胞中蛋白质的磷酸化表明 一种cAMP依赖的蛋白激酶(蛋白激酶A)紊乱 这项建议的目标是确定和描述 系统性红斑狼疮T淋巴细胞蛋白激酶A功能异常 定量cAMP依赖的蛋白激酶活性和 酶的组成亚基I(RI)和类型I(RI) II(RII)调节和催化亚基(C亚基)。具体的 这项提议的目的是(A)比较蛋白激酶A T的非颗粒态和颗粒态的活度 活动期和非活动期淋巴细胞和T淋巴细胞亚群 SLE受试者与对照组。CAMP依赖的蛋白激酶 活性将通过测量磷酸化的方式进行量化 非颗粒和颗粒性T细胞中的组蛋白和克隆肽 萃取物。(B)我们将部分纯化cAMP依赖的蛋白质 DEAE-纤维素层析法测定SLE T淋巴细胞的激活酶 为了确定RI、RII和/或C异常的存在 亚单位。RI或RII亚基异常可表现为cAMP改变 结合，而异常的C亚基可能具有减少的结合 对三磷酸腺苷的亲和力。(C)蛋白激酶A亚单位的数量 将通过酶联免疫吸附试验和硝酸纤维素免疫标记进行定量 确定是否存在一个或多个亚基缺失 解释蛋白激酶活性的改变。中美之间的差异 激酶亚单位的激活度和/或激活量 T细胞亚群之间的关系将被量化以确定 限制性激酶缺陷是存在的。因为T淋巴细胞扮演着一个 在细胞免疫和体液免疫中起着不可或缺的作用 重要的是要发现cAMP通路是否有缺陷 这在一定程度上解释了SLE免疫调节的异常。 因此，我们的长期目标是确定有缺陷的蛋白质 激酶A的功能导致抑制细胞的异常活动。 蛋白激酶A缺陷的鉴定应提供 对这种自身免疫疾病发病机制的重要认识 无序。