T LYMPHOCYTE DYSFUNCTION IN LUPUS ERYTHEMATOSUS

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。

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.