DEVELOPMENTAL AND GENETIC DEFECTS OF IMMUNITY

自Wiskott- Aldrich综合征（WAS）的致病基因被发现以来，我们将研究重点放在WAS蛋白（WASP）上。本提案的目标是确定经典WAS及其温和形式X-linked thrombocytopenia （XLT）的分子基础，并研究WASP在人类和小鼠系统中的许多功能。由于它们对WASP的功能至关重要，我们选择了WASP的pleckstrin同源（PH）结构域和SH3结合结构域进行详细分析。对WAS/XLT患者的突变分析发现，PH结构域中存在许多导致XLT的错义突变。已知PH结构域与膜脂结合（例如，PIP2），因此负责将含有PH结构域的蛋白质定位到细胞膜。使用体外结合系统，我们将研究PH结构域内自然发生的突变是否会干扰WASP与PIP2的结合，以及位点定向诱变是否会产生不再与PIP2结合的PH结构域。相反，WASP的SH3结合域内的突变导致严重的WAS表型。自然发生的突变和通过对WASP的SH3结合域进行定点诱变获得的突变，表达为gst融合蛋白，将用于证明与含有已知与正常WASP相互作用的连接蛋白和激酶的SH3结合的丧失。PH和SH3结构域内的突变对WASP酪氨酸磷酸化的影响也将被研究。我们观察到典型WAS患者的淋巴细胞凋亡加速，而非XLT患者的淋巴细胞凋亡加速，caspase-3活性增加，我们将研究不同的死亡途径，以确定导致这种加速凋亡的机制。最后，我们建立了WAS缺陷（KO）小鼠的繁殖群体，这将使我们能够在体内研究WASP突变引起的免疫缺陷，使用T细胞依赖性抗原，通过不同的途径给予低剂量或高剂量来确定抗体反应，以及体内HSV感染模型来研究抗原特异性ctl的产生。我们将探讨WASP - KO小鼠在体内研究血小板功能异常和加速凋亡的有效性。这些研究的结果将阐明WASP的功能，解释WAS/XLT的表型，并且无疑将对受影响患者的最佳治疗产生影响。

Since the identification of the gene responsible for the Wiskott- Aldrich syndrome (WAS), we have focused our research efforts on the WAS protein (WASP). The goal of this proposal is to define the molecular basis for classic WAS and its milder form, X-linked thrombocytopenia (XLT), and to study the many functions attributed to WASP in human and murine systems. Because of their central importance for the function of WASP, we have selected the pleckstrin homology (PH) domain and the SH3 binding domain of WASP for detailed analysis. Mutation analysis of patients with WAS/XLT has identified many missense mutations within the PH domain that result in XLT. PH domains are known to bind to membrane lipids (e.g., PIP2) and thus are responsible for localizing PH domain containing proteins to the cell membrane. Using an in vitro binding system, we will investigate whether naturally occurring mutations within the PH domain interfere with the binding of WASP to PIP2 and if site directed mutagenesis generates PH domains that no longer bind to PIP2. In contrast, mutations within the SH3 binding domain of WASP result in a severe WAS phenotype. Naturally occurring mutations and mutations obtained by site directed mutagenesis of the SH3 binding domain of WASP, expressed as GST-fusion proteins, will be used to demonstrate a loss of binding to SH3 containing adapter proteins and kinases known to interact with normal WASP. The effect of mutations within the PH and SH3 domain on tyrosine phosphorylation of WASP will also be investigated. Based on the observation that lymphocytes from patients with classic WAS, but not with XLT, show accelerated apoptosis and increased caspase-3 activity, we will investigate different death pathways to identify the mechanisms leading to this accelerated apoptosis. Finally, we have established a breeding colony of WAS deficient (KO) mice that will allows us to study in vivo the immune defect caused by mutations of WASP, using a T cell dependent antigen that is given at low or high doses by different routes to determine antibody responses, and an in vivo HSV infection model to study the generation of antigen-specific CTLs. The usefulness of WASP KO mice to study abnormal platelet function and accelerated apoptosis in vivo will be explored. Results from these investigations will clarify the function of WASP, explain the phenotypes of WAS/XLT and will undoubtedly have implications for optimal therapy of affected patients.