STAT3 Functional Defects and a Novel Therapeutic Approach for Hyper IgE Syndrome

自从Wiskott-Aldrich综合征(Wiskott-Aldrich综合征)的致病基因被发现以来，我们一直致力于对Wiskott-Aldrich综合征蛋白(WASP)的研究。这项建议的目的是确定经典的WAS及其较温和的形式--X-连锁血小板减少症(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AS/XLT患者的突变分析发现，在PH域内存在许多导致XLT的错义突变。已知PH结构域与膜脂(如PIP2)结合，从而负责将含有PH结构域的蛋白质定位到细胞膜上。使用体外结合系统，我们将调查PH域内自然发生的突变是否干扰WASP与PIP2的结合，以及定点突变是否产生不再与PIP2结合的PH域。相反，WASP SH3结合区的突变导致了严重的AS表型。自然发生的突变和通过定点突变WASP的SH3结合域获得的突变，表达为GST-融合蛋白，将被用来证明与含有SH3的适配蛋白和已知与正常WASP相互作用的激酶失去结合。PH和SH3结构域的突变对WASP酪氨酸磷酸化的影响也将被研究。根据观察到的经典型AS患者的淋巴细胞，而不是XLT患者的淋巴细胞，表现出加速的凋亡和caspase-3活性的增加，我们将研究不同的死亡途径，以确定导致这种加速的凋亡的机制。最后，我们建立了一个Was缺陷(KO)小鼠的繁育群体，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细胞依赖抗原通过不同的途径以低或高剂量给予以确定抗体反应，并在体内建立HSV感染模型来研究抗原特异性CTL的产生。将探索WASP KO小鼠在体内研究血小板功能异常和加速细胞凋亡的作用。这些研究的结果将阐明WASP的功能，解释WAS/XLT的表型，并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.