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

STAT3 功能缺陷和高 IgE 综合征的新治疗方法

基本信息

批准号：
7927739
负责人：
HANS D OCHS
金额：
$ 17.89万
依托单位：
SEATTLE CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7927739
关键词：
Abnormal Platelet Affect Antibody Formation Antigens Apoptosis Binding Blood Platelets Breeding Cell membrane Cessation of life Chimeric Proteins Complex DNA Sequence Rearrangement Defect Dose Generations Genes Goals Human Immune In Vitro Infection Investigation Job&apos s Syndrome Knockout Mice Link Lymphocyte Lymphocyte Function Megakaryocytes Membrane Lipids Missense Mutation Modeling Molecular Mus Mutation Mutation Analysis PH Domain Pathway interactions Patients Phenotype Phosphatidylinositol 4,5-Diphosphate Protein Deficiency Protein Kinase Proteins Research Route SH3 Domains STAT3 gene Signal Transduction Simplexvirus Site-Directed Mutagenesis System T-Lymphocyte Thrombocytopenia Tyrosine Phosphorylation Wiskott-Aldrich Syndrome adapter protein base caspase-3 in vivo novel therapeutic intervention platelet protein P47

项目摘要

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.

自从鉴定出导致Wiskott-Aldrich综合征（WAS）的基因以来，我们将研究重点放在WAS蛋白（WASP）上。该提案的目标是定义经典WASP及其较温和形式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SH 3结合结构域进行详细分析。对WAS/XLT患者的突变分析已经确定了导致XLT的PH结构域内的许多错义突变。已知PH结构域结合膜脂质（例如，PIP 2），因此负责将含有PH结构域的蛋白定位于细胞膜。使用体外结合系统，我们将研究PH结构域内的天然突变是否干扰WASP与PIP 2的结合，以及定点诱变是否产生不再与PIP 2结合的PH结构域。相反，WASP的SH 3结合结构域内的突变导致严重的WAS表型。天然存在的突变和通过WASP的SH 3结合结构域的定点诱变获得的突变（表达为GST融合蛋白）将用于证明与已知与正常WASP相互作用的含有衔接子蛋白和激酶的SH 3的结合丧失。还将研究PH和SH 3结构域内的突变对WASP酪氨酸磷酸化的影响。基于观察到经典WAS患者的淋巴细胞（而非XLT患者）显示加速凋亡和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，我们已经建立了WAS缺陷（KO）小鼠的繁殖群体，这将允许我们使用通过不同途径以低或高剂量给予的T细胞依赖性抗原来确定抗体应答，并使用体内HSV感染模型来研究抗原特异性CTL的产生，从而在体内研究由WASP突变引起的免疫缺陷。将探索WASP KO小鼠在体内研究异常血小板功能和加速细胞凋亡的有用性。这些研究的结果将阐明WASP的功能，解释WAS/XLT的表型，无疑将对受影响患者的最佳治疗产生影响。