Deciphering the Role of WASP Family Proteins in T Cell Function

解读 WASP 家族蛋白在 T 细胞功能中的作用

• 批准号: 8147996
• 负责人: Scott B Snapper
• 金额: $ 41.98万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8147996
• 关键词: Actins; Alleles; Binding; Biological Models; Cell Maturation; Cell Shape; Cell physiology; Cell surface; Cells; Chemotaxis; Collaborations; Collection; Complex; Cytoplasmic Protein; Cytoskeleton; Development; Embryo; Family; Family member; Gene Targeting; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Homing; Immunologic Deficiency Syndromes; In Vitro; Knock-in Mouse; Knock-out; Knockout Mice; Leukocytes; Link; Lymphocyte; Lymphocyte Activation; Lymphopoiesis; Mature T-Lymphocyte; Mediating; Modeling; Mus; Mutation; Neutropenia; Phosphatidylinositols; Phosphotransferases; Property; Protein Family; Proteins; Reagent; Regulation; Role; SH3 Domains; Signal Transduction; T-Cell Development; T-Lymphocyte; Tertiary Protein Structure; Transplantation; WASP protein; Wiskott-Aldrich Syndrome; base; embryonic stem cell; in vivo; novel; rho; rho GTP-Binding Proteins; thymocyte

We have recently developed model systems to study the mammalian cellular function of Rho GTPases (e.g., Cdc42) and the Wiskott-Aldrich syndrome proteins (e.g., WASP, N-WASP, WAVEs) - two interacting families of proteins that integrate incoming cell surface signals to mediate cytoskeletal change. WASPs are cytoplasmic proteins that when activated by Rho-family GTPases (Cdc42/Rac) and phosphoinositides directly bind to the Arp2/3 complex, resulting in actin assembly. In this context, coordination of cell shape through cytoskeletal change is required for such diverse properties as cell-cell contact, lymphocyte activation, and chemotaxis. WASP activation is also regulated by interaction with several other proteins including the WASP-interacting protein (WIP), Sh3 domain proteins (e.g. Nek) and Srk family kinases (e.g., Hck). We have employed gene-targeting to generate mice deficient for the Rho-family GTPase Cdc42 as well as several WASP family members: WASP, N-WASP, and WAVE-2. N-WASP- and WAVE-2- deficiencies result in early embryonic lethality whereas WASP-deficient (WKO) mice are viable and fertile, with lymphocytes that develop normally but have signaling and cytoskeletal abnormalities. We have also generated ES cells clones that contained an activated WASP knock-in mutation associated with the recently described novel immunodeficiency, X-linked Neutropenia (XLN). Because N-WASP and WAVE-2 deficiencies were not viable, selective targetings of these alleles were required to assess the role of these proteins in lymphocytes. To this end, we have generated mice with N-WASP that can be conditionally inactivated in leukocytes, and we are currently generating mice with similar mutations in WAVE2. We have generated WASP/N-WASP double knock-out (DKO) mice in T cells and, in collaboration with Dr. Raif Geha (Project 1), WASP/WIP DKO mice; both strains are associated with more severe signaling and cytoskeletal abnormalities in T cells when compared to WKO mice. This collection of novel reagents, in which the various WASP-family members can be inactivated, singularly or in combination and in either cells or mice, provides a powerful basis for our ongoing goals of dissecting the roles of WASP family members in leukocyte function. We propose: A1) To dissect the unique role of WASP and the combined role of WASP/N-WASP in leukocyte development and function. A2) To define the role of constitutively-active mutations in WASP function in T cells.

我们最近开发了模型系统来研究 Rho GTPases 的哺乳动物细胞功能（例如， Cdc42）和 Wiskott-Aldrich 综合征蛋白（例如 WASP、N-WASP、WAVE）——两种相互作用 整合传入细胞表面信号以介导细胞骨架变化的蛋白质家族。 WASP 是 被 Rho 家族 GTPases (Cdc42/Rac) 和磷酸肌醇激活时的细胞质蛋白 直接与 Arp2/3 复合物结合，导致肌动蛋白组装。在这种情况下，细胞形状的协调 通过细胞骨架的变化来实现细胞与细胞接触、淋巴细胞等多种特性 激活和趋化作用。 WASP 的激活也受到与其他几种蛋白质相互作用的调节 包括 WASP 相互作用蛋白 (WIP)、Sh3 结构域蛋白（例如 Nek）和 Srk 家族激酶（例如， 哈克）。我们采用基因靶向技术来产生 Rho 家族 GTPase Cdc42 缺陷的小鼠，如下所示： 以及几个 WASP 家族成员：WASP、N-WASP 和 WAVE-2。 N-WASP- 和 WAVE-2- 缺陷会导致早期胚胎死亡，而 WASP 缺陷 (WKO) 小鼠却能存活并具有生育能力， 淋巴细胞发育正常，但信号传导和细胞骨架异常。我们还有 生成的 ES 细胞克隆包含与最近的相关的激活的 WASP 敲入突变 描述了新型免疫缺陷，X连锁中性粒细胞减少症（XLN）。因为 N-WASP 和 WAVE-2 缺陷是不可行的，需要选择性地针对这些等位基因来评估这些等位基因的作用 淋巴细胞中的蛋白质。为此，我们生成了具有 N-WASP 的小鼠，可以有条件地 在白细胞中失活，我们目前正在培育具有 WAVE2 类似突变的小鼠。我们有 与 Raif Geha 博士合作，在 T 细胞中产生了 WASP/N-WASP 双敲除 (DKO) 小鼠 （项目1），WASP/WIP DKO小鼠；两种菌株都与更严重的信号传导和细胞骨架相关 与 WKO 小鼠相比，T 细胞异常。这一系列新颖的试剂，其中 各种 WASP 家族成员可以在细胞或小鼠中单独或组合失活， 为我们剖析 WASP 家庭成员的角色的持续目标提供了强有力的基础 白细胞功能。我们建议： A1) 剖析 WASP 的独特作用和 WASP 的综合作用 WASP/N-WASP 在白细胞发育和功能中的作用。 A2) 定义本质活性的作用 T 细胞中 WASP 功能的突变。