The F-BAR protein CIP4 in WAS-dependent thrombocytopenia

描述（由申请方提供）：血小板通过其数量、活化以及与炎症和血管系统的相互作用在心血管、中风和出血性疾病中发挥核心作用。Wiskott-Aldrich综合征（WAS）是一种X-连锁疾病，其特征是血小板减少症、免疫缺陷、湿疹和淋巴瘤和自身免疫风险增加。受影响的基因编码细胞骨架蛋白，WASP。通过RhoGTdR，Cdc 42，WASp经历构象变化，导致肌动蛋白聚合。WASp如何导致血小板减少症（WAS最常见的表现）仍然未知。Cdc 42、Src激酶、膜磷脂和SH 3结构域控制WASP功能。本实验室以Src激酶林恩为诱饵，在酵母双杂交筛选中发现了一种新的WASP调节因子CIP 4（Cdc 42 interacting protein 4）。CIP 4是F-BAR蛋白家族的成员，其重塑质膜并促进肌动蛋白聚合。为了确定CIP 4的功能，我们创建了CIP 4-null小鼠。我们发现，CIP 4-/-小鼠显示血小板减少症，类似于在WASP-/-小鼠中观察到的。WAS中血小板减少症的机制尚不清楚。一些研究证明了自身免疫的基础;其他人在前血小板生产的缺陷。因为我们的小鼠没有显示出自身免疫性疾病的迹象，所以我们倾向于CIP 4-WASP形成促进血小板产生的信号传导途径并且CIP 4或WASP的缺乏扰乱巨核细胞中的肌动蛋白聚合的假说（“缺陷性前血小板形成假说”），这导致缺陷性前血小板形成。另一种“免疫破坏假说”指出，该CIP 4-WASP途径中的缺陷促进自身免疫和免疫介导的血小板破坏。我们提出通过以下两个具体目标来解决这些假设：1）通过培养来自野生型、CIP 4-/-、WASP-/-和CIP 4-/-WASP-/-小鼠的巨核细胞前体和巨核细胞并分析它们的前血小板形成来解决有缺陷的前血小板假设。我们将使用这些独特的小鼠品系来分析前血小板形成、CIP 4与巨核细胞中微管的相互作用以及血小板的超微结构特征;以及2）通过标记血小板并测量它们在野生型、CIP 4-/-、WASP-/-和CIP 4-/-WASP-/-小鼠中的清除率以及测量它们的T、B和NK细胞功能来测试免疫破坏假说。我们将使用这些独特的小鼠品系来测量血小板存活率，并与免疫功能的改变相关联。 公共卫生相关性：血小板生成对于正常控制出血至关重要。出血过多和血小板减少是Wiskott-Aldrich综合征（WAS）的特征.我们发现了一种蛋白质CIP 4，它在物理和功能上与WAS蛋白（WASP）相互作用。WAS中血小板减少症的机制尚不清楚。我们将研究CIP 4基因敲除和CIP 4/WASP双基因敲除小鼠的血小板生成，以确定导致血小板减少症的确切机制。由于血小板与心血管、中风和炎症性疾病有关，我们的工作有可能对人类健康产生更广泛的影响。

DESCRIPTION (provided by applicant): Platelets play a central role in cardiovascular, stroke, and bleeding disease through their quantity, activation, and interaction with the inflammatory and vascular systems. The Wiskott -Aldrich syndrome (WAS) is an X- linked disorder characterized by thrombocytopenia, immunodeficiency, eczema and an increased risk of lymphoma and autoimmunity. The affected gene encodes the cytoskeletal protein, WASP. Through the RhoGTPase, Cdc42, WASp undergoes conformational change that leads to actin polymerization. How WASp results in thrombocytopenia, the most common manifestation of WAS, remains unknown. Cdc42, Src kinases, membrane phospholipids, and SH3 domains control WASP function. Our lab has discovered a new regulator of WASP, CIP4 (Cdc42 interacting protein 4), in a yeast two hybrid screen with the Src kinase Lyn as bait. CIP4 is a member of the F-BAR family of proteins, which remodel the plasma membrane and promote actin polymerization. To determine CIP4's function, we created a CIP4-null mouse. We found that CIP4-/- mice display thrombocytopenia, similar to that observed in WASP-/- mice. The mechanism for thrombocytopenia in WAS is not known. Some studies demonstrate an autoimmune basis; others a defect in proplatelet production. Because our mice do not display signs of autoimmune disease, we favor the hypothesis ("defective proplatelet formation hypothesis") that CIP4-WASP forms a signaling pathway that promotes platelet production and that a deficiency of either CIP4 or WASP perturbs actin polymerization in megakaryocytes, which results in defective proplatelet formation. An alternative "immune destruction hypothesis" states that a defect in this CIP4-WASP pathway promotes autoimmunity and immune-mediated destruction of platelets. We propose to address these hypotheses through the following two specific aims: 1) the defective proplatelet hypothesis by culturing megakaryocyte precursors and megakaryocytes from wild-type, CIP4-/-, WASP-/-, and CIP4-/-WASP-/- mice and analyzing their proplatelet formation. We will use these unique mice strains to analyze proplatelet formation, the interaction of CIP4 with microtubules in megakaryocytes, and ultrastructural features of platelets; and 2) Test the immune destruction hypothesis by labeling platelets and measuring their clearance rates in wild-type, CIP4-/-, WASP-/-, and CIP4-/-WASP-/- mice and measuring their T, B, and NK cell functions. We will use these unique mice strains to measure platelet survival and to correlate with altered immune function. PUBLIC HEALTH RELEVANCE: Platelet production is critical for normal control of bleeding. Excessive bleeding and thrombocytopenia characterizes Wiskott - Aldrich syndrome (WAS). We have discovered a protein CIP4 that interacts physically and functionally with WAS Protein (WASP). The mechanism for the thrombocytopenia in WAS is not known. We will study platelet production in CIP4 knockout and CIP4/WASP double knockout mice to determine the precise mechanism that results in thrombocytopenia. Since platelets are involved in cardiovascular, stroke, and inflammatory diseases, our work has the potential to impact more broadly on human health.