Gene and Protein Defects Causing Mastocytosis

导致肥大细胞增多症的基因和蛋白质缺陷

• 批准号: 6909836
• 负责人: YONGSHENG MA
• 金额: $ 13.01万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6909836
• 关键词: Baculoviridae; Sf9 cell line; X ray crystallography; biological signal transduction; clinical research; conformation; enzyme structure; gel filtration chromatography; genetic disorder; human subject; intermolecular interaction; laboratory mouse; laser capture microdissection; loss of heterozygosity; mast cell; mastocytosis; model design /development; nucleic acid sequence; patient oriented research; physical model; polymerase chain reaction; protein tyrosine kinase; protooncogene

DESCRIPTION (provided by applicant): The goal of this proposal is to add to the understanding of the mechanisms underlying the pathogenesis of mastocytosis. They have previously shown that essentially all adult and atypical pediatric mastocytosis are characterized by mutations in codon 816 of c-KIT gene causing constitutive activation of its protein product, KIT, a receptor tyrosine kinase. In contrast, most pediatric cases lack known activating c-KIT mutations. Our recent data indicate that activation of the P13K/AKT pathway downstream of KIT is a general feature of all forms of mastocytosis. We thus hypothesize that most, if not all, cases of mastocytosis are caused by over-activation of the KIT signaling pathway, or another pathway that converges with it at or above the P13K/AKT node. We have found that synthetic compounds that effectively inhibit wild-type KIT are ineffective against KIT containing codon 816 activating mutations at concentrations that can be safely achieved in vivo. Since the residue encoded by codon 816 lies in the active site of the receptor kinase, where the compounds presumably bind, we hypothesize that the conformation of the active site of the mutant receptor differs from that of wild-type KIT. We propose to test the two hypotheses by two specific aims. 1. To determine the mechanisms of oncogenesis in c-KIT mutation negative pediatric mastocytosis, genes encoding molecules affecting P13K/AKT signaling will be examined in laser captured lesional mast cells by PCR sequencing for mutations and by microsatellite analysis for loss of heterozygosity. The functional significance of identified defects will be determined by cDNA expression in mast cells in vitro and in vivo. 2. To determine the three-dimensional structure of KIT kinase domain, wild-type and codon 816 mutated KIT kinase domains will be expressed using the Baculovirus/Sf9 system and purified using anion exchange and gel filtration chromatography. The proteins will be crystallized using vapor diffusion in hanging drops. X-ray diffraction of the crystals will be analyzed using the multiwavelength anomalous dispersion procedure for phase determination. The WARP program will be used to build models, and the models will be refined and rebuilt using the program XPLOR and O. Knowing the structures of the wild-type and mutant kinase domains will allow rational development of drugs targeted specifically at a defined cause of mastocytosis.

描述（由申请人提供）： 该提案的目的是增加对肥大性发病机理的机制的理解。他们先前已经表明，基本上所有成年和非典型小儿肥大症的特征是在C-KIT基因的密码子816中突变，导致其蛋白质产物Kit Kit（一种受体酪氨酸激酶）的组成型激活。相反，大多数儿科病例都缺乏已知的激活C-KIT突变。我们最近的数据表明，盒子下游的P13K/AKT途径的激活是各种形式的肥大性胞吞作用的一般特征。因此，我们假设大多数（如果不是全部）肥大的病例是由试剂盒信号通路过度激活或在P13K/AKT节点上或以上收敛的另一种途径引起的。我们发现，有效抑制野生型试剂盒的合成化合物对含有密码子816激活突变的试剂盒无效，该浓度可以在体内安全实现。由于密码子816编码的残基位于受体激酶的活性位点，其中化合物可能结合，因此我们假设突变受体的活性位点的构象与野生型套件的构象不同。我们建议通过两个特定目标检验两个假设。 1。为了确定C-KIT突变中的肿瘤发生的机制，负小儿肥大症，将通过PCR序列进行突变和微介质分析，从而在激光捕获的病变肥大细胞中检查影响P13K/AKT信号传导的编码分子的基因。鉴定缺陷的功能意义将由体外和体内肥大细胞中的cDNA表达确定。 2。为了确定KIT激酶结构域的三维结构，野生型和密码子816突变的KIT激酶结构域将使用Baculovirus/SF9系统表达，并使用阴离子交换和凝胶过滤色谱法纯化。蛋白质将使用悬挂液中的蒸气扩散来结晶。将使用多波长异常分散程序来分析晶体的X射线衍射。经线计划将用于构建模型，并将使用程序Xplor和O对模型进行完善和重建。了解野生型和突变激酶结构域的结构，将允许合理地开发出针对乳腺癌的定义原因而专门针对的药物。