GPI ANCHOR DEFICIENCY IN HEMATOPOIESIS

造血作用中的 GPI 锚定缺陷

• 批准号: 8212934
• 负责人: ROBERT A BRODSKY
• 金额: $ 28.9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8212934
• 关键词: Aplastic Anemia; Apoptosis; Apoptotic; Biochemical; Blood Cells; CD34 gene; Cell Line; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Ceramides; Characteristics; Clonal Expansion; Clonal Hematopoietic Stem Cell; Complement; Data; Disease; Functional disorder; GPI Membrane Anchors; Gene Mutation; Gene Silencing; Genes; Glycosylphosphatidylinositol-anchor Biosynthesis Pathway; Glycosylphosphatidylinositols; Growth; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hemolysis; Immune; Immunologics; Inositol; Lymphocyte; Malignant - descriptor; Mediating; Membrane; Membrane Microdomains; Modeling; Mutation; Normal Cell; Patients; Phenotype; Play; Protein Deficiency; Proteins; Resistance; Role; Second Messenger Systems; Signal Transduction; Somatic Mutation; Stem cells; Stimulus; T-Lymphocyte; Thrombosis; cancer stem cell; cell type; genetic regulatory protein; killings; mutant; novel; progenitor; promoter; protein expression; rat Piga protein; response; second messenger; stem

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder that is caused by a somatic mutation of the PIG-A gene. The biochemical consequence of PIG-A mutations is a global loss of glycosylphosphatidyl-inositol anchored proteins (GPI-AP). Two GPI-AP (CD55 and CD59) are important complement regulatory proteins, their absence explains the complement-mediated intravascular hemolysis and thrombosis and that occur in PNH patients; however, the mechanism by which PNH cells achieve clonal dominance is not completely understood. All PNH patients have been shown to harbor PIG-A mutations; however, rare PIG-A mutations can also be found in healthy control subjects; thus, the biologic relevance of PIG-A mutations in healthy controls remains to be determined. The overall objective of these studies is to study the relevance of GPI-anchor deficiency in PNH, normal, and malignant lymphohematopoietic cells. Our preliminary data demonstrates that hematopoietic cells can acquire a PNH phenotype (no cell surface GPI-AP) without PIG-A mutations. The mechanism of GPI-AP deficiency in these cells is through transcriptional silencing of genes required for GPI anchor biosynthesis. We have also established a PIGAnull CD34+ cell line with the PIG-A gene under the control of an inducible promoter. This cell line serves as a valuable model to study the mechanism of clonal dominance in PNH. Our preliminary data shows that PNH cells are relatively resistant to T cell mediatied apoptosis, and that the growth advantage of the PNH cells is amplified in the setting of an immune attack. Why PNH cells are resistant to T cell mediated killing is unclear, but our preliminary data suggest it may be related to perturbed signaling through membrane rafts and reduced ability to generate the proapoptotic second messenger, ceramide. Specifically, we will: 1) Investigate the relevance of GPI-AP deficient cells in normals. Hypothesis 1.1: GPI-AP deficiency in lymphocytes from normals can result from a novel mechanism that does not involve PICA mutations. Hypothesis 1.2: In healthy controls, a subset of HSPC in are GPI-APIo/neg, but do not harbor PIG-A mutations. 2) Study the role of GPI-anchor deficiency in cellular resistance to apoptosis. Hypothesis 2.1: GPI-APIo/neg cells are resistant to immune attack via global resistance to apoptosis. Hypothesis 2.2:GPI-anchor expression plays a role in the response of normal cells to apoptotic stimuli. Hypothesis 2.3: Lack of GPI-AP expression disrupts lipid raft signaling.

阵发性夜间血红蛋白尿（PNH）是一种克隆造血干细胞疾病，是由A引起的 猪基因的体细胞突变。 PIG-A突变的生化后果是全球损失 糖基磷脂酰肌醇锚定蛋白（GPI-AP）。两个GPI-AP（CD55和CD59）很重要 补体调节蛋白的缺失解释了补体介导的血管内溶血 和血栓形成以及PNH患者发生的；但是，PNH细胞实现克隆的机制 统治不完全理解。所有PNH患者均显示出猪A突变； 但是，在健康对照受试者中也可以发现罕见的猪突变。因此，生物学相关性 健康对照中的猪突变仍有待确定。这些研究的总体目的是 研究了GPI锚定缺乏症在PNH，正常和恶性淋巴结症细胞中的相关性。 我们的初步数据表明，造血细胞可以获取PNH表型（无细胞表面 GPI-AP）没有猪-A突变。这些细胞中GPI-AP缺乏的机制是通过 GPI锚定生物合成所需的基因的转录沉默。我们还建立了一个Piganull 在诱导型启动子的控制下，具有PIG-A基因的CD34+细胞系。该细胞系作为 研究PNH克隆优势机制的宝贵模型。我们的初步数据表明PNH 细胞对T细胞介导的凋亡相对抗性，PNH细胞的生长优势是 在免疫攻击的情况下放大。为什么PNH细胞对T细胞介导的杀伤具有抗性，尚不清楚 但是我们的初步数据表明，它可能与膜筏的扰动信号和 产生促凋亡的第二信使神经酰胺的能力降低。具体来说，我们将： 1）研究正常元中GPI-AP缺乏细胞的相关性。 假设1.1：正常淋巴细胞的GPI-AP缺乏可能是由一种新型机制引起的 不涉及PICA突变。 假设1.2：在健康对照中，HSPC的子集为GPI-APIO/neg，但不要携带Pig-a 突变。 2）研究GPI锚缺乏症在细胞对细胞凋亡的抗性中的作用。 假设2.1：GPI-APIO/NEG细胞通过全球对凋亡的抗性具有抵抗力的免疫攻击。 假设2.2：GPI锚表达在正常细胞对凋亡刺激的反应中起作用。 假设2.3：缺乏GPI-AP表达会破坏脂质筏信号传导。