GPI ANCHOR DEFICIENCY IN HEMATOPOIESIS

造血作用中的 GPI 锚定缺陷

基本信息

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
项目类别：
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

项目摘要

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.

阵发性睡眠性血红蛋白尿症 (PNH) 是一种克隆性造血干细胞疾病，由 PIG-A基因的体细胞突变。 PIG-A突变的生化后果是整体丧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，它们的缺失解释了补体介导的血管内溶血以及 PNH 患者中发生的血栓形成；然而，PNH 细胞实现克隆的机制统治地位尚未完全被理解。所有 PNH 患者均已被证明携带 PIG-A 突变；然而，在健康对照受试者中也可以发现罕见的 PIG-A 突变；因此，生物学相关性健康对照中的 PIG-A 突变仍有待确定。这些研究的总体目标是研究 PNH、正常和恶性淋巴造血细胞中 GPI 锚定缺陷的相关性。我们的初步数据表明，造血细胞可以获得 PNH 表型（无细胞表面 GPI-AP)，无 PIG-A 突变。这些细胞中 GPI-AP 缺陷的机制是通过 GPI 锚生物合成所需基因的转录沉默。我们还建立了一个PIGAnull CD34+ 细胞系，其 PIG-A 基因受诱导型启动子控制。该细胞系用作是研究 PNH 克隆优势机制的一个有价值的模型。我们的初步数据显示，PNH 细胞对T细胞介导的细胞凋亡具有相对抵抗力，PNH细胞的生长优势是在免疫攻击的情况下被放大。为什么 PNH 细胞能够抵抗 T 细胞介导的杀伤尚不清楚，但我们的初步数据表明，这可能与通过膜筏的扰动信号传导有关产生促凋亡第二信使神经酰胺的能力降低。具体来说，我们将： 1) 研究 GPI-AP 缺陷细胞与正常细胞的相关性。假设 1.1：正常淋巴细胞中 GPI-AP 缺乏可能是由一种新机制引起的，该机制不涉及PICA突变。假设 1.2：在健康对照中，HSPC 的一个子集是 GPI-APIo/neg，但不包含 PIG-A 突变。 2）研究GPI-anchor缺陷在细胞抗凋亡中的作用。假设2.1：GPI-APIo/neg 细胞通过整体抵抗细胞凋亡来抵抗免疫攻击。假设2.2：GPI-anchor表达在正常细胞对凋亡刺激的反应中发挥作用。假设 2.3：GPI-AP 表达的缺乏会破坏脂筏信号传导。