Novel Erythroid Cell Membrane Protein

说明(申请人提供)：虽然促红细胞生成素(EPO)通常用于治疗癌症和肾脏疾病患者的贫血，但最近的研究提出了主要的安全性问题。人们对开发新的策略来调节红细胞生成非常感兴趣，在这方面，我们一直在分析GATA转录因子的功能。GATA-2调节造血干细胞的增殖/存活，而GATA-1促进红系、巨核细胞、嗜酸性粒细胞和肥大细胞的分化。对GATA-1缺失的红系前体细胞中GATA-1功能的分析揭示了一个新的靶基因mFAM55B。GATA-1直接激活mFAM55B转录，mFAM55B在小鼠胎肝和成人骨髓中的表达高度受限于特定的红细胞。虽然mFam55B与细胞间黏附分子-4(ICAM4)有很小的序列相似性，但mFam55B的大部分序列与ICAM4或其他已知蛋白质不同，ICAM4参与了重要红细胞岛的形成。MFAM55B可能是II型膜蛋白，而ICAM是I型膜蛋白。我们发现了四个哺乳动物mFAM55B相关基因，这些基因尚未被描述，但只有mFAM55B受GATA-1调控。MFAM55B被预测有一个跨膜螺旋和两个细胞外免疫球蛋白(Ig)样重复序列。MFAM55B及其类似蛋白定义了一个新的蛋白质家族，我们推测mFAM55B是一种在红系细胞生物学中具有重要功能的红系前体膜蛋白。以下目的描述了开发试剂/模型以确定mFAM55B的功能并研究mFAM55B在红系细胞岛形成中的作用的先导性/探索性研究。目的1-确定mFAM55B是否是GATA-1调节的红系前体细胞跨膜蛋白。我们假设mFAM55B是红系前体细胞中的一种跨膜蛋白，通过细胞间信号发挥作用。我们将测试GATA-1是否调节内源性mFAM55B水平，mFAM55B是否表现出明显的质膜定位，以及它是否位于红细胞岛。目的2-通过斑马鱼和小鼠的功能丧失研究mFam55B的生物学功能。我们发现了5个与mFAM55B有显著序列相似性的斑马鱼新基因。敲除这些基因中的一个会导致严重的贫血。我们将敲除mFam55B相关基因，以确定它们是否调节红细胞生成，定义它们的表达模式，并测试它们的表达是否依赖于GATA-1。我们还将建立一只mFAM55B缺失的小鼠，以开始剖析mFAM55B在红细胞功能和/或红细胞生成中的作用。公共卫生相关性：本项目重点分析了我们发现的一种新的GATA-1调节蛋白mFAM55B，它被预测为红系前体细胞上的一种单程跨膜蛋白，并代表了一个新的蛋白家族的创始成员。由于GATA-1是红细胞生成的基本调节因子，我们推测mFAM55B在调节红细胞发育和/或红系前体细胞功能方面具有重要作用。因此，拟议的先导性/探索性研究有可能产生与刺激红细胞发育的新策略相关的高影响力的发现，阐明红系前体细胞在病理生理状态下的功能是如何改变的，以及解开蛋白质结构/功能的基本方面。

DESCRIPTION (provided by applicant): While erythropoietin (Epo) is commonly used to treat anemia in patients with cancer and kidney disease, recent studies have raised major safety concerns. There is great interest in developing new strategies to modulate erythropoiesis, and in this regard, we have been analyzing GATA transcription factor function. GATA-2 regulates the proliferation/survival of hematopoietic stem cells (HSCs), whereas GATA-1 promotes the differentiation of erythroid, megakaryocyte, eosinophil, and mast cells. Analysis of GATA-1 function in GATA-1-null erythroid precursor cells revealed a novel target gene, mFam55B. GATA-1 directly activates mFam55B transcription, and mFam55B expression is highly restricted to definitive erythroblasts in murine fetal liver and adult bone marrow. Although mFam55B has minor sequence similarity to Intercellular Cell Adhesion Molecule-4 (ICAM4), which is implicated in formation of functionally important erythroblastic islands, the bulk of mFam55B sequence does not resemble ICAM4 or other known proteins. mFam55B appears to be a Type II membrane protein, whereas ICAMs are Type I membrane proteins. We discovered four mammalian mFam55B-related genes, which have not been described, but only mFam55B is GATA-1-regulated. mFam55B is predicted to have a transmembrane helix and two extracellular immunoglobulin (Ig)-like repeats. mFam55B and its paralogs define a new protein family, and we hypothesize that mFam55B is an erythroid precursor membrane protein with important functions in erythroid cell biology. The following aims describe pilot/exploratory studies to develop reagents/models to define mFam55B function and to investigate the role of mFam55B in the formation of erythroblastic islands. Aim 1 - To determine whether mFam55B is a GATA-1-regulated transmembrane protein in erythroid precursor cells. We hypothesize that mFam55B is a transmembrane protein in erythroid precursor cells that functions via intercellular signaling. We will test whether GATA-1 regulates endogenous mFam55B levels, whether mFam55B exhibits predominant plasma membrane localization, and whether it resides in erythroblastic islands. Aim 2 - To investigate biological functions of mFam55B via loss-of-function studies in zebrafish and mice. We identified five novel zebrafish genes with significant sequence similarity to mFam55B. Knocking down one of these genes yielded severe anemia. We will knockdown the mFam55B-related genes to determine if they regulate erythropoiesis, will define their expression patterns, and will test whether their expression is GATA-1-dependent. We will also generate a mFam55B-null mouse to begin to dissect the role of mFam55B in erythroid cell function and/or erythropoiesis. PUBLIC HEALTH RELEVANCE: This project focuses on analyzing a novel GATA-1 regulated protein, mFam55B, that we discovered, which is predicted to be a single-pass transmembrane protein on erythroid precursor cells and to represent the founding member of a new protein family. As GATA-1 is a fundamental regulator of erythropoiesis, we hypothesize that mFam55B has important functions to regulate red blood cell development and/or erythroid precursor cell function. The proposed pilot/exploratory studies therefore have potential to yield high impact findings of relevance to novel strategies to stimulate red blood cell development, to elucidating how erythroid precursor cell function is altered in pathophysiological states, and to unraveling fundamental aspects of protein structure/function.