EPO regulated erythropoiesis

 描述(由申请人提供)：对造血生长因子生物效应和作用机制的研究继续为(Dys)调节的血细胞形成提供重要的见解。促红细胞生成素受体(EPOR)系统是一个信息丰富且具有临床意义的范例。最近应用当代方法的研究表明，该领域对EPO/EPOR/JAK2信号转导及其对红系祖细胞(EPC)形成的调控的理解存在重大差距。为了说明这一点，PI最近发现了一条新的EPOR/JAK2/STAT5途径，在该途径中，EPO诱导的Spi2Aserpin细胞保护EPC免受从ROS受损的溶酶体中提取的执行者组织蛋白的伤害[JEM 210：225-32]。T.Ganz博士的实验室已经将EPOR/JAK2/STAT5诱导的“红铁酮”肿瘤坏死因子细胞因子鉴定为海普西丁抑制因子[NAT Genet]。46：678-84]。通过对R01更新的主要支持研究，我们应用了基于翻译后修饰(PTM)的LC-MS/MS蛋白质组学来发现耐人寻味的EPO/EPOR/JAK2作用的新介质。这些因素包括50个以前与EPO依赖的红细胞生成无关的因子，这些因子存在于不同功能类别的分子适配器、红系细胞骨架蛋白、激酶和磷酸酶以及细胞周期和生存因子中。SA#1将扩展我们对人红系前体细胞的PTM指导的蛋白质组学研究，包括在Py、T*PP和泛素基序修改的广泛靶点，以及对S/T激酶、生存/凋亡因子和细胞周期靶点的更多选择信号节点的分析。将为新的(和已知的)EPO目标和传感器挖掘数百个专门激活的PTM事件的网络(与合作的专业生物信息学家)。SA#2重点定义了作为EPOR/JAK2复合体上游效应因子的三个相互关联的新EPO靶标的功能作用和作用机制。其中两个，C1ORF186/“RHex”和C1ORF150，是一种新的分子接头，在HepC中已进化为EPO信号转导分子(但在小鼠、大鼠和低等脊椎动物中缺失)。第三，PTPN18是一种蛋白酪氨酸磷酸酶，我们证明它可以增加JAK2的活性，降低EPOR的周转，限制Py-RHex的形成。方法将包括GOF，shRNA LOF，以及在UT7epo细胞和原代hEPC中进行突变救援研究。然后，SA#3专注于EPO作用的一种新的下游介体--硫氧还蛋白相互作用蛋白(TXNIP)。EPO调节T末端PT/PS位点的TXNIP，并增强其表达。TXNIP基因敲除抑制了EPC的生长，并显著加速了初级红系前体细胞向KIT-低，GPA-高血红素化红细胞的发育。从机理上讲，TXNIP如何作为一种新的EPO试剂将通过分析EPC的生长、存活、ROS、miRNA种群和代谢特性来确定。总体而言，研究将揭示EPO依赖的人类红细胞生成的重要新介质。某些药物可能可以用来减少EPO的剂量，并限制EPO的主要不良副作用。其他新的EPO靶点可能在功能上与MPN和/或EPO恶化癌症预后的可能性有关。

 DESCRIPTION (provided by applicant): Investigations of hematopoietic growth factor bio-effects and action mechanisms continue to provide important insight into (dys) regulated blood cell formation. The EPO receptor (EPOR) system is an informative and clinically significant paradigm. Recent studies applying contemporary approaches indicate major gaps in the field's understanding of EPO/EPOR/JAK2 signal transducers and their regulation of erythroid progenitor cell (EPC) formation. To illustrate, the PI has recently identified a novel EPOR/JAK2/STAT5 pathway in which an EPO-induced Spi2A serpin cytoprotects EPCs against executioner cathepsins as leached from ROS-compromised lysosomes [JEM 210:225-32]. And Dr. T. Ganz's laboratory has characterized an EPOR/JAK2/STAT5-induced "Erythroferrone" TNF cytokine as a hepcidin suppressor [Nat Genet. 46:678-84]. Via major supporting studies for this R01 renewal, we've applied post-translational modification (PTM) based LC-MS/MS proteomics to discover intriguing new mediators of EPO/EPOR/JAK2 action. These include 50+ factors not previously linked to EPO-dependent erythropoiesis within diverse functional categories of molecular adaptors, erythroid cytoskeletal proteins, kinases & phosphatases, and cell cycle & survival factors. SA#1 will extend our PTM-directed proteomic investigations in human erythroid precursor cells to include broad-based targets as modified at pY, T*PP and ubiquitin motifs, together with analyses of more select signaling nodes for S/T kinases, survival/apoptosis factors and cell cycle targets. Networks for hundreds of specifically activated PTM events for novel (and known) EPO targets and transducers will be mined (with collaborating expert bioinformaticists). SA#2 focuses on defining the functional roles and action mechanisms of three interrelated new EPO targets as upstream effectors of EPOR/JAK2 complexes. Two, C1ORF186/"RHEX" and C1ORF150, are novel molecular adaptors that have evolved as EPO signal transducers in hEPC's (but are absent among mice, rats, lower vertebrates). Third, PTPN18 is a protein tyrosine phosphatase which we demonstrate to increase JAK2 activation, decrease EPOR turnover and limit pY-RHEX formation. Approaches will include GOF, shRNA LOF, and mutant rescue studies in UT7epo cells and primary hEPCs. SA#3 then focuses on a new downstream mediator of EPO action, Thioredoxin-Interacting Protein (TXNIP). EPO modulates TXNIP at C-terminal pT/pS sites, and heightens its expression. TXNIP knockdown attenuates EPC growth, and notably accelerates primary erythroid precursor development to KIT-low, GPA-high hemoglobinizing erythroblasts. Mechanistically how TXNIP acts as a novel EPO agent will be determined by analyzing EPC growth, survival, ROS, miRNA populations and metabolic properties. Overall, studies will reveal important new mediators of EPO-dependent human erythropoiesis. Certain may be druggable with potentials to lessen EPO dosing, and limit EPO's major adverse side effects. Other new EPO targets may functionally relate to MPNs and/or to EPO's potential to worsen cancer outcomes.