Trim58 and the Ubiquitin Proteasome System in Erythro-megakaryopoiesis

描述（由申请人提供）：我们寻求新的，医学相关的见解红细胞（RBC）形成（红细胞生成）的生物学。在红系前体中，泛素蛋白酶体系统（UPS）识别并消除在进行性成熟过程中变得不必要或潜在有害的内源性蛋白。UPS还作为一种保护机制，以消除在RBC疾病中积累的有毒蛋白质，正如我们和其他人已经证明的那样，地中海贫血是一种由血红蛋白合成不平衡引起的常见贫血。虽然UPS被认为是红细胞生成的关键，但对所涉及的特定分子知之甚少。大规模的全基因组关联研究（GWAS）的人群已经确定了许多预测调节红细胞生成的UPS组件。我们将这些GWAS与全局转录组分析相结合，以鉴定在RBC前体中表达的几种潜在重要的UPS蛋白。我们深入研究的一个有趣的候选者是Trim58，这是一种标记其他蛋白质降解的蛋白质，也被GWAS牵连到调节血小板的形成。我们发现，Trim58缺陷型红细胞前体表现出成熟缺陷，包括排出细胞核的能力受损，这是哺乳动物红细胞生成的关键步骤。初步研究表明，Trim58通过消除动力蛋白促进去核，动力蛋白是一种在几乎所有其他细胞类型中具有多种基本功能的分子马达复合物。我们将对纯化的蛋白质进行生化研究，并对培养的红细胞进行遗传操作，以研究Trim58降解动力蛋白的机制以及这如何促进红细胞前体去核。为了研究Trim58的潜在动力蛋白独立功能，我们将进行蛋白质组学研究以鉴定其额外的降解靶点（目的1）。为了检查Trim58在体内的功能，我们将在小鼠中消融该基因，并确定在基线和暴露于各种生理应激后对RBC和血小板形成的影响（目的2）。最后，我们将使用短发夹RNA来抑制培养的初级红系前体细胞中额外的GWAS鉴定的UPS候选物的表达，并确定这如何影响它们的成熟（目的3）。我们的研究旨在阐明通过调节蛋白质降解促进红细胞生成的新途径。通过药物或遗传操作改变这些途径，应该可以加强正在进行的努力，以在体外产生用于输血治疗的红细胞，并治疗由红细胞生成失调引起的各种血液疾病。更一般地说，我们计划的研究与GWAS协同作用，以更好地了解遗传变异如何影响医学相关的表型。

DESCRIPTION (provided by applicant): We seek new, medically relevant insights into the biology of red blood cell (RBC) formation (erythropoiesis). In erythroid precursors, the ubiquitin proteasome system (UPS) identifies and eliminates endogenous proteins that become unnecessary or potentially deleterious during progressive maturation. The UPS also functions as a protective mechanism to eliminate toxic proteins that accumulate in RBC disorders, as we and others have demonstrated for � thalassemia, a common anemia caused by imbalanced hemoglobin synthesis. While the UPS is believed to be critical for erythropoiesis, very little is known regarding the specific molecules involved. Large-scale genome wide association studies (GWAS) of human populations have identified numerous UPS components predicted to regulate erythropoiesis. We combined these GWAS with global transcriptome analyses to identify several potentially important UPS proteins expressed in RBC precursors. One interesting candidate that we have studied in depth is Trim58, a protein that marks other proteins for degradation and has also been implicated by GWAS to regulate the formation of platelets. We showed that Trim58 deficient RBC precursors exhibit faulty maturation, including impaired ability to expel the nucleus, a key step in mammalian erythropoiesis. Preliminary studies indicate that Trim58 facilitates enucleation by eliminating dynein, a molecular motor complex with multiple essential functions in virtually all other cell types. We will perform biochemical studies of purifed proteins and genetic manipulations of cultured RBCs to examine the mechanisms by which Trim58 degrades dynein and how this facilitates RBC precursor enucleation. To investigate potential dynein independent functions of Trim58, we will perform proteomic studies to identify its additional degradation targets (Aim 1). To examine Trim58 functions in vivo, we will ablate the gene in mice and determine the consequences on RBC and platelet formation at baseline and after exposure to various physiological stresses (Aim 2). Finally, we will use short hairpin RNAs to suppress the expression of additional GWAS-identified UPS candidates in cultured primary erythroid precursors and determine how this affects their maturation (Aim 3). Our studies aim to elucidate new pathways that promote erythropoiesis through regulated protein degradation. By altering these pathways through drugs or genetic manipulation, it should be possible to enhance ongoing efforts to generate RBCs in vitro for transfusion therapies and to treat various blood diseases caused by dysregulated erythropoiesis. More generally, our planned investigations synergize with GWAS to better understand how genetic variation influences medically relevant phenotypes.