Trim58 and the Ubiquitin Proteasome System in Erythro-megakaryopoiesis

Trim58 和红细胞巨核细胞生成中的泛素蛋白酶体系统

基本信息

批准号：
8843634
负责人：
Mitchell J Weiss
金额：
$ 38.06万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8843634
关键词：
Ablation Affect Alleles Amino Acids Anemia Animals Autophagocytosis Binding Biochemical Biochemical Pathway Biological Assay Biology Blood Cells Blood Platelets Breeding Cell Nucleus Cells Complex Data Defect Degradation Pathway Development Disease Dynein ATPase Erythro Erythroblasts Erythrocyte Indices Erythrocytes Erythroid Erythroid Cells Erythropoiesis Exhibits Exposure to Family Fetal Liver Gene Expression Profile Genes Genetic Variation Goals Grant Guanine Nucleotide Exchange Factors Guanosine Triphosphate Phosphohydrolases Health Hematological Disease Hematopoiesis Hemoglobin Homeostasis Human Human Genome In Vitro Investigation Knockout Mice Maps Mediating Medicine Megakaryocytes Megakaryocytopoieses Microtubules Molecular Molecular Chaperones Molecular Motors Mus Mutation Pathway interactions Pharmaceutical Preparations Phenotype Physiological Platelet Count measurement Population Production Proteins Proteolysis Proteomics Quality Control Red Blood Cell Count Role Stress System TRIM Motif Testing Thalassemia Thrombopoiesis Transfusion Ubiquitin Variant Work cell type embryonic stem cell genetic manipulation genome wide association study in vitro activity in vivo insight interest juvenile animal loss of function member multicatalytic endopeptidase complex organelle movement postnatal protein complex protein degradation protein function public health relevance scaffold screening small hairpin RNA trait transcription factor ubiquitin ligase ubiquitin-protein ligase

项目摘要

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.

描述（由申请人提供）：我们寻求对红细胞（RBC）形成（红细胞生成）生物学的新的、医学相关的见解。在红细胞前体中，泛素蛋白酶体系统（UPS）识别并消除不必要或潜在的内源性蛋白质。正如我们和其他人已经证明的那样，UPS 还可以作为一种保护机制来消除红细胞疾病中积累的有毒蛋白质。地中海贫血，一种由血红蛋白合成失衡引起的常见贫血症，虽然 UPS 被认为对红细胞生成至关重要，但对人类群体的大规模全基因组关联研究 (GWAS) 却知之甚少。我们将这些 GWAS 与全局转录组分析相结合，以确定红细胞前体中表达的几种潜在重要的 UPS 蛋白，我们深入研究了一个有趣的候选蛋白。一种标记其他蛋白质降解的蛋白质，并且还被 GWAS 认为可以调节血小板的形成。我们发现，Trim58 缺陷的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。表明 Trim58 通过消除动力蛋白来促进去核，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运动复合物。我们将对纯化的蛋白质进行生化研究。对培养的红细胞进行基因操作，以检查 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 潜在的动力蛋白独立功能，我们将进行蛋白质组学研究，以确定其额外的降解目标（目标 1）。 Trim58 在体内发挥作用，我们将在小鼠体内消除该基因，并确定基线时和暴露于各种生理应激后对红细胞和血小板形成的影响（目标2).最后，我们将使用短发夹 RNA 抑制培养的原代红细胞前体细胞中其他 GWAS 鉴定的 UPS 候选物的表达，并确定这如何影响其成熟（目标 3）。通过药物或基因操作改变这些途径，应该有可能加强在体外生成红细胞用于输血治疗和治疗由失调引起的各种血液疾病的持续努力。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表型。