Protein Homeostasis in Hematopoietic Stem Cells

摘要 造血干细胞(HSCs)在一生中再生血细胞。HSC维护中的缺陷可能会导致 导致贫血、免疫力受损、骨髓衰竭和癌症。对HSCs的机械性理解是 对于揭示导致其功能障碍的因素和利用其再生潜力至关重要。我们 最近发现，与其他造血细胞相比，造血干细胞的蛋白质合成率较低，而且 这对于HSC的维持是必要的，因为增加蛋白质合成的基因变化会损害HSC 功能。这提出了一个根本性的问题，即蛋白质合成中细胞类型的特定差异如何促进 HSC功能。在初步研究中，我们确定与其他细胞相比，肝干细胞具有更高的蛋白质质量。 限制祖细胞，增加蛋白质合成会降低HSCs内的蛋白质质量。这引发了 低蛋白质合成通过增强蛋白质稳态(蛋白质稳态)来促进HSC功能的可能性。 在目标1中，我们将测试蛋白质质量的下降是否会损害HSC的功能。我们将检查造血，HSC 具有tRNA编辑缺陷的Aarssti/sti小鼠的功能和衰老，该缺陷导致翻译保真度降低 翻译错误的蛋白质堆积。第二个问题是蛋白质合成增加是如何被 以及它是如何损害它们的功能的。蛋白质合成的高速率会增加翻译错误， 导致蛋白质错误折叠。错误折叠的蛋白质的积累会压倒泛素蛋白酶体。 系统(UPS)。这增加了HSCs通过影响UPS来感知蛋白质合成变化的可能性。 UPS通过调节包括c-Myc在内的几种蛋白质的周转来调节HSC的命运。我们决定 蛋白质合成的增加促进了c-Myc在HSCs中的积累。在目标2中，我们将使用转基因UPS 报告小鼠测试体内蛋白质合成增加是否会扰乱HSCs中的UPS活性。我们将繁衍后代 Mx1-Cre+；Ptenfl/fl小鼠与Mycfl/+小鼠，并测试降低c-Myc表达是否拯救Pten缺陷的HSCs， 它们通常会因蛋白质合成增加而耗尽。最后，在目标3中，我们将研究潜在的 抑制肝星状细胞蛋白质合成的分子机制。在初步研究中，我们发现HSCs 与祖细胞相比，表达低水平的Eif5蛋白。Eif5通过刺激80s促进蛋白质合成 核糖体组装。有趣的是，HSC优先表达Eif5的一个长亚型，它包含一个长的 5‘非编码区预计会形成复杂的二级结构，可能会阻碍其翻译。我们将测试是否 Eif5的差异剪接限制了它的翻译，并限制了HSCs的蛋白质合成。我们将超额表达Eif5 并评估蛋白质合成和HSC功能。我们将测试Eif5 5‘UTRs的翻译效率 在荧光素酶报告分析中。使用一只只表达Eif5短亚型的鼠标，我们将测试 差异剪接在体内影响蛋白质合成、蛋白平衡和HSC功能。这些研究可能 揭示了一种新的机制，即低蛋白质合成增强蛋白平衡以促进HSC功能。

ABSTRACT Hematopoietic stem cells (HSCs) regenerate blood cells throughout life. Defects in HSC maintenance can lead to anemia, impaired immunity, bone marrow failure and cancer. A mechanistic understanding of HSCs is crucial for uncovering the factors that result in their dysfunction and harnessing their regenerative potential. We recently discovered that HSCs have lower rates of protein synthesis than other hematopoietic cells and that this is necessary for HSC maintenance, as genetic changes that increase protein synthesis impair HSC function. This raises a fundamental question of how cell-type specific differences in protein synthesis promote HSC function. In preliminary studies, we determined that HSCs exhibit superior protein quality as compared to restricted progenitors, and increasing protein synthesis reduces protein quality within HSCs. This raises the possibility that low protein synthesis promotes HSC function by enhancing protein homeostasis (proteostasis). In Aim 1, we will test if declines in protein quality impair HSC function. We will examine hematopoiesis, HSC function and aging in Aarssti/sti mice that have a tRNA editing defect that reduces translational fidelity leading to an accumulation of mistranslated proteins. A second question is how increased protein synthesis is sensed by HSCs and how it impairs their function. High rates of protein synthesis can increase translational errors that lead to protein misfolding. An accumulation of misfolded proteins can overwhelm the ubiquitin proteasome system (UPS). This raises the possibility that HSCs sense changes in protein synthesis via effects on the UPS. The UPS regulates HSC fate by modulating the turnover of several proteins, including c-Myc. We determined that increased protein synthesis promotes c-Myc accumulation in HSCs. In Aim 2 we will use transgenic UPS reporter mice to test if increased protein synthesis disrupts UPS activity within HSCs in vivo. We will breed Mx1-Cre+;Ptenfl/fl mice with Mycfl/+ mice and test if reducing c-Myc expression rescues Pten-deficient HSCs, which are normally depleted by increased protein synthesis. Finally, in Aim 3, we will examine the underlying molecular mechanisms that attenuate protein synthesis in HSCs. In preliminary studies we found that HSCs express low levels of Eif5 protein compared to progenitors. Eif5 promotes protein synthesis by stimulating 80S ribosome assembly. Interestingly, HSCs preferentially express a long isoform of Eif5 that contains a long 5’UTR predicted to form complex secondary structures that could impede its translation. We will test if differential splicing of Eif5 limits its translation and restricts protein synthesis in HSCs. We will overexpress Eif5 in HSCs and assess protein synthesis and HSC function. We will test the translational efficiency of Eif5 5’UTRs in luciferase reporter assays. Using a mouse that only expresses the short isoform of Eif5, we will test if differential splicing affects protein synthesis, proteostasis and HSC function in vivo. These studies could unravel a new mechanism whereby low protein synthesis enhances proteostasis to promote HSC function.