Protein Homeostasis in Hematopoietic Stem Cells

摘要 造血干细胞（HSC）在整个生命过程中再生血细胞。HSC维护中的缺陷可能导致 贫血免疫力受损骨髓衰竭和癌症对HSC的机械理解是 这对于揭示导致他们功能障碍的因素和利用他们的再生潜力至关重要。我们 最近发现，HSC的蛋白质合成速率低于其他造血细胞， 这是维持HSC所必需的，因为增加蛋白质合成的遗传变化损害了HSC 功能这就提出了一个基本问题，即蛋白质合成中细胞类型特异性差异如何促进 HSC功能。在初步的研究中，我们确定了HSC表现出上级蛋白质质量， 限制的祖细胞，并且增加的蛋白质合成降低了HSC内的蛋白质质量。这就提出了一个 低蛋白质合成通过增强蛋白质稳态（proteostasis）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功能。我们会检查造血干细胞 Aarssti/sti小鼠的功能和衰老，这些小鼠具有降低翻译保真度的tRNA编辑缺陷， 大量误译的蛋白质第二个问题是， HSC及其如何损害其功能。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可以压倒泛素蛋白酶体 系统（UPS）。这增加了HSC通过对UPS的影响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。我们确定 增加的蛋白质合成促进c-Myc在HSC中的积累。在目标2中，我们将使用转基因UPS 报告小鼠来测试增加的蛋白质合成是否破坏体内HSC内的UPS活性。我们会繁殖 Mx 1-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HSC， 其通常因蛋白质合成增加而耗尽。最后，在目标3中，我们将研究 削弱HSC中蛋白质合成的分子机制。在初步研究中，我们发现HSC 与祖细胞相比，表达低水平的Eif 5蛋白。Eif 5通过刺激80 S促进蛋白质合成 核糖体组装有趣的是，HSC优先表达Eif 5的长同种型，其含有长的 预测5 'UTR形成可能阻碍其翻译的复杂二级结构。我们将测试如果 Eif 5的差异剪接限制了其翻译并限制了HSC中的蛋白质合成。我们将过度表达Eif 5 并评估蛋白质合成和HSC功能。我们将测试Eif 5 5 'UTR的翻译效率 在荧光素酶报告基因测定中。使用仅表达Eif 5的短同种型的小鼠，我们将测试 差异剪接影响体内蛋白质合成、蛋白质稳态和HSC功能。这些研究可以 揭示了一种新的机制，即低蛋白质合成增强蛋白质稳态以促进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.