Mechanism and Function of Endoplasmic Reticulum Protein Quality Control Machinery In The Maintenance of Hematopoietic Stem Cells

摘要 造血干细胞(HSCs)在应激状态下维持组织内环境平衡和补充血液系统。 造血干细胞已经进化出独特的机制来维持整个生命中基因组和蛋白质组的完整性。而当 基因组完整性保护机制已被广泛研究，但对蛋白质组如何 在HSCs中保持完整性，以及休眠HSCs如何在 发育和生理压力。这项提议将解决这一知识差距。我们最近发现 Sel1L内质网相关降解(ERAD)通路在维持 造血干细胞。Erad是主要的蛋白质质量控制机制，负责靶向错误折叠的蛋白质 胞浆蛋白酶体降解的内质网。Sel1L-Hrd1复合体是Hrd1的最保守的分支 埃拉德。使用条件性基因敲除小鼠模型，我们发现造血细胞中的Sel1L缺失 显著降低稳态HSC频率，并导致完全丧失HSC重建能力 应激条件包括骨髓移植和5-氟尿嘧啶(5-flUorouracil，5-FU)介导的骨髓清髓。 有趣的是，Sel1L缺失不会诱导细胞凋亡或损害HSC的植入。相反，我们观察到 在Sel1L基因敲除小鼠中，增加了HSC的循环，减少了静止的HSC数量。这些数据 证明Sel1L ERAD在HSC维持中的关键作用，并提出ER蛋白的新作用 调节干细胞静止和自我更新的质量控制机制。Erad监控和调节 跨膜蛋白的成熟。我们发现CXCR4和CXCR4的表面表达明显减少 在Sel1L基因敲除的HSC中，MPL是HSC静止和生态位相互作用的两个主要调节因子。跟踪HSC 体内单细胞水平的NICs显示Sel1L缺陷的HSCs在骨中的异常定位 骨髓龛。我们假设Sel1L ERAD通过调节HSC来调控HSC的静止和自我更新 跨膜受体成熟和HSC-生态位相互作用。我们将确立生理意义 Sel1L ERAD在维持造血干细胞中的作用(目标1)，确定ERAD的意义和机制- 未折叠蛋白反应(UPR)在造血干细胞中的串扰(AIM 2)，并阐明其机制和意义 在HSC-生态位相互作用中的Sel1L ERAD(目标3)。这项研究将提供重要的洞察后- ER蛋白质量控制对HSC静止、自我更新和生态位相互作用的翻译调控 机制，并进一步确定影响HSC命运的新决定因素。

ABSTRACT Hematopoietic stem cells (HSCs) maintain tissue homeostasis and replenish blood system upon stresses. HSCs have evolved unique mechanisms to maintain genome and proteome integrity throughout life. While the genome integrity safeguard mechanisms have been extensively studied, little is known about how proteome integrity is maintained in HSCs and how dormant HSCs are protected from protein damage during development and physiological stresses. This proposal will address this knowledge gap. We recently found that the Sel1L ER-associated degradation (ERAD) pathway plays an essential role in the maintenance of HSCs. ERAD is the principal protein quality control mechanism responsible for targeting misfolded proteins in the ER for cytosolic proteasomal degradation. The Sel1L-Hrd1 complex is the most conserved branch of ERAD. Using a conditional knockout mouse model, we found that Sel1L deletion in hematopoietic cells significantly reduced steady-state HSC frequency and led to complete loss of HSC reconstitution capacity in stress conditions including bone marrow transplantation and 5-fluorouracil (5-FU) mediated myeloablation. Interestingly, Sel1L deletion did not induce apoptosis or impair HSC engraftment. In contrast, we observed increased HSC cycling and reduced numbers of quiescent HSCs in Sel1L knockout mice. These data demonstrate the critical function of Sel1L ERAD in HSC maintenance and suggest a novel role for ER protein quality control machinery in regulating stem cell quiescence and self-renewal. ERAD monitors and regulates the maturation of transmembrane proteins. We found markedly decreased surface expression of CXCR4 and MPL, two master regulators of HSC quiescence and niche interaction, in Sel1L-knockout HSCs. Tracking HSC and niche cells at the single cell level in vivo showed aberrant localization of Sel1L-deficient HSCs in the bone marrow niche. We hypothesize that Sel1L ERAD governs HSC quiescence and self-renewal by regulating HSC transmembrane receptor maturation and HSC-niche interaction. We will establish the physiological significance of Sel1L ERAD in the maintenance of HSCs (Aim 1), determine the significance and mechanism of the ERAD- Unfolded Protein Response (UPR) crosstalk in HSCs (Aim 2), and elucidate the mechanism and significance of Sel1L ERAD in HSC-niche interactions (Aim 3). This study will provide significant insight into the post- translational regulation of HSC quiescence, self-renewal, and niche interaction by ER protein quality control mechanisms, and further identify novel determinants of HSC fates.