Modulation of protein methylation to improve hematopoietic stem cell engraftment

项目总结： 扩大我们对造血的理解，包括识别关键的生化调节因子，是 对开发新的干预措施和改进的潜在治疗程序(如骨)至关重要 骨髓移植。虽然对激酶的作用已经有了很多研究，但它在造血方面的作用 丝氨酸/苏氨酸，多亚单位PP2A家族磷酸酶，PP2A和PP4，才刚刚开始检测。 PP2Ac在其羧基末端亮氨酸(L309)上被甲基转移酶LCMT-1可逆甲基化，并 甲基酯酶PME-1和这种甲基化调节某些PP2A复合体的形成，因此 亚细胞靶向性和特异性。PP4与PP2A有60%的同源性，它的羧基上也有甲基化- 末端亮氨酸。我们最近发现LCMT-1也是PP4甲基转移酶，并选择性地调节 除精选的PP2A络合物外，还包括某些PP4络合物。通过创造和初步研究一种基因- 诱捕LCMT-1基因敲除(KO)小鼠，随后进行泛造血条件基因敲除(CKO) LCMT-1等位基因，我们显示多系HSC竞争性再种群活动同样显著减少 尽管相对正常的成人HSC动态平衡。因此，LCMT-1可能具有细胞内固有的促进作用 HSC在移植后早期的应激状态下具有独特的适应性。这项提议是一个多人调查的方案 利用两种PI的生物化学和实验血液学专业知识的应用。我们在一起 已发现LCMT-1在造血过程中发挥着关键的内在作用，这种作用是HSCs所特有的。 移植扩张、存活、自我更新阶段。相比之下，LCMT1在 HSC在稳态条件下的维护和循环。因为LCMT-1底物仅限于PP2A 由于LCMT-1必须与特定活性部位和C末端结合的独特方式所致的家族磷酸酶 残基，我们假设LCMT-1促进高水平的“甲基化依赖”PP2A和PP4 异源三聚体，进而调节关键蛋白上的关键调节磷酸化位点，如Raf到 促进早期增殖驱动所需的丝裂原相关蛋白激酶(MAPK)信号通路 在造血再生过程中。在这个提案中，我们将检验这一假设，并开始剖析 利用LCMT-1 CKO小鼠确定成人HSC多系再分化特异性缺陷的机制 以流式细胞术为基础的活性检测。测试将比较稳态和复制压力反应。 LCMT-1催化活性的重要性将被确定，参与的甲基化依赖 将评估PP2A复合体的再生功能，并将制定治疗返老还童的策略 探索过了。实现这些目标将通过提高对信令的理解来推动该领域向前发展 通过为LCMT-1和PP2A家族的调控提供造血调控途径 磷酸酶可加快造血再生的速度。

PROJECT SUMMARY: Expansion of our understanding of hematopoiesis, including identification of key biochemical modulators, is critical to the development of novel interventions and improved potentially curative procedures such as bone marrow transplantation. While much study has occurred on the roles of kinases, the hematopoietic roles of the serine/threonine, multi-subunit PP2A family phosphatases, PP2A and PP4, have only begun to be examined. PP2Ac is reversibly methylated on its carboxy-terminal leucine (L309) by the methyltransferase, LCMT-1, and the methylesterase, PME-1, and this methylation regulates formation of certain PP2A complexes, and thus subcellular targeting and specificity. PP4, which is ~60% identical to PP2A, is also methylated on its carboxyl- terminal leucine. We recently found LCMT-1 is also the PP4 methyltransferase and selectively regulates certain PP4 complexes in addition to select PP2A complexes. Through the creation and initial study of a gene- trap LCMT-1 knockout (KO) mouse and subsequently a pan-hematopoietic conditional knockout (cKO) of the LCMT-1 allele, we show equally striking reductions in multilineage HSC competitive repopulation activity despite relatively normal adult HSC homeostasis. Therefore, LCMT-1 may have cell intrinsic roles in promoting HSC fitness uniquely during the stress at early times post-transplantation. This proposal is a multi-investigator application that leverages the biochemistry and experimental hematology expertise of both PIs. Together we have discovered that LCMT-1 plays a critical intrinsic role in hematopoiesis that is specific to HSCs in the post- transplantation expansion, survival, and self-renewal phase. In contrast, LCMT1 doesn't play a role during HSC maintenance and cycling during steady-state conditions. Because LCMT-1 substrates are limited to PP2A family phosphatases due to the unique way LCMT-1 must interact with both specific active site and C-terminal residues, we hypothesize that LCMT-1 promotes high levels of “methylation-dependent” PP2A and PP4 heterotrimers that in turn modulate key regulatory phosphorylation sites on critical proteins such as Raf to promote mitogen-associated protein kinase (MAPK) pathway signaling required for the early proliferative drive during hematopoietic repopulation. In this proposal, we will test this hypothesis and begin dissecting mechanism by utilizing LCMT-1 cKO mice to define specific defects in adult HSC multilineage repopulating activity using flow cytometry-based assays. Assays will compare steady-state and replicative stress responses. The importance of LCMT-1 catalytic activity will be determined, the involvement of methylation-dependent PP2A complexes in repopulating function will be evaluated, and therapeutic rejuvenation strategies will be explored. Accomplishing these objectives will move the field forward by improving understanding of signaling regulation of hematopoiesis and by providing avenues for manipulation of LCMT-1 and PP2A family phosphatases to accelerate the rate of hematopoietic regeneration.