Modulation of protein methylation to improve hematopoietic stem cell engraftment

项目概要： 扩大我们对造血的理解，包括鉴定关键的生化调节剂， 这对于开发新的干预措施和改进潜在的治疗程序至关重要， 骨髓移植虽然已经对激酶的作用进行了大量研究，但激酶的造血作用仍然是一个未知的问题。 丝氨酸/苏氨酸、多亚基PP 2A家族磷酸酶PP 2A和PP 4才刚刚开始研究。 PP 2Ac在其羧基末端亮氨酸（L309）上被甲基转移酶LCMT-1可逆甲基化， 甲基酯酶PME-1和这种甲基化调节某些PP 2A复合物的形成，因此 亚细胞靶向和特异性。PP 4与PP 2A约60%相同，其羧基也被甲基化。 末端亮氨酸我们最近发现LCMT-1也是PP 4甲基转移酶，并选择性地调节 除了选择PP 2A复合物之外，还可以使用某些PP 4复合物。通过对基因的创造和初步研究- 诱捕LCMT-1敲除（KO）小鼠，随后是LCMT-1的泛造血条件性敲除（cKO）。 LCMT-1等位基因，我们显示多系HSC竞争性再增殖活性同样显著降低 尽管相对正常的成人HSC稳态。因此，LCMT-1可能具有细胞内在的促进作用， HSC适应性在移植后早期的应激期间是独特的。该提案是一个多调查员 该应用程序利用了两名PI的生物化学和实验血液学专业知识。我们一起 已经发现LCMT-1在造血中起着关键的内在作用，这是造血干细胞特异性的， 移植扩展、存活和自我更新阶段。相比之下，LCMT 1不发挥作用， 稳态条件下HSC的维护和循环。由于LCMT-1底物仅限于PP 2A 由于LCMT-1必须与特异性活性位点和C-末端相互作用的独特方式， 我们假设LCMT-1促进高水平的“甲基化依赖性”PP 2A和PP 4， 异源三聚体反过来调节关键蛋白如Raf上的关键调节磷酸化位点， 促进促分裂原相关蛋白激酶（MAPK）通路信号传导，这是早期增殖驱动所需的 在造血重建过程中。在本提案中，我们将检验这一假设，并开始剖析 利用LCMT-1 cKO小鼠确定成年HSC多谱系再增殖中的特定缺陷的机制 使用基于流式细胞术的测定来测定活性。试验将比较稳态和复制应激反应。 LCMT-1催化活性的重要性将被确定，甲基化依赖性的参与将被确定。 将评估PP 2A复合物在再生功能中的作用，并将评估治疗性年轻化策略。 探讨了实现这些目标将通过提高对信号的理解来推动该领域的发展 调节造血并提供操纵LCMT-1和PP 2A家族的途径 磷酸酶，以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.