The regulation of thrombopoietin levels

血小板生成素水平的调节

• 批准号: 10457889
• 负责人: Rami Khoriaty
• 金额: $ 53.93万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10457889
• 关键词: Animals; Autophagocytosis; Binding; Biological; Birth; Blood Platelets; Bone Marrow; Cell Line; Cell Maintenance; Cell Maturation; Cell Proliferation; Cell Surface Receptors; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Complex; Defect; Development; Disease; Dose; Endoplasmic Reticulum; Exhibits; Failure; Flow Cytometry; Foundations; Genetic; Genetic Models; Genetic Transcription; Glycoproteins; Hematopoietic; Hematopoietic stem cells; HepG2; Hepatocyte; Histology; Human; Immunofluorescence Immunologic; Impairment; Interleukin-6; Knock-out; Knowledge; Liver; Lysine; Mediating; Megakaryocytes; Membrane; Messenger RNA; Mus; Physiological; Plasma; Platelet Count measurement; Play; Production; Proteins; Regulation; Ribosomes; Role; Testing; Therapeutic; Thrombocytopenia; Thrombopoietin; Transcriptional Regulation; Ubiquitination; Validation; design; experimental study; extracellular; gain of function mutation; genome-wide; loss of function mutation; mouse genetics; multicatalytic endopeptidase complex; novel; novel strategies; overexpression; progenitor; receptor; response; secretory protein; stem cell survival; thrombocytosis; trafficking; ubiquitin-protein ligase; validation studies

Project Summary Thrombopoietin (TPO), a plasma glycoprotein made in hepatocytes, binds to its cell surface receptor MPL expressed on megakaryocytes and megakaryocyte progenitors, promoting cell proliferation and maturation. In addition to its role in megakaryocyte development and platelet production, TPO also plays a critical role in hematopoietic stem cell survival and maintenance. In humans, gain-of-function mutations in TPO result in autosomal dominant thrombocytosis (high platelet counts), while loss-of-function mutations in TPO (or in its receptor MPL) result in congenital amegakaryocytic thrombocytopenia, a disease characterized by low platelet counts and absence of bone marrow megakaryocytes at birth with subsequent bone marrow aplasia and failure. The transcriptional regulation and plasma clearance of TPO have been well studied. In contrast, the mechanism by which TPO is secreted from hepatocytes and by which TPO is degraded intracellularly remain unknown. We have shown in preliminary results that mice deficient in LMAN1 exhibit thrombocytopenia and that the thrombocytopenia is recapitulated in mice with hepatocyte-specific LMAN1 deletion but not in mice with hematopoietic-specific LMAN1 deletion. We have additionally shown that the plasma TPO level is low in LMAN1 deficient mice (~50% of normal). Taken together with LMAN1’s known function as an endoplasmic reticulum cargo receptor, these results strongly suggest that the thrombocytopenia observed in LMAN1 deficient mice is due to impaired secretion of TPO from hepatocytes. Consistent with these findings in mice, we have shown that LMAN1 deficiency results in intracellular accumulation of TPO in human cells. Therefore, the role of LMAN1 in regulating TPO appears to be conserved in mice and humans. In this proposal, we aim to define the role of LMAN1 in regulating the plasma TPO levels in mice and to dissect the mechanism by which LMAN1 regulates TPO secretion, both under a normal physiological state and in the setting of enhanced TPO production. In additional preliminary results, we have performed an unbiased genome-scale CRISPR knock-out screen to identify novel regulators of the intracellular TPO level. This screen, performed in biological triplicates, followed by validation experiments, demonstrated that deletion of UBE3C results in intracellular accumulation of TPO but no intracellular accumulation of any of the 3 control secretory proteins tested. UBE3C is an E3 ubiquitin ligase, suggesting that TPO is a substrate for UBE3C and that in the absence of UBE3C, TPO ubiquitination is impaired, resulting in reduced degradation. Therefore, an additional aim of this proposal is to define the role of UBE3C in regulating the intracellular TPO level, both under steady state TPO production and in a state of high TPO production. This proposal has important implications for understanding the mechanisms of TPO regulation at the level of its intracellular trafficking/secretion and at the level of its intracellular degradation. Knowledge gained from this proposal may lay the foundation for the development of novel strategies to therapeutically regulate the plasma TPO level.

项目摘要 血小板蛋白（TPO）是一种在肝细胞中制成的血浆糖蛋白，与其细胞表面受体MPL结合 在巨核细胞和巨核细胞祖细胞上表达，促进细胞增殖和成熟。在 除了其在巨核细胞开发和血小板生产中的作用外，TPO在 造血干细胞存活和维持。在人类中，TPO的功能收益突变导致 常染色体显性血小板病（高血小板计数），而功能丧失突变（或在其中） 受体MPL）导致先天性白细胞血小板减少症，这种疾病为特征低血小板 出生时骨髓骨髓的数量和缺失，随后的骨髓性和 失败。 TPO的转录调控和血浆清除率已经很好地研究了。相反， TPO从肝细胞分泌的机制，并通过其细胞内降解TPO 未知。我们在初步结果中表明，LMAN1缺乏暴露血小板减少症和 用肝细胞特异性LMAN1删除在小鼠中概括了血小板减少症，但在小鼠中却没有 带有造血特异性LMAN1缺失。我们还表明，血浆TPO水平很低 LMAN1缺乏小鼠（正常的约50％）。与LMAN1的已知功能一起作为内质功能 网状货物接收器，这些结果强烈表明在LMAN1中观察到的血小板减少症 缺乏小鼠是由于肝细胞中TPO的分泌受损。与小鼠中的这些发现一致，我们 已经表明LMAN1缺乏会导致人类细胞中TPO的细胞内积累。因此， LMAN1在调节中的作用在小鼠和人类中似乎是保守的。在此提案中，我们的目标是 定义LMAN1在调节小鼠血浆TPO水平中的作用，并剖析其机制 LMAN1调节TPO分泌，无论是在正常的物理状态下还是在增强的TPO的情况下 生产。在其他初步结果中，我们进行了公正的基因组尺度CRIS淘汰赛 筛选细胞内TPO水平的新型调节剂。该屏幕以生物学三份执行， 然后进行验证实验，证明UBE3C的缺失导致细胞内积累 TPO但没有测试的3个控制秘密蛋白中的任何一个的细胞内积累。 ube3c是E3 泛素连接酶，表明TPO是UBE3C的底物，并且在不存在UBE3C的情况下TPO 泛素化受损，导致降解降低。因此，该提议的另一个目的是 在稳态TPO产生和 在TPO生产高的状态下。该提议对理解机制具有重要意义 TPO调节在其细胞内贩运/分泌水平和细胞内的水平 降解。从该提议中获得的知识可能为发展新颖的基础奠定了基础 热调节血浆TPO水平的策略。