Integrated Analyses of Taspase 1 (MLL Cleaving Protease)

Taspase 1（MLL 切割蛋白酶）的综合分析

• 批准号: 7225982
• 负责人: JAMES J HSIEH
• 金额: $ 26.21万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7225982
• 关键词: 11q23; Acute Myelocytic Leukemia; Animals; Aspartate; Aspartate Ammonia-Lyase; Biochemical Genetics; Body Patterning; C-terminal; Cell Cycle Progression; Cell Proliferation; Cells; Chromosome Band; Chromosome Banding; Class; Cleaved cell; Complex; Defect; Development; Embryo; Embryonic Development; Endopeptidases; Foundations; Future; Gene Expression; Gene Expression Regulation; General Transcription Factors; Genetic; HRX protein; Hela Cells; Homeobox Genes; Human; In Vitro; Individual; Knock-out; Knockout Mice; Lead; Length; MLL gene; Mass Spectrum Analysis; Mediating; Mus; Myeloid-Lymphoid Leukemia Protein; N-terminal; Normal Cell; Peptide Hydrolases; Phenotype; Physiological; Process; Protein Family; Proteolysis; Proteomics; Reagent; Recurrence; Role; Threonine; Transcription Factor TFIIA; anti-cancer therapeutic; cdc Genes; gel electrophoresis; in vivo; infancy; inhibitor/antagonist; insight; knockout animal; leukemia; nervous system development; null mutation; outcome forecast; polypeptide; postnatal; reconstitution; tumorigenesis

DESCRIPTION (provided by applicant): Chromosome 11q23 translocations disrupting human Mixed Lineage Leukemia (MLL) gene are found in 80% of infantile leukemia and almost all cases of treatment induced secondary acute myeloid leukemia (AML). The MLL gene is required for proper HOX gene expression. Our prior studies demonstrated that full-length 500kD MLL protein undergoes proteolysis to generate N-terminal 320kD (MLLN320) and C-terminal 180kD (MLLC180) fragments. Processed MLL fragments form a complex to regulate the stability and availability of MLLN320 for downstream gene regulation. We subsequently purified and cloned the responsible protease and entitled it Taspase1 (Threonine Aspartase 1). The discovery of Taspase1 initiates a new class of proteases utilizing their N- terminal Threonine of mature ? subunit to cleave polypeptide substrates after P1 aspartate. Preliminary studies in HeLa cells indicated the importance of Taspase1-mediated MLL cleavage in HOX gene expression. Recently, we also identified a basal transcription factor, TFIIA, as a bona fide Taspase1 substrate. To investigate the physiological functions of Taspase1 in vivo, we generated Taspase1 knockout mice. Initial studies on Taspase1 deficient animals indicate the essential role of Taspase1 in body patterning, nervous system development, and cell cycle progression. With these unique reagents, we will further interrogate Taspase1 functions via the following specific aims: Specific Aim 1: We will characterize the role of Taspase1 in mouse embryonic development. It entails the creation of straight and conditional Taspase1 knockout mice to determine whether Taspase1 deficiency in mice results in embryonic lethality, homeotic transformations and/or other developmental abnormalities. We will dissect the mechanisms by which Taspase1 regulates Hox gene expression. Specific Aim 2: We will investigate the requirement of Taspase1 in normal cell cycle progression. We will start with studying cell cycle progression defects in Taspase1 deficient animals and cells, followed by dissecting the mechanisms by which Taspase1 regulates cell cycle progression and perform genetic reconstitutions of processed MLL family proteins into Taspase1 deficient cells to determine whether MLL proteolysis regulates cell proliferation. Specific Aim 3: We will perform studies to identify additional Taspase1 substrates and will validate their importance in vitro and in vivo. We will utilize 2-D difference in-gel electrophoresis in conjunction with mass spectrometry for the initial discovery, followed by phenotypic analyses of individual non-cleavable substrates. Since deregulation of HOX genes and cell cycle genes contribute to tumorigenesis, this combined genetic, biochemical, and proteomic approach to investigate Taspase1 functions will provide further insights regarding MLL leukemia and may lay the foundation for future development of Taspase1 inhibitors as anti-cancer therapeutics.

描述（由申请人提供）：在80％的婴儿白血病中发现了破坏人类混合谱系白血病（MLL）基因的11q23染色体易位，几乎所有治疗诱导的继发性急性髓样白血病（AML）。正确的HOX基因表达需要MLL基因。我们先前的研究表明，全长500KD MLL蛋白经历蛋白水解产生N末端320KD（MLLN320）和C端180KD（MLLC180）片段。加工后的MLL片段形成了一个复杂的，以调节MLLN320在下游基因调节中的稳定性和可用性。随后，我们纯化并克隆了负责任的蛋白酶，并将其标题为taspase1（苏氨酸天冬氨酸1）。 Taspase1的发现启动了一类新的蛋白酶，利用其成熟的N-末端苏氨酸？ P1天冬氨酸后裂解多肽底物的亚基。在HeLa细胞中的初步研究表明，Taspase1介导的MLL裂解在HOX基因表达中的重要性。最近，我们还将基础转录因子TFIIA确定为真正的Taspase1底物。为了研究体内Taspase1的生理功能，我们产生了Taspase1基因敲除小鼠。关于Taspase1动物缺陷动物的初步研究表明，Taspase1在身体模式，神经系统发育和细胞周期进程中的重要作用。使用这些独特的试剂，我们将通过以下特定目的进一步询问taspase1函数：特定目标1：我们将表征taspase1在小鼠胚胎发育中的作用。它需要创建直的和有条件的Taspase1基因敲除小鼠，以确定小鼠的Taspase1缺乏是否会导致胚胎致死性，同源转化和/或其他发育异常。我们将剖析taspase1调节HOX基因表达的机制。具体目标2：我们将研究taspase1在正常细胞周期进程中的需求。我们将首先研究Taspase1缺陷动物和细胞中细胞周期进展缺陷，然后解剖Taspase1调节细胞周期进程的机制，并执行已加工后的MLL家族蛋白的遗传重新施加到TTASPase1缺乏细胞中，以确定MLL蛋白溶解是否确定细胞溶解是否调节细胞扩散。具体目标3：我们将进行研究以识别其他Taspase1底物，并将验证其体外和体内的重要性。我们将利用2-D差异在凝胶电泳中与质谱法一起进行初始发现，然后对单个不可裂解的底物进行表型分析。由于HOX基因和细胞周期基因的放松管制有助于肿瘤发生，因此研究Taspase1功能的这种结合的遗传，生化和蛋白质组学方法将提供有关MLL白血病的进一步见解，并可能为未来的Taspase1抑制剂作为抗癌治疗剂作为抗癌治疗剂的发展奠定基础。