Integrated Analyses of Taspase 1 (MLL Cleaving Protease)

Taspase 1（MLL 切割蛋白酶）的综合分析

基本信息

批准号：
7790791
负责人：
JAMES J HSIEH
金额：
$ 16.94万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-05-01 至 2010-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7790791
关键词：
11q23 Acute Myelocytic Leukemia Animals Aspartate Aspartate Ammonia-Lyase Biochemical Body Patterning C-terminal Cell Cycle Progression Cell Proliferation Cells Chromosome Band Cleaved cell Complex Defect Development Embryo Embryonic Development Foundations Future Gene Expression Gene Expression Regulation General Transcription Factors Genetic 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 mouse development nervous system development null mutation outcome forecast polypeptide postnatal reconstitution

项目摘要

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 (MLLN32¿) and C-terminal 180kD (MLLC18¿) fragments. Processed MLL fragments form a complex to regulate the stability and availability of MLLN32¿ for downstream gene regulation. We subsequently purified and cloned the responsible protease and entitled it Taspasel (Threonine Aspartase 1). The discovery of Taspasel initiates a new class of proteases utilizing their N- terminal Threonine of mature (3 subunit to cleave polypeptide substratesafter P1 aspartate. Preliminary studies in HeLa cells indicated the importance of Taspasel-mediated MLL cleavage in HOX gene expression. Recently, we also identified a basal transcription factor, TFIIA, as a bona fide Taspasel substrate. To investigate the physiological functions of Taspasel in vivo, we generated Taspasel knockout mice. Initial studies on Taspasel deficient animals indicate the essential role of Taspasel in body patterning, nervous system development, and cell cycle progression. With these unique reagents, we will further interrogate Taspasel functions via the following specific aims: Specific Aim 1: We will characterize the role of Taspasel in mouse embryonic development. It entails the creation of straight and conditional Taspasel knockout mice to determine whether Taspasel deficiency in mice results in embryonic lethality, homeotic transformations and/or other developmental abnormalities. We will dissect the mechanisms by which Taspasel regulates Hox gene expression. Specific Aim 2: We will investigate the requirement of Taspasel in normal cell cycle progression. We will start with studying cell cycle progression defects in Taspasel deficient animals and cells, followed by dissecting the mechanisms by which Taspasel regulates cell cycle progression and perform genetic reconstitutions of processed MLL family proteins into Taspasel deficient cells to determine whether MLL proteolysis regulates cell proliferation. Specific Aim 3: We will perform studies to identify additional Taspasel 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 tumorigensis, this combined genetic, biochemical, and proteomic approach to investigate Taspasel functions will provide further insights regarding MLL leukemia and may lay the foundation for future development of Taspasel inhibitors as anti-cancer therapeutics.

染色体11Q23易位破坏人类混合谱系白血病（MLL）基因的易位。基础设施白血病和几乎所有治疗的病例都诱发了继发性髓样白血病（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蛋白经历蛋白水解生成N末端320KD（MLLN32¿）和C末端180KD（MLLC18¿）碎片。加工后的MLL片段形成了一个复杂的调节MLLN32“的稳定性和可用性下游基因调节。随后，我们纯化并克隆了负责任的蛋白酶并有资格 Taspasel（苏氨酸冬季1）。 Taspasel的发现使用其N-启动了一类新的蛋白酶成熟的终末硝酸（3个亚基清除多肽底物P1天冬氨酸。在HeLa细胞中的初步研究表明，hox基因中沙司赛介导的MLL裂解的重要性表达。最近，我们还确定了基本的转录因子TFIIA，是真正的TASPASEL底物。为了研究体内心形的物理功能，我们产生了Taspasel基因敲除小鼠。最初的对沙子赛缺陷动物的研究表明，沙子assel在身体模式，神经系统中的重要作用发育和细胞周期进程。使用这些独特的试剂，我们将进一步询问Taspasel 通过以下特定目的功能：具体目标1：我们将表征伴奏于小鼠胚胎发育中的作用。它需要创建直的和有条件的Taspasel基因敲除小鼠，以确定小鼠的心脏缺乏症是否缺乏导致胚胎致死性，同源转化和/或其他发育异常。我们将剖析心脏调节HOX基因表达的机制。具体目标2：我们将研究心脏在正常细胞周期进程中的需求。我们将开始研究细胞周期进程缺陷在沙子asl不足的动物和细胞中，然后解剖 TASPASEL调节细胞周期进程并执行遗传重构的机制将MLL家族蛋白加工到沙子asl不足的细胞中，以确定MLL蛋白水解是否调节细胞增殖。特定目标3：我们将进行研究以识别其他TASPASEL底物并将验证其体外和体内重要性。我们将利用2-D差与质量结合使用的凝胶电泳初始发现的光谱法，然后进行表型分析，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导致了肿瘤，因此这种结合的遗传学，研究心脏功能的生化和蛋白质组学方法将提供有关有关的进一步见解 MLL白血病，并可能为未来的心脏抑制剂作为抗癌者奠定基础疗法。