LYSOSOMAL PROTEOLYSIS IN HEMATOPOIETIC CELLS

造血细胞中的溶酶体蛋白水解

• 批准号: 6177934
• 负责人: BING LIM
• 金额: $ 26.41万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-17 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177934
• 关键词: binding proteins; cell differentiation; embryonic stem cell; gene expression; hematopoiesis; hematopoietic stem cells; lysosomes; membrane proteins; nuclear factor kappa beta; protein degradation; protein structure function; proteolysis; transcription factor; ubiquitin

DESCRIPTION: (Investigator's abstract) Proteolysis is a fundamental mechanism in all living organisms that provides a sensitive way for titrating levels of active proteins essential for maintaining homeostasis and diverse critical cellular functions. The molecular components of the proteolytic machinery are incompletely understood. The central goal of this proposal is to achieve a better understanding of the intricacies of this machinery through the investigation of a novel lysosomal membrane protein named LAPTm5. In murine animals, LAPTm5 is specifically expressed in hematopoietic cells, and in human, it is preferentially expressed at high levels in hematopoietic cells. The protein was found to bind to ubiquinated proteins. There is accumulating evidence that ubiquination of proteins is an important signal for proteolysis in lysosomes. LAPTm5 has also been found to alter the level of IkB in lysosomes. IkBs are the key regulators of the activation of NF-kB, a major transcriptional factor responsible for regulating an extensive list of important genes including those involved in immune and inflammatory response, cell adhesion, stress response proliferation and differentiation. The process by which IkB is degraded and regulated is therefore an area of intensive research. The immediate goal of this proposal is to investigate the function of LAPTm5 from several approaches indicated by these findings. Aim 1 will determine if LAPTm5 can affect the NF-kB system by analyzing if it regulates IkB degradation in lysosomes, whether it can activate NF-kB and whether it has an influence on the responsiveness of the NF-kB system. Aim 2 will examine the role of LAPTm5 in hematopoietic cell development by using the model of in vitro differentiation of murine embryonal stem cells into hematopoietic cells. The effect of a constitutive expression of the gene and a disruption of the gene by homologous recombination will be analyzed. Aim 3 will use protein chemistry techniques, including 2-D gel analysis to determine the identity of the ubiquinated proteins that bind with LAPTm5. A direct in situ binding assay with purified lysosomes will also be used to identify binding proteins. New findings about this unusual gene will contribute to our knowledge about the molecular basis of the functions of proteolytic organelles and their relevance to hematopoietic cells. The proposal also may lead to new insight about the regulation of a key transcriptional factor that plays a significant role in many human diseases.

描述：（研究者的摘要）蛋白水解是一种基本的 所有生物体中的机制为 滴定维持体内平衡所必需的活性蛋白质的水平 和多种关键的细胞功能。 的分子成分 蛋白水解机制尚未完全了解。 此次活动的中心目标 建议的目的是为了更好地理解这一问题的复杂性 通过研究一种新型溶酶体膜蛋白来研究机械 命名为 LAPTm5。 在小鼠动物中，LAPTm5 特异表达于 造血细胞，在人类中，它优先以高表达 造血细胞中的水平。 发现该蛋白质与泛素化结合 蛋白质。 越来越多的证据表明蛋白质的泛素化是 溶酶体中蛋白水解的重要信号。 LAPTm5 也已 发现可以改变溶酶体中 IkB 的水平。 IkB 是关键调节因子 NF-kB 的激活，NF-kB 是负责的主要转录因子 调节一系列重要基因，包括参与的基因 免疫和炎症反应、细胞粘附、应激反应 增殖和分化。 IkB 降解的过程 因此，监管是一个深入研究的领域。 近期目标是 该提案是从几个方面研究 LAPTm5 的功能 这些发现表明的方法。 目标 1 将确定 LAPTm5 是否可以 通过分析 NF-kB 系统是否调节 IkB 降解来影响 NF-kB 系统 溶酶体是否可以激活NF-kB以及是否有影响 NF-kB 系统的反应能力。 目标 2 将检验以下角色的作用： LAPTm5 在造血细胞发育中的体外模型 小鼠胚胎干细胞分化为造血细胞。 基因组成型表达和破坏的影响 将分析通过同源重组的基因。 目标 3 将使用蛋白质 化学技术，包括用于确定身份的二维凝胶分析 与 LAPTm5 结合的泛素化蛋白。 直接原位结合 纯化的溶酶体测定也将用于鉴定结合 蛋白质。 关于这种不寻常基因的新发现将有助于我们 关于蛋白水解功能的分子基础的知识 细胞器及其与造血细胞的相关性。 该提案还 可能会带来关于关键转录因子调控的新见解 这在许多人类疾病中起着重要作用。