LYSOSOMAL PROTEOLYSIS IN HEMATOPOIETIC CELLS

描述：（研究者摘要）蛋白水解是一种基本的 所有生物体中的一种机制，它提供了一种敏感的方式， 滴定维持体内平衡所必需的活性蛋白质水平 和多种重要的细胞功能。 的分子组成， 蛋白水解机制尚未完全了解。 这个项目的中心目标是 建议是为了更好地理解这一复杂性， 一种新的溶酶体膜蛋白的研究 命名为LAPTm 5。 在鼠动物中，LAPTm 5在小鼠中特异性表达， 在造血细胞中，它优先在高水平表达，在人类中，它优先在高水平表达。 造血细胞的水平。 该蛋白质被发现与泛素化的 proteins. 越来越多的证据表明，蛋白质的泛素化是 是溶酶体中蛋白水解的重要信号。 LAPTm 5也是 发现改变溶酶体中IkB的水平。 IkB是关键的监管机构 NF-kB是一种主要的转录因子， 调节一系列重要基因，包括那些参与 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。 这一近期目标 该建议是从几个方面研究LAPTm 5的功能， 这些研究结果表明的方法。 目标1将确定LAPTm 5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，它是否可以激活NF-κ B，以及它是否对 NF-κ B系统的反应性。 目标2将审查 LAPTm 5在造血细胞体外发育中的作用 小鼠胚胎干细胞向造血细胞的分化。 基因的组成型表达和基因表达的破坏的影响是基因表达的影响。 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，包括二维凝胶分析，以确定身份 与LAPTm 5结合的泛素化蛋白质。 直接原位结合 用纯化的溶酶体进行的测定也将用于鉴定结合 proteins. 关于这种不寻常基因的新发现将有助于我们 了解蛋白水解功能的分子基础 细胞器及其与造血细胞的相关性。 该提案还 可能会导致对一个关键转录因子的调控的新见解， 在许多人类疾病中起着重要作用。

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.