LYSOSOMAL PROTEOLYSIS IN HEMATOPOIETIC CELLS

描述：(研究人员摘要)蛋白质分解是一种基本的 在所有活着的有机体中提供了一种敏感的方式 维持体内平衡所必需的活性蛋白质水平的滴定 和不同的关键细胞功能。它的分子组成 人们对蛋白质分解机制的了解还不完全。这样做的中心目标是 提案的目的是更好地理解这一复杂的问题 一种新的溶酶体膜蛋白的研究进展 命名为LAPTm5。在小鼠动物中，LAPTm5特异性地表达于 造血细胞，在人类中，它优先高表达 在造血细胞中的水平。这种蛋白质被发现与泛素结合。 蛋白质。越来越多的证据表明，蛋白质的泛化是 溶酶体中蛋白质分解的重要信号。LAPTm5也被 发现会改变溶酶体中IKB的水平。IkB是关键的监管者 核因子-kB的激活，这是一个主要的转录因子，负责 调控广泛的重要基因清单，包括那些参与 免疫和炎症反应、细胞黏附、应激反应 增殖分化。IKB被降级的过程和 因此，监管是一个深入研究的领域。眼前的目标是 这项建议是为了从几个方面研究LAPTm5的功能 这些发现所表明的方法。目标1将确定LAPTm5是否可以 通过分析是否调节IKB的降解来影响核因子-kB系统 溶酶体，它是否能激活核因子-kB，是否对 核因子-kB系统的响应性。目标2将考察 LAPTm5在造血细胞发育中的作用 小鼠胚胎干细胞向造血细胞的分化。 基因的结构性表达和基因突变的影响 对同源重组后的基因进行分析。AIM 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LAPTm5结合的泛素蛋白。直接原位结合 用纯化的溶酶体进行的检测也将用于鉴定结合 蛋白质。关于这种不寻常基因的新发现将有助于我们的 对蛋白水解酶功能的分子基础的认识 细胞器及其与造血细胞的关系。该提案还包括 可能导致对一种关键转录因子的调控有新的见解 这在许多人类疾病中扮演着重要的角色。

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.