Analysis of NKLAM: A Novel Gene Associated With Cellular Cytotoxicity

描述(由申请人提供)： 自然杀伤(NK)细胞在宿主抵抗肿瘤和感染性病原体方面发挥着重要作用。 它们主要通过释放含有穿孔素和颗粒酶的细胞毒性颗粒来诱导靶细胞死亡。在鉴定与细胞溶解相关的其他蛋白质的研究中，发现了一种新的蛋白质，其表达在NK细胞的细胞因子刺激下高度增加。这种蛋白质被命名为NK裂解相关分子(NKLAM)，是正在进行的退伍军人事务部奖励金项目的主题。NKLAM是一种定位于NK细胞溶解颗粒膜的环指跨膜蛋白。研究表明，NKLAM在NK介导的肿瘤细胞杀伤中发挥作用。巨噬细胞的NKLAM也在Toll样受体(TLR)刺激下上调，这表明NKLAM在细菌杀灭中也起到了作用。为了进一步评估NKLAM的作用，我们建立了NKLAM缺陷(KO)小鼠。这些小鼠的NK活性比野生型(WT)小鼠低50%，产生的干扰素-？以响应肿瘤细胞的接触。在注射B16黑色素瘤细胞后，NKLAM KO小鼠的肺转移数量也明显高于WT小鼠，并显示出淋巴瘤细胞从原发肿瘤部位更多地扩散到淋巴结。最后，一个关键的发现是NKLAM作为E3泛素连接酶发挥作用。泛素化是调节免疫反应的关键机制。对NKLAM、KO和WT NK细胞和巨噬细胞的体外研究表明，NKLAM参与了细胞因子和TLR介导的信号事件。在这个模型中，NKLAM通过调节效应细胞中的信号事件来调节NK/巨噬细胞的活性。或者，初步研究表明，当效应细胞脱颗粒时，NKLAM被释放到NK细胞的上清中。因此，NKLAM发挥作用的另一种方式是在效应细胞释放后将其输送到靶细胞中。在该模型中，NKLAM作为E3连接酶，将泛素化在靶细胞中的抗凋亡或生长相关分子，导致它们的降解，从而促进靶细胞的凋亡。我们鉴定了一种由NKLAM泛素化的蛋白，尿苷-胞苷激酶样蛋白1(UKL-1)。这种新蛋白质的功能尚不清楚。然而，它与尿苷激酶的同源性和在肿瘤细胞中的过度表达表明了OKL-1在肿瘤生长和/或生存中的作用。为了验证这一点，RNA干扰(RNAi)被用来下调NK敏感的K562红白血病细胞和其他NK靶细胞中UKL-1的表达。K562细胞中UKL-1的表达降低可减缓其增殖，诱导其凋亡，并增强其对NK介导的杀伤的敏感性； 过表达OKL-1可降低其对NK细胞裂解的易感性。初步研究表明，NKLAM可能在效应细胞和/或靶细胞水平起作用。这两种模式并不是相互排斥的，根据情况可能都是有效的。这项应用由三个独立但相互关联的目的组成，目的是研究NKLAM的E3连接酶功能及其在NK细胞、巨噬细胞和靶细胞中的功能后果。 1)确定NKLAM在NK细胞和靶细胞中的作用。将评估NKLAM在NK细胞的信号转导和细胞毒功能中的作用。还将进行研究，以检验这一假设，即在NK细胞脱颗粒后，NKLAM进入目标细胞，并泛素化OKL-1等蛋白质，促进目标细胞死亡。建议进一步研究新蛋白UKL-1在体外和体内的功能。 2)确定NKLAM在巨噬细胞中的作用。NKLAM在TLR介导的信号事件和巨噬细胞吞噬中的作用将被研究。 3)鉴定NKLAM KO小鼠，研究NKLAM在体内的作用。将对各种肿瘤模型进行评估，以确定NKLAM在哪里发挥作用。对NKLAM KO小鼠的分析可能会揭示NKLAM的其他作用，并可能揭示细胞杀伤的替代机制。

DESCRIPTION (provided by applicant): Natural killer (NK) cells play an important role in host defense against tumors and infectious agents. They induce target cell death, primarily by the release of cytotoxic granules containing perforin and granzymes. In studies to characterize additional proteins associated with cytolysis, a novel protein whose expression was highly increased upon cytokine stimulation of NK cells was identified. This protein was named NK lytic associated molecule (NKLAM) and is the subject of this ongoing VA merit grant project. NKLAM is a RING finger transmembrane protein localized to NK cytolytic granule membranes. Studies have shown a role for NKLAM in NK-mediated killing of tumor cells. NKLAM is also up-regulated in macrophages upon toll-like receptor (TLR) stimulation, suggesting a role in bacterial killing as well. To further assess the role of NKLAM, NKLAM deficient (KO) mice were generated. These mice exhibit 50% less NK activity than wild type (WT) mice and produce less interferon-? in response to tumor cell contact. NKLAM KO mice also have substantially higher numbers of lung metastases compared with WT after injection with B16 melanoma cells and show greater dissemination of lymphoma cells to lymph nodes from the primary tumor site. Finally, a critical finding is that NKLAM functions as an E3 ubiquitin ligase. Ubiquitination is a key mechanism for regulating immune responses. In vitro studies of NKLAM KO and WT NK cells and macrophages suggest that NKLAM participates in cytokine and TLR-mediated signaling events. In this model, NKLAM regulates NK/macrophage activity by modulating signaling events in effector cells. Alternatively, preliminary studies have shown that NKLAM is released into the supernatants of NK cells upon degranulation of effector cells. Accordingly, another way NKLAM may function is to be transported into the target cell after release by the effector cell. In this model, NKLAM, acting as an E3 ligase, would ubiquitinate anti-apoptotic or growth associated molecules in the target, resulting in their degradation, thereby promoting target cell apoptosis. We identified a protein, uridine-cytidine kinase-like 1 (UCKL-1), which is ubiquitinated by NKLAM. The function of this novel protein is unknown. However, its homology to uridine kinases and over-expression in tumor cells suggests a role for UCKL-1 in tumor growth and/or survival. To test this, RNA interference (RNAi) was used to down-regulate UCKL-1 expression in NK-sensitive K562 erythroleukemia cells and other NK targets. Decreased expression of UCKL-1 in K562 slows their proliferation, induces apoptosis and enhances their susceptibility to NK-mediated lysis; over-expression of UCKL-1 reduces their susceptibility to NK lysis. Preliminary studies show that NKLAM may act at the level of the effector cell and/or target cell. These two models are not mutually exclusive and may both be functional depending upon the circumstances. This application consists of three independent but interlinked aims to study the E3 ligase function of NKLAM and its functional consequences in NK cells, macrophages and target cells. 1) Determine the role of NKLAM in NK cells and target cells. The role of NKLAM in signaling and cytotoxic function in NK cells will be assessed. Studies will also be performed to test the hypothesis that upon NK cell degranulation, NKLAM enters target cells and ubiquitinates proteins such as UCKL-1, enhancing target cell death. Studies are proposed to further elucidate the function of the novel protein UCKL-1 in vitro and in vivo. 2) Determine the role of NKLAM in macrophages. The role of NKLAM in TLR-mediated signaling events and in macrophage phagocytosis will be investigated. 3) Characterize NKLAM KO mice to study the role of NKLAM in vivo. A variety of tumor models will be evaluated to determine where NKLAM plays a role. Analysis of NKLAM KO mice may reveal additional roles for NKLAM and potentially unveil alternative mechanisms of cell killing.