Analysis of NKLAM: A Novel Gene Associated With Cellular Cytotoxicity

描述（由申请人提供）： 自然杀伤（NK）细胞在宿主防御肿瘤和感染因子中起重要作用。 它们主要通过释放含有穿孔素和颗粒酶的细胞毒性颗粒诱导靶细胞死亡。 在表征与细胞溶解相关的其他蛋白质的研究中，鉴定了一种新的蛋白质，其表达在NK细胞的细胞因子刺激后高度增加。这种蛋白质被命名为NK裂解相关分子（NKLAM），是这个正在进行的VA奖学金项目的主题。NKLAM是定位于NK细胞溶解颗粒膜的环指跨膜蛋白。研究已经显示NKLAM在NK介导的肿瘤细胞杀伤中的作用。NKLAM在巨噬细胞中也在toll样受体（TLR）刺激后上调，这也表明了在细菌杀伤中的作用。为了进一步评估NKLAM的作用，产生NKLAM缺陷型（KO）小鼠。这些小鼠表现出50%的NK活性比野生型（WT）小鼠和产生较少的干扰素-？对肿瘤细胞接触作出反应。在注射B16黑色素瘤细胞后，NKLAM KO小鼠与WT相比也具有显著更高数量的肺转移，并且显示淋巴瘤细胞从原发肿瘤部位向淋巴结的更大播散。最后，一个关键的发现是NKLAM作为E3泛素连接酶发挥作用。泛素化是调节免疫应答的关键机制。NKLAM KO和WT NK细胞和巨噬细胞的体外研究表明，NKLAM参与细胞因子和TLR介导的信号传导事件。在该模型中，NKLAM通过调节效应细胞中的信号传导事件来调节NK/巨噬细胞活性。或者，初步研究已经显示，在效应细胞脱粒后，NKLAM释放到NK细胞的上清液中。因此，NKLAM可以起作用的另一种方式是在被效应细胞释放后被转运到靶细胞中。在该模型中，NKLAM作为E3连接酶，将泛素化靶中的抗凋亡或生长相关分子，导致其降解，从而促进靶细胞凋亡。我们鉴定了一种被NKLAM泛素化的蛋白质，尿苷-胞苷激酶样1（UCKL-1）。这种新蛋白的功能尚不清楚。 然而，其与尿苷激酶的同源性和在肿瘤细胞中的过表达表明UCKL-1在肿瘤生长和/或存活中的作用。为了测试这一点，使用RNA干扰（RNAi）来下调NK敏感性K562红白血病细胞和其他NK靶标中的UCKL-1表达。K562细胞中UCKL-1表达的降低减缓了其增殖，诱导了细胞凋亡，并增强了其对NK介导的裂解的敏感性; UCKL-1的过表达降低了它们对NK细胞溶解的敏感性。 初步研究表明，NKLAM可能在效应细胞和/或靶细胞水平发挥作用。这两种模式并不相互排斥，视情况而定，都可以发挥作用。本申请由三个独立但相互关联的目的组成，旨在研究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细胞中的信号传导和细胞毒性功能中的作用。还将进行研究以检验以下假设：在NK细胞脱粒后，NKLAM进入靶细胞并使蛋白质如UCKL-1泛素化，从而增强靶细胞死亡。本研究旨在进一步阐明新蛋白UCKL-1在体外和体内的功能。 2)确定NKLAM在巨噬细胞中的作用。将研究NKLAM在TLR介导的信号传导事件和巨噬细胞吞噬作用中的作用。 3)表征NKLAM KO小鼠以研究NKLAM在体内的作用。将评估各种肿瘤模型，以确定NKLAM在何处发挥作用。对NKLAM KO小鼠的分析可能揭示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.