Analysis of NKLAM: A Novel Gene Associated With Cellular Cytotoxicity

NKLAM 分析：与细胞毒性相关的新基因

• 批准号: 8696768
• 负责人: JACKI KORNBLUTH
• 金额: --
• 依托单位: ST. LOUIS VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8696768
• 关键词: Acute Erythroblastic Leukemia; Aging; Anti-Bacterial Agents; Antisense Oligonucleotides; Apoptosis; Apoptotic; Binding; Carcinogens; Caspase; Cell Death; Cell Degranulation; Cell-Mediated Cytolysis; Cells; Communicable Diseases; Cytolysis; Cytoplasmic Granules; Digestion; Disease; Effector Cell; Event; Exhibits; Fingers; Genes; Goals; Gram-Negative Bacteria; Grant; Granzyme; Growth; Healthcare Systems; Host Defense; Immune response; Immunity; In Vitro; Incidence; Infection; Infectious Agent; Injection of therapeutic agent; Integral Membrane Protein; Interferons; Interleukin-12; Interleukin-15; Interleukin-2; Interleukins; K-562; Lead; Lung; Lymphoma; Lytic; Malignant Neoplasms; Mediating; Melanoma Cell; Membrane; Messenger RNA; Military Personnel; Modeling; Mus; Names; Natural Killer Cells; Neoplasm Metastasis; Normal Statistical Distribution; Phagocytosis; Play; Population; Predisposition; Primary Neoplasm; Process; Proteins; RNA Interference; Resistance; Role; Signal Transduction; Site; Testing; Toll-like receptors; Ubiquitination; Uridine Kinase; Veterans; Wild Type Mouse; antimicrobial; cell killing; cytokine; cytotoxic; disorder prevention; in vitro activity; in vivo; killings; lymph nodes; macrophage; microbial; neoplastic cell; novel; overexpression; pathogen; perforin; prevent; receptor; receptor-mediated signaling; response; tumor; tumor growth; ubiquitin-protein ligase

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.

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。 在表征与细胞溶解相关的其他蛋白质的研究中，发现了一种新蛋白质，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)，是正在进行的 VA 奖学金项目的主题。 NKLAM 是一种定位于 NK 溶细胞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。在 Toll 样受体 (TLR) 刺激下，巨噬细胞中的 NKLAM 也会上调，这表明 NKLAM 也具有杀灭细菌的作用。为了进一步评估 NKLAM 的作用，产生了 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-α也较少。对肿瘤细胞接触做出反应。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，并且显示出更多的淋巴瘤细胞从原发肿瘤部位扩散至淋巴结。最后，一个重要的发现是 NKLAM 充当 E3 泛素连接酶。泛素化是调节免疫反应的关键机制。 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 介导的信号转导事件。在此模型中，NKLAM 通过调节效应细胞中的信号事件来调节 NK/巨噬细胞活性。或者，初步研究表明，NKLAM 在效应细胞脱颗粒后释放到 NK 细胞的上清液中。因此，NKLAM发挥作用的另一种方式是在效应细胞释放后转运至靶细胞中。在该模型中，NKLAM作为E3连接酶，将靶标中的抗凋亡或生长相关分子泛素化，导致其降解，从而促进靶细胞凋亡。我们鉴定了一种蛋白质，尿苷胞苷激酶样 1 (UCKL-1)，它被 NKLAM 泛素化。这种新型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 UCKL-1 在肿瘤生长和/或存活中发挥作用。为了测试这一点，使用 RNA 干扰 (RNAi) 下调 NK 敏感的 K562 红白血病细胞和其他 NK 靶标中的 UCKL-1 表达。 K562 中 UCKL-1 表达的降低会减慢其增殖、诱导细胞凋亡并增强其对 NK 介导的裂解的敏感性； UCKL-1 的过度表达降低了它们对 NK 裂解的敏感性。 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。这两种模型并不相互排斥，并且可以根据具体情况发挥作用。该应用由三个独立但相互关联的目标组成，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 细胞信号传导和细胞毒性功能中的作用。还将进行研究来测试以下假设：NK 细胞脱粒后，NKLAM 进入靶细胞并泛素化 UCKL-1 等蛋白质，从而增强靶细胞死亡。建议进行研究以进一步阐明新型蛋白质 UCKL-1 的体外和体内功能。 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。将评估多种肿瘤模型以确定 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。