NKLAM: An RBR E3 Ubiquitin Ligase Essential for Regulation of Innate Immunity

先天免疫系统是对病变细胞和感染因子的第一反应。自然 杀伤（NK）细胞和巨噬细胞是该系统的主要组成部分。虽然它们的功能不同，但 两者都需要表达NKLAM（自然杀伤细胞溶解相关分子）以实现最佳功能。 因此，阐明NKLAM在天然免疫中的作用具有重要的生物学和临床意义。 暴露于肿瘤细胞或激活溶细胞因子的细胞因子后，NKLAM在NK细胞中上调 功能在暴露于细菌或促炎细胞因子后，NKLAM在巨噬细胞中上调。我们 产生NKLAM缺陷型（KO）小鼠;它们具有比WT（野生型）小鼠更低的NK活性。他们表现出更大的 肿瘤生长、肿瘤扩散和转移的能力比WT小鼠强。NKLAM KO小鼠也具有以下缺陷： 巨噬细胞在体内和体外的抗菌功能。 NKLAM是一个小的，高度保守的，独特的环间环（RBR）E3组的成员 泛素连接酶。RBR E3泛素连接酶在细胞生理学中起着关键作用，并参与了细胞的生长。 许多疾病的发病机制，包括癌症、自身免疫性疾病和神经系统疾病。有 对药物研发的巨大兴趣。我们确定了NKLAM介导的 泛素化，包括转录因子STAT 1和c-myc，Bcl-2和UCKL-1（尿苷胞苷激酶 Like-1），一种促进肿瘤生长的蛋白质。这些底物在细胞信号传导、生长和代谢中的核心性质是： 存活表明NKLAM的药物调节对于治疗癌症和感染性疾病具有显著的潜力。 疾病 我们发现活化的NK细胞释放含有NKLAM的外泌体。NKLAM+外泌体促进 体外肿瘤细胞死亡。初步数据表明NKLAM+外泌体进入靶细胞并递送NKLAM。 将解决的重要未回答的问题是NKLAM对效应器中关键底物的影响。 细胞和靶细胞。 NKLAM在巨噬细胞中的作用无疑与其在NK细胞中的作用不同， 细菌发生在细胞内。我们正在进行的研究表明，NKLAM调节信号事件， 激活巨噬细胞的抗菌程序这条通路的一个关键调节因子是STAT 1。 我们将检验NKLAM的E3泛素连接酶活性在抗肿瘤中发挥作用的假设。 NK细胞和巨噬细胞的抗微生物功能与以下三个相互关联，但 独立目标： 目的1：阐明NKLAM在外泌体介导的肿瘤细胞杀伤中的作用。我们将测试 假设在含有NKLAM的NK衍生的外泌体进入肿瘤细胞后，NKLAM与 关键底物，导致细胞死亡。我们将测试来自NKLAM WT、NKLAM KO和NKLAM WT的外来体的能力。 NKLAM连接酶缺陷型NK细胞使用多发性骨髓瘤小鼠模型在体内杀死肿瘤细胞， 频率越来越高，尤其是退伍军人。 目的2：确定NKLAM如何在体外和体内影响巨噬细胞的抗菌功能。我们 将用S. pneumoniae和S.金黄色葡萄球菌，病原体是导致 疾病鉴于抗生素耐药性的增加，这些体内研究的重要性被放大。 目的3：确定NKLAM介导的泛素化的机制、位点和类型。我们将研究 在肿瘤生长和抗细菌杀伤中重要的底物。这将有助于更好地理解 NKLAM在抗肿瘤和抗细菌免疫中的作用，并揭示了对治疗敏感的关键调控步骤。 对折磨我们退伍军人的疾病进行干预。

The innate immune system serves is the first response to diseased cells and infectious agents. Natural Killer (NK) cells and macrophages are primary components of this system. Although their functions differ, they both require the expression of NKLAM (Natural Killer Lytic-Associated Molecule) for optimal function. Accordingly, elucidating the role of NKLAM in innate immunity has important biological and clinical significance. NKLAM is up-regulated in NK cells upon exposure to tumor cells or cytokines that activate cytolytic function. NKLAM is up-regulated in macrophages upon exposure to bacteria or pro-inflammatory cytokines. We generated NKLAM-deficient (KO) mice; they have less NK activity than WT (wild type) mice. They exhibit greater tumor growth, tumor dissemination and metastasis than WT mice. NKLAM KO mice also have defects in macrophage anti-bacterial function in vivo and in vitro. NKLAM is a member of a small, highly conserved, unique group of RING-in between-RING (RBR) E3 ubiquitin ligases. RBR E3 ubiquitin ligases play a key role in cellular physiology and are involved in the pathogenesis of many diseases, including cancer, autoimmune diseases and neurological disorders. There is great interest in drug discovery to target them. We identified key potential substrates of NKLAM-mediated ubiquitination, including the transcription factors STAT1 and c-myc, Bcl-2 and UCKL-1 (uridine cytidine kinase like-1), a protein that promotes tumor growth. The central nature of these substrates in cell signaling, growth and survival suggests that drug modulation of NKLAM has significant potential for treating cancer and infectious diseases. We found that activated NK cells releases exosomes containing NKLAM. NKLAM+ exosomes promote tumor cell death in vitro. Preliminary data indicate that NKLAM+ exosomes enter target cells and deliver NKLAM. Important unanswered questions that will be addressed are the effect of NKLAM on critical substrates in effector cells and in target cells. The role of NKLAM in macrophages is undoubtedly different from its role in NK cells in that killing of bacteria occurs intracellularly. Our ongoing studies suggest that NKLAM modulates the signaling events that activate the macrophage anti-bacterial program. A key regulator of this pathway is STAT1. We will test the hypothesis that the E3 ubiquitin ligase activity of NKLAM plays a role in the anti-tumor and anti-microbial functions of NK cells and macrophages with the following three interconnected but independent aims: Aim 1: Elucidate the role of NKLAM in exosome-mediated killing of tumor cells. We will test the hypothesis that upon entry of NK-derived exosomes containing NKLAM into tumor cells, NKLAM interacts with critical substrates, resulting in cell death. We will test the ability of exosomes from NKLAM WT, NKLAM KO and NKLAM ligase defective NK cells to kill tumor cells in vivo using a mouse model of multiple myeloma, a disease increasing in frequency, especially in veterans. Aim 2: Determine how NKLAM affects the anti-bacterial function of macrophages in vitro and in vivo. We will infect NKLAM KO and WT mice with S. pneumoniae and S. aureus, pathogens that are leading causes of disease. In light of increasing antibiotic resistance, the importance of these in vivo studies is magnified. Aim 3: Identify the mechanism, sites and types of ubiquitination mediated by NKLAM. We will examine substrates important in tumor growth and anti-bacterial killing. This will provide a better understanding of the role of NKLAM in anti-tumor and anti-bacterial immunity and reveal key regulatory steps susceptible to therapeutic interventions for diseases that afflict our veteran population.