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

NKLAM：一种 RBR E3 泛素连接酶，对于调节先天免疫至关重要

基本信息

批准号：
9898218
负责人：
JACKI KORNBLUTH
金额：
--
依托单位：
ST. LOUIS VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2021-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9898218
关键词：
Activated Natural Killer Cell Address Affect Aging Anti-Bacterial Agents Antibiotic Resistance Apoptosis Apoptotic Autoimmune Diseases BCL2 gene Bacteria Bacterial Antibiotic Resistance Biological Carcinogens Cell Death Cell Survival Cell physiology Cells Cellular Structures Communicable Diseases Data Defect Disease Drug Modulation Effector Cell Event Exhibits Exposure to Frequencies Genes Goals Growth Healthcare Systems Immune response Immune system Immunity In Vitro Infection Infectious Agent Inflammatory Ingestion Innate Immune System Knockout Mice Laboratories Ligase Light Lytic Malignant - descriptor Malignant Neoplasms Mediating Mediator of activation protein Medical Membrane Military Personnel Molecular Multiple Myeloma Mus Natural Immunity Natural Killer Cells Nature Neoplasm Metastasis Pathogenesis Pathway interactions Play Population Proteins Regulation Rest Risk Role STAT1 gene Signal Transduction Site Staphylococcus aureus Streptococcus pneumoniae Substrate Interaction System Testing Therapeutic Therapeutic Intervention Transcriptional Regulation Ubiquitination Uridine Kinase Veterans Wild Type Mouse antimicrobial assault c-myc Genes cancer cell cell growth clinically significant comorbidity cytokine drug discovery exosome first responder in vivo interest macrophage member microvesicles mouse model neoplastic cell nervous system disorder novel therapeutic intervention pathogen prevent programs response transcription factor tumor tumor growth ubiquitin-protein ligase

项目摘要

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.

先天免疫系统是对患病细胞和感染剂的首次反应。自然的杀手（NK）细胞和巨噬细胞是该系统的主要组成部分。尽管它们的功能不同，但两者都需要表达NKLAM（天然杀手裂解相关的分子）才能获得最佳功能。因此，阐明Nklam在先天免疫中的作用具有重要的生物学和临床意义。暴露于激活细胞溶解的肿瘤细胞或细胞因子后，NKLAM在NK细胞中被上调功能。接触细菌或促炎细胞因子后，巨噬细胞中的NKLAM在巨噬细胞中被上调。我们产生的NKLAM缺陷（KO）小鼠；它们的NK活性少于WT（野生型）小鼠。它们表现更大肿瘤生长，肿瘤传播和转移比WT小鼠。 Nklam KO小鼠也有缺陷巨噬细胞在体内和体外的抗细菌功能。 Nklam是一个小型，高度保守，独特的环环（RBR）E3的成员泛素连接酶。 RBR E3泛素连接酶在细胞生理中起关键作用，并参与许多疾病的发病机理，包括癌症，自身免疫性疾病和神经系统疾病。有对毒品发现的极大兴趣将其瞄准。我们确定了NKLAM介导的关键潜在基材泛素化，包括转录因子STAT1和C-MYC，BCL-2和UCKL-1（尿苷胞苷激酶像1），一种促进肿瘤生长的蛋白质。这些底物在细胞信号，生长和生存表明，NKLAM的药物调节具有治疗癌症和感染性的巨大潜力疾病。我们发现活化的NK细胞释放了含有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提供NKLAM。将要解决的重要未解决的问题是NKLAM对效应子的关键基质的影响细胞和目标细胞。 Nklam在巨噬细胞中的作用无疑与其在NK细胞中的作用有所不同细菌细胞内发生。我们正在进行的研究表明，NKLAM调节了信号事件激活巨噬细胞抗菌计划。该途径的关键调节器是STAT1。我们将检验以下假设：Nklam的E3泛素连接酶活性在抗肿瘤中起作用 NK细胞和巨噬细胞的抗微生物功能与以下三个相互连接但独立目标：目标1：阐明NKLAM在外泌体介导的肿瘤细胞杀死中的作用。我们将测试 NKLAM与NK衍生的外泌体进入肿瘤细胞后的假设，NKLAM与临界底物，导致细胞死亡。我们将测试来自Nklam WT，Nklam Ko和 Nklam连接酶有缺陷的NK细胞使用多发性骨髓瘤的小鼠模型，一种疾病，在体内杀死肿瘤细胞频率增加，尤其是在退伍军人中。 AIM 2：确定NKLAM在体外和体内如何影响巨噬细胞的抗细菌功能。我们将用肺炎链球菌和金黄色葡萄球菌感染Nklam KO和WT小鼠，病原体是导致原因的原因疾病。鉴于抗生素耐药性的增加，这些体内研究的重要性被放大。目标3：确定NKLAM介导的泛素化的机制，地点和类型。我们将检查对肿瘤生长和抗菌杀伤重要的底物。这将更好地了解角色抗肿瘤和抗细菌免疫中NKLAM的作品，并揭示了容易受过治疗的关键调节步骤折磨我们退伍军人人口的疾病的干预措施。