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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.

先天免疫系统是对病态细胞和感染性病原体的第一反应。天然杀伤(NK)细胞和巨噬细胞是这个系统的主要组成部分。虽然它们的功能不同，但它们两者都需要NKLAM(自然杀伤溶解相关分子)的表达才能发挥最佳功能。因此，阐明NKLAM在先天免疫中的作用具有重要的生物学意义和临床意义。 NKLAM在NK细胞暴露于肿瘤细胞或激活细胞溶解的细胞因子时上调功能。NKLAM在暴露于细菌或促炎细胞因子的巨噬细胞中上调。我们产生NKLAM缺陷(KO)小鼠；它们的NK活性低于WT(野生型)小鼠。他们表现出更大的肿瘤生长、肿瘤扩散和转移能力均优于WT小鼠。NKLAM KO小鼠也有缺陷体内和体外巨噬细胞的抗菌功能。 NKLAM是一个小的、高度保守的、独特的环内环间(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发挥关键作用，并参与许多疾病的发病机制，包括癌症、自身免疫性疾病和神经疾病。的确有对药物发现非常感兴趣，以此为目标。我们确定了NKLAM介导的关键潜在底物泛素化，包括转录因子STAT1和c-myc、Bcl2和UKL-1(尿苷胞苷激酶 Like-1)，一种促进肿瘤生长的蛋白质。这些底物的中心性质在细胞信号、生长和存活表明NKLAM的药物调节在治疗癌症和感染性疾病方面具有巨大的潜力疾病。我们发现，激活的NK细胞释放含有NKLAM的外切体。NKLAM+外切体促进肿瘤细胞在体外死亡。初步数据表明，NKLAM+外切体进入靶细胞并运送NKLAM。将解决的重要悬而未决的问题是NKLAM对效应器中关键底物的影响细胞和目标细胞中。 NKLAM在巨噬细胞中作用与其在NK细胞中杀伤巨噬细胞的作用无疑是不同的细菌存在于细胞内。我们正在进行的研究表明，NKLAM调节信号事件，激活巨噬细胞抗菌程序。该途径的一个关键调节因子是STAT1。我们将验证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在外切体介导的肿瘤细胞杀伤中的作用。我们将测试假设当含有NKLAM的NK来源的外切体进入肿瘤细胞时，NKLAM与关键底物，导致细胞死亡。我们将测试NKLAM WT，NKLAM KO和NKLAM KO的外体的能力 NKLAM连接缺陷NK细胞在多发性骨髓瘤小鼠模型中体内杀伤肿瘤细胞频率增加的，尤其是退伍军人。目的2：研究NKLAM在体内外对巨噬细胞抗菌功能的影响。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，这两种病原体是导致疾病。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、部位和类型。我们将研究在肿瘤生长和抗细菌杀灭中很重要的底物。这将提供对角色的更好理解 NKLAM在抗肿瘤和抗细菌免疫中的作用并揭示对治疗敏感的关键调控步骤对折磨我们退伍军人人口的疾病进行干预。