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Elucidating Perforin-2 mediated killing mechanisms against pathogenic bacteria

阐明 Perforin-2 介导的病原菌杀伤机制

基本信息

批准号：
9010907
负责人：
RYAN MICHAEL MCCORMACK
金额：
$ 4.86万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9010907
关键词：
5 year old Adaptor Signaling Protein Amino Acids Anti-Bacterial Agents Bacteria Bacterial Infections Biochemistry Biological Assay Cause of Death Cell Line Cells Cessation of life Characteristics Child Communicable Diseases Complement Membrane Attack Complex Complementary DNA Complex Computer Simulation Coupled Cullin Proteins Cytoplasmic Tail Disease Endoplasmic Reticulum Evolution Genes Gentamicins Goals Golgi Apparatus Health Hematopoietic Hybrids Hydrolase Immune Immune system In Vitro Individual Infection Infectious Agent Intervention Knowledge Lymphocyte Lytic Mammals Mediating Mediator of activation protein Membrane Messenger RNA Molecular Morbidity - disease rate Mycobacterium avium Names Natural Killer Cells Nitric Oxide Pathway interactions Phosphorylation Phosphorylation Site Physiology Post-Translational Modification Site Post-Translational Protein Processing Process Property Proteins Reactive Oxygen Species Recruitment Activity Regulation Salmonella typhimurium Site Site-Directed Mutagenesis Small Interfering RNA Staphylococcus aureus Tertiary Protein Structure Testing Tissues Transmission Electron Microscopy Tyrosine Ubiquitination VDAC1 gene Virus Work World Health Organization Yeasts antimicrobial bacterial genetics base cancer cell cytotoxic extracellular frontier insight killings knock-down loss of function macrophage mortality multicatalytic endopeptidase complex mutant new therapeutic target novel therapeutic intervention pathogen pathogenic bacteria perforin perforin 1 perforin 2 plant fungi polymerization prevent src-Family Kinases stem success trans-Golgi Network yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): The World Health Organization estimates that over seven billion individuals succumb to infectious disease annually and that they are the number one cause of death in the young. Fundamentally, each of these cases stems from an inability of the individual's innate immune system to eliminate infection coupled with the infectious agent capitalizing on the lag period of several days required for the adaptive immune system to prime against the specific pathogen. Despite dramatic advances in the understanding of the innate and adaptive immune systems, the host-pathogen interface is a relatively unexplored frontier. To date, some of the best studied bacterial killing mechanisms at this interface include reactive oxygen species (ROS), nitric oxide (NO), and hydrolases. Notwithstanding this body of knowledge, there is relatively little knowledge of exactly how these components interact with one another to contribute to bacterial killing. A potential explanation for how these effector proteins interact at the host-pathogen interface was recently discovered, with the characterization of macrophage-expressed Mpeg1. It was revealed that this mRNA encodes a protein domain, MACPF, which predicts a pore-forming, perforin-like molecule. Two already described pore-forming molecules of immune defense are the membrane attack complex of complement (MAC) that kills extracellular bacteria, and Perforin-1 of cytotoxic lymphocytes that kills virus-infected and cancer cells. Preliminary studies with Mpeg1 demonstrated that Mpeg-1 cDNA encodes a membrane- associated pore-forming protein, designated Perforin-2 (P2). These studies further characterized P2 as being constitutively expressed in all hematopoietic cells, and inducible in all cells. Furthermore, P2 seems to be a vital component as knockdown of P2 with siRNA blocks intracellular killing of pathogenic bacteria. My goal is to determine the mechanisms of P2 activation, culminating in polymerization and intracellular bacterial killing. I hypothesize that the cytoplasmic domain of P2 is the master regulator of activation and polymerization, that the cytoplasmic tail contains several key domains that undergo post-translational modification to become activated, and that the cytoplasmic tail will interact with several key adaptor proteins to regulate polymerization as well as localization within cells. The objectives of this application are to (i) identify the key cytoplasmic domains and amino acid residues necessary for polymerization to occur utilizing a functional intracellular bacterial killig assay; and (ii) characterize the P2-cytoplasmic domain interacting proteins that promote polymerization of P2 utilizing loss of function studies and transmission electron microscopy. A compelling aspect of this work is the potential to describe the host-pathogen interface with the addition of P2 as a common mediator in the previously described bacterial killing paradigm. By unraveling the molecular mechanisms triggering P2 polymerization and activation of bacterial attack, the proposed studies may provide new targets for drug interventions in the treatment of infections.

描述（由申请人提供）：世界卫生组织估计，每年有超过70亿人死于传染病，并且它们是年轻人死亡的头号原因。从根本上说，这些病例中的每一个都源于个体的先天免疫系统无法消除感染，加上感染因子利用适应性免疫系统针对特定病原体引发所需的几天的滞后期。尽管在先天性和适应性免疫系统的理解方面取得了巨大进展，但宿主-病原体界面是一个相对未探索的前沿。迄今为止，在该界面处研究得最好的一些细菌杀灭机制包括活性氧（ROS）、一氧化氮（NO）和水解酶。尽管有这样的知识体系，但关于这些组分如何相互作用以促进细菌杀灭的确切知识相对较少。最近发现了一个潜在的解释，这些效应蛋白如何在宿主-病原体界面相互作用，与巨噬细胞表达的Mpeg 1的表征。结果表明，这种mRNA编码一个蛋白质结构域，MACPF，它预测一个孔形成，穿孔素样分子。两种已经描述的免疫防御的孔形成分子是补体的膜攻击复合物（MAC），其杀死细胞外细菌，以及细胞毒性淋巴细胞的穿孔蛋白-1，其杀死病毒感染的细胞和癌细胞。对Mpeg 1的初步研究表明，Mpeg-1 cDNA编码一种膜相关的孔形成蛋白，命名为穿孔蛋白-2（P2）。这些研究进一步将P2表征为在所有造血细胞中组成型表达，并且在所有细胞中可诱导。此外，P2似乎是一个重要的组成部分，因为用siRNA敲低P2会阻断病原菌的细胞内杀伤。我的目标是确定P2激活的机制，最终在聚合和细胞内的细菌杀死。我假设P2的胞质结构域是激活和聚合的主调节器，胞质尾包含几个关键结构域，这些结构域经过翻译后修饰而被激活，并且胞质尾将与几个关键衔接蛋白相互作用以调节聚合以及细胞内的定位。本申请的目的是（i）利用功能性细胞内细菌杀灭试验鉴定聚合发生所需的关键胞质结构域和氨基酸残基;以及（ii）利用功能丧失研究和透射电子显微镜表征促进P2聚合的P2-胞质结构域相互作用蛋白。这项工作的一个令人信服的方面是有可能描述的主机-病原体接口与添加P2作为一个共同的调解人在先前描述的细菌杀灭范例。通过揭示触发P2聚合和激活细菌攻击的分子机制，拟议的研究可能为药物干预治疗感染提供新的靶点。