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

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,这是年轻人死亡的头号原因。从根本上说,这些病例都是由于个体先天免疫系统无法消除感染,再加上感染性病原体利用了适应性免疫系统对特定病原体启动所需的几天滞后期。尽管对先天免疫系统和适应性免疫系统的理解取得了巨大进展,但宿主-病原体界面是一个相对未开发的前沿领域。迄今为止,在这一界面上研究得最好的细菌杀伤机制包括活性氧(ROS)、一氧化氮(NO)和水解酶。尽管有这样的知识体系,但对于这些成分是如何相互作用来杀死细菌的,人们知之甚少。随着巨噬细胞表达Mpeg1的表征,最近发现了这些效应蛋白如何在宿主-病原体界面相互作用的潜在解释。结果显示,该mRNA编码一个蛋白质结构域MACPF,该结构域预测成孔的穿孔蛋白样分子。已经描述过的免疫防御的两种成孔分子是杀死细胞外细菌的补体膜攻击复合物(MAC)和杀死病毒感染细胞和癌细胞的细胞毒性淋巴细胞的穿孔素-1。对Mpeg1的初步研究表明,Mpeg-1 cDNA编码一种膜相关的成孔蛋白,称为穿孔素-2 (Perforin-2, P2)。这些研究进一步证实P2在所有造血细胞中均组成性表达,并可在所有细胞中诱导表达。此外,P2似乎是一个至关重要的成分,因为用siRNA敲低P2可以阻断致病菌的细胞内杀伤。我的目标是确定P2激活的机制,最终导致聚合和细胞内细菌杀死。我假设P2的胞质结构域是激活和聚合的主要调节器,胞质尾部包含几个经过翻译后修饰而被激活的关键结构域,并且胞质尾部将与几个关键的接头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确定聚合发生所需的关键细胞质结构域和氨基酸残基;(ii)利用功能损失研究和透射电子显微镜表征促进P2聚合的P2-细胞质结构域相互作用蛋白。这项工作的一个引人注目的方面是有可能描述宿主-病原体界面,添加P2作为先前描述的细菌杀死范式中的常见介质。通过揭示触发P2聚合和激活细菌攻击的分子机制,所提出的研究可能为药物干预治疗感染提供新的靶点。

项目成果

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RYAN MICHAEL MCCORMACK其他文献

RYAN MICHAEL MCCORMACK的其他文献

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{{ truncateString('RYAN MICHAEL MCCORMACK', 18)}}的其他基金

Elucidating Perforin-2 mediated killing mechanisms against pathogenic bacteria
阐明 Perforin-2 介导的病原菌杀伤机制
  • 批准号:
    8627960
  • 财政年份:
    2013
  • 资助金额:
    $ 4.72万
  • 项目类别:
Elucidating Perforin-2 mediated killing mechanisms against pathogenic bacteria
阐明 Perforin-2 介导的病原菌杀伤机制
  • 批准号:
    8811001
  • 财政年份:
    2013
  • 资助金额:
    $ 4.72万
  • 项目类别:
Elucidating Perforin-2 mediated killing mechanisms against pathogenic bacteria
阐明 Perforin-2 介导的病原菌杀伤机制
  • 批准号:
    9252355
  • 财政年份:
    2013
  • 资助金额:
    $ 4.72万
  • 项目类别:
Elucidating Perforin-2 mediated killing mechanisms against pathogenic bacteria
阐明 Perforin-2 介导的病原菌杀伤机制
  • 批准号:
    9010907
  • 财政年份:
    2013
  • 资助金额:
    $ 4.72万
  • 项目类别:
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