Pasteurella multocida toxin: Structure and Activity

描述（由申请方提供）：多杀性巴氏杆菌毒素（PMT）是一种主要毒力因子，与动物进行性萎缩性鼻炎、呼吸道疾病以及人类因咬伤或暴露于受感染动物而导致的皮肤坏死、呼吸道疾病和菌血症相关。PMT是一种1285个氨基酸的蛋白质，可以作用于多种细胞类型。它通过受体介导的内吞作用进入哺乳动物细胞，并激活细胞内信号转导事件，包括磷脂水解、钙动员、蛋白磷酸化、DNA合成和细胞骨架重排，从而引起细胞增殖。我们已经证明，PMT介导的刺激磷脂酶C活性发生通过短暂的，但不可逆的PMT对Gq蛋白的作用。我们已经提出了一个模型PMT的行动。我们还使用PMT表征了一些Gq依赖性途径，并且我们的结果使我们假设PMT对不同细胞的多效性效应是由于Gq靶在不同细胞类型中发挥的不同作用。此外，我们确定PMT的N-末端对细胞内活性是重要的，并且N-和C-末端对于结合和进入哺乳动物细胞是重要的。我们的假设是，PMT进入介导的毒素蛋白上的多个结合决定簇，并通过多种受体。我们的长期目标是在分子和生物化学水平上了解PMT的结构和作用机制，以促进未来对多杀性巴氏杆菌病的治疗干预，并增加我们对涉及类似机制的潜在细菌毒素相关威胁的准备，以及增加我们对Gq依赖性信号传导中涉及的分子信号传导事件的理解。 为了实现我们的目标，我们提出以下目标： (1)通过确定PMT对Gq蛋白的作用是由共价修饰还是由直接或间接的蛋白质相互作用引起，阐明PMT作用于Gq蛋白的分子机制。 (2)确定PMT对Gq偶联信号转导影响的生化基础，（I）通过确定PMT对Gq活性的影响，（II）通过确定PMT对下游Gq信号传导途径的影响。 (3)确定PMT的功能结构域，该结构域负责结合真核细胞受体并将胞内活性结构域转运到胞质溶胶中。 (4)表征细胞受体并阐明PMT进入细胞所利用的内化途径。

DESCRIPTION (provided by applicant): Pasteurella multocida toxin (PMT) is a major virulence factor associated with progressive atrophic rhinitis, respiratory disease in animals, and dermonecrosis, respiratory disease, and bacteremia in humans resulting from bite wounds or exposure to infected animals. PMT is a 1285 amino acid protein that can act on multiple cell types. It enters mammalian cells via receptor-mediated endocytosis and activates intracellular signal transduction events, including phospholipid hydrolysis, calcium mobilization, protein phosphorylation, DNA synthesis, and cytoskeletal rearrangements, which cause cell proliferation. We have demonstrated that the PMT-mediated stimulation of phospholipase C activity occurs through transient, but irreversible PMT action on the Gq protein. We have proposed a model for PMT action. We also characterized a number of the Gq-dependent pathways using PMT, and our results have led us to hypothesize that the pleiotropic effects of PMT on different cells is due to the diverse roles that the Gq target plays in the different cell types. In addition, we determined that the N-terminus of PMT is important for intracellular activity and that both N- and C-termini are important for binding and entry into mammalian cells. Our hypothesis is that PMT entry is mediated through multiple binding determinants on the toxin protein and through multiple receptors. Our long-range goals are to understand the structure and mechanism of action of PMT at the molecular and biochemical level, to facilitate future therapeutic intervention in P. multocida disease and to increase our preparedness against potential bacterial toxin-related threats involving similar mechanisms, as well as to increase our understanding of the molecular signaling events involved in Gq-dependent signaling. To achieve our goals, we propose the following Aims: (1) To elucidate the molecular mechanism by which PMT acts on the Gq-protein, by determining whether the effect of PMT on Gq-protein is caused by covalent modification or by direct or indirect protein interaction. (2) To determine the biochemical basis for the effect of PMT on Gq-coupled signal transduction, (I) by determining the effect of PMT on Gq activity and (II) by determining the effect of PMT on downstream Gq-signaling pathways. (3) To define the functional domain(s) of PMT responsible for binding eukaryotic cell receptors and translocating the intracellular activity domain into the cytosol. (4) To characterize the cellular receptor(s) and to elucidate the internalization pathway(s) utilized by PMT to gain entry into cells.