Pasteurella multocida toxin: Structure and Activity

描述（由申请人提供）：多杀性巴氏杆菌毒素（PMT）是一种主要的毒力因子，与进行性萎缩性鼻炎、动物呼吸道疾病以及人类因咬伤或接触受感染动物而导致的皮肤坏死、呼吸道疾病和菌血症有关。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，激活细胞内信号转导事件，包括磷脂水解、钙动员、蛋白质磷酸化、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介导的磷脂酶C活性的刺激发生在瞬时的，但不可逆的PMT作用于Gq蛋白。我们提出了一个PMT作用的模型。我们还利用PMT描述了一些Gq依赖性通路，我们的结果使我们假设PMT对不同细胞的多效性作用是由于Gq靶点在不同细胞类型中发挥的不同作用。此外，我们确定PMT的N端对细胞内活性很重要，N端和c端对结合和进入哺乳动物细胞都很重要。我们的假设是PMT的进入是通过毒素蛋白上的多个结合决定因素和多个受体介导的。我们的长期目标是在分子和生化水平上了解PMT的结构和作用机制，促进未来对多杀性单胞杆菌疾病的治疗干预，增强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依赖性信号传导所涉及的分子信号事件的理解。

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.