Pasteurella multocida toxin: Structure and Activity

多杀性巴氏杆菌毒素：结构和活性

• 批准号: 7024490
• 负责人: Brenda A. Wilson
• 金额: $ 33.23万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7024490
• 关键词: G protein; Pasteurella multocida; SDS polyacrylamide gel electrophoresis; bacterial proteins; bacterial toxicology; bacterial toxins; biological signal transduction; covalent bond; gene deletion mutation; polymerase chain reaction; protein sequence; protein structure function; receptor binding; receptor coupling; recombinant proteins; transfection; western blottings

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.

描述（由申请人提供）：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、呼吸道疾病以及因咬伤或接触受感染动物而导致的人类皮肤坏死、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、蛋白质磷酸化、DNA合成和细胞骨架重排，从而导致细胞增殖。我们已经证明，PMT 介导的磷脂酶 C 活性刺激是通过短暂但不可逆的 PMT 对 Gq 蛋白的作用而发生的。我们提出了 PMT 行动模型。我们还使用 PMT 表征了许多 Gq 依赖性途径，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 靶标在不同细胞类型中发挥的不同作用。此外，我们确定 PMT 的 N 末端对于细胞内活性很重要，并且 N 末端和 C 末端对于结合和进入哺乳动物细胞都很重要。我们的假设是 PMT 进入是通过毒素蛋白上的多个结合决定簇和多个受体介导的。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，以促进未来对多杀性巴氏杆菌疾病的治疗干预，并提高我们对涉及类似机制的潜在细菌毒素相关威胁的准备程度，以及增加我们对 Gq 依赖性信号传导所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蛋白的作用是通过共价修饰还是通过直接或间接的蛋白质相互作用引起的，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，以及 (II) 通过确定 PMT 对下游 Gq 信号传导途径的影响。 (3) 定义负责结合真核细胞受体并将细胞内活性结构域易位至胞质溶胶的PMT的功能结构域。 (4) 表征细胞受体并阐明 PMT 进入细胞所利用的内化途径。