猪细胞色素P450氧化酶3A46的催化及调控分子机制

Studying the function, structure, catalytic mechanism and regulational mechanism of a porcine CYP isoform, which is involved in the metabolism of important endogenous or exogenous compounds, is important not only for veterinary pharmacology and toxicology but also for new drug development and human health. Our previous research results indicated that porcine CYP3A46 is a critical metabolic enzyme in pig liver, which can metabolize nifedipine, T-2 toxin and HT-2 toxin. To reveal the catalytic and regulational mechanisms of CYP3A46, we will mutate and identify the key amino acid residues contributing to its specific recognition, binding, catalytic activity and allosteric effect towards nifedipne, T-2 toxin and HT-2 toxin, and then propose hypotheses to uncover its catalytic mechanism. Additionally, we will study its regulational mechanism under the treatments of rifampicin, T-2 toxin and HT-2 toxin, through luciferase reporter system, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitaion (Chip). Our project will clarify the catalytic and regulational mechanisms of CYP3A46 and fill gaps at related research field. Furthermore, related research results will probably provide new ideas to reduce the toxicity of T-2 toxin towards livestock and humans.

研究参与重要内源或外源化合物代谢的猪细胞色素P450氧化酶（Cytochrome P450, CYP）的功能、结构、催化机制和调控机理，不但对兽医药理与毒理学研究非常重要，对于新药研发、人类健康也同等重要。我们的前期研究结果显示CYP3A46是猪肝脏中一个重要的代谢酶，它能代谢尼福地平、T-2和HT-2毒素。因此，本项目拟以猪CYP3A46为研究对象，对其可能的关键氨基酸残基进行定点突变，确定影响其对尼福地平、T-2和HT-2毒素的特异性识别、结合、催化活性及别构作用的关键位点，并构建模型假说揭示其催化分子机制；利用荧光素酶报告基因系统、凝胶阻滞、染色质免疫沉淀等技术研究利福平、T-2和HT-2毒素调控CYP3A46表达的分子机制及其差异。本项目的研究结果将最终阐明CYP3A46的催化及调控分子机制，填补相关研究的空白，并可望为减少T-2毒素对畜禽乃至人类的危害提供新的思路。

T-2毒素是由镰刀菌产生的二级代谢产物，具有天然产生、性质稳定和毒性强等特点，广泛污染动物饲料、饲料原料及人类食品。它能直接使畜禽或人中毒甚至死亡，其在畜禽中代谢生成的毒性残留物还可能通过肉、蛋、奶等食物间接对人类健康造成危害。因此，研究T-2毒素在动物体内的代谢转化过程，明确催化关键代谢步骤的酶，阐明其催化机制和调控机理，对于减少T-2毒素对畜禽甚至人类的危害有着重要的理论指导意义。我们的前期研究结果显示CYP3A46是猪肝脏中一个重要的代谢酶，它能代谢尼福地平、T-2和HT-2毒素。然而，CYP3A46的催化分子机制及调控机理仍不清楚。本研究通过体外重组表达、点突变、荧光素酶报告基因系统、凝胶阻滞等多种实验方法和手段，系统研究CYP3A46的催化分子机制及调控机理。通过三年的研究，我们取得了如下成果：揭示Arg105、Ala305和Phe372这三个位点对于CYP3A46代谢T-2毒素至关重要；阐明Sp1和HNF1α分别与CYP3A46近端启动子上的GC-box元件和远端启动子上的HNF1元件结合调控其本底表达，而HNF1α和PXR通过与CYP3A46远端启动子上的HNF1和近端DR4元件结合介导利福平对其的诱导表达。相关研究成果为有效减少T-2毒素对畜禽乃至人类的危害提供了靶标和理论依据。