猪肉成熟过程中的蛋白质亚硝基化及作用机理

Fresh meat quality including meat tenderness and water holding capacity are important traits for consumer acceptance and industry profitability in China. Exploring the basic mechanism that regulates the development of meat quality attributes is critical for delivering consistently high quality fresh meat to consumers. It is well known that early postmortem biochemical and biophysical processes especially protein degradation could contribute to the variation of water-holding capacity and tenderness of pork. Many proteins those are involved in meat quality have been shown to be susceptible substrates of the cysteine protease calpain. Postmortem muscles have decreased ability to maintain their reducing environment resulting in increased accumulations of reactive oxygen species and reactive nitrogen species. These compounds may have significant effects on meat quality through oxidation and nitrosylation or nitration reactions with muscle proteins and lipids. Protein functions can be modified through nitrosylation by nitric oxide and other reactions with some secondary products produced from nitric oxide. These reactions may be involved in muscle metabolism and meat quality mainly through regulating the activation of calpains. However, the presence and the effects of endogenous reactive nitrogen species and their reaction products in postmortem pork have received little attention. The overall hypothesis of this study is that biochemical factors especially protein nitrosylation that influence proteolysis during the antemortem or the early postmortem period can contribute to the variations in fresh meat quality. The overall objective of this study proposed in this application is to determine how nitric oxide reaction products in early postmortem muscle govern protein functions including calpain system and the development of fresh pork quality.

猪肉的嫩度和持水力是影响其受消费者接受程度和猪肉工业经济效益的重要指标。深入探讨猪肉成熟过程中影响其品质形成的基础机制对提高生鲜猪肉的品质具有重要的理论和实践意义。钙蛋白酶引起的蛋白质降解对猪肉的持水力具有重要的调控作用。而钙蛋白酶活性的调控受很多因素的影响其中包括蛋白质氧化和亚硝基化。活性氧类和活性氮类物质会在宰后猪肉中聚集，这些活性物质能够修饰猪肉中的蛋白质特别是钙蛋白酶系统而调控蛋白质的结构和功能。但是，国内外关于蛋白质亚硝基化与猪肉品质形成的关系的相关研究非常少。本研究总的假设是蛋白质亚硝基化通过对蛋白质修饰，影响猪肉成熟过程中品质的形成。总的研究目标是探讨活性氮类物质在猪肉成熟过程中，对钙蛋白酶系统特别是钙蛋白酶1活性和结构的影响，以及研究活性氮类物质对其它肌肉中具有重要作用的蛋白质结构和功能的调控，从而深入探讨猪肉在成熟过程中品质形成的内在机制。

摘要：一氧化氮（nitric oxide, NO）及其介导的蛋白质亚硝基化（protein nitrosylation, SNO）在生物体内起着信号转导，调控多种代谢反应等至关重要的作用。肌肉细胞中，一氧化氮和蛋白质亚硝基化可以调控肌肉收缩，能量代谢及钙离子稳态等，然而在宰后成熟过程中对肉品质的影响却鲜有报道，仅存的几篇研究其结果存在很大的不一致性。因此，本项目旨在研究蛋白质亚硝基化对肌肉成熟过程中关键蛋白质的修饰，从而阐释蛋白质亚硝基化在猪肉成熟过程中的作用机理。主要创新性结果如下：.1.背部最长肌、半膜肌、腰大肌在宰后第0和1天都具有一氧化氮合成酶（NOS）活性，半膜肌的活性和含量在宰后成熟3天内没有显著性变化，而背部最长肌和腰大肌的NOS活性和含量都在成熟第3天消失，不同纤维类型的NOS活性及表达量不同，NOS主要分布于肌细胞膜，但细胞质中也有分布，这为NOS催化产生NO与细胞质蛋白发生亚硝基化反应提供了可能。.2. 蛋白质亚硝基化修饰可调控钙蛋白酶1的自溶及活性，并且抑制其降解肌原纤维蛋白的能力，经鉴定活性亚基上的半胱氨酸位点49，351，384，592和小亚基上位点142的亚硝基化修饰可能是抑制其自溶和活性的原因。.3.通过使用生物素转化法、TMT多样混合标签技术和液质质联用技术，成功鉴定出381个亚硝基化位点，并对应345亚硝基化蛋白，亚硝基化蛋白分布广泛，主要行使酶活性功能参与肌细胞的代谢过程。其中维持细胞质钙离子平衡肌浆内质网钙离子ATP酶第471号位点鉴定为亚硝基化修饰。对所有亚硝基化蛋白的信号网络分析得出影响糖酵解过程最为显著，所有参与糖酵解过程的酶都被亚硝基化修饰。.本项目研究结果表明宰后猪肉成熟过程中多种蛋白质发生亚硝基化修饰，并且可以通过影响多种代谢途径参与成熟过程中肌细胞的生理生化反应，最终可能通过影响pH下降速率和终值、钙离子释放、肌原纤维蛋白降解程度等调控宰后猪肉成熟过程中品质的形成。