组织金属蛋白酶抑制因子-2（TIMP-2）是一种多功能蛋白质，除了抑制金属蛋白酶（MMPs）的酶切活性，还具有细胞因子样的独特功能——介导酶原ProMMP-2激活，从而调控细胞增殖和凋亡等过程，但其作用机制存在较大争议，是目前领域关注的核心问题。近期研究表明，TIMP-2的磷酸化修饰能极大地影响其对MMP-2的调控功能，由于修饰蛋白质来源受到限制，其详细调控机制及生物学意义尚不明确。本项目拟发展蛋白质半化学合成方法，高效获得精确磷酸化修饰的TIMP-2，利用酶动力学及X-射线晶体学等研究其对MMP-2的抑制，利用等温量热法、明胶酶谱法及光交联技术等研究其对酶原激活通路的介导，以期揭示磷酸化修饰在TIMP-2—MMP-2相互作用中的调控机制。本项目的顺利实施将有助于进一步发展修饰蛋白质的制备方法，深化对磷酸化修饰调控机制的认识，并为靶向化学干预奠定基础。

Tissue inhibitors of metalloproteinase-2 (TIMP-2) is a multi-functional protein, besides the endogenous inhibitor of MMP-2, it also has cytokine-like activity - mediating the activation of the zymogen ProMMP-2. As such, TIMP-2 is essential on the regulation of cell growth and apoptosis, the detailed mechanism, however, is controversial, which is a key research area. Recent study shows that phosphorylation of TIMP-2 influences its interaction with MMP-2 dramatically, the detailed mechanism is, however, unknown, mostly because of the lack of modified proteins. The current proposal will develop a semisynthtic approach to obtain precisely phosphorylated TIMP-2. We will investigate its interaction with MMP-2 using enzyme kinetics and X-ray crystallography, and its regulation on the process of ProMMP-2 activation using iTC, gelatin zymography and photo-crosslinking, with the aim of elucidating the influence of phosphorylation on the interaction mechanism. Through this project, we hope to provide an improved methodology for the synthesis of protein modification, to deepen our understanding on the regulation mechanism of phosphorylation in general, and to set the stage for targeted chemical intervention.