Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
Methods Mol Biol. 2024;2747:279-294. doi: 10.1007/978-1-0716-3589-6_21.
Protease inhibitors of the alpha-macroglobulin family (αM) have a unique mechanism that allows them to trap proteases that is dependent not on the protease's class, but rather on its cleavage specificity. Proteases trigger a conformational change in the αM protein by cleaving within a "bait region," resulting in the sequestering of the protease inside the αM molecule. This nonspecific inhibitory mechanism appears to have arisen early in the αM family, and the broad protease-trapping capacity that it allows may play a role in pathogen defense.Human α-macroglobulin (A2M) is a tetrameric αM whose bait region is permissive to cleavage by most proteases, making it a broad-spectrum protease inhibitor. Recent work has demonstrated that the inhibitory capacity of A2M derives directly from its bait region sequence: modifying the bait region sequence to introduce or remove protease cleavage sites will modify A2M's inhibition of the relevant proteases accordingly. Thus, changing the amino acid sequence of the bait region presents an effective avenue for protein engineering of new protease inhibitors if the substrate specificity of the target protease is known. The design of new A2M-based protease inhibitors with tailored inhibitory capacities has potential applications in basic research and the clinic. In this chapter, we describe the general approach and considerations for the bait region engineering of A2M.
α-巨球蛋白家族的蛋白酶抑制剂（αM）具有独特的机制，使其能够捕获蛋白酶，这种机制不依赖于蛋白酶的类别，而是依赖于其切割特异性。蛋白酶通过在“诱饵区域”内切割触发αM 蛋白的构象变化，导致蛋白酶被隔离在αM 分子内。这种非特异性抑制机制似乎在αM 家族早期就出现了，它所允许的广泛蛋白酶捕获能力可能在病原体防御中发挥作用。人α-巨球蛋白（A2M）是一种四聚体αM，其诱饵区域允许大多数蛋白酶切割，使其成为一种广谱蛋白酶抑制剂。最近的研究表明，A2M 的抑制能力直接来自其诱饵区域序列：修饰诱饵区域序列以引入或去除蛋白酶切割位点将相应地改变 A2M 对相关蛋白酶的抑制作用。因此，如果已知靶蛋白酶的底物特异性，改变诱饵区域的氨基酸序列为新型蛋白酶抑制剂的蛋白质工程提供了一种有效途径。具有定制抑制能力的新型基于 A2M 的蛋白酶抑制剂的设计在基础研究和临床应用中具有潜在的应用。在本章中，我们描述了 A2M 的诱饵区域工程的一般方法和注意事项。
