Department of Chemistry & Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854.
Institute for Quantitative Biomedicine, Rutgers, The State University of New Jersey, Piscataway, NJ 08854.
Proc Natl Acad Sci U S A. 2022 Apr 12;119(15):e2116097119. doi: 10.1073/pnas.2116097119. Epub 2022 Apr 4.
Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.
将设计的蛋白质的活性限制在特定的微环境中，将具有广泛的应用，例如通过酶实现细胞类型特异性的治疗作用，同时避免脱靶效应。虽然许多天然酶是作为无活性的酶原合成的，这些酶原可以通过蛋白水解作用激活，但重新设计任何选定的酶使其具有类似的刺激响应性一直具有挑战性。在这里，我们开发了一种大规模并行的计算设计、筛选和基于下一代测序的酶原设计方法。对于一个模型系统，我们采用羧肽酶 G2（CPG2），这是一种临床批准的酶，在癌症治疗和控制药物毒性方面都有应用。对最有效设计的变体进行的详细动力学表征表明，与未修饰的蛋白质相比，它们的抑制率约为 80%，并且在用特异性蛋白酶孵育后，其活性完全恢复。基于设计模型在原结构域和催化结构域之间引入二硫键，将抑制程度提高到 98%，但通过蛋白水解恢复活性的程度降低。在细胞培养中评估时，与完全激活的酶相比，所选的含二硫键的酶原的活性显著降低。结构和热力学表征提供了对原结构域结合和抑制机制的详细见解。所描述的方法是通用的，可以设计具有精确空间调节的各种前蛋白。
