Proteasome-catalyzed protein degradation mediates and regulates critical aspects of many cellular functions and is an important element of proteostasis in health and disease. Proteasome function is determined in part by the types of proteasome holoenzymes formed between the 20S core particle that catalyzes peptide bond hydrolysis and any of multiple regulatory proteins to which it binds. One of these regulators, PI31, was previously identified as an in vitro 20S proteasome inhibitor, but neither the molecular mechanism nor the possible physiologic significance of PI31-mediated proteasome inhibition has been clear. Here we report a high-resolution cryo-EM structure of the mammalian 20S proteasome in complex with PI31. The structure shows that two copies of the intrinsically disordered carboxyl terminus of PI31 are present in the central cavity of the closed-gate conformation of the proteasome and interact with proteasome catalytic sites in a manner that blocks proteolysis of substrates but resists their own degradation. The two inhibitory polypeptide chains appear to originate from PI31 monomers that enter the catalytic chamber from opposite ends of the 20S cylinder. We present evidence that PI31 can inhibit proteasome activity in mammalian cells and may serve regulatory functions for the control of cellular proteostasis.

蛋白酶体催化的蛋白质降解介导和调节许多细胞功能的关键方面，是健康和疾病中蛋白质稳态的重要因素。蛋白酶体功能部分取决于催化肽键水解的 20S 核心颗粒与其结合的多种调节蛋白中的任何一种之间形成的蛋白酶体全酶的类型。其中一个调节因子 PI31 先前被鉴定为体外20S 蛋白酶体抑制剂，但 PI31 介导的蛋白酶体抑制的分子机制和可能的生理意义尚不清楚。在这里，我们报告了哺乳动物 20S 蛋白酶体与 PI31 复合物的高分辨率冷冻电镜结构。该结构表明，本质上无序的 PI31 羧基末端的两个副本存在于蛋白酶体闭门构象的中央空腔中，并以阻止底物蛋白水解但抵抗其自身降解的方式与蛋白酶体催化位点相互作用。两条抑制性多肽链似乎源自从 20S 圆柱体相对两端进入催化室的 PI31 单体。我们提供的证据表明 PI31 可以抑制哺乳动物细胞中的蛋白酶体活性，并可能为控制细胞蛋白质稳态发挥调节功能。