Chinese Academy of Sciences Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
J Immunol. 2013 Feb 1;190(3):1319-30. doi: 10.4049/jimmunol.1202542. Epub 2012 Dec 26.
The granzyme/perforin pathway is a major mechanism for cytotoxic lymphocytes to eliminate virus-infected and tumor cells. The balance between activation and inhibition of the proteolytic cascade must be tightly controlled to avoid self damage. Granzyme H (GzmH) is constitutively expressed in NK cells and induces target cell death; however, how GzmH activity is regulated remains elusive. We reported earlier the crystal structures of inactive D102N-GzmH alone and in complex with its synthetic substrate and inhibitor, as well as defined the mechanisms of substrate recognition and enzymatic activation. In this study, we identified SERPINB1 as a potent intracellular inhibitor for GzmH. Upon cleavage of the reactive center loop at Phe(343), SERPINB1 forms an SDS-stable covalent complex with GzmH. SERPINB1 overexpression suppresses GzmH- or LAK cell-mediated cytotoxicity. We determined the crystal structures of active GzmH and SERPINB1 (LM-DD mutant) in the native conformation to 3.0- and 2.9-Å resolution, respectively. Molecular modeling reveals the possible conformational changes in GzmH for the suicide inhibition. Our findings provide new insights into the inhibitory mechanism of SERPINB1 against human GzmH.
颗粒酶/穿孔素途径是细胞毒性淋巴细胞消除病毒感染和肿瘤细胞的主要机制。为了避免自身损伤，蛋白酶级联反应的激活和抑制之间的平衡必须得到严格控制。颗粒酶 H（GzmH）在 NK 细胞中组成性表达并诱导靶细胞死亡；然而，GzmH 活性如何调节仍不清楚。我们之前报道了单独的无活性 D102N-GzmH 及其与合成底物和抑制剂的复合物的晶体结构，并确定了底物识别和酶激活的机制。在这项研究中，我们鉴定了丝氨酸蛋白酶抑制剂 B1（SERPINB1）作为 GzmH 的一种有效的细胞内抑制剂。在 Phe(343)处的反应中心环被切割后，SERPINB1 与 GzmH 形成 SDS 稳定的共价复合物。SERPINB1 的过表达抑制了 GzmH 或 LAK 细胞介导的细胞毒性。我们以 3.0-和 2.9-Å 的分辨率确定了天然构象下活性 GzmH 和 SERPINB1（LM-DD 突变体）的晶体结构。分子建模揭示了自杀抑制中 GzmH 可能发生的构象变化。我们的研究结果为 SERPINB1 对人 GzmH 的抑制机制提供了新的见解。
