Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, VIC, Australia.
Cell Death Differ. 2018 Aug;25(8):1517-1529. doi: 10.1038/s41418-018-0057-z. Epub 2018 Feb 7.
The pore forming, Ca-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin's cytotoxic function. These results revealed an unexpected role for the Ca-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.
穿孔素是一种依赖于 Ca 的形成孔的蛋白，对于细胞毒性淋巴细胞的功能至关重要，而细胞毒性淋巴细胞是免疫防御病原体和癌症的第一线。穿孔素是一种糖蛋白，在释放到免疫突触前储存在分泌颗粒中。先天性穿孔素缺乏会导致致命的免疫失调，并与各种血液恶性肿瘤有关。至少 50%的穿孔素病理性错义突变导致蛋白质错误折叠和在内质网中滞留。然而，穿孔素的蛋白质稳态调节仍未被探索。通过使用各种评估蛋白质稳定性和获得复杂糖基化的生化测定法，我们证明了 Ca 与 C2 结构域的结合稳定了穿孔素，并调节了它从内质网到分泌颗粒的输出。由于穿孔素是一种热不稳定的蛋白质，我们假设通过改变其 C2 结构域，可能提高蛋白质稳定性。基于穿孔素 C2 结构域的 X 射线晶体结构，我们设计了一个在 Ca 结合环中的突变（T431D）。尽管突变的穿孔素从内质网的运输仍然不变，但突变的穿孔素显示出明显增强的热稳定性和裂解功能。此外，通过将 T431D 突变引入导致蛋白质错误折叠的致病性 A90V 穿孔素中，我们纠正了 A90V 的折叠缺陷，并完全恢复了穿孔素的细胞毒性功能。这些结果揭示了 Ca 依赖性 C2 结构域在维持穿孔素蛋白质稳态中的意外作用，并表明通过稳定 C2 结构域设计具有超生理细胞毒性功能的穿孔素是可能的。
