Dolinski K, Scholz C, Muir R S, Rospert S, Schmid F X, Cardenas M E, Heitman J
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Mol Biol Cell. 1997 Nov;8(11):2267-80. doi: 10.1091/mbc.8.11.2267.
Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 "active-site" mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.
亲环蛋白和FK506结合蛋白（FKBP）可加速顺反肽基脯氨酰异构化，并与免疫抑制剂环孢菌素A和FK506结合并介导其作用。然而，这些蛋白质的正常细胞功能尚不清楚。我们通过引入先前报道可使这些酶失活的突变，改变了酿酒酵母中FKBP12和亲环蛋白的活性位点。令人惊讶的是，这些突变酶中的大多数在体内具有生物活性。与先前的报道一致，在标准肽底物-胰凝乳蛋白酶偶联的体外测定中，所有突变酶几乎没有或没有可检测到的脯氨酰异构酶活性。然而，在该测定的一种变体中，省略了蛋白酶，突变酶表现出相当水平的脯氨酰异构酶活性（野生型的5-20%），这表明这些突变使酶对蛋白酶消化敏感，并且经典的脯氨酰异构酶活性体外测定可能会产生误导。此外，突变酶对两种蛋白质底物二氢叶酸还原酶和核糖核酸酶T1表现出接近野生型的活性，它们的折叠可被脯氨酰异构酶加速。因此，先前鉴定的许多亲环蛋白和FKBP12“活性位点”突变体在很大程度上是有活性的，但对蛋白酶敏感，这与我们的发现一致，即这些突变体在体内表现出野生型功能。一种线粒体亲环蛋白突变体（R73A）以及野生型人FKBP12酶在体外催化蛋白质折叠，但在酵母体内缺乏生物活性。我们的研究结果提供了证据，表明脯氨酰异构酶活性和其他结构特征与FKBP和亲环蛋白的体内功能相关，并建议在使用这些活性位点突变研究FKBP和亲环蛋白功能时要谨慎。
