Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8W 3P6.
Biochem Soc Trans. 2013 Jun;41(3):761-8. doi: 10.1042/BST20130013.
Peptidylprolyl isomerases have been implicated in chromatin regulation through their association with histones, chromatin-modifying enzymes and DNA-binding transcription factors. As with other post-translational modifications to proteins, a mechanistic understanding of the regulation of biological processes is fostered by loss-of-function studies both in vitro and in vivo. For peptidylprolyl isomerases, this can be accomplished with small-molecule inhibitors with high affinity for the isomerase active site or by mutation of amino acid residues that contribute to catalysis. In the present article, we review caveats to each of these approaches, and place emphasis on the thorough characterization of loss-of-function mutations in FKBPs (FK506-binding proteins). Using a case study of mutagenesis of the nuclear FKBP25 peptidylprolyl isomerase enzyme, we demonstrate that certain mutations generate a loss-of-function phenotype because they induce a complete loss of the FKBP domain fold, whereas other mutations are 'surgical' in that they ablate catalytic isomerase activity, while maintaining domain structure. Peptidylprolyl isomerases are thought to have both catalytic and non-catalytic functions, but differentiating between these mechanisms has proved to be challenging. The domain-destabilizing and surgical mutants described will facilitate the characterization of these two reported functions of peptidylprolyl isomerases.
肽基脯氨酰顺反异构酶通过与组蛋白、染色质修饰酶和 DNA 结合转录因子的相互作用而参与染色质调控。与蛋白质的其他翻译后修饰一样，通过体外和体内的功能丧失研究，可以深入了解生物过程的调控机制。对于肽基脯氨酰顺反异构酶，可以使用与异构酶活性位点具有高亲和力的小分子抑制剂，或通过突变参与催化的氨基酸残基来实现。在本文中，我们回顾了这两种方法的注意事项，并重点强调了 FKBP（FK506 结合蛋白）中功能丧失突变的彻底表征。通过对核 FKBP25 肽基脯氨酰顺反异构酶的突变进行案例研究，我们证明某些突变会产生功能丧失表型，因为它们导致 FKBP 结构域折叠完全丧失，而其他突变则是“外科手术式”的，因为它们消除了催化异构酶活性，同时保持了结构域结构。肽基脯氨酰顺反异构酶被认为具有催化和非催化功能，但区分这两种机制一直具有挑战性。所描述的结构域不稳定和外科手术突变将有助于表征这两种报道的肽基脯氨酰顺反异构酶功能。
