Linke Sarah, Hampton-Smith Rachel J, Peet Daniel J
School of Molecular and Biomedical Science and The ARC Special Research Centre for the Molecular Genetics of Development, University of Adelaide, Adelaide, Australia.
Methods Enzymol. 2007;435:61-85. doi: 10.1016/S0076-6879(07)35004-0.
The hypoxia-inducible transcription factors (HIFs) are essential mediators of the genomic response to oxygen deficiency (hypoxia) in multicellular organisms. The HIFs are regulated by four oxygen-sensitive hydroxylases-three prolyl hydroxylases and one asparaginyl hydroxylase. These hydroxylases are all members of the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily and convey changes in cellular oxygen concentration to the HIF-alpha (alpha) subunit, leading to potent accumulation and activity in hypoxia versus degradation and repression in normoxia. HIF-alpha asparaginyl hydroxylation is catalyzed by factor-inhibiting HIF-1 (FIH-1) and directly regulates the transcription activity of the HIF-alpha proteins. Recent work has demonstrated that, in addition to hydroxylating HIF-alpha, FIH-1 can also hydroxylate the ankyrin domains of a wide range of proteins. This paper presents in vitro and cell-based techniques for the preliminary characterization of ankyrin domain-containing proteins as FIH-1 substrates and interacting proteins. Strategies are presented for the expression and purification of FIH-1 from mammalian or bacterial cells. Similar to the HIF-alpha proteins, the ankyrin-containing substrates are examined as purified proteins expressed in bacteria and overexpressed in mammalian cells or in the form of synthetic peptides. Specific conditions for the efficient expression of ankyrin-containing proteins compared with the HIF-alpha substrates in Escherichia coli are detailed. Hydroxylation is rapidly inferred, utilizing the described in vitro CO(2) capture assay. Finally, substrate and non-substrate interactions are examined using in vitro affinity pull-down assays and mammalian cell-based co-immunoprecipitation assays. Together, these methods are rapid and well suited to the preliminary characterization of potential substrates of the therapeutically relevant oxygen-sensing enzyme FIH-1.
缺氧诱导转录因子（HIFs）是多细胞生物中基因组对缺氧反应的重要调节因子。HIFs受四种氧敏感羟化酶调控，即三种脯氨酰羟化酶和一种天冬酰胺酰羟化酶。这些羟化酶均为依赖2-氧代戊二酸（2OG）的双加氧酶超家族成员，可将细胞氧浓度变化传递给HIF-α亚基，导致其在缺氧时大量积累并激活，而在常氧时降解并受到抑制。HIF-α天冬酰胺酰羟化由抑制HIF-1的因子（FIH-1）催化，直接调节HIF-α蛋白的转录活性。最近的研究表明，除了使HIF-α羟化外，FIH-1还能使多种蛋白质的锚蛋白结构域羟化。本文介绍了基于体外和细胞的技术，用于初步鉴定含锚蛋白结构域的蛋白质作为FIH-1底物和相互作用蛋白。文中还介绍了从哺乳动物或细菌细胞中表达和纯化FIH-1的策略。与HIF-α蛋白类似，含锚蛋白的底物以在细菌中表达并在哺乳动物细胞中过表达的纯化蛋白形式或合成肽形式进行检测。详细阐述了与HIF-α底物相比，在大肠杆菌中高效表达含锚蛋白的蛋白质的特定条件。利用所述的体外CO₂捕获试验可快速推断羟化情况。最后，使用体外亲和下拉试验和基于哺乳动物细胞的共免疫沉淀试验检测底物与非底物的相互作用。总之，这些方法快速且非常适合对具有治疗意义的氧感应酶FIH-1的潜在底物进行初步鉴定。
