Cell Signalling Unit, Edinburgh Cancer Research Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Rd South, Edinburgh EH4 2XR, UK.
Biochem J. 2013 Feb 1;449(3):707-17. doi: 10.1042/BJ20121076.
Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.
理解 E3 连接酶组件在泛素化中的特定位置的决定因素，被证明是一个挑战。在本研究中，我们研究了 E3 连接酶对接位点（Mf2 结构域）在 IRF-1 [干扰素（IFN）调节因子-1]的无规则结构域中的作用，IRF-1 是一种短寿命 IFNγ 调节转录因子，在该蛋白的泛素化中起作用。全长 IRF-1 的泛素修饰由 E3 连接酶如 CHIP [Hsc（热休克同源物）70 相互作用蛋白的 C 末端]和 MDM2（鼠双微 2）进行，这些连接酶与 Mf2 结构域对接，特异性地针对赖氨酸残基，这些赖氨酸残基主要存在于从 DNA 结合域延伸的环结构中，而在蛋白质更具构象灵活性的 C 末端则没有检测到修饰。当 IRF-1 处于其 DNA 结合构象时，E3 对接位点不可用，并且同源 DNA 结合序列强烈抑制泛素化，这突出了连接酶结合与 DNA 结合域中特定位置修饰之间的严格关系。非 DNA 结合突变体的过度泛素化支持一种机制，即活性 DNA 结合池的 IRF-1 免受多泛素化和降解的保护。
