Department of Chemistry and Biochemistry, University of Nevada, Las Vegas, Las Vegas, NV, U.S.A.
Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany.
Biochem J. 2023 Nov 29;480(22):1817-1831. doi: 10.1042/BCJ20230373.
Protein ubiquitylation typically involves isopeptide bond formation between the C-terminus of ubiquitin to the side-chain amino group on Lys residues. However, several ubiquitin ligases (E3s) have recently been identified that ubiquitylate proteins on non-Lys residues. For instance, HOIL-1 belongs to the RING-in-between RING (RBR) class of E3s and has an established role in Ser ubiquitylation. Given the homology between HOIL-1 and ARIH1, an RBR E3 that functions with the large superfamily of cullin-RING E3 ligases (CRLs), a biochemical investigation was undertaken, showing ARIH1 catalyzes Ser ubiquitylation to CRL-bound substrates. However, the efficiency of ubiquitylation was exquisitely dependent on the location and chemical environment of the Ser residue within the primary structure of the substrate. Comprehensive mutagenesis of the ARIH1 Rcat domain identified residues whose mutation severely impacted both oxyester and isopeptide bond formation at the preferred site for Ser ubiquitylation while only modestly affecting Lys ubiquitylation at the physiological site. The results reveal dual isopeptide and oxyester protein ubiquitylation activities of ARIH1 and set the stage for physiological investigations into this function of emerging importance.
蛋白质泛素化通常涉及泛素 C 末端与赖氨酸残基侧链氨基之间形成异肽键。然而，最近已经鉴定出几种泛素连接酶（E3）能够在非赖氨酸残基上泛素化蛋白质。例如，HOIL-1 属于 RING-in-between RING（RBR）E3 类，在丝氨酸泛素化中具有确定的作用。鉴于 HOIL-1 和 ARIH1 之间的同源性，ARIH1 是一种与大的 Cullin-RING E3 连接酶（CRL）超家族功能的 RBR E3，进行了生化研究，表明 ARIH1 催化丝氨酸泛素化到 CRL 结合的底物上。然而，泛素化的效率非常依赖于底物一级结构中丝氨酸残基的位置和化学环境。对 ARIH1 Rcat 结构域的全面突变分析确定了残基，其突变严重影响 Ser 泛素化的首选位点的氧酯和异肽键形成，而对生理位点的 Lys 泛素化的影响则适中。结果揭示了 ARIH1 的双重异肽和氧酯蛋白质泛素化活性，并为这一功能的生理研究奠定了基础。
