Khoshnood Behzad, Dacklin Ingrid, Grabbe Caroline
Department of Molecular Biology, Umeå University, Umeå, Sweden.
PLoS One. 2017 Sep 27;12(9):e0185611. doi: 10.1371/journal.pone.0185611. eCollection 2017.
By covalently conjugating to target proteins, ubiquitin-like modifiers (UBLs) act as important regulators of target protein localization and activity, thereby playing a critical role in the orchestration of cellular biology. The most ancient and one of the least studied UBLs is Urm1, a dual-function protein that in parallel to performing similar functions as its prokaryotic ancestors in tRNA modification, also has adopted the capacity to conjugate to cellular proteins analogous to ubiquitin and other UBL modifiers. In order to increase the understanding of Urm1 and its role in multicellular organisms, we have used affinity purification followed by mass spectrometry to identify putative targets of Urm1 conjugation (urmylation) at three developmental stages of the Drosophila melanogaster lifecycle. Altogether we have recovered 79 Urm1-interacting proteins in Drosophila, which include the already established Urm1 binding partners Prx5 and Uba4, together with 77 candidate urmylation targets that are completely novel in the fly. Among these, the majority was exclusively identified during either embryogenesis, larval stages or in adult flies. We further present biochemical evidence that four of these proteins are covalently conjugated by Urm1, whereas the fifth verified Urm1-binding protein appears to interact with Urm1 via non-covalent means. Besides recapitulating the previously established roles of Urm1 in tRNA modification and during oxidative stress, functional clustering of the newly identified Urm1-associated proteins further positions Urm1 in protein networks that control other types of cellular stress, such as immunological threats and DNA damage. In addition, the functional characteristics of several of the candidate targets strongly match the phenotypes displayed by Urm1n123 null animals, including embryonic lethality, reduced fertility and shortened lifespan. In conclusion, this identification of candidate targets of urmylation significantly increases the knowledge of Urm1 and presents an excellent starting point for unravelling the role of Urm1 in the context of a complex living organism.
泛素样修饰因子（UBLs）通过与靶蛋白共价结合，充当靶蛋白定位和活性的重要调节因子，从而在细胞生物学的协调中发挥关键作用。最古老且研究最少的UBLs之一是Urm1，它是一种双功能蛋白，在tRNA修饰中与其原核祖先执行相似功能的同时，还具备了与细胞蛋白结合的能力，类似于泛素和其他UBL修饰因子。为了增进对Urm1及其在多细胞生物中作用的理解，我们利用亲和纯化结合质谱法，在黑腹果蝇生命周期的三个发育阶段鉴定Urm1结合（泛素样修饰）的假定靶标。我们总共在果蝇中鉴定出79种与Urm1相互作用的蛋白，其中包括已确定的Urm1结合伙伴Prx5和Uba4，以及77个在果蝇中全新的候选泛素样修饰靶标。其中，大多数仅在胚胎发育、幼虫阶段或成年果蝇中被鉴定出来。我们进一步提供了生化证据，证明其中四种蛋白被Urm1共价结合，而第五种经证实的Urm1结合蛋白似乎通过非共价方式与Urm1相互作用。除了重现Urm1在tRNA修饰和氧化应激期间先前确定的作用外，新鉴定的与Urm1相关蛋白的功能聚类进一步将Urm1定位在控制其他类型细胞应激（如免疫威胁和DNA损伤）的蛋白网络中。此外，几个候选靶标的功能特征与Urm1n123缺失动物所表现出的表型高度匹配，包括胚胎致死、生育力降低和寿命缩短。总之，泛素样修饰候选靶标的鉴定显著增加了对Urm1的了解，并为阐明Urm1在复杂生物体中的作用提供了一个绝佳的起点。
