Identification of proximal SUMO-dependent interactors using SUMO-ID

Proximity-dependent labelling (PL) methods are based on promiscuous labeling enzymes that produce reactive molecules that covalently bind neighbor proteins. Labeled proteins can be then purified and identified using affinity-purification coupled to mass spectrometry methods39. Proximity-dependent biotin identification (BioID)40 uses a promiscuously active Escherichia coli biotin ligase (BirA*) generated by a point mutation (R118G) to biotinylate lysines in nearby proteins within an estimated range of 10 nm41. By fusing BirA* to specific proteins, BioID efficiently identifies interactors at physiological levels in living cells42, 43. It has been extensively used in the Ub field, for instance, to identify substrates of E3 ligases44-46. Recently, a more efficient version of BioID, termed TurboID, has been developed47. TurboID can proximally biotinylate in 10 minutes to the same levels as BioID does in 18 hours, thus making it more suitable for transient protein-protein interaction (PPI) detection. Several studies have developed split-versions and applied “protein fragment complementation” (PCA) to BioID and TurboID, where proximal biotinylation is dependent on the proximity of the fusion partners, opening new opportunities for spatial and temporal identification of complex-dependent interactomes48-50. To study how SUMOylation and SUMO-SIM interactions can lead to other roles and fates for particular substrates poses particular challenges. SUMOylation occurs transiently and often in a small percentage of a given substrate. Modified proteins can be readily deSUMOylated and SUMO can be recycled and passed to other substrates. SUMO-SIM interactions are also difficult to analyze due to their weak affinity (Kd ranging from 1-100 µM). To overcome those technical issues, we developed SUMO-ID, a new strategy based on Split-TurboID to identify SUMO- interactors of specific substrates dependent on SUMO conjugation or interaction. Using PML as a model, we demonstrate that SUMO-ID can enrich for factors that depend on PML-SUMO interaction. Importantly, those are represented among proximal interactors of PML identified using full-length TurboID. We also applied SUMO-ID to a less-characterized SUMO substrate, Spalt like transcription factor 1 (SALL1), and identified both known and novel interactors that depend on intact SUMOylation sites in SALL1. SUMO-ID is thus a powerful tool to study transient and dynamic SUMO-dependent interaction events.

邻近依赖标记（Proximity-dependent labelling, PL）技术基于一类可产生反应性分子的混杂型标记酶，这类分子可与邻近蛋白质发生共价结合。标记后的蛋白质可通过亲和纯化质谱联用技术完成纯化与鉴定39。邻近依赖生物素识别（Proximity-dependent biotin identification, BioID）40技术使用了经点突变（R118G）获得的混杂活性大肠杆菌生物素连接酶（BirA*），可在约10纳米范围内对邻近蛋白质的赖氨酸残基进行生物素标记41。将BirA*与特定蛋白质融合后，BioID可在活细胞的生理水平下高效鉴定互作蛋白42、43。该技术已在泛素（Ubiquitin, Ub）研究领域得到广泛应用，例如用于鉴定E3泛素连接酶的底物44-46。
近期，研究者开发出了BioID的更高效变体TurboID47。TurboID仅需10分钟即可达到BioID18小时的原位生物素标记效率，因此更适用于瞬时蛋白质-蛋白质互作（Protein-Protein Interaction, PPI）的检测。多项研究已开发出BioID与TurboID的分裂变体，并将"蛋白质片段互补分析（Protein Fragment Complementation Assay, PCA）"技术应用其中，此时近端生物素标记依赖于融合蛋白伙伴的空间邻近性，为时空分辨率下复杂复合物依赖型互作组的鉴定提供了新方向48-50。
探究SUMO化修饰（SUMOylation）与SUMO-SIM互作如何决定特定底物的其他功能与命运，一直存在诸多技术挑战。SUMO化修饰具有瞬时性，且通常仅发生在少量目标底物上。被修饰的蛋白质可快速去SUMO化，SUMO分子也可被循环利用并转移至其他底物。SUMO-SIM互作的亲和力较弱（解离常数Kd介于1~100 μM之间），同样难以进行分析。
为解决上述技术难题，本研究开发出SUMO-ID技术——一种基于分裂TurboID的新策略，可用于鉴定依赖于SUMO结合或修饰的特定底物的SUMO互作蛋白。以早幼粒细胞白血病蛋白（Promyelocytic Leukemia Protein, PML）为模型，我们验证了SUMO-ID可富集依赖于PML-SUMO互作的蛋白因子，且这些因子均存在于使用全长TurboID鉴定出的PML近端互作蛋白组中。我们还将SUMO-ID应用于一个研究较少的SUMO底物——斯帕特样转录因子1（Spalt Like Transcription Factor 1, SALL1），成功鉴定出了依赖于SALL1完整SUMO化位点的已知与新型互作蛋白。综上，SUMO-ID是研究瞬时、动态的SUMO依赖型互作事件的有力工具。