Department of Chemistry and Biomolecular Sciences, Centre for Chemical and Synthetic Biology, Host-Microbe Interactions Laboratory, University of Ottawa, Ottawa, Ontario, Canada.
Department of Biochemistry, Microbiology and Immunology, Centre for Infection, Immunity and Inflammation, University of Ottawa, Ottawa, Ontario, Canada.
mSphere. 2024 Nov 21;9(11):e0055324. doi: 10.1128/msphere.00553-24. Epub 2024 Oct 31.
Promiscuous biotin ligases derived from the bacterial enzyme BirA are used to identify proteins vicinal to a bait protein, thereby defining its proxisome. Despite the popularity of this approach, surprisingly little is known about its use in prokaryotes. Here, we compared the activity of four widely used promiscuous biotin ligases in the cytoplasm of , a pathogenic subgroup of . Our data indicate that the kinetics of TurboID's biotinylating activity is the highest of those tested. In addition, TurboID showed reduced interaction with the natural BirA binding partners, BccP and the biotin operator, when compared to its ancestor BioID. We therefore evaluated the ability of TurboID to probe the proxisome of the type III secretion system (T3SS) chaperone IpgC and the transcriptional activator MxiE. When the T3SS is inactive (off-state), these proteins are inhibited by forming complexes with the T3SS substrates OspD1 and IpaBC, respectively. In contrast, when the T3SS is active (on-state), OspD1 and IpaBC are secreted allowing MxiE and IpgC to interact together and activate their target genes. The results obtained with the IpgC and TurboID fusions capture a good fraction of these known interactions. It also suggests that the availability of IpgC increases in the on-state, resulting in a greater number of proteins detected in its vicinity. Among these is the T3SS ATPase SpaL (also known as Spa47 or SctN), further supporting the notion that chaperones escort their substrate to the T3SS. Interestingly, a specific subset of proteins conserved in completes the IpgC proxisome in the on-state.IMPORTANCEPromiscuous biotin ligases are widely used to study protein function in eukaryotes. Strikingly, their use in prokaryotes has been rare. Indeed, the small volume and the cytoplasmic location of the biotin ligase's natural binding partners in these organisms pose unique challenges that can interfere with the study of the proxisome of proteins of interest. Here, we evaluated four of the most common promiscuous biotin ligases and found TurboID was best suited for use in the cytoplasm of . Using this method, we extended the proxisome of IpgC beyond its known direct binding partners involved in the regulation of the type III secretion system (T3SS) signaling cascade. Of particular interest for further study are transcription factors and housekeeping proteins that are enriched around IpgC when the T3SS is active. We propose a model in which the increased availability of IpgC in the on-state may allow cross-talk of the T3SS with other cellular processes.
尽管这种方法很流行，但人们对其在原核生物中的应用却知之甚少。在这里，我们比较了四种广泛使用的、源自细菌酶 BirA 的、混杂的生物素连接酶在致病性 组 的细胞质中的活性。我们的数据表明，TurboID 的生物素化活性的动力学是测试中最高的。此外，与它的祖先 BioID 相比，TurboID 与天然 BirA 结合伙伴 BccP 和生物素操纵子的相互作用减少。因此，我们评估了 TurboID 探测 III 型分泌系统 (T3SS) 伴侣蛋白 IpgC 和转录激活因子 MxiE 类蛋白酶体的能力。当 T3SS 不活跃（关闭状态）时，这些蛋白质通过与 T3SS 底物 OspD1 和 IpaBC 分别形成复合物而受到抑制。相比之下，当 T3SS 活跃（开启状态）时，OspD1 和 IpaBC 被分泌出来，使 MxiE 和 IpgC 相互作用并激活其靶基因。与 IpgC 和 TurboID 融合蛋白获得的结果捕获了这些已知相互作用的很大一部分。它还表明，IpgC 在开启状态下的可用性增加，导致其附近检测到的蛋白质数量增加。其中包括 T3SS ATPase SpaL（也称为 Spa47 或 SctN），这进一步支持了伴侣蛋白将其底物运送到 T3SS 的观点。有趣的是，一组在 中保守的特定蛋白质在开启状态下完成了 IpgC 类蛋白酶体。
在真核生物中，混杂的生物素连接酶被广泛用于研究蛋白质功能。令人惊讶的是，它们在原核生物中的应用很少。事实上，这些生物体中生物素连接酶的天然结合伙伴的体积小和细胞质位置给研究感兴趣的蛋白质的类蛋白酶体带来了独特的挑战，可能会干扰研究。在这里，我们评估了四种最常见的混杂生物素连接酶，发现 TurboID 最适合用于 细胞质。使用这种方法，我们将 IpgC 的类蛋白酶体扩展到其参与 III 型分泌系统 (T3SS) 信号级联调节的已知直接结合伙伴之外。当 T3SS 活跃时，特别有趣的是转录因子和管家蛋白在 IpgC 周围富集，这是进一步研究的对象。我们提出了一个模型，即 IpgC 在开启状态下的可用性增加可能允许 T3SS 与其他细胞过程进行交叉对话。
