揭示 cAMP 信号在贾第鞭毛虫中的作用:深入了解 PKA 介导的胞吐调控和亚细胞相互作用。

IF 3.7 2区 生物学 Q2 MICROBIOLOGY mSphere Pub Date : 2024-11-21 Epub Date: 2024-10-30 DOI:10.1128/msphere.00723-24
Han-Wei Shih, Germain C M Alas, Alexander R Paredez
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引用次数: 0

摘要

cAMP 作为第二信使在各种原生动物寄生虫的阶段转换中发挥着重要作用。这一信号通路依赖多种效应器,如蛋白激酶 A(PKA)、cAMP 激活的交换蛋白和 cAMP 反应元件结合蛋白转录因子,来启动人体的信号转导。贾第虫基因组只包含两个腺苷酸环化酶(AC)、一个磷酸二酯酶(PDE)和一个已知的 PKA 效应器,这些成分的具体功能还不完全清楚。在我们之前的研究中,我们证明了依赖 AC2 的 cAMP 信号在促进烯化程序中的重要作用。利用 NanoBit 技术,我们强调了 AC2 依赖性 cAMP 生物合成在调节 PKA 调节域(PKAr)和 PKA 催化域(PKAc)解离中的重要作用。在本研究中,我们的目标有两个:首先,我们使用新开发的 Split-Halo 来检测贾第虫中 GlPKAr 和 GlPKAc 的亚细胞相互作用;其次,我们研究了 PKAc 是否调控细胞分裂特异性蛋白。我们的研究结果表明,在滋养体阶段,GlPKAr和GlPKAc在不同的亚细胞位置发生相互作用,包括鞭毛、基底体和胞质。在受到包囊刺激后,相互作用从鞭毛转移到了细胞质。敲除 GlPKAc 会导致胞囊变性特异基因的下调,从而导致产生更少的可存活的耐水囊蚴,这表明 PKA 在胞囊变性的转录调控中发挥作用。这些发现有助于人们更深入地了解 cAMP 信号通路及其在控制贾第虫包囊化过程中的重要作用:相互作用的精确时间和亚细胞区隔在信号转导中起着至关重要的作用。长期以来,共沉淀试验(CO-IP)一直被用来验证蛋白质与蛋白质之间的相互作用;然而,CO-IP 缺乏空间和时间分辨率。我们最近的研究使用了 NanoBit 分析法,该方法展示了 PKAr 和 PKAc 在 cAMP 类似物和瞬时刺激下的可逆蛋白-蛋白相互作用。在此基础上,本研究采用 Split-Halo 分析法揭示了发生 PKAr 和 PKAc 蛋白-蛋白相互作用的亚细胞区室,并揭示了它们对电位刺激的反应。总之,这些分子工具提供了蛋白质-蛋白质相互作用的时空信息,将在该领域大有用武之地。
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Unraveling the role of cAMP signaling in Giardia: insights into PKA-mediated regulation of encystation and subcellular interactions.

cAMP plays an important role as a second messenger in the stage transition of various protozoan parasites. This signaling pathway relies on multiple effectors, such as protein kinase A (PKA), exchange protein activated by cAMP, and cAMP-response element binding protein transcription factors, to initiate signal transduction in humans. The Giardia genome only contains two adenylate cyclases (ACs), a single phosphodiesterase (PDE) and a single known PKA effector, and the specific functions of these components are not fully understood. In our previous research, we demonstrated the important role of AC2-dependent cAMP signaling in promoting the encystation program. Using the NanoBit technology, we emphasized the significance of AC2-dependent cAMP biosynthesis in regulating the dissociation of the PKA regulatory domain (PKAr) and PKA catalytic domain (PKAc). In this study, our objectives are twofold: first, we used the newly developed Split-Halo to examine subcellular interactions of GlPKAr and GlPKAc in Giardia; and second, we investigated whether PKAc regulates encystation-specific proteins. Our findings revealed distinct subcellular locations where GlPKAr and GlPKAc interacted during the trophozoite stage, including the flagella, basal bodies, and cytoplasm. Upon exposure to encystation stimuli, the interaction shifted from the flagella to the cytosol. Knockdown of GlPKAc resulted in the downregulation of encystation-specific genes, leading to the production of fewer viable and water-resistant cysts indicating a role for PKA in the transcriptional regulation of encystation. These discoveries contribute to a deeper understanding of the cAMP signaling pathway and its important role in governing Giardia's encystation process.

Importance: The precise timing of interactions and subcellular compartmentation play crucial roles in signal transduction. The co-immunoprecipitation assay (CO-IP) has long been utilized to validate protein-protein interactions; however, CO-IPs lack spatial and temporal resolutions. Our recent study used the NanoBit assay, which showcased the reversible protein-protein interaction between PKAr and PKAc in response to cAMP analogs and encystation stimuli. Expanding on this groundwork, this study employs the Split-Halo assay to uncover the subcellular compartments where the PKAr and PKAc protein-protein interactions take place and respond to encystation stimuli. Taken together, these molecular tools provide spatiotemporal information on the protein-protein interaction, which will be useful in the field.

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来源期刊
mSphere
mSphere Immunology and Microbiology-Microbiology
CiteScore
8.50
自引率
2.10%
发文量
192
审稿时长
11 weeks
期刊介绍: mSphere™ is a multi-disciplinary open-access journal that will focus on rapid publication of fundamental contributions to our understanding of microbiology. Its scope will reflect the immense range of fields within the microbial sciences, creating new opportunities for researchers to share findings that are transforming our understanding of human health and disease, ecosystems, neuroscience, agriculture, energy production, climate change, evolution, biogeochemical cycling, and food and drug production. Submissions will be encouraged of all high-quality work that makes fundamental contributions to our understanding of microbiology. mSphere™ will provide streamlined decisions, while carrying on ASM''s tradition for rigorous peer review.
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