NUCLEIC ACIDS RESEARCH
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2024
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IF 16.6
Wang Xu,Qin Geng,Yang Jie,Zhao Chuanqi,Ren Jinsong,Qu Xiaogang
G-quadruplexes (G4s), as an important type of non-canonical nucleic acid structure, have received much attention because of their regulations of various biological processes in cells. Identifying G4s-protein interactions is essential for understanding G4s-related biology. However, current strategies for exploring G4 binding proteins (G4BPs) include pull-down assays in cell lysates or photoaffinity labeling, which are lack of sufficient spatial specificity at the subcellular level. Herein, we develop a subcellular selective APEX2-based proximity labeling strategy to investigate the interactome of mitochondrial DNA (mtDNA) G4s in living cells. By this method, we have identified several mtDNA G4BPs. Among them, a previously unrecognized mtDNA G4BP, DHX30 has been selected as an example to explore its important biofunctions. DHX30 localizes both in cytoplasm and mitochondria and can resolve mtDNA G4s. Further studies have demonstrated that DHX30 unfolds mtDNA G4 in living cells, which results in a decrease in glycolysis activity of tumor cells. Besides, RHPS4, a known mtDNA G4 stabilizer, will reverse this inhibition effect. Benefiting from the high spatiotemporal resolution and the ability of genetically encoded systems to perform the labeling with exquisite specificity within living cells, our approach can realize the identification of subcellular localized G4BPs. Our work provides a novel strategy to map protein interactions of specific nucleic acid features in subcellular compartments of living cells.
研究领域:
分子生物学
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遗传学
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癌症研究
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细胞生物学
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产品关联
DOI
PubMed
高影响因子
mSphere
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2022
·Vol. 7
·期 4
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IF 5.029
Hui Li,Cheng-Wen He,Jing Zhu,Zhiping Xie
Genetically encoded tags, such as engineered ascorbate peroxidase APEX2, offer unique advantages for the specific labeling of subcellular structures in electron microscopy (EM). However, the use of APEX2 in EM investigation of yeast has been limited. Here we describe the development of APEX2-based organelle markers for Saccharomyces cerevisiae. We found that with regard to APEX2 -catalyzed formation of diaminobenzidine precipitation, cell wall removal was not essential during sample preparation, yet the presence of fluorescent proteins in APEX2 chimeras had a negative impact. We showed that major organelles including endoplasmic reticulum, early Golgi, late Golgi/early endosomes, late endosomes, mitochondria, peroxisomes, and lipid droplets could be labeled by appropriate APEX2 chimeras. The subcellular localization of our APEX2 chimeras was verified by EM visualization and supplemented with immunofluorescence colocalization analysis when necessary, validating their feasibility as organelle markers.IMPORTANCE Yeast is an excellent single cellular model system for studying basic cellular processes. However, yeast cells are much smaller than most animal and plant cells, making the observation and recognition of yeast subcellular structures challenging. Here we developed a set of yeast organelle markers for use in electron microscopy and documented our technical approach for using this method.
研究领域:
细胞生物学
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分子生物学
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DOI
PubMed
高影响因子
RSC Chemical Biology
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2025
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IF 3.1
Gang Wang,Mo Li,Peng Zou
Subcellular RNA localization is a conserved mechanism in eukaryotic cells and plays critical roles in diverse physiological processes including cell proliferation, differentiation, and embryo development. Nevertheless, the characterization of centrosome-localized mRNAs remains under-explored due to technical difficulties. In this study, we utilize APEX2-mediated proximity labeling to map centrosome-proximal transcriptome, identifying DLGAP5 mRNA as a novel centrosome-localized transcript during mitosis. Using a combination of drug perturbation, truncation, deletion, and mutagenesis, we demonstrate that microtubule binding of nascent MBD1 polypeptide is required for centrosomal transport of DLGAP5 mRNA. Our data also reveals that mRNA targeting efficiency is tightly linked to the coding sequence (CDS) length. Thus, our study provides a transcriptomic resource for future investigation of centrosome-localized RNAs, and sheds light on mechanisms underlying mRNA centrosomal localization.
研究领域:
细胞生物学
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分子生物学
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转录组学
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产品关联
DOI
PubMed
高影响因子
STAR Protocols
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2020
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IF 0
Yi Li,Keke Liu,Ying Zhou,Jing Yang,Peng Zou
Summary Alk-Ph is a clickable APEX2 substrate developed for spatially restricted protein/RNA labeling in intact yeast cells. Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. Using the yeast mitochondria as an example, we provide guidelines to achieve high-resolution mapping of subcellular yeast proteome and transcriptome. For complete details on the use and execution of this protocol, please refer to Li et al. (2020) .
研究领域:
分子生物学
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蛋白质组学
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细胞生物学
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生物化学
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DOI
PubMed
STAR Protocols
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2023
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IF 0
Ran Li,Peng Zou
Summary Knowledge about the spatial organization of RNAs in eukaryotic cells is crucial for understanding their functions. Here, we present a detailed MERR APEX-seq protocol to achieve spatiotemporally resolved mapping of the subcellular transcriptome in cultured mammalian cells. This protocol provides detailed description of constructing cell lines stably expressing APEX2, immunofluorescence characterization, MERR APEX labeling, enrichment of biotinylated RNA, library construction and high-throughput sequencing, and MERR APEX-seq data analysis. For complete details on the use and execution of this protocol, please refer to Li et al. (2022). 1
研究领域:
分子生物学
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细胞生物学
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转录组学
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基因组学
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DOI
PubMed
ACS Nano
·
2025
·
IF 16
Xin-Yu Xiao,Qian-Wei Luo,Wang-Shu Li,Ze-Kun Chen,Zhuo Yang,Ya-Xuan Zhu,Mu-Rong Lei,Fang-Fang Zhuo,Ming Yu,Tian-Tian Wei,Hong-Wei Jin,Zhong-Yao Li,Zhi-Yuan Lu,Zhu-Qing Zhang,Hua Wang,Yong-Cheng Wang,Qing Xia,Wei Yu,Bo Han,Peng-Fei Tu,Ke-Wu Zeng
Nanoparticles bind to proteins in cells selectively and form a protein corona around them. However, the mechanisms of protein conformational changes underlying the interactions between nanoparticles and protein coronas remain poorly understood. In this study, we prepared small molecule self-assembled nanoparticles (Aloin NPs) as a research tool to investigate the allosteric mechanism of protein coronas. Aloin NPs showed a propensity to capture multiple proteins in cells. In particular, Aloin NPs specifically bound to myotrophin (MPTN) as a major protein corona through a multivalent hydrogen bond-mediated nanoprotein interface. Molecular modeling and hydrogen–deuterium exchange mass spectrometry (MS) demonstrated that Aloin NPs promoted a conformational rearrangement of MPTN via a ‘finger-unclasping’ pattern. We then adapted the APEX2 proximity labeling strategy to investigate the conformation-dependent changes in the MPTN interactome and identified peroxiredoxin 6 (PRDX6) as a key substrate protein of MPTN in microglia. Additionally, we observed that MPTN conformational change-dependent PRDX6 release protected the mitochondrial membrane by reducing reactive oxygen species. Consequently, Aloin NPs effectively inhibited the release of mitochondrial DNA to block the downstream cGAS-STING signaling pathway, thereby reprogramming microglial polarization. In translational medicine, Aloin NPs play a role in protecting neurons from microglia-induced inflammatory injury with no significant adverse effects, ultimately improving Parkinson’s disease-associated symptoms. Taken together, our study provides insights into the molecular mechanisms by which nanoparticles regulate the conformational change of protein coronas for human disease therapy.
研究领域:
纳米技术
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生物化学
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细胞生物学
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神经科学
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免疫学
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结构生物学
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药理学
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转化医学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）