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The RNF214-TEAD-YAP signaling axis promotes hepatocellular carcinoma progression via TEAD ubiquitylation

ABSTRACT: We established an APEX proximity labeling strategy in coupled with mass spectrometry to identify interacting proteins of RNF214. In this approach, we first fused an engineered ascorbate peroxidase (APEX2) to either N-terminus or C-terminus of RNF214, expressed these two fusion proteins in HLF, an HCC cell line, near the endogenous level, and generated short-lived radicals around the APEX2-RNF214 fusion proteins to label biotin on nearby interactive proteins by adding hydrogen peroxide (H2O2) and biotin-phenol (also called biotin-tyramide) transiently. Biotinylated proteins were then isolated using Streptavidin resin for protein identification by mass spectrometry.

ORGANISM(S): Homo Sapiens

SUBMITTER: Jianping Jin

PROVIDER: PXD049453 | iProX | Sun Feb 18 00:00:00 GMT 2024

REPOSITORIES: iProX

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The RNF214-TEAD-YAP signaling axis promotes hepatocellular carcinoma progression via TEAD ubiquitylation.

Lin Mengjia M Zheng Xiaoyun X Yan Jianing J Huang Fei F Chen Yilin Y Ding Ran R Wan Jinkai J Zhang Lei L Wang Chenliang C Pan Jinchang J Cao Xiaolei X Fu Kaiyi K Lou Yan Y Feng Xin-Hua XH Ji Junfang J Zhao Bin B Lan Fei F Shen Li L He Xianglei X Qiu Yunqing Y Jin Jianping J

Nature communications 20240611 1

RNF214 is an understudied ubiquitin ligase with little knowledge of its biological functions or protein substrates. Here we show that the TEAD transcription factors in the Hippo pathway are substrates of RNF214. RNF214 induces non-proteolytic ubiquitylation at a conserved lysine residue of TEADs, enhances interactions between TEADs and YAP, and promotes transactivation of the downstream genes of the Hippo signaling. Moreover, YAP and TAZ could bind polyubiquitin chains, implying the underlying m ...[more]

PMID: 38862474

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Mass spectrometry analysis of potential interactors or RNF214

Project description:We established an APEX proximity labeling strategy in coupled with mass spectrometry to identify interacting proteins of RNF214. In this approach, we first fused an engineered ascorbate peroxidase (APEX2) to either N-terminus or C-terminus of RNF214, expressed these two fusion proteins in HLF, an HCC cell line, near the endogenous level, and generated short-lived radicals around the APEX2-RNF214 fusion proteins to label biotin on nearby interactive proteins by adding hydrogen peroxide (H2O2) and biotin-phenol (also called biotin-tyramide) transiently. Biotinylated proteins were then isolated using Streptavidin resin for protein identification by mass spectrometry.

2024-05-18 | PXD052393 |

Proximity labeling via MLF2-APEX2 fusion

Project description:Proximity labeling approach to identify protein inside nuclear envelope blebs arising in Torsin-deficient HeLa cells. WT and TorsinKO cells were engineered to express MLF2-APEX2 fusion. APEX reaction was carried out via 1 mM H2O2 for 1 minute in the presence of biotin phenol. Biotinylated protein were captured via streptavidin beads. Each APEX reaction was accompanied by an untreated (no H2O2) control.

2022-08-25 | PXD036262 | Pride

Defining proximity proteomics of Histone modifications by antibody-mediated protein A-APEX2 labeling

Project description:Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding of post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-APEX2 labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tehers a protein A-APEX2 (pA-APEX2) fusion protein. Activation of APEX2 labels the nearby proteins with biotin; these proteins are then purified using streptavidin beads and are identified by mass spectrometry. We demonstrate the utility of this approach by profiling the binding proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac and H4K12ac, verified the co-localization of these identified proteins with baits proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient tool to identify proteins that are proximal to modified histones.

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Proteomic proximity mapping by rapamycin-dependent targeting of APEX2

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Proteomic proximity mapping by rapamycin-dependent targeting of APEX2 to tail-anchored membrane proteins

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2019-09-09 | PXD012157 | Pride

Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

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2018-10-26 | GSE121575 | GEO

APEX-seq Captures the Protein-RNA Interaction Patterns of DDX3X and DDX3Y

Project description:Although stress granules (SGs) are composed of a stable core structure, they are surrounded by a dynamic shell with assembly, disassembly, and transitions of proteins and RNA between the core and shell. Different SGs have distinct proteins and RNA constituents, which raises the possibility that different SGs might perform different biological functions. The RNA compositions of DDX3X- or DDX3Y-positive SGs are not known, nor is it known what differential effects DDX3X or DDX3Y may exert on these RNA components. To understand the biological function and the compositional differences between DDX3X- or DDX3Y-positive SGs, we first determined the RNA constituents in these SGs using an adapted Ascorbate-peroxidase (APEX)-based proximity labeling method. APEX converts exogenously supplied biotin-phenol (BP) to biotin-phenoxyl radicals, which in turn covalently labels protein and RNA in nanometers. APEX-mediated biotinylation has been widely used in studies of protein-protein interactions and recently in studying protein-RNA interactions. Here, we applied the APEX-mediated biotinylation in DDX3X and DDX3Y SGs to dissect their different RNA composition.

2022-06-01 | GSE171792 | GEO

Cell Surface Engineering Enables Surfaceome Profiling

Project description:Understanding which proteins are presented at the cell surface is essential for ongoing therapeutic development, and to expand our basic understanding of biology. We envisioned the integration of cell surface engineering with radical-mediated protein biotinylation to profile cell surface proteins (CSPs). This method relies on the pre-functionalization of cells with cholesterol lipid groups, followed by their conjugation with an APEX2 ascorbate peroxidase enzyme. The APEX2 enzyme allows for the covalent conjugation of biotin moieties to nearby residues for subsequent enrichment, and serves as a tool for the enrichment and subsequent MS analysis of CSPs.

2022-05-06 | PXD031845 | Pride

APEX2-MLF2 (Table S1, Prophet et al.)

Project description:MLF2-APEX2 fusion-based proximity labeling of nuclear envelope bleb composition. APEX reactions were conducted in WT and TorsinKO HeLa cells.

2022-08-22 | MSV000090177 | MassIVE

Charecterization of RBM42 interacting proteins in HCT116 cells with and without DNA damage

Project description:RNA binding motif protein 42 (RBM42) is an understudied gene mapped to 19q13.12 region and codes for a protein that consists of 480 amino acids, containing one RNA recognition motif (RRM) at its C-terminal region. We mapped the RBM42 interactome using ascorbate peroxidase (APEX2)-based proximity labelling combined with mass spectrometry. We utilized CRISPR-Cas9 methodology for biallelic knock-in APEX2 at the C-terminus of the endogenous RBM42 coding sequence (CDS) and established an HCT116 cell line expressing RBM42-APEX2 fusion. RBM42 interactome mapping was performed by using Control and and HCT116 expressing RBM42-APEX2 cells that were treated with DMSO or Etoposide (VP-16) that induce DNA repair and incubated in media containing biotin phenol, followed by H2O2 treatment, to activate APEX2 peroxidase activity. Pathway enrichment analysis revealed that the majority of proteins that appeared in proximity to RBM42 are implicated in mRNA splicing and processing. Furthermore, we observed a significant enrichment of proteins involved in cell cycle checkpoints and regulation, further highlighting the role of RBM42 in DNA damage response. Together, RBM42 interactome analysis substantiates its function as a splicing regulator and implicates it in additional cellular pathways related to mRNA metabolism and DNA damage response.

2023-11-22 | PXD034854 | Pride

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