Dataset Information

Directed Evolution of APOX for Proximity Labeling Using Electron-Deficient Phenols

ABSTRACT: Proximity labeling identifies endogenous proteins in specific subcellular regions. APEX2 is an enzyme that enables PL with high temporal resolution by converting biotin phenol (BP) into a radical that tags nearby proteins. However, the BP radical has a relatively large diffusion radius, limiting spatial resolution. Replacing BP with an electron-deficient nitrophenol (NP) probe could potentially improve the spatial resolution by generating more reactive radicals, but APEX2 cannot efficiently activate electron-deficient phenols. Here, we report the directed evolution of APOX, a quadruple mutant of APEX2 with a higher reduction potential, exhibiting 6-fold and 2.5-fold faster oxidation of NPs and unsubstituted phenols, respectively. Using APOX with a membrane-permeable alkyne-NP probe, we demonstrate PL in living mammalian cells, including proteomic mapping with excellent subcellular-compartment specificity. APOX expands heme peroxidase PL by accessing electron-deficient probes, opening opportunities to further tune the diffusion radius and enhance the tagging of biomolecules beyond proteins.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: Alexander Solivais

LAB HEAD: Lloyd M Smith

PROVIDER: PXD060753 | Pride | 2026-04-10

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	1-9-25_putativeEluate_2p5ul_tr1_S13.mzID	Mzid
	1-9-25_putativeEluate_2p5ul_tr1_S13.mzML	Mzml
	1-9-25_putativeEluate_2p5ul_tr1_S13.raw	Raw
	1-9-25_putativeEluate_2p5ul_tr2_S13.mzID	Mzid
	1-9-25_putativeEluate_2p5ul_tr2_S13.mzML	Mzml

Items per page:

1 - 5 of 130

Similar Datasets

Project description:The core purpose of this project is to identify the novel epigenetic factors which play a crucial role on regulating stem cells developed from embryo development to ageing through the longitudinal analysis of their transcriptome, protein interactome, and post- translational modification network. Thus, this study would not only advance our understanding of epigenetic nature of stem cells through whole life, but also provide the novel strategy to develop the next generation stem cell therapeutics. According to our previous reports, we found that DNA methylation, particularly mediated by Dnmt3L could play a common epigenetic signature for modulating the stemness potency of both embryonic and adult stem cells. To investigate the post-translational modifications of Dnmt3L protein, we performed mass spectrometry analysis of Flag-tagged Dnmt3L proteins in murine embryonic stem cells (ESCs) and then identified several acetylated lysine residues. To examine their functional roles, we manufactured the lentivirus containing wild type Dnmt3L or the acetylated lysine mutants and they established the ESC cell lines stably harboring these constructs. To focus on epigenetic mechanism for stem cell biology, we will narrow down the key acetylated lysine sites of Dnmt3L, which can characteristically regulate the epigenetic status or transcription gene ontology. To get mechanistic insights, the transcriptome and DNA methylome features of the established ESC cell lines will be analyzed. The functional significance was examined by a series of experiments for in vitro and in vivo self-renewal and differentiation assays. Finally, we would extend these results to develop the efficient ex vivo expansion protocol for adult stem cells, particularly focusing on human mesenchymal stem cells. We expect that this proposal will not only advance our understating the epigenetic landscape of stem cell population but also contribute to establish the clinically best-suitable stem cell population. In addition, this study can be appliable to get a novel conceptional advances in other biological research such as cancer biology and ageing research.

Dataset Information

Directed Evolution of APOX for Proximity Labeling Using Electron-Deficient Phenols

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets