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Global profiling of functional histidines in live cells using small-molecule photosensitizer and chemical probe relay labelling

Abstract

Recent advances in chemical proteomics have focused on developing chemical probes that react with nucleophilic amino acid residues. Although histidine is an attractive candidate due to its importance in enzymatic catalysis, metal binding and protein–protein interaction, its moderate nucleophilicity poses challenges. Its modification is frequently influenced by cysteine and lysine, which results in poor selectivity and narrow proteome coverage. Here we report a singlet oxygen and chemical probe relay labelling method that achieves high selectivity towards histidine. Libraries of small-molecule photosensitizers and chemical probes were screened to optimize histidine labelling, enabling histidine profiling in live cells with around 7,200 unique sites. Using NMR spectroscopy and X-ray crystallography, we characterized the reaction mechanism and the structures of the resulting products. We then applied this method to discover unannotated histidine sites key to enzymatic activity and metal binding in select metalloproteins. This method also revealed the accessibility change of histidine mediated by protein–protein interaction that influences select protein subcellular localization, underscoring its capability in discovering functional histidines.

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Fig. 1: Development of global histidine labelling via a singlet-oxygen-based chemical proteomic method.
Fig. 2: Characterization of histidine sites in live cells.
Fig. 3: Mechanistic study and product characterization of histidine labelling.
Fig. 4: Bioinformatics analysis of the functions of labelled histidines.
Fig. 5: Characterization of unannotated, functional histidines in metalloproteins.
Fig. 6: Discovery of functional histidine in mitophagy.

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Data availability

The mass spectrometry data generated in this study have been deposited to the ProteomeXchange Consortium via the iProX (ref. 82) partner repository with the dataset identifier PXD042377 (Histidine_Profiling_MS dataset). Crystallographic data for small molecule 7 reported in this Article have been deposited at the Cambridge Crystallographic Data Centre, under deposition number CCDC 2312673. Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures. The dataset corresponding to the Python and R codes are available via Zenodo at https://doi.org/10.5281/zenodo.10867769 (ref. 83) or from the corresponding author upon request. Source data are provided with this paper.

Code availability

The Python code used for cleaning up histidine-containing peptides data, SASA analysis, secondary structure distribution analysis and distance measurement, along with the R code for domain enrichment analysis, are available via Zenodo at https://doi.org/10.5281/zenodo.10867769 (ref. 83) or from the corresponding author upon request.

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Acknowledgements

We thank the mass spectrometry, imaging, sequencing and NMR core facility in Shenzhen Bay Laboratory for their assistance in running samples and collecting data. We thank X. Li, Y. Liu, C. Wang and W. Zhong for helpful discussion and proofreading assistance. We thank Z. Li (Peking University Shenzhen Graduate School) for providing the thioacetal alkyne (TAA) probe. We are grateful for financial support of this work from Shenzhen Bay Laboratory Startup (21240041 to G.L.) and Grant from Shenzhen Bay Laboratory Open Fund (SZBL2020090501008 to G.L.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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All authors reviewed the manuscript. G.L. conceived and supervised the research. Y.Z., X.Z., X.W., Y.H., Y.-H.T. and G.L. designed and analysed biological experiments. Z.C., L.Z., T.L. and G.L. designed and analysed chemical experiments. D.Y., W.S. and D.W. provided the photosensitizers. X.Y. wrote the Python program for data processing and generated the figures. K.T. conducted the domain enrichment analysis. Z.Z. performed the solvent-accessible surface area analysis, secondary structure distribution analysis and distance measurement between histidines and active sites. Y.Z. and G.L. wrote the manuscript with input from all the authors.

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Correspondence to Gang Li.

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Extended data

Extended Data Fig. 1 Chemical structures.

a) Structures of small molecule photosensitizers. Eosin B (EB); Eosin Y (EY); Hypocrellin A (HA); Hypocrellin B (HB); Riboflavin (RF); Rose Bengal (RB); Dibromofluorescein (DBF); TCy5-CHO (T5C); TCy5-Btz (T5B); TCy5-Ph-3F (T5P); Methylene blue (MB); Icy-OH (IO); Methyl pyropheophorbide-a (MP); Chlorin e6 trimethyl ester (CE); TTPy-alkyne (TA); TTPy-OH (TO); DPA-SCPI (DS); b) Structures of chemical probes. N-(2-aminophenyl)pent-4-ynamide (NPA); Propargylamine (PA); 2-ethynylaniline (2-EA); 3-ethynylaniline (3-EA); 4-ethynylaniline (4-EA); 2-ethynylphenol (2-EP); 3-ethynylphenol (3-EP); 4-ethynylphenol (4-EP); 3-ethynyl-4-methylaniline (3E4MA); 3-ethynyl-4-fluoroaniline (3E-4FA); 5-ethynyl-2-fluoroaniline (5E-2FA); thioacetal alkyne (TAA); 3-ethynyl-N-methylaniline (3E-MA); 3-ethynylpyrazin-2-amine (3EP-2A); 5-ethynylpyridin-3-amine (5EP-3A); 4-ethynylpiperidine (4-EPD).

Extended Data Fig. 2 Analysis of the labeling specificity.

a) The distribution analysis of the PSMs for different photosensitizers in the open search. b) The distribution of singlet oxygen-sensitive amino acids in enriched peptide and human proteome. c) Labeling sites in closed search, where histidine (H) and one of the other 19 amino acids, as well as the N or C protein terminal, were jointly searched with differential masses of 229 and 247 Da. d) Box plot of percentage of histidine sites for combinations in c. The center line shows the median, while the box hinges mark the first and third quartiles. Whiskers indicate the full data range. n = 21 closed search analyses.

Source data

Extended Data Fig. 3

X-ray structure of the acyl-histamine oxidation product 7 (CCDC 2312673).

Extended Data Fig. 4 Bioinformatic analysis of non-identified and whole proteome histidiens.

a) Histogram plot showing the distribution of distances of non-identified histidine sites to the active sites for the identified proteins. b) Calculation of the proportions of identified histidine sites, non-identified histidine sites, and histidine residues in the active site for the identified proteins, as well as the proportion of histidine residues in the active site for the human proteome. c) The distribution of solvent accessibility of non-identified histidine residues in the identified proteins. d) The distribution of solvent accessibility of histidine residues in human proteome. e) The secondary structures analysis of the distribution of non-identified histidine sites in the identified proteome. f) The secondary structures analysis of the distribution of histidine residues in human proteome.

Source data

Extended Data Fig. 5 EDTA-sensitive histidines discovery.

a) Forward and reverse SILAC experiments were used to identify the metal-binding dependent histidine sites using the three additional photosensitizers: T5C, T5B, TO. b) Venn diagram illustrating the overlapping proteins identified with different photosensitizers.

Source data

Extended Data Fig. 6 Identification of differentially interacting proteins after mitophagy and validation of the interacting proteins knockdown.

a) Proteins consistently exhibiting over 1.5-fold increase in at least two forward and reverse experiments were listed. The overlapping proteins between forward and reverse experiments were selected, indicating their increased interaction with PARK7 after mitophagy. b) Western blot analysis confirming the knockdown of LUC7L3, BAG2, TMOD3, PLEC. c) Real-time PCR analysis was performed for BANF1 knockdown due to the unavailability of a suitable antibody. Data are presented as mean values +/− SD. n = 3 biologically independent experiments.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–34 and Tables 1–7.

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Supplementary Data

Statistical source data of supplementary figures.

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Source Data Fig. 1

Unprocessed gels of Fig. 1.

Source Data Fig. 2

Statistical source data of Fig. 2.

Source Data Fig. 4

Statistical source data of Fig. 4.

Source Data Fig. 5

Unprocessed western blots and gels; statistical source data of Fig. 5.

Source Data Fig. 6

Unprocessed western blots and gels; statistical source data of Fig. 6.

Source Data Extended Data Fig. 2

Statistical source data of Extended Data Fig. 2.

Source Data Extended Data Fig. 4

Statistical source data of Extended Data Fig. 4.

Source Data Extended Data Fig. 5

Statistical source data of Extended Data Fig. 5.

Source Data Extended Data Fig. 6

Unprocessed western blots and statistical source data of Extended Data Fig. 6.

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Zhai, Y., Zhang, X., Chen, Z. et al. Global profiling of functional histidines in live cells using small-molecule photosensitizer and chemical probe relay labelling. Nat. Chem. (2024). https://doi.org/10.1038/s41557-024-01545-6

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