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Photocatalytic zero-length proximity crosslinking reconstructs dynamic protein degradation networks in living cells

This article is a preprint and has not been certified by peer review.

Authors

    Xinyu Zhang,  
    Xinyu Zhang
    • Shenzhen Medical Academy of Research and Translation
    • Shenzhen Bay Laboratory
    Pengzhi Mao,  
    Pengzhi Mao
    • Institute of Computing Technology
    • University of Chinese Academy of Sciences
    Chaohong Bao,  
    Chaohong Bao
    Zuye Fang,  
    Zuye Fang
    Fenfen Zhang,  
    Fenfen Zhang
    Tianyi Deng,  
    Tianyi Deng
    • Shenzhen Medical Academy of Research and Translation
    • Shenzhen Bay Laboratory
    Xi Yang,  
    Xi Yang
    Min Guo,  
    Min Guo
    Min Zheng,  
    Min Zheng
    • Shenzhen Bay Laboratory
    Gang Li
    Gang Li
Categories
Chemical Biology
Keywords
protein-protein interaction; proteomics; proximity crosslinking; F-box proteins; protein degradation

Abstract

Protein interaction networks are dynamically remodeled during signaling and proteostasis, but many transient, low-stoichiometry, or degradation-coupled contacts are lost during affinity purification and difficult to distinguish from proximity-labeling backgrounds. Here, we introduce photocatalytic zero-length proximity crosslinking (PZPC), a genetically encoded, blue-light-controlled strategy that covalently captures protein-of-interest-containing covalent complexes in living cells. We further develop LinkMasser, a blind-search crosslinking mass spectrometry algorithm that identifies unknown crosslink-associated mass offsets, revealing an oxidative Lys–His zero-length coupling signature underlying PZPC-mediated crosslinking. Using the ubiquitin-independent degradation factor midnolin as a benchmark system, PZPC recovered inducible transcription factors and short-lived regulatory proteins largely missed by conventional AP–MS, leading to the identification of five novel midnolin substrates. We next applied PZPC to 63 human F-box proteins, generating a family-wide proximity atlas of 554 candidate interactors and functionally validating 81 candidate substrates from 257 tested proteins. By coupling this atlas to an antiviral signaling screen, we identified FBXO44 as a positive regulator of the RIG-I–MAVS–IFN pathway and degraded ADAR1, EIF3B, and TUFM as FBXO44-regulated negative modulators of antiviral signaling. These results establish PZPC as a scalable framework for resolving dynamic protein networks and uncovering E3-regulated signaling circuits in living cells.

References

1 Larance, M. & Lamond, A. I. Multidimensional proteomics for cell biology. Nat Rev Mol Cell Biol 16, 269-280, doi:10.1038/nrm3970 (2015).

2 Huttlin, E. L. et al. Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell 184, 3022-3040 e3028, doi:10.1016/j.cell.2021.04.011 (2021).

3 Bonetta, L. Protein-protein interactions: Tools for the search. Nature 468, 852, doi:10.1038/468852a (2010).

4 Gingras, A. C., Abe, K. T. & Raught, B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles. Curr. Opin. Chem. Biol. 48, 44-54, doi:10.1016/j.cbpa.2018.10.017 (2019).

5 Zhou, Y. & Zou, P. The evolving capabilities of enzyme-mediated proximity labeling. Curr Opin Chem Biol 60, 30-38, doi:10.1016/j.cbpa.2020.06.013 (2021).

6 McCutcheon, D. C., Lee, G., Carlos, A., Montgomery, J. E. & Moellering, R. E. Photoproximity Profiling of Protein-Protein Interactions in Cells. J. Am. Chem. Soc. 142, 146-153, doi:10.1021/jacs.9b06528 (2020).

7 Yang, B. et al. Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry. Proc. Natl. Acad. Sci. U S A 115, 11162-11167, doi:10.1073/pnas.1813574115 (2018).

8 Liu, J., Yang, B. & Wang, L. Residue selective crosslinking of proteins through photoactivatable or proximity-enabled reactivity. Curr Opin Chem Biol 74, 102285, doi:10.1016/j.cbpa.2023.102285 (2023).

9 Rhee, H. W. et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328-1331, doi:10.1126/science.1230593 (2013).

10 Roux, K. J., Kim, D. I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801-810, doi:10.1083/jcb.201112098 (2012).

11 Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880-887, doi:10.1038/nbt.4201 (2018).

12 Zheng, F., Yu, C., Zhou, X. & Zou, P. Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome. Nat Commun 14, 2978, doi:10.1038/s41467-023-38565-8 (2023).

13 Hananya, N., Ye, X., Koren, S. & Muir, T. W. A genetically encoded photoproximity labeling approach for mapping protein territories. Proc Natl Acad Sci U S A 120, e2219339120, doi:10.1073/pnas.2219339120 (2023).

14 Zhai, Y. et al. Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling. Nat Commun 13, 4906, doi:10.1038/s41467-022-32689-z (2022).

15 Geri, J. B. et al. Microenvironment mapping via Dexter energy transfer on immune cells. Science 367, 1091-1097, doi:10.1126/science.aay4106 (2020).

16 Seath, C. P., Trowbridge, A. D., Muir, T. W. & MacMillan, D. W. C. Reactive intermediates for interactome mapping. Chem. Soc. Rev. 50, 2911-2926, doi:10.1039/d0cs01366h (2021).

17 Lobingier, B. T. et al. An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells. Cell 169, 350-360.e312, doi:10.1016/j.cell.2017.03.022 (2017).

18 Oakley, J. V. et al. Radius measurement via super-resolution microscopy enables the development of a variable radii proximity labeling platform. 119, e2203027119, doi:doi:10.1073/pnas.2203027119 (2022).

19 Larios, J. M., Budhiraja, R., Fanburg, B. L. & Thannickal, V. J. Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts. J Biol Chem 276, 17437-17441, doi:10.1074/jbc.M100426200 (2001).

20 Barroso-Gomila, O. et al. BioE3 identifies specific substrates of ubiquitin E3 ligases. Nat Commun 14, 7656, doi:10.1038/s41467-023-43326-8 (2023).

21 Tamura, T., Takato, M., Shiono, K. & Hamachi, I. Development of a Photoactivatable Proximity Labeling Method for the Identification of Nuclear Proteins. Chem. Lett. 49, 145-148, doi:10.1246/cl.190804 (2020).

22 Wheat, A. et al. Protein interaction landscapes revealed by advanced in vivo cross-linking-mass spectrometry. Proc Natl Acad Sci U S A 118, doi:10.1073/pnas.2023360118 (2021).

23 O'Reilly, F. J. & Rappsilber, J. Cross-linking mass spectrometry: methods and applications in structural, molecular and systems biology. Nat Struct Mol Biol 25, 1000-1008, doi:10.1038/s41594-018-0147-0 (2018).

24 Piersimoni, L., Kastritis, P. L., Arlt, C. & Sinz, A. Cross-Linking Mass Spectrometry for Investigating Protein Conformations and Protein-Protein Interactions horizontal line A Method for All Seasons. Chem Rev 122, 7500-7531, doi:10.1021/acs.chemrev.1c00786 (2022).

25 Yu, C. & Huang, L. New advances in cross-linking mass spectrometry toward structural systems biology. Curr Opin Chem Biol 76, 102357, doi:10.1016/j.cbpa.2023.102357 (2023).

26 Ester, M., Kriegel, H., Sander, J. & Xu, X. A density-based algorithm for discovering clusters in large spatial databases with noise. KDD'96: Proceedings of the Second International Conference on Knowledge Discovery and Data Mining, 226-231 (1996).

27 Chi, H. et al. Comprehensive identification of peptides in tandem mass spectra using an efficient open search engine. Nature Biotechnology 36, 1059-1061, doi:10.1038/nbt.4236 (2018).

28 Guo, H. et al. LipoID profiles lipid droplet interactions and identifies interorganelle regulators. Nat Chem Biol, doi:10.1038/s41589-025-02127-4 (2026).

29 Ahn, J. et al. Structural basis for lamin assembly at the molecular level. Nat Commun 10, 3757, doi:10.1038/s41467-019-11684-x (2019).

30 Zhang, Y. et al. Protenix-v1: Toward High-Accuracy Open-Source Biomolecular Structure Prediction. Bio Archiv., doi:https://doi.org/10.64898/2026.02.05.703733 (2026).

31 Gu, X. et al. The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation. Science 381, eadh5021, doi:10.1126/science.adh5021 (2023).

32 Skaar, J. R., Pagan, J. K. & Pagano, M. SCF ubiquitin ligase-targeted therapies. Nat Rev Drug Discov 13, 889-903, doi:10.1038/nrd4432 (2014).

33 Feng, T., Wang, P. & Zhang, X. Skp2: A critical molecule for ubiquitination and its role in cancer. Life Sciences 338, 122409, doi:https://doi.org/10.1016/j.lfs.2023.122409 (2024).

34 Zheng, J. et al. CAND1 Binds to Unneddylated CUL1 and Regulates the Formation of SCF Ubiquitin E3 Ligase Complex. Molecular cell 10, 1519-1526, doi:https://doi.org/10.1016/S1097-2765(02)00784-0 (2002).

35 Liu, J., Furukawa, M., Matsumoto, T. & Xiong, Y. NEDD8 Modification of CUL1 Dissociates p120CAND1, an Inhibitor of CUL1-SKP1 Binding and SCF Ligases. Molecular cell 10, 1511-1518, doi:https://doi.org/10.1016/S1097-2765(02)00783-9 (2002).

36 Pierce, Nathan W. et al. Cand1 Promotes Assembly of New SCF Complexes through Dynamic Exchange of F Box Proteins. Cell 153, 206-215, doi:10.1016/j.cell.2013.02.024 (2013).

37 Royle, S. J., Bright, N. A. & Lagnado, L. Clathrin is required for the function of the mitotic spindle. Nature 434, 1152-1157, doi:10.1038/nature03502 (2005).

38 Zhao, X. et al. The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein. Nature Cell Biology 10, 643-653, doi:10.1038/ncb1727 (2008).

39 Han, X. et al. Destabilizing LSD1 by Jade-2 Promotes Neurogenesis: An Antibraking System in Neural Development. Molecular cell 55, 482-494, doi:https://doi.org/10.1016/j.molcel.2014.06.006 (2014).

40 Apriamashvili, G. et al. Ubiquitin ligase STUB1 destabilizes IFNγ-receptor complex to suppress tumor IFNγ signaling. Nature Communications 13, 1923, doi:10.1038/s41467-022-29442-x (2022).

41 Corcoran, C. A. et al. Identification and Characterization of Two Novel Isoforms of Pirh2 Ubiquitin Ligase That Negatively Regulate p53 Independent of RING Finger Domains*. Journal of Biological Chemistry 284, 21955-21970, doi:https://doi.org/10.1074/jbc.M109.024232 (2009).

42 Pérez-Oliva, A. B., Olivares, C., Jiménez-Cervantes, C. & García-Borrón, J. C. Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with Gαs*. Journal of Biological Chemistry 284, 31714-31725, doi:https://doi.org/10.1074/jbc.M109.028100 (2009).

43 Lam, S. T. T. & Lim, C. J. in Cellular Biology of the Endoplasmic Reticulum (eds Luis B. Agellon & Marek Michalak) 181-196 (Springer International Publishing, 2021).

44 Kozlov, G. & Gehring, K. Calnexin cycle – structural features of the ER chaperone system. The FEBS Journal 287, 4322-4340, doi:https://doi.org/10.1111/febs.15330 (2020).

45 Wu, S. A., Li, Z. J. & Qi, L. Endoplasmic reticulum (ER) protein degradation by ER-associated degradation and ER-phagy. Trends in Cell Biology 35, 576-591, doi:https://doi.org/10.1016/j.tcb.2025.01.002 (2025).

46 Wang, Q. et al. A Ubiquitin Ligase-Associated Chaperone Holdase Maintains Polypeptides in Soluble States for Proteasome Degradation. Molecular cell 42, 758-770, doi:https://doi.org/10.1016/j.molcel.2011.05.010 (2011).

47 Ishimoto, K. et al. Degradation of human Lipin-1 by BTRC E3 ubiquitin ligase. Biochem Biophys Res Commun 488, 159-164, doi:10.1016/j.bbrc.2017.04.159 (2017).

48 Mavrommati, I. et al. β-TrCP- and Casein Kinase II-Mediated Degradation of Cyclin F Controls Timely Mitotic Progression. Cell Rep 24, 3404-3412, doi:10.1016/j.celrep.2018.08.076 (2018).

49 Li, J., Chai, Q. Y. & Liu, C. H. The ubiquitin system: a critical regulator of innate immunity and pathogen-host interactions. Cell Mol Immunol 13, 560-576, doi:10.1038/cmi.2016.40 (2016).

50 Bhoj, V. G. & Chen, Z. J. Ubiquitylation in innate and adaptive immunity. Nature 458, 430-437, doi:10.1038/nature07959 (2009).

51 Onomoto, K., Onoguchi, K. & Yoneyama, M. Regulation of RIG-I-like receptor-mediated signaling: interaction between host and viral factors. Cellular & Molecular Immunology 18, 539-555, doi:10.1038/s41423-020-00602-7 (2021).

52 Rehwinkel, J. & Gack, M. U. RIG-I-like receptors: their regulation and roles in RNA sensing. Nat Rev Immunol 20, 537-551, doi:10.1038/s41577-020-0288-3 (2020).

53 Shang, J. et al. Quantitative Proteomics Identified TTC4 as a TBK1 Interactor and a Positive Regulator of SeV-Induced Innate Immunity. Proteomics 18, 1700403, doi:https://doi.org/10.1002/pmic.201700403 (2018).

54 Vogel, O. A. et al. The p150 Isoform of ADAR1 Blocks Sustained RLR signaling and Apoptosis during Influenza Virus Infection. Plos Pathog 16, e1008842, doi:10.1371/journal.ppat.1008842 (2020).

55 Lei, Y. et al. The mitochondrial proteins NLRX1 and TUFM form a complex that regulates type I interferon and autophagy. Immunity 36, 933-946, doi:10.1016/j.immuni.2012.03.025 (2012).

56 Xu, Y. et al. Multiplexed Photo-Cross-Linking Reveals Comprehensive Midnolin Interactome: Insights into Ubiquitin-Independent Degradation and Functional Diversity. J Am Chem Soc 148, 11962-11973, doi:10.1021/jacs.5c22099 (2026).

57 Coassolo, S. et al. Template-driven scaffolding of SCF(FBXO42) regulates PP2A degradation. Nature, doi:10.1038/s41586-026-10368-z (2026).

58 Muhar, M. F. et al. C-terminal amides mark proteins for degradation via SCF-FBXO31. Nature 638, 519-527, doi:10.1038/s41586-024-08475-w (2025).

59 Zhang, C. et al. Harnessing FBXO31 with Terminal Amide-Functionalized Molecules for Targeted Protein Degradation. J Am Chem Soc 148, 15391-15396, doi:10.1021/jacs.6c02580 (2026).

60 Chung, H. et al. Human ADAR1 Prevents Endogenous RNA from Triggering Translational Shutdown. Cell 172, 811-824.e814, doi:10.1016/j.cell.2017.12.038 (2018).

61 Shiromoto, Y., Sakurai, M., Minakuchi, M., Ariyoshi, K. & Nishikura, K. ADAR1 RNA editing enzyme regulates R-loop formation and genome stability at telomeres in cancer cells. Nature Communications 12, 1654, doi:10.1038/s41467-021-21921-x (2021).

62 Karki, R. & Kanneganti, T. D. ADAR1 and ZBP1 in innate immunity, cell death, and disease. Trends Immunol 44, 201-216, doi:10.1016/j.it.2023.01.001 (2023).

63 Rice, G. I. et al. Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon signature. Nat Genet 44, 1243-1248, doi:10.1038/ng.2414 (2012).

64 Beveridge, R., Stadlmann, J., Penninger, J. M. & Mechtler, K. A synthetic peptide library for benchmarking crosslinking-mass spectrometry search engines for proteins and protein complexes. Nature Communications 11, 742, doi:10.1038/s41467-020-14608-2 (2020).

65 Matzinger, M. et al. Mimicked synthetic ribosomal protein complex for benchmarking crosslinking mass spectrometry workflows. Nature Communications 13, 3975, doi:10.1038/s41467-022-31701-w (2022).

66 Ma, J. et al. iProX: an integrated proteome resource. Nucleic Acids Res 47, D1211-D1217, doi:10.1093/nar/gky869 (2019).

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https://doi.org/10.65215/LTSpreprints.2026.05.17.000244

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2026-05-18

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Zhang, X., Mao, P., Bao, C., Fang, Z., Zhang, F., Deng, T., Yang, X., Guo, M., Zheng, M., & Li, G. (2026). Photocatalytic zero-length proximity crosslinking reconstructs dynamic protein degradation networks in living cells. LangTaoSha Preprint Server. https://doi.org/10.65215/LTSpreprints.2026.05.17.000244

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Declaration of Competing Interests

Details of all competing interests to be disclosed are as follows:

G.L. and X.Z. are inventors on a patent related to photoactivation-dependent proximity crosslinking methods and applications (patent no. ZL 2022 1 0820043.9). The remaining authors declare no competing interests.

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