Abstract
It is unclear how Toll-like receptor (TLR) 4 signaling affects protein succinylation in the brain after intracerebral hemorrhage (ICH). Here, we constructed a mouse ICH model to investigate the changes in ICH-associated brain protein succinylation, following a treatment with a TLR4 antagonist, TAK242, using a high-resolution mass spectrometry-based, quantitative succinyllysine proteomics approach. We characterized the prevalence of approximately 6700 succinylation events and quantified approximately 3500 sites, highlighting 139 succinyllysine site changes in 40 pathways. Further analysis showed that TAK242 treatment induced an increase of 29 succinyllysine sites on 28 succinylated proteins and a reduction of 24 succinyllysine sites on 23 succinylated proteins in the ICH brains. TAK242 treatment induced both protein hypersuccinylations and hyposuccinylations, which were mainly located in the mitochondria and cytoplasm. GO analysis showed that TAK242 treatment-induced changes in the ICH-associated succinylated proteins were mostly located in synapses, membranes and vesicles, and enriched in many cellular functions/compartments, such as metabolism, synapse, and myelin. KEGG analysis showed that TAK242-induced hyposuccinylation was mainly linked to fatty acid metabolism, including elongation and degradation. Moreover, a combined analysis of the succinylproteomic data with previously published transcriptome data revealed that most of the differentially succinylated proteins induced by TAK242 treatment were mainly distributed throughout neurons, astrocytes, and endothelial cells, and the mRNAs of seven and three succinylated proteins were highly expressed in neurons and astrocytes, respectively. In conclusion, we revealed that several TLR4 signaling pathways affect the succinylation processes and pathways in mouse ICH brains, providing new insights on the ICH pathophysiological processes. Data are available via ProteomeXchange with identifier PXD025622.
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Acknowledgements
This work was supported by the Science Foundation for Distinguished Young Scholars of Science and Technology Department of Sichuan Province (2020JDJQ0046) and the National Natural Science Foundation of China (81701292). We thank the bioinformatic analysis team from Jingjie PTM BioLab Co. Ltd (Hangzhou, China) for the analysis of and advice on the succinylproteome data in this study. We thank Prof. Jia Qian Wu (University of Texas Medical School at Houston) for providing us with the transcriptome data.
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SGY and XYX designed the research studies and wrote the manuscript. YJL, YRY, and CYT conducted the experiments and analyzed the data. SHY, XXZ, JY, YHD, and ZQZ analyzed the data.
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10571_2021_1144_MOESM1_ESM.pdf
Supplementary file1 Fig. 1 Analysis of the succinylproteomic data of ICH brains. (A) NDS in the Sham, ICH + vehicle, and ICH + TAK242 groups at 1 and 3 days after ICH. (B) Overview of protein and succinylation identification. (C and D) Data quality control of the peptide length distribution (C) and peptide mass tolerance distribution (D). (E) PCA analysis shows the dispersion degree of the succinylproteome between the sham, TAK242-treated, and vehicle-treated ICH brains. (F) Amino acid sequence properties of the succinyllysine sites. The heat map shows the significant position-specific under-representation or over-representation of the amino acids flanking the succinyllysine sites. (G) Succinylation motifs and the conservation of the succinyllysine sites. The height of each letter corresponds to the frequency of that amino acid residue at that position. The central K refers to the succinylated Lys. (PDF 3065 kb)
10571_2021_1144_MOESM2_ESM.pdf
Supplementary file2 Fig. 2 Flow chart of the definition of highly expressed genes in nerve cells. When all the FPKM of gene A in neurons/the FPKM of gene A in the other three neural cell types (astrocyte, microglia, and endothelial cell) ratios were > 1.0, we considered gene A to be highly expressed in neurons; and when the ratio > 10.0, we considered gene A to be highly specific to neurons. (PDF 774 kb)
10571_2021_1144_MOESM3_ESM.pdf
Supplementary file3 Fig. 3 MS spectrum of the representative succinylated sites. MS/MS spectra of Stxbp1_K120su (A), Cend1_K16su (B), Cntn1_K757su (C), Ina_K438su (D), Atp1a2_K658su (E), and Slc6a11_K610su (F) (PDF 997 kb)
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Liang, YJ., Yang, YR., Tao, CY. et al. Deep Succinylproteomics of Brain Tissues from Intracerebral Hemorrhage with Inhibition of Toll-Like Receptor 4 Signaling. Cell Mol Neurobiol 42, 2791–2804 (2022). https://doi.org/10.1007/s10571-021-01144-w
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DOI: https://doi.org/10.1007/s10571-021-01144-w