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.
Highlights
Protein posttranslational modifications (PTMs) are one of the most efficient biological mechanisms due to the expansion of protein functions (Walsh et al 2005; Witze et al 2007; Karve and Cheema 2011) and have been shown to be involved in regulating biological processes of human diseases, such as Alzheimer's Disease (Marcelli et al 2018), brain injury (Klimova et al 2018), cancer (Hsu et al 2018), etc
TLR4-mediated inflammation is the major contribution to the secondary brain injury after intracerebral hemorrhage (ICH) (Fang et al 2013), and we previously showed that inhibiting TLR4 signaling by its antagonist TAK242 significantly reduced the brain injury via downregulation of inflammation response (Wang et al 2013)
We are the first to investigate the contribution of TLR4 signaling in protein succinylation in ICH brains using a high-resolution LC-MS/MS-based quantitative succinylproteomic approach
Summary
Protein posttranslational modifications (PTMs) are one of the most efficient biological mechanisms due to the expansion of protein functions (Walsh et al 2005; Witze et al 2007; Karve and Cheema 2011) and have been shown to be involved in regulating biological processes of human diseases, such as Alzheimer's Disease (Marcelli et al 2018), brain injury (Klimova et al 2018), cancer (Hsu et al 2018), etc. Succinate as a metabolite plays an important role in contributing innate immune signaling might via increasing protein succinylation (Tannahill et al 2013), suggesting that protein succinylation is an important mechanism for the activation of inflammatory immune cells (e.g., macrophages, dendritic cells, and T cells), because which would display a metabolic switching from oxidative phosphorylation (OXPHO) to aerobic glycolysis enabling rapid cell proliferation when they activated (Everts et al 2014; Jha et al 2015; Shi et al 2011). Protein succinylation has been shown might contribute to cellular energy regulation via increasing the activity of enzymes involved in glucose and lipid metabolism (Park et al 2013; Mills and O'Neill 2014). Protein succinylation through the addition of succinyl groups to lysine residues is critical to regulate metabolic processes, and immunity and inflammation (Mills and O'Neill 2014; Liu et al 2016)
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