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Global Phosphoproteomic Analysis Reveals Significant Metabolic Reprogramming in the Termination of Liver Regeneration in Mice.

ABSTRACT: Phosphorylation is crucial in regulating various biological processes. However, comprehensive phosphoproteomic profiling in the termination of liver regeneration (LR) is still missing. Here, we used Tandem Mass Tag (TMT) labeling coupled with phosphopeptide enrichment and two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS)/MS analysis to establish a global phosphoproteomic map in the liver of mice at day 5 after partial hepatectomy (PH). Altogether, 9731 phosphosites from 3443 proteins were identified and 7802 phosphosites from 2980 proteins were quantified. Motif analysis of the identified phosphosites revealed a diverse array of consensus sequences, suggesting that multiple kinase families including ERK/MAPK, PKA/PKC, CaMK-II, CKII, and CDK may be involved in the termination of LR. Functional clustering analysis of proteins with dysregulated phosphosites showed that they mainly participate in metabolic pathways, DNA replication, and tight junction. More importantly, the deletion of PP2Ac? in the liver remarkably changes the overall phosphorylation profile, indicating its critical role in regulating the termination of LR. Finally, several differentially phosphorylated sites were validated by co-immunoprecipitation and Western blot. Taken together, our data unravel the first comprehensive phosphoproteomic map in the termination of LR in mice, which greatly expands our knowledge in the complicated regulation of this process and provides new directions for the treatment of liver cancer using liver resection.

SUBMITTER: Zhang J

PROVIDER: S-EPMC7205775 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Global Phosphoproteomic Analysis Reveals Significant Metabolic Reprogramming in the Termination of Liver Regeneration in Mice.

Zhang Jingzi J Tang Neng N Zhao Yinjuan Y Zhao Ruoyu R Fu Xiao X Zhao Dandan D Zhao Yue Y Huang Lan L Li Chaojun C Qiu Yudong Y Xue Bin B Fang Lei L

Journal of proteome research 20200310 4

Phosphorylation is crucial in regulating various biological processes. However, comprehensive phosphoproteomic profiling in the termination of liver regeneration (LR) is still missing. Here, we used Tandem Mass Tag (TMT) labeling coupled with phosphopeptide enrichment and two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS)/MS analysis to establish a global phosphoproteomic map in the liver of mice at day 5 after partial hepatectomy (PH). Altogether, 9731 phosphosites from 3443 p ...[more]

PMID: 32105074

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Project description:Phosphorylation is well-known in regulating various biological processes. However, comprehensively phosphoproteomic profiling in the termination of liver regeneration (LR) is still missing. Here, we used TMT labeling coupled with phosphopeptides enrichment and 2D LC-MS/MS analysis to establish a global phosphoproteomic map in mice liver at day 5 after partial hepatectomy. Altogether, 619 phosphorylation sites in 437 proteins were significantly up-regulated and 503 phosphorylation sites in 358 proteins were down-regulated. The differentially phosphorylated proteins were further classified according to their biological process, cellular components, molecular function and subcellular localization. KEGG pathway analysis showed that they mainly participate in metabolic pathways, DNA replication, tight junction and focal adhesion. Motif analysis of the identified phosphorylation sites has revealed a diverse array of consensus sequences, suggesting multiple kinase families such as MAPK, PKA/PKC, CaMK-II, CKII and CDK, may be involved in the termination of LR. Finally, several differentially phosphorylated proteins were validated by Co-immunoprecipitation and Western blot. Taken together, our data unravels the first comprehensive phosphoproteomic map in the termination of liver regeneration in mice, which greatly expands our knowledge in the complicate regulation of this process and provides new directions on the treatment of liver cancer using liver resection and donor liver transplantation. Biological significance: The novelty of this study is that we have established for the first time a global phosphoproteomic map in mice liver at day 5 after partial hepatectomy, which represents the termination of liver regeneration. Altogether, 9,775 phosphorylation sites in 3,458 proteins were identified, among which 7,738 phosphorylation site in 2,971 proteins were quantified. KEGG pathway analysis demonstrates markedly metabolic reprograming in termination of LR, which is further validated by selected validation of several differentially phosphorylated metabolic enzymes such as PDHA, ACACA and FASN. The consistent high phosphorylation of PDHA in the first 4 days and its rapidly decreased phosphorylation at day 5 after PH might be a key signal in controlling the switch between glycolysis and oxidative

Project description:Background & aimsAcute-on-chronic liver failure (ACLF) is a clinical syndrome defined by liver failure on pre-existing chronic liver disease. It is often associated with bacterial infection and high short-term mortality. Experimental models that fully reproduce ACLF are lacking, so too are effective pharmacological therapies for this condition.MethodsTo mimic ACLF conditions, we developed a severe liver injury model by combining chronic injury (chronic carbon tetrachloride [CCl4] injection), acute hepatic insult (injection of a double dose of CCl4), and bacterial infection (intraperitoneal injection of bacteria). Serum and liver samples from patients with ACLF or acute drug-induced liver injury (DILI) were used. Liver injury and regeneration were assessed to ascertain the potential benefits of interleukin-22 (IL-22Fc) administration.ResultsThis severe liver injury model recapitulated some of the key features of clinical ACLF, including acute-on-chronic liver injury, bacterial infection, multi-organ injury, and high mortality. Liver regeneration in this model was severely impaired because of a shift from the activation of the pro-regenerative IL-6/STAT3 pathway to the anti-regenerative IFN-γ/STAT1 pathway. The impaired IL-6/STAT3 activation was due to the inability of Kupffer cells to produce IL-6; whereas the enhanced STAT1 activation was due to a strong innate immune response and subsequent production of IFN-γ. Compared to patients with DILI, patients with ACLF had higher levels of IFN-γ but lower liver regeneration. IL-22Fc treatment improved survival in ACLF mice by reversing the STAT1/STAT3 pathway imbalance and enhancing expression of many antibacterial genes in a manner involving the anti-apoptotic protein BCL2.ConclusionsAcute-on-chronic liver injury or bacterial infection is associated with impaired liver regeneration due to a shift from a pro-regenerative to an anti-regenerative pathway. IL-22Fc therapy reverses this shift and attenuates bacterial infection, thus IL-22Fc may have therapeutic potential for ACLF treatment.Lay summaryA mouse model combining chronic liver injury, acute hepatic insult, and bacterial infection recapitulates some of the key features of acute-on-chronic liver failure (ACLF) in patients. Both fibrosis and bacterial infection contribute to the impaired regenerative capacity of the liver in patients with ACLF. Herein, we show that IL-22Fc therapy improves ACLF by reprogramming impaired regenerative pathways and attenuating bacterial infection. Thus, it may have therapeutic potential for patients with ACLF.

Project description:Hepatic expression of A20, including in hepatocytes, increases in response to injury, inflammation and resection. This increase likely serves a hepatoprotective purpose. The characteristic unfettered liver inflammation and necrosis in A20 knockout mice established physiologic upregulation of A20 as integral to the anti-inflammatory and anti-apoptotic armamentarium of hepatocytes. However, the implication of physiologic upregulation of A20 in modulating hepatocytes' proliferative responses following liver resection remains controversial. To resolve the impact of A20 on hepatocyte proliferation and the liver's regenerative capacity, we examined whether decreased A20 expression, as in A20 heterozygous knockout mice, affects outcome following two-third partial hepatectomy. A20 heterozygous mice do not demonstrate a striking liver phenotype, indicating that their A20 expression levels are still sufficient to contain inflammation and cell death at baseline. However, usually benign partial hepatectomy provoked a staggering lethality (>40%) in these mice, uncovering an unsuspected phenotype. Heightened lethality in A20 heterozygous mice following partial hepatectomy resulted from impaired hepatocyte proliferation due to heightened levels of cyclin-dependent kinase inhibitor, p21, and deficient upregulation of cyclins D1, E and A, in the context of worsened liver steatosis. A20 heterozygous knockout minimally affected baseline liver transcriptome, mostly circadian rhythm genes. Nevertheless, this caused differential expression of >1000 genes post hepatectomy, hindering lipid metabolism, bile acid biosynthesis, insulin signaling and cell cycle, all critical cellular processes for liver regeneration. These results demonstrate that mere reduction of A20 levels causes worse outcome post hepatectomy than full knockout of bona fide liver pro-regenerative players such as IL-6, clearly ascertaining A20's primordial role in enabling liver regeneration. Clinical implications of these data are of utmost importance as they caution safety of extensive hepatectomy for donation or tumor in carriers of A20/TNFAIP3 single nucleotide polymorphisms alleles that decrease A20 expression or function, and prompt the development of A20-based liver pro-regenerative therapies.

Dataset Information

Global Phosphoproteomic Analysis Reveals Significant Metabolic Reprogramming in the Termination of Liver Regeneration in Mice.

Publications

Global Phosphoproteomic Analysis Reveals Significant Metabolic Reprogramming in the Termination of Liver Regeneration in Mice.

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