Apolipoprotein B binds to enolase-1 and aggravates inflammation in rheumatoid arthritis
Abstract
Background: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and joint destruction. Monocytes and synovial macrophages are key players in inflammatory process of RA. Enolase-1 (ENO1) is a multifunctional glycolytic enzyme in cytoplasm of cells and it is also found on the cell surface as plasminogen receptor. The majority of cells expressing ENO1 in peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) derived from RA patients were known to be CD14-positive monocytes and macrophages.
Objective: This study was aimed to discover and investigate a novel ligand of cell surface expressed ENO1 and biological role of the interaction between ENO1 and its novel ligand, apolipoprotein B (apoB).
Methods: ENO1 binding protein was identified present in RA synovial fluid (SF) using affinity-base mass spectrometry analysis. The interaction between ENO1 and apoB was evaluated using physical characterization, such as ligand blotting assay, ligand binding assay, surface plasmon resonance (SPR), and confocal microscopy. The production of pro-inflammatory cytokines in PBMCs from RA or healthy control (HC) after stimulation with apoB were evaluated using cytokines ELISA. The pro-inflammatory effect of apoB was evaluated in K/BxN serum transfer arthritis mouse model.
Results: Characterization of physical interaction between ENO1 and apoB using various binding assay, ligand blotting assay, ligand binding assay, SPR, and confocal microscopy showed that apoB is a novel ligand of ENO1. Interaction between surface ENO1 and apoB induced higher levels of pro-inflammatory cytokines in RA PBMCs than HC PBMCs. When surface ENO1 expression was down-regulated after transfection with ENO1-specific siRNA, production of inflammatory cytokines by RA PBMCs in response to apoB stimulation decreased. Moreover, in K/BxN serum transfer arthritis model, mice were immunized with K/BxN serum and apoB induced exacerbation of arthritis with increased ankle thickness, arthritis score, and production of pro-inflammatory cytokines. LDLR knockout mice are known to have high levels of serum LDL and apoB. The arthritis score and ankle thickness were markedly higher in low density lipoproteins receptor (LDLR) knockout mice compared to wild type after K/BxN serum transfer.
Conclusion: In this study, we discovered apoB as a novel ligand of ENO1. ApoB may enhance chronic inflammation in RA patients.
Post-Translational Modification of PPAR-gamma in metabolic diseases
Abstract
As an important metabolic organ in the human, the live and adipose tissues play a major role in the regulation of lipid metabolism and glucose metabolism such as gluconeogenesis. Especially, Hepatic steatosis is an early pathological step of the liver diseases and can cause steatohepatitis, cirrhosis, hepatocellular carcinoma, and serious cardiovascular diseases. Some evidence indicates that hepatic steatosis occurs in individuals with obesity and insulin resistance. In the liver, overloaded freeIn this suty fatty acid into hepatocyte by peroxisome proliferator-activated receptor gamma (PPARγ) results in lipid accumulation in hepatocyte. It suggests that excessive uptake of free fatty acid into hepatocyte, insulin resistance, and systemic pro-inflammatory response are interconnected pathological events that are usually found in NAFLD patients. Peroxisome proliferator-activated receptor gamma (PPAR) is a nuclear receptor that plays a key role in lipid metabolism. PPAR is activated by ligands, binds to the PPAR response element (PPRE), and increases expression of target genes. PPAR is known as a major transcription factor for adipocyte differentiation, and many studies have reported that PPAR expression and activity regulate adipocyte differentiation. Therefore, the regulation of PPAR is important in metabolic diseases. In this study, we demonstrated post-translational modification of PPAR by mass spectrometry and which mlecules regulates these modification. As a result, the meaning in detection of post-translational modification is a critical approach to find out physiological and pathological events in NAFLD patients.
Reactive oxygen species (ROS) generated in a wide range of physiological and pathological processes can promote cell damage, but also can activate cell regulatory and signaling pathways as a signaling molecule. Site-specific modification of cysteinyl thiols on oxidation-sensitive proteins represents a unique molecular mechanism for transducing ROS signals into biological responses. Recent advances in redox proteomics have greatly expanded the repertoire of oxidation-sensitive proteins. Nevertheless, it has proven challenging to directly identify specific-type of oxidation (e.g., sulfenic acid, sulfinic acid, etc.), so as to quantify the potency and specificity of these oxidations in complex proteomes. Here we describe two types of site-specific chemoproteomic strategies for global profiling of oxidation-sensitive proteins. Using these strategies, we are able to (1) site-specifically map and quantify hundreds of S-sulfenylation or S-sulfinylation events in cells, (2) to quantify changes of thousands of reactive cysteine residues in cellular or tissue proteomes in response to exogenous and endogenous redox perturbations. These studies not only greatly expand the catalogue of thiol proteins that undergo these modifications in cells, but also provide a new view of the landscape of thiol modifications in proteomes and suggests new hypotheses for future exploration of redox signaling and oxidative stress. We also developed a web portal database, OXID, as a compendium of proteome-wide analyses to identify and quantify oxidation-sensitive proteins.
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