Inflammatory Proteomic Network Analysis of Statin-treated and Lipopolysaccharide-activated Macrophages

Abstract

A significant component of immune biology research is the investigation of protein encoding genes that play central roles in contributing inflammatory response. A gel-free quantitative bottom-up proteomics study was performed on immune cell macrophages after the combined treatment of lipopolysaccharide (LPS) and statin drugs using mass spectrometry and a detailed bioinformatics analyses were conducted. Systematic bioinformatics analysis was applied for discovering novel relationships among proteins and effects of statin and lipopolysaccharide in macrophage cells. Based on gene ontology, majority of protein encoding genes was involved in metabolic and cellular processes and are actively associated with binding, structural molecular, and catalytic activity. Notably, proteomic data analyzed by Ingenuity Pathway Analysis (IPA), discovered the plectin and prohibitin 2 protein interactions network and inflammatory-disease based protein networks. Two up-regulated proteins, plectin and prohibitin 2, were further validated by immunoblotting. Plectin was also cross-validated by immunocytochemistry, since its expression was highly modulated by statin but inhibited during LPS-stimulation. Collectively, the significant up-regulation of plectin due to the treatment of statin, suggests that statin has a significant impact on the cytoskeletal networks of cells. Plectin might have a significant role in the intermediate filament assembly and dynamics, and possibly stabilizing and crosslinking intermediate filament networks.

Multi-tissue epigenetic analysis of the osteoarthritis susceptibility locus mapping to the plectin gene PLEC

“…This model should incorporate plectin deficient differentiated joint cells, rather than the undifferentiated MSCs investigated here, to more accurately recapitulate the articulating joint. Furthermore, and despite our inability to undertake PLEC AEI on blood cells, we should not exclude circulating immune cells as potential targets of the rs11780978 association signal, particularly in light of the altered expression of plectin in macrophages in response to immunomodulatory agents 43 .…”

Discovery of protein modifications using high resolution differential mass spectrometry proteomics

“…To explore this idea, we sought to map protein modifications induced during mammalian cell differentiation. We chose to study the response of mouse RAW264.7 macrophage cells to lipopolysaccharide (LPS), a potent inducer of macrophage activation and differentiation that involves extensive protein and metabolic signaling (Alasoo et al 2015;Kamal et al 2018;Rattigan et al 2018;Seim et al 2019). We treated RAW264.7 cells with LPS using established methods and analyzed the resultant proteomes by two-dimensional nanoscale liquid chromatography and high-resolution mass spectrometry proteomics (Cifani et al 2018).…”

Crosslinking Proteomics Indicates Effects of Simvastatin on the TLR2 interactome and Reveals ACTR1A as a Novel Regulator of the TLR2 Signal Cascade

“…SDS-PAGE gel bands were excised, squeezed with acetonitrile, and dried at room temperature. Proteins were then reduced and alkylated and digested with trypsin (porcine) (MS Grade) at 37°C for overnight (30). Formic acid to pH < 3 was added to the resulting peptides, followed by drying by speed vacuum, and then dissolution in 0.1% formic acid.…”

“…Additional criteria were applied to increase confidence that PSMs must be present in all three biological replicates samples. Normalization of identified PSMs among LC-MS/MS runs was done by dividing individual PSMs of proteins with total PSMs and average of % PSM count was utilized for calculating fold changes for different treatment conditions (30,31). For contrasting relative intensities of proteins between control, P3C, statin-P3C, and statin groups, samples were evaluated using cumulative confident normalized PSMs value.…”

“…Following this, the cells were collected for mRNA analysis. Total RNA was extracted by TRIzol ® (Invitrogen) (30). First-strand cDNA synthesis was performed using one-step cDNA synthesis kit (Origene, MD, USA).…”