Proteomic analysis of microbial induced redox-dependent intestinal signaling

Abstract

Intestinal homeostasis is regulated in-part by reactive oxygen species (ROS) that are generated in the colonic mucosa following contact with certain lactobacilli. Mechanistically, ROS can modulate protein function through the oxidation of cysteine residues within proteins. Recent advances in cysteine labeling by the Isotope Coded Affinity Tags (ICATs) technique has facilitated the identification of cysteine thiol modifications in response to stimuli. Here, we used ICATs to map the redox protein network oxidized upon initial contact of the colonic mucosa with Lactobacillus rhamnosus GG (LGG). We detected significant LGG-specific redox changes in over 450 proteins, many of which are implicated to function in cellular processes such as endosomal trafficking, epithelial cell junctions, barrier integrity, and cytoskeleton maintenance and formation. We particularly noted the LGG-specific oxidation of Rac1, which is a pleiotropic regulator of many cellular processes. Together, these data reveal new insights into lactobacilli-induced and redox-dependent networks involved in intestinal homeostasis.

Commensal microbiota-induced redox signaling activates proliferative signals in the intestinal stem cell microenvironment

“…We previously reported that lactobacilli with gut epithelium induce ROS generation, with downstream influences on cell proliferation by mechanisms that require their mucus binding properties (Ardita et al, 2014). Indeed, epithelial ROS that is generated in response to bacteria functions in a signaling role, by which ROS-sensitive cellular enzymes could be influenced by bacterial-induced changes in redox status (Matthews et al, 2018). This hypothesis is supported by reports that reversible oxidative inactivation of a wide range of regulatory enzymes is a recognized mechanism of signal transduction (Ray et al, 2012).…”

Oxidized LDL Disrupts Metabolism and Inhibits Macrophage Survival by Activating a miR-9/Drp1/Mitochondrial Fission Signaling Pathway

“…Therefore, increased intake and decreased efflux of cholesterol from macrophages have been identified as a key contributor to macrophage dysfunction, which is associated with the development of AS [ 5 , 6 ]. However, cholesterol metabolism homeostasis in macrophages under hyperlipidemia stress conditions has not been well studied [ 7 , 8 ].…”

Neutrophil-Derived Reactive Oxygen Orchestrates Epithelial Cell Signaling Events during Intestinal Repair

“…The biotin-iodoacetamide (BIAM) pull-down assay was performed, as described previously, 27 without modification. Briefly, epithelial cell monolayers were lysed, the insoluble material was removed by centrifugation, and the lysates were adjusted to 25 mmol/L (biotinylated iodoacetamide) BIAM and incubated on ice in the dark for 45 minutes.…”

“…The Cleavable ICAT Reagent for Protein Labeling (stock keeping unit number 4339036) was obtained from SCIEX (Framingham, MA). Isotope-coded affinity tag (ICAT) and tandem mass spectrometry analysis was performed essentially, as previously described, 27 with minor modification. Briefly, trichloroacetic acid precipitated proteins from cell lysates were incubated with a heavy ICAT reagent to label reduced cysteines, followed by a second trichloroacetic acid precipitation, disulfide reduction with tris(2-carboxyethyl) phosphine (TCEP), and then labeling of newly reduced cysteines with a light ICAT reagent.…”

“…To validate the findings of the ICAT analysis in Figure 2 that elements involved in the formation of focal adhesions within cells are sensitive to reactive oxygen, SK-CO-1 cells were subjected to 1 mmol/L H 2 O 2 for 15 minutes. Cells were harvested and oxidized proteins were identified using a BIAM cysteine labeling pull-down assay, 27 where BIAM reacts with reduced thiols. If the thiol is oxidized after the addition of hydrogen peroxide, then BIAM will not react with the thiol on that protein and, therefore, the protein will not be pulled down by streptavidin agarose.…”