Oxidation is an underappreciated post-translational modification in the regulation of immune responses associated with changes in phosphorylation.

Although macrophages are known to be affected by their redox status, oxidation is not yet a well-recognized post-translational modification (PTM) in regulating macrophages and immune cells in general. While it has been described that the redox status of single cysteines in specific proteins is relevant for macrophage functions, global oxidation information is scarce. Hence, we globally assessed the impact of oxidation on macrophage activation using untargeted proteomics and PTM-omics. We exposed THP-1 macrophages to lipopolysaccharide (LPS) for 4 h and 24 h and applied a sequential iodoTMT labeling approach to get information on overall oxidation as well as reversible oxidation of cysteines. Thus, we identified 10452 oxidation sites, which were integratively analyzed with 5057 proteins and 7148 phosphorylation sites to investigate their co-occurance with other omics layers. Based on this integrative analysis, we found significant upregulation of several immune-related pathways, e.g. toll-like receptor 4 (TLR4) signaling, for which 19 proteins, 7 phosphorylation sites, and 39 oxidation sites were significantly affected, highlighting the relevance of oxidations in TLR4-induced macrophage activation. Co-regulation of oxidation and phosphorylation was observed, as evidenced by multiply modified proteins related to inflammatory pathways. Additionally, we observed time-dependent effects, with differences in the dynamics of oxidation sites compared to proteins and phosphorylation sites. Overall, this study highlights the importance of oxidation in regulating inflammatory processes and provides a method that can be readily applied to study the cellular redoxome globally.

Proteomic Characterization of the Cellular Effects of AhR Activation by Microbial Tryptophan Catabolites in Endotoxin-Activated Human Macrophages.

Sensing microbial tryptophan catabolites by the aryl hydrocarbon receptor (AhR) plays a pivotal role in host-microbiome homeostasis by modulating the host immune response. Nevertheless, the involved cellular processes triggered by the metabolites are mainly unknown. Here, we analyzed proteomic changes in macrophages after treatment with the tryptophan metabolites indole-3-acetic acid (I3AA) or indole-3-aldehyde (IAld), as well as the prototypic exogenous AhR-ligand benzo(a)pyrene (BaP) in the absence and presence of lipopolysaccharide (LPS) to identify affected cellular processes and pathways. The AhR-ligands regulated metabolic and immunologic processes in dependency of LPS co-stimulation. All investigated ligands time-dependently enhanced fatty acid ß-oxidation. Differences due to the combination with LPS were observed for all three ligands. Additionally, oxidative phosphorylation was significantly increased by IAld and I3AA in a time and LPS-dependent manner. Immunoregulatory processes were affected in distinct ways. While BaP and I3AA up-regulated IL-8 signaling, IL-6 signaling was decreased by IAld. BaP decreased the inflammasome pathway. Thus, AhR-ligand-dependent regulations were identified, which may modulate the response of macrophages to bacterial infections, but also the commensal microbiota through changes in immune cell signaling and metabolic pathways that may also alter functionality. These findings highlight the relevance of AhR for maintaining microbial homeostasis and, consequently, host health.

Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

Non-Genomic AhR-Signaling Modulates the Immune Response in Endotoxin-Activated Macrophages After Activation by the Environmental Stressor BaP.

Emerging studies revealed that the Aryl hydrocarbon receptor (AhR), a receptor sensing environmental contaminants, is executing an immunomodulatory function. However, it is an open question to which extent this is achieved by its role as a transcription factor or via non-genomic signaling. We utilized a multi-post-translational modification-omics approach to examine non-genomic AhR-signaling after activation with endogenous (FICZ) or exogenous (BaP) ligand in endotoxin-activated (LPS) monocyte-derived macrophages. While AhR activation affected abundances of few proteins, regulation of ubiquitination and phosphorylation were highly pronounced. Although the number and strength of effects depended on the applied AhR-ligand, both ligands increased ubiquitination of Rac1, which participates in PI3K/AKT-pathway-dependent macrophage activation, resulting in a pro-inflammatory phenotype. In contrast, co-treatment with ligand and LPS revealed a decreased AKT activity mediating an anti-inflammatory effect. Thus, our data show an immunomodulatory effect of AhR activation through a Rac1ubiquitination-dependent mechanism that attenuated AKT-signaling, resulting in a mitigated inflammatory response.

Comparison of quantitation methods in proteomics to define relevant toxicological information on AhR activation of HepG2 cells by BaP.

The application of quantitative proteomics provides a new and promising tool for standardized toxicological research. However, choosing a suitable quantitative method still puzzles many researchers because the optimal method needs to be determined. In this study, we investigated the advantages and limitations of two of the most commonly used global quantitative proteomics methods, namely label-free quantitation (LFQ) and tandem mass tags (TMT). As a case study, we exposed hepatocytes (HepG2) to the environmental contaminant benzo[a]pyrene (BaP) using a concentration of 2 µM. Our results revealed that both methods yield a similar proteome coverage, in which for LFQ a wider range of fold changes was observed but with less significant p-values compared to TMT. We detected 37 and 47 significantly enriched pathways by LFQ and TMT, respectively, with 17 overlapping pathways. To define the minimally required effort in proteomics as a benchmark, we artificially reduced the LFQ, and TMT data sets stepwise and compared the pathway enrichment. Thereby, we found that fewer proteins are necessary for detecting significant enrichment of pathways in TMT compared to LFQ, which might be explained by the higher reproducibility of the TMT data that was observed. In summary, we showed that the TMT approach is the preferable one when investigating toxicological questions because it offers a high reproducibility and sufficient proteome coverage in a comparably short time.