Coordinating Role of RXR alpha in Downregulating Hepatic Detoxification during Inflammation Revealed by Fuzzy-Logic Modeling

During various inflammatory processes circulating cytokines including IL-6, IL-1 beta, and TNF alpha elicit a broad and clinically relevant impairment of hepatic detoxification that is based on the simultaneous downregulation of many drug metabolizing enzymes and transporter genes. To address the question whether a common mechanism is involved we treated human primary hepatocytes with IL-6, the major mediator of the acute phase response in liver, and characterized acute phase and detoxification responses in quantitative gene expression and (phospho-)proteomics data sets. Selective inhibitors were used to disentangle the roles of JAK/STAT, MAPK, and PI3K signaling pathways. A prior knowledge-based fuzzy logic model comprising signal transduction and gene regulation was established and trained with perturbation-derived gene expression data from five hepatocyte donors. Our model suggests a greater role of MAPK/PI3K compared to JAK/STAT with the orphan nuclear receptor RXR alpha playing a central role in mediating transcriptional downregulation. Validation experiments revealed a striking similarity of RXRa gene silencing versus IL-6 induced negative gene regulation (r(s) = 0.79;P<0.0001). These results concur with RXRa functioning as obligatory heterodimerization partner for several nuclear receptors that regulate drug and lipid metabolism

Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system

Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system

Kinase analysis in alcoholic hepatitis identifies p90RSK as a potential mediator of liver fibrogenesis

Alcoholic hepatitis (AH) is often associated with advanced fibrosis, which negatively impacts survival. We aimed at identifying kinases deregulated in livers from patients with AH and advanced fibrosis in order to discover novel molecular targets

Kinase analysis in alcoholic hepatitis identifies p90RSK as a potential mediator of liver fibrogenesis

Objective Alcoholic hepatitis (AH) is often associated with advanced fibrosis, which negatively impacts survival. We aimed at identifying kinases deregulated in livers from patients with AH and advanced fibrosis in order to discover novel molecular targets. Design Extensive phosphoprotein analysis by reverse phase protein microarrays was performed in AH (n=12) and normal human livers (n=7). Ribosomal S6 kinase (p90RSK) hepatic expression was assessed by qPCR, Western blot and immunohistochemistry. Kaempferol was used as a selective pharmacological inhibitor of the p90RSK pathway to assess the regulation of experimentally-induced liver fibrosis and injury, using in vivo and in vitro approaches. Results Proteomic analysis identified p90RSK as one of the most deregulated kinases in AH. Hepatic p90RSK gene and protein expression was also upregulated in livers with chronic liver disease. Immunohistochemistry studies showed increased p90RSK staining in areas of active fibrogenesis in cirrhotic livers. Therapeutic administration of kaempferol to carbon tetrachloride-treated mice resulted in decreased hepatic collagen deposition, and expression of profibrogenic and proinflammatory genes, compared to vehicle administration. In addition, kaempferol reduced the extent of hepatocellular injury and degree of apoptosis. In primary hepatic stellate cells, kaempferol and small interfering RNA decreased activation of p90RSK, which in turn regulated key profibrogenic actions. In primary hepatocytes, kaempferol attenuated proapoptotic signalling. Conclusions p90RSK is upregulated in patients with chronic liver disease and mediates liver fibrogenesis in vivo and in vitro. These results suggest that the p90RSK pathway could be a new therapeutic approach for liver diseases characterised by advanced fibrosis

Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry

<p>Abstract</p> <p>Background</p> <p>Mass spectrometry (MS) based protein profiling has become one of the key technologies in biomedical research and biomarker discovery. One bottleneck in MS-based protein analysis is sample preparation and an efficient fractionation step to reduce the complexity of the biological samples, which are too complex to be analyzed directly with MS. Sample preparation strategies that reduce the complexity of tryptic digests by using immunoaffinity based methods have shown to lead to a substantial increase in throughput and sensitivity in the proteomic mass spectrometry approach. The limitation of using such immunoaffinity-based approaches is the availability of the appropriate peptide specific capture antibodies. Recent developments in these approaches, where subsets of peptides with short identical terminal sequences can be enriched using antibodies directed against short terminal epitopes, promise a significant gain in efficiency.</p> <p>Results</p> <p>We show that the minimal set of terminal epitopes for the coverage of a target protein list can be found by the formulation as a set cover problem, preceded by a filtering pipeline for the exclusion of peptides and target epitopes with undesirable properties.</p> <p>Conclusions</p> <p>For small datasets (a few hundred proteins) it is possible to solve the problem to optimality with moderate computational effort using commercial or free solvers. Larger datasets, like full proteomes require the use of heuristics.</p