pSTIING: a ‘systems’ approach towards integrating signalling pathways, interaction and transcriptional regulatory networks in inflammation and cancer

pSTIING () is a new publicly accessible web-based application and knowledgebase featuring 65 228 distinct molecular associations (comprising protein–protein, protein–lipid, protein–small molecule interactions and transcriptional regulatory associations), ligand–receptor–cell type information and signal transduction modules. It has a particular major focus on regulatory networks relevant to chronic inflammation, cell migration and cancer. The web application and interface provide graphical representations of networks allowing users to combine and extend transcriptional regulatory and signalling modules, infer molecular interactions across species and explore networks via protein domains/motifs, gene ontology annotations and human diseases. pSTIING also supports the direct cross-correlation of experimental results with interaction information in the knowledgebase via the CLADIST tool associated with pSTIING, which currently analyses and clusters gene expression, proteomic and phenotypic datasets. This allows the contextual projection of co-expression patterns onto prior network information, facilitating the identification of functional modules in physiologically relevant systems

Ubiquitin Accumulation in Autophagy-Deficient Mice is Dependent on the Nrf2-Mediated Stress Response Pathway: A Potential Role for Protein Aggregation in Autophagic Substrate Selection

Genetic ablation of autophagy in mice leads to liver and brain degeneration accompanied by the appearance of ubiquitin (Ub) inclusions, which has been considered to support the hypothesis that ubiquitination serves as a cis-acting signal for selective autophagy. We show that tissue-specific disruption of the essential autophagy genes Atg5 and Atg7 leads to the accumulation of all detectable Ub–Ub topologies, arguing against the hypothesis that any particular Ub linkage serves as a specific autophagy signal. The increase in Ub conjugates in Atg7$^{−/−}$ liver and brain is completely suppressed by simultaneous knockout of either p62 or Nrf2. We exploit a novel assay for selective autophagy in cell culture, which shows that inactivation of Atg5 leads to the selective accumulation of aggregation-prone proteins, and this does not correlate with an increase in substrate ubiquitination. We propose that protein oligomerization drives autophagic substrate selection and that the accumulation of poly-Ub chains in autophagy-deficient circumstances is an indirect consequence of activation of Nrf2-dependent stress response pathways

TMEM258 Is a Component of the Oligosaccharyltransferase Complex Controlling ER Stress and Intestinal Inflammation

Summary - Significant insights into disease pathogenesis have been gleaned from population-level genetic studies; however, many loci associated with complex genetic disease contain numerous genes, and phenotypic associations cannot be assigned unequivocally. In particular, a gene-dense locus on chromosome 11 (61.5–61.65 Mb) has been associated with inflammatory bowel disease, rheumatoid arthritis, and coronary artery disease. Here, we identify TMEM258 within this locus as a central regulator of intestinal inflammation. Strikingly, Tmem258 haploinsufficient mice exhibit severe intestinal inflammation in a model of colitis. At the mechanistic level, we demonstrate that TMEM258 is a required component of the oligosaccharyltransferase complex and is essential for N-linked protein glycosylation. Consequently, homozygous deficiency of Tmem258 in colonic organoids results in unresolved endoplasmic reticulum (ER) stress culminating in apoptosis. Collectively, our results demonstrate that TMEM258 is a central mediator of ER quality control and intestinal homeostasis.Leona M. and Harry B. Helmsley Charitable Trust (2014PG-IBD016)Crohn's and Colitis Foundation of AmericaNational Institutes of Health (U.S.) (grant DK043351)National Institutes of Health (U.S.) (grant DK097485

Comprehensive Identification of Host Modulators of HIV-1 Replication using Multiple Orthologous RNAi Reagents

SummaryRNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene’s phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1–host cell interactions

Identification of Atg5-dependent transcriptional changes and increases in mitochondrial mass in Atg5-deficient T lymphocytes

Autophagy is implicated in many functions of mammalian cells such as organelle recycling, survival and differentiation, and is essential for the maintenance of T and B lymphocytes. Here, we demonstrate that autophagy is a constitutive process during T cell development. Deletion of the essential autophagy genes Atg5 or Atg7 in T cells resulted in decreased thymocyte and peripheral T cell numbers, and Atg5-deficient T cells had a decrease in cell survival. We employed functional-genetic and integrative computational analyses to elucidate specific functions of the autophagic process in developing T-lineage lymphocytes. Our whole-genome transcriptional profiling identified a set of 699 genes differentially expressed in Atg5-deficient and Atg5-sufficient thymocytes (Atg5-dependent gene set). Strikingly, the Atg5-dependent gene set was dramatically enriched in genes encoding proteins associated with the mitochondrion. In support of a role for autophagy in mitochondrial maintenance in T lineage cells, the deletion of Atg5 led to increased mitochondrial mass in peripheral T cells. We also observed a correlation between mitochondrial mass and Annexin-V staining in peripheral T cells. We propose that autophagy is critical for mitochondrial maintenance and T cell survival. We speculate that, similar to its role in yeast or mammalian liver cells, autophagy is required in T cells for the removal of damaged or aging mitochondria and that this contributes to the cell death of autophagy-deficient T cells

Allergic asthma is distinguished by sensitivity of allergen-specific CD4+ T cells and airway structural cells to type 2 inflammation

Despite systemic sensitization, not all allergic individuals develop asthma symptoms upon airborne allergen exposure. Determination of the factors that lead to the asthma phenotype in allergic individuals could guide treatment and identify novel therapeutic targets. In this study, we utilized segmental allergen challenge (SAC) of allergic asthmatics (AA) and allergic non-asthmatic controls (AC) to determine if there are differences in the airway immune response or airway structural cells that could drive the development of asthma. Both groups developed prominent allergic airway inflammation in response to allergen. However, asthmatic subjects had markedly higher levels of innate type 2 receptors on allergen-specific CD4+ T cells recruited into the airway. There were also increased levels of type 2 cytokines, increased total mucin and increased MUC5AC in response to allergen in the airways of AA subjects. Furthermore, type 2 cytokine levels correlated with the mucin response in AA but not AC subjects, suggesting differences in the airway epithelial response to inflammation. Finally, AA subjects had increased airway smooth muscle mass at baseline measured in vivo using novel orientation-registered optical coherence tomography (OR-OCT). Our data demonstrate that the development of allergic asthma is dependent on the responsiveness of allergen-specific CD4+ T cells to innate type 2 mediators as well as increased sensitivity of airway epithelial cells and smooth muscle to type 2 inflammation