Membrane proximal ectodomain cleavage of MUC16 occurs in the acidifying Golgi/post-Golgi compartments.

MUC16, precursor of the most widely used ovarian cancer biomarker CA125, is up regulated in multiple malignancies and is associated with poor prognosis. While the pro-tumorigenic and metastatic roles of MUC16 are ascribed to the cell-associated carboxyl-terminal MUC16 (MUC16-Cter), the exact biochemical nature of MUC16 cleavage generating MUC16-Cter has remained unknown. Using different lengths of dual-epitope (N-terminal FLAG- and C-terminal HA-Tag) tagged C-terminal MUC16 fragments, we demonstrate that MUC16 cleavage takes place in the juxta-membrane ectodomain stretch of twelve amino acids that generates a ~17 kDa cleaved product and is distinct from the predicted sites. This was further corroborated by domain swapping experiment. Further, the cleavage of MUC16 was found to take place in the Golgi/post-Golgi compartments and is dependent on the acidic pH in the secretory pathway. A similar pattern of ~17 kDa cleaved MUC16 was observed in multiple cell types eliminating the possibility of cell type specific phenomenon. MUC16-Cter translocates to the nucleus in a cleavage dependent manner and binds to the chromatin suggesting its involvement in regulation of gene expression. Taken together, we demonstrate for the first time the oft-predicted cleavage of MUC16 that is critical in designing successful therapeutic interventions based on MUC16

Impaired expression of protein phosphatase 2A subunits enhances metastatic potential of human prostate cancer cells through activation of AKT pathway.

BACKGROUND: Protein phosphatase 2A (PP2A) is a dephosphorylating enzyme, loss of which can contribute to prostate cancer (PCa) pathogenesis. The aim of this study was to analyse the transcriptional and translational expression patterns of individual subunits of the PP2A holoenzyme during PCa progression. METHODS: Immunohistochemistry (IHC), western blot, and real-time PCR was performed on androgen-dependent (AD) and androgen-independent (AI) PCa cells, and benign and malignant prostate tissues for all the three PP2A (scaffold, regulatory, and catalytic) subunits. Mechanistic and functional studies were performed using various biochemical and cellular techniques. RESULTS: Through immunohistochemical analysis we observed significantly reduced levels of PP2A-A and -B\u27γ subunits (P CONCLUSION: We conclude that loss of expression of scaffold and regulatory subunits of PP2A is responsible for its altered function during PCa pathogenesis

Retinoic Acid and Its Role in Modulating Intestinal Innate Immunity

Vitamin A (VA) is amongst the most well characterized food-derived nutrients with diverse immune modulatory roles. Deficiency in dietary VA has not only been associated with immune dysfunctions in the gut, but also with several systemic immune disorders. In particular, VA metabolite all-trans retinoic acid (atRA) has been shown to be crucial in inducing gut tropism in lymphocytes and modulating T helper differentiation. In addition to the widely recognized role in adaptive immunity, increasing evidence identifies atRA as an important modulator of innate immune cells, such as tolerogenic dendritic cells (DCs) and innate lymphoid cells (ILCs). Here, we focus on the role of retinoic acid in differentiation, trafficking and the functions of innate immune cells in health and inflammation associated disorders. Lastly, we discuss the potential involvement of atRA during the plausible crosstalk between DCs and ILCs

An Escherichia coli strain for expression of the connexin45 carboxyl terminus attached to the 4th transmembrane domain

A major problem for structural characterization of membrane proteins, such as connexins, by nuclear magnetic resonance (NMR) occurs at the initial step of the process, the production of sufficient amounts of protein. This occurs because proteins must be expressed in minimal based media. Here, we describe an expression system for membrane proteins that significantly improves yield by addressing two common problems, cell toxicity caused by protein translation and codon bias between genomes. This work provides researchers with a cost-effective tool for NMR and other biophysical studies, to use when faced with little-to-no expression of eukaryotic membrane proteins in Escherichia coli expression systems

Perfluorooctanesulfonic acid modulates barrier function and systemic T cell homeostasis during intestinal inflammation.

The intestinal epithelium is continuously exposed to deleterious environmental factors which might cause aberrant immune responses leading to inflammatory disorders. However, what environmental factors might contribute to disease are yet poorly understood. Here, to overcome the lack of in vivo models suitable for screening of environmental factors we used zebrafish reporters of intestinal inflammation. Using zebrafish, we interrogated the immunomodulatory effects of polyfluoroalkyl substances (PFAS), which have been positively associated with ulcerative colitis incidence. Exposure with perfluorooctanesulfonic acid (PFOS) during TNBS-induced inflammation enhances the expression of proinflammatory cytokines as well as neutrophil recruitment to the intestine of zebrafish larvae, which was validated in TNBS-induced colitis mice models. Moreover, PFOS exposure in mice undergoing colitis resulted in neutrophil-dependent increased intestinal permeability and enhanced PFOS translocation into circulation. Finally, this was associated with a neutrophil dependent expansion of systemic CD4+ T cells. Thus, our results indicate that PFOS worsens inflammation-induced intestinal damage with disruption of T cell homeostasis beyond the gut and provides a novel in vivo toolbox to screen for pollutants affecting intestinal homeostasis