Ubiquitin-like protein 5 is a novel player in the UPR-PERK arm and ER stress-induced cell death.

Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In Caenorhabditis elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPR). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-UPR in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like ER kinase arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells in vivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-protein kinase R-like ER kinase pathway, linking ER stress to cell death.

Histopathological Diagnosis of Nonalcoholic Steatohepatitis (NASH).

Fatty acid beta oxidation (FAO) is a predominant bioenergetic pathway in mammals. Substantial investigations have demonstrated that FAO activity is dysregulated in many pathophysiological conditions including nonalcoholic steatohepatitis (NASH). Convenient and quantitative assays of FAO activities are important for studies of cell metabolism and the biological relevance of FAO to health and diseases. However, most current FAO assays are based on non-physiological culture conditions, measure FAO activity indirectly or lack adequate quantification. We herein describe details of practical protocols for measurement of basal and genetically or pharmacologically regulated FAO activities in the mammalian system. We also discuss the advantages and disadvantages of these assays in the context of experimental purposes.

Lysophosphatidic acid induces tumor necrosis factor-alpha to regulate a pro-inflammatory cytokine network in ovarian cancer.

Epithelial ovarian carcinoma tissues express high levels of tumor necrosis factor-alpha (TNF-α) and other inflammatory cytokines. The underlying mechanism leading to the abnormal TNF-α expression in ovarian cancer remains poorly understood. In the current study, we demonstrated that lysophosphatidic acid (LPA), a lipid mediator present in ascites of ovarian cancer patients, induced expression of TNF-α mRNA and release of TNF-α protein in ovarian cancer cells. LPA also induced expression of interleukin-1ß (IL-1ß) mRNA but no significant increase in IL-1ß protein was detected. LPA enhanced TNF-α mRNA through NF-κB-mediated transcriptional activation. Inactivation of ADAM17, a disintegrin and metalloproteinase, with a specific inhibitor TMI-1 or by shRNA knockdown prevented ovarian cancer cells from releasing TNF-α protein in response to LPA, indicating that LPA-mediated TNF-α production relies on both transcriptional upregulations of the TNF-α gene and the activity of ADAM17, the membrane-associated TNF-α-converting enzyme. Like many other biological responses to LPA, induction of TNF-α by LPA also depended on the transactivation of the epidermal growth factor receptor (EGFR). Interestingly, our results revealed that ADAM17 was also the shedding protease responsible for the transactivation of EGFR by LPA in ovarian cancer cells. To explore the biological outcomes of LPA-induced TNF-α, we examined the effects of a TNF-α neutralizing antibody and recombinant TNF-α soluble receptor on LPA-stimulated expression of pro-tumorigenic cytokines and chemokines overexpressed in ovarian cancer. Blockade of TNF-α signaling significantly reduced the production of IL-8, IL-6, and CXCL1, suggesting a hierarchy of mechanisms contributing to the robust expression of the inflammatory mediators in response to LPA in ovarian cancer cells. In contrast, TNF-α inhibition did not affect LPA-dependent cell proliferation. Taken together, our results establish that the bioactive lipid LPA drives the expression of TNF-α to regulate an inflammatory network in ovarian cancer.