Showing papers by "Fumihiko Urano published in 2011"

Measuring ER stress and the unfolded protein response using mammalian tissue culture system

Abstract: The endoplasmic reticulum (ER) functions to properly fold and process secreted and transmembrane proteins. Environmental and genetic factors that disrupt ER function cause an accumulation of misfolded and unfolded proteins in the ER lumen, a condition termed ER stress. ER stress activates a signaling network called the Unfolded Protein Response (UPR) to alleviate this stress and restore ER homeostasis, promoting cell survival and adaptation. However, under unresolvable ER stress conditions, the UPR promotes apoptosis. Here, we discuss the current methods to measure ER stress levels, UPR activation, and subsequent pathways in mammalian cells. These methods will assist us in understanding the UPR and its contribution to ER stress-related disorders such as diabetes and neurodegeneration.

Endoplasmic reticulum stress and pancreatic β-cell death

Abstract: In pancreatic β-cells, the endoplasmic reticulum (ER) is an important cellular compartment for insulin biosynthesis, which accounts for half of the total protein production in these cells Protein flux through the ER must be carefully monitored to prevent dysregulation of ER homeostasis and stress ER stress elicits a signaling cascade known as the unfolded protein response (UPR), which influences both life and death decisions in cells β-cell loss is a pathological component of both type 1 and type 2 diabetes, and recent findings suggest that ER stress is involved In this review, we address the transition from the physiological ER stress response to the pathological response, and explore the mechanisms of ER stress-mediated β-cell loss during the progression of diabetes

The IRE1α–XBP1 pathway is essential for osteoblast differentiation through promoting transcription of Osterix

Abstract: During skeletal development, osteoblasts produce large amounts of extracellular matrix proteins and must therefore increase their secretory machinery to handle the deposition. The accumulation of unfolded protein in the endoplasmic reticulum induces an adoptive mechanism called the unfolded protein response (UPR). We show that one of the most crucial UPR mediators, inositol-requiring protein 1α (IRE1α), and its target transcription factor X-box binding protein 1 (XBP1), are essential for bone morphogenic protein 2-induced osteoblast differentiation. Furthermore, we identify Osterix (Osx, a transcription factor that is indispensible for bone formation) as a target gene of XBP1. The promoter region of the Osx gene encodes two potential binding motifs for XBP1, and we show that XBP1 binds to these regions. Thus, the IRE1α-XBP1 pathway is involved in osteoblast differentiation through promoting Osx transcription.

IRE1-Dependent Activation of AMPK in Response to Nitric Oxide

Abstract: While there can be detrimental consequences of nitric oxide production at pathological concentrations, eukaryotic cells have evolved protective mechanisms to defend themselves against this damage. The unfolded-protein response (UPR), activated by misfolded proteins and oxidative stress, is one adaptive mechanism that is employed to protect cells from stress. Nitric oxide is a potent activator of AMP-activated protein kinase (AMPK), and AMPK participates in the cellular defense against nitric oxide-mediated damage in pancreatic β-cells. In this study, the mechanism of AMPK activation by nitric oxide was explored. The known AMPK kinases LKB1, CaMKK, and TAK1 are not required for the activation of AMPK by nitric oxide. Instead, this activation is dependent on the endoplasmic reticulum (ER) stress-activated protein IRE1. Nitric oxide-induced AMPK phosphorylation and subsequent signaling to AMPK substrates, including Raptor, acetyl coenzyme A carboxylase, and PGC-1α, is attenuated in IRE1α-deficient cells. The endoribonuclease activity of IRE1 appears to be required for AMPK activation in response to nitric oxide. In addition to nitric oxide, stimulation of IRE1 endoribonuclease activity with the flavonol quercetin leads to IRE1-dependent AMPK activation. These findings indicate that the RNase activity of IRE1 participates in AMPK activation and subsequent signaling through multiple AMPK-dependent pathways in response to nitrosative stress.

Subcellular Fractionation Using Step-Wise Density Extraction for Mass Spectrometry Analysis

Abstract: In recent years, significant technological advances in mass spectrometry instrumentation have moved towards higher sensitivity, resolution, and throughput. However, the ability to generate quantitative information on complex biological samples, especially for identification of low abundance proteins, subcellular compartment-specific protein expression and biomarker discovery, still depends upon the quality and the reproducibility of the sample preparation. Upstream fractionation of tissue or cell culture samples can play a key role in reducing the complexity of the sample. The ability to enrich selective fractions with proteins of interest can significantly expedite the identification of those proteins. We have developed a novel technology (Edge™) and instrumentation (Edge 200) platform for upstream proteomics sample preparation and fractionation, which uses stepwise density extraction of biological particles within a sample, based upon the densities of those particles.