Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress

Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson's disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death

IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress

IRE1β is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1β have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1β diminishes expression and inhibits signaling by the closely related stress sensor IRE1α. IRE1β can assemble with and inhibit IRE1α to suppress stress-induced XBP1 splicing, a key mediator of the unfolded protein response. In comparison to IRE1α, IRE1β has relatively weak XBP1 splicing activity, largely explained by a nonconserved amino acid in the kinase domain active site that impairs its phosphorylation and restricts oligomerization. This enables IRE1β to act as a dominant-negative suppressor of IRE1α and affect how barrier epithelial cells manage the response to stress at the host–environment interface

Ge quantum dot arrays grown by ultrahigh vacuum molecular beam epitaxy on the Si(001) surface: nucleation, morphology and CMOS compatibility

Issues of morphology, nucleation and growth of Ge cluster arrays deposited by ultrahigh vacuum molecular beam epitaxy on the Si(001) surface are considered. Difference in nucleation of quantum dots during Ge deposition at low (<600 deg C) and high (>600 deg. C) temperatures is studied by high resolution scanning tunneling microscopy. The atomic models of growth of both species of Ge huts---pyramids and wedges---are proposed. The growth cycle of Ge QD arrays at low temperatures is explored. A problem of lowering of the array formation temperature is discussed with the focus on CMOS compatibility of the entire process; a special attention is paid upon approaches to reduction of treatment temperature during the Si(001) surface pre-growth cleaning, which is at once a key and the highest-temperature phase of the Ge/Si(001) quantum dot dense array formation process. The temperature of the Si clean surface preparation, the final high-temperature step of which is, as a rule, carried out directly in the MBE chamber just before the structure deposition, determines the compatibility of formation process of Ge-QD-array based devices with the CMOS manufacturing cycle. Silicon surface hydrogenation at the final stage of its wet chemical etching during the preliminary cleaning is proposed as a possible way of efficient reduction of the Si wafer pre-growth annealing temperature.Comment: 30 pages, 11 figure

Ire1α-Regulated Rate of mRNA Translation is Required for Acquisition of Identity and Polarity in Upper Layer Cortical Neurons

Evolutionary expansion of the neocortex is associated with the increase in upper layer neurons. Here, we present Inositol-Requiring Enzyme 1α, Ire1α, as an essential determinant of upper layer fate, neuronal polarization and cortical lamination. We demonstrate a non-canonical function of Ire1α in the regulation of global translation rates in the developing neocortex through its dynamic interaction with the ribosome and regulation of eIF4A1 and eEF-2 expression. Inactivation of Ire1α engenders lower protein synthesis rates associated with stalled ribosomes and decreased number of translation start sites. We show unique sensitivity of upper layer fate to translation rates. Whereas eEF-2 is required for cortical lamination, eIF4A1 regulates acquisition of upper layer fate downstream of Ire1α in a mechanism of translational control dependent on 5’UTR-embedded structural elements in fate determinant genes. Our data unveil developmental regulation of ribosome dynamics as post-transcriptional mechanisms orchestrating neuronal diversity establishment and assembly of cortical layers

A transgenic mouse model for monitoring oxidative stress

Oxidative stress conditions enhance the production of reactive oxygen species resulting from a variety of stimuli, and are associated with various human diseases, including neurodegenerative disorders, inflammation, and various cancers. Though such associations have been closely studied using animal models, there has been no in vivo system for monitoring oxidative stress. We have developed an oxidative stress indicator that is dually regulated by induction at the transcriptional level, and by protein stabilisation at the post-translational level in Keap1-Nrf2 pathway. In vitro, our indicator elicited an intense and specific signal to oxidative stress among various agents, in a Keap1-Nrf2-dependent manner. Moreover, the transgenic animal expressing the indicator exhibited significant signals upon oxidative stress. These results indicate the usefulness of our system as an indicator of oxidative stress both in vitro and in vivo

Uterine selection of human embryos at implantation

Human embryos frequently harbor large-scale complex chromosomal errors that impede normal development. Affected embryos may fail to implant although many first breach the endometrial epithelium and embed in the decidualizing stroma before being rejected via mechanisms that are poorly understood. Here we show that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells. A stress response was also evident upon in vivo exposure of mouse uteri to culture medium conditioned by low-quality human embryos. By contrast, signals emanating from developmentally competent embryos activated a focused gene network enriched in metabolic enzymes and implantation factors. We further show that trypsin, a serine protease released by pre-implantation embryos, elicits Ca2+ signaling in endometrial epithelial cells. Competent human embryos triggered short-lived oscillatory Ca2+ fluxes whereas low-quality embryos caused a heightened and prolonged Ca2+ response. Thus, distinct positive and negative mechanisms contribute to active selection of human embryos at implantation

ER stress transcription factor Xbp1 suppresses intestinal tumorigenesis and directs intestinal stem cells

Unresolved endoplasmic reticulum (ER) stress in the epithelium can provoke intestinal inflammation. Hypomorphic variants of ER stress response mediators, such as X-box–binding protein 1 (XBP1), confer genetic risk for inflammatory bowel disease. We report here that hypomorphic Xbp1 function instructs a multilayered regenerative response in the intestinal epithelium. This is characterized by intestinal stem cell (ISC) expansion as shown by an inositol-requiring enzyme 1α (Ire1α)–mediated increase in Lgr5+ and Olfm4+ ISCs and a Stat3-dependent increase in the proliferative output of transit-amplifying cells. These consequences of hypomorphic Xbp1 function are associated with an increased propensity to develop colitis-associated and spontaneous adenomatous polyposis coli (APC)–related tumors of the intestinal epithelium, which in the latter case is shown to be dependent on Ire1α. This study reveals an unexpected role for Xbp1 in suppressing tumor formation through restraint of a pathway that involves an Ire1α- and Stat3-mediated regenerative response of the epithelium as a consequence of ER stress. As such, Xbp1 in the intestinal epithelium not only regulates local inflammation but at the same time also determines the propensity of the epithelium to develop tumors

CMOS-compatible dense arrays of Ge quantum dots on the Si(001) surface: hut cluster nucleation, atomic structure and array life cycle during UHV MBE growth

We report a direct observation of Ge hut nucleation on Si(001) during UHV molecular beam epitaxy at 360°C. Nuclei of pyramids and wedges were observed on the wetting layer (WL) (M × N) patches starting from the coverage of 5.1 Å and found to have different structures. Atomic models of nuclei of both hut species have been built as well as models of the growing clusters. The growth of huts of each species has been demonstrated to follow generic scenarios. The formation of the second atomic layer of a wedge results in rearrangement of its first layer. Its ridge structure does not repeat the nucleus. A pyramid grows without phase transitions. A structure of its vertex copies the nucleus. Transitions between hut species turned out to be impossible. The wedges contain point defects in the upper corners of the triangular faces and have preferential growth directions along the ridges. The derived structure of the {105} facet follows the paired dimer model. Further growth of hut arrays results in domination of wedges, and the density of pyramids exponentially drops. The second generation of huts arises at coverages >10 Å; new huts occupy the whole WL at coverages ~14 Å. Nanocrystalline Ge 2D layer begins forming at coverages >14 Å