Lucy E. Dalton

Diabetes as a disease of endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is an integral part of life for all professional secretory cells, but it has been studied to greatest depth in the pancreatic β‐cell. This reflects both the crucial role played by ER stress in the pathogenesis of diabetes and also the exquisite vulnerability of these cells to ER dysfunction. The adaptive cellular response to ER stress, the unfolded protein response, comprises mechanisms to both regulate new protein translation and a transcriptional program to allow adaptation to the stress. The core of this response is a triad of stress‐sensing proteins: protein kinase R‐like endoplasmic reticulum kinase (PERK), inositol‐requiring enzyme 1 (IRE1) and activating transcription factor 6. All three regulate portions of the transcriptional unfolded protein response, while PERK also attenuates protein synthesis during ER stress and IRE1 interacts directly with the c‐Jun amino‐terminal kinase stress kinase pathway. In this review we shall discuss these processes in detail, with emphasis given to their impact on diabetes and how recent findings indicate that ER stress may be responsible for the loss of β‐cell mass in the disease. Copyright

The endoplasmic reticulum stress marker CHOP predicts survival in malignant mesothelioma

Background:Mesothelioma is an incurable cancer originating from the mesothelial cells that line the pleural, peritoneal and pericardial cavities. These cells synthesise large quantities of surface glycoproteins, rendering them dependent upon efficient endoplasmic reticulum (ER) function. When faced with elevated levels of secretory protein load, cells are said to experience ER stress, which has been implicated in the pathogenesis of many human diseases including cancer.Method:We set out to measure markers of ER stress in malignant mesothelioma and to determine whether ER stress signalling correlates with clinical parameters.Results:We observed that expression of the ER stress-responsive transcription factor C/EBP homologous protein (CHOP) correlated with patient survival and remained an independent prognostic variable in pairwise comparisons with all clinical variables tested. The most parsimonious multivariate model in our study comprised only performance status and CHOP staining. In contrast, expression of the ER stress-responsive phosphatase growth arrest and DNA damage 34 (GADD34) correlated with the degree of mesothelial differentiation, being lost progressively in biphasic and sarcomatoid mesotheliomas.Conclusion:Our findings suggest that staining for CHOP provides prognostic information that may be useful in the stratification of patients with mesothelioma. Staining for GADD34 may prove useful in classification of mesothelioma histopathology.

Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation

Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actins role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR. DOI: http://dx.doi.org/10.7554/eLife.04872.001

Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to “ER stress” and activation of the “unfolded protein response” (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.

Phosphoproteins in stress-induced disease.

The integrated stress response (ISR) is an evolutionarily conserved homeostatic program activated by specific pathological states. These include amino acid deprivation, viral infection, iron deficiency, and the misfolding of proteins within the endoplasmic reticulum (ER), the so-called ER stress. Although apparently disparate, each of these stresses induces phosphorylation of a translation initiation factor, eIF2α, to attenuate new protein translation while simultaneously triggering a transcriptional program. This is achieved by four homologous stress-sensing kinases: GCN2, PKR, HRI, and PERK. In addition to these kinases, mammals possess two specific eIF2α phosphatases, GADD34 and CReP, which play crucial roles in the recovery of protein synthesis following the initial insult. They are not only important in embryonic development but also appear to play important roles in disease, particularly cancer. In this chapter, we discuss each of the eIF2α kinases, in turn, with particular emphasis on their regulation and the new insights provided by recent structural studies. We also discuss the potential for developing novel drug therapies that target the ISR.

p53 and translation attenuation regulate distinct cell cycle checkpoints during endoplasmic reticulum (ER) stress.

Background: ER stress impairs progression through G1 and G2 phases of the cell cycle. Results: G2 arrest is enhanced in p53 mutant cells but is not enhanced by expression of the p53/47 isoform. Conclusion: Early G2 arrest in ER stress is a response to translation attenuation. Significance: Understanding cell cycle regulation in ER stress has implications for rational cancer therapy. Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G1 phase, although recent studies have identified a novel ER stress G2 checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G2 delay involving CHK1 and a later induction of G1 arrest associated both with the induction of p53 target genes and loss of cyclin D1. We show that substitution of p53/47 for p53 impairs the ER stress G1 checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G2 progression prior to ultimate G1 arrest.

Coordinate regulation of eIF2α phosphorylation by PPP1R15 and GCN2 is required during Drosophila development

Summary Phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2&agr;) by the kinase GCN2 attenuates protein synthesis during amino acid starvation in yeast, whereas in mammals a family of related eIF2&agr; kinases regulate translation in response to a variety of stresses. Unlike single-celled eukaryotes, mammals also possess two specific eIF2&agr; phosphatases, PPP1R15a and PPP1R15b, whose combined deletion leads to a poorly understood early embryonic lethality. We report the characterisation of the first non-mammalian eIF2&agr; phosphatase and the use of Drosophila to dissect its role during development. The Drosophila protein demonstrates features of both mammalian proteins, including limited sequence homology and association with the endoplasmic reticulum. Of note, although this protein is not transcriptionally regulated, its expression is controlled by the presence of upstream open reading frames in its 5′UTR, enabling induction in response to eIF2&agr; phosphorylation. Moreover, we show that its expression is necessary for embryonic and larval development and that this is to oppose the inhibitory effects of GCN2 on anabolic growth.

Interactions between N-linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum

The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site‐directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early‐onset dementia. Our findings indicate that N401 glycosylation reports lability of the C‐terminal end of neuroserpin in its native state. This C‐terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant‐specific N‐linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin.

Unravelling the story of protein misfolding in diabetes mellitus

Both environmental and genetic factors contribute to the development of diabetes mellitus and although monogenic disorders are rare, they offer unique insights into the fundamental biology underlying the disease. Mutations of the insulin gene or genes involved in the response to protein misfolding cause early onset diabetes. These have revealed an important role for endoplasmic reticulum stress in β-cell survival. This form of cellular stress occurs when secretory proteins fail to fold efficiently. Of all the professional secretory cells we possess, β-cells are the most sensitive to endoplasmic reticulum stress because of the large fluctuations in protein synthesis they face daily. Studies of endoplasmic reticulum stress signaling therefore offer the potential to identify new drug targets to treat diabetes.

Spontaneous pneumothorax can be associated with TGFBR2 mutation.

Primary pneumothorax affects 0.01% of the population. 10% of cases have a family history of pneumothorax but in the majority, a definitive genetic diagnosis is not made. We report a 26-year-old, white British woman who presented with left apical pneumothorax (figure 1a). Previously, she had migraines, multiple stress fractures in her right foot, myopia, easy bruising, lumbar scoliosis and spontaneous dislocation of the right patella. She had no previous history of pneumothoraces or any other respiratory problems, and had never smoked. TGFBR2 mutations that cause Loeys-Dietz syndrome can present as pneumothorax http://ow.ly/SlMkS