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1.  A conformational RNA zipper promotes intron ejection during non‐conventional XBP1 mRNA splicing 
EMBO Reports  2015;16(12):1688-1698.
Abstract
The kinase/endonuclease IRE1 is the most conserved signal transducer of the unfolded protein response (UPR), an intracellular signaling network that monitors and regulates the protein folding capacity of the endoplasmic reticulum (ER). Upon sensing protein folding perturbations in the ER, IRE1 initiates the unconventional splicing of XBP1 mRNA culminating in the production of the transcription factor XBP1s, which expands the ER's protein folding capacity. We show that an RNA‐intrinsic conformational change causes the intron of XBP1 mRNA to be ejected and the exons to zipper up into an extended stem, juxtaposing the RNA ends for ligation. These conformational rearrangements are important for XBP1 mRNA splicing in vivo. The features that point to such active participation of XBP1 mRNA in the splicing reaction are highly conserved throughout metazoan evolution, supporting their importance in orchestrating XBP1 mRNA processing with efficiency and fidelity.
doi:10.15252/embr.201540955
PMCID: PMC4687415  PMID: 26483401
endoribonuclease; ER stress; RNA conformational change; unfolded protein response; XBP1 splicing; Protein Biosynthesis & Quality Control; RNA Biology
2.  Dynamics of co-translational protein targeting 
Most membrane and secretory proteins are delivered co-translationally to protein translocation channels in their destination membrane by the signal recognition particle (SRP) and its receptor. This co-translational molecular machinery is conserved across all kingdoms of life, though it varies in composition and function. Here we report recent progress towards understanding the mechanism of SRP function, focusing on findings about E. coli SRP’s conformational dynamics throughout the targeting process. These insights shed light on a key checkpoint in the targeting cycle: how SRP regulates engagement of an actively translating ribosome with the translocation machinery at the membrane.
doi:10.1016/j.cbpa.2015.09.016
PMCID: PMC4684440  PMID: 26517565
3.  Galvanic Exchange in Colloidal Metal/Metal-Oxide Core/Shell Nanocrystals 
While galvanic exchange is commonly applied to metallic nanoparticles, recently its applicability was expanded to metal-oxides. Here the galvanic exchange is studied in metal/metal-oxide core/shell nanocrystals. In particular Sn/SnO2 is treated by Ag+, Pt2+, Pt4+, and Pd2+. The conversion dynamics is monitored by in situ synchrotron X-ray diffraction. The Ag+ treatment converts the Sn cores to the intermetallic AgxSn (x ∼ 4) phase, by changing the core’s crystal structure. For the analogous treatment by Pt2+, Pt4+, and Pd2+, such a galvanic exchange is not observed. This different behavior is caused by the semipermeability of the naturally formed SnO2 shell, which allows diffusion of Ag+ but protects the nanocrystal cores from oxidation by Pt and Pd ions.
doi:10.1021/acs.jpcc.6b06405
PMCID: PMC5018861  PMID: 27635186
4.  Calorie for calorie, dietary fat restriction results in more body fat loss than carbohydrate restriction in people with obesity 
Cell metabolism  2015;22(3):427-436.
Summary
Dietary carbohydrate restriction has been purported to cause endocrine adaptations that promote body fat loss more than dietary fat restriction. We selectively restricted dietary carbohydrate versus fat for 6 days following a 5 day baseline diet in 19 adults with obesity confined to a metabolic ward where they exercised daily. Subjects received both isocaloric diets in random order during each of two inpatient stays. Body fat loss was calculated as the difference between daily fat intake and net fat oxidation measured while residing in a metabolic chamber. Whereas carbohydrate restriction led to sustained increases in fat oxidation and loss of 53±6 g/d of body fat, fat oxidation was unchanged by fat restriction leading to 89±6 g/d of fat loss and was significantly greater than carbohydrate restriction (p=0.002). Mathematical model simulations agreed with these data, but predicted that the body acts to minimize body fat differences with isocaloric diets varying in carbohydrate and fat.
doi:10.1016/j.cmet.2015.07.021
PMCID: PMC4603544  PMID: 26278052
5.  Financial burden and quality of life of informal caregivers of patients with wet age-related macular degeneration 
Purpose
The purpose of this research is to quantify the cost burden, care times and the impact on the quality of life (QoL) of informal caring relatives caring for patients with wet age-related macular degeneration (wet AMD). Moreover we investigated the impact of care times on the QoL.
Methods
Through a specifically designed questionnaire, 150 caring relatives were interviewed retrospectively on all accrued financial costs, caring times incurred and the current QoL, assessed by a Visual Analogue Scale for happiness (VAS).
Results
The caring time incurred was on average 6.4 ± 8.5 (mean +/- SD) hours per week. The QoL was on average rated at 6.7 ± 1.9 on a ten point scale. Financial strain was incurred by the direct non-medical costs of on average € 405 ± 1104 and the direct medical costs of on average € 134 ± 340 per year. Indirect costs were stated by two caregivers as amounting to € 2400 and € 6000 net income loss per year respectively. Caregivers of privately insured patients with wet AMD carried a financial cost burden which was up to six times higher than caregivers of patients who were on state insurance while showing the same visual acuity.
Conclusion
The evaluation shows that caregivers of privately insured patients with wet AMD have higher costs than caregivers of patients with state insurance coverage. This burden seems to be a factor to be considered independently since it does not appear to have any relation to patients AMD acuity.
doi:10.1186/s13561-016-0116-4
PMCID: PMC4999382  PMID: 27562805
Age-related macular degeneration; Caregivers; Costs; Quality of life
6.  Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation 
eLife  null;5:e15550.
Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases.
DOI: http://dx.doi.org/10.7554/eLife.15550.001
eLife digest
Newly made proteins must be folded into specific three-dimensional shapes before they can perform their roles in cells. Many proteins are folded in a compartment called the endoplasmic reticulum before being transported to their final location. However, if a cell suddenly needs to make a large number of new proteins, it can overwhelm the endoplasmic reticulum and unfolded proteins may accumulate. The cell responds to this stress by activating the unfolded protein response, which increases the folding capacity of the endoplasmic reticulum to match the demand. However, if the stress persists, then the unfolded protein response instructs the cell to die to protect the rest of the body.
A protein called ATF6 is involved in one branch of the unfolded protein response. Endoplasmic reticulum stress causes ATF6 to move from the endoplasmic reticulum to another cell compartment where certain enzymes are able to cut the protein. A fragment of ATF6 then moves to the nucleus of the cell to activate genes needed to increase the cell’s capacity to fold proteins.
Errors in protein folding can cause serious diseases in humans and other animals. Drugs that target ATF6 might be able to regulate part of the unfolded protein response to treat these diseases. However, no drugs that act on ATF6 had been identified. Now, two groups of researchers have independently identified small molecules that specifically target ATF6.
Plate et al. used a new approach to screen over 600,000 small molecules and identified a small number that could activate ATF6-regulated genes without inducing global endoplasmic reticulum stress. Further experiments tested whether any of these ATF6 drug candidates could prevent the release of incorrectly folded versions of two particular proteins from cells that are associated with types of amyloid disease in humans. One of the small molecules tested effectively reduced the release of these proteins and prevented harmful deposits of the proteins forming in the spaces surrounding cells.
In an independent study, Gallagher et al. identified a type of small molecule that can inhibit the activity of ATF6. Together, these findings may lead to further development of new drugs for treating diseases associated with incorrect protein folding in the endoplasmic reticulum.
DOI: http://dx.doi.org/10.7554/eLife.15550.002
doi:10.7554/eLife.15550
PMCID: PMC4954754  PMID: 27435961
patient-derived plasma cells; HEK293 cells; HepG2 cells; mouse embryonic fibroblasts; None
7.  Ceapins inhibit ATF6α signaling by selectively preventing transport of ATF6α to the Golgi apparatus during ER stress 
eLife  null;5:e11880.
The membrane-bound transcription factor ATF6α is activated by proteolysis during endoplasmic reticulum (ER) stress. ATF6α target genes encode foldases, chaperones, and lipid biosynthesis enzymes that increase protein-folding capacity in response to demand. The off-state of ATF6α is maintained by its spatial separation in the ER from Golgi-resident proteases that activate it. ER stress induces trafficking of ATF6α. We discovered Ceapins, a class of pyrazole amides, as selective inhibitors of ATF6α signaling that do not inhibit the Golgi proteases or other UPR branches. We show that Ceapins block ATF6α signaling by trapping it in ER-resident foci that are excluded from ER exit sites. Removing the requirement for trafficking by pharmacological elimination of the spatial separation of the ER and Golgi apparatus restored cleavage of ATF6α in the presence of Ceapins. Washout of Ceapins resensitized ATF6α to ER stress. These results suggest that trafficking of ATF6α is regulated by its oligomeric state.
DOI: http://dx.doi.org/10.7554/eLife.11880.001
eLife digest
Newly made proteins must be folded into specific three-dimensional shapes before they can perform their roles in cells. Many proteins are folded in a cell compartment called the endoplasmic reticulum. The cell closely monitors the quality of the work done by this compartment. If the endoplasmic reticulum has more proteins to fold than it can handle, unfolded or misfolded proteins accumulate and trigger a stress response called the unfolded protein response. This increases the capacity of the endoplasmic reticulum to fold proteins to match the demand. However, if the stress persists, then the unfolded protein response instructs the cell to die to protect the rest of the body.
A protein called ATF6α is one of three branches of the unfolded protein response. This protein is found in the endoplasmic reticulum where it is inactive. Endoplasmic stress causes ATF6α to move from the endoplasmic reticulum to another compartment called the Golgi apparatus. There, two enzymes cut ATF6α to release a fragment of the protein that then moves to the nucleus to increase the production of the machinery needed to fold proteins in the endoplasmic reticulum.
In a related study, Gallagher et al. identified a group of small molecules called Ceapins, which inhibit ATF6α activity. Here, Gallagher and Walter investigate how Ceapins act on ATF6α. The experiments show that Ceapin causes ATF6α molecules to form clusters that prevent the protein from moving to the Golgi apparatus by keeping it away from the machinery that moves proteins between these compartments. When the enzymes that cut ATF6α are sent to the endoplasmic reticulum, Ceapin treatment no longer prevents ATF6α activation, which shows that these small molecules specifically inhibit the stress-induced movement of ATF6α. When Ceapins are washed out of cells, the ATF6α clusters fall apart and ATF6α can now move to the Golgi.
These experiments show that ATF6α is actively held in the endoplasmic reticulum by a mechanism that is stabilized by Ceapins. Gallagher and Walter propose that the small clusters of ATF6α in unstressed cells act to keep this protein in the endoplasmic reticulum. However, when cells experience stress, the ATF6α clusters fall apart to allow the protein to move to the Golgi. The next steps following on from this work are to find out what these clusters are, how they are influenced by endoplasmic reticulum stress and exactly how the Ceapins stabilize these clusters.
DOI: http://dx.doi.org/10.7554/eLife.11880.002
doi:10.7554/eLife.11880
PMCID: PMC4954756  PMID: 27435962
endoplasmic reticulum; unfolded protein response; ATF6-alpha; ER to Golgi trafficking; small molecule inhibitors; ER stress; Human
8.  Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch 
eLife  null;5:e11878.
The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6β or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination.
DOI: http://dx.doi.org/10.7554/eLife.11878.001
eLife digest
Newly made proteins must be folded into specific three-dimensional shapes before they can perform their roles in cells. Many proteins are folded in a cell compartment called the endoplasmic reticulum. The cell closely monitors the quality of the work done by this compartment. If the endoplasmic reticulum has more proteins to fold than it can handle, unfolded or misfolded proteins accumulate and trigger a stress response called the unfolded protein response. This increases the capacity of the endoplasmic reticulum to fold proteins to match the demand. However, if the stress persists, then the unfolded protein response instructs the cell to die to protect the rest of the body.
A protein called ATF6α is one of three branches of the unfolded protein response. This protein is found in the endoplasmic reticulum where it is inactive. Endoplasmic stress causes ATF6α to move from the endoplasmic reticulum to another compartment called the Golgi apparatus. There, two enzymes cut ATF6α to release a fragment of the protein that then moves to the nucleus to increase the production of the machinery needed to fold proteins in the endoplasmic reticulum.
Errors in protein folding can cause serious diseases in humans and other animals. Drugs that target ATF6α might be able to regulate part of the unfolded protein response to treat these diseases. However, no drugs that act on ATF6α had been identified. Now, two groups of researchers have independently identified small molecules that specifically target ATF6α.
Gallagher et al. screened over 100,000 compounds for their ability to reduce the activity of ATF6α-regulated genes. The experiments reveal that a class of small molecules termed Ceapins can selectively block the activity of ATF6α during endoplasmic reticulum stress, but had no effect on other proteins involved in the unfolded protein response. Furthermore, when human cells experiencing stress were treated with Ceapins, a greater number of cells died in comparison to cells that had not received Ceapins. An accompanying study by Gallagher and Walter reports on the mechanism by which Ceapins act on ATF6α.
Independently, Plate et al. identified a type of small molecule that can activate ATF6. Together, the findings of Gallagher et al. and Plate et al. may lead to the development of new drugs for treating diseases associated with incorrect protein folding in the endoplasmic reticulum.
DOI: http://dx.doi.org/10.7554/eLife.11878.002
doi:10.7554/eLife.11878
PMCID: PMC4954757  PMID: 27435960
endoplasmic reticulum; unfolded protein response; small molecule screening; ATF6-alpha; site-1-protease; ER to Golgi trafficking; Human
9.  Translation from the 5′ untranslated region shapes the integrated stress response 
Science (New York, N.Y.)  2016;351(6272):aad3867.
Translated regions distinct from annotated coding sequences have emerged as essential elements of the proteome. This includes upstream open reading frames (uORFs) present in mRNAs controlled by the integrated stress response (ISR) that show “privileged” translation despite inhibited eukaryotic initiation factor 2–guanosine triphosphate–initiator methionyl transfer RNA (eIF2·GTP·Met-tRNAiMet). We developed tracing translation by T cells to directly measure the translation products of uORFs during the ISR. We identified signature translation events from uORFs in the 5′ untranslated region of binding immunoglobulin protein (BiP) mRNA (also called heat shock 70-kilodalton protein 5mRNA) that were not initiated at the start codon AUG. BiP expression during the ISR required both the alternative initiation factor eIF2A and non–AUG-initiated uORFs. We propose that persistent uORF translation, for a variety of chaperones, shelters select mRNAs from the ISR, while simultaneously generating peptides that could serve as major histocompatibility complex class I ligands, marking cells for recognition by the adaptive immune system.
doi:10.1126/science.aad3867
PMCID: PMC4882168  PMID: 26823435
10.  Properties of Retinal Precursor Cells Grown on Vertically Aligned Multiwalled Carbon Nanotubes Generated for the Modification of Retinal Implant-Embedded Microelectrode Arrays 
Journal of Ophthalmology  2016;2016:2371021.
Background. To analyze the biocompatibility of vertically aligned multiwalled carbon nanotubes (MWCNT), used as nanomodification to optimize the properties of prostheses-embedded microelectrodes that induce electrical stimulation of surviving retinal cells. Methods. MWCNT were synthesized on silicon wafers. Their growth was achieved by iron particles (Fe) or mixtures of iron-platinum (Fe-Pt) and iron-titanium (Fe-Ti) acting as catalysts. Viability, growth, adhesion, and gene expression of L-929 and retinal precursor (R28) cells were analyzed after nondirect and direct contact. Results. Nondirect contact had almost no influence on cell growth, as measured in comparison to reference materials with defined levels of cytotoxicity. Both cell types exhibited good proliferation properties on each MWCNT-coated wafer. Viability ranged from 95.9 to 99.8%, in which better survival was observed for nonfunctionalized MWCNT generated with the Fe-Pt and Fe-Ti catalyst mixtures. R28 cells grown on the MWCNT-coated wafers showed a decreased gene expression associated with neural and glial properties. Expression of the cell cycle-related genes CCNC, MYC, and TP53 was slightly downregulated. Cultivation on plasma-treated MWCNT did not lead to additional changes. Conclusions. All tested MWCNT-covered slices showed good biocompatibility profiles, confirming that this nanotechnology is a promising tool to improve prostheses bearing electrodes which connect with retinal tissue.
doi:10.1155/2016/2371021
PMCID: PMC4856943  PMID: 27200182
11.  ER–mitochondrial junctions can be bypassed by dominant mutations in the endosomal protein Vps13 
The Journal of Cell Biology  2015;210(6):883-890.
Substitutions in Vps13 suppress all measured phenotypic consequences of ERMES deficiency, and Vps13 dynamically localizes to vacuole–mitochondria and to vacuole–nucleus contact sites depending on growth conditions, suggesting that ERMES function can be bypassed by the activity of other contact sites, and that contact sites establish a growth condition–regulated organelle network.
The endoplasmic reticulum–mitochondria encounter structure (ERMES) complex tethers the endoplasmic reticulum and the mitochondria. It is thought to facilitate interorganelle lipid exchange and influence mitochondrial dynamics and mitochondrial DNA maintenance. Despite this important role, ERMES is not found in metazoans. Here, we identified single amino acid substitutions in Vps13 (vacuolar protein sorting 13), a large universally conserved eukaryotic protein, which suppress all measured phenotypic consequences of ERMES deficiency. Combined loss of VPS13 and ERMES is lethal, indicating that Vps13 and ERMES function in redundant pathways. Vps13 dynamically localizes to vacuole–mitochondria and to vacuole–nucleus contact sites depending on growth conditions, suggesting that ERMES function can be bypassed by the activity of other contact sites, and that contact sites establish a growth condition–regulated organelle network.
doi:10.1083/jcb.201502105
PMCID: PMC4576869  PMID: 26370498
12.  Translational control of nicotine-evoked synaptic potentiation in mice and neuronal responses in human smokers by eIF2α 
eLife  null;5:e12056.
Adolescents are particularly vulnerable to nicotine, the principal addictive component driving tobacco smoking. In a companion study, we found that reduced activity of the translation initiation factor eIF2α underlies the hypersensitivity of adolescent mice to the effects of cocaine. Here we report that nicotine potentiates excitatory synaptic transmission in ventral tegmental area dopaminergic neurons more readily in adolescent mice compared to adults. Adult mice with genetic or pharmacological reduction in p-eIF2α-mediated translation are more susceptible to nicotine’s synaptic effects, like adolescents. When we investigated the influence of allelic variability of the Eif2s1 gene (encoding eIF2α) on reward-related neuronal responses in human smokers, we found that a single nucleotide polymorphism in the Eif2s1 gene modulates mesolimbic neuronal reward responses in human smokers. These findings suggest that p-eIF2α regulates synaptic actions of nicotine in both mice and humans, and that reduced p-eIF2α may enhance susceptibility to nicotine (and other drugs of abuse) during adolescence.
DOI: http://dx.doi.org/10.7554/eLife.12056.001
eLife digest
Nicotine addiction is a serious public health problem. People who start using nicotine during adolescence are more likely to become addicted to it during adulthood, but the reasons for this are not well understood. Nicotine causes long-lasting changes in the brain that are responsible for the feelings of pleasure and reward. In particular, nicotine strengthens the connections between neurons at structures called synapses and increases communication between reward-related neurons in key reward areas of the brain. This hijacking of the natural reward system requires new proteins to be made. However, the relationship between protein synthesis and adolescents being particularly vulnerable to nicotine addiction was not known.
In a related study, Huang et al. found that the reduced activity of a protein called eIF2α, which controls the production of new proteins, accounts for why adolescents are more likely to become addicted to cocaine than adults. Thus, Placzek et al. wanted to know whether the same was true for nicotine and whether the proteins controlled by eIF2α are involved in the way human nicotine addicts experience reward.
Placzek et al. found that adolescent mice are more susceptible than adult mice to the changes in synaptic strength that are caused by nicotine. This increased susceptibility results from reduced activity levels of the protein eIF2α. Reducing the activity of eIF2α in adult mice made their synapses as likely to change strength in response to nicotine as the synapses of adolescent mice.
Placzek et al. also used a technique called functional magnetic resonance imaging and found that compared to non-smokers, the brain activity of human smokers was significantly reduced when given a natural reward. Further studies revealed a variation in the gene encoding the eIF2α protein that affects how smokers respond to a reward, suggesting that this variant is linked to the likelihood that a person will be addicted to nicotine.
This work raises several important questions. In addition to regulating the initial adaptive changes induced in the brain by nicotine, does eIF2α activity affect compulsive nicotine use? If so, could targeting parts of the eIF2α pathway help treat nicotine addiction? Finally, further studies could explore whether the gene variant identified by Placzek et al. affects how users of other drugs (such as cocaine or alcohol) respond to natural rewards.
DOI: http://dx.doi.org/10.7554/eLife.12056.002
doi:10.7554/eLife.12056
PMCID: PMC4786418  PMID: 26928076
nicotine-induced plasticity; protein synthesis; ventral tegmental area; human fMRI; adolescents; Human; Mouse
13.  Translational control by eIF2α phosphorylation regulates vulnerability to the synaptic and behavioral effects of cocaine 
eLife  null;5:e12052.
Adolescents are especially prone to drug addiction, but the underlying biological basis of their increased vulnerability remains unknown. We reveal that translational control by phosphorylation of the translation initiation factor eIF2α (p-eIF2α) accounts for adolescent hypersensitivity to cocaine. In adolescent (but not adult) mice, a low dose of cocaine reduced p-eIF2α in the ventral tegmental area (VTA), potentiated synaptic inputs to VTA dopaminergic neurons, and induced drug-reinforced behavior. Like adolescents, adult mice with reduced p-eIF2α-mediated translational control were more susceptible to cocaine-induced synaptic potentiation and behavior. Conversely, like adults, adolescent mice with increased p-eIF2α became more resistant to cocaine's effects. Accordingly, metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD)—whose disruption is postulated to increase vulnerability to drug addiction—was impaired in both adolescent mice and adult mice with reduced p-eIF2α mediated translation. Thus, during addiction, cocaine hijacks translational control by p-eIF2α, initiating synaptic potentiation and addiction-related behaviors. These insights may hold promise for new treatments for addiction.
DOI: http://dx.doi.org/10.7554/eLife.12052.001
eLife digest
Drug addiction a is major mental health problem that presents a huge financial, social and legal burden worldwide. Adolescents are notoriously prone to drug abuse and addicts typically begin using drugs at a young age. However, an explanation for why young people are particularly vulnerable to the effects of addictive substances remains elusive.
Addictive drugs change how the brain works, in particular by strengthening the connections (synapses) between brain cells (neurons) and making it easier for neurons to communicate with each other. Such strengthening of synaptic connections, which can be observed when the activity of the neurons is recorded with microelectrodes, relies on new proteins being made in the brain. Since adolescents have a greater capacity than adults to make new proteins, Huang et al. hypothesized that changes in synaptic strength might occur more easily in the brain of adolescents, explaining why they are more likely to become addicted to drugs than adults.
A protein called eIF2α plays a key role in regulating the production of new proteins. Huang et al. discovered that reduced eIF2α activity accounts for why adolescents are particularly vulnerable to the synaptic and behavioral effects of cocaine. Giving adolescent mice a low dose of cocaine reduced the activity of eIF2α, caused an increase in the strength of synaptic connections in a part of the brain that processes pleasurable feelings, and promoted drug-reinforced behavior. This did not occur in adult mice.
Reducing the activity of eIF2α using either genetics or pharmacological methods caused adult mice to become as vulnerable as adolescents to cocaine-induced changes in synaptic strength and addiction-related behavior. Conversely, increasing the activity of eIF2α made adolescent mice more resistant to cocaine’s effects; in other words, adolescents responded to cocaine more like adults.
Huang et al. also found that other drugs of abuse, including alcohol, methamphetamine and nicotine, all reduce eIF2α activity, suggesting that eIF2α is a common target of different drugs of abuse. In a related study, Placzek et al. investigated the role of eIF2α in nicotine addiction in mice and humans.
These findings raise several intriguing questions. How do cocaine and other drugs of abuse reduce eIF2α activity? Could variations in the activity of eIF2α or other components of the eIF2α pathway in the brain explain why some people are more likely to abuse drugs? Finally, could compounds that regulate the activity of eIF2α be useful for treating addiction?
DOI: http://dx.doi.org/10.7554/eLife.12052.002
doi:10.7554/eLife.12052
PMCID: PMC4786430  PMID: 26928234
ventral tegemental area; long-term potentiation (LTP); long-term depression (LTD); protein synthesis; cocaine; Mouse
14.  Validation of the Hsp70-Bag3 Protein-Protein Interaction as a Potential Therapeutic Target in Cancer 
Molecular cancer therapeutics  2015;14(3):642-648.
Heat shock protein 70 (Hsp70) is a stress-inducible molecular chaperone that is required for cancer development at several steps. Targeting the active site of Hsp70 has proven relatively challenging, driving interest in alternative approaches. Hsp70 collaborates with the Bcl2-associated athanogene 3 (Bag3) to promote cell survival through multiple pathways, including FoxM1. Therefore, inhibitors of the Hsp70-Bag3 protein-protein interaction (PPI) may provide a non-canonical way to target this chaperone. We report that JG-98, an allosteric inhibitor of this PPI, indeed has anti-proliferative activity (EC50 values between 0.3 and 4 μM) across cancer cell lines from multiple origins. JG-98 destabilized FoxM1 and relieved suppression of downstream effectors, including p21 and p27. Based on these findings, JG-98 was evaluated in mice for pharmacokinetics, tolerability and activity in two xenograft models. The results suggested that the Hsp70-Bag3 interaction may be a promising, new target for anti-cancer therapy.
doi:10.1158/1535-7163.MCT-14-0650
PMCID: PMC4456214  PMID: 25564440
15.  A conformational RNA zipper promotes intron ejection during non-conventional XBP1 mRNA splicing 
EMBO Reports  2015;16(12):1688-1698.
The kinase/endonuclease IRE1 is the most conserved signal transducer of the unfolded protein response (UPR), an intracellular signaling network that monitors and regulates the protein folding capacity of the endoplasmic reticulum (ER). Upon sensing protein folding perturbations in the ER, IRE1 initiates the unconventional splicing of XBP1 mRNA culminating in the production of the transcription factor XBP1s, which expands the ER's protein folding capacity. We show that an RNA-intrinsic conformational change causes the intron of XBP1 mRNA to be ejected and the exons to zipper up into an extended stem, juxtaposing the RNA ends for ligation. These conformational rearrangements are important for XBP1 mRNA splicing in vivo. The features that point to such active participation of XBP1 mRNA in the splicing reaction are highly conserved throughout metazoan evolution, supporting their importance in orchestrating XBP1 mRNA processing with efficiency and fidelity.
doi:10.15252/embr.201540955
PMCID: PMC4687415  PMID: 26483401
endoribonuclease; ER stress; RNA conformational change; unfolded protein response; XBP1 splicing
16.  Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo 
BioMed Research International  2015;2015:863845.
Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth.
doi:10.1155/2015/863845
PMCID: PMC4678073  PMID: 26697494
17.  The Role of Specifically Tailored Communication Training Among Factors Influencing Consent for Cornea Donation Requested Via Telephone 
Transplantation  2015;99(10):2223-2229.
Background
Consent for cornea donation from non–heart-beating donors is often requested by means of telephone interviews with relatives of the deceased. The purpose of this study was to identify the effect of specifically tailored interviewer communications training among other factors related to obtaining consent.
Methods
A retrospective analysis of consent requests made by the Aachen Cornea Bank was performed. Interviews were conducted via telephone by 26 ophthalmic residents or fellows in accordance with German and European laws and guidelines. Multiple logistic regression was used to identify factors related to the consent. Results were expressed as odds ratios (OR), 95% confidence interval (95% CI), and Wald P value.
Results
In 1694 interviews, the overall consent rate was 50.12%. Multivariate analysis identified 4 significant factors associated with the donation decision. Completion of a specific communication training seminar increased odds of consent by 1.533 (95% CI, 1.250-1.880; P < 0.0001). The individual interviewer had an impact on the OR of up to 1.255 (95% CI, 1.113-1.417; P = 0.0002). The odds for consent were inversely correlated with the clinical experience of the interviewer (OR, 0.884; 95% CI, 0.831-0.938; P < 0.0001). The consulted family member was 0.894 times less likely to grant consent for donation with each increase in degree of blood relation (95% CI, 0.849-0.940; P < 0.0001).
Conclusions
A telephone interview is effective for obtaining consent for cornea donation. The consent decision may be associated with factors relating to the interviewer as well as the consulted family member. Specific training for cornea bank staff may increase the odds of obtaining consent.
Can specific training assist people seeking consent to corneal donation by telephone? Some people are just better at it, but training, clinical experience and who is on each end of the phone are shown to influence the consent rate.
doi:10.1097/TP.0000000000000707
PMCID: PMC4617284  PMID: 25839709
18.  Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits 
eLife  null;4:e08153.
Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM patients invariably acquire resistance to these drugs. Using a next-generation shRNA platform, we found that proteostasis factors, including chaperones and stress-response regulators, controlled the response to carfilzomib. Paradoxically, 19S proteasome regulator knockdown induced resistance to carfilzomib in MM and non-MM cells. 19S subunit knockdown did not affect the activity of the 20S subunits targeted by carfilzomib nor their inhibition by the drug, suggesting an alternative mechanism, such as the selective accumulation of protective factors. In MM patients, lower 19S levels predicted a diminished response to carfilzomib-based therapies. Together, our findings suggest that an understanding of network rewiring can inform development of new combination therapies to overcome drug resistance.
DOI: http://dx.doi.org/10.7554/eLife.08153.001
eLife digest
Cells have several mechanisms for removing proteins that have been damaged or are no longer needed. One of these mechanisms is carried out by a large protein complex called the proteasome. Drugs that block the proteasome are toxic to all cells, and a type of blood cancer called multiple myeloma is particularly sensitive to these ‘proteasome inhibitors’. However, tumors in patients with multiple myeloma can also become resistant to these drugs.
Using a genetic approach, Acosta-Alvear et al. identified the factors that control the sensitivity of cells to proteasome inhibitors. In particular, reducing the levels of other factors that contribute to protein balance made the cells more sensitive. Using a combination of proteasome inhibitors and drugs that target these other factors could prove to be useful in the fight against multiple myeloma.
The proteasome complex contains two types of subunits: regulatory subunits that recognize the proteins that need to be degraded, and catalytic subunits that degrade the proteins. The results of Acosta-Alvear et al. revealed how varying the levels of these two subunits influenced the sensitivity of cells to inhibitors. While decreasing the levels of catalytic subunits made the cells more sensitive, as expected, decreasing the level of regulatory subunits surprisingly made the cells resistant to the inhibitors. A possible explanation for this paradoxical result is that certain proteins are less effectively degraded by the proteasome in these cells, and that the buildup of these proteins protects the cells against the drugs.
Acosta-Alvear et al. also found that lower levels of regulatory subunits desensitized multiple myeloma patients to therapy based on proteasome inhibition, suggesting that results from the genetic screen carried out in cells can predict clinical resistance mechanisms and guide the development of future therapies to increase patient survival.
DOI: http://dx.doi.org/10.7554/eLife.08153.002
doi:10.7554/eLife.08153
PMCID: PMC4602331  PMID: 26327694
proteasome; cancer; proteostasis; carfilzomib; myeloma; human
19.  Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic 
eLife  null;4:e05434.
Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.
DOI: http://dx.doi.org/10.7554/eLife.05434.001
eLife digest
Cells contain thousands of proteins that carry out the essential tasks needed for survival. Before they can work, proteins must first fold into specific three-dimensional shapes. The endoplasmic reticulum, a cellular compartment that specializes in properly folding newly made proteins into their native states, is critical for this protein maturation process. If folding-enzymes in the endoplasmic reticulum are not properly balanced with the load of proteins they must fold, the endoplasmic reticulum can be overwhelmed with unfolded proteins that accumulate, leading to ‘endoplasmic reticulum stress’.
The cell copes with endoplasmic reticulum stress by triggering the ‘unfolded protein response’ (UPR). This response helps to clear the unfolded proteins by increasing the size of the endoplasmic reticulum and the concentration of folding enzymes within it, and by decreasing the influx of newly made protein into the endoplasmic reticulum. The UPR engages signaling molecules in the endoplasmic reticulum membrane, among them two signaling enzymes called IRE1 and PERK. Drugs that activate these signaling enzymes could help the cell to deal with unfolded proteins, prevent toxicity resulting from endoplasmic reticulum stress, and ward off the diseases that result from it.
Mendez, Alfaro, Morales-Soto et al. developed a small molecule, called IPA (short for IRE1/PERK Activator), that was designed to bind to and activate IRE1. Serendipitously, IPA not only activated IRE1 but also activated PERK. Surprisingly, PERK activation was only observed at low IPA concentrations in which IPA occupied the active sites in only a few PERK molecules, whereas at higher concentrations and full occupancy IPA completely inhibited PERK. Mendez, Alfaro, Morales-Soto et al. proposed that, under conditions of partial IPA occupancy, a minority of IPA-bound PERK molecules assume an activated state that propagates to adjacent PERK molecules that have no IPA bound to them, and activates them.
Similar dose-dependent activation was previously observed for a clinically used drug designed to inhibit a similar signaling enzyme that is important in cancer progression. Together with the observations of Mendez, Alfaro, Morales-Soto et al., these results suggest that research into similar treatments must consider that a ‘minimal dose’ can exist, below which drugs may have the opposite effect to what is desired. Further work is still needed to fully understand the mechanisms that produce such behavior.
DOI: http://dx.doi.org/10.7554/eLife.05434.002
doi:10.7554/eLife.05434
PMCID: PMC4436593  PMID: 25986605
protein kinase; human cells; IPA; IRE1; PERK; E. coli; human; mouse
20.  Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response 
eLife  null;4:e07314.
The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases.
DOI: http://dx.doi.org/10.7554/eLife.07314.001
eLife digest
Proteins are often described as life's ‘workhorse’ molecules, and cells must be able to build new proteins to stay alive. This ability is also vital for storing new memories. A protein called eIF2 carries out a critical step in the process that cells use to make proteins; and a decrease in the activity of eIF2 has been linked with memory loss in diseases such as Parkinson's and Alzheimer's disease.
When a cell experiences stressful conditions—such as virus infection or starvation—it triggers the ‘integrated stress response’. This response helps the cell conserve its resources and take corrective steps to restore its normal working conditions. As part of the integrated stress response, an enzyme adds a phosphate group onto eIF2. The ‘phosphorylated’ eIF2 blocks protein production, which causes the cell to make fewer proteins. In 2013, researchers revealed that a small drug-like molecule, called ISRIB, could prevent this decline in protein production following eIF2 phosphorylation; and when ISRIB was administered to mice and rats, it enhanced their long-term memories. However, this early work did not identify the molecule that is targeted by ISRIB.
Now Sidrauski, Tsai et al.—including many of researchers involved in the 2013 work—have used a combination of techniques including genetics, chemistry and biochemistry to reveal the target of ISRIB. The experiments show that ISRIB's molecular target is a protein complex called eIF2B. Artificially reducing the production of eIF2B made cells resistant to the effects of ISRIB. The eIF2B protein normally works to activate eIF2; Sidrauski, Tsai et al. observed that ISRIB boosts the activity of eIF2B and renders it insensitive to blockage by phosphorylated eIF2. This in turn increases protein production in the cell.
But how does ISRIB activate eIF2B? It was known that two copies of eIF2B can bind to each other; and Sidrauski, Tsai et al. found that ISRIB acts by stabilizing these larger protein complexes that are more active and less sensitive to inhibition by phosphorylated eIF2. Finally, in further experiments, new versions of ISRIB were synthesized that are ten-times as active as the original molecule inside cells.
Importantly, the discovery that eIF2B is the molecular target for ISRIB has recently been independently validated by other researchers, and it looks promising that this discovery will guide future efforts to develop clinically useful drugs to treat memory disorders.
DOI: http://dx.doi.org/10.7554/eLife.07314.002
doi:10.7554/eLife.07314
PMCID: PMC4426669  PMID: 25875391
eIF2B; eIF2; integrated stress response; ISRIB; unfolded protein response; protein synthesis; human
21.  Beamline P02.1 at PETRA III for high-resolution and high-energy powder diffraction 
Journal of Synchrotron Radiation  2015;22(Pt 3):675-687.
By providing the capabilities for high-resolution, high-energy and time-resolved powder X-ray diffraction, beamline P02.1 is a versatile tool to tackle various problems in materials science, crystallography and chemistry.
Powder X-ray diffraction techniques largely benefit from the superior beam quality provided by high-brilliance synchrotron light sources in terms of photon flux and angular resolution. The High Resolution Powder Diffraction Beamline P02.1 at the storage ring PETRA III (DESY, Hamburg, Germany) combines these strengths with the power of high-energy X-rays for materials research. The beamline is operated at a fixed photon energy of 60 keV (0.207 Å wavelength). A high-resolution monochromator generates the highly collimated X-ray beam of narrow energy bandwidth. Classic crystal structure determination in reciprocal space at standard and non-ambient conditions are an essential part of the scientific scope as well as total scattering analysis using the real space information of the pair distribution function. Both methods are complemented by in situ capabilities with time-resolution in the sub-second regime owing to the high beam intensity and the advanced detector technology for high-energy X-rays. P02.1’s efficiency in solving chemical and crystallographic problems is illustrated by presenting key experiments that were carried out within these fields during the early stage of beamline operation.
doi:10.1107/S1600577515002222
PMCID: PMC4416682  PMID: 25931084
synchrotron powder diffraction; high-energy X-rays; high angular resolution; time-resolved experiments; X-ray total scattering
22.  Endoplasmic Reticulum Stress Sensing in the Unfolded Protein Response 
Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation. The unfolded protein response (UPR) maintains the protein-folding homeostasis within the ER, ensuring that the protein-folding capacity of the ER meets the load of client proteins. Activation of the UPR depends on three ER stress sensor proteins, Ire1, PERK, and ATF6. Although the consequences of activation are well understood, how these sensors detect ER stress remains unclear. Recent evidence suggests that yeast Ire1 directly binds to unfolded proteins, which induces its oligomerization and activation. BiP dissociation from Ire1 regulates this oligomeric equilibrium, ultimately modulating Ire1’s sensitivity and duration of activation. The mechanistic principles of ER stress sensing are the focus of this review.
Activation of the UPR depends on three stress sensor proteins: Ire1, PERK, and ATF6. In yeast, Ire1 appears to bind directly to unfolded proteins.
doi:10.1101/cshperspect.a013169
PMCID: PMC3578356  PMID: 23388626
23.  The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly 
eLife  null;4:e05033.
Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.
DOI: http://dx.doi.org/10.7554/eLife.05033.001
doi:10.7554/eLife.05033
PMCID: PMC4341466  PMID: 25719440
integrated stress response; eIF2; unfolded protein response; ISRIB; protein synthesis; ribosome profiling; Human; Mouse
24.  Translational control of mGluR-dependent long-term depression and object-place learning by eIF2α 
Nature neuroscience  2014;17(8):1073-1082.
At hippocampal synapses, activation of group I metabotropic glutamate receptors (mGluRs) induces long-term depression (LTD), which requires new protein synthesis. However, the underlying mechanism remains elusive. Here we describe the translational program that underlies mGluR-LTD and identify the translation factor eIF2α as its master effector. Genetically reducing eIF2α phosphorylation, or specifically blocking the translation controlled by eIF2α phosphorylation, prevented mGluR-LTD and the internalization of surface AMPA receptors (AMPARs). Conversely, direct phosphorylation of eIF2α, bypassing mGluR activation, triggered a sustained LTD and removal of surface AMPARs. Combining polysome profiling and RNA sequencing, we identified the mRNAs translationally upregulated during mGluR-LTD. Translation of one of these mRNAs, oligophrenin-1, mediates the LTD induced by eIF2α phosphorylation. Mice deficient in phospho-eIF2α–mediated translation are impaired in object-place learning, a behavioral task that induces hippocampal mGluR-LTD in vivo. Our findings identify a new model of mGluR-LTD, which promises to be of value in the treatment of mGluR-LTD-linked cognitive disorders.
doi:10.1038/nn.3754
PMCID: PMC4340591  PMID: 24974795
25.  Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis 
Science (New York, N.Y.)  2014;345(6192):98-101.
Protein folding by the endoplasmic reticulum (ER) is physiologically critical, while its disruption causes ER stress and augments disease. ER stress activates the unfolded protein response (UPR) to restore homeostasis. If stress persists, the UPR induces apoptotic cell death, but the mechanisms remain elusive. Here we find that unmitigated ER stress promotes apoptosis through cell-autonomous, UPR-controlled activation of death receptor 5 (DR5). ER stressors induced DR5 transcription via the UPR mediator CHOP; however, the UPR sensor IRE1α transiently catalyzed DR5 mRNA decay, allowing time for adaptation. Persistent ER stress built up intracellular DR5 protein, driving ligand-independent DR5 activation and apoptosis engagement via caspase-8. Thus, DR5 integrates opposing UPR signals to couple ER stress and apoptotic cell fate.
doi:10.1126/science.1254312
PMCID: PMC4284148  PMID: 24994655

Results 1-25 (97)