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1.  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
2.  Specificity in endoplasmic reticulum-stress signaling in yeast entails a step-wise engagement of HAC1 mRNA to clusters of the stress sensor Ire1 
eLife  null;3:e05031.
Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.
DOI: http://dx.doi.org/10.7554/eLife.05031.001
eLife digest
Proteins are built based on instructions in template molecules called messenger RNAs (or mRNAs), which are copied from the DNA of genes. As they are made, proteins must fold into a specific three-dimensional shape and some proteins pass into a compartment in the cell, called the endoplasmic reticulum, in which they fold. So-called molecular chaperone proteins assist this folding process. From the endoplasmic reticulum, most proteins travel to other destinations within or outside of the cell.
If the molecular chaperones in the endoplasmic reticulum are overwhelmed by their protein folding task, unfolded proteins accumulate; a situation that can be harmful to the cell. In eukaryotic cells including yeast, a sensor protein called Ire1 detects when unfolded proteins build up in the endoplasmic reticulum. As a result, the Ire1 sensor proteins join together to form clusters and an mRNA molecule called HAC1 is specifically recruited to the Ire1 clusters. The portions of the Ire1 protein that extend out from the endoplasmic reticulum into the cell proper then bind to HAC1 mRNA and cut a piece out of it. This edited mRNA encodes the instructions to build a protein that in turn boosts the expression of various components—including the appropriate molecular chaperones—that are needed to alleviate the stress caused by an excess of unfolded proteins.
Within clusters, individual Ire1 proteins interact through the portions of the protein found on the inside of the endoplasmic reticulum. Now, van Anken et al. show that these interactions are sufficient for forming and maintaining clusters. The interactions between the portions of the Ire1 proteins outside of the endoplasmic reticulum are needed for editing the HAC1 mRNA but not for forming and maintaining the clusters or for recruiting the HAC1 mRNA molecule to bind to Ire1. Instead, van Anken et al. discovered an mRNA binding site on the Ire1 clusters, which is separate from the part of the Ire1 protein that cuts the mRNA molecules. The Ire1 protein needs to first bind the HAC1 mRNA molecule at this binding site before it can cut it; van Anken et al. suggest that this two-step process helps ensure accurate and efficient editing of the HAC1 mRNA by Ire1. This process could also help to minimize the chance of other mRNA molecules being edited by mistake.
It will be of interest to investigate if similar safety measures are key for endoplasmic reticulum stress signaling mechanisms in humans, and whether these newly discovered steps can be targeted by drugs to treat disease.
DOI: http://dx.doi.org/10.7554/eLife.05031.002
doi:10.7554/eLife.05031
PMCID: PMC4279078  PMID: 25549299
stress signaling; endoplasmic reticulum; unfolded protein response; mRNA targeting; mRNA processing; S. cerevisiae
3.  Influence of the prediction error of the first eye undergoing cataract surgery on the refractive outcome of the fellow eye 
Introduction
In addition to measurement errors, individual anatomical conditions could be made responsible for unexpected prediction errors in the determination of the correct intraocular lens power for cataract surgery. Obviously, such anatomical conditions might be relevant for both eyes. The purpose of this study was to evaluate whether the postoperative refractive error of the first eye has to be taken in account for the biometry of the second.
Methods
In this retrospective study, we included 670 eyes of 335 patients who underwent phacoemulsification and implantation of a foldable intraocular lens in both eyes. According to the SRK/T formula, the postoperative refractive error of each eye was determined and compared with its fellow eye.
Results
Of 670 eyes, 622 showed a postoperative refractive error within ±1.0 D (93%), whereas the prediction error was 0.5 D or less in 491 eyes (73%). The postoperative difference between both eyes was within 0.5 D in 71% and within 1.0 D in 93% of the eyes. Comparing the prediction error of an eye and its fellow eye, the error of the fellow eye was about half the value of the other.
Conclusion
Our results imply that substitution of half of the prediction error of the first eye into the calculation of the second eye may be useful to reduce the prediction error in the second eye. However, prospective studies should be initiated to demonstrate an improved accuracy for the second eye’s intraocular lens power calculation by partial adjustment.
doi:10.2147/OPTH.S69255
PMCID: PMC4222621  PMID: 25382967
cataract surgery; biometry; IOL power calculation; refractive error; fellow eye
4.  Real-time observation of signal recognition particle binding to actively translating ribosomes 
eLife  2014;3:e04418.
The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.
DOI: http://dx.doi.org/10.7554/eLife.04418.001
eLife digest
Genes contain the instructions needed to make proteins from smaller building blocks called amino acids. These instructions are first transcribed to produce molecules of messenger RNA, which are then translated by a ribosome. This ‘molecular machine’ translates the instructions in the messenger RNA into the sequence of amino acids needed to make the protein.
For some proteins to carry out their role, they need to be delivered to the outside of the cell, or inserted into one of the cell's membranes. As they are being built, these proteins are identified by a so-called ‘signal recognition particle’, which is often called an SRP for short. The SRP attaches to the new protein when it is still joined to the ribosome, and pulls the protein-ribosome complex to an opening in the target membrane. The new protein chain then enters this opening and either passes through to the other side of the membrane, or ends up embedded within it.
To date, most studies that have investigated this process have involved scientists stalling the building of the new protein to see how SRP interacts with inactivated protein-ribosome complexes. Unfortunately, this means that some of the details of what happens during this process have likely been missed.
Now, Noriega et al. have addressed this problem by developing a method to watch, in real-time, a single active protein-ribosome complex interacting with individual SRPs. This was achieved by attaching fluorescent molecules to SRP and protein-ribosome complexes purified from the bacterium E. coli. The distance between the two fluorescent molecules was then tracked over time. This revealed that the SRP typically binds to the protein-ribosome complex after 40–55 amino acids have been built into the protein. At this point, a so-called ‘signal sequence’ of amino acids has emerged from the complex and can be recognized by the SRP.
Earlier studies had suggested that signal sequences might tell the SRP when to bind, but this had not been demonstrated in experiments using active protein-ribosome complexes. The strategy of using fluorescent molecules to follow single molecules undergoing this process in real-time could now be used by other scientists to re-examine and determine new properties of the protein-ribosome complex in action.
DOI: http://dx.doi.org/10.7554/eLife.04418.002
doi:10.7554/eLife.04418
PMCID: PMC4213662  PMID: 25358118
signal recognition particle; ribosome translation; protein targeting; single molecule fluorescence; E. coli
5.  Topography of retinal recovery processes in humans 
Background
The purpose of this study was to examine retinal recovery processes to pographically by the application of three flash sequences with specific interstimulus intervals.
Methods
Twelve healthy subjects underwent multifocal electroretinography with a light-emitting diode stimulator. Every flash sequence consisted of three flashes with 25 msec between the first and the second flash and 35 msec between the second and the third flash. The interval between the third and the first flash of the next step was 85 msec. The interstimulus interval-dependent amplitude reductions of the multifocal electroretinographic response for these three intervals yielded three data points that were used to determine the complete curve of the recovery kinetics.
Results
Amplitude reductions were higher with shorter interstimulus intervals. The mean half-life periods of the recovery kinetics for the different concentric rings and all subjects were: ring 1, 29.3±5.9 msec; ring 2, 24.2±6.4 msec; ring 3, 23±4.1 msec; ring 4, 23.1±4.6 msec; and ring 5, 22.3±4.4 msec. The differences between the first and all other rings were statistically significant (P<0.05).
Conclusion
The kinetics of the amplitude recovery after short interstimulus intervals showed a spatial distribution, with faster recovery toward the macular periphery.
doi:10.2147/OPTH.S49708
PMCID: PMC4208419  PMID: 25349472
multifocal; electroretinography; recovery; LED stimulator; interstimulus interval
6.  ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery 
Journal of Cell Science  2014;127(18):4078-4088.
ABSTRACT
Selective autophagy of damaged or redundant organelles is an important mechanism for maintaining cell homeostasis. We found previously that endoplasmic reticulum (ER) stress in the yeast Saccharomyces cerevisiae causes massive ER expansion and triggers the formation of large ER whorls. Here, we show that stress-induced ER whorls are selectively taken up into the vacuole, the yeast lysosome, by a process termed ER-phagy. Import into the vacuole does not involve autophagosomes but occurs through invagination of the vacuolar membrane, indicating that ER-phagy is topologically equivalent to microautophagy. Even so, ER-phagy requires neither the core autophagy machinery nor several other proteins specifically implicated in microautophagy. Thus, autophagy of ER whorls represents a distinct type of organelle-selective autophagy. Finally, we provide evidence that ER-phagy degrades excess ER membrane, suggesting that it contributes to cell homeostasis by controlling organelle size.
doi:10.1242/jcs.154716
PMCID: PMC4163648  PMID: 25052096
Endoplasmic reticulum; Stress response; Autophagy
7.  Pharmacological Analysis of Intrinsic Neuronal Oscillations in rd10 Retina 
PLoS ONE  2014;9(6):e99075.
In the widely used mouse model of retinal degeneration, rd1, the loss of photoreceptors leads to rhythmic electrical activity of around 10–16 Hz in the remaining retinal network. Recent studies suggest that this oscillation is formed within the electrically coupled network of AII amacrine cells and ON-bipolar cells. A second mouse model, rd10, displays a delayed onset and slower progression of degeneration, making this mouse strain a better model for human retinitis pigmentosa. In rd10, oscillations occur at a frequency of 3–7 Hz, raising the question whether oscillations have the same origin in the two mouse models. As rd10 is increasingly being used as a model to develop experimental therapies, it is important to understand the mechanisms underlying the spontaneous rhythmic activity. To study the properties of oscillations in rd10 retina we combined multi electrode recordings with pharmacological manipulation of the retinal network. Oscillations were abolished by blockers for ionotropic glutamate receptors and gap junctions. Frequency and amplitude of oscillations were modulated strongly by blockers of inhibitory receptors and to a lesser extent by blockers of HCN channels. In summary, although we found certain differences in the pharmacological modulation of rhythmic activity in rd10 compared to rd1, the overall pattern looked similar. This suggests that the generation of rhythmic activity may underlie similar mechanisms in rd1 and rd10 retina.
doi:10.1371/journal.pone.0099075
PMCID: PMC4053359  PMID: 24918437
8.  The Unfolded Protein Response Element IRE1α Senses Bacterial Proteins Invading the ER to Activate RIG-I and Innate Immune Signaling 
Cell host & microbe  2013;13(5):558-569.
SUMMARY
The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1α (IRE1α), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1α-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1α to degrade endogenous mRNA, a process termed regulated IRE1α-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cyto-solic sensor of RNA viruses, to activate NF-κB and interferon pathways. We propose IRE1α provides for a generalized mechanism of innate immune surveillance originating within the ER lumen.
doi:10.1016/j.chom.2013.03.011
PMCID: PMC3766372  PMID: 23684307
9.  Molecular Mechanisms of Hypoxic Responses via Unique Roles of Ras1, Cdc24 and Ptp3 in a Human Fungal Pathogen Cryptococcus neoformans 
PLoS Genetics  2014;10(4):e1004292.
Cryptococcus neoformans encounters a low oxygen environment when it enters the human host. Here, we show that the conserved Ras1 (a small GTPase) and Cdc24 (the guanine nucleotide exchange factor for Cdc42) play an essential role in cryptococcal growth in hypoxia. Suppressor studies indicate that PTP3 functions epistatically downstream of both RAS1 and CDC24 in regulating hypoxic growth. Ptp3 shares sequence similarity to the family of phosphotyrosine-specific protein phosphatases and the ptp3Δ strain failed to grow in 1% O2. We demonstrate that RAS1, CDC24 and PTP3 function in parallel to regulate thermal tolerance but RAS1 and CDC24 function linearly in regulating hypoxic growth while CDC24 and PTP3 reside in compensatory pathways. The ras1Δ and cdc24Δ strains ceased to grow at 1% O2 and became enlarged but viable single cells. Actin polarization in these cells, however, was normal for up to eight hours after transferring to hypoxic conditions. Double deletions of the genes encoding Rho GTPase Cdc42 and Cdc420, but not of the genes encoding Rac1 and Rac2, caused a slight growth retardation in hypoxia. Furthermore, growth in hypoxia was not affected by the deletion of several central genes functioning in the pathways of cAMP, Hog1, or the two-component like phosphorylation system that are critical in the cryptococcal response to osmotic and genotoxic stresses. Interestingly, although deletion of HOG1 rescued the hypoxic growth defect of ras1Δ, cdc24Δ, and ptp3Δ, Hog1 was not hyperphosphorylated in these three mutants in hypoxic conditions. RNA sequencing analysis indicated that RAS1, CDC24 and PTP3 acted upon the expression of genes involved in ergosterol biosynthesis, chromosome organization, RNA processing and protein translation. Moreover, growth of the wild-type strain under low oxygen conditions was affected by sub-inhibitory concentrations of the compounds that inhibit these biological processes, demonstrating the importance of these biological processes in the cryptococcal hypoxia response.
Author Summary
When Cryptococcus neoformans, an environmental fungal pathogen, enters the human host, it encounters a low oxygen condition. The well conserved Ras1 and Cdc24 proteins are known for their key roles in maintenance of the actin cytoskeletal integrity in eukaryotic cells. In this work, we show a unique role of RAS1 and CDC24 in the growth of C. neoformans in a low oxygen environment. Actin polarization, however, appeared normal in the ras1Δ and cdc24Δ strains under hypoxic conditions for up to eight hours. We show that PTP3 is required for hypoxic growth and it can rescue the hypoxic growth defect in ras1Δ and cdc24Δ. Genetic analysis suggested that RAS1 and CDC24 function linearly while CDC24 and PTP3 function parallelly in regulating hypoxic growth. RNA sequencing combined with analysis by small molecular inhibitors revealed that RAS1, CDC24 and PTP3 regulate several biological processes such as ergosterol biosynthesis, chromosome organization, RNA processing and protein translation which are required in the cryptococcal response to hypoxic conditions.
doi:10.1371/journal.pgen.1004292
PMCID: PMC3998916  PMID: 24762475
10.  The cost-effectiveness of the Argus II retinal prosthesis in Retinitis Pigmentosa patients 
BMC Ophthalmology  2014;14:49.
Background
Retinitis Pigmentosa (RP) is a hereditary genetic disease causing bilateral retinal degeneration. RP is a leading cause of blindness resulting in incurable visual impairment and drastic reduction in the Quality of life of the patients. Second Sight Medical Products Inc. developed Argus II, a retinal prosthesis system for treating RP. Argus II is the world’s first ever-commercial implant intended to restore some vision in the blind patients. The objective of this study was to assess the cost-effectiveness of the Argus® II Retinal Prosthesis System (Argus II) in Retinitis Pigmentosa (RP) patients.
Method
A multi -state transition Markov model was developed to determine the cost-effectiveness of Argus II versus usual care in RP from the perspective of healthcare payer. A hypothetical cohort of 1000 RP patients aged 46 years followed up over a (lifetime) 25-year time horizon. Health outcomes were expressed as quality adjusted life years (QALYs) and direct healthcare costs expressed in 2012 €. Results are reported as incremental cost per ratios (ICERs) with outcomes and costs discounted at an annual rate of 3.5%.
Results
The ICER for Argus II was €14,603/QALY. Taking into account the uncertainty in model inputs the ICER was €14,482/QALY in the probabilistic analysis. In the scenarios of an assumption of no reduction on cost across model visual acuity states or a model time horizon as short as 10 years the ICER increased to €31,890/QALY and €49,769/QALY respectively.
Conclusion
This economic evaluation shows that Argus II is a cost-effective intervention compared to usual care of the RP patients. The lifetime analysis ICER for Argus II falls below the published societal willingness to pay of EuroZone countries.
doi:10.1186/1471-2415-14-49
PMCID: PMC3990272  PMID: 24731533
Retinitis Pigmentosa; Retinal prosthesis; Cost-effectiveness analysis; Decision analytic modelling
11.  Clinical Observations and Occurrence of Complications following Heavy Silicone Oil Surgery 
BioMed Research International  2014;2014:706809.
Purpose. To demonstrate development and complications in heavy silicone oil (HSO) surgery in 100 eyes following primary vitreoretinal surgery. Methods. 100 eyes were included in this retrospective study that underwent vitreoretinal surgery using HSO as endotamponade. Indication diagnoses were retinal detachments (n = 76), complicated macular holes (MH) (n = 20), and others (n = 4). HSO removal was performed after a mean period of 20.2 ± 19.0 weeks. In 18 eyes with poor functional prognosis the silicone oil remained permanently for stabilisation. Overall follow-up time was 35.9 ± 51.8 weeks. Results. The mean IOP before HSO surgery was 13.3 ± 5.6 mmHg and raised to an average maximum of 23.3 ± 8.5 mmHg postoperatively and decreased to 13.7 ± 7.2 mmHg after removal. Secondary IOP raise due to emulsification of the silicone oil endotamponade was seen in 29 eyes after 7.8 ± 4.5 weeks. Other complications being observed with HSO installed were persistent corneal erosion (n = 3) and prolonged anterior chamber inflammation (n = 29). In 13 eyes recurrent retinal detachments occurred during followup. Conclusions. According to our analysis HSO surgery might deliver satisfying results in complicated cases of ophthalmological surgery. However, potential complications should always be taken into account when making the decision if to use and when to remove HSO in complicated retinal surgery.
doi:10.1155/2014/706809
PMCID: PMC4009140  PMID: 24829913
12.  Partial Coherence Laser Interferometry in Highly Myopic versus Emmetropic Eyes 
Purpose
To investigate the reliability of partial coherence laser interferometry for optical biometry in highly myopic eyes.
Methods
Axial length measurements by the IOLMaster (Carl Zeiss Meditec, Germany) with signal-to-noise ratio (SNR) ≥2 were performed in 52 consecutive myopic subjects with axial length ≥26.5 mm and 45 emmetropic patients before cataract surgery. Axial length measurements and SNR were analyzed and compared among the two study groups.
Results
Axial length measurements were feasible in 46 of 52 (88.5%) highly myopic eyes and in 41 of 45 (91.1%) eyes with normal axial length. To achieve two reliable axial length values with SNR ≥2, a mean number of 2.06±0.25 measurements was necessary in myopic eyes and 2.10±0.37 in emmetropic counterparts. Mean SNR after two measurements was 4.98±2.44 in myopic eyes versus 5.56±2.32 in control eyes. Even though successful measurement was independent of preoperative visual acuity, patients with visual acuity better than 20/63 showed significantly higher SNR values.
Conclusions
Partial coherence laser interferometry shows satisfying feasibility and good signal quality for axial length determination in highly myopic eyes with stable retinal condition and clear media.
PMCID: PMC4181198  PMID: 25279117
Axial Length; Partial Coherence Interferometry; Biometry; High Myopia
13.  Development of very large electrode arrays for epiretinal stimulation (VLARS) 
Background
Retinal implants have been developed to treat blindness causing retinal degenerations such as Retinitis pigmentosa (RP). The retinal stimulators are covering only a small portion of the retina usually in its center. To restore not only central vision but also a useful visual field retinal stimulators need to cover a larger area of the retina. However, large area retinal stimulators are much more difficult to implant into an eye. Some basic questions concerning this challenge should be answered in a series of experiments.
Methods
Large area retinal stimulators were fabricated as flexible multielectrode arrays (MEAs) using silicon technology with polyimide as the basic material for the substrate. Electrodes were made of gold covered with reactively sputtered iridium oxide. Several prototype designs were considered and implanted into enucleated porcine eyes. The prototype MEAs were also used as recording devices.
Results
Large area retinal stimulator MEAs were fabricated with a diameter of 12 mm covering a visual angle of 37.6° in a normal sighted human eye. The structures were flexible enough to be implanted in a folded state through an insertion nozzle. The implants could be positioned onto the retinal surface and fixated here using a retinal tack. Recording of spontaneous activity of retinal neurons was possible in vitro using these devices.
Conclusions
Large flexible MEAs covering a wider area of the retina as current devices could be fabricated using silicon technology with polyimide as a base material. Principal surgical techniques were established to insert such large devices into an eye and the devices could also be used for recording of retinal neural activity.
doi:10.1186/1475-925X-13-11
PMCID: PMC3976033  PMID: 24502253
Retinal prosthesis; Artificial vision; Retinitis pigmentosa; Blindness; Rehabilitation; Vitreoretinal surgery; Silicon wafer Technology; Polyimide; Neurostimulation
14.  Correlations between ERG, OCT, and Anatomical Findings in the rd10 Mouse 
Journal of Ophthalmology  2014;2014:874751.
Background. To evaluate the correlation between ERG, OCT, and microscopic findings in the rd10 mouse. Methods. C57BL/6J wild type mice and rd10 mice were compared at the age of 2, 3, 5, 7, 9, 12, 24, and 48 weeks (each age group n = 3) using full-field electroretinography (ERG), spectral domain Optical Coherence Tomography (sd-OCT), fluorescein angiography (FA), Hematoxylin & Eosin histology (HE), and immunohistology (IH). Results. While in wild type mice, the amplitude of a- and b-wave increased with light intensity and with the age of the animals, the rd10 mice showed extinction of the ERG beginning with the age of 5 weeks. In OCT recordings, the thickness of the retina decreased up to 9 weeks of age, mainly based on the degradation of the outer nuclear layer (ONL). Afterwards, the ONL was no longer visible in the OCT. HE staining and immunohistological findings confirmed the in vivo data. Conclusion. ERG and OCT are useful methods to evaluate the retinal function and structure in vivo. The retinal changes seen in the OCT closely match those observed in histological staining.
doi:10.1155/2014/874751
PMCID: PMC3941775  PMID: 24683495
15.  Heat Shock Transcription Factor σ32 Co-opts the Signal Recognition Particle to Regulate Protein Homeostasis in E. coli 
PLoS Biology  2013;11(12):e1001735.
The bacterial heat shock transcription factor, σ32, maintains proper protein homeostasis only after it is targeted to the inner membrane by the signal recognition particle (SRP), thereby enabling integration of protein folding information from both the cytoplasm and cell membrane.
All cells must adapt to rapidly changing conditions. The heat shock response (HSR) is an intracellular signaling pathway that maintains proteostasis (protein folding homeostasis), a process critical for survival in all organisms exposed to heat stress or other conditions that alter the folding of the proteome. Yet despite decades of study, the circuitry described for responding to altered protein status in the best-studied bacterium, E. coli, does not faithfully recapitulate the range of cellular responses in response to this stress. Here, we report the discovery of the missing link. Surprisingly, we found that σ32, the central transcription factor driving the HSR, must be localized to the membrane rather than dispersed in the cytoplasm as previously assumed. Genetic analyses indicate that σ32 localization results from a protein targeting reaction facilitated by the signal recognition particle (SRP) and its receptor (SR), which together comprise a conserved protein targeting machine and mediate the cotranslational targeting of inner membrane proteins to the membrane. SRP interacts with σ32 directly and transports it to the inner membrane. Our results show that σ32 must be membrane-associated to be properly regulated in response to the protein folding status in the cell, explaining how the HSR integrates information from both the cytoplasm and bacterial cell membrane.
Author Summary
All cells have to adjust to frequent changes in their environmental conditions. The heat shock response is a signaling pathway critical for survival of all organisms exposed to elevated temperatures. Under such conditions, the heat shock response maintains enzymes and other proteins in a properly folded state. The mechanisms for sensing temperature and the subsequent induction of the appropriate transcriptional response have been extensively studied. Prior to this work, however, the circuitry described in the best studied bacterium E. coli could not fully explain the range of cellular responses that are observed following heat shock. We report the discovery of this missing link. Surprisingly, we find that σ32, a transcription factor that induces gene expression during heat shock, needs to be localized to the membrane, rather than being active as a soluble cytoplasmic protein as previously thought. We show that, equally surprisingly, σ32 is targeted to the membrane by the signal recognition particle (SRP) and its receptor (SR). SRP and SR constitute a conserved protein targeting machine that normally only operates on membrane and periplasmic proteins that contain identifiable signal sequences. Intriguingly, σ32 does not have any canonical signal sequence for export or membrane-integration. Our results indicate that membrane-associated σ32, not soluble cytoplasmic σ32, is the preferred target of regulatory control in response to heat shock. Our new model thus explains how protein folding status from both the cytoplasm and bacterial cell membrane can be integrated to control the heat shock response.
doi:10.1371/journal.pbio.1001735
PMCID: PMC3866087  PMID: 24358019
16.  IRE1 Signaling Affects Cell Fate During the Unfolded Protein Response 
Science (New York, N.Y.)  2007;318(5852):944-949.
Endoplasmic reticulum (ER) stress activates a set of signaling pathways, collectively termed the unfolded protein response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if ER stress is not alleviated. How the UPR integrates its cytoprotective and proapoptotic outputs to select between life or death cell fates is unknown. We found that IRE1 and ATF6 activities were attenuated by persistent ER stress in human cells. By contrast, PERK signaling, including translational inhibition and proapoptotic transcription regulator Chop induction, was maintained. When IRE1 activity was sustained artificially, cell survival was enhanced, suggesting a causal link between the duration of UPR branch signaling and life or death cell fate after ER stress. Key findings from our studies in cell culture were recapitulated in photoreceptors expressing mutant rhodopsin in animal models of retinitis pigmentosa.
doi:10.1126/science.1146361
PMCID: PMC3670588  PMID: 17991856
17.  Pharmacological brake-release of mRNA translation enhances cognitive memory 
eLife  2013;2:e00498.
Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the ‘integrated stress response’ (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders.
DOI: http://dx.doi.org/10.7554/eLife.00498.001
eLife digest
The synthesis of proteins is an essential step in many biological processes, including memory, and drugs that inhibit protein synthesis are known to impair memory in rodents. It is thought that the brain needs these proteins to convert short-term memories into long-term memories through a process known as consolidation.
A protein called EIF2α has a key role in the regulation of protein synthesis, and has also been implicated in memory. EIF2α can be activated as a result of being phosphorylated by any of four protein kinases: these are in turn activated by processes that subject cells to stress, such as viral infection, UV light or—in the case of a kinase known as PERK—the accumulation of unfolded proteins in a cellular organelle called the endoplasmic reticulum. Activation of EIF2α downregulates most protein synthesis inside the cell, but upregulates the production of a small number of key regulatory molecules: these changes help cells to cope with whatever stressful event they have just experienced.
To obtain further insight into the cellular stress response, Sidrauski et al. screened a large library of compounds in search of one that inhibits PERK. They identified a molecule—known as ISRIB—which acts downstream of all four protein kinases by reversing the effects of EIF2α phosphorylation. ISRIB is the first molecule shown to have this effect, and thus represents an important tool for investigating the stress response inside cells.
When Sidrauski et al. injected ISRIB into mice, the animals showed improved memory: for example, they learnt to locate a hidden platform in a water maze more rapidly than controls. This suggests that ISRIB could be used to explore the mechanisms that underlie memory consolidation, and possibly even as a memory enhancer. Moreover, given that many tumor cells exploit the cellular stress response to aid their own growth, ISRIB may have potential as a novel chemotherapeutic agent.
DOI: http://dx.doi.org/10.7554/eLife.00498.002
doi:10.7554/eLife.00498
PMCID: PMC3667625  PMID: 23741617
eIF2; eIF2B; ATF4; integrated stress response; unfolded protein response; memory consolidation; Human; Mouse; Rat
18.  ER-associated mitochondrial division links the distribution of mitochondria and mitochondrial DNA in yeast 
eLife  2013;2:e00422.
Mitochondrial division is important for mitochondrial distribution and function. Recent data have demonstrated that ER–mitochondria contacts mark mitochondrial division sites, but the molecular basis and functions of these contacts are not understood. Here we show that in yeast, the ER–mitochondria tethering complex, ERMES, and the highly conserved Miro GTPase, Gem1, are spatially and functionally linked to ER-associated mitochondrial division. Gem1 acts as a negative regulator of ER–mitochondria contacts, an activity required for the spatial resolution and distribution of newly generated mitochondrial tips following division. Previous data have demonstrated that ERMES localizes with a subset of actively replicating mitochondrial nucleoids. We show that mitochondrial division is spatially linked to nucleoids and that a majority of these nucleoids segregate prior to division, resulting in their distribution into newly generated tips in the mitochondrial network. Thus, we postulate that ER-associated division serves to link the distribution of mitochondria and mitochondrial nucleoids in cells.
DOI: http://dx.doi.org/10.7554/eLife.00422.001
eLife digest
Mitochondria generate most of the energy used by cells, and they also play key roles in cellular growth, death, and differentiation. They are evolutionarily derived from bacteria and have retained their own DNA and protein translation system, but they are also dependent on the cell for their growth and replication.
A significant portion of the outer membrane of a mitochondrion is in contact with the endoplasmic reticulum (ER)—an organelle that is the starting point for the synthesis of secreted proteins, and is also critical for the synthesis of lipids and other organelles. Recent work suggests that mitochondria–ER contact points mark sites of mitochondrial division, but it is unclear exactly how this process occurs.
Here, Murley et al. use the budding yeast and model organism Saccharomyces cerevisiae to show that at mitochondrial division sites, a multiprotein complex called ERMES promotes the formation of ER–mitochondrial contact points, while an evolutionarily conserved enzyme, Gem1, antagonizes these contacts to aid mitochondrial segregation. The contact points are found adjacent to nucleoids (which are complexes of mitochondrial DNA and proteins)—an observation suggesting that ER-associated mitochondrial division evolved to help distribute nucleoids between newly formed mitochondria.
The present study also reveals a novel role for the conserved protein Gem1 and could lead researchers to reinvestigate the functions of Miro1/2—the equivalent of Gem1 in higher eukaryotes. Miro1/2 is thought to connect mitochondria to motor proteins, which transports them through the cell along microtubules. Dysfunction of Miro1/2 reduces the mobility of mitochondria, and the work of Murley et al. suggests that this could be a consequence of enhanced contacts between mitochondria and the ER.
DOI: http://dx.doi.org/10.7554/eLife.00422.002
doi:10.7554/eLife.00422
PMCID: PMC3654481  PMID: 23682313
ERMES; Gem1; Miro; mitochondrial DNA; mitochondria; S. cerevisiae
19.  Endoplasmic Reticulum Stress in Disease Pathogenesis 
Annual review of pathology  2008;3:399-425.
The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins, which, collectively, represent a large fraction of the total protein output of a mammalian cell. Therefore, the protein flux through the ER must be carefully monitored for abnormalities, including the buildup of misfolded proteins. Mammalian cells have evolved an intricate set of signaling pathways from the ER to the cytosol and nucleus, to allow the cell to respond to the presence of misfolded proteins within the ER. These pathways, known collectively as the unfolded protein response, are important for normal cellular homeostasis and organismal development and may also play key roles in the pathogenesis of many diseases. This review provides background information on the unfolded protein response and discusses a selection of diseases whose pathogenesis involves ER stress.
doi:10.1146/annurev.pathmechdis.3.121806.151434
PMCID: PMC3653419  PMID: 18039139
protein misfolding; unfolded protein response; IRE1; PERK; ATF-6
21.  Predicting where Small Molecules Bind at Protein-Protein Interfaces 
PLoS ONE  2013;8(3):e58583.
Small molecules that bind at protein-protein interfaces may either block or stabilize protein-protein interactions in cells. Thus, some of these binding interfaces may turn into prospective targets for drug design. Here, we collected 175 pairs of protein-protein (PP) complexes and protein-ligand (PL) complexes with known three-dimensional structures for which (1) one protein from the PP complex shares at least 40% sequence identity with the protein from the PL complex, and (2) the interface regions of these proteins overlap at least partially with each other. We found that those residues of the interfaces that may bind the other protein as well as the small molecule are evolutionary more conserved on average, have a higher tendency of being located in pockets and expose a smaller fraction of their surface area to the solvent than the remaining protein-protein interface region. Based on these findings we derived a statistical classifier that predicts patches at binding interfaces that have a higher tendency to bind small molecules. We applied this new prediction method to more than 10 000 interfaces from the protein data bank. For several complexes related to apoptosis the predicted binding patches were in direct contact to co-crystallized small molecules.
doi:10.1371/journal.pone.0058583
PMCID: PMC3591369  PMID: 23505538
22.  Seg1 controls eisosome assembly and shape 
The Journal of Cell Biology  2012;198(3):405-420.
Seg1 establishes a platform for the assembly of eisosomes and is important for determining their length.
Eisosomes are stable domains at the plasma membrane of the budding yeast Saccharomyces cerevisiae and have been proposed to function in endocytosis. Eisosomes are composed of two main cytoplasmic proteins, Pil1 and Lsp1, that form a scaffold around furrow-like plasma membrane invaginations. We show here that the poorly characterized eisosome protein Seg1/Ymr086w is important for eisosome biogenesis and architecture. Seg1 was required for efficient incorporation of Pil1 into eisosomes and the generation of normal plasma membrane furrows. Seg1 preceded Pil1 during eisosome formation and established a platform for the assembly of other eisosome components. This platform was further shaped and stabilized upon the arrival of Pil1 and Lsp1. Moreover, Seg1 abundance controlled the shape of eisosomes by determining their length. Similarly, the Schizosaccharomyces pombe Seg1-like protein Sle1 was necessary to generate the filamentous eisosomes present in fission yeast. The function of Seg1 in the stepwise biogenesis of eisosomes reveals striking architectural similarities between eisosomes in yeast and caveolae in mammals.
doi:10.1083/jcb.201202097
PMCID: PMC3413353  PMID: 22869600
23.  A first line of defense against ER stress 
The Journal of Cell Biology  2012;198(3):277-279.
BiP is the predominant DnaK/Hsp70-type chaperone protein in the ER. It is required for folding and assembling newly synthesized ER client proteins, yet having too much BiP inhibits folding. In this issue, Chambers et al. (2012. J. Cell Biol. doi:10.1083/jcb.201202005) report that ADP ribosylation of BiP provides a reversible switch that fine tunes BiP activity according to need.
doi:10.1083/jcb.201207076
PMCID: PMC3413362  PMID: 22869593
24.  Structural Basis for Mobility in the 1.1 Å Crystal Structure of the NG Domain of Thermus aquaticus Ffh 
Journal of molecular biology  2002;320(4):783-799.
The NG domain of the prokaryotic signal recognition protein Ffh is a two-domain GTPase that comprises part of the prokaryotic signal recognition particle (SRP) that functions in co-translational targeting of proteins to the membrane. The interface between the N and G domains includes two highly conserved sequence motifs and is adjacent in sequence and structure to one of the conserved GTPase signature motifs. Previous structural studies have shown that the relative orientation of the two domains is dynamic. The N domain of Ffh has been proposed to function in regulating the nucleotide-binding interactions of the G domain. However, biochemical studies suggest a more complex role for the domain in integrating communication between signal sequence recognition and interaction with receptor. Here, we report the structure of the apo NG GTPase of Ffh from Thermus aquaticus refined at 1.10 Å resolution. Although the G domain is very well ordered in this structure, the N domain is less well ordered, reflecting the dynamic relationship between the two domains previously inferred. We demonstrate that the anisotropic displacement parameters directly visualize the underlying mobility between the two domains, and present a detailed structural analysis of the packing of the residues, including the critical α4 helix, that comprise the interface. Our data allows us to propose a structural explanation for the functional significance of sequence elements conserved at the N/G interface.
PMCID: PMC3542393  PMID: 12095255
ultrahigh resolution; SRP; Ffh; GTPase; X-ray crystallography
25.  The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis 
eLife  2012;1:e00048.
The unfolded protein response (UPR) monitors the protein folding capacity of the endoplasmic reticulum (ER). In all organisms analyzed to date, the UPR drives transcriptional programs that allow cells to cope with ER stress. The non-conventional splicing of Hac1 (yeasts) and XBP1 (metazoans) mRNA, encoding orthologous UPR transcription activators, is conserved and dependent on Ire1, an ER membrane-resident kinase/endoribonuclease. We found that the fission yeast Schizosaccharomyces pombe lacks both a Hac1/XBP1 ortholog and a UPR-dependent-transcriptional-program. Instead, Ire1 initiates the selective decay of a subset of ER-localized-mRNAs that is required to survive ER stress. We identified Bip1 mRNA, encoding a major ER-chaperone, as the sole mRNA cleaved upon Ire1 activation that escapes decay. Instead, truncation of its 3′ UTR, including loss of its polyA tail, stabilized Bip1 mRNA, resulting in increased Bip1 translation. Thus, S. pombe uses a universally conserved stress-sensing machinery in novel ways to maintain homeostasis in the ER.
DOI: http://dx.doi.org/10.7554/eLife.00048.001
eLife digest
Protein folding—the process by which a sequence of amino acids adopts the precise shape that is needed to perform a specific biological function—is one of the most important processes in all of biology. Any sequence of amino acids has the potential to fold into a large number of different shapes, and misfolded proteins can lead to toxicity and other problems. For example, all cells rely on signaling proteins in the membranes that enclose them to monitor their environment so that they can adapt to changing conditions and, in multicellular organisms, communicate with neighboring cells: without properly folded signaling proteins, chaos would ensue. Moreover, many diseases—including diabetes, cancer, viral infection and neurodegenerative disease—have been linked to protein folding processes. It is not surprising, therefore, that cells have evolved elaborate mechanisms to exert exquisite quality control over protein folding.
One of these mechanisms, called the unfolded protein response (UPR), operates in a compartment within the cell known as the endoplasmic reticulum (ER). The ER is a labyrinthine network of tubes and sacs within all eukaryotic cells, and most proteins destined for the cell surface or outside the cell adopt their properly folded shapes within this compartment. If the ER does not have enough capacity to fold all of the proteins that are delivered there, the UPR switches on to increase the protein folding capacity, to expand the surface area and volume of the compartment, and to degrade misfolded proteins. If the UPR cannot adequately adjust the folding capacity of the ER to meet the demands of the cell, the UPR triggers a program that kills the cell to prevent putting the whole organism at risk.
Researchers have identified the cellular components that monitor the protein folding conditions inside the ER. All eukaryotic cells, from unicellular yeasts to mammalian cells, contain a highly conserved protein-folding sensor called Ire1. In all species analyzed to date, Ire1 is known to activate the UPR through an messenger RNA (mRNA) splicing mechanism. This splicing event provides the switch that drives a gene expression program in which the production of ER components is increased to boost the protein folding capacity of the compartment.
Kimmig, Diaz et al. now report the first instance of an organism in which the UPR does not involve mRNA splicing or the initiation of a gene expression program. Rather, the yeast Schizosaccharomyces pombe utilizes Ire1 to an entirely different end. The authors find that the activation of Ire1 in S. pombe leads to the selective decay of a specific class of mRNAs that all encode proteins entering the ER. Thus, rather than increasing the protein folding capacity of the ER when faced with an increased protein folding load, S. pombe cells correct the imbalance by decreasing the load.
The authors also show that a lone mRNA—the mRNA that encodes the molecular chaperone BiP, which is one of the major protein-folding components in the ER—uniquely escapes this decay. Rather than being degraded, Ire1 truncates BiP mRNA and renders it more stable. By studying the UPR in a divergent organism, the authors shed new light on the evolution of a universally important process and illustrate how conserved machinery has been repurposed.
DOI: http://dx.doi.org/10.7554/eLife.00048.002
doi:10.7554/eLife.00048
PMCID: PMC3470409  PMID: 23066505
Unfolded Protein Response; Ire1; selective mRNA decay; Bip1 mRNA stabilization; ER homeostasis; S. pombe

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