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1.  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.
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.
PMCID: PMC4436593  PMID: 25986605
protein kinase; human cells; IPA; IRE1; PERK; E. coli; human; mouse
2.  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.
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.
PMCID: PMC4426669  PMID: 25875391
eIF2B; eIF2; integrated stress response; ISRIB; unfolded protein response; protein synthesis; human
3.  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.
PMCID: PMC4416682  PMID: 25931084
synchrotron powder diffraction; high-energy X-rays; high angular resolution; time-resolved experiments; X-ray total scattering
4.  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.
PMCID: PMC3578356  PMID: 23388626
5.  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.
PMCID: PMC4341466  PMID: 25719440
integrated stress response; eIF2; unfolded protein response; ISRIB; protein synthesis; ribosome profiling; Human; Mouse
6.  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.
PMCID: PMC4340591  PMID: 24974795
7.  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.
PMCID: PMC4284148  PMID: 24994655
8.  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.
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.
PMCID: PMC4279078  PMID: 25549299
stress signaling; endoplasmic reticulum; unfolded protein response; mRNA targeting; mRNA processing; S. cerevisiae
9.  Influence of the prediction error of the first eye undergoing cataract surgery on the refractive outcome of the fellow eye 
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.
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.
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.
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.
PMCID: PMC4222621  PMID: 25382967
cataract surgery; biometry; IOL power calculation; refractive error; fellow eye
10.  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.
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.
PMCID: PMC4213662  PMID: 25358118
signal recognition particle; ribosome translation; protein targeting; single molecule fluorescence; E. coli
11.  Topography of retinal recovery processes in humans 
The purpose of this study was to examine retinal recovery processes to pographically by the application of three flash sequences with specific interstimulus intervals.
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.
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).
The kinetics of the amplitude recovery after short interstimulus intervals showed a spatial distribution, with faster recovery toward the macular periphery.
PMCID: PMC4208419  PMID: 25349472
multifocal; electroretinography; recovery; LED stimulator; interstimulus interval
12.  ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery 
Journal of Cell Science  2014;127(18):4078-4088.
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.
PMCID: PMC4163648  PMID: 25052096
Endoplasmic reticulum; Stress response; Autophagy
13.  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.
PMCID: PMC4053359  PMID: 24918437
14.  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.
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.
PMCID: PMC3766372  PMID: 23684307
15.  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.
PMCID: PMC3998916  PMID: 24762475
16.  The cost-effectiveness of the Argus II retinal prosthesis in Retinitis Pigmentosa patients 
BMC Ophthalmology  2014;14:49.
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.
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%.
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.
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.
PMCID: PMC3990272  PMID: 24731533
Retinitis Pigmentosa; Retinal prosthesis; Cost-effectiveness analysis; Decision analytic modelling
17.  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.
PMCID: PMC4009140  PMID: 24829913
18.  Partial Coherence Laser Interferometry in Highly Myopic versus Emmetropic Eyes 
To investigate the reliability of partial coherence laser interferometry for optical biometry in highly myopic eyes.
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.
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.
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
19.  Development of very large electrode arrays for epiretinal stimulation (VLARS) 
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.
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.
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.
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.
PMCID: PMC3976033  PMID: 24502253
Retinal prosthesis; Artificial vision; Retinitis pigmentosa; Blindness; Rehabilitation; Vitreoretinal surgery; Silicon wafer Technology; Polyimide; Neurostimulation
20.  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.
PMCID: PMC3941775  PMID: 24683495
21.  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.
PMCID: PMC3866087  PMID: 24358019
22.  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.
PMCID: PMC3670588  PMID: 17991856
23.  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.
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.
PMCID: PMC3667625  PMID: 23741617
eIF2; eIF2B; ATF4; integrated stress response; unfolded protein response; memory consolidation; Human; Mouse; Rat
24.  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.
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.
PMCID: PMC3654481  PMID: 23682313
ERMES; Gem1; Miro; mitochondrial DNA; mitochondria; S. cerevisiae
25.  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.
PMCID: PMC3653419  PMID: 18039139
protein misfolding; unfolded protein response; IRE1; PERK; ATF-6

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