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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  Unfolded Proteins are Ire1-Activating Ligands that Directly Induce the Unfolded Protein Response 
Science (New York, N.Y.)  2011;333(6051):1891-1894.
The unfolded protein response (UPR) detects the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and adjusts the protein folding capacity to the needs of the cell. Under conditions of ER stress, the transmembrane protein Ire1 oligomerizes to activate its cytoplasmic kinase and RNase domains. It is unclear what feature of ER stress Ire1 detects. Here we found that the core ER-lumenal domain (cLD) of yeast Ire1 binds to unfolded proteins in vivo and to peptides primarily composed of basic and hydrophobic residues in vitro. Mutation of amino acid side chains exposed in a putative peptide-binding groove of Ire1 cLD impaired peptide binding. Peptide binding caused Ire1 cLD oligomerization in vitro, suggesting that direct binding to unfolded proteins activates the UPR.
doi:10.1126/science.1209126
PMCID: PMC3202989  PMID: 21852455
20.  Bioinformatics Training Network (BTN): a community resource for bioinformatics trainers 
Briefings in Bioinformatics  2011;13(3):383-389.
Funding bodies are increasingly recognizing the need to provide graduates and researchers with access to short intensive courses in a variety of disciplines, in order both to improve the general skills base and to provide solid foundations on which researchers may build their careers. In response to the development of ‘high-throughput biology’, the need for training in the field of bioinformatics, in particular, is seeing a resurgence: it has been defined as a key priority by many Institutions and research programmes and is now an important component of many grant proposals. Nevertheless, when it comes to planning and preparing to meet such training needs, tension arises between the reward structures that predominate in the scientific community which compel individuals to publish or perish, and the time that must be devoted to the design, delivery and maintenance of high-quality training materials. Conversely, there is much relevant teaching material and training expertise available worldwide that, were it properly organized, could be exploited by anyone who needs to provide training or needs to set up a new course. To do this, however, the materials would have to be centralized in a database and clearly tagged in relation to target audiences, learning objectives, etc. Ideally, they would also be peer reviewed, and easily and efficiently accessible for downloading. Here, we present the Bioinformatics Training Network (BTN), a new enterprise that has been initiated to address these needs and review it, respectively, to similar initiatives and collections.
doi:10.1093/bib/bbr064
PMCID: PMC3357490  PMID: 22110242
Bioinformatics; training; end users; bioinformatics courses; learning bioinformatics
21.  Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity 
The Journal of Cell Biology  2011;193(1):171-184.
Uncoupling of Ire1’s RNAse and kinase activities reveals that its auto-phosphorylation is important for resolution of the unfolded protein response. (See also a related paper by Chawla et al. in this issue).
Accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). Ire1, an ER-resident transmembrane kinase/RNase, senses the protein folding status inside the ER. When activated, Ire1 oligomerizes and trans-autophosphorylates, activating its RNase and initiating a nonconventional mRNA splicing reaction. Splicing results in production of the transcription factor Hac1 that induces UPR target genes; expression of these genes restores ER homeostasis by increasing its protein folding capacity and allows abatement of UPR signaling. Here, we uncouple Ire1’s RNase from its kinase activity and find that cells expressing kinase-inactive Ire1 can regulate Ire1’s RNase, splice HAC1 mRNA, produce Hac1 protein, and induce UPR target genes. Unlike wild-type IRE1, kinase-inactive Ire1 cells display defects in Ire1 deactivation. Failure to properly inactivate Ire1 causes chronic ER stress and reduces cell survival under UPR-inducing conditions. Thus, Ire1-catalyzed phosphoryl-transfer aids disassembly of Ire1 signaling complexes and is a critical component of the UPR homeostatic feedback loop.
doi:10.1083/jcb.201007077
PMCID: PMC3082176  PMID: 21444684
22.  Antimyeloma Effects of the Heat Shock Protein 70 Molecular Chaperone Inhibitor MAL3-101 
Journal of Oncology  2011;2011:232037.
Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable, primarily due to the treatment-refractory/resistant nature of the disease. A rational approach to this compelling challenge is to develop new drugs that act synergistically with existing effective agents. This approach will reduce drug concentrations, avoid treatment resistance, and also improve treatment effectiveness by targeting new and nonredundant pathways in MM. Toward this goal, we examined the antimyeloma effects of MAL3-101, a member of a new class of non-ATP-site inhibitors of the heat shock protein (Hsp) 70 molecular chaperone. We discovered that MAL3-101 exhibited antimyeloma effects on MM cell lines in vitro and in vivo in a xenograft plasmacytoma model, as well as on primary tumor cells and bone marrow endothelial cells from myeloma patients. In combination with a proteasome inhibitor, MAL3-101 significantly potentiated the in vitro and in vivo antimyeloma effects. These data support a preclinical rationale for small molecule inhibition of Hsp70 function, either alone or in combination with other agents, as an effective therapeutic strategy for MM.
doi:10.1155/2011/232037
PMCID: PMC3184436  PMID: 21977030
23.  A CHOP-regulated microRNA controls rhodopsin expression 
The Journal of Cell Biology  2011;192(6):919-927.
ER stress induces expression of miR-708, which suppresses the production of rhodopsin to prevent ER overloading in retinal epithelial cells.
Using genome-wide microribonucleic acid (microRNA [miRNA]) expression profiling, bioinformatics, and biochemical analyses, we identified miR-708, an endoplasmic reticulum (ER) stress-inducible miRNA whose expression is regulated by the transcription factor CCAAT enhancer-binding protein homologous protein (CHOP) in vertebrates. miR-708 is encoded within an intron of the CHOP-regulated gene Odz4, a member of the highly conserved teneurin family of developmental regulators. Odz4 and mir-708 expression is coregulated by CHOP, and the two transcripts are coexpressed in the brain and eyes of mice, suggesting common physiological functions in these tissues. We validated rhodopsin as a target of miR-708 through loss- and gain-of-function experiments. Together, our data implicate miR-708 in the homeostatic regulation of ER function in mammalian rod photoreceptors, whereby miR-708 may help prevent an excessive rhodopsin load from entering the ER. Hence, miR-708 may function analogously to other unfolded protein response controls that throttle protein influx into the ER to avoid ER stress through mechanisms, such as general translational attenuation by protein kinase RNA–like ER kinase or membrane-bound messenger RNA decay by inositol-requiring enzyme 1.
doi:10.1083/jcb.201010055
PMCID: PMC3063143  PMID: 21402790
24.  Structural and functional basis for RNA cleavage by Ire1 
BMC Biology  2011;9:47.
Background
The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis.
Results
This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase.
Conclusions
Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.
doi:10.1186/1741-7007-9-47
PMCID: PMC3149027  PMID: 21729333
25.  Cofactor-mediated conformational control in the bifunctional kinase/RNase Ire1 
BMC Biology  2011;9:48.
Background
Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity.
Results
Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal β-phosphate group of a potent cofactor ADP by sulfur results in ADPβS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn2+ and Cd2+, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors.
Conclusions
Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on protein-protein interactions involving Ire1's kinase domain suggests that protein kinases and pseudokinases encoded in metazoan genomes may use ATP pocket-binding ligands similarly to exert signaling roles other than phosphoryl transfer.
doi:10.1186/1741-7007-9-48
PMCID: PMC3158555  PMID: 21729334

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