Endoplasmic Reticulum Lumen Research Articles

Single-Molecule Displacement Mapping Unveils Sign-Asymmetric Protein Charge Effects on Intraorganellar Diffusion.

Using single-molecule displacement/diffusivity mapping (SMdM), an emerging super-resolution microscopy method, here we quantify, at nanoscale resolution, the diffusion of a typical fluorescent protein (FP) in the endoplasmic reticulum (ER) and mitochondrion of living mammalian cells. We thus show that the diffusion coefficients D in both organelles are ∼40% of that in the cytoplasm, with the latter exhibiting higher spatial inhomogeneities. Moreover, we unveil that diffusions in the ER lumen and the mitochondrial matrix are markedly impeded when the FP is given positive, but not negative, net charges. Calculation shows most intraorganellar proteins as negatively charged, hence a mechanism to impede the diffusion of positively charged proteins. However, we further identify the ER protein PPIB as an exception with a positive net charge and experimentally show that the removal of this positive charge elevates its intra-ER diffusivity. We thus unveil a sign-asymmetric protein charge effect on the nanoscale intraorganellar diffusion.

Two nucleotide sugar transporters are important for cell wall integrity and full virulence of Magnaporthe oryzae.

Cell wall polysaccharides play key roles in fungal development, virulence, and resistance to the plant immune system, and are synthesized from many nucleotide sugars in the endoplasmic reticulum (ER)-Golgi secretory system. Nucleotide sugar transporters (NSTs) are responsible for transporting cytosolic-derived nucleotide sugars to the ER lumen for processing, but their roles in plant-pathogenic fungi remain to be revealed. Here, we identified two important NSTs, NST1 and NST2, in the rice blast fungus Magnaporthe oryzae. Both NSTs were localized in the ER, which was consistent with a function in transporting nucleotide sugar for processing in the ER. Sugar transport property analysis suggested that NST1 is involved in transportation of mannose and glucose, while NST2 is only responsible for mannose transportation. Accordingly, deletion ofNSTs resulted in a significant decrease in corresponding soluble saccharides abundance and defect in sugar utilization. Moreover, bothNSTs played important roles in cell wall integrity, were involved in asexual development, and were required for full virulence. The NST mutants exhibited decreasing external glycoproteins and exposure of inner chitin, which resulted in activation of the host defence response. Altogether, our results revealed that two sugar transporters are required for fungal cell wall polysaccharides accumulation and full virulence of M.oryzae.

Apolipoprotein B in the Rough Endoplasmic Reticulum: Translation, Translocation and the Initiation of Lipoprotein Assembly

Apolipoprotein (apo) B and the microsomal triglyceride transfer protein are essential for the hepatic assembly and secretion of triglyceride-rich VLDL. To understand how apoB initiates the process of lipoprotein formation, interest has focused on the biogenesis of its amino terminal globular domain (α1 domain). When only this domain is expressed in hepatoma cells, no lipoprotein particle will form. However, proper folding of the α1 domain is essential for the internal lipophilic regions of apoB to engage in cotranslational lipid recruitment. The essential function of this domain may be related to its capacity to promote a specific physical interaction with the microsomal triglyceride transfer protein, necessary for apoB's proper folding and lipidation. Alternatively, this domain may promote an autonomous lipid recruitment step that nucleates microsomal triglyceride transfer protein-dependent lipid sequestration by apoB. Forms of apoB that fail to initiate particle assembly or forms associated with aberrant underlipidated particles are targeted for intracellular turnover. Two sites of apoB degradation have been identified. In hepatocarcinoma-derived cells, misassembled apoB may undergo progressive reverse translocation from the endoplasmic reticulum lumen to the cytosol, a process that is mechanistically coupled to polyubiquitination and proteasome-mediated degradation on the cytosolic side of the membrane. Alternatively, studies in primary hepatocytes reveal that apoB may undergo sorting to a post-endoplasmic reticulum compartment for presecretory degradation. In either case, the balance between assembly and presecretory degradation of apoB may represent a control point for the production of hepatic VLDL.

Knockout of ENO1 leads to metabolism reprogramming and tumor retardation in pancreatic cancer.

The shift in glucose utilization from oxidative phosphorylation to glycolysis is the hallmark of tumor cells. The overexpression of ENO1, one of the key enzymes in the glycolysis process, has been identified in several cancers, however, its role in pancreatic cancer (PC) is yet unclear. This study identifies ENO1 as an indispensable factor in the progression of PC. Interestingly, ENO1-knockout could inhibit cell invasion and migration and prevent cell proliferation in pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1 and MIA PaCa-2); meanwhile, tumor cell glucose uptake and lactate excretion also decreased significantly. Furthermore, ENO1-knockout reduced colony formation and tumorigenesis in both in vitro and in vivo tests. In total, after ENO1 knockout, 727 differentially expressed genes (DEGs) were identified in PDAC cells by RNA-seq. Gene Ontology enrichment analysis revealed that these DEGs are mainly associated with components such as the 'extracellular matrix' and 'endoplasmic reticulum lumen', and participate in the regulation of signal receptor activity. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the identified DEGs are associated with pathways, such as 'fructose and mannose metabolism', 'pentose phosphate pathway, and 'sugar metabolism for amino and nucleotide. Gene Set Enrichment Analysis showed that ENO1 knockout promoted the upregulation of oxidative phosphorylation and lipid metabolism pathways-related genes. Altogether, these results indicated that ENO1-knockout inhibited tumorigenesis by reducing cell glycolysis and activating other metabolic pathways by altering the expression of G6PD, ALDOC, UAP1, as well as other related metabolic genes. Concisely, ENO1, which plays a vital role in the abnormal glucose metabolism in PC, can be exploited as a target to control carcinogenesis by reducing aerobic glycolysis.

Selenoprotein S Interacts with the Replication and Transcription Complex of SARS-CoV-2 by Binding nsp7

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replicates and evades detection using ER membranes and their associated protein machinery. Among these hijacked human proteins is selenoprotein S (selenos). This selenoprotein takes part in the protein quality control, signaling, and the regulation of cytokine secretion. While the role of selenos in the viral life cycle is not yet known, it has been reported to interact with SARS-CoV-2 nonstructural protein 7 (nsp7), a viral protein essential for the replication of the virus. We set to study whether selenos and nsp7 interact directly and if they can still bind when nsp7 is bound to the replication and transcription complex of the virus. Using biochemical assays, we show that selenos binds directly to nsp7. In addition, we found that selenos can bind to nsp7 when it is in a complex with the coronavirus's minimal replication and transcription complex, comprised of nsp7, nsp8, and the RNA-dependent RNA polymerase nsp12. In addition, through crosslinking experiments, we mapped the interaction sites of selenos and nsp7 in the replication complex and showed that the hydrophobic segment of selenos is essential for binding to nsp7. This arrangement leaves an extended helix and the intrinsically disordered segment of selenos—including the reactive selenocysteine—exposed and free to potentially recruit additional proteins to the replication and transcription complex.

Surf4 (Surfeit Locus Protein 4) Deficiency Reduces Intestinal Lipid Absorption and Secretion and Decreases Metabolism in Mice.

Postprandial dyslipidemia is a causative risk factor for cardiovascular disease. The majority of absorbed dietary lipids are packaged into chylomicron and then delivered to circulation. Previous studies showed that Surf4 (surfeit locus protein 4) mediates very low-density lipoprotein secretion from hepatocytes. Silencing hepatic Surf4 markedly reduces the development of atherosclerosis in different mouse models of atherosclerosis without causing hepatic steatosis. However, the role of Surf4 in chylomicron secretion is unknown. We developed inducible intestinal-specific Surf4 knockdown mice (Surf4IKO) using Vil1Cre-ERT2 and Surf4flox mice. Metabolic cages were used to monitor mouse metabolism. Enzymatic kits were employed to measure serum and tissue lipid levels. The expression of target genes was detected by qRT-PCR and Western Blot. Transmission electron microscopy and radiolabeled oleic acid were used to assess the structure of enterocytes and intestinal lipid absorption and secretion, respectively. Proteomics was performed to determine changes in protein expression in serum and jejunum. Surf4IKO mice, especially male Surf4IKO mice, displayed significant body weight loss, increased mortality, and reduced metabolism. Surf4IKO mice exhibited lipid accumulation in enterocytes and impaired fat absorption and secretion. Lipid droplets and small lipid vacuoles were accumulated in the cytosol and the endoplasmic reticulum lumen of the enterocytes of Surf4IKO mice, respectively. Surf4 colocalized with apoB and co-immunoprecipitated with apoB48 in differentiated Caco-2 cells. Intestinal Surf4 deficiency also significantly reduced serum triglyceride, cholesterol, and free fatty acid levels in mice. Proteomics data revealed that diverse pathways were altered in Surf4IKO mice. In addition, Surf4IKO mice had mild liver damage, decreased liver size and weight, and reduced hepatic triglyceride levels. Our findings demonstrate that intestinal Surf4 plays an essential role in lipid absorption and chylomicron secretion and suggest that the therapeutic use of Surf4 inhibition requires highly cell/tissue-specific targeting.

Interactions between cadmium, lead, mercury, and arsenic and depression: A molecular mechanism involved

BackgroundWe aimed to assess the interactions between mixed heavy metals, genes, and miRNAs implicated in depression development and to design and create miRNA sponges. MethodsThe key data-mining approaches in this study were the Comparative Toxicogenomics Database (CTD), MIENTURNET, GeneMania, Metascape, Webgestalt, miRNAsong, and Cytoscape software. ResultsA mixture of cadmium, lead, mercury, and arsenic was related to the development of depression. Even though the genes acquired from the heavy metals of depression studied were different, the “selenium micronutrient network”, “vitamin B12 and folate metabolism”, and “positive regulation of peptidyl-serine phosphorylation” pathways were highlighted. The heavy metal mixture altered the genes SOD1, IL6, PTGS2, PON1, BDNF, and ALB, highlighting the role of oxidative stress, pro-inflammatory cytokines, paraoxonase activity, neurotrophic factors, and antioxidants related to depression, as well as the possibility of targeting these genes in prospective depressive treatment. Chr1q31.1, five transcription factors (NR4A3, NR1H4, ATF3, CREB3L3, and NR1I3), the “endoplasmic reticulum lumen,” “blood microparticle,” and “myelin sheath”, were found to be important chromosomal locations, transcription factors, and cellular parts linked to depression and affected by mixed heavy metals. Furthermore, we developed a network-based approach to detect significant genes, miRNA, pathways, and illnesses related to depression development. We also observed eight important miRNAs related to depression induced by mixed heavy metals (hsa-miR-16-5p, hsa-miR-132-3p, hsa-miR-1-3p, hsa-miR-204-5p, hsa-miR-206, hsa-miR-124-3p, hsa-miR-146a-5p, and hsa-miR-26a-5p). In addition, we created and evaluated miRNA sponge sequences for these miRNAs in silico. LimitationsA toxicogenomic design in silico was used. ConclusionsOur findings highlight the importance of oxidative stress, notably SOD1 and the selenium micronutrient network, in depression caused by heavy metal mixtures and provide additional insights into common molecular pathways implicated in depression pathogenesis.

S-acylation of SARAF regulates store-operated calcium entry.

The endoplasmic reticulum (ER) is the primary intracellular calcium storage site. Upon cellular stimulation by an agonist, the production of inositol 1,4,5-trisphosphate (IP3) leads to activation of IP3 receptor channels on the ER membrane and calcium efflux from ER lumen. This leads to ER calcium store depletion. Upon store depletion, the ER-resident calcium sensor STIM1 and plasma membrane calcium channel Orai1 bind to each other to form the calcium release-activated calcium (CRAC) channel. These channels lead to calcium entry from extracellular milieu. This entry is known as store-operated calcium entry (SOCE). Previously, we have shown that Orai1 and STIM1 are S-acylated upon store-depletion and this process is crucial for CRAC channel formation and SOCE. TMEM66, also known as SARAF, is a ER transmembrane protein that is known to associate with STIM1 in resting conditions, and subsequently with Orai1 after store-depletion. This binding of SARAF with Orai1 upon store-depletion is important for the inactivation of CRAC currents. We hypothesize S-acylation of SARAF is required for the inactivation of SOCE. Here we show that SARAF is S-acylated in Jurkat T cells upon activation of the T cell receptor (TCR) and subsequent store-depletion. We also show the residue where SARAF undergoes this S-acylation. Importantly, a C320S mutant version of SARAF that cannot undergo S-acylation has deficits in CRAC channel formation and SOCE. Put together, these data suggest SARAF is a crucial component of SOCE.

Atlas of interactions between SARS-CoV-2 macromolecules and host proteins.

The proteins and RNAs of viruses extensively interact with host proteins after infection. We collected and reanalyzed all available datasets of protein-protein and RNA-protein interactions related to SARS-CoV-2. We investigated the reproducibility of those interactions and made strict filters to identify highly confident interactions. We systematically analyzed the interaction network and identified preferred subcellular localizations of viral proteins, some of which such as ORF8 in ER and ORF7A/B in ER membrane were validated using dual fluorescence imaging. Moreover, we showed that viral proteins frequently interact with host machinery related to protein processing in ER and vesicle-associated processes. Integrating the protein- and RNA-interactomes, we found that SARS-CoV-2 RNA and its N protein closely interacted with stress granules including 40 core factors, of which we specifically validated G3BP1, IGF2BP1, and MOV10 using RIP and Co-IP assays. Combining CRISPR screening results, we further identified 86 antiviral and 62 proviral factors and associated drugs. Using network diffusion, we found additional 44 interacting proteins including two proviral factors previously validated. Furthermore, we showed that this atlas could be applied to identify the complications associated with COVID-19. All data are available in the AIMaP database (https://mvip.whu.edu.cn/aimap/) for users to easily explore the interaction map.

Structural flexibility of huntingtin oligomers plays a key role in directly binding lipid membranes.

Huntingtin disease (HD) is a neurodegenerative disease caused by expansion of a polyglutamine (polyQ) tract within the huntingtin (htt) protein, leading to aggregation into a variety of species from oligomers to fibrils. The ability of htt to directly bind and damage cellular membranes represents a fundamental step in a variety of toxic mechanisms. For example, membrane damage associated with the ER, mitochondria, and nuclear membrane occurs in several HD models. However, how distinct htt aggregates interact with lipid membranes remains unclear. Directly preceding the polyQ domain, the first seventeen amino acids of htt (Nt17) functions as an amphipathic α-helix lipid-binding domain. Thus, Nt17 plays a prominent role in the direct interaction of htt with membranes. Nt17 also facilitates the formation of α-helical oligomers that serve as intermediates in fibril formation. Here, the membrane activity of different aggregate species was systematically determined using lipid-binding assays. While htt monomers readily bound membranes, oligomer formation enhanced the htt/lipid interaction several fold. Htt fibrils displayed minimal membrane activity. To elucidate mechanisms by which oligomers bind membranes, two strategies were implemented: 1) stabilizing oligomers with small peptides based on Nt17 and 2) chemically cross-linking oligomers via lysine residues within Nt17. Some of the small Nt17 peptides contained point mutations that alter the total charge of the oligomer. While changing charge slightly reduced the apparent affinity of oligomers for membranes, structural flexibility within the oligomer appeared to play a larger role in the oligomer/membrane interaction. In particular, chemical cross-linking effectively eliminated the ability of oligomers to bind lipid membranes. Additionally, cross-link stabilized oligomers lost their toxic gain of function when exogenously added to cell culture. Collectively, this suggests that targeting structural flexibility of htt oligomers may be an effective therapeutic strategy.

Mechanism for membrane contact formation with full-length Osh4 in dual-membrane environment.

Membrane contacts sites (MCSs) play important roles in lipid trafficking across cellular compartments and maintain the widespread structural diversity of organelles. Osh4 is the most abundantly expressed oxysterol binding protein (Osh) in yeast and facilitates the sterol and lipid transport via a non-vesicular pathway between cellular organelles. The functional mechanism of Osh4 in lipid transport is elusive as several experimental studies have suggested that Osh proteins operate at MCSs and shuttle lipids between membranes in a direct fashion or via diffusing through cytoplasm. We have utilized microsecond long all-atom molecular dynamics simulations and enhanced sampling techniques to unravel the functional mechanism of Osh4 protein in dual-membrane environment mimicking the interface of ER and TGN membranes using CHARMM36m forcefield with additional CUFIX parameters for lipid-protein electrostatic interactions. In dual-membrane environment, unbiased MD simulations indicate that Osh4 initially rotates approaching a single membrane and eventually establishes contact with that membrane yielding a β-crease membrane bound conformation similar to our previous observation based on single-membrane approach. We have further used targeted MD simulations based on our past work with α6-α7 region of Osh4 to accelerate the conformational transition of Osh4 enabling simultaneous binding on two opposing membranes at MCS. Interestingly, we have identified a bound state of Osh4 at MCS where the protein is interacting with the lower membrane through the β-crease surface with its PHE-239 residue located below the membrane phosphate plane and other part of the protein is simultaneously establishing contact with the opposite membrane though extended α6-α7 region. These dual membrane contacts are maintained concurrently throughout microsecond long MD simulations. Nevertheless, Osh4 structural determination at MCS utilizing NMR and cryoEM techniques are planned to support our computational findings and shed light on the functional mechanism of Osh4 at MCS.

A structurally derived universal mechanism for the catalytic cycle of the tail-anchored targeting factor Get3.

Tail-anchored (TA) membrane proteins, accounting for ∼2% of proteomes, are primarily targeted post-translationally to the ER membrane by the Guided Entry of TA proteins (GET) pathway. For this complicated process, it remains unknown how the central targeting factor Get3 uses nucleotide to facilitate large conformational changes to recognize then bind clients while also preventing exposure of hydrophobic surfaces. We have identified the GET pathway in Giardia intestinalis. Using x-ray crystallography and cryo-EM, I will present a series of structures including the first structure of the Get3/client complex in the critical post-nucleotide-hydrolysis state, demonstrating that Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix. Four additional structures of GiGet3, spanning the nucleotide-free (apo) open to closed transition and the ATP-bound state, reveal the details of nucleotide stabilization and occluded CBD. This work provides the first direct data of the GET pathway outside of opishthokonts and resolves key conundrums, allowing for a complete model of the dramatic conformational landscape of Get3. I will further discuss an exciting Get3-like protein found associated with photosynthesis.

Tex2 is required for lysosomal functions at TMEM55-dependent ER membrane contact sites.

ER tubules form and maintain membrane contact sites (MCSs) with late endosomes/lysosomes (LE/lys). The molecular composition and cellular functions of these MCSs are poorly understood. Here, we find that Tex2, an SMP domain-containing lipid transfer protein conserved in metazoan and yeast, is a tubular ER protein and is recruited to ER-LE/lys MCSs by TMEM55, phosphatases that convert PI(4,5)P2 to PI5P on LE/lys. We show that the Tex2-TMEM55 interaction occurs between an N-terminal region of Tex2 and a catalytic motif in the PTase domain of TMEM55. The Tex2-TMEM55 interaction can be regulated by endosome-resident type 2 PI4K activities. Functionally, Tex2 knockout results in defects in lysosomal trafficking, digestive capacity, and lipid composition of LE/lys membranes. Together, our data identify Tex2 as a tubular ER protein that resides at TMEM55-dependent ER-LE/lys MCSs required for lysosomal functions.

Renal Ischemia Tolerance Mediated by eIF5A Hypusination Inhibition Is Regulated by a Specific Modulation of the Endoplasmic Reticulum Stress.

Through kidney transplantation, ischemia/reperfusion is known to induce tissular injury due to cell energy shortage, oxidative stress, and endoplasmic reticulum (ER) stress. ER stress stems from an accumulation of unfolded or misfolded proteins in the lumen of ER, resulting in the unfolded protein response (UPR). Adaptive UPR pathways can either restore protein homeostasis or can turn into a stress pathway leading to apoptosis. We have demonstrated that N1-guanyl-1,7-diamineoheptane (GC7), a specific inhibitor of eukaryotic Initiation Factor 5A (eIF5A) hypusination, confers an ischemic protection of kidney cells by tuning their metabolism and decreasing oxidative stress, but its role on ER stress was unknown. To explore this, we used kidney cells pretreated with GC7 and submitted to either warm or cold anoxia. GC7 pretreatment promoted cell survival in an anoxic environment concomitantly to an increase in xbp1 splicing and BiP level while eiF2α phosphorylation and ATF6 nuclear level decreased. These demonstrated a specific modulation of UPR pathways. Interestingly, the pharmacological inhibition of xbp1 splicing reversed the protective effect of GC7 against anoxia. Our results demonstrated that eIF5A hypusination inhibition modulates distinctive UPR pathways, a crucial mechanism for the protection against anoxia/reoxygenation.

Ectopic RING activity at the ER membrane differentially impacts ERAD protein quality control pathways

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that ensures misfolded proteins are removed from the ER and destroyed. In ERAD, membrane and luminal substrates are ubiquitylated by ER-resident RING-type E3 ubiquitin ligases, retrotranslocated into the cytosol, and degraded by the proteasome. Overexpression of ERAD factors is frequently used in yeast and mammalian cells to study this process. Here we analyze the impact of ERAD E3 overexpression on substrate turnover in yeast, where there are three ERAD E3 complexes (Doa10, Hrd1, and Asi1-3). Elevated Doa10 or Hrd1 (but not Asi1) RING activity at the ER membrane resulting from protein overexpression inhibits the degradation of specific Doa10 substrates. The ERAD E2 ubiquitin-conjugating enzyme Ubc6 becomes limiting under these conditions, and UBC6 overexpression restores Ubc6-mediated ERAD. Using a subset of the dominant-negative mutants, which contain the Doa10 RING domain but lack the E2-binding region, we show that they induce degradation of membrane tail-anchored Ubc6 independently of endogenous Doa10 and the other ERAD E3 complexes. This remains true even if the cells lack the Dfm1 rhomboid pseudoprotease, which is also a proposed retrotranslocon. Hence, rogue RING activity at the ER membrane elicits a highly specific off-pathway defect in the Doa10 pathway, and the data point to an additional ERAD E3-independent retrotranslocation mechanism.

Single-cell RNA sequencing reveals the suppressive effect of PPP1R15A inhibitor Sephin1 in antitumor immunity

Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, Tcell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific Tcells was inhibited by Sephin1. A special TCR+ macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.

SELENOM Knockout Induces Synaptic Deficits and Cognitive Dysfunction by Influencing Brain Glucose Metabolism.

Selenium, a trace element associated with memory impairment and glucose metabolism, mainly exerts its function through selenoproteins. SELENOM is a selenoprotein located in the endoplasmic reticulum (ER) lumen. Our study demonstrates for the first time that SELENOM knockout decreases synaptic plasticity and causes memory impairment in 10-month-old mice. In addition, SELENOM knockout causes hyperglycaemia and disturbs glucose metabolism, which is essential for synapse formation and transmission in the brain. Further research reveals that SELENOM knockout leads to inhibition of the brain insulin signaling pathway [phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR/p70 S6 kinase pathway], which may impair synaptic plasticity in mice. High-fat diet (HFD) feeding suppresses the brain insulin signaling pathway in SELENOM knockout mice and leads to earlier onset of cognitive impairment at 5 months of age. In general, our study demonstrates that SELENOM knockout induces synaptic deficits via the brain insulin signaling pathway, thus leading to cognitive dysfunction in mice. These data strongly suggest that SELENOM plays a vital role in brain glucose metabolism and contributes substantially to synaptic plasticity.

Sec61 channel subunit Sbh1/Sec61β promotes ER translocation of proteins with suboptimal targeting sequences and is fine-tuned by phosphorylation

The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one nonessential subunit, Sec61β/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen, we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T-specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation.

Endoplasmic Reticulum Stress Causing Apoptosis in a Mouse Model of an Ischemic Spinal Cord Injury.

A spinal cord injury (SCI) is the devastating trauma associated with functional deterioration due to apoptosis. Most laboratory SCI models are generated by a direct impact on an animal's spinal cord; however, our model does not involve the direct impact on the spinal cord. Instead, we use a clamp compression to create an ischemia in the descending aortas of mice. Following the success of inducing an ischemic SCI (ISCI), we hypothesized that this model may show apoptosis via an endoplasmic reticulum (ER) stress pathway. This apoptosis by the ER stress pathway is enhanced by the inducible nitric oxide synthase (iNOS). The ER is used for the protein folding in the cell. When the protein folding capacity is overloaded, the condition is termed the ER stress and is characterized by the accumulation of misfolded proteins inside the ER lumen. The unfolded protein response (UPR) signaling pathways that deal with the ER stress response then become activated. This UPR activates the three signal pathways that are regulated by the inositol-requiring enzyme 1α (IRE1α), the activating transcription factor 6 (ATF6), and the protein kinase RNA-like ER kinase (PERK). IRE1α and PERK are associated with the expression of the apoptotic proteins. Apoptosis caused by an ISCI is assessed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. An ISCI also reduces synaptophysin and the neuronal nuclear protein (NeuN) in the spinal cord. In conclusion, an ISCI increases the ER stress proteins, resulting in apoptosis in neuronal cells in the spinal cord.

Can 900 MHz and 2100 MHz radiofrequency radiation exposure induce endoplasmic reticulum stress and apoptosis in rat thymus?

Objectives: Electrical appliances are source of radiofrequency radiation (RFR). The effects of RFR on the organism are not fully understood. Endoplasmic reticulum (ER) stress is appeared by the accumulation of misfolded proteins in ER lumen. The aim of this study was to investigate the effects of 900 and 2100 MHz RFR exposure on the ER stress pathway in rat thymus. Methods: Rats were divided into six groups: 1 week (w) and 10 w Sham rats were kept in plexiglass tubes for 2 hours/day without RFR, experiment groups were created as 1-w (acute) and 10 w (chronic) rats which exposed to 900 and 2100 MHz RFR for 2 h/day. There were 20 male Wistar rats in each group. Immunohistochemistry stainings were performed GRP78, CHOP, Cleaved (Clv.) Caspase 3 and Caspase 12. Results: Expressions of GRP78 and Clv. Caspase3 in RFR groups is significantly higher than sham groups (p < 0.001). In 900 MHZ-1 w rats, high levels of GRP78 expressions were at the cytoplasm of epithelial reticular cells. In other groups, GRP78 expressions were seen also at thymocytes. Expressions of CHOP in RFR rats were higher than sham rats (2100 MHz /Sham for 10 w; p < 0.001, 900 MHz /Sham for 10 w; p = 0.004, 900 MHz /Sham for 1 w; p = 0.003). Localization of CHOP expressions was at the nucleus membrane and cytoplasm. The expression of Caspase 12 in RFR rats was higher than sham rats (900 MHz /Sham for 1 w; p = 0.006, other groups; p < 0.001). Conclusions: This study demonstrates RFR exposure could increase levels of ER stress pathway proteins and could cause apoptosis.