The effects of biofeedback training for efficient activation of infraspinatus on proprioception and EMG activity during shoulder external rotation

The purpose of this study investigated which biofeedback (BF) training enables efficient activation of the infraspinatus muscle that affect joint position sense (JPS) and force sense (FS) of the shoulder joint. Twenty healthy males participated and performed three external rotation (ER) exercises under three randomly assigned training conditions: 1) non-biofeedback (NBF), 2) BF and 3) force biofeedback (FBF). Each exercise was performed at intervals of one week between training conditions. After performed the ER exercise under each training condition, the relative error (RE) was calculated at shoulder ER 45° and 80°, and then shoulder ER force were measured to determine the JPS error and FS error, respectively. Muscle activity of infraspinatus and posterior deltoid were measured and compared between training conditions. The RE of shoulder ER 45° and 80° were significantly lower under the FBF conditions than other training conditions (P < 0.05). The RE of shoulder ER force were also significantly lower under the FBF conditions compared to those under the other training conditions (P < 0.05). The activity of the infraspinatus muscle was significantly higher under the FBF conditions during all three ER exercises than other training conditions (p < 0.05). We suggest that BF trainings can be useful to improve the proprioception of shoulder joint as well as activation of infraspinatus muscle while performing the ER exercises.     

Expression of OsBiP4 and OsBiP5 is highly correlated with the endoplasmic reticulum stress response in rice

Binding protein (BiP) is a chaperone protein involved in the folding of secretory proteins in the ER lumen. OsBiP1 is constitutively expressed in various tissues, whereas the expression of OsBiP4 and OsBiP5 (OsBiP4&5) is not detected in any tissue under normal conditions. However, expression of OsBiP4&5 was highly and specifically activated under ER stress conditions induced by DTT treatment, OsBiP1 knockdown, OsBiP1 overexpression, OsIRE1 overexpression, or various exogenous recombinant proteins in transgenic rice. In contrast, OsBiP4&5 did not accumulate in OsIRE1 knockdown transgenic rice even after DTT treatment. When the subcellular localization of OsBiP4&5 was investigated in seed endosperm cells under the ER stress condition, OsBiP4&5 were localized to the ER, but did not participate in ER-derived protein body (PB-I) formation in a different manner to OsBiP1. These results indicate that OsBiP4&5 levels were positively correlated with stress levels in the ER. Taken together, these results suggest that OsBiP4&5 are ER stress-related BiP proteins that are regulated by OsIRE1/OsbZIP50 pathway and that they may have a distinct function from that of OsBiP1 in rice.     

Direct monitoring of in vivo ER stress during the development of insulin resistance with ER stress-activated indicator transgenic mice

Type 2 diabetes is one of the most prevalent and serious metabolic diseases in the world, and insulin resistance and pancreatic β-cell dysfunction are the hallmarks of the disease. It has been suggested that endoplasmic reticulum (ER) stress is provoked under diabetic conditions and is possibly involved in the development of insulin resistance. In this study, using ER stress-activated indicator (ERAI) transgenic mice which express green fluorescent protein (GFP) under ER stress conditions, we directly monitored in vivo ER stress in various insulin target tissues such as liver, fat, and muscle in diabetic mice with insulin resistance induced by high fat and high sucrose (HF/HS) diet treatment. In the liver of the ERAI transgenic mice, ERAI fluorescence activity was clearly observed as early as after 4 weeks of HF/HS diet treatment, whereas it was not detected at all in the fat and muscle even after 12 weeks of HF/HS diet treatment. These results suggest that induction of ER stress is associated with the development of insulin resistance and that ER stress in the liver may facilitate the development of insulin resistance in the whole body. This is the first report to directly monitor in vivo ER stress in various insulin target tissues during the development of insulin resistance. In addition, our present results suggest that ERAI transgenic mice are very useful for evaluating in vivo ER stress, especially in the liver, during the development of insulin resistance.     

Targeting EDEM protects against ER stress and improves development and survival in C. elegans

EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival.     

Arabidopsis Stromal-derived Factor2 (SDF2) Is a Crucial Target of the Unfolded Protein Response in the Endoplasmic Reticulum

Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 Å resolution revealed the typical β-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.     

Calnexin is involved in apoptosis induced by endoplasmic reticulum stress in the fission yeast

Stress conditions affecting the functions of the endoplasmic reticulum (ER) cause the accumulation of unfolded proteins. ER stress is counteracted by the unfolded-protein response (UPR). However, under prolonged stress the UPR initiates a proapoptotic response. Mounting evidence indicate that the ER chaperone calnexin is involved in apoptosis caused by ER stress. Here, we report that overexpression of calnexin in Schizosaccharomyces pombe induces cell death with apoptosis markers. Cell death was partially dependent on the Ire1p ER-stress transducer. Apoptotic death caused by calnexin overexpression required its transmembrane domain (TM), and involved sequences on either side of the ER membrane. Apoptotic death caused by tunicamycin was dramatically reduced in a strain expressing endogenous levels of calnexin lacking its TM and cytosolic tail. This demonstrates the involvement of calnexin in apoptosis triggered by ER stress. A genetic screen identified the S. pombe homologue of the human antiapoptotic protein HMGB1 as a suppressor of apoptotic death due to calnexin overexpression. Remarkably, overexpression of human calnexin in S. pombe also provoked apoptotic death. Our results argue for the conservation of the role of calnexin in apoptosis triggered by ER stress, and validate S. pombe as a model to elucidate the mechanisms of calnexin-mediated cell death.     

Heterologous expression of rice calnexin (OsCNX) confers drought tolerance in Nicotiana tabacum

Calnexin (CNX) is an integral membrane protein of endoplasmic reticulum (ER) and is a critical component of ER quality control machinery. It acts as a chaperone and ensures proper folding of newly synthesised glycoproteins. CNX shares a considerable homology with its luminal counterpart calreticulin (CRT). Together, they constitute CNX/CRT cycle which is imperative for proper folding of nascent proteins. CNX deficient organisms develop severe complications because of improper folding of proteins and consequently ER stress. CNX maintains calcium homeostasis by binding to the Ca²⁺ which is a central node in various signaling pathways. Phosphorylation of cytoplasmic tail of CNX controls the sarco endoplasmic reticulum calcium ATPase and thus the movement of Ca²⁺ in and out of its store-house, i.e. ER. Our studies on Oryza sativa CNX (OsCNX) reveal constitutive expression at various developmental stages and various tissues, thereby proving its requirement throughout the plant development. Further, its expression under various stress conditions gives an insight of the crosstalk existing between ER stress and abiotic stress signaling. This was confirmed by heterologous expression of OsCNX (OsCNX-HE) in tobacco and the OsCNX-HE lines were observed to exhibit better germination under mannitol stress and survival under dehydration stress conditions. The dehydration tolerance conferred by OsCNX appears to be ABA-dependent pathway.     

Glyceollin improves endoplasmic reticulum stress-induced insulin resistance through CaMKK-AMPK pathway in L6 myotubes

Glyceollin has been shown to have antidiabetic properties, although its molecular mechanism is not known. Here, we have investigated the metabolic effects of glyceollin in animal models of insulin resistance and in endoplasmic reticulum (ER) stress-responsive muscle cells. db/db mice were treated with glyceollin for 4 weeks and triglycerides, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were measured. Glyceollin reduced serum insulin and triglycerides and increased HDL levels in db/db mice. Furthermore, glyceollin caused a significant improvement in glucose homeostasis without altering body weight and food intake in db/db mice. In muscle cells, glyceollin increased the activity of AMP-activated protein kinase (AMPK) as well as cellular glucose uptake. Fatty acid oxidation was also increased. In parallel, phosphorylation of acetyl-CoA carboxylase (ACC) at Ser-79 was increased, consistent with decreased ACC activity. An insulin-resistant state was induced by exposing cells to 5 μg/ml of tunicamycin as indicated by decreased insulin-mediated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and glucose uptake. Inhibition of insulin-mediated tyrosine phosphorylation of IRS-1 and glucose uptake under ER stress was prevented by glyceollin. Strikingly, glyceollin reduced ER stress-induced, c-Jun NH₂-terminal kinase activation and subsequently increased insulin signaling via stimulation of AMPK activity in L6 myotubes. Pharmacologic inhibition or knockdown of Ca²⁺/calmodulin-dependent protein kinase kinase blocked glyceollin-increased AMPK phosphorylation and insulin sensitivity under ER stress conditions. Taken together, these results indicate that glyceollin-mediated enhancement of insulin sensitivity under ER stress conditions is predominantly accomplished by activating AMPK, thereby having beneficial effects on hyperglycemia and insulin resistance.     

Activation of the Mitogen-activated Protein Kinase,Slt2p,at Bud Tips Blocks a Late Stage of Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae

Inheritance of the endoplasmic reticulum (ER) requires Ptc1p, a type 2C protein phosphatase of Saccharomyces cerevisiae. Genetic analysis indicates that Ptc1p is needed to inactivate the cell wall integrity (CWI) MAP kinase, Slt2p. Here we show that under normal growth conditions, Ptc1p inactivates Slt2p just as ER tubules begin to spread from the bud tip along the cortex. In ptc1Δ cells, the propagation of cortical ER from the bud tip to the periphery of the bud is delayed by hyperactivation of Slt2p. The pool of Slt2p that controls ER inheritance requires the CWI pathway scaffold, Spa2p, for its retention at the bud tip, and a mutation within Slt2p that prevents its association with the bud tip blocks its role in ER inheritance. These results imply that Slt2p inhibits a late step in ER inheritance by phosphorylating a target at the tip of daughter cells. The PI4P5-kinase, Mss4p, is an upstream activator of this pool of Slt2p. Ptc1p-dependant inactivation of Slt2p is also needed for mitochondrial inheritance; however, in this case, the relevant pool of Slt2p is not at the bud tip.     

Endoplasmic reticulum oxidoreductin provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics

Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. Here, we isolated viable Arabidopsis thaliana ero1 ero2 double mutants that are highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER in vivo, we expressed the glutathione redox potential (E_GSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of −240 mV in the ER of Arabidopsis plants. We found E_GSH values of −241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal E_GSH was reduced further to −253 mV. Recovery to reductive ER stress induced by dithiothreitol was delayed in ero1 ero2. The characteristic signature of E_GSH dynamics in the ER lumen triggered by hypoxia was affected in ero1 ero2 reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis.

Dynamic monitoring of the ER luminal glutathione redox potential highlights the role of ER oxidoreductins in defining redox conditions and the interplay between different redox inputs during hypoxia and reductive stress.

IN A NUTSHELL Background: Most secreted proteins contain disulfide bridges that are essential for their structure and function. Those disulfides are introduced into the nascent polypeptide through the oxidation of cysteines in the endoplasmic reticulum (ER) lumen. Oxidative protein folding requires molecular oxygen (O₂) as ultimate electron acceptor. The final electron transfer is catalyzed by thiol oxidases called ER oxidoreductins (EROs). Question: What is the role of EROs in maintaining ER redox homeostasis at steady state and when oxygen supply is limiting? Finding: Arabidopsis thaliana contains two ERO genes. An ero1 ero2 double mutant generated by combining a null allele for ERO1 with a knockdown allele for ERO2 showed enhanced sensitivity towards thiol-based reductive challenge and hypoxia. By monitoring the glutathione redox potential E_GSH in the ER lumen using the redox biosensor variant roGFP2iL we measured −241 mV in the wild-type, which is a less oxidizing value than previously thought. A good match between the midpoint potential of the biosensor variant and the physiological E_GSH in the ER lumen enabled dynamic measurements indicating ERO activity in vivo. Diminished ERO activity in ero1 ero2 caused a reductive shift to −253 mV and delayed recovery after reductive challenge. The dynamics of luminal E_GSH under hypoxia in ero1 ero2 differed from the response obtained in wild-type plants, indicating that ERO activity plays a key role in luminal redox homeostasis. Next steps: Monitoring luminal E_GSH represents a platform for evaluating ER redox dynamics and allows assessing other candidates for their potential contribution to oxidative protein folding and maintaining luminal redox homeostasis. Future research may focus on the integration of ER redox homeostasis and phytohormone signaling especially under stress situations or during developmental phases associated with hypoxic conditions.     

Cu, Zn Superoxide Dismutase and NADP(H) Homeostasis Are Required for Tolerance of Endoplasmic Reticulum Stress in Saccharomyces cerevisiae

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.     

Proline Biosynthesis Is Required for Endoplasmic Reticulum Stress Tolerance in Saccharomyces cerevisiae

The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP⁺/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.