Stress-induced Degradation Research Articles

Abstract 607: Modulating VEGFR3 Signaling by Epsins Regulates Lymphatic Dysfuntion in Diabetes

Background: Impaired lymphatic function in diabetes is a major cause of lymphedema and immune suppression. VEGFR3 signaling is a master regulator of lymphatic development; however, whether alteration of VEGFR3 signaling regulates lymphatic function in diabetes is unknown. We uncovered that loss of epsins in lymphatic endothelial cells (LECs) increases VEGFR3 signaling by hindering VEGFR3 downregulation (Science Signaling, 2014). The goals for the present study were (1) to determine if hyperglycemia inhibits VEGFR3 signaling and impairs lymphangiogenesis, (2) identify critical molecular pathways that drive the upregulation of epsin, leading to enhanced degradation of VEGFR3 in diabetes, (3) to ascertain if elevated VEGFR3 signaling caused by epsin loss is crucial for restoring impaired lymphatic circulation in diabetes. Methods and Results: Using innovative near-infrared imaging and tail lymphedema models, we showed that epsin loss accelerates recovery from diabetic secondary lymphedema. We also found that hyperglycemia-induced oxidative stress attenuates VEGFR3 signaling, delays diabetic secondary lymphedema resolution, however, elevates epsin expression in LECs. Mechanistically, using ChIP and luciferase reporter analyses, we discovered that hyperglycemia driven oxidative stress-induced Src activation stimulates AP1 activity, which in turn binds epsin promoter and upregulates epsin transcription. Consequently, heightened epsin expression in primary LECs isolated from diabetic mice suppresses VEGFR3 signaling and prohibits lymphangiogenesis by facilitating VEGFR3 downregulation. Conversely, epsin deficiency rescues high-glucose-induced VEGFR3 loss, amends defective lymphangiogenesis, and ameliorates diabetic secondary lymphedema. Conclusions: Epsin depletion promotes diabetic secondary lymphedema resolution by protecting VEGFR3 from oxidative stress-induced degradation, highlighting the therapeutic potential for targeted inhibition of epsins in the lymphatic endothelium as a treatment for lymphatic dysfunction in diabetes.

Electro-magnetic stress-induced degradation of insulation vacuum of a large cryo-magnetic system

In superconducting magnets, the cold mass is placed in a vacuum vessel to reduce heat load to the liquid helium system. Helium leaks into the vacuum vessel can degrade the insulation vacuum, which can, in turn, cause an increase in the heat load to the liquid helium system. These leaks are called cold leaks, as they show up when the coil is cooled with liquid helium. K500 superconducting cyclotron magnet at Variable Energy Cyclotron Centre, Kolkata has such cold leaks in the helium vessel that developed during cool down. The leak rate increases with the increase of current in the superconducting coils. This paper describes a series of experiments carried out on the superconducting cyclotron magnet to find the level of degradation of insulation vacuum and measure the increase in heat load with magnet current. The leak rate was also measured and the leak size was estimated analytically. Detail magneto-structural analysis was done using Finite Element Method (FEM) to identify highly stressed zones in the helium vessel and found out that highly stressed zones coincide with the weld zones. The magneto-structural stress was applied on an estimated size of single crack and found that crack tip stress could reach beyond elastic limit of the material. We can predict that the full design current may be unachievable in this situation. Mitigation of increased heat load was also done using an additional vacuum pump for the insulation vacuum space.

Characterizing Radiation and Stress-Induced Degradation in an Embedded Split-Gate NOR Flash Memory

Radiation and stress-induced degradation are characterized in split-gate NOR flash cells through a set of unique experiments. Radiation and program/erase stress on the bit cells is shown to create both positive and negative traps in the oxide around the floating gate cell. The annealing temperature following radiation determines the rate at which oxide traps are neutralized. To analyze both program/erase and radiation induced damage in greater detail; partial program and erase operations are performed. The implications of this work for both radiation hardness assurance testing and device reliability are discussed.

Gate Reliability Investigation in Normally-Off p-Type-GaN Cap/AlGaN/GaN HEMTs Under Forward Bias Stress

Gate reliability of normally-off p-type-GaN/AlGaN/GaN high-electron mobility transistors grown on Si substrate subjected to forward bias stress at different gate voltages and temperatures was analyzed. Stress-induced gate current degradation was found to be consistent with the percolation process. Obtained time-to-breakdown data were interpreted using the Weibull statistics, and the maximum allowed gate operating voltage was estimated. The gate degradation was found to be weakly dependent on temperature with an activation energy of 0.1 eV.

Stress-induced endocytosis and degradation of epidermal growth factor receptor are two independent processes.

BackgroundEpidermal growth factor receptor (EGFR) is an important oncogenic protein in multiple types of cancer. Endocytosis and degradation of epidermal growth factor receptor (EGFR) are two key steps for down-regulation of cell surface level of EGFR and modulation of EGFR signaling. Stress conditions induce ligand-independent endocytosis and degradation of EGFR. However, it is not clear whether stress-induced endocytosis and degradation are consequential or two independent events.MethodsEndocytosis and degradation of EGFR in response to stress treatment and effects of the p38 inhibitor, the Caspase-3 inhibitor and the proteasomal inhibitor in cervical cancer HeLa cells were determined using immunoblotting and immunofluorescent staining assays.ResultsStress conditions, such as protein biosynthesis inhibition, UV light irradiation, and hyper-osmosis, induced both ligand-independent endocytosis and degradation of EGFR. Stress-induced endocytosis of EGFR relies on p38 kinase activity, while stress-induced degradation of EGFR is catalyzed by Caspase-3 activity. Inhibiting p38 kinase impairs only the endocytosis but not the degradation, while inhibiting Caspase-3 results in the opposite effect to inhibiting p38. Furthermore, proteasomal activity is required for stress-induced degradation of EGFR and cell death, but not for endocytosis.ConclusionsThe results indicate that stress-induced endocytosis and degradation are two independent events and suggest stress signaling may utilize a double-secure mechanism to down-regulate cell surface EGFR in cancer cells.

Reciprocal Degradation of YME1L and OMA1 Adapts Mitochondrial Proteolytic Activity during Stress.

The mitochondrial inner membrane proteases YME1L and OMA1 are critical regulators of essential mitochondrial functions, including inner membrane proteostasis maintenance and mitochondrial dynamics. Here, we show that YME1L and OMA1 are reciprocally degraded in response to distinct types of cellular stress. OMA1 is degraded through a YME1L-dependent mechanism in response to toxic insults that depolarize the mitochondrial membrane. Alternatively, insults that depolarize mitochondria and deplete cellular ATP stabilize active OMA1 and promote YME1L degradation. We show that the differential degradation of YME1L and OMA1 alters their proteolytic processing of the dynamin-like GTPase OPA1, a critical regulator of mitochondrial inner membrane morphology, which influences the recovery of tubular mitochondria following membrane-depolarization-induced fragmentation. Our results reveal the differential stress-induced degradation of YME1L and OMA1 as a mechanism for sensitively adapting mitochondrial inner membrane protease activity and function in response to distinct types of cellular insults.

Characterization of IRE1 ribonuclease-mediated mRNA decay in plants using transient expression analyses in rice protoplasts.

In some eukaryotes, endoplasmic reticulum (ER) stress induces regulated inositol-requiring enzyme 1 (IRE1)-dependent decay (RIDD) of mRNAs. Recently, the expression levels of the mRNAs encoding some secretory proteins were reported to be downregulated by RIDD in the vegetative tissues of plants. However, the characteristics of plant RIDD have been insufficiently investigated due to difficulty of in planta analyses. Here, the RIDD susceptibilities of various mRNAs that are difficult to analyze in planta were examined using transient expression analyses of rice protoplasts. In this system, the mRNAs encoding three rice seed storage proteins (SSPs) - namely α-globulin, 16-kDa prolamin and 10-kDa prolamin - were downregulated in response to ER stress. The rapid ER stress-induced degradation of these mRNAs was repressed in cells in which the ribonuclease activity of IRE1 was specifically abolished by genome editing, suggesting that the mRNAs encoding certain SSPs are strong targets of RIDD. Furthermore, we investigated whether these RIDD targets are substrates of the IRE1 ribonuclease using a recombinant IRE1 protein, and identified candidate IRE1-mediated cleavage sites. Overall, the results demonstrate the existence of a post-transcriptional mechanism of regulation of SSPs, and illustrate the basic and multifaceted characteristics of RIDD in higher plants.

Tropomyosin-1 protects transformed alveolar epithelial cells against cigaret smoke extract through the stabilization of F-actin-dependent cell–cell junctions

The aim of the study was to estimate the effect of tropomyosin-1-based structural stabilization of F-actin in transformed human alveolar epithelial line H1299 cells subjected to high oxidative stress induced by cigaret smoke extract. We demonstrated here that cigaret smoke extract induces cell shrinking and detachment as a consequence of F-actin cytoskeleton degradation in H1299 cells not overexpressing tropomyosin-1. Furthermore, the treatment of these cells with cigaret smoke extract resulted in the loss of peripheral localization of ZO-1 and initiated apoptosis. In contrast, structural stabilization of F-actin, by overexpression of tropomyosin-1, preserved cell to cell interactions through the attenuation of cortical actin organization into thin fibers and thus protected these cells against oxidative stress-induced degradation of actin cytoskeleton and cell death. In conclusion, we suggest that structural stabilization of thin cortical F-actin fibers increases link between tight junctions proteins and actin cytoskeleton and thus protects H1299 cells against cigaret smoke extract.

Structural stress responses and degradation of dictyosomes in algae analysed by TEM and FIB‐SEM tomography

Stress-induced physiological deficiencies in cells are reflected in structural, morphological and functional reactions of organelles. Although numerous investigations have focused on chloroplasts and mitochondria as main targets of different stressors in plant cells, there is insufficient information on the plant Golgi apparatus as stress sensor. By using the advantages of field emission scanning electron microscopy tomography in combination with classical ultrathin sectioning and transmission electron microscopic analyses, we provide structural evidence for common stress responses of the large and highly stable dictyosomes in the algal model system Micrasterias. Stress is induced by different metals such as manganese and lead, by starvation in 9 weeks of darkness or by inhibiting photosynthesis or glycolysis and by disturbing ionic homeostasis via KCl. For the first time a stress-induced degradation pathway of dictyosomes is described that does not follow "classical" autophagy but occurs by disintegration of cisternae into single membrane balls that seem to be finally absorbed by the endoplasmic reticulum (ER). Comparison of the morphological features that accompany dictyosomal degradation in Micrasterias to similar reactions observed during the same stress application in Nitella indicates an ubiquitous degradation process at least in algae. As the algae investigated belong to the closest relatives of higher land plants these results may also be relevant for understanding dictyosomal stress and degradation responses in the latter phylogenetic group. In addition, this study shows that two-dimensional transmission electron microscopy is insufficient for elucidating complex processes such as organelle degradation, and that information from three-dimensional reconstructions as provided by field emission scanning electron microscopy tomography is absolutely required for a comprehensive understanding of the phenomenon.

A new vertebrate SUMO enzyme family reveals insights into SUMO-chain assembly.

SUMO chains act as stress-induced degradation tags or repair factor-recruiting signals at DNA lesions. Although E1 activating, E2 conjugating and E3 ligating enzymes efficiently assemble SUMO chains, specific chain-elongation mechanisms are unknown. E4 elongases are specialized E3 ligases that extend a chain but are inefficient in the initial conjugation of the modifier. We identified ZNF451, a representative member of a new class of SUMO2 and SUMO3 (SUMO2/3)-specific enzymes that execute catalysis via a tandem SUMO-interaction motif (SIM) region. One SIM positions the donor SUMO while a second SIM binds SUMO on the back side of the E2 enzyme. This tandem-SIM region is sufficient to extend a back side-anchored SUMO chain (E4 elongase activity), whereas efficient chain initiation also requires a zinc-finger region to recruit the initial acceptor SUMO (E3 ligase activity). Finally, we describe four human proteins sharing E4 elongase activities and their function in stress-induced SUMO2/3 conjugation.

Conditional Proteolysis of the Membrane Protein YfgM by the FtsH Protease Depends on a Novel N-terminal Degron

Regulated proteolysis efficiently and rapidly adapts the bacterial proteome to changing environmental conditions. Many protease substrates contain recognition motifs, so-called degrons, that direct them to the appropriate protease. Here we describe an entirely new degron identified in the cytoplasmic N-terminal end of the membrane-anchored protein YfgM of Escherichia coli. YfgM is stable during exponential growth and degraded in stationary phase by the essential FtsH protease. The alarmone (p)ppGpp, but not the previously described YfgM interactors RcsB and PpiD, influence YfgM degradation. By scanning mutagenesis, we define individual amino acids responsible for turnover of YfgM and find that the degron does not at all comply with the known N-end rule pathway. The YfgM degron is a distinct module that facilitates FtsH-mediated degradation when fused to the N terminus of another monotopic membrane protein but not to that of a cytoplasmic protein. Several lines of evidence suggest that stress-induced degradation of YfgM relieves the response regulator RcsB and thereby permits cellular protection by the Rcs phosphorelay system. On the basis of these and other results in the literature, we propose a model for how the membrane-spanning YfgM protein serves as connector between the stress responses in the periplasm and cytoplasm.

Reliability study of organic complementary logic inverters using constant voltage stress

We performed constant voltage stresses with different bias conditions on all-organic complementary inverters. We found a 20% maximum variation of DC inverter parameters after a 104-s stress. However, the largest stress-induced degradation was found in the delay times, which increased by a factor as high as 7. This is mainly due to the threshold voltage variation of the p-type thin-film-transistor and the mobility reduction of the n-type thin-film transistors, which both decrease the saturation drain current.

Beyond a Model of Polymer Processing-Triggered Shear: Reconciling Shish-Kebab Formation and Control of Chain Degradation in Sheared Poly(l-lactic acid)

Here we disclose an unprecedented methodology toward high-performance poly(l-lactic acid) (PLLA) through generation of dense shish-kebabs, while the normal shear stress-induced chain degradation is controlled. The key elements involve the application of a pulse of strong shear and controlled crystallization. Specifically, the shear featuring a short duration of 1 s and a shear rate high to 100 s–1 was employed to create shish precursors, which was followed by high-temperature crystallization (at 130, 135, and 140 °C) to render the prevailing development of shish-kebabs rather than spherulites. The direct observation of the overgrown shish afforded the demonstration of its origin from shear-aligned bundles of fibrillar chains, implying the crucial importance of chain entanglements in driving the alignment of neighboring chains along the transient shear. For the first time, the shear-aligned shish was revealed to present much higher conformational order, compared to the neighboring kebabs or spherulites. It...

Annealing influence on recovery of electrically stressed power vertical double-diffused metal oxide semiconductor transistors

The effect of annealing on the recovery of electrically stressed power vertical double-diffused metal oxide semiconductor (VDMOS) transistors has been investigated. Behaviors of device threshold voltage and channel carrier mobility have been analyzed in terms of underlying changes in the densities of gate oxide charges and interface traps. The mechanisms responsible for the above-mentioned changes during the recovery of stressed devices have been analyzed as well. The annealing resulted in achieving stable threshold voltage and mobility, but did not ensure full recovery, as part of the stress-induced degradation remained permanent and could not be annealed. Namely, the annealing was found to enhance the neutralization of stress-induced oxide charges and the passivation of stress-induced interface traps, while the stability upon its completion resulted from the significantly decreased density of gate oxide charges and the possible balance between the processes of trap passivation and creation at the SiO2–Si interface.

Emerging roles of autophagy in metabolism and metabolic disorders.

The global prevalence of metabolic disorders is an immediate threat to human health. Genetic features, environmental aspects and lifestyle changes are the major risk factors determining metabolic dysfunction in the body. Autophagy is a housekeeping stress-induced lysosomal degradation pathway, which recycles macromolecules and metabolites for new protein synthesis and energy production and regulates cellular homeostasis by clearance of damaged protein or organelles. Recently, a dramatically increasing number of literatures has shown that defects of the autophagic machinery is associated with dysfunction of multiple metabolic tissues including pancreatic β cells, liver, adipose tissue and muscle, and is implicated in metabolic disorders such as obesity and insulin resistance. Here in this review, we summarize the representative works on these topics and discuss the versatile roles of autophagy in the regulation of cellular metabolism and its possible implication in metabolic diseases.

Oxidative challenge enhances REGγ–proteasome-dependent protein degradation

Elimination of oxidized proteins is important to cells as accumulation of damaged proteins causes cellular dysfunction, disease, and aging. Abundant evidence shows that the 20S proteasome is largely responsible for degradation of oxidative proteins in both ubiquitin-dependent and ubiquitin-independent pathways. However, the role of the REGγ–proteasome in degrading oxidative proteins remains unclear. Here, we focus on two of the well-known REGγ–proteasome substrates, p21Waf1/Cip1 and hepatitis C virus (HCV) core protein, to analyze the impact of oxidative stress on REGγ–proteasome functions. We demonstrate that REGγ–proteasome is essential for oxidative stress-induced rapid degradation of p21 and HCV proteins. Silencing REGγ abrogated this response in multiple cell lines. Furthermore, pretreatment with proteasome inhibitor MG132 completely blunted oxidant-induced p21 degradation, indicating a proteasome-dependent action. Cellular oxidation promoted REGγ–proteasome-dependent trypsin-like activity by enhancing the interaction between REGγ and 20S proteasome. Antioxidant could counteract oxidation-induced protein degradation, indicating that REGγ–proteasome activity may be regulated by redox state. This study provides further insights into the actions of a unique proteasome pathway in response to an oxidative stress environment, implying a novel molecular basis for REGγ–proteasome functions in antioxidation.

Significant Reduction of Dynamic Negative Bias Stress-Induced Degradation in Bridged-Grain Poly-Si TFTs

The device degradation of bridged-grain (BG) polycrystalline silicon thin-film transistors under dynamic negative bias stress (NBS) is investigated for the first time. By employing a BG structure in the active channel, dynamic NBS-induced hot carrier degradation could be significantly reduced from -99.9% to -2.4% (10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sup> s dynamic NBS), which is attributed to a sharing of the lateral electric field across the multiple p-n junctions inherent in the structure. The nonequilibrium junction degradation model is employed and developed, incorporated with TCAD simulations.

Progressive failure site generation in AlGaN/GaN high electron mobility transistors under OFF-state stress: Weibull statistics and temperature dependence

Gate leakage degradation of AlGaN/GaN high electron mobility transistors under OFF-state stress is investigated using a combination of electrical, optical, and surface morphology characterizations. The generation of leakage “hot spots” at the edge of the gate is found to be strongly temperature accelerated. The time for the formation of each failure site follows a Weibull distribution with a shape parameter in the range of 0.7–0.9 from room temperature up to 120 °C. The average leakage per failure site is only weakly temperature dependent. The stress-induced structural degradation at the leakage sites exhibits a temperature dependence in the surface morphology, which is consistent with a surface defect generation process involving temperature-associated changes in the breakdown sites.

Stress-induced chloroplast degradation in Arabidopsis is regulated via a process independent of autophagy and senescence-associated vacuoles.

Two well-known pathways for the degradation of chloroplast proteins are via autophagy and senescence-associated vacuoles. Here, we describe a third pathway that was activated by senescence- and abiotic stress-induced expression of Arabidopsis thaliana CV (for chloroplast vesiculation). After targeting to the chloroplast, CV destabilized the chloroplast, inducing the formation of vesicles. CV-containing vesicles carrying stromal proteins, envelope membrane proteins, and thylakoid membrane proteins were released from the chloroplasts and mobilized to the vacuole for proteolysis. Overexpression of CV caused chloroplast degradation and premature leaf senescence, whereas silencing CV delayed chloroplast turnover and senescence induced by abiotic stress. Transgenic CV-silenced plants displayed enhanced tolerance to drought, salinity, and oxidative stress. Immunoprecipitation and bimolecular fluorescence complementation assays demonstrated that CV interacted with photosystem II subunit PsbO1 in vivo through a C-terminal domain that is highly conserved in the plant kingdom. Collectively, our work indicated that CV plays a crucial role in stress-induced chloroplast disruption and mediates a third pathway for chloroplast degradation. From a biotechnological perspective, silencing of CV offers a suitable strategy for the generation of transgenic crops with increased tolerance to abiotic stress.

Determination of stress-induced degradation products of cetirizine dihydrochloride by a stability-indicating RP-HPLC method.

BackgroundA new, simple and accurate stability-indicating reverse phase high performance liquid chromatography method was developed and validated during the early stage of drug development of an oral lyophilizate dosage form of cetirizine dihydrochloride.MethodsFor RP-HPLC analysis it was used an Eclipse XDB C8 column 150 mm × 4.6 mm, 5 μm (Agilent columns, Barcelona, Spain) as the stationary phase with a mobile phase consisted of a mixture of 0.2 M K2HPO4 pH 7.00 and acetonitrile (65:35, v/v) at a flow rate of 1 mL min −1. Detection was performed at 230 nm using diode array detector. The method was validated in accordance with ICH guidelines with respect to linearity, accuracy, precision, specificity, limit of detection and quantification.ResultsThe method results in excellent separation between the drug substance and its stress-induced degradation products. The peak purity factor is >950 for the drug substance after all types of stress, which confirms the complete separation of the drug substance peak from its stress induced degradation products.Regression analysis showed r2 > 0.999 for cetirizine dihydrochloride in the concentration range of 650 μg mL −1 to 350 μg mL−1 for drug substance assay and a r2 > 0.999 in the concentration range of 0.25 μg mL−1 to 5 μg mL−1 for degradation products. The method presents a limit of detection of 0.056 μg mL −1 and a limit of quantification of 0.25 μg mL−1. The obtained results for precision and accuracy for drug substance and degradation products are within the specifications established for the validation of the method.ConclusionsThe proposed stability-indicating method developed in the early phase of drug development proved to be a simple, sensitive, accurate, precise, reproducible and therefore useful for the following stages of the cetirizine dihydrochloride oral lyophilizate dosage form development.