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Sample records for accelerated protein degradation

  1. Accelerated degradation of caspase-8 protein correlates with TRAIL resistance in a DLD1 human colon cancer cell line.

    PubMed

    Zhang, Lidong; Zhu, Hongbo; Teraishi, Fuminori; Davis, John J; Guo, Wei; Fan, Zhen; Fang, Bingliang

    2005-06-01

    The tumor-selective cytotoxic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) makes TRAIL an attractive candidate as an anticancer agent. However, resistance to TRAIL poses a challenge in anticancer therapy with TRAIL. Therefore, characterizing the mechanisms of resistance and developing strategies to overcome the resistance are important steps toward successful TRAIL-mediated cancer therapy. In this study, we investigated mechanisms of acquired TRAIL resistance in a colon cancer DLD1 cell line. Compared with the TRAIL-susceptible DLD1 cell line, TRAIL-resistant DLD1/TRAIL-R cells have a low level of caspase-8 protein, but not its mRNA. Suppression of caspase-8 expression by siRNA in parental DLD1 cells led to TRAIL resistance. Restoration of caspase-8 protein expression by stable transfection rendered the DLD1/TRAIL-R cell line fully sensitive to TRAIL protein, suggesting that the low level of caspase-8 protein expression might be the culprit in TRAIL resistance in DLD1/TRAIL-R cells. Sequencing analysis of the caspase-8 coding region revealed a missense mutation that is present in both TRAIL-sensitive and TRAIL-resistant DLD1 cells. Subsequent study showed that the degradation of caspase-8 protein was accelerated in DLD1/TRAIL-R cells compared to parental DLD1 cells. Thus, accelerated degradation of caspase-8 protein is one of the mechanisms that lead to TRAIL resistance. PMID:16036110

  2. Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice.

    PubMed

    Fan, Ming-Hui; Zhu, Qiang; Li, Hui-Hua; Ra, Hyun-Jeong; Majumdar, Sonali; Gulick, Dexter L; Jerome, Jacob A; Madsen, Daniel H; Christofidou-Solomidou, Melpo; Speicher, David W; Bachovchin, William W; Feghali-Bostwick, Carol; Puré, Ellen

    2016-04-01

    Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2-4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface-associated serine protease up-regulated in the lungs of patients with idiopathic pulmonary fibrosis as well as in wound healing and cancer. We postulate that FAP is not only a marker of disease but influences the development of pulmonary fibrosis after lung injury. In two different models of pulmonary fibrosis, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent within vitrostudies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180. Pharmacologic FAP inhibition decreases collagen internalization as expected. Conversely, restoration of FAP expression in the lungs of FAP-deficient mice decreases lung hydroxyproline content after intratracheal bleomycin to levels comparable with that of wild-type controls. Our findings indicate that FAP participates directly, in concert with MMPs, in collagen catabolism and clearance and is an important factor in resolving scar after injury and restoring lung homeostasis. Our study identifies FAP as a novel endogenous regulator of fibrosis and is the first to show FAP's protective effects in the lung. PMID:26663085

  3. Flow accelerated organic coating degradation

    NASA Astrophysics Data System (ADS)

    Zhou, Qixin

    Applying organic coatings is a common and the most cost effective way to protect metallic objects and structures from corrosion. Water entry into coating-metal interface is usually the main cause for the deterioration of organic coatings, which leads to coating delamination and underfilm corrosion. Recently, flowing fluids over sample surface have received attention due to their capability to accelerate material degradation. A plethora of works has focused on the flow induced metal corrosion, while few studies have investigated the flow accelerated organic coating degradation. Flowing fluids above coating surface affect corrosion by enhancing the water transport and abrading the surface due to fluid shear. Hence, it is of great importance to understand the influence of flowing fluids on the degradation of corrosion protective organic coatings. In this study, a pigmented marine coating and several clear coatings were exposed to the laminar flow and stationary immersion. The laminar flow was pressure driven and confined in a flow channel. A 3.5 wt% sodium chloride solution and pure water was employed as the working fluid with a variety of flow rates. The corrosion protective properties of organic coatings were monitored inline by Electrochemical Impedance Spectroscopy (EIS) measurement. Equivalent circuit models were employed to interpret the EIS spectra. The time evolution of coating resistance and capacitance obtained from the model was studied to demonstrate the coating degradation. Thickness, gloss, and other topography characterizations were conducted to facilitate the assessment of the corrosion. The working fluids were characterized by Fourier Transform Infrared Spectrometer (FTIR) and conductivity measurement. The influence of flow rate, fluid shear, fluid composition, and other effects in the coating degradation were investigated. We conclude that flowing fluid on the coating surface accelerates the transport of water, oxygen, and ions into the coating, as

  4. The chloroplast protein LTO1/AtVKOR is involved in the xanthophyll cycle and the acceleration of D1 protein degradation.

    PubMed

    Yu, Zhi-Bo; Lu, Ying; Du, Jia-Jia; Peng, Jun-Jie; Wang, Xiao-Yun

    2014-01-01

    The thylakoid protein LTO1/AtVKOR-DsbA is recently found to be an oxidoreductase involved in disulfide bond formation and the assembly of photosystem II (PSII) in Arabidopsis thaliana. In this study, experimental evidence showed that LTO1 deficiency caused severe photoinhibition which was related to the xanthophyll cycle and D1 protein degradation. The lto1-2 mutant was more sensitive to intense irradiance than wild type. When treated with different concentrations of dithiothreitol (DTT), an inhibitor of violaxanthin de-epoxidase (VDE) in the xanthophyll cycle, there was a larger reduction in NPQ in the wild type than in the lto1-2 mutant under high irradiance, indicating that lto1-2 had a lower sensitivity to DTT gradients than did the wild type. Zeaxanthin in the xanthophyll cycle, which participates in the thermal dissipation of excess absorbed light energy, was much less active in lto1-2 than in the wild type under intense light levels, and the de-epoxidation state of the xanthophyll cycle was consistent with the susceptibility of NPQ. Together these observations indicated that aggravated photoinhibition in lto1-2 was related to a reduction in xanthophyll cycle-associated energy dissipation. When D1 protein synthesis was suppressed by an inhibitor of chloroplast protein synthesis (streptomycin sulfate), the levels of D1 protein decreased more in the lto1-2 mutant than in the wild type when exposed to intense light levels, implying that a deficiency in LTO1 accelerated the degradation of D1 and thus affected D1 turnover. Transgenic complementation of plants with lto1-2 ultimately allowed for the recovery of the photoinhibition properties of leaves. PMID:24300993

  5. Accelerated Testing Of Photothermal Degradation Of Polymers

    NASA Technical Reports Server (NTRS)

    Kim, Soon Sam; Liang, Ranty Hing; Tsay, Fun-Dow

    1989-01-01

    Electron-spin-resonance (ESR) spectroscopy and Arrhenius plots used to determine maximum safe temperature for accelerated testing of photothermal degradation of polymers. Aging accelerated by increasing illumination, temperature, or both. Results of aging tests at temperatures higher than those encountered in normal use valid as long as mechanism of degradation same throughout range of temperatures. Transition between different mechanisms at some temperature identified via transition between activation energies, manifesting itself as change in slope of Arrhenius plot at that temperature.

  6. Accelerated degradation testing of a photovoltaic module

    NASA Astrophysics Data System (ADS)

    Charki, Abdérafi; Laronde, Rémi; Bigaud, David

    2013-01-01

    There are a great many photovoltaic (PV) modules installed around the world. Despite this, not enough is known about the reliability of these modules. Their electrical power output decreases with time mainly as a result of the effects of corrosion, encapsulation discoloration, and solder bond failure. The failure of a PV module is defined as the point where the electrical power degradation reaches a given threshold value. Accelerated life tests (ALTs) are commonly used to assess the reliability of a PV module. However, ALTs provide limited data on the failure of a module and these tests are expensive to carry out. One possible solution is to conduct accelerated degradation tests. The Wiener process in conjunction with the accelerated failure time model makes it possible to carry out numerous simulations and thus to determine the failure time distribution based on the aforementioned threshold value. By this means, the failure time distribution and the lifetime (mean and uncertainty) can be evaluated.

  7. Degradation mechanisms and accelerated aging test design

    SciTech Connect

    Clough, R L; Gillen, K T

    1985-01-01

    The fundamental mechanisms underlying the chemical degradation of polymers can change as a function of environmental stress level. When this occurs, it greatly complicates any attempt to use accelerated tests for predicting long-term material degradation behaviors. Understanding how degradation mechanisms can change at different stress levels facilitates both the design and the interpretation of aging tests. Oxidative degradation is a predominant mechanism for many polymers exposed to a variety of different environments in the presence of air, and there are two mechanistic considerations which are widely applicable to material oxidation. One involves a physical process, oxygen diffusion, as a rate-limiting step. This mechanism can predominate at high stress levels. The second is a chemical process, the time-dependent decomposition of peroxide species. This leads to chain branching and can become a rate-controlling factor at lower stress levels involving time-scales applicable to use environments. The authors describe methods for identifying the operation of these mechanisms and illustrate the dramatic influence they can have on the degradation behaviors of a number of polymer types. Several commonly used approaches to accelerated aging tests are discussed in light of the behaviors which result from changes in degradation mechanisms. 9 references, 4 figures.

  8. Cellular senescence and protein degradation

    PubMed Central

    Deschênes-Simard, Xavier; Lessard, Frédéric; Gaumont-Leclerc, Marie-France; Bardeesy, Nabeel; Ferbeyre, Gerardo

    2014-01-01

    Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells. PMID:24866342

  9. Redox control of protein degradation

    PubMed Central

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H.K.; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E.; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-01-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  10. Tunable protein degradation in bacteria.

    PubMed

    Cameron, D Ewen; Collins, James J

    2014-12-01

    Tunable control of protein degradation in bacteria would provide a powerful research tool. Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems and can be transferred to diverse bacteria with minimal modification. PMID:25402616

  11. Acceleration of purine degradation by periodontal diseases.

    PubMed

    Barnes, V M; Teles, R; Trivedi, H M; Devizio, W; Xu, T; Mitchell, M W; Milburn, M V; Guo, L

    2009-09-01

    Periodontal diseases, such as gingivitis and periodontitis, are characterized by bacterial plaque accumulation around the gingival crevice and the subsequent inflammation and destruction of host tissues. To test the hypothesis that cellular metabolism is altered as a result of host-bacteria interaction, we performed an unbiased metabolomic profiling of gingival crevicular fluid (GCF) collected from healthy, gingivitis, and periodontitis sites in humans, by liquid and gas chromatography mass spectrometry. The purine degradation pathway, a major biochemical source for reactive oxygen species (ROS) production, was significantly accelerated at the disease sites. This suggests that periodontal-disease-induced oxidative stress and inflammation are mediated through this pathway. The complex host-bacterial interaction was further highlighted by depletion of anti-oxidants, degradation of host cellular components, and accumulation of bacterial products in GCF. These findings provide new mechanistic insights and a panel of comprehensive biomarkers for periodontal disease progression. PMID:19767584

  12. Accelerated degradation of silicon metallization systems

    NASA Technical Reports Server (NTRS)

    Lathrop, J. W.

    1983-01-01

    Clemson University has been engaged for the past five years in a program to determine the reliability attributes of solar cells by means of accelerated test procedures. The cells are electrically measured and visually inspected and then subjected for a period of time to stress in excess of that normally encountered in use, and then they are reinspected. Changes are noted and the process repeated. This testing has thus far involved 23 different unencapsulated cell types from 12 different manufacturers, and 10 different encapsulated cell types from 9 different manufacturers. Reliability attributes of metallization systems can be classified as major or minor, depending on the severity of the effects observed. As a result of the accelerated testing conducted under the Clemson program, major effects have been observed related to contact resistance and to mechanical adherence and solderability. This paper does not attempt a generalized survey of accelerated test results, but rather concentrates on one particular attribute of metallization that has been observed to cause electrical degradation - increased contact resistance due to Schottky barrier formation. In this example basic semiconductor theory was able to provide an understanding of the electrical effects observed during accelerated stress testing.

  13. Protein Degradation and Iron Homeostasis

    PubMed Central

    Thompson, Joel W.; Bruick, Richard K.

    2013-01-01

    Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron’s privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. PMID:22349011

  14. Protein degradation and iron homeostasis.

    PubMed

    Thompson, Joel W; Bruick, Richard K

    2012-09-01

    Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron's privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. This article is part of a Special Issue entitled: Cell Biology of Metals. PMID:22349011

  15. Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

    SciTech Connect

    Jegerschoeld, C.V.; Virgin, I.; Styring, S. )

    1990-07-03

    Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.

  16. Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover

    PubMed Central

    Farkas, Zoltán; Horvath, Peter; Bódi, Zoltán; Daraba, Andreea; Szamecz, Béla; Gut, Ivo; Bayes, Mónica; Santos, Manuel A. S.; Pál, Csaba

    2015-01-01

    Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome–mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that

  17. The Small C-terminal Domain Phosphatase 1 Inhibits Cancer Cell Migration and Invasion by Dephosphorylating Ser(P)68-Twist1 to Accelerate Twist1 Protein Degradation.

    PubMed

    Sun, Tong; Fu, Junjiang; Shen, Tao; Lin, Xia; Liao, Lan; Feng, Xin-Hua; Xu, Jianming

    2016-05-27

    Twist1 is a basic helix-loop-helix transcription factor that strongly promotes epithelial-to-mesenchymal transition, migration, invasion, and metastasis of cancer cells. The MAPK-phosphorylated Twist1 on its serine 68 (Ser(P)(68)-Twist1) has a significantly enhanced stability and function to drive cancer cell invasion and metastasis. However, the phosphatase that dephosphorylates Ser(P)(68)-Twist1 and destabilizes Twist1 has not been identified and characterized. In this study, we screened a serine/threonine phosphatase cDNA expression library in HEK293T cells with ectopically coexpressed Twist1. We found that the small C-terminal domain phosphatase 1 (SCP1) specifically dephosphorylates Ser(P)(68)-Twist1 in both cell-free reactions and living cells. SCP1 uses its amino acid residues 43-63 to interact with the N terminus of Twist1. Increased SCP1 expression in cells decreased Ser(P)(68)-Twist1 and total Twist1 proteins, whereas knockdown of SCP1 increased Ser(P)(68)-Twist1 and total Twist1 proteins. Furthermore, the levels of SCP1 are negatively correlated with Twist1 protein levels in several cancer cell lines. SCP1-dephosphorylated Twist1 undergoes fast degradation via the ubiquitin-proteasome pathway. Importantly, an increase in SCP1 expression in breast cancer cells with either endogenous or ectopically expressed Twist1 largely inhibits the Twist1-induced epithelial-to-mesenchymal transition phenotype and the migration and invasion capabilities of these cells. These results indicate that SCP1 is the phosphatase that counterregulates the MAPK-mediated phosphorylation of Ser(68)-Twist1. Thus, an increase in SCP1 expression and activity may be a useful strategy for eliminating the detrimental roles of Twist1 in cancer cells. PMID:26975371

  18. Involvement of microorganisms in accelerated degradation of EPTC in soil

    SciTech Connect

    Tal, A.; Rubin, B.; Katan, J. ); Aharonson, N. )

    1990-04-01

    Accelerated EPTC (S-ethyl dipropylcarbamothioate) degradation was confirmed in a mixed culture of microorganisms derived from a soil with enhanced degradation (history soil) by using {sup 14}C-labeled EPTC. The antibacterial agent chloramphenicol (D-({minus})-threo-2,2-dichloro-N-({beta}-hydroxy-{alpha}-(hydroxymethyl)-p-nitrophenethyl)acetamide) markedly suppressed {sup 14}CO{sub 2} evolution while the antifungal agent cycloheximide (4-((2R)-2((1S,3S,5S)-3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl)glutarimide) did not, suggesting that soil bacteria play a significant role in enhanced EPTC degradation. A fast EPTC bacterial degrader (FD1) strain and a slower one (SD1), which were isolated by a soil enrichment technique from a history soil, were capable of utilizing EPTC as a sole carbon source. Vernolate (S-propyl dipropylcarbamothioate), butylate (S-ethyl bis(2-methylpropyl)carbamothioate), or cycloate (S-ethyl cyclohexylethylcarbamothioate) were also degraded by these bacteria in a pattern similar to that in a soil with enhanced degradation. Inoculation of nonhistory soil with FD1 strain induced accelerated degradation of the herbicide in the soil at rates similar to those in field soils exhibiting EPTC accelerated degradation.

  19. Reliability evaluation of a photovoltaic module using accelerated degradation model

    NASA Astrophysics Data System (ADS)

    Laronde, Rémi; Charki, Abdérafi; Bigaud, David; Excoffier, Philippe

    2011-09-01

    Many photovoltaic modules are installed all around the world. However, the reliability of this product is not enough really known. The electrical power decreases in time due mainly to corrosion, encapsulation discoloration and solder bond failure. The failure of a photovoltaic module is obtained when the electrical power degradation reaches a threshold value. Accelerated life tests are commonly used to estimate the reliability of the photovoltaic module. However, using accelerated life tests, few data on the failure of this product are obtained and the realization of this kind of tests is expensive. As a solution, an accelerated degradation test can be carried out using only one stress if parameters of the acceleration model are known. The Wiener process associated with the accelerated failure time model permits to carry out many simulations and to determine the failure time distribution when the threshold value is reached. So, the failure time distribution and the lifetime (mean and uncertainty) can be evaluated.

  20. Accelerating degradation rate of pure iron by zinc ion implantation.

    PubMed

    Huang, Tao; Zheng, Yufeng; Han, Yong

    2016-12-01

    Pure iron has been considered as a promising candidate for biodegradable implant applications. However, a faster degradation rate of pure iron is needed to meet the clinical requirement. In this work, metal vapor vacuum arc technology was adopted to implant zinc ions into the surface of pure iron. Results showed that the implantation depth of zinc ions was about 60 nm. The degradation rate of pure iron was found to be accelerated after zinc ion implantation. The cytotoxicity tests revealed that the implanted zinc ions brought a slight increase on cytotoxicity of the tested cells. In terms of hemocompatibility, the hemolysis of zinc ion implanted pure iron was lower than 2%. However, zinc ions might induce more adhered and activated platelets on the surface of pure iron. Overall, zinc ion implantation can be a feasible way to accelerate the degradation rate of pure iron for biodegradable applications. PMID:27482462

  1. Accelerating degradation rate of pure iron by zinc ion implantation

    PubMed Central

    Huang, Tao; Zheng, Yufeng; Han, Yong

    2016-01-01

    Pure iron has been considered as a promising candidate for biodegradable implant applications. However, a faster degradation rate of pure iron is needed to meet the clinical requirement. In this work, metal vapor vacuum arc technology was adopted to implant zinc ions into the surface of pure iron. Results showed that the implantation depth of zinc ions was about 60 nm. The degradation rate of pure iron was found to be accelerated after zinc ion implantation. The cytotoxicity tests revealed that the implanted zinc ions brought a slight increase on cytotoxicity of the tested cells. In terms of hemocompatibility, the hemolysis of zinc ion implanted pure iron was lower than 2%. However, zinc ions might induce more adhered and activated platelets on the surface of pure iron. Overall, zinc ion implantation can be a feasible way to accelerate the degradation rate of pure iron for biodegradable applications. PMID:27482462

  2. Statistical Modeling of Photovoltaic Reliability Using Accelerated Degradation Techniques (Poster)

    SciTech Connect

    Lee, J.; Elmore, R.; Jones, W.

    2011-02-01

    We introduce a cutting-edge life-testing technique, accelerated degradation testing (ADT), for PV reliability testing. The ADT technique is a cost-effective and flexible reliability testing method with multiple (MADT) and Step-Stress (SSADT) variants. In an environment with limited resources, including equipment (chambers), test units, and testing time, these techniques can provide statistically rigorous prediction of lifetime and other interesting parameters, such as failure rate, warranty time, mean time to failure, degradation rate, activation energy, acceleration factor, and upper limit level of stress. J-V characterization can be used for degradation data and the generalized Eyring model can be used for the thermal-humidity stress condition. The SSADT model can be constructed based on the cumulative damage model (CEM), which assumes that the remaining test united are failed according to cumulative density function of current stress level regardless of the history on previous stress levels.

  3. Step-Stress Accelerated Degradation Testing for Solar Reflectors: Preprint

    SciTech Connect

    Jones, W.; Elmore, R.; Lee, J.; Kennedy, C.

    2011-09-01

    To meet the challenge to reduce the cost of electricity generated with concentrating solar power (CSP) new low-cost reflector materials are being developed including metalized polymer reflectors and must be tested and validated against appropriate failure mechanisms. We explore the application of testing methods and statistical inference techniques for quantifying estimates and improving lifetimes of concentrating solar power (CSP) reflectors associated with failure mechanisms initiated by exposure to the ultraviolet (UV) part of the solar spectrum. In general, a suite of durability and reliability tests are available for testing a variety of failure mechanisms where the results of a set are required to understand overall lifetime of a CSP reflector. We will focus on the use of the Ultra-Accelerated Weathering System (UAWS) as a testing device for assessing various degradation patterns attributable to accelerated UV exposure. Depending on number of samples, test conditions, degradation and failure patterns, test results may be used to derive insight into failure mechanisms, associated physical parameters, lifetimes and uncertainties. In the most complicated case warranting advanced planning and statistical inference, step-stress accelerated degradation (SSADT) methods may be applied.

  4. Degradable PEGylated Protein Conjugates Utilizing RAFT Polymerization

    PubMed Central

    Decker, Caitlin G.; Maynard, Heather D.

    2015-01-01

    Poly(ethylene glycol) (PEG)-protein therapeutics exhibit enhanced pharmacokinetics, but have drawbacks including decreased protein activities and polymer accumulation in the body. Therefore a major aim for second-generation polymer therapeutics is to introduce degradability into the backbone. Herein we describe the synthesis of poly(poly(ethylene glycol methyl ether methacrylate)) (pPEGMA) degradable polymers with protein-reactive end-groups via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the subsequent covalent attachment to lysozyme through a reducible disulfide linkage. RAFT copolymerization of cyclic ketene acetal (CKA) monomer 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) with PEGMA yielded two polymers with number-average molecular weight (Mn) (GPC) of 10.9 and 20.9 kDa and molecular weight dispersities (Ð) of 1.34 and 1.71, respectively. Hydrolytic degradation of the polymers was analyzed by 1H-NMR and GPC under basic and acidic conditions. The reversible covalent attachment of these polymers to lysozyme, as well as the hydrolytic and reductive cleavage of the polymer from the protein, was analyzed by gel electrophoresis and mass spectrometry. Following reductive cleavage of the polymer, an increase in activity was observed for both conjugates, with the released protein having full activity. This represents a method to prepare PEGylated proteins, where the polymer is readily cleaved from the protein and the main chain of the polymer is degradable. PMID:25937643

  5. A Complex of Htm1 and the Oxidoreductase Pdi1 Accelerates Degradation of Misfolded Glycoproteins.

    PubMed

    Pfeiffer, Anett; Stephanowitz, Heike; Krause, Eberhard; Volkwein, Corinna; Hirsch, Christian; Jarosch, Ernst; Sommer, Thomas

    2016-06-01

    A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal. We show that a complex encompassing the yeast lectin-like protein Htm1 and the oxidoreductase Pdi1 converts Man8GlcNAc2 on glycoproteins into the Man7GlcNAc2 signal. In vitro the Htm1-Pdi1 complex processes both unfolded and native proteins albeit with a preference for the former. In vivo, elevated expression of HTM1 causes glycan trimming on misfolded and folded proteins, but only degradation of the non-native species is accelerated. Thus, modification with a Man7GlcNAc2 structure does not inevitably commit a protein for ER-associated protein degradation. The function of Htm1 in ERAD relies on its association with Pdi1, which appears to regulate the access to substrates. Our data support a model in which the balanced activities of Pdi1 and Htm1 are crucial determinants for the efficient removal of misfolded secretory glycoproteins. PMID:27053108

  6. Mercury BLASTP: Accelerating Protein Sequence Alignment.

    PubMed

    Jacob, Arpith; Lancaster, Joseph; Buhler, Jeremy; Harris, Brandon; Chamberlain, Roger D

    2008-06-01

    Large-scale protein sequence comparison is an important but compute-intensive task in molecular biology. BLASTP is the most popular tool for comparative analysis of protein sequences. In recent years, an exponential increase in the size of protein sequence databases has required either exponentially more running time or a cluster of machines to keep pace. To address this problem, we have designed and built a high-performance FPGA-accelerated version of BLASTP, Mercury BLASTP. In this paper, we describe the architecture of the portions of the application that are accelerated in the FPGA, and we also describe the integration of these FPGA-accelerated portions with the existing BLASTP software. We have implemented Mercury BLASTP on a commodity workstation with two Xilinx Virtex-II 6000 FPGAs. We show that the new design runs 11-15 times faster than software BLASTP on a modern CPU while delivering close to 99% identical results. PMID:19492068

  7. Mercury BLASTP: Accelerating Protein Sequence Alignment

    PubMed Central

    Jacob, Arpith; Lancaster, Joseph; Buhler, Jeremy; Harris, Brandon; Chamberlain, Roger D.

    2008-01-01

    Large-scale protein sequence comparison is an important but compute-intensive task in molecular biology. BLASTP is the most popular tool for comparative analysis of protein sequences. In recent years, an exponential increase in the size of protein sequence databases has required either exponentially more running time or a cluster of machines to keep pace. To address this problem, we have designed and built a high-performance FPGA-accelerated version of BLASTP, Mercury BLASTP. In this paper, we describe the architecture of the portions of the application that are accelerated in the FPGA, and we also describe the integration of these FPGA-accelerated portions with the existing BLASTP software. We have implemented Mercury BLASTP on a commodity workstation with two Xilinx Virtex-II 6000 FPGAs. We show that the new design runs 11-15 times faster than software BLASTP on a modern CPU while delivering close to 99% identical results. PMID:19492068

  8. Degradation mechanisms and accelerated testing in PEM fuel cells

    SciTech Connect

    Borup, Rodney L; Mukundan, Rangachary

    2010-01-01

    The durability of PEM fuel cells is a major barrier to the commercialization of these systems for stationary and transportation power applications. Although there has been recent progress in improving durability, further improvements are needed to meet the commercialization targets. Past improvements have largely been made possible because of the fundamental understanding of the underlying degradation mechanisms. By investigating component and cell degradation modes; defining the fundamental degradation mechanisms of components and component interactions new materials can be designed to improve durability. Various factors have been shown to affect the useful life of PEM fuel cells. Other issues arise from component optimization. Operational conditions (such as impurities in either the fuel and oxidant stream), cell environment, temperature (including subfreezing exposure), pressure, current, voltage, etc.; or transient versus continuous operation, including start-up and shutdown procedures, represent other factors that can affect cell performance and durability. The need for Accelerated Stress Tests (ASTs) can be quickly understood given the target lives for fuel cell systems: 5000 hours ({approx} 7 months) for automotive, and 40,000 hrs ({approx} 4.6 years) for stationary systems. Thus testing methods that enable more rapid screening of individual components to determine their durability characteristics, such as off-line environmental testing, are needed for evaluating new component durability in a reasonable turn-around time. This allows proposed improvements in a component to be evaluated rapidly and independently, subsequently allowing rapid advancement in PEM fuel cell durability. These tests are also crucial to developers in order to make sure that they do not sacrifice durability while making improvements in costs (e.g. lower platinum group metal [PGM] loading) and performance (e.g. thinner membrane or a GDL with better water management properties). To

  9. Tamoxifen Inhibits ER-negative Breast Cancer Cell Invasion and Metastasis by Accelerating Twist1 Degradation

    PubMed Central

    Ma, Gang; He, Jianjun; Yu, Yang; Xu, Yixiang; Yu, Xiaobin; Martinez, Jarrod; Lonard, David M.; Xu, Jianming

    2015-01-01

    Twist1 is a transcription factor driving epithelial-mesenchymal transition, invasion and metastasis of breast cancer cells. Mice with germ-line Twist1 knockout are embryonic lethal, while adult mice with inducible Twist1 knockout have no obvious health problems, suggesting that Twist1 is a viable therapeutic target for the inhibition of invasion and metastasis of breast cancer in adult patients. In this study, we expressed a luciferase protein or a Twist1-luciferase fusion protein in HeLa cells as part of a high throughput system to screen 1280 compounds in the Library of Pharmacologically Active Compounds (LOPAC) from Sigma-Aldrich for their effects on Twist1 protein expression. One of the most interesting compounds identified is tamoxifen, a selective estrogen receptor (ER) modulator used to treat ER-positive breast cancer. Tamoxifen treatment significantly accelerated Twist1 degradation in multiple cell lines including HEK293 human kidney cells, 4T1 and 168FARN mouse mammary tumor cells with either ectopically or endogenously expressed Twist1. Tamoxifen-induced Twist1 degradation could be blocked by the MG132 proteasome inhibitor, suggesting that tamoxifen induces Twist1 degradation through the ubiquitination-proteasome pathway. However, tamoxifen-induced Twist1 degradation was independent of Twist1 mRNA expression, estrogen signaling and MAPK-mediated Twist1 phosphorylation in these cells. Importantly, tamoxifen also significantly inhibited invasive behavior in Matrigel and lung metastasis in SCID-bg mice of ER-negative 4T1 mammary tumor cells, which depend on endogenous Twist1 to invade and metastasize. These results indicate that tamoxifen can significantly accelerate Twist1 degradation to suppress cancer cell invasion and metastasis, suggesting that tamoxifen can be used not only to treat ER-positive breast cancers but also to reduce Twist1-mediated invasion and metastasis in ER-negative breast cancers. PMID:25892968

  10. Tamoxifen inhibits ER-negative breast cancer cell invasion and metastasis by accelerating Twist1 degradation.

    PubMed

    Ma, Gang; He, Jianjun; Yu, Yang; Xu, Yixiang; Yu, Xiaobin; Martinez, Jarrod; Lonard, David M; Xu, Jianming

    2015-01-01

    Twist1 is a transcription factor driving epithelial-mesenchymal transition, invasion and metastasis of breast cancer cells. Mice with germ-line Twist1 knockout are embryonic lethal, while adult mice with inducible Twist1 knockout have no obvious health problems, suggesting that Twist1 is a viable therapeutic target for the inhibition of invasion and metastasis of breast cancer in adult patients. In this study, we expressed a luciferase protein or a Twist1-luciferase fusion protein in HeLa cells as part of a high throughput system to screen 1280 compounds in the Library of Pharmacologically Active Compounds (LOPAC) from Sigma-Aldrich for their effects on Twist1 protein expression. One of the most interesting compounds identified is tamoxifen, a selective estrogen receptor (ER) modulator used to treat ER-positive breast cancer. Tamoxifen treatment significantly accelerated Twist1 degradation in multiple cell lines including HEK293 human kidney cells, 4T1 and 168FARN mouse mammary tumor cells with either ectopically or endogenously expressed Twist1. Tamoxifen-induced Twist1 degradation could be blocked by the MG132 proteasome inhibitor, suggesting that tamoxifen induces Twist1 degradation through the ubiquitination-proteasome pathway. However, tamoxifen-induced Twist1 degradation was independent of Twist1 mRNA expression, estrogen signaling and MAPK-mediated Twist1 phosphorylation in these cells. Importantly, tamoxifen also significantly inhibited invasive behavior in Matrigel and lung metastasis in SCID-bg mice of ER-negative 4T1 mammary tumor cells, which depend on endogenous Twist1 to invade and metastasize. These results indicate that tamoxifen can significantly accelerate Twist1 degradation to suppress cancer cell invasion and metastasis, suggesting that tamoxifen can be used not only to treat ER-positive breast cancers but also to reduce Twist1-mediated invasion and metastasis in ER-negative breast cancers. PMID:25892968

  11. Protein degradation and protection against misfolded or damaged proteins

    NASA Astrophysics Data System (ADS)

    Goldberg, Alfred L.

    2003-12-01

    The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

  12. Step-Stress Accelerated Degradation Testing (SSADT) for Photovoltaic (PV) Devices and Cells (Presentation)

    SciTech Connect

    Lee, J.; Elmore, R.; Suh, C.; Jones, W.

    2010-10-01

    Presentation on step-stress accelerated degradation testing (SSADT) for photovoltaics (PV). Developed are a step-stress degradation test (SSADT) for PV reliability tests and a lifetime prediction model for PV products.

  13. Non-degradative Ubiquitination of Protein Kinases

    PubMed Central

    Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

    2016-01-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

  14. Hycrest crested wheatgrass accelerates the degradation of pentachlorophenol in soil

    NASA Technical Reports Server (NTRS)

    Ferro, A. M.; Sims, R. C.; Bugbee, B.

    1994-01-01

    We investigated the effects of vegetation on the fate of pentachlorophenol (PCP) in soil using a novel high-flow sealed test system. Pentachlorophenol has been widely used as a wood preservative, and this highly toxic biocide contaminates soil and ground water at many sites. Although plants are known to accelerate the rates of degradation of certain soil contaminants, this approach has not been thoroughly investigated for PCP. The fate of [14C]PCP, added to soil at a concentration of 100 mg/kg, was compared in three unplanted and three planted systems. The plant used was Hycrest, a perennial, drought-tolerant cultivar of crested wheatgrass [Agropyron desertorum (Fischer ex Link) Schultes]. The flow-through test system allowed us to maintain a budget for 14C-label as well as monitor mineralization (breakdown to 14CO2) and volatilization of the test compound in a 155-d trial. In the unplanted systems, an average of 88% of the total radiolabel remained in the soil and leachate and only 6% was mineralized. In the planted system, 33% of the radiolabel remained in the soil plus leachate, 22% was mineralized, and 36% was associated with plant tissue (21% with the root fraction and 15% with shoots). Mineralization rates were 23.1 mg PCP mineralized kg-1 soil in 20 wk in the planted system, and for the unplanted system 6.6 mg PCP kg-1 soil for the same time period. Similar amounts of volatile organic material were generated in the two systems (1.5%). Results indicated that establishing crested wheatgrass on PCP-contaminated surface soils may accelerate the removal of the contaminant.

  15. Degradation properties of protein and carbohydrate during sludge anaerobic digestion.

    PubMed

    Yang, Guang; Zhang, Panyue; Zhang, Guangming; Wang, Yuanyuan; Yang, Anqi

    2015-09-01

    Degradation of protein and carbohydrate is vital for sludge anaerobic digestion performance. However, few studies focused on degradation properties of protein and carbohydrate. This study investigated detailed degradation properties of sludge protein and carbohydrate in order to gain insight into organics removal during anaerobic digestion. Results showed that carbohydrate was more efficiently degraded than protein and was degraded prior to protein. The final removal efficiencies of carbohydrate and protein were 49.7% and 32.2%, respectively. The first 3 days were a lag phase for protein degradation since rapid carbohydrate degradation in this phase led to repression of protease formation. Kinetics results showed that, after initial lag phase, protein degradation followed the first-order kinetic with rate constants of 0.0197 and 0.0018 d(-1) during later rapid degradation phase and slow degradation phase, respectively. Carbohydrate degradation also followed the first-order kinetics with a rate constant of 0.007 d(-1) after initial quick degradation phase. PMID:26025350

  16. Light-triggered chemical amplification to accelerate degradation and release from polymeric particles.

    PubMed

    Olejniczak, Jason; Nguyen Huu, Viet Anh; Lux, Jacques; Grossman, Madeleine; He, Sha; Almutairi, Adah

    2015-12-11

    We describe a means of chemical amplification to accelerate triggered degradation of a polymer and particles composed thereof. We designed a light-degradable copolymer containing carboxylic acids masked by photolabile groups and ketals. Photolysis allows the unmasked acidic groups in the polymer backbone to accelerate ketal hydrolysis even at neutral pH. PMID:26445896

  17. Polymer degradation and molecular relaxation during accelerated weathering of coatings

    NASA Astrophysics Data System (ADS)

    Fernando, B. Malcolm Dilhan

    2011-12-01

    A model polyester-urethane coating similar to those on USAF aircraft was the focus in this research. It was studied for physical property changes during accelerated weathering. Isothermal aging and natural weathering were utilized as control studies. Coatings subjected to accelerated weathering had an increase in tensile modulus, glass transition temperature and surface stiffness. DSC analysis of these coatings clearly showed evidence for 'physical aging'. This phenomenon was pursued further to find out the impact of macromolecular relaxation on the polymer physical properties. The unique feature of this research is the investigation of kinetics of macromolecular relaxation whilst a polymer undergoes simultaneous degradation. Assessment was done for some material parameters as found in theoretical models. Fictive temperature (Tf), apparent activation energy (Deltah*/R) and non linearity parameter ( x) found in Tool-Narayanswamy-Moyniham (TNM) model were explored. Tf was found to be decreasing with weathering and explained the increasingly aged 'state' of the structure. Deltah*/R was found to be increasing and explains an increased energy barrier to overcome to attain relaxation. DSC peak-shift method was used to characterize x. At early stages there is a stronger non linearity of relaxation (lower x) with a stronger structure dependence and later the relaxation kinetics seems more temperature dependent (higher x). MDSC was done to characterize the non exponentiality parameter (beta) as found in the Kohlrauch-Williams-Watts (KWW) equation. Decreasing beta value with exposure implies an increasingly broad distribution of relaxation times. The Cooperatively Rearranging Regions (CRR) concept of Adams and Gibbs was also examined. Molecular weight (Ma) of the volume (Va) represented by a CRR was compared with Mc, the molecular weight between crosslinks. Nanoindentation was done to explore the coatings' surface mechanical properties. During accelerated weathering the

  18. Protein Degradation and the Stress Response

    PubMed Central

    Flick, Karin; Kaiser, Peter

    2012-01-01

    Environmental stresses are manifold and so are the responses they elicit. This is particularly true for higher eukaryotes where various tissues and cell types are differentially affected by the insult. Type and scope of the stress response can therefore differ greatly among cell types. Given the importance of the Ubiquitin Proteasome System (UPS) for most cellular processes, it comes as no surprise that the UPR plays a pivotal role in counteracting the effects of stressors. Here we outline contributions of the UPS to stress sensing, signaling, and response pathways. We make no claim to comprehensiveness but choose selected examples to illustrate concepts and mechanisms by which protein modification with ubiquitin and proteasomal degradation of key regulators ensures cellular integrity during stress situations. PMID:22414377

  19. Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Kansal, Rita; Bartels, Scott R; Hamilton, David J; Shaaban, Salwa; Fleckenstein, James M

    2011-08-26

    Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development. PMID:21757737

  20. High Modulus Biodegradable Polyurethanes for Vascular Stents: Evaluation of Accelerated in vitro Degradation and Cell Viability of Degradation Products

    PubMed Central

    Sgarioto, Melissa; Adhikari, Raju; Gunatillake, Pathiraja A.; Moore, Tim; Patterson, John; Nagel, Marie-Danielle; Malherbe, François

    2015-01-01

    We have recently reported the mechanical properties and hydrolytic degradation behavior of a series of NovoSorb™ biodegradable polyurethanes (PUs) prepared by varying the hard segment (HS) weight percentage from 60 to 100. In this study, the in vitro degradation behavior of these PUs with and without extracellular matrix (ECM) coating was investigated under accelerated hydrolytic degradation (phosphate buffer saline; PBS/70°C) conditions. The mass loss at different time intervals and the effect of aqueous degradation products on the viability and growth of human umbilical vein endothelial cells (HUVEC) were examined. The results showed that PUs with HS 80% and below completely disintegrated leaving no visual polymer residue at 18 weeks and the degradation medium turned acidic due to the accumulation of products from the soft segment (SS) degradation. As expected the PU with the lowest HS was the fastest to degrade. The accumulated degradation products, when tested undiluted, showed viability of about 40% for HUVEC cells. However, the viability was over 80% when the solution was diluted to 50% and below. The growth of HUVEC cells is similar to but not identical to that observed with tissue culture polystyrene standard (TCPS). The results from this in vitro study suggested that the PUs in the series degraded primarily due to the SS degradation and the cell viability of the accumulated acidic degradation products showed poor viability to HUVEC cells when tested undiluted, however particles released to the degradation medium showed cell viability over 80%. PMID:26000274

  1. Cardiac protein synthesis and degradation during thyroxine-induced left ventricular hypertrophy.

    PubMed

    Parmacek, M S; Magid, N M; Lesch, M; Decker, R S; Samarel, A M

    1986-11-01

    Assessment of cardiac protein metabolism in thyroxine-induced left ventricular hypertrophy requires measurements of both protein synthesis and degradation. In vivo protein degradative rates can best be measured as the difference between rates of protein synthesis and growth. Accordingly, rates of left ventricular protein accumulation were determined in growing rabbits, and in animals administered intravenous L-thyroxine (200 micrograms X kg-1 X day-1) for up to 15 days. Left ventricular protein fractional synthetic rates in euthyroid and thyroxine-treated rabbits were measured by continuous infusion of [3H]leucine (200 mu Ci/h X 6 h), and results converted to milligrams protein synthesized and degraded per day. Thyroxine administration produced left ventricular hypertrophy by increasing the rate of total protein synthesis (35.7 +/- 2.0, 71.0 +/- 7.0, and 62.6 +/- 4.0 mg of left ventricular protein synthesized per day for 0-, 3-, and 9-day, thyroxine-treated rabbits, respectively). However, the increased rate of total protein synthesis was greater than the measured rate of total protein accumulation (8.1 vs. 15.9 mg protein/day for euthyroid and thyroxine-treated animals), indicating that left ventricular protein degradative rates were increased as well. These studies indicate that accelerated proteolysis may be important in the molecular and architectural remodeling of the rapidly hypertrophying heart during thyrotoxicosis. PMID:2946236

  2. Degradation of SsrA-tagged proteins in streptococci.

    PubMed

    Tao, Liang; Biswas, Indranil

    2015-04-01

    In prokaryotes, a conserved small RNA molecule, called tmRNA, rescues ribosomes from proteins that are abnormally truncated due to the presence of rare codons or degraded mRNA. During the rescue process, a peptide tag (SsrA) encoded by tmRNA is cotranslationally added to the truncated polypeptides, thereby targeting these proteins for proteolytic degradation. In Escherichia coli, ClpXP and ClpAP proteases primarily degrade SsrA-tagged proteins. Other proteases such as Lon and FtsH also participate in the degradation in E. coli. However, in Bacillus subtilis, ClpXP is the major protease that degrades the SsrA-tagged proteins. Degradation of SsrA-tagged protein in streptococci is not well understood except that ClpXP is responsible for the majority of the degradation. Here we show that in Streptococcus mutans, in addition to ClpXP, two other Clp complexes, ClpCP and ClpEP, are also involved in the degradation. We also found that ClpCP- and ClpEP-mediated proteolysis of SsrA-tagged substrates is induced by heat stress. As ClpCP and ClpEP proteins are highly conserved in streptococci, we predicted that ClpEP- and ClpCP-mediated degradation of SsrA-tagged proteins might be operational in other streptococci. PMID:25645948

  3. Degradation of SsrA-tagged proteins in streptococci

    PubMed Central

    Tao, Liang

    2015-01-01

    In prokaryotes, a conserved small RNA molecule, called tmRNA, rescues ribosomes from proteins that are abnormally truncated due to the presence of rare codons or degraded mRNA. During the rescue process, a peptide tag (SsrA) encoded by tmRNA is cotranslationally added to the truncated polypeptides, thereby targeting these proteins for proteolytic degradation. In Escherichia coli, ClpXP and ClpAP proteases primarily degrade SsrA-tagged proteins. Other proteases such as Lon and FtsH also participate in the degradation in E. coli. However, in Bacillus subtilis, ClpXP is the major protease that degrades the SsrA-tagged proteins. Degradation of SsrA-tagged protein in streptococci is not well understood except that ClpXP is responsible for the majority of the degradation. Here we show that in Streptococcus mutans, in addition to ClpXP, two other Clp complexes, ClpCP and ClpEP, are also involved in the degradation. We also found that ClpCP- and ClpEP-mediated proteolysis of SsrA-tagged substrates is induced by heat stress. As ClpCP and ClpEP proteins are highly conserved in streptococci, we predicted that ClpEP- and ClpCP-mediated degradation of SsrA-tagged proteins might be operational in other streptococci. PMID:25645948

  4. Detecting and Confirming Accelerated Atrazine Degradation in Illinois Soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Technical abstract: Enhanced degradation of atrazine has been documented in many parts of the world where the herbicide has been extensively used. Atrazine is widely used in corn in Illinois, but enhanced degradation in the field has not been documented. In this study, the dissipation of atrazine...

  5. Investigation of accelerated stress factors and failure/degradation mechanisms

    NASA Astrophysics Data System (ADS)

    Lathrop, J. W.

    1984-03-01

    Results of the performance tests on unencapsulated cells are described. Equivalent circuit parameters; characteristics of a degraded solar cell; and atomic and molecular species at the cell surface are detailed.

  6. Loosening xyloglucan accelerates the enzymatic degradation of cellulose in wood.

    PubMed

    Kaida, Rumi; Kaku, Tomomi; Baba, Kei'ichi; Oyadomari, Masafumi; Watanabe, Takashi; Nishida, Koji; Kanaya, Toshiji; Shani, Ziv; Shoseyov, Oded; Hayashi, Takahisa

    2009-09-01

    In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood. PMID:19825667

  7. Curcumin inhibits HIV-1 by promoting Tat protein degradation.

    PubMed

    Ali, Amjad; Banerjea, Akhil C

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  8. Curcumin inhibits HIV-1 by promoting Tat protein degradation

    PubMed Central

    Ali, Amjad; Banerjea, Akhil C.

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  9. Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Aβ Generation and Amyloid Plaque Pathogenesis

    PubMed Central

    Xiao, Qingli; Yan, Ping; Ma, Xiucui; Liu, Haiyan; Perez, Ronaldo; Zhu, Alec; Gonzales, Ernesto; Tripoli, Danielle L.; Czerniewski, Leah; Ballabio, Andrea; Cirrito, John R.

    2015-01-01

    In AD, an imbalance between Aβ production and removal drives elevated brain Aβ levels and eventual amyloid plaque deposition. APP undergoes nonamyloidogenic processing via α-cleavage at the plasma membrane, amyloidogenic β- and γ-cleavage within endosomes to generate Aβ, or lysosomal degradation in neurons. Considering multiple reports implicating impaired lysosome function as a driver of increased amyloidogenic processing of APP, we explored the efficacy of targeting transcription factor EB (TFEB), a master regulator of lysosomal pathways, to reduce Aβ levels. CMV promoter-driven TFEB, transduced via stereotactic hippocampal injections of adeno-associated virus particles in APP/PS1 mice, localized primarily to neuronal nuclei and upregulated lysosome biogenesis. This resulted in reduction of APP protein, the α and β C-terminal APP fragments (CTFs), and in the steady-state Aβ levels in the brain interstitial fluid. In aged mice, total Aβ levels and amyloid plaque load were selectively reduced in the TFEB-transduced hippocampi. TFEB transfection in N2a cells stably expressing APP695, stimulated lysosome biogenesis, reduced steady-state levels of APP and α- and β-CTFs, and attenuated Aβ generation by accelerating flux through the endosome-lysosome pathway. Cycloheximide chase assays revealed a shortening of APP half-life with exogenous TFEB expression, which was prevented by concomitant inhibition of lysosomal acidification. These data indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce Aβ generation. Activation of TFEB in neurons is an effective strategy to attenuate Aβ generation and attenuate amyloid plaque deposition in AD. SIGNIFICANCE STATEMENT A key driver for AD pathogenesis is the net balance between production and clearance of Aβ, the major component of amyloid plaques. Here we demonstrate that lysosomal degradation of holo-APP influences Aβ production by limiting the availability of

  10. Quantitating protein synthesis, degradation, and endogenous antigen processing.

    PubMed

    Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

    2003-03-01

    Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

  11. Clipping or Extracting: Two Ways to Membrane Protein Degradation.

    PubMed

    Avci, Dönem; Lemberg, Marius K

    2015-10-01

    Protein degradation is a fundamentally important process that allows cells to recognize and remove damaged protein species and to regulate protein abundance according to functional need. A fundamental challenge is to understand how membrane proteins are recognized and removed from cellular organelles. While most of our understanding of this mechanism comes from studies on p97/Cdc48-mediated protein dislocation along the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, recent studies have revealed intramembrane proteolysis to be an additional mechanism that can extract transmembrane segments. Here, we review these two principles in membrane protein degradation and discuss how intramembrane proteolysis, which introduces an irreversible step in protein dislocation, is used to drive regulated protein turnover. PMID:26410407

  12. Protein degradation in bovine milk caused by Streptococcus agalactiae.

    PubMed

    Åkerstedt, Maria; Wredle, Ewa; Lam, Vo; Johansson, Monika

    2012-08-01

    Streptococcus (Str.) agalactiae is a contagious mastitis bacterium, often associated with cases of subclinical mastitis. Different mastitis bacteria have been evaluated previously from a diagnostic point of view, but there is a lack of knowledge concerning their effect on milk composition. Protein composition is important in achieving optimal yield and texture when milk is processed to fermented products, such as cheese and yoghurt, and is thus of great economic value. The aim of this in vitro study was to evaluate protein degradation mainly caused by exogenous proteases originating from naturally occurring Str. agalactiae. The samples were incubated at 37°C to imitate degradation caused by the bacteria in the udder. Protein degradation caused by different strains of Str. agalactiae was also investigated. Protein degradation was observed to occur when Str. agalactiae was added to milk, but there were variations between strains of the bacteria. Caseins, the most economically important proteins in milk, were degraded up to 75% in milk inoculated with Str. agalactiae in relation to sterile ultra-high temperature (UHT) milk, used as control milk. The major whey proteins, α-lactalbumin and β-lactoglobulin, were degraded up to 21% in relation to the sterile control milk. These results suggest that different mastitis bacteria but also different strains of mastitis bacteria should be evaluated from a milk quality perspective to gain knowledge about their ability to degrade the economically important proteins in milk. PMID:22850579

  13. Degradation Mechanisms and Accelerated Testing in PEM Fuel Cells

    SciTech Connect

    Borup, Rodney L.

    2011-01-01

    The durability of PEM fuel cells is a major barrier to the commercialization of these systems for stationary and transportation power applications. Although there has been recent progress in improving durability, further improvements are needed to meet the commercialization targets. Past improvements have largely been made possible because of the fundamental understanding of the underlying degradation mechanisms. By investigating component and cell degradation modes; defining the fundamental degradation mechanisms of components and component interactions new materials can be designed to improve durability. Various factors have been shown to affect the useful life of PEM fuel cells. Other issues arise from component optimization. Operational conditions (such as impurities in either the fuel or oxidant stream), cell environment, temperature (including subfreezing exposure), pressure, current, voltage, etc.; or transient versus continuous operation, including start-up and shutdown procedures, represent other factors that can affect cell performance and durability.

  14. Error-Induced Beam Degradation in Fermilab's Accelerators

    SciTech Connect

    Yoon, Phil S.; /Rochester U.

    2007-08-01

    In Part I, three independent models of Fermilab's Booster synchrotron are presented. All three models are constructed to investigate and explore the effects of unavoidable machine errors on a proton beam under the influence of space-charge effects. The first is a stochastic noise model. Electric current fluctuations arising from power supplies are ubiquitous and unavoidable and are a source of instabilities in accelerators of all types. A new noise module for generating the Ornstein-Uhlenbeck (O-U) stochastic noise is first created and incorporated into the existing Object-oriented Ring Beam Injection and Tracking (ORBIT-FNAL) package. After being convinced with a preliminary model that the noise, particularly non-white noise, does matter to beam quality, we proceeded to measure directly current ripples and common-mode voltages from all four Gradient Magnet Power Supplies (GMPS). Then, the current signals are Fourier-analyzed. Based upon the power spectra of current signals, we tune up the Ornstein-Uhlnbeck noise model. As a result, we are able to closely match the frequency spectra between current measurements and the modeled O-U stochastic noise. The stochastic noise modeled upon measurements is applied to the Booster beam in the presence of the full space-charge effects. This noise model, accompanied by a suite of beam diagnostic calculations, manifests that the stochastic noise, impinging upon the beam and coupled to the space-charge effects, can substantially enhance the beam degradation process throughout the injection period. The second model is a magnet misalignment model. It is the first time to utilize the latest beamline survey data for building a magnet-by-magnet misalignment model. Given as-found survey fiducial coordinates, we calculate all types of magnet alignment errors (station error, pitch, yaw, roll, twists, etc.) are implemented in the model. We then follow up with statistical analysis to understand how each type of alignment errors are

  15. POLYPHENOL AND CONDITIONING EFFECTS ON FORAGE PROTEIN SOLUBILITY AND DEGRADABILITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasing the degree of tissue disruption during mechanical harvesting of forages may augment protein interactions with polyphenols and other cellular constituents, enhancing protein utilization by reducing protein solubility and shifting its degradation from the rumen to the intestine. In 2002 and...

  16. Sulforaphane promotes murine hair growth by accelerating the degradation of dihydrotestosterone.

    PubMed

    Sasaki, Mari; Shinozaki, Shohei; Shimokado, Kentaro

    2016-03-25

    Dihydrotestosterone (DHT) causes the regression of human hair follicles in the parietal scalp, leading to androgenic alopecia (AGA). Sulforaphane (SFN) increases the expression of DHT degrading enzymes, such as 3α-hydroxysteroid dehydrogenases (3α-HSDs), and, therefore, SFN treatment may improve AGA. To determine the effects of SFN on hair growth, we administered SFN (10 mg/kg BW, IP) or vehicle (DMSO) to ob/ob mice for six weeks and examined hair regeneration and the plasma levels of testosterone and DHT. We also tested the effects of SFN on the expression of two forms of 3α-HSD, aldo-keto reductase 1c21 and dehydrogenase/reductase (SDR family) member 9, both in vitro and in vivo. SNF significantly enhanced hair regeneration in ob/ob mice. The mice treated with SFN showed lower plasma levels of testosterone and DHT than those treated with vehicle. SFN increased the mRNA and protein levels of the two forms of 3α-HSD in the liver of the mice and in cultured murine hepatocyte Hepa1c1c7 cells. These results suggest that SFN treatment increases the amount of 3α-HSDs in the liver, accelerates the degradation of blood DHT, and subsequently blocks the suppression of hair growth by DHT. PMID:26923074

  17. Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics*

    PubMed Central

    Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J.; Ly, Tony; Lamond, Angus I.

    2013-01-01

    Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ∼5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution. PMID:23242552

  18. Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae.

    PubMed

    Buchanan, Bryce W; Lloyd, Michael E; Engle, Sarah M; Rubenstein, Eric M

    2016-01-01

    Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation. PMID:27167179

  19. Accelerated in vitro Degradation of Optically Clear Low β-sheet Silk Films by Enzyme-Mediated Pretreatment

    PubMed Central

    Shang, Ke; Rnjak-Kovacina, Jelena; Lin, Yinan; Hayden, Rebecca S.; Hu, Tao; Kaplan, David L.

    2013-01-01

    Purpose To design patterned, transparent silk films with fast degradation rates for the purpose of tissue engineering corneal stroma, Methods β-sheet (crystalline) content of silk films was decreased significantly by using a short water annealing time. Additionally, a protocol combining short water annealing time with enzymatic pretreatment of silk films with protease XIV was developed. Results Low β-sheet content (17–18%) and enzymatic pre-treatment provided film stability in aqueous environments and accelerated degradation of the silk films in the presence of human corneal fibroblasts in vitro. The results demonstrate a direct relationship between reduced β-sheet content and enzymatic pre-treatment and overall degradation rate of the protein films. Conclusions The novel protocol developed here provides new approaches to modulate the regeneration rate of silk biomaterials for corneal tissue regeneration needs. Translational relevance Patterned silk protein films possess desirable characteristics for corneal tissue engineering, including optical transparency, biocompatibility, cell alignment and tunable mechanical properties, but current fabrication protocols do not provide adequate degradation rates to match the regeneration properties of the human cornea. This novel processing protocol makes silk films more suitable for the construction of human corneal stroma tissue and a promising way to tune silk film degradation properties to match corneal tissue regeneration. PMID:23579493

  20. Contribution of Accelerated Degradation to Feedback Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase and Cholesterol Metabolism in the Liver.

    PubMed

    Hwang, Seonghwan; Hartman, Isamu Z; Calhoun, Leona N; Garland, Kristina; Young, Gennipher A; Mitsche, Matthew A; McDonald, Jeffrey; Xu, Fang; Engelking, Luke; DeBose-Boyd, Russell A

    2016-06-24

    Accumulation of sterols in endoplasmic reticulum membranes stimulates the ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), which catalyzes a rate-limiting step in synthesis of cholesterol. This ubiquitination marks HMGCR for proteasome-mediated degradation and constitutes one of several mechanisms for feedback control of cholesterol synthesis. Mechanisms for sterol-accelerated ubiquitination and degradation of HMGCR have been elucidated through the study of cultured mammalian cells. However, the extent to which these reactions modulate HMGCR and contribute to control of cholesterol metabolism in whole animals is unknown. Here, we examine transgenic mice expressing in the liver the membrane domain of HMGCR (HMGCR (TM1-8)), a region necessary and sufficient for sterol-accelerated degradation, and knock-in mice in which endogenous HMGCR harbors mutations that prevent sterol-induced ubiquitination. Characterization of transgenic mice revealed that HMGCR (TM1-8) is appropriately regulated in the liver of mice fed a high cholesterol diet or chow diet supplemented with the HMGCR inhibitor lovastatin. Ubiquitination-resistant HMGCR protein accumulates in the liver and other tissues disproportionately to its mRNA, indicating that sterol-accelerated degradation significantly contributes to feedback regulation of HMGCR in vivo Results of these studies demonstrate that HMGCR is subjected to sterol-accelerated degradation in the liver through mechanisms similar to those established in cultured cells. Moreover, these studies designate sterol-accelerated degradation of HMGCR as a potential therapeutic target for prevention of atherosclerosis and associated cardiovascular disease. PMID:27129778

  1. Small-molecule control of protein degradation using split adaptors.

    PubMed

    Davis, Joseph H; Baker, Tania A; Sauer, Robert T

    2011-11-18

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  2. Small-molecule control of protein degradation using split adaptors

    PubMed Central

    Davis, Joseph H.; Baker, Tania A.; Sauer, Robert T.

    2011-01-01

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly-defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  3. Nylon 6.6 accelerated aging studies : thermal-oxidative degradation and its interaction with hydrolysis.

    SciTech Connect

    Bernstein, Robert; Derzon, Dora Kay; Gillen, Kenneth T.

    2004-06-01

    Accelerated aging of Nylon 6.6 fibers used in parachutes has been conducted by following the tensile strength loss under both thermal-oxidative and 100% relative humidity conditions. Thermal-oxidative studies (air circulating ovens) were performed for time periods of weeks to years at temperatures ranging from 37 C to 138 C. Accelerated aging humidity experiments (100% RH) were performed under both an argon atmosphere to examine the 'pure' hydrolysis pathway, and under an oxygen atmosphere (oxygen partial pressure close to that occurring in air) to mimic true aging conditions. As expected the results indicated that degradation caused by humidity is much more important than thermal-oxidative degradation. Surprisingly when both oxygen and humidity were present the rate of degradation was dramatically enhanced relative to humidity aging in the absence of oxygen. This significant and previously unknown phenomena underscores the importance of careful accelerated aging that truly mimics real world storage conditions.

  4. Protein-Linked Glycan Degradation in Infants Fed Human Milk

    PubMed Central

    Dallas, David C.; Sela, David; Underwood, Mark A.; German, J. Bruce; Lebrilla, Carlito

    2014-01-01

    Many human milk proteins are glycosylated. Glycosylation is important in protecting bioactive proteins and peptide fragments from digestion. Protein-linked glycans have a variety of functions; however, there is a paucity of information on protein-linked glycan degradation in either the infant or the adult digestive system. Human digestive enzymes can break down dietary disaccharides and starches, but most of the digestive enzymes required for complex protein-linked glycan degradation are absent from both human digestive secretions and the external brush border membrane of the intestinal lining. Indeed, complex carbohydrates remain intact throughout their transit through the stomach and small intestine, and are undegraded by in vitro incubation with either adult pancreatic secretions or intact intestinal brush border membranes. Human gastrointestinal bacteria, however, produce a wide variety of glycosidases with regio- and anomeric specificities matching those of protein-linked glycan structures. These bacteria degrade a wide array of complex carbohydrates including various protein-linked glycans. That bacteria possess glycan degradation capabilities, whereas the human digestive system, perse, does not, suggests that most dietary protein-linked glycan breakdown will be of bacterial origin. In addition to providing a food source for specific bacteria in the colon, protein-linked glycans from human milk may act as decoys for pathogenic bacteria to prevent invasion and infection of the host. The composition of the intestinal microbiome may be particularly important in the most vulnerable humans-the elderly, the immunocompromised, and infants (particularly premature infants). PMID:24533224

  5. Hydrolytically degradable thiol–ene hydrogels for protein release

    PubMed Central

    Rehmann, Matthew S.; Garibian, Andrew C.; Kloxin, April M.

    2014-01-01

    Summary A new degradable PEG-diester-dinorbornene/PEG-triester-trithiol hydrogel was evaluated for protein release. The hydrogel polymerized rapidly with seconds of UV irradiation and subsequently hydrolytically degraded in aqueous buffer over the course of approximately 3 weeks. Further, the hydrogel enabled the encapsulation and release of a model protein, bovine serum albumin (BSA), over 7 days with ~ 90% released at 48 h. This study serves as a proof-of-concept for the creation of hydrolytically degradable, PEG-ester-thiol-based hydrogels by a photoinitiated step growth mechanism for protein release. With this approach, degradation and release rates could be tuned by varying the monomer molecular weight and functionality in future studies. PMID:25309103

  6. Degradation of microinjected proteins: the role of substrate flexibility

    SciTech Connect

    Rote, K.V.

    1985-01-01

    RB-mediated microinjection was used to introduce radioiodinated proteins of similar structure, but diverse flexibilities, into HeLa cells. Rates of intracellular degradation were then measured by release of /sup 125/I-tyrosine into the media. Ribonuclease-A was much more stable to degradation by trypsin, pepsin, or papain than its relatively flexible derivatives ribonuclease-S and S-protein. Likewise, ribonuclease-S and S-protein were degraded more quickly in reticulocyte lysates than ribonuclease-A. In contrast, all three proteins displayed similar, if not identical, half-lives in vivo. Similarly, intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were in the same range whether they were measured in the free state or following complex formation, which drastically decreases flexibility. Trypsinogen, which contains a relatively flexible activation domain, was degraded more slowly than anhydrotrypsin. Nondenaturing agarose or polyacrylamide gel electrophoresis of microinjected cell lysates revealed that complexes of trypsin and its inhibitors remained intact following radioiodination and introduction into cells, and are therefore degraded as a unit. All microinjected proteins remained in their unbound, unprocessed forms prior to degradation.

  7. The Cdc48 machine in endoplasmic reticulum associated protein degradation.

    PubMed

    Wolf, Dieter H; Stolz, Alexandra

    2012-01-01

    The AAA-type ATPase Cdc48 (named p97/VCP in mammals) is a molecular machine in all eukaryotic cells that transforms ATP hydrolysis into mechanic power to unfold and pull proteins against physical forces, which make up a protein's structure and hold it in place. From the many cellular processes, Cdc48 is involved in, its function in endoplasmic reticulum associated protein degradation (ERAD) is understood best. This quality control process for proteins of the secretory pathway scans protein folding and discovers misfolded proteins in the endoplasmic reticulum (ER), the organelle, destined for folding of these proteins and their further delivery to their site of action. Misfolded lumenal and membrane proteins of the ER are detected by chaperones and lectins and retro-translocated out of the ER for degradation. Here the Cdc48 machinery, recruited to the ER membrane, takes over. After polyubiquitylation of the protein substrate, Cdc48 together with its dimeric co-factor complex Ufd1-Npl4 pulls the misfolded protein out and away from the ER membrane and delivers it to down-stream components for degradation by a cytosolic proteinase machine, the proteasome. The known details of the Cdc48-Ufd1-Npl4 motor complex triggered process are subject of this review article. PMID:21945179

  8. Protein Quality Control and Degradation in Cardiomyocytes

    PubMed Central

    Wang, Xuejun; Su, Huabo; Ranek, Mark J.

    2008-01-01

    The heart is constantly under stress and cardiomyocytes face enormous challenges to correctly fold nascent polypeptides and keep mature proteins from denaturing. To meet the challenge, cardiomyocytes have developed multi-layered protein quality control (PQC) mechanisms which are carried out primarily by chaperones and ubiquitin-proteasome system mediated proteolysis. Autophagy may also participate in PQC in cardiomyocytes, especially under pathological conditions. Cardiac PQC often becomes inadequate in heart disease, which may play an important role in the development of congestive heart failure. PMID:18495153

  9. Regulation of protein degradation in muscle by calcium

    NASA Technical Reports Server (NTRS)

    Zeman, Richard J.; Kameyama, Tsuneo; Matsumoto, Kazue; Bernstein, Paul; Etlinger, Joseph D.

    1985-01-01

    Calcium-dependent regulation of intracellular protein degradation was studied in isolated rat skeletal muscles incubated in vitro in the presence of a large variety of agents known to affect calcium movement and distribution. The effect of different classes of protease inhibitors was tested to determine the responsible proteolytic systems involved in calcium-dependent degradation. The results suggest that nonlysosomal leupetin- and E-64-c-sensitive proteases are resposible for calcium-dependent proteolysis in muscle.

  10. Results of metallographical diagnostic examination of Navy half-watt thermoelectric converters degraded by accelerated tests

    NASA Technical Reports Server (NTRS)

    Rosell, F. E., Jr.; Rouklove, P. G.

    1977-01-01

    To verify the 15-year reliability of the Navy half-watt radioisotope thermoelectric generator (RTG), bismuth-telluride thermoelectric converters were submitted to testing at high temperatures which accelerated the degradation and caused failure of the converters. Metallographic diagnostic examination of failed units verified failure mechanisms. Results of diagnostic examinations are presented.

  11. Prion protein degradation by lichens of the genus Cladonia

    USGS Publications Warehouse

    Bennett, James P.; Rodriguez, Cynthia M.; Johnson, Christopher J.

    2012-01-01

    It has recently been discovered that lichens contain a serine protease capable of degrading the pathogenic prion protein, the etiological agent of prion diseases such as sheep scrapie and cervid chronic wasting disease. Limited methods are available to degrade or inactivate prion disease agents, especially in the environment, and lichens or their serine protease could prove important for management of these diseases. Scant information is available regarding the presence or absence of the protease responsible for degrading prion protein (PrP) in lichen species and, in this study, we tested the hypothesis that PrP degradation activity in lichens is phylogenetically-based by testing 44 species of Cladonia lichens, a genus for which a significant portion of the phylogeny is well established. We categorized PrP degradation activity among the 44 species (high, moderate, low or none) and found that activity in Cladonia species did not correspond with phylogenetic position of the species. Degradation of PrP did correspond, however, with three classical taxonomic characters within the genus: species with brown apothecia, no usnic acid, and the presence of a cortex. Of the 44 species studied, 18 (41%) had either high or moderate PrP degradation activity, suggesting the protease may be frequent in this genus of lichens.

  12. Uniform and accelerated degradation of pure iron patterned by Pt disc arrays

    PubMed Central

    Huang, Tao; Zheng, Yufeng

    2016-01-01

    Pure iron has been confirmed as a promising biodegradable metal. However, the degradation rate of pure iron should be accelerated to meet the clinical requirements. In this work, two different designs of platinum disc arrays, including sizes of Φ20 μm × S5 μm and Φ4 μm × S4 μm, have been coated on the surface of pure iron. Corrosion tests showed the platinum discs formed plenty of galvanic cells with the iron matrix which significantly accelerated the degradation of pure iron. Simultaneously, due to the designability of the shape, size as well as distribution of Pt discs, the degradation rate as well as degradation uniformity of pure iron can be effectively controlled by coating with platinum discs. The cytotoxicity test results unveiled that Pt discs patterned pure iron exhibited almost no toxicity to human umbilical vein endothelial cells, but a significant inhibition on proliferation of vascular smooth muscle cells. In addition, the hemolysis rate of Pt discs patterned pure iron was lower than 1%. Moreover, Pt discs also effectively reduced the number of adhered platelets. All these results indicated that Pt discs patterning is an effective way to accelerate degradation and improve biocompatibility of pure iron. PMID:27033380

  13. Uniform and accelerated degradation of pure iron patterned by Pt disc arrays

    NASA Astrophysics Data System (ADS)

    Huang, Tao; Zheng, Yufeng

    2016-04-01

    Pure iron has been confirmed as a promising biodegradable metal. However, the degradation rate of pure iron should be accelerated to meet the clinical requirements. In this work, two different designs of platinum disc arrays, including sizes of Φ20 μm × S5 μm and Φ4 μm × S4 μm, have been coated on the surface of pure iron. Corrosion tests showed the platinum discs formed plenty of galvanic cells with the iron matrix which significantly accelerated the degradation of pure iron. Simultaneously, due to the designability of the shape, size as well as distribution of Pt discs, the degradation rate as well as degradation uniformity of pure iron can be effectively controlled by coating with platinum discs. The cytotoxicity test results unveiled that Pt discs patterned pure iron exhibited almost no toxicity to human umbilical vein endothelial cells, but a significant inhibition on proliferation of vascular smooth muscle cells. In addition, the hemolysis rate of Pt discs patterned pure iron was lower than 1%. Moreover, Pt discs also effectively reduced the number of adhered platelets. All these results indicated that Pt discs patterning is an effective way to accelerate degradation and improve biocompatibility of pure iron.

  14. Uniform and accelerated degradation of pure iron patterned by Pt disc arrays.

    PubMed

    Huang, Tao; Zheng, Yufeng

    2016-01-01

    Pure iron has been confirmed as a promising biodegradable metal. However, the degradation rate of pure iron should be accelerated to meet the clinical requirements. In this work, two different designs of platinum disc arrays, including sizes of Φ20 μm × S5 μm and Φ4 μm × S4 μm, have been coated on the surface of pure iron. Corrosion tests showed the platinum discs formed plenty of galvanic cells with the iron matrix which significantly accelerated the degradation of pure iron. Simultaneously, due to the designability of the shape, size as well as distribution of Pt discs, the degradation rate as well as degradation uniformity of pure iron can be effectively controlled by coating with platinum discs. The cytotoxicity test results unveiled that Pt discs patterned pure iron exhibited almost no toxicity to human umbilical vein endothelial cells, but a significant inhibition on proliferation of vascular smooth muscle cells. In addition, the hemolysis rate of Pt discs patterned pure iron was lower than 1%. Moreover, Pt discs also effectively reduced the number of adhered platelets. All these results indicated that Pt discs patterning is an effective way to accelerate degradation and improve biocompatibility of pure iron. PMID:27033380

  15. Phospholipases as GTPase activity accelerating proteins (GAPs) in plants.

    PubMed

    Pandey, Sona

    2016-05-01

    GTPase activity accelerating proteins (GAPs) are key regulators of the G-protein signaling cycle. By facilitating effective hydrolysis of the GTP bound on Gα proteins, GAPs control the timing and amplitude of the signaling cycle and ascertain the availability of the inactive heterotrimer for the next round of activation. Until very recently, the studies of GAPs in plants were focused exclusively on the regulator of G-protein signaling (RGS) protein. We now show that phospholipase Dα1 (PLDα1) is also a bona fide GAP in plants and together with the RGS protein controls the level of active Gα protein. PMID:27124090

  16. Dual degradation signals control Gli protein stability and tumor formation

    PubMed Central

    Huntzicker, Erik G.; Estay, Ivette S.; Zhen, Hanson; Lokteva, Ludmila A.; Jackson, Peter K.; Oro, Anthony E.

    2006-01-01

    Regulated protein destruction controls many key cellular processes with aberrant regulation increasingly found during carcinogenesis. Gli proteins mediate the transcriptional effects of the Sonic hedgehog pathway, which is implicated in up to 25% of human tumors. Here we show that Gli is rapidly destroyed by the proteasome and that mouse basal cell carcinoma induction correlates with Gli protein accumulation. We identify two independent destruction signals in Gli1, DN and DC, and show that removal of these signals stabilizes Gli1 protein and rapidly accelerates tumor formation in transgenic animals. These data argue that control of Gli protein accumulation underlies tumorigenesis and suggest a new avenue for antitumor therapy. PMID:16421275

  17. Accelerated Testing of UH-60 Viscous Bearings for Degraded Grease Fault

    NASA Technical Reports Server (NTRS)

    Dykas, Brian; Hood, Adrian; Krantz, Timothy; Klemmer, Marko

    2015-01-01

    An accelerated aging investigation of critical aviation bearings lubricated with MIL-PRF- 81322 grease was conducted to derive an understanding of the mechanisms of grease degradation and loss of lubrication over time. The current study focuses on UH-60 Black Hawk viscous damper bearings supporting the tail rotor driveshaft, which were subjected to more than 5800 hours of testing in a heated environment to accelerate the deterioration of the grease. The mechanism of grease degradation is a reduction in the oil/thickener ratio rather than the expected chemical degradation of grease constituents. Over the course of testing, vibration and temperature monitoring of bearings was conducted and trends for failing bearings are presented.

  18. Degradation of Akt Using Protein Catalyzed Capture Agents

    PubMed Central

    Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M.; Heath, James R.

    2016-01-01

    Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry we recently developed multiple protein catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein based assay. Here we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare Proteolysis Targeting Chimeric molecules (PROTACs) that are shown to promote the rapid degradation of Akt in live cancer cells. These novel PROTACs demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. PMID:26880702

  19. Acceleration of potential-induced degradation by salt-mist preconditioning in crystalline silicon photovoltaic modules

    NASA Astrophysics Data System (ADS)

    Suzuki, Soh; Nishiyama, Naoki; Yoshino, Seiji; Ujiro, Takumi; Watanabe, Shin; Doi, Takuya; Masuda, Atsushi; Tanahashi, Tadanori

    2015-08-01

    We examined the sequential effects of salt-mist stress followed by high-system-voltage stress on the power loss of crystalline silicon photovoltaic (PV) modules to determine whether a crucial failure as potential-induced degradation (PID) is accelerated by material-property changes caused by the long-term effects of a less harmful stress such as salt-mist spraying. Degradation profiles confirmed in this study show that PID is accelerated by certain types of salt-mist preconditioning. For the acceleration of PID, the contribution of sodium ions liberated from the front glass of the PV module seems to be excluded. Therefore, we consider that the sodium ions penetrating into the PV modules from the ambient environment may also cause degradation according to the proposed mechanisms of PID, as the sodium ions existing in the front glass cause PID. Furthermore, this type of degradation may indicate the wear-out phenomenon after a long-term exposure in the field (especially near the coast).

  20. Lifetime Prediction for Degradation of Solar Mirrors using Step-Stress Accelerated Testing (Presentation)

    SciTech Connect

    Lee, J.; Elmore, R.; Kennedy, C.; Gray, M.; Jones, W.

    2011-09-01

    This research is to illustrate the use of statistical inference techniques in order to quantify the uncertainty surrounding reliability estimates in a step-stress accelerated degradation testing (SSADT) scenario. SSADT can be used when a researcher is faced with a resource-constrained environment, e.g., limits on chamber time or on the number of units to test. We apply the SSADT methodology to a degradation experiment involving concentrated solar power (CSP) mirrors and compare the results to a more traditional multiple accelerated testing paradigm. Specifically, our work includes: (1) designing a durability testing plan for solar mirrors (3M's new improved silvered acrylic "Solar Reflector Film (SFM) 1100") through the ultra-accelerated weathering system (UAWS), (2) defining degradation paths of optical performance based on the SSADT model which is accelerated by high UV-radiant exposure, and (3) developing service lifetime prediction models for solar mirrors using advanced statistical inference. We use the method of least squares to estimate the model parameters and this serves as the basis for the statistical inference in SSADT. Several quantities of interest can be estimated from this procedure, e.g., mean-time-to-failure (MTTF) and warranty time. The methods allow for the estimation of quantities that may be of interest to the domain scientists.

  1. Protein unfolding and degradation by the AAA+ Lon protease.

    PubMed

    Gur, Eyal; Vishkautzan, Marina; Sauer, Robert T

    2012-02-01

    AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases. PMID:22162032

  2. Degradation of antenna chlrophyll-binding protein CP43 during photoinhibition of photosystem II

    SciTech Connect

    Yamamoto, Yasusi; Akasaka, Tatsuya

    1995-07-18

    When photosystem II (PS II) membranes from spinach were treated with Tris (0.8 M, pH 9.0) and illuminated with white light (5000 {mu}E m{sup -2} s{sup -1}) under aerobic conditions at 25{degrees}C, not only were the reaction center-forming D1 and D2 proteins degraded but the antenna chlorophyll-binding protein CP43 was also degraded. Three products of the degradation of CP43, with molecular masses of 17.0, 15.5, and 14 kDa, respectively, were identified by sodium dodecyl sulfate/urea polyacrylamide gel electrophoresis and Western blotting with a specific antibody. Degradation products of another antenna chlorophyll-binding protein of PS II, CP47, were not detected under the same conditions. Concomitant with the damage to the D1 and D2 proteins and CP43, cross-linked products of the D1 protein, CP43, and CP47 were formed. These products were identified as slow-moving smeared bands in the higher molecular weight range of the gel during electrophoresis. Both the degradation and the cross-linking of these proteins were prevented by the addition of electron donors to PS II, a result that suggests that these processes were caused by the donor-side mechanism of photoinhibition. The photoinduced degradation of CP43 and the cross-linking among the D1 protein, CP43, and CP47 were less obvious in the PS II membranes that had been treated with hydroxylamine rather than Tris and in the membranes that had been treated with Tris and reconstituted by addition of an extrinsic 33-kDa protein (OEC33). These results indicate that removal of OEC33, which is closely associated with CP43, from the PS II complex accelerates the degradation and cross-linking of CP43 during photoinhibition. It is suggested that OEC33 is involved in the stabilization of the antenna chlorophyll-binding proteins in PS II during photoinhibition. 55 refs., 7 figs.

  3. Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.

    PubMed Central

    Loo, M A; Jensen, T J; Cui, L; Hou, Y; Chang, X B; Riordan, J R

    1998-01-01

    Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface. PMID:9843494

  4. Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.

    PubMed

    Loo, M A; Jensen, T J; Cui, L; Hou, Y; Chang, X B; Riordan, J R

    1998-12-01

    Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface. PMID:9843494

  5. ATP-dependent degradation of ubiquitin-protein conjugates.

    PubMed Central

    Hershko, A; Leshinsky, E; Ganoth, D; Heller, H

    1984-01-01

    Previous studies have indicated that the ATP-requiring conjugation of ubiquitin with proteins plays a role in the energy-dependent degradation of intracellular proteins. To examine whether such conjugates are indeed intermediates in protein breakdown, conjugates of 125I-labeled lysozyme with ubiquitin were isolated and incubated with a fraction of reticulocyte extract that lacks the enzymes that carry out ubiquitin-protein conjugation. ATP markedly stimulated degradation of the lysozyme moiety of ubiquitin conjugates to products soluble in trichloroacetic acid. By contrast, free 125I-labeled lysozyme was not degraded under these conditions, unless ubiquitin and the three enzymes required for ubiquitin conjugation were supplemented. Mg2+ was absolutely required for conjugate breakdown. Of various nucleotides, only CTP replaced ATP. Nonhydrolyzable analogs of ATP were not effective. In the absence of ATP, free lysozyme is released from ubiquitin-lysozyme conjugates by isopeptidases present in the extract. Thus, ATP is involved in both the formation and the breakdown of ubiquitin-protein conjugates. Images PMID:6324208

  6. Temperature compensation via cooperative stability in protein degradation

    NASA Astrophysics Data System (ADS)

    Peng, Yuanyuan; Hasegawa, Yoshihiko; Noman, Nasimul; Iba, Hitoshi

    2015-08-01

    Temperature compensation is a notable property of circadian oscillators that indicates the insensitivity of the oscillator system's period to temperature changes; the underlying mechanism, however, is still unclear. We investigated the influence of protein dimerization and cooperative stability in protein degradation on the temperature compensation ability of two oscillators. Here, cooperative stability means that high-order oligomers are more stable than their monomeric counterparts. The period of an oscillator is affected by the parameters of the dynamic system, which in turn are influenced by temperature. We adopted the Repressilator and the Atkinson oscillator to analyze the temperature sensitivity of their periods. Phase sensitivity analysis was employed to evaluate the period variations of different models induced by perturbations to the parameters. Furthermore, we used experimental data provided by other studies to determine the reasonable range of parameter temperature sensitivity. We then applied the linear programming method to the oscillatory systems to analyze the effects of protein dimerization and cooperative stability on the temperature sensitivity of their periods, which reflects the ability of temperature compensation in circadian rhythms. Our study explains the temperature compensation mechanism for circadian clocks. Compared with the no-dimer mathematical model and linear model for protein degradation, our theoretical results show that the nonlinear protein degradation caused by cooperative stability is more beneficial for realizing temperature compensation of the circadian clock.

  7. Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation.

    PubMed

    Itakura, Eisuke; Zavodszky, Eszter; Shao, Sichen; Wohlever, Matthew L; Keenan, Robert J; Hegde, Ramanujan S

    2016-07-01

    We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions. PMID:27345149

  8. Accelerated Arctic land warming and permafrost degradation during rapid sea ice loss

    NASA Astrophysics Data System (ADS)

    Lawrence, D. M.; Slater, A. G.; Tomas, R. A.; Holland, M. M.; Deser, C.

    2008-12-01

    Coupled climate models and recent observational evidence suggest that Arctic sea ice may undergo abrupt periods of loss during the next fifty years. Here, we evaluate how rapid sea ice loss affects terrestrial Arctic climate and ground thermal state in the Community Climate System Model. We find that simulated western Arctic land warming trends during rapid sea ice loss are 3.5 times greater than secular 21st century climate- change trends. The accelerated warming signal penetrates up to 1500km inland and is apparent throughout most of the year, peaking in autumn. Idealized experiments using the Community Land Model, with improved permafrost dynamics, indicate that an accelerated warming period substantially increases ground heat accumulation. Enhanced heat accumulation leads to rapid degradation of warm permafrost and may increase the vulnerability of colder permafrost to degradation under continued warming. Taken together, these results imply a direct link between rapid sea ice loss and permafrost health.

  9. Accelerated Arctic land warming and permafrost degradation during rapid sea ice loss

    NASA Astrophysics Data System (ADS)

    Lawrence, David M.; Slater, Andrew G.; Tomas, Robert A.; Holland, Marika M.; Deser, Clara

    2008-06-01

    Coupled climate models and recent observational evidence suggest that Arctic sea ice may undergo abrupt periods of loss during the next fifty years. Here, we evaluate how rapid sea ice loss affects terrestrial Arctic climate and ground thermal state in the Community Climate System Model. We find that simulated western Arctic land warming trends during rapid sea ice loss are 3.5 times greater than secular 21st century climate-change trends. The accelerated warming signal penetrates up to 1500 km inland and is apparent throughout most of the year, peaking in autumn. Idealized experiments using the Community Land Model, with improved permafrost dynamics, indicate that an accelerated warming period substantially increases ground heat accumulation. Enhanced heat accumulation leads to rapid degradation of warm permafrost and may increase the vulnerability of colder permafrost to degradation under continued warming. Taken together, these results imply a link between rapid sea ice loss and permafrost health.

  10. Experimental design and analysis for accelerated degradation tests with Li-ion cells.

    SciTech Connect

    Doughty, Daniel Harvey; Thomas, Edward Victor; Jungst, Rudolph George; Roth, Emanuel Peter

    2003-08-01

    This document describes a general protocol (involving both experimental and data analytic aspects) that is designed to be a roadmap for rapidly obtaining a useful assessment of the average lifetime (at some specified use conditions) that might be expected from cells of a particular design. The proposed experimental protocol involves a series of accelerated degradation experiments. Through the acquisition of degradation data over time specified by the experimental protocol, an unambiguous assessment of the effects of accelerating factors (e.g., temperature and state of charge) on various measures of the health of a cell (e.g., power fade and capacity fade) will result. In order to assess cell lifetime, it is necessary to develop a model that accurately predicts degradation over a range of the experimental factors. In general, it is difficult to specify an appropriate model form without some preliminary analysis of the data. Nevertheless, assuming that the aging phenomenon relates to a chemical reaction with simple first-order rate kinetics, a data analysis protocol is also provided to construct a useful model that relates performance degradation to the levels of the accelerating factors. This model can then be used to make an accurate assessment of the average cell lifetime. The proposed experimental and data analysis protocols are illustrated with a case study involving the effects of accelerated aging on the power output from Gen-2 cells. For this case study, inadequacies of the simple first-order kinetics model were observed. However, a more complex model allowing for the effects of two concurrent mechanisms provided an accurate representation of the experimental data.

  11. Limitations of predicting in vivo biostability of multiphase polyurethane elastomers using temperature-accelerated degradation testing.

    PubMed

    Padsalgikar, Ajay; Cosgriff-Hernandez, Elizabeth; Gallagher, Genevieve; Touchet, Tyler; Iacob, Ciprian; Mellin, Lisa; Norlin-Weissenrieder, Anna; Runt, James

    2015-01-01

    Polyurethane biostability has been the subject of intense research since the failure of polyether polyurethane pacemaker leads in the 1980s. Accelerated in vitro testing has been used to isolate degradation mechanisms and predict clinical performance of biomaterials. However, validation that in vitro methods reproduce in vivo degradation is critical to the selection of appropriate tests. High temperature has been proposed as a method to accelerate degradation. However, correlation of such data to in vivo performance is poor for polyurethanes due to the impact of temperature on microstructure. In this study, we characterize the lack of correlation between hydrolytic degradation predicted using a high temperature aging model of a polydimethylsiloxane-based polyurethane and its in vivo performance. Most notably, the predicted molecular weight and tensile property changes from the accelerated aging study did not correlate with clinical explants subjected to human biological stresses in real time through 5 years. Further, DMTA, ATR-FTIR, and SAXS experiments on samples aged for 2 weeks in PBS indicated greater phase separation in samples aged at 85°C compared to those aged at 37°C and unaged controls. These results confirm that microstructural changes occur at high temperatures that do not occur at in vivo temperatures. In addition, water absorption studies demonstrated that water saturation levels increased significantly with temperature. This study highlights that the multiphase morphology of polyurethane precludes the use of temperature accelerated biodegradation for the prediction of clinical performance and provides critical information in designing appropriate in vitro tests for this class of materials. PMID:24810790

  12. Accelerated Degradation of Aldicarb and Its Metabolites in Cotton Field Soils

    PubMed Central

    Lawrence, K. S.; Feng, Yucheng; Lawrence, G. W.; Burmester, C. H.; Norwood, S. H.

    2005-01-01

    The degradation of aldicarb, and the metabolites aldicarb sulfoxide and aldicarb sulfone, was evaluated in cotton field soils previously exposed to aldicarb. A loss of efficacy had been observed in two (LM and MS) of the three (CL) field soils as measured by R. reniformis population development and a lack of cotton yield response. Two soils were compared for the first test—one where aldicarb had been effective (CL) and the second where aldicarb had lost its efficacy (LM). The second test included all three soils: autoclaved, non-autoclaved and treated with aldicarb at 0.59 kg a.i./ha, or not treated with aldicarb. The degradation of aldicarb to aldicarb sulfoxide and then to aldicarb sulfone was measured using high-performance liquid chromatography (HPLC) in both tests. In test one, total degradation of aldicarb and its metabolites occurred within 12 days in the LM soil. Aldicarb sulfoxide and aldicarb sulfone were both present in the CL soil at the conclusion of the test at 42 days after aldicarb application. Autoclaving the LM and MS soils extended the persistence of the aldicarb metabolites as compared to the same soils not autoclaved. The rate of degradation was not changed when the CL natural soil was autoclaved. The accelerated degradation was due to more rapid degradation of aldicarb sulfoxide and appears to be biologically mediated. PMID:19262860

  13. Stuxnet Facilitates the Degradation of Polycomb Protein during Development.

    PubMed

    Du, Juan; Zhang, Junzheng; He, Tao; Li, Yajuan; Su, Ying; Tie, Feng; Liu, Min; Harte, Peter J; Zhu, Alan Jian

    2016-06-20

    Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. Here, we report a Drosophila gene, stuxnet (stx), that controls Pc protein stability. We find that heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Our biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; Stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. Our results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity. PMID:27326929

  14. α-Synuclein and protein degradation systems: a reciprocal relationship.

    PubMed

    Xilouri, Maria; Brekk, Oystein Rod; Stefanis, Leonidas

    2013-04-01

    An increasing wealth of data indicates a close relationship between the presynaptic protein alpha-synuclein and Parkinson's disease (PD) pathogenesis. Alpha-synuclein protein levels are considered as a major determinant of its neurotoxic potential, whereas secreted extracellular alpha-synuclein has emerged as an additional important factor in this regard. However, the manner of alpha-synuclein degradation in neurons remains contentious. Both the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP)-mainly macroautophagy and chaperone-mediated autophagy-have been suggested to contribute to alpha-synuclein turnover. Additionally, other proteases such as calpains, neurosin, and metalloproteinases have been also proposed to have a role in intracellular and extracellular alpha-synuclein processing. Both UPS and ALP activity decline with aging and such decline may play a pivotal role in many neurodegenerative conditions. Alterations in these major proteolytic pathways may result in alpha-synuclein accumulation due to impaired clearance. Conversely, increased alpha-synuclein protein burden promotes the generation of aberrant species that may impair further UPS or ALP function, generating thus a bidirectional positive feedback loop leading to neuronal death. In the current review, we summarize the recent findings related to alpha-synuclein degradation, as well as to alpha-synuclein-mediated aberrant effects on protein degradation systems. Identifying the factors that regulate alpha-synuclein association to cellular proteolytic pathways may represent potential targets for therapeutic interventions in PD and related synucleinopathies. PMID:22941029

  15. Investigation of accelerated stress factors and failure/degradation mechanisms in terrestrial solar cells

    NASA Technical Reports Server (NTRS)

    Lathrop, J. W.

    1983-01-01

    Results of an ongoing research program into the reliability of terrestrial solar cells are presented. Laboratory accelerated testing procedures are used to identify failure/degradation modes which are then related to basic physical, chemical, and metallurgical phenomena. In the most recent tests, ten different types of production cells, both with and without encapsulation, from eight different manufacturers were subjected to a variety of accelerated tests. Results indicated the presence of a number of hitherto undetected failure mechanisms, including Schottky barrier formation at back contacts and loss of adhesion of grid metallization. The mechanism of Schottky barrier formation is explained by hydrogen, formed by the dissociation of water molecules at the contact surface, diffusing to the metal semiconductor interface. This same mechanism accounts for the surprising increase in sensitivity to accelerated stress conditions that was observed in some cells when encapsulated.

  16. Control of protein function through regulated protein degradation: biotechnological and biomedical applications.

    PubMed

    Nagpal, Jyotsna; Tan, Ju Lin; Truscott, Kaye N; Heras, Begoña; Dougan, David A

    2013-01-01

    Targeted protein degradation is crucial for the correct function and maintenance of a cell. In bacteria, this process is largely performed by a handful of ATP-dependent machines, which generally consist of two components - an unfoldase and a peptidase. In some cases, however, substrate recognition by the protease may be regulated by specialized delivery factors (known as adaptor proteins). Our detailed understanding of how these machines are regulated to prevent uncontrolled degradation within a cell has permitted the identification of novel antimicrobials that dysregulate these machines, as well as the development of tunable degradation systems that have applications in biotechnology. Here, we focus on the physiological role of the ClpP peptidase in bacteria, its role as a novel antibiotic target and the use of protein degradation as a biotechnological approach to artificially control the expression levels of a protein of interest. PMID:23920496

  17. Attomole quantitation of protein separations with accelerator mass spectrometry

    SciTech Connect

    Vogel, J S; Grant, P G; Buccholz, B A; Dingley, K; Turteltaub, K W

    2000-12-15

    Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to sub-attomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5% . Micro-proton-induced-xray-emission quantifies elemental abundances in separated metalloprotein samples to nanogram amounts and is capable of quantifying phosphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.

  18. Degradable thermoresponsive nanogels for protein encapsulation and controlled release.

    PubMed

    Bhuchar, Neha; Sunasee, Rajesh; Ishihara, Kazuhiko; Thundat, Thomas; Narain, Ravin

    2012-01-18

    Reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used for the fabrication of stable core cross-linked micelles (CCL) with thermoresponsive and degradable cores. Well-defined poly(2-methacryloyloxyethyl phosphorylcholine), poly(MPC) macroRAFT agent, was first synthesized with narrow molecular weight distribution via the RAFT process. These CCL micelles (termed as nanogels) with hydrophilic poly(MPC) shell and thermoresponsive core consisting of poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM) and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA) were then obtained in a one-pot process by RAFT polymerization in the presence of an acid degradable cross-linker. These acid degradable nanogels were efficiently synthesized with tunable sizes and low polydispersities. The encapsulation efficiencies of the nanogels with different proteins such as insulin, BSA, and β-galactosidase were studied and found to be dependent of the cross-linker concentration, size of protein, and the cationic character of the nanogels imparted by the presence of AEMA in the core. The thermoresponsive nature of the synthesized nanogels plays a vital role in protein encapsulation: the hydrophilic core and shell of the nanogels at low temperature allow easy diffusion of the proteins inside out and, with an increase in temperature, the core becomes hydrophobic and the nanogels are easily separated out with entrapped protein. The release profile of insulin from nanogels at low pH was studied and results were analyzed using bicinchoninic assay (BCA). Controlled release of protein was observed over 48 h. PMID:22171688

  19. Protein/Protein Interactions in the Mammalian Heme Degradation Pathway

    PubMed Central

    Spencer, Andrea L. M.; Bagai, Ireena; Becker, Donald F.; Zuiderweg, Erik R. P.; Ragsdale, Stephen W.

    2014-01-01

    Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence. PMID:25196843

  20. Performance Degradation of Encapsulated Monocrystalline-Si Solar Cells upon Accelerated Weathering Exposures: Preprint

    SciTech Connect

    Glick, S. H.; Pern, F. J.; Watson, G. L.; Tomek, D.; Raaff, J.

    2001-10-01

    Presented at 2001 NCPV Program Review Meeting: Performed accelerated exposures to study performance reliability/materials degradation of encapsulated c-Si cells using weathering protocols in 2 weatherometers. We have performed accelerated exposures to study performance reliability and materials degradation of a total of forty-one 3-cm x 3-cm monocrystalline-Si (c-Si) solar cells that were variously encapsulated using accelerated weathering protocols in two weatherometers (WOMs), with and without front specimen water sprays. Laminated cells (EVA/c-Si/EVA, ethylene vinyl acetate) with one of five superstrate/substrate variations and other features including with and without: (i) load resistance, (ii) Al foil light masks, and (iii) epoxy edge-sealing were studied. Three additional samples, omitting EVA, were exposed under a full-spectrum solar simulator, or heated in an oven, for comparison. After exposures, cell performance decreased irregularly, but to a relatively greater extent for samples exposed in WOM where light, heat, and humidity cycles were present (solar simulator or oven lacked such cycles). EVA laminates in the samples masked with aluminum (Al) foils were observed to retain moisture in WOM with water spray. Moisture effects caused substantial efficiency losses probably related in part to increasing series resistance.

  1. A macromolecular delivery vehicle for protein-based vaccines: Acid-degradable protein-loaded microgels

    PubMed Central

    Murthy, Niren; Xu, Mingcheng; Schuck, Stephany; Kunisawa, Jun; Shastri, Nilabh; Fréchet, Jean M. J.

    2003-01-01

    The development of protein-based vaccines remains a major challenge in the fields of immunology and drug delivery. Although numerous protein antigens have been identified that can generate immunity to infectious pathogens, the development of vaccines based on protein antigens has had limited success because of delivery issues. In this article, an acid-sensitive microgel material is synthesized for the development of protein-based vaccines. The chemical design of these microgels is such that they degrade under the mildly acidic conditions found in the phagosomes of antigen-presenting cells (APCs). The rapid cleavage of the microgels leads to phagosomal disruption through a colloid osmotic mechanism, releasing protein antigens into the APC cytoplasm for class I antigen presentation. Ovalbumin was encapsulated in microgel particles, 200–500 nm in diameter, prepared by inverse emulsion polymerization with a synthesized acid-degradable crosslinker. Ovalbumin is released from the acid-degradable microgels in a pH-dependent manner; for example, microgels containing ovalbumin release 80% of their encapsulated proteins after 5 h at pH 5.0, but release only 10% at pH 7.4. APCs that phagocytosed the acid-degradable microgels containing ovalbumin were capable of activating ovalbumin-specific cytoxic T lymphocytes. The acid-degradable microgels developed in this article should therefore find applications as delivery vehicles for vaccines targeted against viruses and tumors, where the activation of cytoxic T lymphocytes is required for the development of immunity. PMID:12704236

  2. Phytoferritin association induced by EGCG inhibits protein degradation by proteases.

    PubMed

    Wang, Aidong; Zhou, Kai; Qi, Xin; Zhao, Guanghua

    2014-12-01

    Phytoferritin is a promising resource of non-heme iron supplementation, but it is not stable against degradation by proteases in the gastrointestinal tract. Therefore, how to improve the stability of ferritin in the presence of proteases is a challenge. Since (-)-epigallocatechin-3-gallate (EGCG) is rich in phenolic-hydroxyl groups, it could interact with ferritin through hydrogen bonds, thereby preventing protein from degradation. To confirm this idea, we focus on the interaction between EGCG and phytoferritin, and the consequence of such interaction. Results demonstrated that EGCG did interact with ferritin, and such interaction induced the change in the tertiary/quaternary structure of protein but not in its secondary structure. Furthermore, stopped-flow and dynamic light scattering (DLS) results showed that EGCG could trigger ferritin association. Consequently, such protein association markedly inhibited protein digestion by pepsin at pH 4.0 and by trypsin at pH 7.5. These findings raise the possibility to improve the stability of phytoferritin in the presence of proteases. PMID:25384342

  3. Effect of Rapid Chilling on Beef Quality and Cytoskeletal Protein Degradation in M. longissimus of Chinese Yellow Crossbred Bulls

    PubMed Central

    Mao, Yanwei; Zhang, Yimin; Liang, Rongrong; Ren, Lulu; Zhu, He; Li, Ke; Zhu, Lixian; Luo, Xin

    2012-01-01

    The objective of this study was to investigate the effect of rapid chilling (RC) on beef quality and the degradation of cytoskeletal proteins. Twenty Chinese Yellow crossbred bulls were selected and randomly divided into two groups. RC and conventional chilling (CC) were applied to left and right sides of the carcasses respectively after slaughtering. To determine whether electrical stimulation (ES) treatment can alleviate the potential hazard of RC on meat quality, ES was applied to one group. The effects of RC and ES were determined by meat color, shear force and cytoskeletal protein degradation postmortem (PM). The results showed that RC decreased beef tenderness at 1 d and 3 d postmortem, but had no detrimental effect on meat color. Western blotting showed that RC decreased the degradation rate of desmin and troponin-T, but the effects weakened gradually as postmortem aging extended. Degradation rates of both desmin and troponin-T were accelerated by ES. The combination of RC and ES could improve beef color, accelerate degradation rate of cytoskeletal protein and improve beef tenderness. PMID:25049681

  4. Fluctuation Flooding Method (FFM) for accelerating conformational transitions of proteins

    NASA Astrophysics Data System (ADS)

    Harada, Ryuhei; Takano, Yu; Shigeta, Yasuteru

    2014-03-01

    A powerful conformational sampling method for accelerating structural transitions of proteins, "Fluctuation Flooding Method (FFM)," is proposed. In FFM, cycles of the following steps enhance the transitions: (i) extractions of largely fluctuating snapshots along anisotropic modes obtained from trajectories of multiple independent molecular dynamics (MD) simulations and (ii) conformational re-sampling of the snapshots via re-generations of initial velocities when re-starting MD simulations. In an application to bacteriophage T4 lysozyme, FFM successfully accelerated the open-closed transition with the 6 ns simulation starting solely from the open state, although the 1-μs canonical MD simulation failed to sample such a rare event.

  5. Design Principles of a Universal Protein Degradation Machine

    PubMed Central

    Matyskiela, Mary E.; Martin, Andreas

    2012-01-01

    The 26S proteasome is a 2.5 MDa, 32-subunit ATP-dependent protease that is responsible for the degradation of ubiquitinated protein targets in all eukaryotic cells. This proteolytic machine consists of a barrel-shaped peptidase capped by a large regulatory particle, which contains a heterohexameric AAA+ unfoldase as well as several structural modules of previously unknown function. Recent electron microscopy studies have allowed major breakthroughs in understanding the architecture of the regulatory particle, revealing that the additional modules provide a structural framework to position critical, ubiquitin-interacting subunits and thus allow the 26S proteasome to function as a universal degradation machine for a wide variety of protein substrates. The electron microscopy studies have also uncovered surprising asymmetries in the spatial arrangement of proteasome subunits, yet the functional significance of these architectural features remains unclear. This review will summarize the recent findings on 26S proteasome structure and discuss the mechanistic implications for substrate binding, deubiquitination, unfolding, and degradation. PMID:23147216

  6. Effect of cyclodextrin derivation and amorphous state of complex on accelerated degradation of ziprasidone.

    PubMed

    Hong, Jinyang; Shah, Jaymin C; Mcgonagle, Maura D

    2011-07-01

    Inclusion complexes of ziprasidone with several β-cyclodextrins [β-CDs; sulfobutylether-β-cyclodextrins (SBEβCD), hydroxypropyl-β-cyclodextrins (HPβCD), methyl-β-cyclodextrins (MβCD), and carboxyethyl-β-cyclodextrins (CEβCD)] were prepared and solution stability was evaluated at elevated temperature. Solid-state stability was assessed by subjecting various CD complexes of ziprasidone, spray-dried dispersion (SDD), partially crystalline ziprasidone-SBEβCD salts, and the physical mixture of ziprasidone-SBEβCD to γ-irradiation. Degradant I was formed by oxidation of ziprasidone, which upon aldol condensation with ziprasidone formed degradant II in both solution and solid states. In the solution state, CD complexes with electron-donating side chains, such as SBEβCD and CEβCD, produced the highest oxidative degradation followed by HPβCD with 6, 3, and 4 degrees of substitution. In the solid state, crystalline drug substance and physical mixture of crystalline drug-SBEβCD showed very little to no degradation. In contrast, amorphous βCD, MβCD, CEβCD, and SBEβCD complexes as well as the amorphous SDD exhibited greatest extent of oxidative degradation. Results suggest that electron-donating side chains of the derivatized CD interact with transition state of the oxidation reaction and catalyze drug degradation in solution, However, higher mobility in the amorphous state of CD-drug complexes promoted chemical instability of ziprasidone under accelerated conditions irrespective of the chemical nature of the side chain on CD. PMID:21283987

  7. Effects of UV on power degradation of photovoltaic modules in combined acceleration tests

    NASA Astrophysics Data System (ADS)

    Ngo, Trang; Heta, Yushi; Doi, Takuya; Masuda, Atsushi

    2016-05-01

    UV exposure and other factors such as high/low temperature, humidity and mechanical stress have been reported to degrade photovoltaic (PV) module materials. By focusing on the combined effects of UV stress and moisture on PV modules, two new acceleration tests of light irradiation and damp heat (DH) were designed and conducted. The effects of UV exposure were validated through a change in irradiation time (UV dosage) and a change of the light irradiation side (glass side vs backsheet side) in the UV-preconditioned DH and cyclic sequential tests, respectively. The chemical corrosion of finger electrodes in the presence of acetic acid generated from ethylene vinyl acetate used as an encapsulant was considered to be the main origin of degradation. The module performance characterized by electroluminescence images was confirmed to correlate with the measured acetic acid concentration and Ag finger electrode resistance.

  8. Investigation of accelerated stress factors and failure/degradation mechanisms in terrestrial solar cells

    NASA Technical Reports Server (NTRS)

    Lathrop, J. W.

    1984-01-01

    Research on the reliability of terrestrial solar cells was performed to identify failure/degradation modes affecting solar cells and to relate these to basic physical, chemical, and metallurgical phenomena. Particular concerns addressed were the reliability attributes of individual single crystalline, polycrystalline, and amorphous thin film silicon cells. Results of subjecting different types of crystalline cells to the Clemson accelerated test schedule are given. Preliminary step stress results on one type of thin film amorphous silicon (a:Si) cell indicated that extraneous degradation modes were introduced above 140 C. Also described is development of measurement procedures which are applicable to the reliability testing of a:Si solar cells as well as an approach to achieving the necessary repeatability of fabricating a simulated a:Si reference cell from crystalline silicon photodiodes.

  9. Intracellular protein degradation in mammalian cells: recent developments.

    PubMed

    Knecht, Erwin; Aguado, Carmen; Cárcel, Jaime; Esteban, Inmaculada; Esteve, Juan Miguel; Ghislat, Ghita; Moruno, José Félix; Vidal, José Manuel; Sáez, Rosana

    2009-08-01

    In higher organisms, dietary proteins are broken down into amino acids within the digestive tract but outside the cells, which incorporate the resulting amino acids into their metabolism. However, under certain conditions, an organism loses more nitrogen than is assimilated in the diet. This additional loss was found in the past century to come from intracellular proteins and started an intensive research that produced an enormous expansion of the field and a dispersed literature. Therefore, our purpose is to provide an updated summary of the current knowledge on the proteolytic machinery involved in intracellular protein degradation and its physiological and pathological relevance, especially addressed to newcomers in the field who may find further details in more specialized reviews. However, even providing a general overview, this is an extremely wide field and, therefore, we mainly focus on mammalian cells, while other cells will be mentioned only for comparison purposes. PMID:19399586

  10. Synthesis and protein degradation capacity of photoactivated enediynes.

    PubMed

    Fouad, Farid S; Wright, Justin M; Plourde, Gary; Purohit, Ajay D; Wyatt, Justin K; El-Shafey, Ahmed; Hynd, George; Crasto, Curtis F; Lin, Yiqing; Jones, Graham B

    2005-11-25

    [structure: see text] The viability of proteins as targets of thermally and photoactivated enediynes has been confirmed at the molecular level. Model studies using a labeled substrate confirmed the efficacy of atom transfer from diyl radicals produced from enediynes to form captodatively stabilized carbon centered aminoacyl radicals, which then undergo either fragmentation or dimerization. To exploit this finding, a family of enediynes was developed using an intramolecular coupling strategy. Derivatives were prepared and used to target specific proteins, showing good correlation between affinity and photoinduced protein degrading activity. The findings have potential applications in the design of artificial chemical proteases and add to our understanding of the mechanism of action of the clinically important enediyne antitumor antibiotics. PMID:16292807

  11. Fructose Accelerates UV-C Induced Photochemical Degradation of Pentachlorophenol in Low and High Salinity Water.

    PubMed

    Nayak, Shaila; O'Donnell, Sean-Erik; Sales, Christopher M; Tikekar, Rohan V

    2016-06-01

    A novel process involving 254 nm UV-C and fructose to degrade pentachlorophenol (PCP), a pollutant, in low and high salinity (0-10 g/L salt) solutions is presented. The first order rate constants in the presence of 0, 300, and 500 mM fructose were 0.23 ± 0.04, 0.54 ± 0.01, and 1.18 ± 0.03 min(-1), respectively. Experimental evidence has shown generation of hydrogen peroxide and singlet oxygen from the UV-C exposure of fructose, which may have accelerated PCP degradation. Although salts (sodium, potassium, and calcium chloride, 1101:6.4:1) are expected to enhance the degradation rate due to generation of reactive halide species (RHS) from exposure to UV-C light, 10 g/L salt decreased the degradation rates in both the absence and presence of fructose. An LC-ESI-MS spectrum of the reaction mixture revealed a high relative abundance at m/z of 215 that corresponds to a fructose-chlorine adduct, indicating that fructose may have scavenged these RHS and prevented their reaction with PCP. PMID:27160945

  12. The cell-in-series method: A technique for accelerated electrode degradation in redox flow batteries

    DOE PAGESBeta

    Pezeshki, Alan M.; Sacci, Robert L.; Veith, Gabriel M.; Zawodzinski, Thomas A.; Mench, Matthew M.

    2015-11-21

    Here, we demonstrate a novel method to accelerate electrode degradation in redox flow batteries and apply this method to the all-vanadium chemistry. Electrode performance degradation occurred seven times faster than in a typical cycling experiment, enabling rapid evaluation of materials. This method also enables the steady-state study of electrodes. In this manner, it is possible to delineate whether specific operating conditions induce performance degradation; we found that both aggressively charging and discharging result in performance loss. Post-mortem x-ray photoelectron spectroscopy of the degraded electrodes was used to resolve the effects of state of charge (SoC) and current on the electrodemore » surface chemistry. For the electrode material tested in this work, we found evidence that a loss of oxygen content on the negative electrode cannot explain decreased cell performance. Furthermore, the effects of decreased electrode and membrane performance on capacity fade in a typical cycling battery were decoupled from crossover; electrode and membrane performance decay were responsible for a 22% fade in capacity, while crossover caused a 12% fade.« less

  13. The cell-in-series method: A technique for accelerated electrode degradation in redox flow batteries

    SciTech Connect

    Pezeshki, Alan M.; Sacci, Robert L.; Veith, Gabriel M.; Zawodzinski, Thomas A.; Mench, Matthew M.

    2015-11-21

    Here, we demonstrate a novel method to accelerate electrode degradation in redox flow batteries and apply this method to the all-vanadium chemistry. Electrode performance degradation occurred seven times faster than in a typical cycling experiment, enabling rapid evaluation of materials. This method also enables the steady-state study of electrodes. In this manner, it is possible to delineate whether specific operating conditions induce performance degradation; we found that both aggressively charging and discharging result in performance loss. Post-mortem x-ray photoelectron spectroscopy of the degraded electrodes was used to resolve the effects of state of charge (SoC) and current on the electrode surface chemistry. For the electrode material tested in this work, we found evidence that a loss of oxygen content on the negative electrode cannot explain decreased cell performance. Furthermore, the effects of decreased electrode and membrane performance on capacity fade in a typical cycling battery were decoupled from crossover; electrode and membrane performance decay were responsible for a 22% fade in capacity, while crossover caused a 12% fade.

  14. Accelerated aromatic compounds degradation in aquatic environment by use of interaction between Spirodela polyrrhiza and bacteria in its rhizosphere.

    PubMed

    Toyama, Tadashi; Yu, Ning; Kumada, Hirohide; Sei, Kazunari; Ike, Michihiko; Fujita, Masanori

    2006-04-01

    Accelerated degradation of organic chemicals by aquatic plant-bacterial associations was reported for the first time with elucidation of the role and contribution of aquatic plant and bacteria in its rhizosphere using a fast-growing giant duckweed, Spirodela polyrrhiza. The results clearly showed the accelerated degradation of all the three aromatic compounds (phenol, aniline and 2,4-dichlorophenol [2,4-DCP]) tested by aquatic plant-bacterial associations. In phenol degradation system, phenol-degrading bacteria indigenous to the rhizosphere fraction of S. polyrrhiza mainly contributed, while in aniline degradation system S. polyrrhiza mainly contributed by stimulating aniline-degrading bacteria both in the rhizosphere and balk water fraction. On the other hand in 2,4-DCP degradation system, S. polyrrhiza itself mainly contributed to its removal by uptake and degradation. Thus, the mechanisms for accelerated removal of aromatic compounds were quite different depending on the substrates. S. polyrrhiza showed selective accumulation of phenol-degrading bacteria in its rhizosphere fraction, while aniline- and 2,4-DCP-degrading bacteria were not much accumulated. S. polyrrhiza secreted peroxidase and laccase. However, both of the enzymatic activities increased with the addition of aromatic compounds, degrading ability of S. polyrrhiza itself should be owing to the production of peroxidase rather than laccase because the change of peroxidase activity and concentration of each aromatic compound well concurred. From the results obtained in the present study, it can be concluded that the feasibility of the use of aquatic plant-bacterial associations to accelerate the degradation of organic chemicals especially recalcitrant compounds in aquatic environment was shown. PMID:16716944

  15. Acceleration of degradation by highly accelerated stress test and air-included highly accelerated stress test in crystalline silicon photovoltaic modules

    NASA Astrophysics Data System (ADS)

    Suzuki, Soh; Tanahashi, Tadanori; Doi, Takuya; Masuda, Atsushi

    2016-02-01

    We examined the effects of hyper-hygrothermal stresses with or without air on the degradation of crystalline silicon (c-Si) photovoltaic (PV) modules, to shorten the required duration of a conventional hygrothermal-stress test [i.e., the “damp heat (DH) stress test”, which is conducted at 85 °C/85% relative humidity for 1,000 h]. Interestingly, the encapsulant within a PV module becomes discolored under the air-included hygrothermal conditions achieved using DH stress test equipment and an air-included highly accelerated stress test (air-HAST) apparatus, but not under the air-excluded hygrothermal conditions realized using a highly accelerated stress test (HAST) machine. In contrast, the reduction in the output power of the PV module is accelerated irrespective of air inclusion in hyper-hygrothermal test atmosphere. From these findings, we conclude that the required duration of the DH stress test will at least be significantly shortened using air-HAST, but not HAST.

  16. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers.

    PubMed

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-15

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. PMID:26609072

  17. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

    PubMed Central

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K.; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-01

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. PMID:26609072

  18. Effects of manipulation of the caspase system on myofibrillar protein degradation in vitro.

    PubMed

    Kemp, C M; Wheeler, T L

    2011-10-01

    Apoptosis via the intrinsic caspase 9 pathway can be induced by oxidative stressors hydrogen peroxide (H₂O₂) and N-(4 hydroxyphenol) rentinamide (fenretinide), a synthetic retinoid. Accelerated muscle atrophy and proteolysis in muscle-wasting conditions have been linked to oxidative stress and activated protease systems. Therefore, the hypothesis of this study was that proteolysis of myofibrillar proteins could be manipulated through the induction or inhibition of the caspase system. After slaughter, LM and supraspinatus muscles from callipyge (n = 5) and normal (n = 3) lambs were excised, finely diced, and incubated with treatment buffers containing oxidative stressors fenretinide or H₂O₂, recombinant caspase 3, caspase-specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (DEVD), or control solution. Muscle samples were incubated for 1, 2, 7, and 21 d at 4°C. Activation of the initiator caspase, caspase 9, and myofibrillar protein degradation was determined by SDS-PAGE and Western blotting. Results showed that fenretinide, H₂O₂, and recombinant caspase 3 increased (P < 0.05) proteolysis of myofibril proteins, whereas DEVD inhibited degradation (P < 0.05). Proteolysis of myofibrillar proteins increased with incubation time (P < 0.0001), and incubation time × treatment interactions (P < 0.05) indicated that the treatment effects did not all occur at the same rate. This study has shown that manipulation of the caspase system through induction or inhibition of activity can affect degradation of myofibrillar proteins, providing further evidence that the caspase system could be involved in postmortem proteolysis and tenderization. However, these stimulated changes were not sufficient to overcome the lack of proteolysis that is characteristic of muscle from callipyge lambs. PMID:21622882

  19. Irradiation imposed degradation of the mechanical and electrical properties of electrical insulation for future accelerator magnets

    SciTech Connect

    Polinski, J.; Chorowski, M.; Bogdan, P.; Strychalski, M.; Rijk, G. de

    2014-01-27

    Future accelerators will make extensive use of superconductors made of Nb{sub 3}Sn, which allows higher magnetic fields than NbTi. However, the wind-and-react technology of Nb{sub 3}Sn superconducting magnet production makes polyimide Kapton® non applicable for the coils' electrical insulation. A Nb{sub 3}Sn technology compatible insulation material should be characterized by high radiation resistivity, good thermal conductivity, and excellent mechanical properties. Candidate materials for the electrical insulation of future accelerator's magnet coils have to be radiation certified with respect to potential degradation of their electrical, thermal, and mechanical properties. This contribution presents procedures and results of tests of the electrical and mechanical properties of DGEBA epoxy + D400 hardener, which is one of the candidates for the electrical insulation of future magnets. Two test sample types have been used to determine the material degradation due to irradiation: a untreated one (unirradiated) and irradiated at 77 K with 11 kGy/min intense, 4MeV energy electrons beam to a total dose of 50 MGy.

  20. Evidence of oleuropein degradation by olive leaf protein extract.

    PubMed

    De Leonardis, Antonella; Macciola, Vincenzo; Cuomo, Francesca; Lopez, Francesco

    2015-05-15

    The enzymatic activity of raw protein olive leaf extract has been investigated in vivo, on olive leaf homogenate and, in vitro with pure oleuropein and other phenolic substrates. At least two types of enzymes were found to be involved in the degradation of endogenous oleuropein in olive leaves. As for the in vitro experiments, the presence of active polyphenoloxidase and β-glucosidase was determined by HPLC and UV-Visible spectroscopy. Interestingly, both the enzymatic activities were found to change during the storage of olive leaves. Specifically, the protein extracts obtained from fresh leaves showed the presence of both the enzymatic activities, because oleuropein depletion occurred simultaneously with the formation of the oleuropein aglycon, 3,4-DHPEA-EA. In comparison leaves subjected to the drying process showed a polyphenoloxidase activity leading exclusively to the formation of oxidation products responsible for the typical brown coloration of the reaction solution. PMID:25577121

  1. Protein phosphatase 1 suppresses androgen receptor ubiquitylation and degradation.

    PubMed

    Liu, Xiaming; Han, Weiwei; Gulla, Sarah; Simon, Nicholas I; Gao, Yanfei; Cai, Changmeng; Yang, Hongmei; Zhang, Xiaoping; Liu, Jihong; Balk, Steven P; Chen, Shaoyong

    2016-01-12

    The phosphoprotein phosphatases are emerging as important androgen receptor (AR) regulators in prostate cancer (PCa). We reported previously that the protein phosphatase 1 catalytic subunit (PP1α) can enhance AR activity by dephosphorylating a site in the AR hinge region (Ser650) and thereby decrease AR nuclear export. In this study we show that PP1α increases the expression of wildtype as well as an S650A mutant AR, indicating that it is acting through one or more additional mechanisms. We next show that PP1α binds primarily to the AR ligand binding domain and decreases its ubiquitylation and degradation. Moreover, we find that the PP1α inhibitor tautomycin increases phosphorylation of AR ubiquitin ligases including SKP2 and MDM2 at sites that enhance their activity, providing a mechanism by which PP1α may suppress AR degradation. Significantly, the tautomycin mediated decrease in AR expression was most pronounced at low androgen levels or in the presence of the AR antagonist enzalutamide. Consistent with this finding, the sensitivity of LNCaP and C4-2 PCa cells to tautomycin, as assessed by PSA synthesis and proliferation, was enhanced at low androgen levels or by treatment with enzalutamide. Together these results indicate that PP1α may contribute to stabilizing AR protein after androgen deprivation therapies, and that targeting PP1α or the AR-PP1α interaction may be effective in castration-resistant prostate cancer (CRPC). PMID:26636645

  2. Induced oligomerization targets Golgi proteins for degradation in lysosomes

    PubMed Central

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D.

    2015-01-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130’s cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. PMID:26446839

  3. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    PubMed

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. PMID:26446839

  4. Chemical methods for degradation of target proteins using designed light-activatable organic molecules.

    PubMed

    Tanimoto, Shuho; Takahashi, Daisuke; Toshima, Kazunobu

    2012-08-11

    Molecular design, chemical synthesis, and biological evaluation of several designed organic molecules, which target-selectively degrade proteins upon photo-irradiation, are introduced. The designed molecules for protein photo-degradation include 2-phenylquinoline-steroid hormone hybrids and porphyrin derivatives, both of which selectively photo-degrade estrogen receptor-α, and fullerene-sugar and -sulfonic acid hybrids, which selectively photo-degrade HIV-1 protease and amyloid β, respectively. The information will provide a novel and effective way to control specific functions of proteins, and contribute to the molecular design of novel protein photo-degrading agents, which should find wide application in chemistry, biology, and medicine. PMID:22739361

  5. Cyclophilin A Restricts Influenza A Virus Replication through Degradation of the M1 Protein

    PubMed Central

    Xu, Chongfeng; Sun, Lei; Chen, Jilong; Zhang, Lianfeng; Liu, Wenjun

    2012-01-01

    Cyclophilin A (CypA) is a typical member of the cyclophilin family of peptidyl-prolyl isomerases and is involved in the replication of several viruses. Previous studies indicate that CypA interacts with influenza virus M1 protein and impairs the early stage of the viral replication. To further understand the molecular mechanism by which CypA impairs influenza virus replication, a 293T cell line depleted for endogenous CypA was established. The results indicated that CypA inhibited the initiation of virus replication. In addition, the infectivity of influenza virus increased in the absence of CypA. Further studies indicated that CypA had no effect on the stages of virus genome replication or transcription and also did not impair the nuclear export of the viral mRNA. However, CypA decreased the viral protein level. Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway. Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein. PMID:22347431

  6. Cytokinin inhibits the proteasome-mediated degradation of carbonylated proteins in Arabidopsis leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Under normal conditions, plants contain numerous carbonylated proteins, which are thought to be indicative of oxidative stress damage. Conditions that promote formation of reactive oxygen species (ROS) enhance protein carbonylation, and protein degradation is required to reverse the damage. However,...

  7. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins

    PubMed Central

    Ju Shin, Yeong; Kyun Park, Seung; Jung Jung, Yoo; Na Kim, Ye; Sung Kim, Ki; Kyu Park, Ok; Kwon, Seung-Hae; Ho Jeon, Sung; Trinh, Le A.; Fraser, Scott E.; Kee, Yun; Joon Hwang, Byung

    2015-01-01

    Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein. PMID:26373678

  8. Lack of response to addition of degradable protein to a low protein diet fed to midlactation dairy cows.

    PubMed

    Armentano, L E; Bertics, S J; Riesterer, J

    1993-12-01

    Midlactation Holstein cows (n = 24, 12 primiparous) were subjected to four dietary treatments arranged in six Latin squares. Cows were assigned to squares according to parity and previous production within parity. Diets contained 53 to 55% DM from corn silage, and the remaining DM was from concentrates. The basal diet contained 13.9% CP with 9.5% rumen-degraded and 4.4% undegraded intake protein. Three other treatments were formulated to give one diet with more degradable true protein than the basal (11.9% degraded intake protein, 4.3% undegraded intake protein); another with urea added to the basal (12.2% degraded intake protein, 4.5% undegraded intake protein); and a third with additional undegraded protein added to the basal (8.3% degraded intake protein, 7.2% undegraded intake protein). Milk fat and protein concentration were unaffected by diet in all squares. Only the highest producing cows responded significantly to increased undegraded intake protein; milk production was 30.8, 30.9, 31.6, and 33.2 kg/d for basal, added degradable true protein, added urea, and added undegraded protein, respectively. Corresponding protein productions were 913, 929, 927, and 1004 g/d for these cows. Neither degradable true protein nor degradable N increased milk production in the highest producing cows, suggesting that microbial protein production was not limited by the amount of degradable protein in the basal diet. For midlactation, multiparous, and primiparous cows producing < 30 and 25 kg of milk/d, respectively, the protein content of the basal ration appeared to be adequate. PMID:8132882

  9. HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress

    NASA Astrophysics Data System (ADS)

    Zhang, Liyong; Chen, Xin; Sharma, Parveen; Moon, Mark; Sheftel, Alex D.; Dawood, Fayez; Nghiem, Mai P.; Wu, Jun; Li, Ren-Ke; Gramolini, Anthony O.; Sorensen, Poul H.; Penninger, Josef M.; Brumell, John H.; Liu, Peter P.

    2014-03-01

    The HECT E3 ubiquitin ligase HACE1 is a tumour suppressor known to regulate Rac1 activity under stress conditions. HACE1 is increased in the serum of patients with heart failure. Here we show that HACE1 protects the heart under pressure stress by controlling protein degradation. Hace1 deficiency in mice results in accelerated heart failure and increased mortality under haemodynamic stress. Hearts from Hace1-/- mice display abnormal cardiac hypertrophy, left ventricular dysfunction, accumulation of LC3, p62 and ubiquitinated proteins enriched for cytoskeletal species, indicating impaired autophagy. Our data suggest that HACE1 mediates p62-dependent selective autophagic turnover of ubiquitinated proteins by its ankyrin repeat domain through protein-protein interaction, which is independent of its E3 ligase activity. This would classify HACE1 as a dual-function E3 ligase. Our finding that HACE1 has a protective function in the heart in response to haemodynamic stress suggests that HACE1 may be a potential diagnostic and therapeutic target for heart disease.

  10. Nudix hydrolases degrade protein-conjugated ADP-ribose.

    PubMed

    Daniels, Casey M; Thirawatananond, Puchong; Ong, Shao-En; Gabelli, Sandra B; Leung, Anthony K L

    2015-01-01

    ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD(+) to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR. PMID:26669448

  11. Nudix hydrolases degrade protein-conjugated ADP-ribose

    PubMed Central

    Daniels, Casey M.; Thirawatananond, Puchong; Ong, Shao-En; Gabelli, Sandra B.; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP—the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR. PMID:26669448

  12. Amyloid precursor protein modulates β-catenin degradation

    PubMed Central

    Chen, Yuzhi; Bodles, Angela M

    2007-01-01

    Background The amyloid precursor protein (APP) is genetically associated with Alzheimer's disease (AD). Elucidating the function of APP should help understand AD pathogenesis and provide insights into therapeutic designs against this devastating neurodegenerative disease. Results We demonstrate that APP expression in primary neurons induces β-catenin phosphorylation at Ser33, Ser37, and Thr41 (S33/37/T41) residues, which is a prerequisite for β-catenin ubiquitinylation and proteasomal degradation. APP-induced phosphorylation of β-catenin resulted in the reduction of total β-catenin levels, suggesting that APP expression promotes β-catenin degradation. In contrast, treatment of neurons with APP siRNAs increased total β-catenin levels and decreased β-catenin phosphorylation at residues S33/37/T41. Further, β-catenin was dramatically increased in hippocampal CA1 pyramidal cells from APP knockout animals. Acute expression of wild type APP or of familial AD APP mutants in primary neurons downregulated β-catenin in membrane and cytosolic fractions, and did not appear to affect nuclear β-catenin or β-catenin-dependent transcription. Conversely, in APP knockout CA1 pyramidal cells, accumulation of β-catenin was associated with the upregulation of cyclin D1, a downstream target of β-catenin signaling. Together, these data establish that APP downregulates β-catenin and suggest a role for APP in sustaining neuronal function by preventing cell cycle reactivation and maintaining synaptic integrity. Conclusion We have provided strong evidence that APP modulates β-catenin degradation in vitro and in vivo. Future studies may investigate whether APP processing is necessary for β-catenin downregulation, and determine if excessive APP expression contributes to AD pathogenesis through abnormal β-catenin downregulation. PMID:18070361

  13. Reliability and Lifetime Prediction of Remote Phosphor Plates in Solid-State Lighting Applications Using Accelerated Degradation Testing

    NASA Astrophysics Data System (ADS)

    Mehr, M. Yazdan; van Driel, W. D.; Zhang, G. Q.

    2016-01-01

    A methodology, based on accelerated degradation testing, is developed to predict the lifetime of remote phosphor plates used in solid-state lighting (SSL) applications. Both thermal stress and light intensity are used to accelerate degradation reaction in remote phosphor plates. A reliability model, based on the Eyring relationship, is also developed in which both acceleration factors (light intensity and temperature) are incorporated. Results show that the developed methodology leads to a significant decay of the luminous flux, correlated colour temperature (CCT) and chromatic properties of phosphor plates within a practically reasonable period of time. The combination of developed acceleration testing and a generalized Eyring equation-based reliability model is a very promising methodology which can be applied in the SSL industry.

  14. Requirement for alanine in the amino acid control of deprivation-induced protein degradation in liver.

    PubMed Central

    Pösö, A R; Mortimore, G E

    1984-01-01

    Protein degradation in liver is actively controlled by a small group of inhibitory amino acids--leucine, tyrosine (or phenylalanine), glutamine, proline, histidine, tryptophan, and methionine. Other evidence, however, suggests that one or more of the remaining 12 noninhibitory amino acids is also required for suppression of proteolysis at normal concentrations. This question was investigated in livers of fed rats perfused in the single-pass mode. The deletion of alanine at normal (1x), but not at 4x or 10x normal, plasma amino acid concentrations evoked a near-maximal acceleration of protein degradation. No other noninhibitory amino acid was effective. Because alanine alone was not directly inhibitory and its omission was not associated with a decrease in inhibitory amino acid pools, alanine was presumed to act as a coregulator in the expression of inhibitory activity. When tested alone, the inhibitory group was as effective as the complete mixture at 0.5x and 4x levels, but it lost its suppressive ability within a narrow zone of concentration centered slightly above 1x. The addition of 1x (0.48 mM) alanine completely restored the inhibition. Pyruvate and lactate could be effectively substituted, but only at concentrations 10-20 times greater than that of alanine. These, together with earlier findings, indicate the existence of a regulatory complex that recognizes specific amino acids and transmits positive and negative signals to proteolytic sites. The results also suggest that alanine can provide an important regulatory link between energy demands and protein degradation. PMID:6589593

  15. Accelerated chemical synthesis of peptides and small proteins

    PubMed Central

    Miranda, Les P.; Alewood, Paul F.

    1999-01-01

    The chemical synthesis of peptides and small proteins is a powerful complementary strategy to recombinant protein overexpression and is widely used in structural biology, immunology, protein engineering, and biomedical research. Despite considerable improvements in the fidelity of peptide chain assembly, side-chain protection, and postsynthesis analysis, a limiting factor in accessing polypeptides containing greater than 50 residues remains the time taken for chain assembly. The ultimate goal of this work is to establish highly efficient chemical procedures that achieve chain-assembly rates of approximately 10–15 residues per hour, thus underpinning the rapid chemical synthesis of long polypeptides and proteins, including cytokines, growth factors, protein domains, and small enzymes. Here we report Boc chemistry that employs O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU)/dimethyl sulfoxide in situ neutralization as the coupling agent and incorporates a protected amino acid residue every 5 min to produce peptides of good quality. This rapid coupling chemistry was successfully demonstrated by synthesizing several small to medium peptides, including the “difficult” C-terminal sequence of HIV-1 proteinase (residues 81–99); fragment 65–74 of the acyl carrier protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor antagonist; and the pro-inflammatory chemotactic protein CP10, an 88-residue protein, by means of native chemical ligation. The benefits of this approach include enhanced ability to identify and characterize “difficult couplings,” rapid access to peptides for biological and structure–activity studies, and accelerated synthesis of tailored large peptide segments (<50 residues) for use in chemoselective ligation methods. PMID:9989998

  16. Internal loop photo-biodegradation reactor used for accelerated quinoline degradation and mineralization.

    PubMed

    Chang, Ling; Zhang, Yongming; Gan, Lu; Xu, Hua; Yan, Ning; Liu, Rui; Rittmann, Bruce E

    2014-07-01

    Biofilm biodegradation was coupled with ultra-violet photolysis using the internal loop photobiodegradation reactor for degradation of quinoline. Three protocols-photolysis alone (P), biodegradation alone (B), and intimately coupled photolysis and biodegradation (P&B)-were used for degradation of quinoline in batch and continuous-flow experiments. For a 1,000 mg/L initial quinoline concentration, the volumetric removal rate for quinoline was 38 % higher with P&B than with B in batch experiments, and the P&B kinetics were the sum of kinetics from the P and B experiments. Continuous-flow experiments with an influent quinoline concentration of 1,000 mg/L also gave significantly greater quinoline removal in P&B, and the quinoline-removal kinetics for P&B were approximately equal to the sum of the removal kinetics for P and B. P&B similarly increased the rate and extent of quinoline mineralization, for which the kinetics for P&B were nearly equal to the sum of kinetics for P and B. These findings support that the rate-limiting step for mineralization was transformation of quinoline, which was accelerated by the simultaneous action of photolysis and biodegradation. PMID:24488551

  17. Visualization of TlBr ionic transport mechanism by the Accelerated Device Degradation technique

    NASA Astrophysics Data System (ADS)

    Datta, Amlan; Becla, Piotr; Motakef, Shariar

    2015-06-01

    Thallium Bromide (TlBr) is a promising gamma radiation semiconductor detector material. However, it is an ionic semiconductor and suffers from polarization. As a result, TlBr devices degrade rapidly at room temperature. Polarization is associated with the flow of ionic current in the crystal under electrical bias, leading to the accumulation of charged ions at the device's electrical contacts. We report a fast and reliable direct characterization technique to identify the effects of various growth and post-growth process modifications on the polarization process. The Accelerated Device Degradation (ADD) characterization technique allows direct observation of nucleation and propagation of ionic transport channels within the TlBr crystals under applied bias. These channels are observed to be initiated both directly under the electrode as well as away from it. The propagation direction is always towards the anode indicating that Br- is the mobile diffusing species within the defect channels. The effective migration energy of the Br- ions was calculated to be 0.33±0.03 eV, which is consistent with other theoretical and experimental results.

  18. Rapid degradation of abnormal proteins in vacuoles from Acer pseudoplatanus L. cells

    SciTech Connect

    Canut, H.; Alibert, G.; Carrasco, A.; Boudet, A.M.

    1986-06-01

    In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ((/sup 14/C)p-fluorophenylalanine), were degraded more rapidly than normal ones ((/sup 14/C)phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole.

  19. Small Molecule-facilitated Degradation of ANO1 Protein

    PubMed Central

    Bill, Anke; Hall, Michelle Lynn; Borawski, Jason; Hodgson, Catherine; Jenkins, Jeremy; Piechon, Philippe; Popa, Oana; Rothwell, Christopher; Tranter, Pamela; Tria, Scott; Wagner, Trixie; Whitehead, Lewis; Gaither, L. Alex

    2014-01-01

    ANO1, a calcium-activated chloride channel, is highly expressed and amplified in human cancers and is a critical survival factor in these cancers. The ANO1 inhibitor CaCCinh-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCCinh-A01 on cell proliferation using a combined experimental and in silico approach. We show that inhibition of ANO1 function is not sufficient to diminish proliferation of ANO1-dependent cancer cells. We report that CaCCinh-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCCinh-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCCinh-A01-resistant cell pools was not affected by CaCCinh-A01 as compared with the parental cells. Consistently, CaCCinh-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCCinh-A01, indicating that CaCCinh-A01 inhibits cell proliferation by reducing ANO1 protein levels. Furthermore, we employed in silico methods to elucidate novel biological functions of ANO1 inhibitors. Specifically, we derived a pharmacophore model to describe inhibitors capable of promoting ANO1 degradation and report new inhibitors of ANO1-dependent cell proliferation. In summary, our data demonstrate that inhibition of the channel activity of ANO1 is not sufficient to inhibit ANO1-dependent cell proliferation, indicating that the role of ANO1 in cancer only partially depends on its function as a channel. Our results provide an impetus for gaining a deeper understanding of ANO1 modulation in cells and introduce a new targeting approach for antitumor therapy in ANO1-amplified cancers. PMID:24599954

  20. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly

    PubMed Central

    Mueller, Susanne; Wahlander, Asa; Selevsek, Nathalie; Otto, Claudia; Ngwa, Elsy Mankah; Poljak, Kristina; Frey, Alexander D.; Aebi, Markus; Gauss, Robert

    2015-01-01

    Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation. PMID:25995378

  1. Heat-induced Protein Structure and Subfractions in Relation to Protein Degradation Kinetics and Intestinal Availability in Dairy Cattle

    SciTech Connect

    Doiron, K.; Yu, P; McKinnon, J; Christensen, D

    2009-01-01

    The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included (1) protein structure a-helix to e-sheet ratio; (2) protein subfractions profiles; (3) protein degradation kinetics and effective degradability; (4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein was used as a model feed protein and was autoclave-heated at 120C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120C for 40 and 60 min increased the protein structure a-helix to e-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value), but there were no differences

  2. Arabidopsis DELLA Protein Degradation Is Controlled by a Type-One Protein Phosphatase, TOPP4

    PubMed Central

    Qin, Qianqian; Wang, Wei; Guo, Xiaola; Yue, Jing; Huang, Yan; Xu, Xiufei; Li, Jia; Hou, Suiwen

    2014-01-01

    Gibberellins (GAs) are a class of important phytohormones regulating a variety of physiological processes during normal plant growth and development. One of the major events during GA-mediated growth is the degradation of DELLA proteins, key negative regulators of GA signaling pathway. The stability of DELLA proteins is thought to be controlled by protein phosphorylation and dephosphorylation. Up to date, no phosphatase involved in this process has been identified. We have identified a dwarfed dominant-negative Arabidopsis mutant, named topp4-1. Reduced expression of TOPP4 using an artificial microRNA strategy also resulted in a dwarfed phenotype. Genetic and biochemical analyses indicated that TOPP4 regulates GA signal transduction mainly via promoting DELLA protein degradation. The severely dwarfed topp4-1 phenotypes were partially rescued by the DELLA deficient mutants rga-t2 and gai-t6, suggesting that the DELLA proteins RGA and GAI are required for the biological function of TOPP4. Both RGA and GAI were greatly accumulated in topp4-1 but significantly decreased in 35S-TOPP4 transgenic plants compared to wild-type plants. Further analyses demonstrated that TOPP4 is able to directly bind and dephosphorylate RGA and GAI, confirming that the TOPP4-controlled phosphorylation status of DELLAs is associated with their stability. These studies provide direct evidence for a crucial role of protein dephosphorylation mediated by TOPP4 in the GA signaling pathway. PMID:25010794

  3. Acceleration of calcite kinetics by abalone nacre proteins

    SciTech Connect

    Fu, G; Qiu, S R; Orme, C A; Morse, D E; De Yoreo, J J

    2005-06-09

    The fascinating shapes and hierarchical designs of biomineralized structures have long been an inspiration to materials scientists because of the potential they suggest for biomolecular control over synthesis of crystalline materials. One prevailing view is that mineral-associated macromolecules are responsible for initiating and stabilizing non-equilibrium crystal polymorphs and morphologies through interactions between anionic moieties and cations in solution or at mineral surfaces. Indeed, numerous studies have demonstrated that bio-organic additives can dramatically alter crystal shapes and growth-rates in vitro. However, previous molecular-scale studies revealing mechanisms of growth modification focused on small molecules such as amino acids or peptides and always observed growth inhibition. In contrast, studies using full proteins were non-quantitative and underlying sources of growth modification were ill-defined. Here we investigate interactions between proteins isolated from abalone shell nacre and growing surfaces of calcite. We find that these proteins significantly accelerate the molecular-scale kinetics and, though much larger than atomic steps, alter growth morphology through step-specific interactions that lower their free energies. We propose that these proteins act as surfactants to promote ion attachment at calcite surfaces.

  4. An Inducible System for Rapid Degradation of Specific Cellular Proteins Using Proteasome Adaptors

    PubMed Central

    Wilmington, Shameika R.; Matouschek, Andreas

    2016-01-01

    A common way to study protein function is to deplete the protein of interest from cells and observe the response. Traditional methods involve disrupting gene expression but these techniques are only effective against newly synthesized proteins and leave previously existing and stable proteins untouched. Here, we introduce a technique that induces the rapid degradation of specific proteins in mammalian cells by shuttling the proteins to the proteasome for degradation in a ubiquitin-independent manner. We present two implementations of the system in human culture cells that can be used individually to control protein concentration. Our study presents a simple, robust, and flexible technology platform for manipulating intracellular protein levels. PMID:27043013

  5. The different roles of selective autophagic protein degradation in mammalian cells

    PubMed Central

    Wang, Da-wei; Peng, Zhen-ju; Ren, Guang-fang; Wang, Guang-xin

    2015-01-01

    Autophagy is an intracellular pathway for bulk protein degradation and the removal of damaged organelles by lysosomes. Autophagy was previously thought to be unselective; however, studies have increasingly confirmed that autophagy-mediated protein degradation is highly regulated. Abnormal autophagic protein degradation has been associated with multiple human diseases such as cancer, neurological disability and cardiovascular disease; therefore, further elucidation of protein degradation by autophagy may be beneficial for protein-based clinical therapies. Macroautophagy and chaperone-mediated autophagy (CMA) can both participate in selective protein degradation in mammalian cells, but the process is quite different in each case. Here, we summarize the various types of macroautophagy and CMA involved in determining protein degradation. For this summary, we divide the autophagic protein degradation pathways into four categories: the post-translational modification dependent and independent CMA pathways and the ubiquitin dependent and independent macroautophagy pathways, and describe how some non-canonical pathways and modifications such as phosphorylation, acetylation and arginylation can influence protein degradation by the autophagy lysosome system (ALS). Finally, we comment on why autophagy can serve as either diagnostics or therapeutic targets in different human diseases. PMID:26415220

  6. [The 2004 Nobel Prize in Chemistry for the discovery of ubiquitin-mediated protein degradation].

    PubMed

    Neefjes, J; Groothuis, T A M; Dantuma, N P

    2004-12-25

    This year's Nobel Prize in Chemistry has been awarded to Aaron Ciechanover, Avram Herskho and Irwin Rose for the discovery of ubiquitin-mediated protein degradation. In a series of groundbreaking experiments these scientists described the basic principles for a unique posttranslational modification based on the conjugation of the small protein ubiquitin to proteins deemed for degradation. Although ubiquitin started in 1980 as an unusual modification of certain proteins, it is now clear that it functions as a signal for degradation when it forms a polymer. Hundreds of proteins are involved in the controlled destruction of ubiquitin-labelled proteins in the cell. And hundreds of other proteins are involved in protein modification by mono-ubiquitin, so that other processes, such as the formation of another degradation compartment, the lysosome, can proceed normally. PMID:15646859

  7. Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates.

    PubMed

    Gorovits, Rena; Fridman, Lilia; Kolot, Mikhail; Rotem, Or; Ghanim, Murad; Shriki, Oz; Czosnek, Henryk

    2016-02-01

    Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted by the whitefly Bemisia tabaci to tomato and other crops. TYLCV proteins are endangered by the host defenses. We have analyzed the capacity of the tomato plant and of the whitefly insect vector to degrade the six proteins encoded by the TYLCV genome. Tomato and whitefly demonstrated the highest proteolytic activity in the fractions containing soluble proteins, less-in large protein aggregates; a significant decrease of TYLCV proteolysis was detected in the intermediate-sized aggregates. All the six TYLCV proteins were differently targeted by the cytoplasmic and nuclear degradation machineries (proteases, ubiquitin 26S proteasome, autophagy). TYLCV could confront host degradation by sheltering in small/midsized aggregates, where viral proteins are less exposed to proteolysis. Indeed, TYLCV proteins were localized in aggregates of various sizes in both host organisms. This is the first study comparing degradation machinery in plant and insect hosts targeting all TYLCV proteins. PMID:26654789

  8. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling.

    PubMed

    De Los Rios, Paolo; Ben-Zvi, Anat; Slutsky, Olga; Azem, Abdussalam; Goloubinoff, Pierre

    2006-04-18

    Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation. PMID:16606842

  9. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling

    PubMed Central

    De Los Rios, Paolo; Ben-Zvi, Anat; Slutsky, Olga; Azem, Abdussalam; Goloubinoff, Pierre

    2006-01-01

    Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation. PMID:16606842

  10. HIV-1 proteins accelerate HPA axis habituation in female rats.

    PubMed

    Panagiotakopoulos, Leonidas; Kelly, Sean; Neigh, Gretchen N

    2015-10-15

    Congenital infection by the Human Immunodeficiency Virus (HIV) has been shown to lead to multiple co-morbidities, and people living with HIV have a higher incidence of affective and anxiety disorders. A marked increase in mood disorders is evident during the sensitive phase of adolescence and this is further pronounced in females. Depression has been linked to dysfunction of the intracellular response system to corticosteroids at the level of the hippocampus (HC) and prefrontal cortex (PFC) with a notable role of the glucocorticoid receptor (GR) and its co-chaperones (FKBP5 and FKBP4). The current study examined the extent to which HIV protein expression in adolescent female rats altered the stress response at both the level of corticosterone output and molecular regulation of the glucocorticoid receptor in the brain. WT and HIV-1 genotype female rats were randomly allocated in control, acute stress and repeat stress groups. Corticosterone plasma levels and expression of GR, FKBP4, and FKBP5 in the HC and PFC were measured. The presence of HIV-1 proteins facilitates habituation of the corticosterone response to repeated stressors, such that HIV-1 TG rats habituated to repeated restraint and WT rats did not. This was reflected by interactions between stress exposure and HIV-1 protein expression at the level of GR co-chaperones. Although expression of the GR was similarly reduced after acute and repeat stress in both genotypes, expression of FKBP5 and FKBP4 was altered in a brain-region specific manner depending on the duration of the stress exposure and the presence or absence of HIV-1 proteins. Collectively, the data presented demonstrate that HIV-1 proteins accelerate habituation to repeated stressors and modify the influence of acute and repeat stressors on GR co-chaperones in a brain region-specific manner. PMID:25666308

  11. Gaucher disease due to saposin C deficiency is an inherited lysosomal disease caused by rapidly degraded mutant proteins.

    PubMed

    Motta, Marialetizia; Camerini, Serena; Tatti, Massimo; Casella, Marialuisa; Torreri, Paola; Crescenzi, Marco; Tartaglia, Marco; Salvioli, Rosa

    2014-11-01

    Saposin (Sap) C is an essential cofactor for the lysosomal degradation of glucosylceramide (GC) by glucosylceramidase (GCase) and its functional impairment underlies a rare variant form of Gaucher disease (GD). Sap C promotes rearrangement of lipid organization in lysosomal membranes favoring substrate accessibility to GCase. It is characterized by six invariantly conserved cysteine residues involved in three intramolecular disulfide bonds, which make the protein remarkably stable to acid environment and degradation. Five different mutations (i.e. p.C315S, p.342_348FDKMCSKdel, p.L349P, p.C382G and p.C382F) have been identified to underlie Sap C deficiency. The molecular mechanism by which these mutations affect Sap C function, however, has not been delineated in detail. Here, we characterized biochemically and functionally four of these gene lesions. We show that all Sap C mutants are efficiently produced, and exhibit lipid-binding properties, modulatory behavior on GCase activity and subcellular localization comparable with those of the wild-type protein. We then delineated the structural rearrangement of these mutants, documenting that most proteins assume diverse aberrant disulfide bridge arrangements, which result in a substantial diminished half-life, and rapid degradation via autophagy. These findings further document the paramount importance of disulfide bridges in the stability of Sap C and provide evidence that accelerated degradation of the Sap C mutants is the underlying pathogenetic mechanism of Sap C deficiency. PMID:24925315

  12. Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies

    PubMed Central

    Ciechanover, Aaron; Kwon, Yong Tae

    2015-01-01

    Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons. PMID:25766616

  13. Thermally Accelerated Oxidative Degradation of Quercetin Using Continuous Flow Kinetic Electrospray-Ion Trap-Time of Flight Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Barnes, Jeremy S.; Foss, Frank W.; Schug, Kevin A.

    2013-10-01

    Thermally accelerated oxidative degradation of aqueous quercetin at pH 5.9 and 7.4 was kinetically measured using an in-house built online continuous flow device made of concentric capillary tubes, modified to fit to the inlet of an electrospray ionization-ion trap-time-of-flight-mass spectrometer (ESI-IT-TOF-MS). Time-resolved mass spectral measurements ranging from 2 to 21 min were performed in the negative mode to track intermediate degradation products and to evaluate the degradation rate of the deprotonated quercetin ion, [Q-H]-. Upon heating solutions in the presence of dissolved oxygen, degradation of [Q-H]- was observed and was accelerated by an increase in pH and temperature. Regardless of the condition, the same degradation pathways were observed. Degradation mechanisms and structures were determined using higher order tandem mass spectrometry (up to MS3) and high mass accuracy. The observed degradation mechanisms included oxidation, hydroxylation, and ring-cleavage by nucleophilic attack. A chalcan-trione structure formed by C-ring opening after hydroxylation at C2 was believed to be a precursor for other degradation products, formed by hydroxylation at the C2, C3, and C4 carbons from attack by nucleophilic species. This resulted in A-type and B-type ions after cross-ring cleavage of the C-ring. Based on time of appearance and signal intensity, nucleophilic attack at C3 was the preferred degradation pathway, which generated 2,4,6-trihydroxymandelate and 2,4,6-trihydroxyphenylglyoxylate ions. Overall, 23 quercetin-related ions were observed.

  14. MIIP accelerates epidermal growth factor receptor protein turnover and attenuates proliferation in non-small cell lung cancer

    PubMed Central

    Wen, Jing; Fu, Jianhua; Ling, Yihong; Zhang, Wei

    2016-01-01

    The migration and invasion inhibitory protein (MIIP) has been discovered recently to have inhibitory functions in cell proliferation and migration. Overexpression of MIIP reduced the intracellular steady-state level of epidermal growth factor receptor (EGFR) protein in lung cancer cells with no effect on EGFR mRNA expression compared to that in the control cells. This MIIP-promoted EGFR protein degradation was reversed by proteasome and lysosome inhibitors, suggesting the involvement of both proteasomal and lysosomal pathways in this degradation. This finding was further validated by pulse-chase experiments using 35S-methionine metabolic labeling. We found that MIIP accelerates EGFR protein turnover via proteasomal degradation in the endoplasmic reticulum and then via the lysosomal pathway after its entry into endocytic trafficking. MIIP-stimulated downregulation of EGFR inhibits downstream activation of Ras and blocks the MEK signal transduction pathway, resulting in inhibition of cell proliferation. The negative correlation between MIIP and EGFR protein expression was validated in lung adenocarcinoma samples. Furthermore, the higher MIIP protein expression predicts a better overall survival of Stage IA-IIIA lung adenocarcinoma patients who underwent radical surgery. These findings reveal a new mechanism by which MIIP inhibits cell proliferation. PMID:26824318

  15. Using co-metabolism to accelerate synthetic starch wastewater degradation and nutrient recovery in photosynthetic bacterial wastewater treatment technology.

    PubMed

    Lu, Haifeng; Zhang, Guangming; Lu, Yufeng; Zhang, Yuanhui; Li, Baoming; Cao, Wei

    2016-01-01

    Starch wastewater is a type of nutrient-rich wastewater that contains numerous macromolecular polysaccharides. Using photosynthetic bacteria (PSB) to treat starch wastewater can reduce pollutants and enhance useful biomass production. However, PSB cannot directly degrade macromolecular polysaccharides, which weakens the starch degradation effect. Therefore, co-metabolism with primary substances was employed in PSB wastewater treatment to promote starch degradation. The results indicated that co-metabolism is a highly effective method in synthetic starch degradation by PSB. When malic acid was used as the optimal primary substrate, the chemical oxygen demand, total sugar, macromolecules removal and biomass yield were considerably higher than when primary substances were not used, respectively. Malic acid was the primary substrate that played a highly important role in starch degradation. It promoted the alpha-amylase activity to 46.8 U and the PSB activity, which induced the degradation of macromolecules. The products in the wastewater were ethanol, acetic acid and propionic acid. Ethanol was the primary product throughout the degradation process. The introduction of co-metabolism with malic acid to treat wastewater can accelerate macromolecules degradation and bioresource production and weaken the acidification effect. This method provides another pathway for bioresource recovery from wastewater. This approach is a sustainable and environmentally friendly wastewater treatment technology. PMID:26360302

  16. Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration.

    PubMed

    Hwang, J-Y; Lee, J; Oh, C-K; Kang, H W; Hwang, I-Y; Um, J W; Park, H C; Kim, S; Shin, J-H; Park, W-Y; Darnell, R B; Um, H-D; Chung, K C; Kim, K; Oh, Y J

    2016-01-01

    Cerebellar degeneration-related protein 2 (cdr2) is expressed in the central nervous system, and its ectopic expression in tumor cells of patients with gynecological malignancies elicits immune responses by cdr2-specific autoantibodies and T lymphocytes, leading to neurological symptoms. However, little is known about the regulation and function of cdr2 in neurodegenerative diseases. Because we found that cdr2 is highly expressed in the midbrain, we investigated the role of cdr2 in experimental models of Parkinson's disease (PD). We found that cdr2 levels were significantly reduced after stereotaxic injection of 1-methyl-4-phenylpyridinium (MPP(+)) into the striatum. cdr2 levels were also decreased in the brains of post-mortem PD patients. Using primary cultures of mesencephalic neurons and MN9D cells, we confirmed that MPP(+) reduces cdr2 in tyrosine hydroxylase-positive dopaminergic neuronal cells. The MPP(+)-induced decrease of cdr2 was primarily caused by calpain- and ubiquitin proteasome system-mediated degradation, and cotreatment with pharmacological inhibitors of these enzymes or overexpression of calcium-binding protein rendered cells less vulnerable to MPP(+)-mediated cytotoxicity. Consequently, overexpression of cdr2 rescued cells from MPP(+)-induced cytotoxicity, whereas knockdown of cdr2 accelerated toxicity. Collectively, our findings provide insights into the novel regulatory mechanism and potentially protective role of onconeural protein during dopaminergic neurodegeneration. PMID:27253404

  17. N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli

    PubMed Central

    Sekar, Karthik; Gentile, Andrew M.; Bostick, John W.; Tyo, Keith E. J.

    2016-01-01

    Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag) strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits. PMID:26900850

  18. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Goldberg, A. L.

    1992-01-01

    To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

  19. POLYPHENOLS AND MECHANICAL MACERATION SHIFT PROTEIN FRACTIONS IN LEGUME HAYS FROM RAPIDLY TO SLOWLY DEGRADED FORMS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid proteolysis of forage protein during rumen fermentation can impair protein use by dairy cattle. The severity of conditioning at harvest may influence protein degradability in forages, particularly if protein-binding polyphenols are present. In 2002 and 2003, first and second cuttings of alfalf...

  20. Effects of starvation for potassium and other inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli.

    PubMed

    St John, A C; Goldberg, A L

    1980-09-01

    Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition of ribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change. The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined. Upon starvation for amino acids or amino acyl-transfer RNA (tRNA), protein breakdown increases in relA+ (but not relA) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). However, a lack of amino acyl-tRNA does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both relA+ and relA cultures, and since a large excess of amino acids did not affect this response. In bacteria in which energy production is restricted, ppGpp levels also rise, and protein breakdown increases. The ion-deprived cultures did show a 40 to 75% reduction in adenosine-5'-triphosphate levels,l similar to that seen upon glucose starvation. However, this decrease in ATP content does not appear to cause the increase in protein breakdown or lead to an accumulation of ppGpp. No consistent change in intracellular ppGpp levels was found in relA+ or relA cells starved for these ions. In addition, in relX mutants, removal of these ions led to accelerated protein degradation even though relX cells are unable to increase ppGpp levels or proteolysis when deprived of a carbon source. In the potassium-, phosphate-, and magnesium-deprived cultures, the addition of choramphenicol or tetracycline caused a reduction in protein breakdown toward basal levels. Such findings, however, do not indicate that protein synthesis is essential for the

  1. Chemical degradation of proteins in the solid state with a focus on photochemical reactions.

    PubMed

    Mozziconacci, Olivier; Schöneich, Christian

    2015-10-01

    Protein pharmaceuticals comprise an increasing fraction of marketed products but the limited solution stability of proteins requires considerable research effort to prepare stable formulations. An alternative is solid formulation, as proteins in the solid state are thermodynamically less susceptible to degradation. Nevertheless, within the time of storage a large panel of kinetically controlled degradation reactions can occur such as, e.g., hydrolysis reactions, the formation of diketopiperazine, condensation and aggregation reactions. These mechanisms of degradation in protein solids are relatively well covered by the literature. Considerably less is known about oxidative and photochemical reactions of solid proteins. This review will provide an overview over photolytic and non-photolytic degradation reactions, and specially emphasize mechanistic details on how solid structure may affect the interaction of protein solids with light. PMID:25481682

  2. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

    PubMed Central

    Young, Barry P.; Loewen, Christopher J.; Mayor, Thibault

    2016-01-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

  3. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

    PubMed

    Comyn, Sophie A; Young, Barry P; Loewen, Christopher J; Mayor, Thibault

    2016-07-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

  4. A cellular system that degrades misfolded proteins and protects against neurodegeneration.

    PubMed

    Guo, Lili; Giasson, Benoit I; Glavis-Bloom, Alex; Brewer, Michael D; Shorter, James; Gitler, Aaron D; Yang, Xiaolu

    2014-07-01

    Misfolded proteins compromise cellular function and cause disease. How these proteins are detected and degraded is not well understood. Here we show that PML/TRIM19 and the SUMO-dependent ubiquitin ligase RNF4 act together to promote the degradation of misfolded proteins in the mammalian cell nucleus. PML selectively interacts with misfolded proteins through distinct substrate recognition sites and conjugates these proteins with the small ubiquitin-like modifiers (SUMOs) through its SUMO ligase activity. SUMOylated misfolded proteins are then recognized and ubiquitinated by RNF4 and are subsequently targeted for proteasomal degradation. We further show that PML deficiency exacerbates polyglutamine (polyQ) disease in a mouse model of spinocerebellar ataxia 1 (SCA1). These findings reveal a mammalian system that removes misfolded proteins through sequential SUMOylation and ubiquitination and define its role in protection against protein-misfolding diseases. PMID:24882209

  5. Accelerating the degradation of green plant waste with chemical decomposition agents.

    PubMed

    Kejun, Sun; Juntao, Zhang; Ying, Chen; Zongwen, Liao; Lin, Ruan; Cong, Liu

    2011-10-01

    Degradation of green plant waste is often difficult, and excess maturity times are typically required. In this study, we used lignin, cellulose and hemicellulose assays; scanning electron microscopy; infrared spectrum analysis and X-ray diffraction analysis to investigate the effects of chemical decomposition agents on the lignocellulose content of green plant waste, its structure and major functional groups and the mechanism of accelerated degradation. Our results showed that adding chemical decomposition agents to Ficus microcarpa var. pusillifolia sawdust reduced the contents of lignin by 0.53%-11.48% and the contents of cellulose by 2.86%-7.71%, and increased the contents of hemicellulose by 2.92%-33.63% after 24 h. With increasing quantities of alkaline residue and sodium lignosulphonate, the lignin content decreased. Scanning electron microscopy showed that, after F. microcarpa var. pusillifolia sawdust was treated with chemical decomposition agents, lignocellulose tube wall thickness increased significantlyIncreases of 29.41%, 3.53% and 34.71% were observed after treatment with NaOH, alkaline residue and sodium lignosulphonate, respectively. Infrared spectroscopy showed that CO and aromatic skeleton stretching absorption peaks were weakened and the C-H vibrational absorption peak from out-of-plane in positions 2 and 6 (S units) (890-900 cm(-1)) was strengthened after F. microcarpa var. pusillifolia sawdust was treated with chemical decomposition agents, indicating a reduction in lignin content. Several absorption peaks [i.e., C-H deformations (asymmetry in methyl groups, -CH(3)- and -CH(2)-) (1450-1460 cm(-1)); Aliphatic C-H stretching in methyl and phenol OH (1370-1380 cm(-1)); CO stretching (cellulose and hemicellulose) (1040-1060 cm(-1))] that indicate the presence of a chemical bond between lignin and cellulose was reduced, indicating that the chemical bond between lignin and cellulose had been partially broken. X-ray diffraction analysis showed that Na

  6. Accelerated degradation of 14C-atrazine in an atrazine adapted field soil from Belgium

    NASA Astrophysics Data System (ADS)

    Hamacher, Georg; Jablonowski, Nicolai David; Martinazzo, Rosane; Accinelli, Cesare; Köppchen, Stephan; Langen, Ulrike; Linden, Andreas; Krause, Martina; Burauel, Peter

    2010-05-01

    Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] is one of the most widely used herbicides in the world. Atrazine is considered to be mobile in soil and has often been characterized as a rather recalcitrant compound in the environment. In the present study the accelerated atrazine degradation in an agriculturally used soil was examined. Soil samples were collected from a Belgian field which was used for corn-plantations and was regularly treated with atrazine during the last 30 years. The experiment was conducted under controlled laboratory conditions (GLP) using 14C-labelled and unlabelled atrazine in accordance to the reported field application dose of 1 mg kg-1. Triplicates of treated subsamples were incubated at 50% WHCmax and under slurry conditions (1:4 soil:solution ratio, using distilled water) in the dark at 20° C. Control samples were collected at an adjacent pear orchard where no atrazine or other triazine pesticides application was reported. After 92 days of incubation, the mineralized amount of atrazine reached 83% of the initially applied 14C-activity in the atrazine treated soil for the slurry setup. A maximum of atrazine mineralization was observed in the treated field soil between 6 and 7 days of incubation for both, 50% WHCmax and slurry setups. The total 14C-atrazine mineralization was equally high for 50% WHCmax in the atrazine treated soil. After an extended lag-phase in comparison to the treated soil the overall mineralization of 14C-atrazine of 81% was observed in the atrazine untreated soil under slurry conditions. This observation might be due to a possible cross adaption of the microflora. These results could be attributed to an atrazine drift during application since the control samples were taken in an adjacent pear orchard with no atrazine application history. These results demonstrate an adaption of the microflora to mineralize atrazine rapidly. The formation of desorbable metabolites as well as the formation of

  7. Bone morphogenetic protein-7 accelerates fracture healing in osteoporotic rats

    PubMed Central

    Diwan, Ashish D; Leong, Anthony; Appleyard, Richard; Bhargav, Divya; Fang, Zhi Ming; Wei, Aiqun

    2013-01-01

    Background: Osteoporosis is characterized by low bone mass, bone fragility and increased susceptibility to fracture. Fracture healing in osteoporosis is delayed and rates of implant failure are high with few biological treatment options available. This study aimed to determine whether a single dose of bone morphogenetic protein-7 (BMP-7) in a collagen/carboxy-methyl cellulose (CMC) composite enhanced fracture healing in an osteoporotic rat model. Materials and Methods: An open femoral midshaft osteotomy was performed in female rats 3 months post-ovarectomy. Rats were randomized to receive either BMP-7 composite (n = 30) or composite alone (n = 30) at the fracture site during surgery. Thereafter calluses were collected on days 12, 20 and 31. Callus cross-sectional area, bone mineral density, biomechanical stiffness and maximum torque, radiographic bony union and histological callus maturity were evaluated at each time point. Results: There were statistically significant increases in bone mineral density and callus cross-section area at all time points in the BMP-7 group as compared to controls and biomechanical readings showed stronger bones at day 31 in the BMP-7 group. Histological and radiographic evaluation indicated significant acceleration of bony union in the BMP-7 group as compared to controls. Conclusion: This study demonstrated that BMP-7 accelerates fracture healing in an oestrogen-deficient environment in a rat femoral fracture healing model to scientific relevance level I. The use of BMP-7 composite could offer orthopedic surgeons an advantage over oestrogen therapy, enhancing osteoporotic fracture healing with a single, locally applied dose at the time of surgery, potentially overcoming delays in healing caused by the osteoporotic state. PMID:24379457

  8. In vitro degradation of the 32kDa PS II reaction centre protein

    SciTech Connect

    Eckenswiller, L.C.; Greenberg, B.M. )

    1989-04-01

    The 32kDa thylakoid membrane protein is an integral component of the PS II reaction centre. The protein, although stable in the dark, undergoes light dependent turnover. Light from the UV, visible and far-red spectral regions induce 32kDa protein degradation. To better understand 32kDa protein metabolism, an in vitro degradation system is being developed. It consists of isolated thylakoid membranes than contain radiolabelled protein. The 32kDa protein is actively and specifically degraded when the thylakoid preparation is exposed to UV or visible radiation. The protein is stable in the dark. The herbicides (atrazine and DCMU) inhibit degradation in the in vitro system as they do in vivo. Additionally, several methods of isolating thylakoids are being compared to optimize the 32kDa protein degradation reaction. The preparations will be evaluated based on their ability to permit light dependent degradation of the 32kDa protein without affecting the other membrane components.

  9. Mass Spectrometry Analysis of Proteome-wide Proteolytic Post-translational Degradation of Proteins

    SciTech Connect

    Shen, Yufeng; Hixson, Kim K.; Tolic, Nikola; Camp, David G.; Purvine, Samuel O.; Moore, Ronald J.; Smith, Richard D.

    2008-08-01

    Protein proteolysis is an essential component to proper cell function. Here, we demonstrate a method for studying protein degradation by detection of intermediate intracellular peptides with a high-precision tandem mass spectrometry de novo sequencing-based approach. From a Saccharomyces cerevisiae lysate, we identified >1,200 peptides containing 6-100 amino acids without random false positives and ascribed most identifications as being products of protein degradation. Most protein degradation observed was located in the cytoplasm, and multiple types of cleavage were found to exist in addition to the expected trypsin-like and chymotrypsin-like preferences. The yeast nucleus was found as a proteolysis-inert organelle under the conditions studied and the V-ATPase to be degraded during disassembly. Additionally, matrix associated mitochondrial proteins functioning as transport carriers and gates were found to be commonly degraded. Determining these protein degradation events could eventually aid in understanding of cell biology and detection and treatment of protein degradation-related diseases.

  10. Forage Management Effects on Protein and Fiber Fractions, Protein Degradability, and Dry Matter Yield of Red Clover Conserved as Silage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Due to the action of o-quinones formed via polyphenol oxidase, conserved red clover (Trifolium pratense L.) contains abundant rumen undegradable protein (RUP), but inadequate rumen degradable protein (RDP) for dairy cattle. This study examined how forage management influences RDP, RUP, crude protein...

  11. Protein degradation to low-molecular compounds after death and during reanimation.

    PubMed

    Konikova, A S; Vinarskaya, A A; Nikulin, V I; Pogossova, A V; Petukhova, L M

    1975-09-11

    The process of protein degradation to amino acids and peptides in rabbits following death and during reanimation in terms of the effects of artificial postmortem cooling on that process has been studied. Protein degradation was judged by increase of low-molecular nitrogenous compounds in serum and in organs by increase in soluble radioactivity with time in animals the proteins of which had been marked in vivo with radioisotopes. It has been found that immediately after death resulting from acute anoxia the processes of protein degradation to amino acids as well as synthesis stops in liver, skeletal and cardiac muscles, spleen, brain and spinal cord. Similar phenomenon takes place in the case of deep hypothermy. During reanimation the process of protein degradation to low-molecular compounds in organs restores. PMID:809921

  12. Chain and pore-blocking effects on matrix degradation in protein-loaded microgels.

    PubMed

    Widenbring, Ronja; Frenning, Göran; Malmsten, Martin

    2014-10-13

    Factors affecting matrix degradation in protein-loaded microgels were investigated for dextran-based microgels, the sugar-binding protein Concanavalin A (ConA), and the dextran-degrading enzyme Dextranase. For this system, effects of enzyme, protein, and glucose concentrations, as well as pH, were considered. Microgel network degradation was monitored by micromanipulator-assisted light microscopy, whereas enzyme and protein distributions were monitored by confocal microscopy. Results show that Dextranase-mediated microgel degradation increased with increasing enzyme concentration, whereas an increased ConA loading in the dextran microgels caused a concentration-dependent decrease in microgel degradation. In the presence of glucose, competitive release of microgel-bound ConA restored the microgel degradation observed in the absence of ConA. To clarify effects of mass transport limitations, microgel degradation was compared to that of non-cross-linked dextran, demonstrating that ConA limits enzyme substrate access in dextran microgels primarily through pore blocking and induction of pore shrinkage. The experimentally observed effects were qualitatively captured by a modified Michaelis-Menten approach for spherical symmetry, in which network blocking by ConA was included. Taken together, the results demonstrate that matrix degradation of protein-loaded microgels depends sensitively on a number of factors, which need to be considered in the use of microgels in biomedical applications. PMID:25144139

  13. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    PubMed

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation. PMID:25837301

  14. The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein.

    PubMed

    Chai, Qian; Wang, Zhaoshuai; Webb, Stacy R; Dutch, Rebecca E; Wei, Yinan

    2016-04-26

    ATP-dependent degradation plays a critical role in the quality control and recycling of proteins in cells. However, complete degradation of membrane proteins by ATP-dependent proteases in bacteria is not well-studied. We discovered that the degradation of a multidomain and multispan integral membrane protein AcrB could be facilitated by the introduction of a ssrA-tag at the C-terminus of the protein sequence and demonstrated that the cytoplasmic unfoldase-protease complex ClpXP was involved in the degradation. This is the first report to our knowledge to reveal that the ClpXP complex is capable of degrading integral membrane proteins. The chaperone SspB also played a role in the degradation. Using purified proteins, we demonstrated that the addition of the ssrA-tag did not drastically affect the structure of AcrB, and the degradation of detergent solubilized AcrB by purified ClpXP could be observed in vitro. PMID:27078234

  15. Redox modulation of cellular metabolism through targeted degradation of signaling proteins by the proteasome

    SciTech Connect

    Squier, Thomas C.

    2006-02-01

    Under conditions of oxidative stress, the 20S proteasome plays a critical role in maintaining cellular homeostasis through the selective degradation of oxidized and damaged proteins. This adaptive stress response is distinct from ubiquitin-dependent pathways in that oxidized proteins are recognized and degraded in an ATP-independent mechanism, which can involve the molecular chaperone Hsp90. Like the regulatory complexes 19S and 11S REG, Hsp90 tightly associates with the 20S proteasome to mediate the recognition of aberrant proteins for degradation. In the case of the calcium signaling protein calmodulin, proteasomal degradation results from the oxidation of a single surface exposed methionine (i.e., Met145); oxidation of the other eight methionines has a minimal effect on the recognition and degradation of calmodulin by the proteasome. Since cellular concentrations of calmodulin are limiting, the targeted degradation of this critical signaling protein under conditions of oxidative stress will result in the downregulation of cellular metabolism, serving as a feedback regulation to diminish the generation of reactive oxygen species. The targeted degradation of critical signaling proteins, such as calmodulin, can function as sensors of oxidative stress to downregulate global rates of metabolism and enhance cellular survival.

  16. Protein-Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery.

    PubMed

    Milhas, Sabine; Raux, Brigitt; Betzi, Stéphane; Derviaux, Carine; Roche, Philippe; Restouin, Audrey; Basse, Marie-Jeanne; Rebuffet, Etienne; Lugari, Adrien; Badol, Marion; Kashyap, Rudra; Lissitzky, Jean-Claude; Eydoux, Cécilia; Hamon, Véronique; Gourdel, Marie-Edith; Combes, Sébastien; Zimmermann, Pascale; Aurrand-Lions, Michel; Roux, Thomas; Rogers, Catherine; Müller, Susanne; Knapp, Stefan; Trinquet, Eric; Collette, Yves; Guillemot, Jean-Claude; Morelli, Xavier

    2016-08-19

    Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse "PPI-like" modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using homogeneous time-resolved fluorescence (HTRF) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with nonoriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets. PMID:27219844

  17. [6]-Shogaol inhibits α-MSH-induced melanogenesis through the acceleration of ERK and PI3K/Akt-mediated MITF degradation.

    PubMed

    Huang, Huey-Chun; Chang, Shu-Jen; Wu, Chia-Yin; Ke, Hui-Ju; Chang, Tsong-Min

    2014-01-01

    [6]-Shogaol is the main biologically active component of ginger. Previous reports showed that [6]-shogaol has several pharmacological characteristics, such as antioxidative, anti-inflammatory, antimicrobial, and anticarcinogenic properties. However, the effects of [6]-shogaol on melanogenesis remain to be elucidated. The study aimed to evaluate the potential skin whitening mechanisms of [6]-shogaol. The effects of [6]-shogaol on cell viability, melanin content, tyrosinase activity, and the expression of the tyrosinase and microphthalmia-associated transcription factor (MITF) were measured. The results revealed that [6]-shogaol effectively suppresses tyrosinase activity and the amount of melanin and that those effects are more pronounced than those of arbutin. It was also found that [6]-shogaol decreased the protein expression levels of tyrosinase-related protein 1 (TRP-1) and microphthalmia-associated transcriptional factor (MITF). In addition, the MITF mRNA levels were also effectively decreased in the presence of 20 μM [6]-shogaol. The degradation of MITF protein was inhibited by the MEK 1-inhibitor (U0126) or phosphatidylinositol-3-kinase inhibitor (PI3K inhibitor) (LY294002). Further immunofluorescence staining assay implied the involvement of the proteasome in the downregulation of MITF by [6]-shogaol. Our confocal assay results also confirmed that [6]-shogaol inhibited α-melanocyte stimulating hormone- (α-MSH-) induced melanogenesis through the acceleration of extracellular responsive kinase (ERK) and phosphatidylinositol-3-kinase- (PI3K/Akt-) mediated MITF degradation. PMID:25045707

  18. [6]-Shogaol Inhibits α-MSH-Induced Melanogenesis through the Acceleration of ERK and PI3K/Akt-Mediated MITF Degradation

    PubMed Central

    Huang, Huey-Chun; Chang, Shu-Jen; Wu, Chia-Yin; Ke, Hui-Ju; Chang, Tsong-Min

    2014-01-01

    [6]-Shogaol is the main biologically active component of ginger. Previous reports showed that [6]-shogaol has several pharmacological characteristics, such as antioxidative, anti-inflammatory, antimicrobial, and anticarcinogenic properties. However, the effects of [6]-shogaol on melanogenesis remain to be elucidated. The study aimed to evaluate the potential skin whitening mechanisms of [6]-shogaol. The effects of [6]-shogaol on cell viability, melanin content, tyrosinase activity, and the expression of the tyrosinase and microphthalmia-associated transcription factor (MITF) were measured. The results revealed that [6]-shogaol effectively suppresses tyrosinase activity and the amount of melanin and that those effects are more pronounced than those of arbutin. It was also found that [6]-shogaol decreased the protein expression levels of tyrosinase-related protein 1 (TRP-1) and microphthalmia-associated transcriptional factor (MITF). In addition, the MITF mRNA levels were also effectively decreased in the presence of 20 μM [6]-shogaol. The degradation of MITF protein was inhibited by the MEK 1-inhibitor (U0126) or phosphatidylinositol-3-kinase inhibitor (PI3K inhibitor) (LY294002). Further immunofluorescence staining assay implied the involvement of the proteasome in the downregulation of MITF by [6]-shogaol. Our confocal assay results also confirmed that [6]-shogaol inhibited α-melanocyte stimulating hormone- (α-MSH-) induced melanogenesis through the acceleration of extracellular responsive kinase (ERK) and phosphatidylinositol-3-kinase- (PI3K/Akt-) mediated MITF degradation. PMID:25045707

  19. Polyphenol, Conditioning, and Conservation Effects on Protein Fractions and Degradability in Forage Legumes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forage legume proteins were fractionated by the Cornell Net Carbohydrate and Protein System or ruminally incubated to assess how conditioning and conservation methods interact with polyphenols (condensed tannins or o-quinones) to alter protein degradability. The presence of polyphenols, conditioning...

  20. Thalidomide Accelerates the Degradation of Extracellular Matrix in Rat Hepatic Cirrhosis via Down-Regulation of Transforming Growth Factor-β1

    PubMed Central

    Meng, Qingshun; Liu, Jie; Wang, Chuanfang

    2015-01-01

    Purpose The degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis. Materials and Methods Cirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and α-smooth muscle actin (α-SMA) protein in the liver, transforming growth factor β1 (TGF-β1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-β1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction. Results Liver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-β1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group. Conclusion Thalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-β1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats. PMID:26446639

  1. Characterization of the proteostasis roles of glycerol accumulation, protein degradation and protein synthesis during osmotic stress in C. elegans.

    PubMed

    Burkewitz, Kristopher; Choe, Keith P; Lee, Elaine Choung-Hee; Deonarine, Andrew; Strange, Kevin

    2012-01-01

    Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50-70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50-80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70-180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage brought

  2. The Arabidopsis CROWDED NUCLEI genes regulate seed germination by modulating degradation of ABI5 protein.

    PubMed

    Zhao, Wenming; Guan, Chunmei; Feng, Jian; Liang, Yan; Zhan, Ni; Zuo, Jianru; Ren, Bo

    2016-07-01

    In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination. PMID:26564029

  3. Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins

    PubMed Central

    Das, Indrajit; Png, Chin Wen; Oancea, Iulia; Hasnain, Sumaira Z.; Lourie, Rohan; Proctor, Martina; Eri, Rajaraman D.; Sheng, Yong; Crane, Denis I.; Florin, Timothy H.

    2013-01-01

    Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca2+ or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX’s suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER. PMID:23650437

  4. Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

    PubMed

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells. PMID:23325696

  5. Toscana Virus NSs Protein Promotes Degradation of Double-Stranded RNA-Dependent Protein Kinase

    PubMed Central

    Kalveram, Birte

    2013-01-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses—i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus—has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells. PMID:23325696

  6. Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Zubenko, George S.; Jones, Elizabeth W.

    1981-01-01

    During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation. PMID:7021321

  7. Degradation-mediated protein quality control at the inner nuclear membrane

    PubMed Central

    Boban, Mirta; Foisner, Roland

    2016-01-01

    abstract An intricate machinery protects cells from the accumulation of misfolded, non-functional proteins and protein aggregates. Protein quality control pathways have been best described in the cytoplasm and the endoplasmic reticulum, however, recent findings indicate that the nucleus is also an important compartment for protein quality control. Several nuclear ubiquitinylation pathways target soluble and membrane proteins in the nucleus and mediate their degradation through nuclear proteasomes. In addition, emerging data suggest that nuclear envelope components are also degraded by autophagy, although the mechanisms by which cytoplasmic autophagy machineries get access to nuclear targets remain unclear. In this minireview we summarize the nuclear ubiquitin-proteasome pathways in yeast, focusing on pathways involved in the protein degradation at the inner nuclear membrane. In addition, we discuss potential mechanisms how nuclear targets at the nuclear envelope may be delivered to the cytoplasmic autophagy pathways in yeast and mammals. PMID:26760377

  8. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation.

    PubMed

    Chen, Chao; Cui, Zhenling; Song, Xiangfei; Liu, Ya-Jun; Cui, Qiu; Feng, Yingang

    2016-03-01

    Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions, which are the most efficient system for the degradation of lignocellulosic resources in nature. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins, Clocl_1298 and Clocl_1862, contain a dockerin module, which suggests that they are components of the cellulosome. Bacterial expansin-like proteins do not have hydrolytic activities, but can facilitate the degradation of cellulosic biomass via synergistic effects with cellulases. In this study, the synergistic effect of the expansin-like proteins with both native and designer cellulosomes was investigated. The free expansin-like proteins, including expansin-like domains of Clocl_1298 and Clocl_1862, as well as a well-studied bacterial expansin-like protein BsEXLX1 from Bacillus subtilis, promoted the cellulose degradation by native cellulosomes, indicating the cellulosomal expansin-like proteins have the synergistic function. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. These results indicated that non-catalytic expansin-like proteins in the cellulosome can enhance the activity of the cellulosome in lignocellulose degradation. The involvement of functional expansin-like proteins in the cellulosome also implies new physiological functions of bacterial expansin-like proteins and cellulosomes. PMID:26521249

  9. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast.

    PubMed

    Maurer, Matthew J; Spear, Eric D; Yu, Allen T; Lee, Evan J; Shahzad, Saba; Michaelis, Susan

    2016-01-01

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic "degron library" in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3 About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

  10. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast

    PubMed Central

    Maurer, Matthew J.; Spear, Eric D.; Yu, Allen T.; Lee, Evan J.; Shahzad, Saba; Michaelis, Susan

    2016-01-01

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic “degron library” in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3. About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

  11. Effects of grain source, grain processing, and protein degradability on rumen kinetics and microbial protein synthesis in Boer kids.

    PubMed

    Brassard, M-E; Chouinard, P Y; Berthiaume, R; Tremblay, G F; Gervais, R; Martineau, R; Cinq-Mars, D

    2015-11-01

    Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM). PMID:26641055

  12. Accelerated Testing and Modeling of Potential-Induced Degradation as a Function of Temperature and Relative Humidity

    SciTech Connect

    Hacke, Peter; Spataru, Sergiu; Terwilliger, Kent; Perrin, Greg; Glick, Stephen; Kurtz, Sarah; Wohlgemuth, John

    2015-06-14

    An acceleration model based on the Peck equation was applied to power performance of crystalline silicon cell modules as a function of time and of temperature and humidity, the two main environmental stress factors that promote potential-induced degradation. This model was derived from module power degradation data obtained semi-continuously and statistically by in-situ dark current-voltage measurements in an environmental chamber. The modeling enables prediction of degradation rates and times as functions of temperature and humidity. Power degradation could be modeled linearly as a function of time to the second power; additionally, we found that coulombs transferred from the active cell circuit to ground during the stress test is approximately linear with time. Therefore, the power loss could be linearized as a function of coulombs squared. With this result, we observed that when the module face was completely grounded with a condensed phase conductor, leakage current exceeded the anticipated corresponding degradation rate relative to the other tests performed in damp heat.

  13. Effects of gamma irradiation on chemical composition and ruminal protein degradation of canola meal

    NASA Astrophysics Data System (ADS)

    Shawrang, P.; Nikkhah, A.; Zare-Shahneh, A.; Sadeghi, A. A.; Raisali, G.; Moradi-Shahrebabak, M.

    2008-07-01

    Gamma irradiation of canola meal (at doses of 25, 50 and 75 kGy) could alter its ruminal protein degradation characteristics by cross-linking of the polypeptide chains. This processing resulted in decrease (linear effect, P<0.001) of ruminal protein degradation and increase (linear effect, P<0.001) of intestinal protein digestibility. The results showed that gamma irradiation at doses higher than 25 kGy can be used as a cross-linking agent to improve protein properties of supplements in ruminant nutrition.

  14. Critical lysine residues of Klf4 required for protein stabilization and degradation

    SciTech Connect

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  15. Performance degradation studies on an poly 2,5-benzimidazole high-temperature proton exchange membrane fuel cell using an accelerated degradation technique

    NASA Astrophysics Data System (ADS)

    Jung, Guo-Bin; Chen, Hsin-Hung; Yan, Wei-Mon

    2014-02-01

    In this work, the performance degradation of a poly 2,5-benzimidazole (ABPBI) based high-temperature proton exchange membrane fuel cell (HT-PEMFC) was examined using an accelerated degradation technique (ADT). Experiments using an ADT with 30 min intervals were performed by applying 1.5 V to a membrane electrode assembly (MEA) with hydrogen and nitrogen feeding to the anode and cathode, respectively, to simulate the high voltage generated during fuel cell shutdown and restart. The characterization of the MEAs was performed using in-situ and ex-situ electrochemical methods, such as polarization curves, AC impedance, and cyclic voltammetry (CV), and TEM imaging before and after the ADT experiments. The measured results demonstrated that the ADT testing could be used to dramatically reduce the duration of the degradation. The current output at 0.4 V decreased by 48% after performing ADT testing for 30 min. From the AC impedance, CV and RTGA measurements, the decline in cell performance was found to be primarily due to corrosion and thinning of the catalyst layer (or carbon support) during the first 30 min, leading to the dissolution and agglomeration of the platinum catalyst.

  16. THE DELICATE BALANCE BETWEEN SECRETED PROTEIN FOLDING AND ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION IN HUMAN PHYSIOLOGY

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.

    2014-01-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates. PMID:22535891

  17. Analysis of junction temperature and modification of luminous flux degradation for white LEDs in a thermal accelerated reliability test.

    PubMed

    Ke, Hong-Liang; Jing, Lei; Hao, Jian; Gao, Qun; Wang, Yao; Wang, Xiao-Xun; Sun, Qiang; Xu, Zhi-Jun

    2016-08-01

    An accelerated aging test is the main method in evaluation of the reliability of light-emitting diodes (LEDs), and the first goal of this study is to investigate how the junction temperature (Tj) of the LED varies during accelerated aging. The Tj measured by the forward voltage method shows an upward trend over the aging time, which gives a variation about 6°C-8°C after 3,000 h of aging under an ambient temperature of 80°C. The second goal is to investigate how the variation of Tj affects the lifetime estimation. It is verified that at a certain aging stage, as Tj increases, the normalized luminous flux linearly decreases with variation rate of microns (μ) (1/°C). Then, we propose a method to modify the luminous flux degradation with the Tj and μ to meet the requirements of a constant degradation rate in the data fitting. The experimental results show that with the proposed method, the accelerated lifetimes of samples are bigger than that of the current method with increment values from 8.8% to 21.4% in this research. PMID:27505370

  18. Differential degradation for small heat shock proteins IbpA and IbpB is synchronized in Escherichia coli: implications for their functional cooperation in substrate refolding.

    PubMed

    Shi, Xiaodong; Yan, Linxuan; Zhang, Hanlin; Sun, Kai; Chang, Zengyi; Fu, Xinmiao

    2014-09-26

    Small heat shock proteins (sHSPs), as a conserved family of ATP-independent molecular chaperones, are known to bind non-native substrate proteins and facilitate the substrate refolding in cooperation with ATP-dependent chaperones (e.g., DnaK and ClpB). However, how different sHSPs function in coordination is poorly understood. Here we report that IbpA and IbpB, the two sHSPs of Escherichia coli, are coordinated by synchronizing their differential in vivo degradation. Whereas the individually expressed IbpA and IbpB are respectively degraded slowly and rapidly in cells cultured under both heat shock and normal conditions, their simultaneous expression leads to a synchronized degradation at a moderate rate. Apparently, such synchronization is linked to their hetero-oligomerization and cooperation in binding substrate proteins. In addition, truncation of the flexible N- and C-terminal tails dramatically suppresses the IbpB degradation, and somehow accelerates the IbpA degradation. In view of these in vivo data, we propose that the synchronized degradation for IbpA and IbpB are crucial for their synergistic promoting effect on DnaK/ClpB-mediated substrate refolding, conceivably via the formation of IbpA-IbpB-substrate complexes. This scenario may be common for different sHSPs that interact with each other in cells. PMID:25173932

  19. Effects of Synchronicity of Carbohydrate and Protein Degradation on Rumen Fermentation Characteristics and Microbial Protein Synthesis

    PubMed Central

    Seo, J. K.; Kim, M. H.; Yang, J. Y.; Kim, H. J.; Lee, C. H.; Kim, K. H.; Ha, Jong K.

    2013-01-01

    A series of in vitro studies were carried out to determine i) the effects of enzyme and formaldehyde treatment on the degradation characteristics of carbohydrate and protein sources and on the synchronicity of these processes, and ii) the effects of synchronizing carbohydrate and protein supply on rumen fermentation and microbial protein synthesis (MPS) in in vitro experiments. Untreated corn (C) and enzyme-treated corn (EC) were combined with soy bean meal with (ES) and without (S) enzyme treatment or formaldehyde treatment (FS). Six experimental feeds (CS, CES, CFS, ECS, ECES and ECFS) with different synchrony indices were prepared. Highly synchronous diets had the greatest dry matter (DM) digestibility when untreated corn was used. However, the degree of synchronicity did not influence DM digestibility when EC was mixed with various soybean meals. At time points of 12 h and 24 h of incubation, EC-containing diets showed lower ammonia-N concentrations than those of C-containing diets, irrespective of the degree of synchronicity, indicating that more efficient utilization of ammonia-N for MPS was achieved by ruminal microorganisms when EC was offered as a carbohydrate source. Within C-containing treatments, the purine base concentration increased as the diets were more synchronized. This effect was not observed when EC was offered. There were significant effects on VFA concentration of both C and S treatments and their interactions. Similar to purine concentrations, total VFA production and individual VFA concentration in the groups containing EC as an energy source was higher than those of other groups (CS, CES and CFS). The results of the present study suggested that the availability of energy or the protein source are the most limiting factors for rumen fermentation and MPS, rather than the degree of synchronicity. PMID:25049798

  20. Kinetic and multidimensional profiling of accelerated degradation of oil sludge by biostimulation.

    PubMed

    Dong, Yijie; Lang, Zhe; Kong, Xian; Lu, Diannan; Liu, Zheng

    2015-04-01

    Biostimulation, which employs nutrients to enhance the proliferation of indigenous microorganisms and therefore the degradation of contaminants, is an effective tool for treatment of oil-contaminated soil. However, the evolution of microbial ecology, which responds directly to stimulation procedures and intrinsically determines the degradation of oil contaminants, has rarely been explored, particularly in the context of biostimulation. In this study, the effects of biostimulation procedures including the regulation of the C : N : P ratio, as well as application of surfactants and electron acceptors in the degradation of crude oil contaminants and the evolution of the microbial community were examined simultaneously to provide ecological insights into the biostimulation. The real-time PCR showed that biostimulation promoted the proliferation of bacteria, with Gammaproteobacteria showing the greatest increase. However, the proliferation of fungi was inhibited by the accumulation of the degradation products. The degradation of polar compounds of crude oil contaminants was characterized by negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (negative-ion ESI FT-ICR MS), showing a biased increase in the relative abundance of naphthenic acids. Principal component analysis (PCA) showed that different species in oil sludge have different degradation rates during biostimulation. The addition of fertilizers with surfactants and electron acceptors profoundly stimulated the indigenous microorganisms with N1, O1 and O2 species as substrates while those with O3 and O4 species were little affected. An enriched abundance of alkB genes was observed during the degradation of saturated hydrocarbons. Monitoring the kinetics of the microbial community, functional genes and degradation offers a comprehensive view for the understanding and optimization of the biostimulation process. PMID:25699544

  1. Accelerated stress factors and failure/degradation mechanisms in terrestrial solar cells

    NASA Technical Reports Server (NTRS)

    Lathrop, J. W.

    1984-01-01

    Plans for the development of amorphous cell accelerated test measurement instrumentation are outlined. Diagrams for an 11-lamp ELH solar simulator and ac light source instrumentation are given. Examples of ac and dc analysis graphs are also provided.

  2. Data for accelerated degradation of calcium phosphate surface-coated polycaprolactone and polycaprolactone/bioactive glass composite scaffolds

    PubMed Central

    Poh, Patrina S.P.; Hutmacher, Dietmar W.; Holzapfel, Boris M.; Solanki, Anu K.; Woodruff, Maria A.

    2016-01-01

    Polycaprolactone (PCL)-based composite scaffolds containing 50 wt% of 45S5 bioactive glass (45S5) or strontium-substituted bioactive glass (SrBG) particles were fabricated into scaffolds using melt-extrusion based additive manufacturing technique. Additionally, the PCL scaffolds were surface coated with a layer of calcium phosphate (CaP). For a comparison of the scaffold degradation, the scaffolds were then subjected to in vitro accelerated degradation by immersion in 5 M sodium hydroxide (NaOH) solution for up to 7 days. The scaffold׳s morphology was observed by means of SEM imaging and scaffold mass loss was recorded over the experimental period. PMID:27081669

  3. Data for accelerated degradation of calcium phosphate surface-coated polycaprolactone and polycaprolactone/bioactive glass composite scaffolds.

    PubMed

    Poh, Patrina S P; Hutmacher, Dietmar W; Holzapfel, Boris M; Solanki, Anu K; Woodruff, Maria A

    2016-06-01

    Polycaprolactone (PCL)-based composite scaffolds containing 50 wt% of 45S5 bioactive glass (45S5) or strontium-substituted bioactive glass (SrBG) particles were fabricated into scaffolds using melt-extrusion based additive manufacturing technique. Additionally, the PCL scaffolds were surface coated with a layer of calcium phosphate (CaP). For a comparison of the scaffold degradation, the scaffolds were then subjected to in vitro accelerated degradation by immersion in 5 M sodium hydroxide (NaOH) solution for up to 7 days. The scaffold׳s morphology was observed by means of SEM imaging and scaffold mass loss was recorded over the experimental period. PMID:27081669

  4. Immunolocalization of Tom1 in relation to protein degradation systems in Alzheimer's disease.

    PubMed

    Makioka, Kouki; Yamazaki, Tsuneo; Takatama, Masamitsu; Ikeda, Masaki; Murayama, Shigeo; Okamoto, Koichi; Ikeda, Yoshio

    2016-06-15

    Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Its pathological hallmarks are senile plaques (SPs), which contain extracellular deposits of amyloid β (Aβ) protein fibrils and dystrophic neurites (DNs), and neurofibrillary tangles (NFTs) containing hyperphosphorylated tau. Impairment of protein-degradation systems, including the ubiquitin-proteasome and the autophagy-lysosome systems, has been proposed as one of the causes of the accumulation of these aberrant proteins in AD brains. Tom1 (target of Myb1) was originally identified by the induction of its expression by the v-Myb oncogene and is a part of two major protein-degradation systems. The present study was conducted by immunohistochemical and immunofluorescent stainings to show that Tom1 was localized in DNs, perisomatic granules (PSGs), and NFTs in AD brains. Moreover, in DNs, Tom1 colocalized with ubiquitin, lysosomal proteins, and Tom1-related proteins (Tollip and myosin VI), which act in both protein-degradation systems via Tom1. These results indicate that Tom1 plays important roles in protein-degradation systems in AD pathogenesis. PMID:27206884

  5. Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the curre...

  6. Degradation of HaloTag-fused nuclear proteins using bestatin-HaloTag ligand hybrid molecules.

    PubMed

    Tomoshige, Shusuke; Naito, Mikihiko; Hashimoto, Yuichi; Ishikawa, Minoru

    2015-10-14

    We have developed a protein knockdown technology using hybrid small molecules designed as conjugates of a ligand for the target protein and a ligand for ubiquitin ligase cellular inhibitor of apoptosis protein 1 (cIAP1). However, this technology has several limitations. Here, we report the development of a novel protein knockdown system to address these limitations. In this system, target proteins are fused with HaloTag to provide a common binding site for a degradation inducer. We designed and synthesized small molecules consisting of alkyl chloride as the HaloTag-binding degradation inducer, which binds to HaloTag, linked to BE04 (2), which binds to cIAP1. Using this system, we successfully knocked down HaloTag-fused cAMP responsive element binding protein 1 (HaloTag-CREB1) and HaloTag-fused c-jun (HaloTag-c-jun), which are ligand-unknown nuclear proteins, in living cells. HaloTag-binding degradation inducers can be synthesized easily, and are expected to be useful as biological tools for pan-degradation of HaloTag-fused proteins. PMID:26338696

  7. Senescence-Associated Vacuoles, a Specific Lytic Compartment for Degradation of Chloroplast Proteins?

    PubMed Central

    Carrión, Cristian A.; Martínez, Dana E.; Costa, M. Lorenza; Guiamet, Juan José

    2014-01-01

    Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s) involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, “senescence-associated vacuoles” (SAVs), characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves. PMID:27135516

  8. A synthetic defect in protein degradation caused by loss of Ufd4 and Rad23

    SciTech Connect

    Ju Donghong; Xie Youming . E-mail: xiey@karmanos.org

    2006-03-10

    The UFD (ubiquitin fusion degradation) pathway is responsible for multiubiquitination of the fusion proteins that bear a 'non-removable' N-terminal ubiquitin moiety. Previous reports have shown that the UFD pathway is conserved from yeast to human. The essential elements of the UFD pathway have also been identified in Saccharomyces cerevisiae. These studies, however, are limited to use of engineered UFD substrates. The biological significance of the UFD pathway remains unknown. Here we demonstrate that Ufd4, the E3 component of the UFD pathway, is involved in controlling the degradation of Rad4, a nucleotide excision repair protein. Moreover, simultaneous loss of Ufd4 and Rad23 exhibits a synthetic inhibitory effect on Rad4 degradation, presenting First example that a UBA/UBL-domain protein functionally overlaps with a ubiquitin ligase in determining the turnover rate of a protein substrate. The current work also provides a direction for further investigation of the physiological functions of the UFD pathway.

  9. Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis

    PubMed Central

    Kaushik, Susmita; Cuervo, Ana Maria

    2015-01-01

    Chaperone-mediated autophagy (CMA) selectively degrades a subset of cytosolic proteins in lysosomes. A potent physiological activator of CMA is nutrient deprivation, a condition in which intracellular triglyceride stores or lipid droplets (LD) also undergo hydrolysis (lipolysis) to generate free fatty acids for energetic purposes. Here we report that LD-associated proteins perilipin 2 (PLIN2) and perilipin 3 (PLIN3) are CMA substrates and their degradation via CMA precedes lipolysis. In vivo studies revealed that CMA degradation of PLIN2 and PLIN3 was enhanced during starvation, concurrent with elevated levels of cytosolic adipose triglyceride lipase (ATGL) and macroautophagy proteins on LD. CMA blockage both in cultured cells and mouse liver or expression of CMA-resistant PLINs lead to reduced association of ATGL and macrolipophagy-related proteins with LD and the subsequent decrease in lipid oxidation and accumulation of LD. We propose a role of CMA in LD biology and in the maintenance of lipid homeostasis. PMID:25961502

  10. Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity.

    PubMed

    Lambert, Nevin A; Johnston, Christopher A; Cappell, Steven D; Kuravi, Sudhakiranmayi; Kimple, Adam J; Willard, Francis S; Siderovski, David P

    2010-04-13

    G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G betagamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of "regulator of G-protein signaling" (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a G alpha switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP x AlF(4)(-)-bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A) x GDP assumed a divergent conformation more closely resembling the GDP x AlF(4)(-)-bound state. When placed within Saccharomyces cerevisiae G alpha subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context. PMID:20351284

  11. Catalase protects HepG2 cells from apoptosis induced by DNA-damaging agents by accelerating the degradation of p53.

    PubMed

    Bai, Jingxiang; Cederbaum, Arthur I

    2003-02-14

    Oxidants such as H(2)O(2) play a role in the toxicity of certain DNA-damaging agents, a process that often involves the tumor suppressor p53. H(2)O(2) is rapidly degraded by catalase, which protects cells against oxidant injury. To study the effect of catalase on apoptosis induced by DNA-damaging agents, HepG2 cells were infected with adenovirus containing the cDNA of catalase (Ad-Cat). Forty-eight hours after infection, catalase protein and activity was increased 7-10-fold compared with control cells infected with Ad-LacZ. After treatment with Vp16 or mitomycin C, control cells underwent apoptosis in a p53-dependent manner; however, overexpression of catalase inhibited this apoptosis. Basal levels as well as Vp16- or mitomycin C-stimulated levels of p53 and p21 protein were decreased in the catalase-overexpressing cells as compared with control cells; however, p53 mRNA levels were not decreased by catalase. There was no difference in p53 protein synthesis between catalase-overexpressing cells and control cells. However, pulse-chase experiments indicated that p53 protein degradation was enhanced in the catalase-overexpressing cells. Proteasome inhibitors but not calpeptin prevented the catalase-mediated decrease of p53 content. Whereas Vp16 increased, catalase overexpression decreased the phosphorylation of p53. The protein phosphatase inhibitor okadaic acid did not prevent the catalase-mediated down-regulation of p53 or phosphorylated p53. These results demonstrate that catalase protects HepG2 cells from apoptosis induced by DNA-damaging agents in association with decreasing p53 phosphorylation; the latter may lead to an acceleration in the degradation of p53 protein by the proteasome complex. This suggests that the level of catalase may play a critical role in cell-induced resistance to the effects of anti-cancer drugs which up-regulate p53. PMID:12468545

  12. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.

    PubMed

    Antunes, Ana T; Goos, Yvonne J; Pereboom, Tamara C; Hermkens, Dorien; Wlodarski, Marcin W; Da Costa, Lydie; MacInnes, Alyson W

    2015-07-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  13. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway

    PubMed Central

    Hermkens, Dorien; Wlodarski, Marcin W.; Da Costa, Lydie; MacInnes, Alyson W.

    2015-01-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  14. Ribosomal proteins produced in excess are degraded by the ubiquitin-proteasome system.

    PubMed

    Sung, Min-Kyung; Reitsma, Justin M; Sweredoski, Michael J; Hess, Sonja; Deshaies, Raymond J

    2016-09-01

    Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality control (QC) mechanisms remain poorly characterized. Here we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin-proteasome system and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and ubiquitinated and degraded within the nuclear compartment. PMID:27385339

  15. Reactive oxygen species accelerate degradation of anion exchange membranes based on polyphenylene oxide in alkaline environments.

    PubMed

    Parrondo, Javier; Wang, Zhongyang; Jung, Min-Suk J; Ramani, Vijay

    2016-07-20

    Anion exchange membranes (AEM) based on polyphenylene oxide (PPO) suffered quaternary-ammonium-cation-site degradation in alkaline environments. Surprisingly, the degradation rate was considerably faster in the presence of molecular oxygen. We postulated that the AEM cation-site catalyzes the reduction of dioxygen by hydroxide ions to yield the superoxide anion radical and the highly reactive hydroxyl free radical. We substantiated our hypothesis by using a phosphorous-containing spin trap (5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide) to detect the adducts for both free radicals in situ using (31)P-NMR spectroscopy. PMID:27381009

  16. In vitro ruminal degradation and synthesis of protein on fractions extracted from alfalfa hay and silage.

    PubMed

    Peltekova, V D; Broderick, G A

    1996-04-01

    Net release of degraded N as NH3 and total AA plus microbial protein synthesis, quantified from incorporation of 15NH3 into microbial protein, was used to estimate the rate and extent of in vitro degradation of protein fractions isolated from alfalfa hay and silage. Seven proteins (casein, alfalfa hay, alfalfa silage, extracts from alfalfa hay and silage, and residues from alfalfa hay and silage) were studied. Results from (NH4)2SO4 and SDS-PAGE fractionations suggested that soluble proteins in alfalfa hay and silage differed in susceptibility to proteolytic attack. Although the net release of NH3 plus total AA N from alfalfa silage and alfalfa silage extract was twofold greater than that from alfalfa hay and alfalfa hay extract, net microbial protein synthesis on alfalfa hay and alfalfa hay extract was 33 and 43% greater. Despite greater NPN content in alfalfa silage, protein degradation rate and estimated escape were similar for intact alfalfa hay (0.103/h and 43%) and silage (0.067/h and 43%). This result might be explained by the less efficient microbial utilization of silage NPN, greater protozoal numbers on hay, greater soluble true protein in hay, or differences in molecular mass and stability of soluble proteins in hay versus silage. Use of a two-compartment model, based on water-soluble and insoluble CP fractions assumed to pass with the liquid and solid phases, respectively, yielded RUP estimates for alfalfa hay and silage that were similar to NRC estimates. PMID:8744226

  17. Aphid salivary proteases are capable of degrading sieve-tube proteins.

    PubMed

    Furch, Alexandra C U; van Bel, Aart J E; Will, Torsten

    2015-02-01

    Sieve tubes serve as transport conduits for photo-assimilates and other resources in angiosperms and are profitable targets for piercing-sucking insects such as aphids. Sieve-tube sap also contains significant amounts of proteins with diverse functions, for example in signalling, metabolism, and defence. The identification of salivary proteases in Acyrthosiphon pisum led to the hypothesis that aphids might be able to digest these proteins and by doing so suppress plant defence and access additional nitrogen sources. Here, the scarce knowledge of proteases in aphid saliva is briefly reviewed. In order to provide a better platform for discussion, we conducted a few tests on in vitro protease activity and degradation of sieve-tube sap proteins of Cucurbita maxima by watery saliva. Inhibition of protein degradation by EDTA indicates the presence of different types of proteases (e.g. metalloproteses) in saliva of A. pisum. Proteases in the watery saliva from Macrosiphum euphorbiae and A. pisum were able to degrade the most abundant phloem protein, which is phloem protein 1. Our results provide support for the breakdown of sieve-element proteins by aphid saliva in order to suppress/neutralize the defence responses of the plant and to make proteins of sieve-tube sap accessible as a nitrogen source, as is discussed in detail. Finally, we discuss whether glycosylation of sieve-element proteins and the presence of protease inhibitors may confer partial protection against the proteolytic activity of aphid saliva. PMID:25540441

  18. Cathepsins L and Z Are Critical in Degrading Polyglutamine-containing Proteins within Lysosomes*

    PubMed Central

    Bhutani, Nidhi; Piccirillo, Rosanna; Hourez, Raphael; Venkatraman, Prasanna; Goldberg, Alfred L.

    2012-01-01

    In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins. PMID:22451661

  19. Small-Molecule Hydrophobic Tagging Induced Degradation of HaloTag Fusion Proteins

    PubMed Central

    Neklesa, Taavi K.; Tae, Hyun Seop; Schneekloth, Ashley R.; Stulberg, Michael J.; Corson, Timothy W.; Sundberg, Thomas B.; Raina, Kanak; Holley, Scott A.; Crews, Craig M.

    2011-01-01

    The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules that bind a bacterial dehalogenase (HaloTag protein) and present a hydrophobic group on its surface. Remarkably, hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated, and transmembrane fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting RasG12V-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models. PMID:21725302

  20. Conductive iron oxide minerals accelerate syntrophic cooperation in methanogenic benzoate degradation.

    PubMed

    Zhuang, Li; Tang, Jia; Wang, Yueqiang; Hu, Min; Zhou, Shungui

    2015-08-15

    Recent studies have suggested that conductive iron oxide minerals can facilitate syntrophic metabolism of the methanogenic degradation of organic matter, such as ethanol, propionate and butyrate, in natural and engineered microbial ecosystems. This enhanced syntrophy involves direct interspecies electron transfer (DIET) powered by microorganisms exchanging metabolic electrons through electrically conductive minerals. Here, we evaluated the possibility that conductive iron oxides (hematite and magnetite) can stimulate the methanogenic degradation of benzoate, which is a common intermediate in the anaerobic metabolism of aromatic compounds. The results showed that 89-94% of the electrons released from benzoate oxidation were recovered in CH4 production, and acetate was identified as the only carbon-bearing intermediate during benzoate degradation. Compared with the iron-free controls, the rates of methanogenic benzoate degradation were enhanced by 25% and 53% in the presence of hematite and magnetite, respectively. This stimulatory effect probably resulted from DIET-mediated methanogenesis in which electrons transfer between syntrophic partners via conductive iron minerals. Phylogenetic analyses revealed that Bacillaceae, Peptococcaceae, and Methanobacterium are potentially involved in the functioning of syntrophic DIET. Considering the ubiquitous presence of iron minerals within soils and sediments, the findings of this study will increase the current understanding of the natural biological attenuation of aromatic hydrocarbons in anaerobic environments. PMID:25827267

  1. Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins

    PubMed Central

    Boname, Jessica M.; Bloor, Stuart; Wandel, Michal P.; Nathan, James A.; Antrobus, Robin; Dingwell, Kevin S.; Thurston, Teresa L.; Smith, Duncan L.; Smith, James C.; Randow, Felix

    2014-01-01

    The regulated turnover of endoplasmic reticulum (ER)–resident membrane proteins requires their extraction from the membrane lipid bilayer and subsequent proteasome-mediated degradation. Cleavage within the transmembrane domain provides an attractive mechanism to facilitate protein dislocation but has never been shown for endogenous substrates. To determine whether intramembrane proteolysis, specifically cleavage by the intramembrane-cleaving aspartyl protease signal peptide peptidase (SPP), is involved in this pathway, we generated an SPP-specific somatic cell knockout. In a stable isotope labeling by amino acids in cell culture–based proteomics screen, we identified HO-1 (heme oxygenase-1), the rate-limiting enzyme in the degradation of heme to biliverdin, as a novel SPP substrate. Intramembrane cleavage by catalytically active SPP provided the primary proteolytic step required for the extraction and subsequent proteasome-dependent degradation of HO-1, an ER-resident tail-anchored protein. SPP-mediated proteolysis was not limited to HO-1 but was required for the dislocation and degradation of additional tail-anchored ER-resident proteins. Our study identifies tail-anchored proteins as novel SPP substrates and a specific requirement for SPP-mediated intramembrane cleavage in protein turnover. PMID:24958774

  2. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins

    PubMed Central

    Bezawork-Geleta, Ayenachew; Brodie, Erica J.; Dougan, David A.; Truscott, Kaye N.

    2015-01-01

    Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPRmt). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPRmt. Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPRmt, this pathway may preferentially act to promote chaperone mediated refolding of proteins. PMID:26627475

  3. E6-AP promotes misfolded polyglutamine proteins for proteasomal degradation and suppresses polyglutamine protein aggregation and toxicity.

    PubMed

    Mishra, Amit; Dikshit, Priyanka; Purkayastha, Sudarshana; Sharma, Jaiprakash; Nukina, Nobuyuki; Jana, Nihar Ranjan

    2008-03-21

    The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most age-related neurodegenerative disorders including polyglutamine diseases. Appearance of aggregates of the misfolded mutant disease proteins suggest that cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. Recently, the quality control ubiquitin ligase CHIP has been shown to suppress the polyglutamine protein aggregation and toxicity. Here we have identified another ubiquitin ligase, called E6-AP, which is able to promote the proteasomal degradation of misfolded polyglutamine proteins and suppress the polyglutamine protein aggregation and polyglutamine protein-induced cell death. E6-AP interacts with the soluble misfolded polyglutamine protein and associates with their aggregates in both cellular and transgenic mouse models. Partial knockdown of E6-AP enhances the rate of aggregate formation and cell death mediated by the polyglutamine protein. Finally, we have demonstrated the up-regulation of E6-AP in the expanded polyglutamine protein-expressing cells as well as cells exposed to proteasomal stress. These findings suggest that E6-AP is a critical mediator of the neuronal response to misfolded polyglutamine proteins and represents a potential therapeutic target in the polyglutamine diseases. PMID:18201976

  4. Reduction-oxidation state and protein degradation in skeletal muscle of fasted and refed rats

    NASA Technical Reports Server (NTRS)

    Fagan, Julie M.; Tischler, Marc E.

    1986-01-01

    Redox state and protein degradation were measured in isolated muscles of fasted (up to 10 d) and refed (up to 4 d) 7- to 14-wk-old rats. Protein degradation in the extensor digitorum longus muscle, but not in the soleus muscle, was greater in the fasted rats than in weight-matched muscle from fed rats. The NAD couple was more oxidized in incubated and fresh extensor digitorum longus muscles and in some incubated soleus muscles of fasted rats than in weight-matched muscle from fed rats. In the extensor digitorum longus muscle of refed or prolonged fasted rats, protein degradation was slower and the NAD couple was more reduced than in the fed state. Therefore, oxidation of the NAD couple was associated with increased muscle breakdown during fasting, whereas reduction of the NAD couple was associated with muscle conservation and deposition.

  5. Degradation versus aggregation of misfolded maltose-binding protein in the periplasm of Escherichia coli.

    PubMed

    Betton, J M; Sassoon, N; Hofnung, M; Laurent, M

    1998-04-10

    The periplasmic fates of misfolded MalE31, a defective folding mutant of the maltose-binding protein, were determined by manipulating two cellular activities affecting the protein folding pathway in host cells: (i) the malEp promoter activity, which is controlled by the transcriptional activator MalT, and (ii) the DegP and Protease III periplasmic proteolytic activity. At a low level of expression, the degradation of misfolded MalE31 was partially impaired in cells lacking DegP or Protease III. At a high level of expression, misfolded MalE31 rapidly formed periplasmic inclusion bodies and thus escaped degradation. However, the manipulated host cell activities did not enhance the production of periplasmic, soluble MalE31. A kinetic competition between folding, aggregation, and degradation is proposed as a general model for the biogenesis of periplasmic proteins. PMID:9535871

  6. Active Degradation Explains the Distribution of Nuclear Proteins during Cellular Senescence

    PubMed Central

    Giampieri, Enrico; De Cecco, Marco; Remondini, Daniel; Sedivy, John; Castellani, Gastone

    2015-01-01

    The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF) undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction), and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome. PMID:26115222

  7. Structural basis of Ornithine Decarboxylase inactivation and accelerated degradation by polyamine sensor Antizyme1

    PubMed Central

    Wu, Donghui; Kaan, Hung Yi Kristal; Zheng, Xiaoxia; Tang, Xuhua; He, Yang; Vanessa Tan, Qianmin; Zhang, Neng; Song, Haiwei

    2015-01-01

    Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step of polyamine biosynthesis in humans. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Excessive accumulation of polyamines has a cytotoxic effect on cells and elevated level of ODC activity is associated with cancer development. To maintain normal cellular proliferation, regulation of polyamine synthesis is imposed by Antizyme1 (AZ1). The expression of AZ1 is induced by a ribosomal frameshifting mechanism in response to increased intracellular polyamines. AZ1 regulates polyamine homeostasis by inactivating ODC activity and enhancing its degradation. Here, we report the structure of human ODC in complex with N-terminally truncated AZ1 (cAZ1). The structure shows cAZ1 binding to ODC, which occludes the binding of a second molecule of ODC to form the active homodimer. Consequently, the substrate binding site is disrupted and ODC is inactivated. Structural comparison shows that the binding of cAZ1 to ODC causes a global conformational change of ODC and renders its C-terminal region flexible, therefore exposing this region for degradation by the 26S proteasome. Our structure provides the molecular basis for the inactivation of ODC by AZ1 and sheds light on how AZ1 promotes its degradation. PMID:26443277

  8. Mechanochemical basis of protein degradation by a double-ring AAA+ machine.

    PubMed

    Olivares, Adrian O; Nager, Andrew R; Iosefson, Ohad; Sauer, Robert T; Baker, Tania A

    2014-10-01

    Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins. We demonstrate that ClpA unfolds some protein substrates substantially faster than does the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates. PMID:25195048

  9. Regulated protein depletion by the auxin-inducible degradation system in Drosophila melanogaster.

    PubMed

    Trost, Martina; Blattner, Ariane C; Lehner, Christian F

    2016-01-01

    The analysis of consequences resulting after experimental elimination of gene function has been and will continue to be an extremely successful strategy in biological research. Mutational elimination of gene function has been widely used in the fly Drosophila melanogaster. RNA interference is used extensively as well. In the fly, exceptionally precise temporal and spatial control over elimination of gene function can be achieved in combination with sophisticated transgenic approaches and clonal analyses. However, the methods that act at the gene and transcript level cannot eliminate protein products which are already present at the time when mutant cells are generated or RNA interference is started. Targeted inducible protein degradation is therefore of considerable interest for controlled rapid elimination of gene function. To this end, a degradation system was developed in yeast exploiting TIR1, a plant F box protein, which can recruit proteins with an auxin-inducible degron to an E3 ubiquitin ligase complex, but only in the presence of the phytohormone auxin. Here we demonstrate that the auxin-inducible degradation system functions efficiently also in Drosophila melanogaster. Neither auxin nor TIR1 expression have obvious toxic effects in this organism, and in combination they result in rapid degradation of a target protein fused to the auxin-inducible degron. PMID:27010248

  10. Drosophila IAP1-Mediated Ubiquitylation Controls Activation of the Initiator Caspase DRONC Independent of Protein Degradation

    PubMed Central

    Wang, Shiuan; Srivastava, Mayank; Broemer, Meike; Meier, Pascal; Bergmann, Andreas

    2011-01-01

    Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition (“undead” cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in “undead” cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs. PMID:21909282

  11. Dual Positive Feedback Regulation of Protein Degradation of an Extra-cytoplasmic Function σ Factor for Cell Differentiation in Streptomyces coelicolor *

    PubMed Central

    Mao, Xu-Ming; Sun, Ning; Wang, Feng; Luo, Shuai; Zhou, Zhan; Feng, Wei-Hong; Huang, Fang-Liang; Li, Yong-Quan

    2013-01-01

    Here we report that in Streptomyces coelicolor, the protein stability of an ECF σ factor SigT, which is involved in the negative regulation of cell differentiation, was completely dependent on its cognate anti-σ factor RstA. The degradation of RstA caused a ClpP/SsrA-dependent degradation of SigT during cell differentiation. This was consistent with the delayed morphological development or secondary metabolism in the ΔclpP background after rstA deletion or sigT overexpression. Meanwhile, SigT negatively regulated clpP/ssrA expression by directly binding to the clpP promoter (clpPp). The SigT-clpPp interaction could be disrupted by secondary metabolites, giving rise to the stabilized SigT protein and retarded morphological development in a non-antibiotic-producing mutant. Thus a novel regulatory mechanism was revealed that the protein degradation of the ECF σ factor was initiated by the degradation of its anti-σ factor, and was accelerated in a dual positive feedback manner, through regulation by secondary metabolites, to promote rapid and irreversible development of the secondary metabolism. This ingenious cooperation of intracellular components can ensure economical and exquisite control of the ECF σ factor protein level for the proper cell differentiation in Streptomyces. PMID:24014034

  12. Systematic Protein Level Regulation via Degradation Machinery Induced by Genotoxic Drugs.

    PubMed

    Kume, Kohei; Ishida, Kazushige; Ikeda, Miyuki; Takemoto, Kazuhiro; Shimura, Tsutomu; Young, Lynn; Nishizuka, Satoshi S

    2016-01-01

    In this study we monitored protein dynamics in response to cisplatin, 5-fluorouracil, and irinotecan with different concentrations and administration modes using "reverse-phase" protein arrays (RPPAs) in order to gain comprehensive insight into the protein dynamics induced by genotoxic drugs. Among 666 protein time-courses, 38% exhibited an increasing trend, 32% exhibited a steady decrease, and 30% fluctuated within 24 h after drug exposure. We analyzed almost 12,000 time-course pairs of protein levels based on the geometrical similarity by correlation distance (dCor). Twenty-two percent of the pairs showed dCor > 0.8, which indicates that each protein of the pair had similar dynamics. These trends were disrupted by a proteasome inhibitor, MG132, suggesting that the protein degradation system was activated in response to the drugs. Among the pairs with high dCor, the average dCor of pairs with apoptosis-related protein was significantly higher than those without, indicating that regulation of protein levels was induced by the drugs. These results suggest that the levels of numerous functionally distinct proteins may be regulated by common degradation machinery induced by genotoxic drugs. PMID:26625007

  13. Small ruminant lentiviral Vif proteins commonly utilize cyclophilin A, an evolutionarily and structurally conserved protein, to degrade ovine and caprine APOBEC3 proteins.

    PubMed

    Yoshikawa, Rokusuke; Izumi, Taisuke; Nakano, Yusuke; Yamada, Eri; Moriwaki, Miyu; Misawa, Naoko; Ren, Fengrong; Kobayashi, Tomoko; Koyanagi, Yoshio; Sato, Kei

    2016-06-01

    Mammals have co-evolved with retroviruses, including lentiviruses, over a long period. Evidence supporting this contention is that viral infectivity factor (Vif) encoded by lentiviruses antagonizes the anti-viral action of cellular apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) of the host. To orchestrate E3 ubiquitin ligase complex for APOBEC3 degradation, Vifs utilize mammalian proteins such as core-binding factor beta (CBFB; for primate lentiviruses) or cyclophilin A (CYPA; for Maedi-Visna virus [MVV]). However, the co-evolutionary relationship between lentiviral Vif and the mammalian proteins associated with Vif-mediated APOBEC3 degradation is poorly understood. Moreover, it is unclear whether Vif proteins of small ruminant lentiviruses (SRLVs), including MVV and caprine arthritis encephalitis virus (CAEV), commonly utilize CYPA to degrade the APOBEC3 of their hosts. In this study, molecular phylogenetic and protein homology modeling revealed that Vif co-factors are evolutionarily and structurally conserved. It was also found that not only MVV but also CAEV Vifs degrade APOBEC3 of both sheep and goats and that CAEV Vifs interact with CYPA. These findings suggest that lentiviral Vifs chose evolutionarily and structurally stable proteins as their partners (e.g., CBFB or CYPA) for APOBEC3 degradation and, particularly, that SRLV Vifs evolved to utilize CYPA as their co-factor in degradation of ovine and caprine APOBEC3. PMID:27193350

  14. Accelerated protein engineering for chemical biotechnology via homologous recombination.

    PubMed

    Nordwald, Erik M; Garst, Andrew; Gill, Ryan T; Kaar, Joel L

    2013-12-01

    Protein engineering has traditionally relied on random mutagenesis strategies to generate diverse libraries, which require high-throughput screening or selection methods to identify rare variants. Alternatively, approaches to semi-rational library construction can be used to minimize the screening load and enhance the efficiency by which improved mutants may be identified. Such methods are typically limited to characterization of relatively few variants due to the difficulties in generating large rational libraries. New tools from synthetic biology, namely multiplexed DNA synthesis and homologous recombination, provide a promising avenue to rapidly construct large, rational libraries. These technologies also enable incorporation of synthetically encoded features that permit efficient characterization of the fitness of each mutant. Extension of these tools to protein library design could complement rational protein design cycles in an effort to more systematically search complex fitness landscapes. The highly parallelized nature with which such libraries can be generated also has the potential to expand directed protein evolution from single protein targets to protein networks whose concerted activities are required for the biological function of interest. PMID:23540421

  15. Accelerated Nucleation of Hydroxyapatite Using an Engineered Hydrophobin Fusion Protein.

    PubMed

    Melcher, Melanie; Facey, Sandra J; Henkes, Thorsten M; Subkowski, Thomas; Hauer, Bernhard

    2016-05-01

    Calcium phosphate mineralization is of particular interest in dental repair. A biomimetic approach using proteins or peptides is a highly promising way to reconstruct eroded teeth. In this study, the screening of several proteins is described for their binding and nucleating activities toward hydroxyapatite. Out of 27 tested candidates, only two hydrophobin fusion proteins showed binding abilities to hydroxyapatite in a mouthwash formulation and an increased nucleation in artificial saliva. Using a semirational approach, one of the two candidates (DEWA_5), a fusion protein consisting of a truncated section of the Bacillus subtilis synthase YaaD, the Aspergillus nidulans hydrophobin DEWA, and the rationally designed peptide P11-4 described in the literature, could be further engineered toward a faster mineral formation. The variants DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA) were able to enhance the nucleation activity without losing the ability to form hydroxyapatite. In the case of variant DEWA_5b, an additional increase in the binding toward hydroxyapatite could be achieved. Especially with the variant DEWA_5a, the protein engineering of the rationally designed peptide sequence resulted in a resemblance of an amino acid motif that is found in nature. The engineered peptide resembles the amino acid motif in dentin phosphoprotein, one of the major proteins involved in dentinogenesis. PMID:27010648

  16. Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri

    PubMed Central

    Ramírez-Rico, Gerardo; Serrano-Luna, Jesús; Shibayama, Mineko

    2015-01-01

    Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium) from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C) employing conditioned medium (CM) and total crude extracts (TCEs) of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system. PMID:26090408

  17. Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri.

    PubMed

    Martínez-Castillo, Moisés; Ramírez-Rico, Gerardo; Serrano-Luna, Jesús; Shibayama, Mineko

    2015-01-01

    Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium) from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C) employing conditioned medium (CM) and total crude extracts (TCEs) of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system. PMID:26090408

  18. Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination.

    PubMed

    Braten, Ori; Livneh, Ido; Ziv, Tamar; Admon, Arie; Kehat, Izhak; Caspi, Lilac H; Gonen, Hedva; Bercovich, Beatrice; Godzik, Adam; Jahandideh, Samad; Jaroszewski, Lukasz; Sommer, Thomas; Kwon, Yong Tae; Guharoy, Mainak; Tompa, Peter; Ciechanover, Aaron

    2016-08-01

    The "canonical" proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established-in both human and yeast cells-a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes. PMID:27385826

  19. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.

    PubMed Central

    Pittman, R C; Carew, T E; Glass, C K; Green, S R; Taylor, C A; Attie, A D

    1983-01-01

    We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples. PMID:6882394

  20. Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease

    PubMed Central

    Shin, Yongdae; Davis, Joseph H.; Brau, Ricardo R.; Martin, Andreas; Kenniston, Jon A.; Baker, Tania A.; Sauer, Robert T.; Lang, Matthew J.

    2009-01-01

    ClpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. ClpX, an AAA+ enzyme, recognizes specific proteins, and then uses cycles of ATP hydrolysis to denature any native structure and to translocate the unfolded polypeptide into ClpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by ClpXP. These assays employ a single-chain variant of the ClpX hexamer, linked via a single biotin to a streptavidin-coated surface, and fusion substrates with an N-terminal fluorophore and a C-terminal GFP-titin-ssrA module. In the presence of adenosine 5′-[γ-thio]triphosphate (ATPγS), ClpXP degrades the titin-ssrA portion of these substrates but stalls when it encounters GFP. Exchange into ATP then allows synchronous resumption of denaturation and degradation of GFP and any downstream domains. GFP unfolding can be monitored directly, because intrinsic fluorescence is quenched by denaturation. The time required for complete degradation coincides with loss of the substrate fluorophore from the protease complex. Fitting single-molecule data for a set of related substrates provides time constants for ClpX unfolding, translocation, and a terminal step that may involve product release. Comparison of these single-molecule results with kinetics measured in bulk solution indicates similar levels of microscopic and macroscopic ClpXP activity. These results support a stochastic engagement/unfolding mechanism that ultimately results in highly processive degradation and set the stage for more detailed single-molecule studies of machine function. PMID:19892734

  1. GPU-accelerated visualization of protein dynamics in ribbon mode

    NASA Astrophysics Data System (ADS)

    Wahle, Manuel; Birmanns, Stefan

    2011-01-01

    Proteins are biomolecules present in living organisms and essential for carrying out vital functions. Inherent to their functioning is folding into different spatial conformations, and to understand these processes, it is crucial to visually explore the structural changes. In recent years, significant advancements in experimental techniques and novel algorithms for post-processing of protein data have routinely revealed static and dynamic structures of increasing sizes. In turn, interactive visualization of the systems and their transitions became more challenging. Therefore, much research for the efficient display of protein dynamics has been done, with the focus being space filling models, but for the important class of abstract ribbon or cartoon representations, there exist only few methods for an efficient rendering. Yet, these models are of high interest to scientists, as they provide a compact and concise description of the structure elements along the protein main chain. In this work, a method was developed to speed up ribbon and cartoon visualizations. Separating two phases in the calculation of geometry allows to offload computational work from the CPU to the GPU. The first phase consists of computing a smooth curve along the protein's main chain on the CPU. In the second phase, conducted independently by the GPU, vertices along that curve are moved to set up the final geometrical representation of the molecule.

  2. Effect of cleaning agents and additives on Protein A ligand degradation and chromatography performance.

    PubMed

    Yang, Lihua; Harding, Jason D; Ivanov, Alexander V; Ramasubramanyan, Natarajan; Dong, Diane D

    2015-03-13

    Protein A chromatography, employing the recombinant Protein A ligand, is widely used as a capture step for antibody and Fc-fusion proteins manufacture. Protein A ligands in these matrices are susceptible to degradation/loss when exposed to cleaning agents such as sodium hydroxide, resulting in loss of capacity on reuse. In this study, MabSelect Protein A ligand and MabSelect SuRe Protein A ligand were chosen to evaluate the impact of alkaline cleaning solutions on the ligands and the packed columns. The Protein A ligands alone and the Protein A columns were incubated or cycled in different concentrations of sodium hydroxide solutions with and without additives, respectively. Ligand integrity (degradation) and ligand function (binding affinity) were studied using SDS-PAGE and customized Biacore technology, surface plasma resonance (SPR) and were successfully correlated with column performance measurement in terms of static binding capacity (SBC), dynamic binding capacity (DBC) and recovery as a function of exposure to cleaning agents with and without additives. The findings and the methodology presented in this study are not only able to determine appropriate cleaning conditions for Protein A chromatography, but also provided tools to enable systematic and rapid study of the cleaning solutions and conditions. PMID:25680549

  3. Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins.

    PubMed

    Armenti, Stephen T; Lohmer, Lauren L; Sherwood, David R; Nance, Jeremy

    2014-12-01

    The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional loss-of-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events. PMID:25377555

  4. Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

    PubMed Central

    Wagner, I; Arlt, H; van Dyck, L; Langer, T; Neupert, W

    1994-01-01

    ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria. Images PMID:7957078

  5. The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation.

    PubMed

    Baumberger, Nicolas; Tsai, Ching-Hsui; Lie, Miranda; Havecker, Ericka; Baulcombe, David C

    2007-09-18

    Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group. PMID:17869110

  6. Stability of CIGS Solar Cells and Component Materials Evaluated by a Step-Stress Accelerated Degradation Test Method: Preprint

    SciTech Connect

    Pern, F. J.; Noufi, R.

    2012-10-01

    A step-stress accelerated degradation testing (SSADT) method was employed for the first time to evaluate the stability of CuInGaSe2 (CIGS) solar cells and device component materials in four Al-framed test structures encapsulated with an edge sealant and three kinds of backsheet or moisture barrier film for moisture ingress control. The SSADT exposure used a 15oC and then a 15% relative humidity (RH) increment step, beginning from 40oC/40%RH (T/RH = 40/40) to 85oC/70%RH (85/70) as of the moment. The voluminous data acquired and processed as of total DH = 3956 h with 85/70 = 704 h produced the following results. The best CIGS solar cells in sample Set-1 with a moisture-permeable TPT backsheet showed essentially identical I-V degradation trend regardless of the Al-doped ZnO (AZO) layer thickness ranging from standard 0.12 μm to 0.50 μm on the cells. No clear 'stepwise' feature in the I-V parameter degradation curves corresponding to the SSADT T/RH/time profile was observed. Irregularity in I-V performance degradation pattern was observed with some cells showing early degradation at low T/RH < 55/55 and some showing large Voc, FF, and efficiency degradation due to increased series Rs (ohm-cm2) at T/RH ≥ 70/70. Results of (electrochemical) impedance spectroscopy (ECIS) analysis indicate degradation of the CIGS solar cells corresponded to increased series resistance Rs (ohm) and degraded parallel (minority carrier diffusion/recombination) resistance Rp, capacitance C, overall time constant Rp*C, and 'capacitor quality' factor (CPE-P), which were related to the cells? p-n junction properties. Heating at 85/70 appeared to benefit the CIGS solar cells as indicated by the largely recovered CPE-P factor. Device component materials, Mo on soda lime glass (Mo/SLG), bilayer ZnO (BZO), AlNi grid contact, and CdS/CIGS/Mo/SLG in test structures with TPT showed notable to significant degradation at T/RH ≥ 70/70. At T/RH = 85/70, substantial blistering of BZO layers on CIGS

  7. Stability of CIGS solar cells and component materials evaluated by a step-stress accelerated degradation test method

    NASA Astrophysics Data System (ADS)

    Pern, F. J.; Noufi, R.

    2012-10-01

    A step-stress accelerated degradation testing (SSADT) method was employed for the first time to evaluate the stability of CuInGaSe2 (CIGS) solar cells and device component materials in four Al-framed test structures encapsulated with an edge sealant and three kinds of backsheet or moisture barrier film for moisture ingress control. The SSADT exposure used a 15°C and then a 15% relative humidity (RH) increment step, beginning from 40°C/40%RH (T/RH = 40/40) to 85°C/70%RH (85/70) as of the moment. The voluminous data acquired and processed as of total DH = 3956 h with 85/70 = 704 h produced the following results. The best CIGS solar cells in sample Set-1 with a moisture-permeable TPT backsheet showed essentially identical I-V degradation trend regardless of the Al-doped ZnO (AZO) layer thickness ranging from standard 0.12 μm to 0.50 μm on the cells. No clear "stepwise" feature in the I-V parameter degradation curves corresponding to the SSADT T/RH/time profile was observed. Irregularity in I-V performance degradation pattern was observed with some cells showing early degradation at low T/RH < 55/55 and some showing large Voc, FF, and efficiency degradation due to increased series Rs (ohm-cm2) at T/RH >= 70/70. Results of (electrochemical) impedance spectroscopy (ECIS) analysis indicate degradation of the CIGS solar cells corresponded to increased series resistance Rs (ohm) and degraded parallel (minority carrier diffusion/recombination) resistance Rp, capacitance C, overall time constant Rp*C, and "capacitor quality" factor (CPE-P), which were related to the cells' p-n junction properties. Heating at 85/70 appeared to benefit the CIGS solar cells as indicated by the largely recovered CPE-P factor. Device component materials, Mo on soda lime glass (Mo/SLG), bilayer ZnO (BZO), AlNi grid contact, and CdS/CIGS/Mo/SLG in test structures with TPT showed notable to significant degradation at T/RH >= 70/70. At T/RH = 85/70, substantial blistering of BZO layers on CIGS

  8. Carbohydrate Source and Protein Degradability Alter Lactation, Ruminal, and Blood Measures.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Effects of nonfiber carbohydrate source (NFC) and protein degradability (RDP) in the diets of lactating dairy cattle on intake, production, efficiency, and ruminal measures were evaluated in a three period (21 d) partially balanced incomplete latin square design with a 3x2 factorial arrangement of t...

  9. EFFECTS OF FEEDING DAIRY COWS PROTEIN SUPPLEMENTS OF VARYING RUMINAL DEGRADABILITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty-five (10 ruminally cannulated) Holstein cows averaging 82 ± 34 days in milk were assigned to 5x5 Latin squares (21-d periods) and fed diets supplemented with one of four different proteins to assess effects on production, ruminal metabolism, omasal flow of N fractions, and degradation rates o...

  10. Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation.

    PubMed

    Shao, Jia; Choe, Vitnary; Cheng, Haili; Tsai, Yien Che; Weissman, Allan M; Luo, Shiwen; Rao, Hai

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis. PMID:24714645

  11. Protein Degradation by Ubiquitin-Proteasome System in Formation and Labilization of Contextual Conditioning Memory

    ERIC Educational Resources Information Center

    Fustiñana, María Sol; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-01-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this…

  12. Proteasomal Degradation of Nod2 Protein Mediates Tolerance to Bacterial Cell Wall Components*

    PubMed Central

    Lee, Kyoung-Hee; Biswas, Amlan; Liu, Yuen-Joyce; Kobayashi, Koichi S.

    2012-01-01

    The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during Gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock. PMID:23019338

  13. Developing an in vitro method for determining feed soluble protein degradation rate by mixed ruminal microorganisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For the purposes of ration balancing using predictive computer models, more accurate content and rumen soluble protein degradability values are needed, especially for highly processed feeds. Consequently, a standardized method of determination is needed. Hence, a novel ruminal in vitro method is d...

  14. Leucine and isoleucine reduce protein degradation in rainbow trout (Oncorhynchus mykiss) primary myoblast cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myogenic precursor cells were isolated from rainbow trout skeletal muscle and incubated in media containing 10% fetal bovine serum for 7 days, thereby differentiating into myoblasts. Rates of protein degradation were determined in response to minimal essential media (MEM) of various amino acid (AA)...

  15. Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C*

    PubMed Central

    Lum, Michelle A.; Balaburski, Gregor M.; Murphy, Maureen E.; Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    Although alterations in stimulus-induced degradation of PKC have been implicated in disease, mechanistic understanding of this process remains limited. Evidence supports the existence of both proteasomal and lysosomal mechanisms of PKC processing. An established pathway involves rate-limiting priming site dephosphorylation of the activated enzyme and proteasomal clearance of the dephosphorylated protein. However, here we show that agonists promote down-regulation of endogenous PKCα with minimal accumulation of a nonphosphorylated species in multiple cell types. Furthermore, proteasome and lysosome inhibitors predominantly protect fully phosphorylated PKCα, pointing to this form as a substrate for degradation. Failure to detect substantive dephosphorylation of activated PKCα was not due to rephosphorylation because inhibition of Hsp70/Hsc70, which is required for re-priming, had only a minor effect on agonist-induced accumulation of nonphosphorylated protein. Thus, PKC degradation can occur in the absence of dephosphorylation. Further analysis revealed novel functions for Hsp70/Hsc70 and Hsp90 in the control of agonist-induced PKCα processing. These chaperones help to maintain phosphorylation of activated PKCα but have opposing effects on degradation of the phosphorylated protein; Hsp90 is protective, whereas Hsp70/Hsc70 activity is required for proteasomal processing of this species. Notably, down-regulation of nonphosphorylated PKCα shows little Hsp70/Hsc70 dependence, arguing that phosphorylated and nonphosphorylated species are differentially targeted for proteasomal degradation. Finally, lysosomal processing of activated PKCα is not regulated by phosphorylation or Hsps. Collectively, these data demonstrate that phosphorylated PKCα is a direct target for agonist-induced proteasomal degradation via an Hsp-regulated mechanism, and highlight the existence of a novel pathway of PKC desensitization in cells. PMID:23900841

  16. Sequence heterogeneity accelerates protein search for targets on DNA

    NASA Astrophysics Data System (ADS)

    Shvets, Alexey A.; Kolomeisky, Anatoly B.

    2015-12-01

    The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity, and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry, and heterogeneity of a genome.

  17. Sequence heterogeneity accelerates protein search for targets on DNA

    SciTech Connect

    Shvets, Alexey A.; Kolomeisky, Anatoly B.

    2015-12-28

    The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity, and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry, and heterogeneity of a genome.

  18. Effect of TiO2-Crystal Forms on the Photo-Degradation of EVA/PLA Blend Under Accelerated Weather Testing

    NASA Astrophysics Data System (ADS)

    Van Cong, Do; Trang, Nguyen Thi Thu; Giang, Nguyen Vu; Lam, Tran Dai; Hoang, Thai

    2016-05-01

    Photo-degradation of poly (ethylene-co-vinyl acetate) (EVA)/poly (lactic acid) (PLA) blend and EVA/PLA/TiO2 nanocomposites was carried out under accelerated weather testing conditions by alternating cycles of ultraviolet (UV) light and moisture at controlled and elevated temperatures. The characters, properties, and morphology of these materials before and after accelerated weather testing were determined by Fourier transform infrared spectroscopy, colour changes, viscosity, tensile test, thermogravimetric analysis, and field emission scanning electron microscopy. The increases in the content of oxygen-containing groups, colour changes; the decreases in viscosity, tensile properties, and thermal stability of these materials after accelerated weather testing are the evidence for the photo-degradation of the blend and nanocomposites. After accelerated weather testing, the appearance of many micro-holes and micro-pores on the surface of the collected samples was observed. The photo-degradation degree of the nanocomposites depended on the TiO2-crystal form. Rutile TiO2 do not enhance the degradation, but anatase and mixed crystals TiO2 nanoparticles promoted the degradation of the nanocomposites. Particularly, the mixed crystals TiO2 nanoparticles showed the highest photo-catalytic activity of the nanocomposites.

  19. Staphylococcus aureus protects its immune-evasion proteins against degradation by neutrophil serine proteases.

    PubMed

    Stapels, D A C; Kuipers, A; von Köckritz-Blickwede, M; Ruyken, M; Tromp, A T; Horsburgh, M J; de Haas, C J C; van Strijp, J A G; van Kessel, K P M; Rooijakkers, S H M

    2016-04-01

    Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti-staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune-evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune-escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus. PMID:26418545

  20. Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif.

    PubMed

    Belizario, José E; Alves, Juliano; Garay-Malpartida, Miguel; Occhiucci, João Marcelo

    2008-06-01

    The degradation is critical to activation and deactivation of regulatory proteins involved in signaling pathways to cell growth, differentiation, stress responses and physiological cell death. Proteins carry domains and sequence motifs that function as prerequisite for their proteolysis by either individual proteases or the 26S multicomplex proteasomes. Two models for entry of substrates into the proteasomes have been considered. In one model, it is proposed that the ubiquitin chain attached to the protein serves as recognition element to drag them into the 19S regulatory particle, which promotes the unfolding required to its access into the 20S catalytic chamber. In second model, it is proposed that an unstructured tail located at amino or carboxyl terminus directly track proteins into the 26S/20S proteasomes. Caspases are cysteinyl aspartate proteases that control diverse signaling pathways, promoting the cleavage at one or two sites of hundreds of structural and regulatory protein substrates. Caspase cleavage sites are commonly found within PEST motifs, which are segments rich in proline (P), glutamic acid (D), aspartic acid (E) and serine (S) or threonine (T) residues. Considering that N- and C- terminal peptide carrying PEST motifs form disordered loops in the globular proteins after caspase cleavage, it is postulated here that these exposed termini serve as unstructured initiation site, coupling caspase cleavage and ubiquitin-proteasome dependent and independent degradation of short-lived proteins. This could explain the inherent susceptibility to proteolysis among proteins containing PEST motif. PMID:18537676

  1. Surface degradation of polymer insulators under accelerated climatic aging in weather-ometer

    SciTech Connect

    Xu, G.; McGrath, P.B.; Burns, C.W.

    1996-12-31

    Climatic aging experiments were conducted on two types of outdoor polymer insulators by using a programmable weather-ometer. The housing materials for the insulators were silicone rubber (SR) and ethylene propylene diene monomer (EPDM). The accelerated aging stresses were comprised of ultraviolet radiation, elevated temperature, temperature cycling, thermal shock and high humidity. Their effects on the insulator surface conditions and electrical performance wee examined through visual inspection and SEM studies, contact angle measurements, thermogravimetric analysis (TGA), energy dispersive spectroscopy (EDS) analysis, and 50% impulse flashover voltage tests. The results showed a significant damage on the insulator surface caused by some of the imposed aging stresses. The EDS analysis suggested a photooxidation process that happened on the insulator surface during the aging period.

  2. Modelling of Zircaloy-4 accelerated degradation kinetics in nitrogen-oxygen mixtures at 850 °C

    NASA Astrophysics Data System (ADS)

    Lasserre, M.; Peres, V.; Pijolat, M.; Coindreau, O.; Duriez, C.; Mardon, J.-P.

    2015-07-01

    Zirconium-based alloys used in PWR cladding show an acceleration of their oxidation kinetics in air at high temperature compared to their behaviour under oxygen or steam alone. This paper presents an analysis of the oxidation kinetics in order to explain the role of nitrogen during the accelerated corrosion. Isothermal thermogravimetry on alloy thin plates was used to collect kinetic data during the reaction of Zircaloy-4 at 850 °C in oxygen and nitrogen mixtures. The influence of oxygen and nitrogen partial pressure on the degradation kinetics was studied by a jump method. The presence of nitrogen in the reacting gas enables the formation of zirconium nitride near the oxide-metal interface which acts as a catalytic phase. A three steps reaction path composed of nitride oxidation, α-Zr(O) nitridation and oxidation is proposed. A detailed mechanism and the rate-determining step of the overall process are proposed that account for the experimentally observed dependence of the kinetic rate with the oxygen and nitrogen partial pressures; a kinetic model based on surface nucleation and growth of regions attacked by nitrogen was successful in describing the mass variations with time of exposure at 850 °C.

  3. Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems

    PubMed Central

    Siwaszek, Aleksandra; Ukleja, Marta; Dziembowski, Andrzej

    2014-01-01

    The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5’-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3’>5’direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals. PMID:25483043

  4. Pathogenic prion protein is degraded by a manganese oxide mineral found in soils

    USGS Publications Warehouse

    Russo, F.; Johnson, C.J.; McKenzie, D.; Aiken, Judd M.; Pedersen, J.A.

    2009-01-01

    Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO2) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrPTSE) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO2 suspensions degraded the PrPTSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrPTSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO2. Exposure to 5.6 mg MnO2 ml-1 (PrPTSE:MnO2=1 : 110) decreased PrPTSE levels by ???4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals. ?? 2009 SGM.

  5. HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation

    SciTech Connect

    Okumura, Atsushi; Alce, Tim; Lubyova, Barbora; Ezelle, Heather; Strebel, Klaus; Pitha, Paula M.

    2008-03-30

    The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication.

  6. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation.

    PubMed

    Wang, Qian Qian; Zhou, Xin Yi; Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-09-22

    Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RARα oncoprotein by As2O3, which results in initiation of PML-RARα degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAs(III)) and its intermediate metabolites (i.e., MMA(III)/DMA(III)) in NB4 cells. Inorganic iAs(III) predominantly showed induction of cell differentiation, while MMA(III) and DMA(III) specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAs(III), MMA(III) showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RARα fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  7. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation

    PubMed Central

    Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-01-01

    Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RARα oncoprotein by As2O3, which results in initiation of PML-RARa degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAsIII) and its intermediate metabolites (i.e., MMAIII/DMAIII) in NB4 cells. Inorganic iAsIII predominantly showed induction of cell differentiation, while MMAIII and DMAIII specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAsIII, MMAIII showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RARa fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  8. Identification of Disulfide Bonds in Protein Proteolytic Degradation Products Using de Novo-Protein Unique Sequence Tags Approach

    SciTech Connect

    Shen, Yufeng; Tolic, Nikola; Purvine, Samuel O.; Smith, Richard D.

    2010-08-01

    Disulfide bonds are a form of posttranslational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, and specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags) we unambiguously correlated the spectra to specific database proteins. Examination of the UStags’ prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to ~10 kDa.

  9. Identification of disulfide bonds in protein proteolytic degradation products using de novo-protein unique sequence tags approach.

    PubMed

    Shen, Yufeng; Tolić, Nikola; Purvine, Samuel O; Smith, Richard D

    2010-08-01

    Disulfide bonds are a form of post-translational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags), we unambiguously correlated the spectra to specific database proteins. Examination of the UStags' prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to approximately 10 kDa. PMID:20590115

  10. Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli.

    PubMed

    Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R; Liu, Yu; Powers, David L; Kelly, Jeffery W; Gierasch, Lila M; Powers, Evan T

    2015-04-14

    The folding fate of a protein in vivo is determined by the interplay between a protein's folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. PMID:25843722

  11. Determinants of rodent longevity in the chaperone-protein degradation network.

    PubMed

    Rodriguez, Karl A; Valentine, Joseph M; Kramer, David A; Gelfond, Jonathan A; Kristan, Deborah M; Nevo, Eviatar; Buffenstein, Rochelle

    2016-05-01

    Proteostasis is an integral component of healthy aging, ensuring maintenance of protein structural and functional integrity with concomitant impact upon health span and longevity. In most metazoans, increasing age is accompanied by a decline in protein quality control resulting in the accrual of damaged, self-aggregating cytotoxic proteins. A notable exception to this trend is observed in the longest-lived rodent, the naked mole-rat (NMR, Heterocephalus glaber) which maintains proteostasis and proteasome-mediated degradation and autophagy during aging. We hypothesized that high levels of the proteolytic degradation may enable better maintenance of proteostasis during aging contributing to enhanced species maximum lifespan potential (MLSP). We test this by examining proteasome activity, proteasome-related HSPs, the heat-shock factor 1 (HSF1) transcription factor, and several markers of autophagy in the liver and quadriceps muscles of eight rodent species with divergent MLSP. All subterranean-dwelling species had higher levels of proteasome activity and autophagy, possibly linked to having to dig in soils rich in heavy metals and where underground atmospheres have reduced oxygen availability. Even after correcting for phylogenetic relatedness, a significant (p < 0.02) positive correlation between MLSP, HSP25, HSF1, proteasome activity, and autophagy-related protein 12 (ATG12) was observed, suggesting that the proteolytic degradation machinery and maintenance of protein quality play a pivotal role in species longevity among rodents. PMID:26894765

  12. An efficient parallel algorithm for accelerating computational protein design

    PubMed Central

    Zhou, Yichao; Xu, Wei; Donald, Bruce R.; Zeng, Jianyang

    2014-01-01

    Motivation: Structure-based computational protein design (SCPR) is an important topic in protein engineering. Under the assumption of a rigid backbone and a finite set of discrete conformations of side-chains, various methods have been proposed to address this problem. A popular method is to combine the dead-end elimination (DEE) and A* tree search algorithms, which provably finds the global minimum energy conformation (GMEC) solution. Results: In this article, we improve the efficiency of computing A* heuristic functions for protein design and propose a variant of A* algorithm in which the search process can be performed on a single GPU in a massively parallel fashion. In addition, we make some efforts to address the memory exceeding problem in A* search. As a result, our enhancements can achieve a significant speedup of the A*-based protein design algorithm by four orders of magnitude on large-scale test data through pre-computation and parallelization, while still maintaining an acceptable memory overhead. We also show that our parallel A* search algorithm could be successfully combined with iMinDEE, a state-of-the-art DEE criterion, for rotamer pruning to further improve SCPR with the consideration of continuous side-chain flexibility. Availability: Our software is available and distributed open-source under the GNU Lesser General License Version 2.1 (GNU, February 1999). The source code can be downloaded from http://www.cs.duke.edu/donaldlab/osprey.php or http://iiis.tsinghua.edu.cn/∼compbio/software.html. Contact: zengjy321@tsinghua.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24931991

  13. A Role of Protein Degradation in Memory Consolidation after Initial Learning and Extinction Learning in the Honeybee ("Apis mellifera")

    ERIC Educational Resources Information Center

    Felsenberg, Johannes; Dombrowski, Vincent; Eisenhardt, Dorothea

    2012-01-01

    Protein degradation is known to affect memory formation after extinction learning. We demonstrate here that an inhibitor of protein degradation, MG132, interferes with memory formation after extinction learning in a classical appetitive conditioning paradigm. In addition, we find an enhancement of memory formation when the same inhibitor is…

  14. Sequence Heterogeneity Accelerates Protein Search for Targets on DNA

    NASA Astrophysics Data System (ADS)

    Shvets, Alexey; Kolomeisky, Anatoly

    The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry and heterogeneity of a genome. The work was supported by the Welch Foundation (Grant C-1559), by the NSF (Grant CHE-1360979), and by the Center for Theoretical Biological Physics sponsored by the NSF (Grant PHY-1427654).

  15. Degradation of the disease-associated prion protein by a serine protease from lichens

    USGS Publications Warehouse

    Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J.C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.

    2011-01-01

    The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  16. Degradation of the disease-associated prion protein by a serine protease from lichens

    USGS Publications Warehouse

    Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J. C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.

    2011-01-01

    The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  17. Degradation of the disease-associated prion protein by a serine protease from lichens.

    USGS Publications Warehouse

    Johnson, C.J.; Bennett, J.P.; Biro, S.M.; Duque-Velasquez, J. C.; Rodriguez, C.M.; Bessen, R.A.; Rocke, T.E.

    2011-01-01

    The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

  18. Degradation of the Disease-Associated Prion Protein by a Serine Protease from Lichens

    PubMed Central

    Johnson, Christopher J.; Bennett, James P.; Biro, Steven M.; Duque-Velasquez, Juan Camilo; Rodriguez, Cynthia M.; Bessen, Richard A.; Rocke, Tonie E.

    2011-01-01

    The disease-associated prion protein (PrPTSE), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrPTSE inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrPTSE. Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrPTSE-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrPTSE and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted. PMID:21589935

  19. The Role of the Ubiquitin Proteasome Pathway in Keratin Intermediate Filament Protein Degradation

    PubMed Central

    Rogel, Micah R.; Jaitovich, Ariel; Ridge, Karen M.

    2010-01-01

    Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation—causes, mechanisms, and consequences—will allow for a greater understanding of epithelial cell biology and lung pathology alike. PMID:20160151

  20. Effects of feeding dairy cows protein supplements of varying ruminal degradability.

    PubMed

    Reynal, S M; Broderick, G A

    2003-03-01

    Twenty-five (10 ruminally cannulated) Holstein cows averaging 82 +/- 34 d in milk were assigned to 5 x 5 Latin squares (21-d periods) and fed diets supplemented with one of four different proteins to assess effects on production, ruminal metabolism, omasal flow of N fractions, and degradation rates of protein supplements. Total mixed diets contained (dry matter basis) 44% corn silage, 22% alfalfa silage, 2% urea, and 31% concentrate. Five concentrate mixes were fed: 31% high-moisture shelled corn (HMSC; basal); 9% solvent soybean meal (SSBM), 22% HMSC; 10% expeller soybean meal (ESBM), 21% HMSC; 5.5% blood meal (BM), 25.5% HMSC; and 7% corn gluten meal (CGM), 24% HMSC. Diets averaged, respectively, 15.8, 19.1, 19.7, 20.3, and 19.3% crude protein. Feeding the basal diet reduced intake and yield of milk, fat-corrected milk (FCM), and all milk components compared to the protein-supplemented diets. Milk yield was higher for cows fed ESBM and CGM, fat yield was higher for cows fed SSBM and CGM, but FCM and protein yields were not different among cows fed supplemental protein. Based on omasal sampling, mean in vivo estimates of ruminal degradation rate for the crude protein in SSBM, ESBM, BM, and CGM was, respectively, 0.417, 0.179, 0.098, and 0.051/h (computed using passage rates observed for the small particle phase; mean = 0.14/h), and 0.179, 0.077, 0.042, and 0.026/h (computed using a passage rate of 0.06/h). The in vivo degradation rate computed for SSBM at a passage rate = 0.06/h was similar to that estimated using the inhibitor in vitro method. However, in vivo degradation rates computed at passage rate = 0.06/h for ESBM, BM, and CGM were about two, four, and three times more rapid than those estimated by inhibitor in vitro. Experimental proteins fed in this trial will be used as standards for developing in vitro methods for predicting rates of ruminal protein degradation. PMID:12703620

  1. Involvement of Two Latex-Clearing Proteins during Rubber Degradation and Insights into the Subsequent Degradation Pathway Revealed by the Genome Sequence of Gordonia polyisoprenivorans Strain VH2

    PubMed Central

    Hiessl, Sebastian; Schuldes, Jörg; Thürmer, Andrea; Halbsguth, Tobias; Bröker, Daniel; Angelov, Angel; Liebl, Wolfgang; Daniel, Rolf

    2012-01-01

    The increasing production of synthetic and natural poly(cis-1,4-isoprene) rubber leads to huge challenges in waste management. Only a few bacteria are known to degrade rubber, and little is known about the mechanism of microbial rubber degradation. The genome of Gordonia polyisoprenivorans strain VH2, which is one of the most effective rubber-degrading bacteria, was sequenced and annotated to elucidate the degradation pathway and other features of this actinomycete. The genome consists of a circular chromosome of 5,669,805 bp and a circular plasmid of 174,494 bp with average GC contents of 67.0% and 65.7%, respectively. It contains 5,110 putative protein-coding sequences, including many candidate genes responsible for rubber degradation and other biotechnically relevant pathways. Furthermore, we detected two homologues of a latex-clearing protein, which is supposed to be a key enzyme in rubber degradation. The deletion of these two genes for the first time revealed clear evidence that latex-clearing protein is essential for the microbial utilization of rubber. Based on the genome sequence, we predict a pathway for the microbial degradation of rubber which is supported by previous and current data on transposon mutagenesis, deletion mutants, applied comparative genomics, and literature search. PMID:22327575

  2. Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    PubMed Central

    Cazanave, S C; Wang, X; Zhou, H; Rahmani, M; Grant, S; Durrant, D E; Klaassen, C D; Yamamoto, M; Sanyal, A J

    2014-01-01

    Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte lipoapoptosis. This lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1−/− mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte lipoapoptosis. PMID:24769730

  3. Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy

    PubMed Central

    Li, Honghao; Chen, Qi; Liu, Fuchen; Zhang, Xuemei; Li, Wei; Liu, Shuping; Zhao, Yuying; Gong, Yaoqin; Yan, Chuanzhu

    2013-01-01

    Although intracellular beta amyloid (Aβ) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation. PMID:23472144

  4. Accelerated light-induced degradation for detecting copper contamination in p-type silicon

    SciTech Connect

    Inglese, Alessandro Savin, Hele; Lindroos, Jeanette

    2015-08-03

    Copper is a harmful metal impurity that significantly impacts the performance of silicon-based devices if present in active regions. In this contribution, we propose a fast method consisting of simultaneous illumination and annealing for the detection of copper contamination in p-type silicon. Our results show that, within minutes, such method is capable of producing a significant reduction of the minority carrier lifetime. A spatial distribution map of copper contamination can then be obtained through the lifetime values measured before and after degradation. In order to separate the effect of the light-activated copper defects from the other metastable complexes in low resistivity Cz-silicon, we carried out a dark anneal at 200 °C, which is known to fully recover the boron-oxygen defect. Similar to the boron-oxygen behavior, we show that the dark anneal also recovers the copper defects. However, the recovery is only partial and it can be used to identify the possible presence of copper contamination.

  5. Isotope Coded Labeling for Accelerated Protein Interaction Profiling using MS

    PubMed Central

    Venable, John D.; Steckler, Caitlin; Ou, Weijia; Grünewald, Jan; Agarwalla, Sanjay; Brock, Ansgar

    2015-01-01

    Protein interaction surface mapping using MS is widely applied but comparatively resource intensive. Here a workflow adaptation for use of isotope coded tandem mass tags for the purpose is reported. The key benefit of improved throughput derived from sample acquisition multiplexing and automated analysis is shown to be maintained in the new application. Mapping of the epitopes of two monoclonal antibodies on their respective targets serves to illustrate the novel approach. We conclude that the approach enables mapping of interactions by MS at significantly larger scales than hereto possible. PMID:26151661

  6. Pipe degradation investigations for optimization of flow-accelerated corrosion inspection location selection

    SciTech Connect

    Chandra, S.; Habicht, P.; Chexal, B.; Mahini, R.; McBrine, W.; Esselman, T.; Horowitz, J.

    1995-12-01

    A large amount of piping in a typical nuclear power plant is susceptible to Flow-Accelerated Corrosion (FAC) wall thinning to varying degrees. A typical PAC monitoring program includes the wall thickness measurement of a select number of components in order to judge the structural integrity of entire systems. In order to appropriately allocate resources and maintain an adequate FAC program, it is necessary to optimize the selection of components for inspection by focusing on those components which provide the best indication of system susceptibility to FAC. A better understanding of system FAC predictability and the types of FAC damage encountered can provide some of the insight needed to better focus and optimize the inspection plan for an upcoming refueling outage. Laboratory examination of FAC damaged components removed from service at Northeast Utilities` (NU) nuclear power plants provides a better understanding of the damage mechanisms involved and contributing causes. Selected results of this ongoing study are presented with specific conclusions which will help NU to better focus inspections and thus optimize the ongoing FAC inspection program.

  7. Degradation mechanism of LiCoO2/mesocarbon microbeads battery based on accelerated aging tests

    NASA Astrophysics Data System (ADS)

    Guan, Ting; Zuo, Pengjian; Sun, Shun; Du, Chunyu; Zhang, Lingling; Cui, Yingzhi; Yang, Lijie; Gao, Yunzhi; Yin, Geping; Wang, Fuping

    2014-12-01

    A series of LiCoO2/mesocarbon microbeads (MCMB) commercial cells cycled at different rates (0.6C, 1.2C, 1.5C, 1.8C, 2.4C and 3.0C) are disassembled and the capacity fade mechanism is proposed by analyzing the structure, morphology and electrochemical performance evolution at the capacity retention of 95%, 90%, 85%, 80%. The capacity deterioration of the commercial cell is mainly caused by the decay of the reversible capacity of LiCoO2 cathode, the irreversible loss of active lithium and the lithium remaining in anode. The proportions of effects by the above three factors are calculated accurately. The consumption of the active lithium leads to a cell imbalance between the anode and the cathode. The electrochemical test results indicate that the capacity fade of the active materials at the low rate is more obvious than that at the high rate. The influence of the active lithium is gradually increscent with the increasing rate. The rate of 1.5C is the optimal value to accelerate the aging of the full cell by comparing the testing results at different capacity retentions in the specific condition of low charge/discharge rate and shallow depth of discharge.

  8. HPLC and HPLC/MS/MS Studies on Stress, Accelerated and Intermediate Degradation Tests of Antivirally Active Tricyclic Analog of Acyclovir.

    PubMed

    Lesniewska, Monika A; Dereziński, Paweł; Klupczyńska, Agnieszka; Kokot, Zenon J; Ostrowski, Tomasz; Zeidler, Joanna; Muszalska, Izabela

    2015-01-01

    The degradation behavior of a tricyclic analog of acyclovir [6-(4-MeOPh)-TACV] was determined in accordance with International Conference on Harmonization guidelines for good clinical practice under different stress conditions (neutral hydrolysis, strong acid/base degradation, oxidative decomposition, photodegradation, and thermal degradation). Accelerated [40±2°C/75%±5% relative humidity (RH)] and intermediate (30±2°C/65%±5% RH) stability tests were also performed. For observation of the degradation of the tested compound the RP-HPLC was used, whereas for the analysis of its degradation products HPLC/MS/MS was used. Degradation of the tested substance allowed its classification as unstable in neutral environment, acidic/alkaline medium, and in the presence of oxidizing agent. The tested compound was also light sensitive and was classified as photolabile both in solution and in the solid phase. However, the observed photodegradation in the solid phase was at a much lower level than in the case of photodegradation in solution. The study showed that both air temperature and RH had no significant effect on the stability of the tested substance during storage for 1 month at 100°C (dry heat) as well as during accelerated and intermediate tests. Based on the HPLC/MS/MS analysis, it can be concluded that acyclovir was formed as a degradation product of 6-(4-MeOPh)-TACV. PMID:26525242

  9. A structural overview of GH61 proteins - fungal cellulose degrading polysaccharide monooxygenases.

    PubMed

    Lo Leggio, Leila; Welner, Ditte; De Maria, Leonardo

    2012-01-01

    Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full. PMID:24688660

  10. A structural overview of GH61 proteins – fungal cellulose degrading polysaccharide monooxygenases

    PubMed Central

    Leggio, Leila Lo; Welner, Ditte; De Maria, Leonardo

    2012-01-01

    Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full. PMID:24688660

  11. Individual and Collective Contributions of Chaperoning and Degradation to Protein Homeostasis in E. coli

    PubMed Central

    Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R.; Liu, Yu; Powers, David L.; Kelly, Jeffery W.; Gierasch, Lila M.; Powers, Evan T.

    2015-01-01

    SUMMARY The folding fate of a protein in vivo is determined by the interplay between a protein’s folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding vs. aggregation vs. degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. PMID:25843722

  12. Accelerated degradation of PAHs using edaphic biostimulants obtained from sewage sludge and chicken feathers.

    PubMed

    Rodríguez-Morgado, Bruno; Gómez, Isidoro; Parrado, Juan; García, Carlos; Hernández, Teresa; Tejada, Manuel

    2015-12-30

    We studied in the laboratory the bioremediation effects over a 100-day period of three edaphic biostimulants (BS) obtained from sewage sludge (SS) and from two different types of chicken feathers (CF1 and CF2), in a soil polluted with three polycyclic aromatic hydrocarbons (PAH) (phenanthrene, Phe; pyrene, Py; and benzo(a)pyrene, BaP), at a concentration of 100 mg kg(-1) soil. We determined their effects on enzymatic activities and on soil microbial community. Those BS with larger amounts of proteins and a higher proportion of peptides (<300 daltons), exerted a greater stimulation on the soil biochemical properties and microbial community, possibly because low molecular weight proteins can be easily assimilated by soil microorganisms. The soil dehydrogenase, urease, β-glucosidase and phosphatase activities and microbial community decreased in PAH-polluted soil. This decrease was more pronounced in soils contaminated with BaP than with Py and Phe. The application of the BS to PAH-polluted soils decreased the inhibition of the soil biological properties, principally at 7 days into the experiment. This decrease was more pronounced in soils contaminated with BaP than with Py and Phe and was higher in polluted soils amended with CF2, followed by SS and CF1, respectively. PMID:26188866

  13. Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation.

    PubMed

    Pyeon, Dohun; Timani, Khalid Amine; Gulraiz, Fahad; He, Johnny J; Park, In-Woo

    2016-09-01

    HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation. PMID:27460547

  14. Inhibitors of cholesterol biosynthesis increase hepatic low-density lipoprotein receptor protein degradation.

    PubMed

    Ness, G C; Zhao, Z; Lopez, D

    1996-01-15

    Inhibitors of cholesterol biosynthesis are believed to lower serum cholesterol levels by enhancing the removal of serum low-density lipoprotein (LDL) by increasing hepatic LDL receptor function. Thus, the effects of several different inhibitors of cholesterol biosynthesis were examined for their effects on the expression of the hepatic LDL receptor in rats. We found that administration of inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase such as lovastatin, pravastatin, fluvastatin, and rivastatin resulted in increased hepatic LDL receptor mRNA levels. Surprisingly, these agents failed to increase levels of immunoreactive LDL receptor protein in rat liver even when the dose and length of treatment were increased. Treatment of rats with zaragozic acid A, an inhibitor of squalene synthase, caused even greater increases in hepatic LDL receptor mRNA levels, but did not increase levels of immunoreactive protein. Further investigation revealed that the rate of degradation of the hepatic LDL receptor was increased in rats given inhibitors of cholesterol biosynthesis. The greatest increase in the rate of degradation was seen in animals treated with zaragozic acid A which caused the largest increase in hepatic LDL receptor mRNA levels. In contrast, hepatic LDL receptor protein was stabilized in cholesterol-fed rats. It appears that increased potential for LDL receptor protein synthesis, reflected in increased mRNA levels, is offset by a corresponding increase in the rate of receptor protein degradation resulting in constant steady-state levels of hepatic LDL receptor protein. These findings are suggestive of increased cycling of the hepatic LDL receptor. This postulated mechanism can provide for enhanced hepatic uptake of lipoproteins without increasing steady-state levels of LDL receptor protein. PMID:8561503

  15. Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A.

    PubMed

    Wan, Xiaofeng; Ru, Yanfei; Chu, Chen; Ni, Zimei; Zhou, Yuchuan; Wang, Shoulin; Zhou, Zuomin; Zhang, Yonglian

    2016-06-01

    Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility. PMID:27174873

  16. c-myc and c-myb protein degradation: effect of metabolic inhibitors and heat shock.

    PubMed Central

    Lüscher, B; Eisenman, R N

    1988-01-01

    The proteins encoded by both viral and cellular forms of the c-myc oncogene have been previously demonstrated to have exceptionally short in vivo half-lives. In this paper we report a comparative study on the parameters affecting turnover of nuclear oncoproteins c-myc, c-myb, and the rapidly metabolized cytoplasmic enzyme ornithine decarboxylase. The degradation of all three proteins required metabolic energy, did not result in production of cleavage intermediates, and did not involve lysosomes or ubiquitin. A five- to eightfold increase in the half-life of c-myc proteins, and a twofold increase in the half-life of c-myb proteins was detected after heat-shock treatment at 46 degrees C. In contrast, heat shock had no effect on the turnover of ornithine decarboxylase. Heat shock also had the effect of increasing the rate of c-myc protein synthesis twofold, whereas c-myb protein synthesis was decreased nearly fourfold. The increased stability and synthesis of c-myc proteins led to an overall increase in the total level of c-myc proteins in response to heat-shock treatment. Furthermore, treatments which reduced c-myc and c-myb protein turnover, such as heat shock and exposure to inhibitors of metabolic energy production, resulted in reduced detergent solubility of both proteins. The recovery from heat shock, as measured by increased turnover and solubility, was energy dependent and considerably more rapid in thermotolerant cells. Images PMID:3043180

  17. From start to finish: amino-terminal protein modifications as degradation signals in plants.

    PubMed

    Gibbs, Daniel J; Bailey, Mark; Tedds, Hannah M; Holdsworth, Michael J

    2016-09-01

    Contents 1188 I. 1188 II. 1189 III. 1190 IV. 1191 V. 1192 1192 References 1192 SUMMARY: The amino- (N-) terminus (Nt) of a protein can undergo a diverse array of co- and posttranslational modifications. Many of these create degradation signals (N-degrons) that mediate protein destruction via the N-end rule pathway of ubiquitin-mediated proteolysis. In plants, the N-end rule pathway has emerged as a major system for regulated control of protein stability. Nt-arginylation-dependent degradation regulates multiple growth, development and stress responses, and recently identified functions of Nt-acetylation can also be linked to effects on the in vivo half-lives of Nt-acetylated proteins. There is also increasing evidence that N-termini could act as important protein stability determinants in plastids. Here we review recent advances in our understanding of the relationship between the nature of protein N-termini, Nt-processing events and proteolysis in plants. PMID:27439310

  18. TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis

    PubMed Central

    Aizawa, Sayaka; Okamoto, Toru; Sugiyama, Yukari; Kouwaki, Takahisa; Ito, Ayano; Suzuki, Tatsuya; Ono, Chikako; Fukuhara, Takasuke; Yamamoto, Masahiro; Okochi, Masayasu; Hiraga, Nobuhiko; Imamura, Michio; Chayama, Kazuaki; Suzuki, Ryosuke; Shoji, Ikuo; Moriishi, Kohji; Moriya, Kyoji; Koike, Kazuhiko; Matsuura, Yoshiharu

    2016-01-01

    Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin–proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells. PMID:27142248

  19. TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis.

    PubMed

    Aizawa, Sayaka; Okamoto, Toru; Sugiyama, Yukari; Kouwaki, Takahisa; Ito, Ayano; Suzuki, Tatsuya; Ono, Chikako; Fukuhara, Takasuke; Yamamoto, Masahiro; Okochi, Masayasu; Hiraga, Nobuhiko; Imamura, Michio; Chayama, Kazuaki; Suzuki, Ryosuke; Shoji, Ikuo; Moriishi, Kohji; Moriya, Kyoji; Koike, Kazuhiko; Matsuura, Yoshiharu

    2016-01-01

    Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin-proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells. PMID:27142248

  20. The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome.

    PubMed

    Peth, Andreas; Nathan, James A; Goldberg, Alfred L

    2013-10-01

    The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2-3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub5-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub5-DHFR molecule (∼13 s) and the energy expenditure (50-80 ATPs/Ub5-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost. PMID:23965995

  1. Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP.

    PubMed

    Falkevall, Annelie; Alikhani, Nyosha; Bhushan, Shashi; Pavlov, Pavel F; Busch, Katrin; Johnson, Kenneth A; Eneqvist, Therese; Tjernberg, Lars; Ankarcrona, Maria; Glaser, Elzbieta

    2006-09-29

    Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid beta-protein (Abeta). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Abeta. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu(78) in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Abeta-(1-42), Abeta-(1-40), and Abeta Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 A resolution of AtPreP allowed us to identify Cys(90) and Cys(527) that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Abeta by hPreP may potentially be of importance in the pathology of Alzheimer disease. PMID:16849325

  2. Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

    PubMed Central

    2014-01-01

    Background The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the current study, we identified mechanisms involved in protein degradation regulation. In experiment 1, 6- and 26-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic, 2) euinsulinemic-euglycemic-hyperaminoacidemic, and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps for 2 h. In experiment 2, 5-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic-euleucinemic, 2) euinsulinemic-euglycemic-hypoaminoacidemic-hyperleucinemic, and 3) euinsulinemic-euglycemic-euaminoacidemic-hyperleucinemic clamps for 24 h. We determined in muscle indices of ubiquitin-proteasome, i.e., atrogin-1 (MAFbx) and muscle RING-finger protein-1 (MuRF1) and autophagy-lysosome systems, i.e., unc51-like kinase 1 (UKL1), microtubule-associated protein light chain 3 (LC3), and lysosomal-associated membrane protein 2 (Lamp-2). For comparison, we measured ribosomal protein S6 (rpS6) and eukaryotic initiation factor 4E (eIF4E) activation, components of translation initiation. Results Abundance of atrogin-1, but not MuRF1, was greater in 26- than 6-d-old pigs and was not affected by insulin, amino acids, or leucine. Abundance of ULK1 and LC3 was higher in younger pigs and not affected by treatment. The LC3-II/LC3-I ratio was reduced and ULK1 phosphorylation increased by insulin, amino acids, and leucine. These responses were more profound in younger pigs. Abundance of Lamp-2 was not affected by treatment or development. Abundance of eIF4E, but not rpS6, was higher in 6- than 26-d-old-pigs but unaffected by treatment. Phosphorylation of eIF4E was not affected by treatment, however, insulin, amino acids, and leucine stimulated rpS6 phosphorylation, and the

  3. Acute ER stress regulates amyloid precursor protein processing through ubiquitin-dependent degradation.

    PubMed

    Jung, Eun Sun; Hong, HyunSeok; Kim, Chaeyoung; Mook-Jung, Inhee

    2015-01-01

    Beta-amyloid (Aβ), a major pathological hallmark of Alzheimer's disease (AD), is derived from amyloid precursor protein (APP) through sequential cleavage by β-secretase and γ-secretase enzymes. APP is an integral membrane protein, and plays a key role in the pathogenesis of AD; however, the biological function of APP is still unclear. The present study shows that APP is rapidly degraded by the ubiquitin-proteasome system (UPS) in the CHO cell line in response to endoplasmic reticulum (ER) stress, such as calcium ionophore, A23187, induced calcium influx. Increased levels of intracellular calcium by A23187 induces polyubiquitination of APP, causing its degradation. A23187-induced reduction of APP is prevented by the proteasome inhibitor MG132. Furthermore, an increase in levels of the endoplasmic reticulum-associated degradation (ERAD) marker, E3 ubiquitin ligase HRD1, proteasome activity, and decreased levels of the deubiquitinating enzyme USP25 were observed during ER stress. In addition, we found that APP interacts with USP25. These findings suggest that acute ER stress induces degradation of full-length APP via the ubiquitin-proteasome proteolytic pathway. PMID:25740315

  4. Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system

    PubMed Central

    Guharoy, Mainak; Bhowmick, Pallab; Sallam, Mohamed; Tompa, Peter

    2016-01-01

    Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation. PMID:26732515

  5. Multiple Sclerosis Autoantigen Myelin Basic Protein Escapes Control by Ubiquitination during Proteasomal Degradation*

    PubMed Central

    Belogurov, Alexey; Kudriaeva, Anna; Kuzina, Ekaterina; Smirnov, Ivan; Bobik, Tatyana; Ponomarenko, Natalia; Kravtsova-Ivantsiv, Yelena; Ciechanover, Aaron; Gabibov, Alexander

    2014-01-01

    The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome. PMID:24739384

  6. Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation

    PubMed Central

    Li, Jun; Chen, Cancan; Ma, Xiancai; Geng, Guannan; Liu, Bingfeng; Zhang, Yijun; Zhang, Shaoyang; Zhong, Fudi; Liu, Chao; Yin, Yue; Cai, Weiping; Zhang, Hui

    2016-01-01

    Long noncoding RNAs (lncRNAs) play multiple key regulatory roles in various cellular pathways. However, their functions in HIV-1 latent infection remain largely unknown. Here we show that a lncRNA named NRON, which is highly expressed in resting CD4+ T lymphocytes, could be involved in HIV-1 latency by specifically inducing Tat protein degradation. Our results suggest that NRON lncRNA potently suppresses the viral transcription by decreasing the cellular abundance of viral transactivator protein Tat. NRON directly links Tat to the ubiquitin/proteasome components including CUL4B and PSMD11, thus facilitating Tat degradation. Depletion of NRON, especially in combination with a histone deacetylase (HDAC) inhibitor, significantly reactivates the viral production from the HIV-1-latently infected primary CD4+ T lymphocytes. Our data indicate that lncRNAs play a role in HIV-1 latency and their manipulation could be a novel approach for developing latency-reversing agents. PMID:27291871

  7. The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans.

    PubMed

    Zhang, Liangyu; Ward, Jordan D; Cheng, Ze; Dernburg, Abby F

    2015-12-15

    Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3' UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism. PMID:26552885

  8. The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans

    PubMed Central

    Zhang, Liangyu; Ward, Jordan D.; Cheng, Ze; Dernburg, Abby F.

    2015-01-01

    Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3′ UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism. PMID:26552885

  9. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    NASA Astrophysics Data System (ADS)

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2014-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

  10. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    PubMed Central

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2013-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits HIV-1 infection of myeloid-lineage cells 1,2 as well as resting CD4+ T cells 3,4 by reducing the cellular dNTP concentration to a level where the viral reverse transcriptase cannot function 5,6. In other lentiviruses, including HIV-2 and related SIVs, SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation 7,8. The molecular mechanism by which these viral proteins are able to usurp the host cell’s ubiquitination machinery to destroy the cell’s protection against these viruses has not been defined. We present here the crystal structure of a ternary complex of Vpx with the host cell’s E3 ligase substrate adaptor DCAF1 and the C-terminal region of SAMHD1. Vpx is made up of a three-helical bundle, stabilised by a zinc finger motif and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C-terminus making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure provides the first description of how a lentiviral accessory protein is able to subvert the cell’s normal protein degradation pathway to inactivate the cellular viral defence system. PMID:24336198

  11. Degradation of the 32 kD herbicide binding protein in far red light. [Spirodella oligorrhiza

    SciTech Connect

    Gaba, V.; Marder, J.B.; Greenberg, B.M.; Mattoo, A.K.; Edelman, M.

    1987-06-01

    White light (400-700 nanometers) supports the activity of photosystem I (PSI) and photosystem II while far red light (greater than or equal to700 nanometers) supports PSI almost exclusively. In intact fronds of Spirodela oligorrhiza, turnover of the 32 kilodaltons herbicide binding protein is stimulated under both these light conditions, although not in the dark or at wavelengths > 730 nanometers. As is the case in white light, the far red light induced degradation of the protein is inhibited by DCMU. The means by which far red light operates is unclear. Hypotheses considered include: PSI activated proteolysis, PSI-induced formation of semiquinone anions, and PSI-generated free radicals.

  12. ClpXP, an ATP-powered unfolding and protein-degradation machine

    PubMed Central

    Baker, Tania A.; Sauer, Robert T.

    2011-01-01

    ClpXP is a AAA+ protease that uses the energy of ATP binding and hydrolysis to perform mechanical work during targeted protein degradation within cells. ClpXP consists of hexamers of a AAA+ ATPase (ClpX) and a tetradecameric peptidase (ClpP). Asymmetric ClpX hexamers bind unstructured peptide tags in protein substrates, unfold stable tertiary structure in the substrate, and then translocate the unfolded polypeptide chain into an internal proteolytic compartment in ClpP. Here, we review our present understanding of ClpXP structure and function, as revealed by two decades of biochemical and biophysical studies. PMID:21736903

  13. PBRM1 (BAF180) protein is functionally regulated by p53-induced protein degradation in renal cell carcinomas.

    PubMed

    Macher-Goeppinger, Stephan; Keith, Martina; Tagscherer, Katrin E; Singer, Stephan; Winkler, Juliane; Hofmann, Thomas G; Pahernik, Sascha; Duensing, Stefan; Hohenfellner, Markus; Kopitz, Juergen; Schirmacher, Peter; Roth, Wilfried

    2015-12-01

    About 40% of clear-cell renal cell carcinomas (ccRCC) harbour mutations in Polybromo-1 (PBRM1), encoding the BAF180 subunit of a SWI/SNF chromatin remodelling complex. This qualifies PBRM1 as a major cancer gene in ccRCC. The PBRM1 protein alters chromatin structure and its known functions include transcriptional regulation by controlling the accessibility of DNA and influencing p53 transcriptional activity. Since little is known about the regulation of PBRM1, we studied possible mechanisms and interaction partners involved in the regulation of PBRM1 expression. Activation of p53 in RCC cells resulted in a marked decrease of PBRM1 protein levels. This effect was abolished by siRNA-mediated down-regulation of p53, and transcriptional activity was not crucial for p53-dependent PBRM1 regulation. Pulse-chase experiments determined post-translational protein degradation to be the underlying mechanism for p53-dependent PBRM1 regulation, which was accordingly inhibited by proteasome inhibitors. The effects of p53 activation on PBRM1 expression were confirmed in RCC tissue ex vivo. Our results demonstrate that PBRM1 is a target of p53-induced proteasomal protein degradation and provide further evidence for the influence of PBRM1 on p53 function in RCC tumour cells. Considering the paramount role of p53 in carcinogenesis and the presumptive impact of PBRM1 in RCC development, this novel regulation mechanism might be therapeutically exploited in the future. PMID:26178300

  14. Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes.

    PubMed

    Yu, Jun; Lan, Xiang; Chen, Xia; Yu, Chao; Xu, Yiwen; Liu, Yujuan; Xu, Lingna; Fan, Heng-Yu; Tong, Chao

    2016-08-15

    The homeostasis of self-renewal and differentiation in stem cells is controlled by intrinsic signals and their niche. We conducted a large-scale RNA interference (RNAi) screen in Drosophila testes and identified 221 genes required for germline stem cell (GSC) maintenance or differentiation. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in the protein degradation pathway in cyst cells leads to testis tumors consisting of overproliferated germ cells. Importantly, in the Cullin 4-RING E3 ubiquitin ligase (CRL4) complex, we identified multiple proteins that are crucial to GSC self-renewal: pic/DDB1, a CRL4 linker protein, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice. PMID:27471256

  15. Salicylic acid signaling controls the maturation and localization of the arabidopsis defense protein ACCELERATED CELL DEATH6.

    PubMed

    Zhang, Zhongqin; Shrestha, Jay; Tateda, Chika; Greenberg, Jean T

    2014-08-01

    ACCELERATED CELL DEATH6 (ACD6) is a multipass membrane protein with an ankyrin domain that acts in a positive feedback loop with the defense signal salicylic acid (SA). This study implemented biochemical approaches to infer changes in ACD6 complexes and localization. In addition to forming endoplasmic reticulum (ER)- and plasma membrane (PM)-localized complexes, ACD6 forms soluble complexes, where it is bound to cytosolic HSP70, ubiquitinated, and degraded via the proteasome. Thus, ACD6 constitutively undergoes ER-associated degradation. During SA signaling, the soluble ACD6 pool decreases, whereas the PM pool increases. Similarly, ACD6-1, an activated version of ACD6 that induces SA, is present at low levels in the soluble fraction and high levels in the PM. However, ACD6 variants with amino acid substitutions in the ankyrin domain form aberrant, inactive complexes, are induced by a SA agonist, but show no PM localization. SA signaling also increases the PM pools of FLAGELLIN SENSING2 (FLS2) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). FLS2 forms complexes ACD6; both FLS2 and BAK1 require ACD6 for maximal accumulation at the PM in response to SA signaling. A plausible scenario is that SA increases the efficiency of productive folding and/or complex formation in the ER, such that ACD6, together with FLS2 and BAK1, reaches the cell surface to more effectively promote immune responses. PMID:24923602

  16. Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

    SciTech Connect

    Radulescu, Razvan T.; Duckworth, William C.; Levy, Jennifer L.; Fawcett, Janet

    2010-04-30

    Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

  17. Light-triggered chemical amplification to accelerate degradation and release from polymeric particles† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc06143a Click here for additional data file.

    PubMed Central

    Olejniczak, Jason; Nguyen Huu, Viet Anh; Lux, Jacques; Grossman, Madeleine; He, Sha

    2015-01-01

    We describe a means of chemical amplification to accelerate triggered degradation of a polymer and particles composed thereof. We designed a light-degradable copolymer containing carboxylic acids masked by photolabile groups and ketals. Photolysis allows the unmasked acidic groups in the polymer backbone to accelerate ketal hydrolysis even at neutral pH. PMID:26445896

  18. Individual expression of influenza virus PA protein induces degradation of coexpressed proteins.

    PubMed Central

    Sanz-Ezquerro, J J; de la Luna, S; Ortín, J; Nieto, A

    1995-01-01

    In the process of in vivo reconstitution of influenza virus transcriptase-replicase complex, an inhibitory effect was observed when the level of PA protein expression was increased. This inhibition was paralleled by a decrease in the accumulation of the other influenza virus core proteins. The sole expression of PA protein was sufficient to reduce the accumulation level of the proteins encoded by the coexpressed genes. The PA effect was observed upon influenza virus and non-influenza virus proteins and independently of the expression system chosen and the origin of cell line used. The expression of PA protein did not induce variations in the translation of the target proteins but did induce variations on their half-lives, which were clearly reduced. A functional PA subunit seems to be necessary to induce this negative effect, because an inactive point mutant was unable to decrease the steady-state levels or the half-lives of the reporter proteins. The PA effect was observed as early as 5 h after its expression, and continuous synthesis of proteins was not required for performance of its biological activity. The results presented represent the first biological activity of individually expressed PA polymerase subunit. PMID:7884889

  19. Insulin attenuates atrophy of unweighted soleus muscle by amplified inhibition of protein degradation

    NASA Technical Reports Server (NTRS)

    Tischler, M. E.; Satarug, S.; Aannestad, A.; Munoz, K. A.; Henriksen, E. J.

    1997-01-01

    Unweighting atrophy of immature soleus muscle occurs rapidly over the first several days, followed by slower atrophy coinciding with increased sensitivity to insulin of in vitro protein metabolism. This study determined whether this increased sensitivity might account for the diminution of atrophy after 3 days of tall-cast hindlimb suspension. The physiological significance of the increased response to insulin in unweighted muscle was evaluated by analyzing in vivo protein metabolism for day 3 (48 to 72 hours) and day 4 (72 to 96 hours) of unweighting in diabetic animals either injected with insulin or not treated. Soleus from nontreated diabetic animals showed a similar loss of protein during day 3 (-16.2%) and day 4 (-14.5%) of unweighting, whereas muscle from insulin-treated animals showed rapid atrophy (-14.5%) during day 3 only, declining to just -3.1% the next day. Since fractional protein synthesis was similar for both day 3 (8.6%/d) and day 4 (7.0%/d) of unweighting in insulin-treated animals, the reduction in protein loss must be accounted for by a slowing of protein degradation due to circulating insulin. Intramuscular (IM) injection of insulin (600 nmol/L) stimulated in situ protein synthesis similarly in 4-day unweighted (+56%) and weight-bearing (+90%) soleus, even though unweighted muscle showed a greater in situ response of 2-deoxy-[3H]glucose uptake to IM injection of either insulin (133 nmol/L) or insulin-like growth factor-I (IGF-I) (200 nmol/L) than control muscle. These findings suggest that unweighted muscle is selectively more responsive in vivo to insulin, and that the slower atrophy after 3 days of unweighting was due to an increased effect of insulin on inhibiting protein degradation.

  20. In vitro protein degradation of 38 sainfoin accessions and its relationship to tannin content by different assays.

    PubMed

    Lorenz, Martin M; Hayot Carbonero, Christine; Smith, Lydia; Udén, Peter

    2012-05-23

    This study compared 38 sainfoin and 2 Lotus accessions to their respective tannin contents, N buffer solubility, and in vitro protein degradation. Tannin contents were measured by a protein precipitation method using either bovine serum albumin or Rubisco and by the colorimetric HCl/butanol method. Precipitation of bovine serum albumin and Rubisco was highly correlated (R(2) = 0.939). Correlations between the protein precipitation variants and the HCl/butanol method were relatively low (R(2) < 0.6). Protein degradation was measured at 4 h of incubation in an inhibited in vitro system and could not be explained by any of the tannin assays (R(2) < 0.03) and only partially by N buffer solubility (R(2) ≤ 0.433). Decisive factors other than the quantity of tannins or their ability to precipitate proteins must be considered. Resistance of soluble protein toward degradation can possibly be caused by tannin protein binding. PMID:22494200

  1. Quorum sensing controls hyphal initiation in Candida albicans through Ubr1-mediated protein degradation

    PubMed Central

    Lu, Yang; Su, Chang; Unoje, Ohimai; Liu, Haoping

    2014-01-01

    Candida albicans is the most common cause of invasive fungal infections in humans. Its ability to undergo the morphological transition from yeast to hyphal growth forms is critical for its pathogenesis. Hyphal initiation requires the activation of the cAMP-PKA pathway, which down-regulates the expression of NRG1, the major repressor of hyphal development. Hyphal initiation also requires inoculation of a small amount of C. albicans cells from overnight culture to fresh medium. This inoculation releases the inhibition from farnesol, a quorum-sensing molecule of C. albicans, that accumulated in the spent medium. Here, we show that farnesol inhibits hyphal initiation mainly through blocking the protein degradation of Nrg1. Through screening a kinase mutant library, we identified Sok1 as the kinase required for Nrg1 degradation during inoculation. SOK1 expression is transiently activated on inoculation during hyphal initiation, and overexpression of SOK1 overcomes the farnesol-mediated inhibition of hyphal initiation. Screening a collection of transcription factor mutants, the homeodomain-containing transcription repressor Cup9 is found to be responsible for the repression of SOK1 expression in response to farnesol inhibition. Interestingly, farnesol inhibits Cup9 degradation mediated by the N-end rule E3 ubiquitin ligase, Ubr1. Therefore, hyphal initiation requires both the cAMP-PKA pathway-dependent transcriptional down-regulation of NRG1 and Sok1-mediated degradation of Nrg1 protein. The latter is triggered by the release from farnesol inhibition of Cup9 degradation and consequently, derepression of SOK1 transcription. Neither pathway alone is sufficient for hyphal initiation. PMID:24449897

  2. Cbl-b accelerates trypsin-induced cell detachment through ubiquitination and degradation of proline-rich tyrosine kinase 2.

    PubMed

    Fan, Yibo; Qu, Xiujuan; Ma, Yanju; Qu, Jinglei; Liu, Yunpeng; Hu, Xuejun

    2014-11-01

    Trypsin is a digestive enzyme that is widely used for cell detachment, which is the first stage of tumor metastasis. Recent studies show that adhesion-related kinases are involved in cell detachment. Proline-rich tyrosine kinase 2 (Pyk2) is a crucial kinase in the regulation of cell adhesion and detachment. However, the effect of Pyk2 on cell detachment is controversial. In the present study, we found that Pyk2 expression was rapidly decreased after trypsin treatment in gastric cancer, breast cancer, colon cancer, lung cancer, and human gastric epithelial cells. Knockdown of Pyk2 accelerated cell detachment. Furthermore, lysosome inhibitor NH4CL suppressed cell detachment and increased ubiquitination of Pyk2. Cbl-b is a type of E3 ubiquitin ligase that interacted with Pyk2, reduced the expression of Pyk2, and promoted trypsin-induced degradation of Pyk2. These findings suggest that Cbl-b promoted cell detachment through mono-ubiquitination of Pyk2. Our data provide a new insight into the role of Cbl-b in cell detachment. PMID:25099615

  3. Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation.

    PubMed Central

    Pagès, S; Gal, L; Bélaïch, A; Gaudin, C; Tardif, C; Bélaïch, J P

    1997-01-01

    The role of a miniscaffolding protein, miniCipC1, forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated. The parameters of the binding of miniCipC1, which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined. At saturating concentrations, about 8.2 micromol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, colloidal Avicel, and phosphoric acid-swollen cellulose bound 0.28, 0.38, and 0.55 micromol of miniCipC1 per g, respectively. The dissociation constants measured varied between 1.3 x 10(-7) and 1.5 x 10(-8) M. These results are discussed with regard to the properties of the various substrates. The synergistic action of miniCipC1 and two forms of endoglucanase CelA (with and without the dockerin domain [CelA2 and CelA3, respectively]) in cellulose degradation was also studied. Although only CelA2 interacted with miniCipC1 (K(d), 7 x 10(-9) M), nonhydrolytic miniCipC1 enhanced the activities of endoglucanases CelA2 and CelA3 with all of the insoluble substrates tested. This finding shows that miniCipC1 plays two roles: it increases the enzyme concentration on the cellulose surface and enhances the accessibility of the enzyme to the substrate by modifying the structure of the cellulose, leading to an increased available cellulose surface area. In addition, the data obtained with a hybrid protein, CelA3-CBD(CipC), which was more active towards all of the insoluble substrates tested confirm that the CBD of the scaffolding protein plays an essential role in cellulose degradation. PMID:9139893

  4. Displacement of plasma protein and conduction velocity in rats under action of acceleration forces and hypokinesia

    NASA Technical Reports Server (NTRS)

    Baranski, S.; Edelwejn, Z.; Wojtkowiak, M.

    1980-01-01

    The permeability of capillary vessels was investigated in order to determine if acceleration alone or following prolonged hypokinesia would induce changes in the vascular wall leading to the penetration by l-albumins and/or proteins with larger molecules. In rats undergoing action of +5 Gz accelerations, no increase in vascular permeability, as tested with the use of (Cr-5k)-globulin, was demostrated. In rats immobilized for 4 weeks before centrifugation, rather weak migration of (Cr-51)-globulin from the vessels was observed. Immobilization resulted also in lowering of conduction velocity in the sciatic nerve.

  5. Proteolytic degradation of ewe milk proteins during fermentation of yoghurts and storage.

    PubMed

    El-Zahar, Khaled; Chobert, Jean-Marc; Sitohy, Mahmoud; Dalgalarrondo, Michèle; Haertlé, Thomas

    2003-06-01

    Yoghurts are mostly produced from cow milk and to a very limited extent from ewe milk. The evolution of caseins and whey proteins in ovine milk submitted to different thermal treatments (63 degrees C/30 min; 73 degrees C/15 min; 85 degrees C/10 min or 96 degrees C/5 min) was followed during fermentation of yoghurts and during their storage up to 14 days, using two different sets of starters. One set of starter LAB was a "ropy" culture (YC-191), which is a well-defined mixed strain culture containing Streptococcus thermophilus ST-143 and Lactobacillus delbrueckii subsp. bulgaricus (LB-18 and LB-CH2). The other set of starter bacteria (YC-460) was a standard yoghurt culture("non-ropy") containing mixed strain culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Contents of free amino groups in produced yoghurts increased gradually during the fermentation, up to a maximal value obtained after 4 h fermentation, then they did not change significantly during storage of yoghurt produced with YC-191 starter. In contrary, a large drop in the amount of free amino groups was observed in the first 24 h of storage in the case of yoghurt made with YC-460 indicating that microorganisms continue still to grow in low temperatures. During fermentation and storage of both yoghurt types, alpha-lactalbumin was hydrolyzed to a slightly bigger extent than beta-lactoglobulin. During fermentation, beta-casein was slightly more degraded than alpha(s)-caseins; however, the opposite was observed during storage up to 14 days. Generally, a more intense heat pretreatment led to a higher degradation of whey proteins and caseins during fermentation and storage. Differences in proteolytic activity between the two starters used (whey proteins more degraded by YC-191; caseins more degraded by YC-460) may lead to improvement in production and formulation of yoghurts differing in their physicochemical and rheological properties. PMID:12866624

  6. Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis

    PubMed Central

    Briz, Victor; Hsu, Yu-Tien; Li, Yi; Lee, Erin; Bi, Xiaoning; Baudry, Michel

    2013-01-01

    Memory consolidation has been suggested to be protein synthesis-dependent. Recent data indicate that BDNF-induced dendritic protein synthesis is a key event in memory formation through activation of the mammalian target of rapamycin (mTOR) pathway. BDNF also activates calpain, a calcium-dependent cysteine protease, which has been shown to play a critical role in learning and memory. This study was therefore directed at testing the hypothesis that calpain activity is required for BDNF-stimulated local protein synthesis, and at identifying the underlying molecular mechanism. In rat hippocampal slices, cortical synaptoneurosomes, and cultured neurons, BDNF-induced mTOR pathway activation and protein translation were blocked by calpain inhibition. BDNF treatment rapidly reduced levels of hamartin and tuberin, negative regulators of mTOR, in a calpain-dependent manner. Treatment of brain homogenates with purified calpain-1 and calpain-2 truncated both proteins. BDNF treatment increased phosphorylation of both Akt and ERK, but only the effect on Akt was blocked by calpain inhibition. Levels of PTEN (phosphatase and tensin homolog deleted on chromosome ten), a phosphatase that inactivates Akt, were decreased following BDNF treatment, and calpain inhibition reversed this effect. Calpain-2 but not calpain-1 treatment of brain homogenates resulted in PTEN degradation. In cultured cortical neurons, knock-down of calpain-2 but not calpain-1 by siRNA completely suppressed the effect of BDNF on mTOR activation. Our results reveal a critical role for calpain-2 in BDNF-induced mTOR signaling and dendritic protein synthesis via PTEN, hamartin and tuberin degradation. This mechanism therefore provides a link between proteolysis and protein synthesis that might contribute to synaptic plasticity. PMID:23467348

  7. Effect of feed deprivation and insulin-like growth hormone on indices of protein degradation in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin-like growth factor-I (IGF-I) is a hormone that promotes growth by both increasing protein synthesis and decreasing protein degradation. This study utilizes a comparative slaughter approach to determine the effect of feed deprivation and IGF-I treatment on weight loss and indices of protein ...

  8. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    PubMed Central

    Chakrabarti, Sanjukta; Barrow, Colin J.; Kanwar, Rupinder K.; Ramana, Venkata; Kanwar, Jagat R.

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

  9. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization.

    PubMed

    Chakrabarti, Sanjukta; Barrow, Colin J; Kanwar, Rupinder K; Ramana, Venkata; Kanwar, Jagat R

    2016-01-01

    Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1-D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities. PMID:27294920

  10. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis.

    PubMed

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKY(Y115E) phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  11. Transient degradation of NF-kappaB proteins in macrophages after interaction with mast cell granules.

    PubMed Central

    Ito, N; Li, Y; Suzuki, T; Stechschulte, D J; Dileepan, K N

    1998-01-01

    The exposure of the macrophage cell line, J774 to mast cell granules (MCG) led to the formation of altered nuclear transcription factor proteins (NF-kappaBx), which had faster electrophoretic mobility than the p50 homodimer of NF-KB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-kappaBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBalpha remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of mouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-KB proteins. The alterations in NF-KB proteins did not affect the expression of inducible nitric oxide synthase (iNOS) or TNF-alpha mRNA inJ774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-KB proteins with associated inhibition of iNOS or TNF-alpha mRNA expression. PMID:9927232

  12. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis

    PubMed Central

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKYY115E phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  13. Degradation of gangliosides by the lysosomal sialidase requires an activator protein.

    PubMed

    Fingerhut, R; van der Horst, G T; Verheijen, F W; Conzelmann, E

    1992-09-15

    Lysosomal sialidase, which was formerly believed to degrade only water-soluble substrates but not glycolipids, cleaves ganglioside substrates II3NeuNAc-LacCer, IV3NeuNAc, II3NeuNAc-GgOse4Cer, IV3 NeuNAc, II3(NeuNAc)2-GgOse4Cer when these are dispersed either with an appropriate detergent (taurodeoxycholate) or with the sulfatide activator protein, a physiologic lipid solubilizer required for the lysosomal hydrolysis of other glycolipids by water-soluble hydrolases. In the presence of the activator protein, time and protein dependence were linear within wide limits, while the detergent rapidly inactivated the enzyme. The disialo group of the b-series gangliosides was only poorly attacked by the enzyme when the lipids were dispersed with the activator protein, whereas in the presence of the detergent, they were hydrolyzed as fast as terminal sialic acid residues. With the appropriate assay method, significant ganglioside sialidase activity could be demonstrated in the secondary lysosome fraction of normal skin fibroblasts but not of sialidosis fibroblasts. Our results support the notion that there is only one lysosomal sialidase, which degrades both the water-soluble and the membrane-bound sialyl glycoconjugates. PMID:1396669

  14. β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes*

    PubMed Central

    Jakhria, Toral; Hellewell, Andrew L.; Porter, Morwenna Y.; Jackson, Matthew P.; Tipping, Kevin W.; Xue, Wei-Feng; Radford, Sheena E.; Hewitt, Eric W.

    2014-01-01

    Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β2-microglobulin (β2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β2m fibrils did not, however, cause cell death. Instead, fragmented β2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway. PMID:25378395

  15. Mathematical aspects of the kinetics of formation and degradation of linear peptide or protein aggregates.

    PubMed

    Zhdanov, Vladimir P

    2016-08-01

    In cells, peptides and proteins are sometimes prone to aggregation. In neurons, for example, amyloid β peptides form plaques related to Alzheimer's disease (AD). The corresponding kinetic models either ignore or do not pay attention to degradation of these species. Here, the author proposes a generic kinetic model describing formation and degradation of linear aggregates. The process is assumed to occur via reversible association of monomers and attachment of monomers to or detachment from terminal parts of aggregates. Degradation of monomers is described as a first-order process. Degradation of aggregates is considered to occur at their terminal and internal parts with different rates and these steps are described by first-order equations as well. Irrespective of the choice of the values of the rate constants, the model predicts that eventually the system reaches a stable steady state with the aggregate populations rapidly decreasing with increasing size at large sizes. The corresponding steady-state size distributions of aggregates are illustrated in detail. The transient kinetics are also shown. The observation of AD appears, however, to indicate that the peptide production becomes eventually unstable, i.e., the growth of the peptide population is not properly limited. This is expected to be related to the specifics of the genetic networks controlling the peptide production. Following this line, two likely general networks with, respectively, global negative and positive feedbacks in the peptide production are briefly discussed. PMID:27132946

  16. Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

    PubMed Central

    Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Takatsuki, Hanae; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrPSc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrPSc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrPSc degradation were evaluated. The accumulation of PrPSc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann–Sträussler–Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrPSc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrPSc from either the 22L or the Chandler strain, indicating that the degradation of PrPSc in host cells might be strain-dependent. PMID:26368533

  17. Protein binding mediation of biomaterial-dependent monocyte activation on a degradable polar hydrophobic ionic polyurethane.

    PubMed

    Battiston, Kyle G; Labow, Rosalind S; Santerre, J Paul

    2012-11-01

    Protein adsorption is an important phenomenon influencing the cellular response to biomaterials. Previous studies comparing monocyte activation on a degradable polar hydrophobic ionic polyurethane (D-PHI) indicated a reduced pro-inflammatory monocyte response relative to tissue culture polystyrene (TCPS) and poly(lactide-co-glycolide) (PLGA) substrates. The present study investigated the influence of protein binding in order to gain further insight into the observed differential monocyte activation. Several proteins, identified in different relative amounts within the bound protein layers on D-PHI vs. PLGA and TCPS, were evaluated for their effect on monocyte activation. It was found that, in general, both non-coated and protein pre-adsorbed D-PHI supported a reduced pro-inflammatory response relative to PLGA, as indicated by lower levels of tumor necrosis factor-α (TNF-α) release. An initial increase in TNF-α release occurred when α(2)-macroglobulin (A2M) was pre-adsorbed to D-PHI, which was shown to involve the α(2)-macroglobulin receptor and was active on D-PHI but not on the two other biomaterials. This response was not observed during competitive protein binding in the presence of fetal bovine serum (FBS), suggesting that a more complex arrangement of the bound proteins and their interactions with one another, as well as with the surface chemistry of the individual biomaterials, resulted in the low-activating character of D-PHI when interacting with human monocytes. PMID:22940217

  18. Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation.

    PubMed

    van der Goot, Annemieke T; Zhu, Wentao; Vázquez-Manrique, Rafael P; Seinstra, Renée I; Dettmer, Katja; Michels, Helen; Farina, Francesca; Krijnen, Jasper; Melki, Ronald; Buijsman, Rogier C; Ruiz Silva, Mariana; Thijssen, Karen L; Kema, Ido P; Neri, Christian; Oefner, Peter J; Nollen, Ellen A A

    2012-09-11

    Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer's diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra L-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases. PMID:22927396

  19. Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation

    PubMed Central

    van der Goot, Annemieke T.; Zhu, Wentao; Vázquez-Manrique, Rafael P.; Seinstra, Renée I.; Dettmer, Katja; Michels, Helen; Farina, Francesca; Krijnen, Jasper; Melki, Ronald; Buijsman, Rogier C.; Ruiz Silva, Mariana; Thijssen, Karen L.; Kema, Ido P.; Neri, Christian; Oefner, Peter J.; Nollen, Ellen A. A.

    2012-01-01

    Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer’s diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra l-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases. PMID:22927396

  20. The TSG101 protein binds to connexins and is involved in connexin degradation

    SciTech Connect

    Auth, Tanja Schlueter, Sharazad; Urschel, Stephanie; Kussmann, Petra; Sonntag, Stephan; Hoeher, Thorsten; Kreuzberg, Maria M.; Dobrowolski, Radoslaw; Willecke, Klaus

    2009-04-01

    Gap junctions mediate electrical and metabolic communication between cells in almost all tissues and are proposed to play important roles in cellular growth control, differentiation and embryonic development. Gap junctional communication and channel assembly were suggested to be regulated by interaction of connexins with different proteins including kinases and phosphatases. Here, we identified the tumor susceptibility gene 101 (TSG101) protein to bind to the carboxyterminal tail of connexin45 in a yeast two-hybrid protein interaction screen. Glutathione S-transferase pull down experiments and immunoprecipitation revealed that not only connexin45 but also connexin30.2, -36, and -43 carboxyterminal regions were associated with TSG101 protein in pull down analyses and that connexin31, -43 and -45 co-precipitate with endogenous TSG101 protein in lysates from HM1 embryonic stem cells. TSG101 has been shown to be involved in cell cycle control, transcriptional regulation and turnover of endocytosed proteins. Thus, we decided to study the functional role of this interaction. SiRNA mediated knock down of TSG101 in HM1 embryonic stem cells led to increased levels of connexin43 and -45, prolonged half life of these connexins and increased transfer of microinjected Lucifer yellow. Our results suggest that TSG101 is involved in the degradation of connexins via interaction with connexin proteins.

  1. Homeodomain protein Dlx3 induces phosphorylation-dependent p63 degradation

    PubMed Central

    Di Costanzo, Antonella; Festa, Luisa; Duverger, Olivier; Vivo, Maria; Guerrlal, Luisa; La Mantia, Girolama; Morasso, Marla I.; Calabro, Viola

    2009-01-01

    The epidermis is a stratified epithelium which develops depending on the transcription factor p63, a member of the p53 family of transcription factors. p63 is strongly expressed in the innermost basal layer where highly proliferative epithelial cells reside. p63 functions as a molecular switch that initiates epithelial stratification or cell fate determination while regulating proliferation and differentiation of developmentally mature keratinocytes. p63 acts upstream of Dlx3 homeobox gene in a transcriptional regulatory pathway relevant to ectodermal dysplasia. Here we show that Dlx3 triggers p63 protein degradation by a proteasome-dependent pathway. Mutant ΔNp63α in which Threonine397 and Serine383 were replaced with Alanine as well as C-terminal truncated versions of ΔNp63α are resistant to Dlx3-mediated degradation. Transient expression of Dlx3 is associated with Raft phosphorylation. Dlx3 is unable to promote p63 degradation in Raft depleted MEF cells or upon pharmacological knockdown of Raft. Our data support a previously unrecognized role for Dlx3 in posttranslational regulation of ΔNp63α protein level, a mechanism that may contribute to reduce the abundance of ΔNp63α during differentiation of stratified epithelia. PMID:19282665

  2. Homeodomain protein Dlx3 induces phosphorylation-dependent p63 degradation.

    PubMed

    Di Costanzo, Antonella; Festa, Luisa; Duverger, Olivier; Vivo, Maria; Guerrini, Luisa; La Mantia, Girolama; Morasso, Maria I; Calabrò, Viola

    2009-04-15

    The epidermis is a stratified epithelium which develops depending on the transcription factor p63, a member of the p53 family of transcription factors. p63 is strongly expressed in the innermost basal layer where highly proliferative epithelial cells reside. p63 functions as a molecular switch that initiates epithelial stratification or cell fate determination while regulating proliferation and differentiation of developmentally mature keratinocytes. p63 acts upstream of Dlx3 homeobox gene in a transcriptional regulatory pathway relevant to ectodermal dysplasia. Here we show that Dlx3 triggers p63 protein degradation by a proteasome-dependent pathway. Mutant DeltaNp63alpha in which Threonine397 and Serine383 were replaced with Alanine as well as C-terminal truncated versions of DeltaNp63alpha are resistant to Dlx3-mediated degradation. Transient expression of Dlx3 is associated with Raf1 phosphorylation. Dlx3 is unable to promote p63 degradation in Raf1 depleted MEF cells or upon pharmacological knockdown of Raf1. Our data support a previously unrecognized role for Dlx3 in posttranslational regulation of DeltaNp63alpha protein level, a mechanism that may contribute to reduce the abundance of DeltaNp63alpha during differentiation of stratified epithelia. PMID:19282665

  3. Postmortem degradation of skeletal muscle proteins: a novel approach to determine the time since death.

    PubMed

    Pittner, Stefan; Monticelli, Fabio C; Pfisterer, Alexander; Zissler, Angela; Sänger, Alexandra M; Stoiber, Walter; Steinbacher, Peter

    2016-03-01

    Estimating the time since death is a very important aspect in forensic sciences which is pursued by a variety of methods. The most precise method to determine the postmortem interval (PMI) is the temperature method which is based on the decrease of the body core temperature from 37 °C. However, this method is only useful in the early postmortem phase (~0-36 h). The aim of the present work is to develop an accurate method for PMI determination beyond this present limit. For this purpose, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and casein zymography to analyze the time course of degradation of selected proteins and calpain activity in porcine biceps femoris muscle until 240 h postmortem (hpm). Our results demonstrate that titin, nebulin, desmin, cardiac troponin T, and SERCA1 degraded in a regular and predictable fashion in all samples investigated. Similarly, both the native calpain 1 and calpain 2 bands disintegrate into two bands subsequently. This degradation behavior identifies muscular proteins and enzymes as promising substrates for future molecular-based PMI determination technologies. PMID:26041514

  4. Hepatitis B Virus X Protein Promotes Degradation of SMC5/6 to Enhance HBV Replication.

    PubMed

    Murphy, Christopher M; Xu, Yanping; Li, Feng; Nio, Kouki; Reszka-Blanco, Natalia; Li, Xiaodong; Wu, Yaxu; Yu, Yanbao; Xiong, Yue; Su, Lishan

    2016-09-13

    The hepatitis B virus (HBV) regulatory protein X (HBx) activates gene expression from the HBV covalently closed circular DNA (cccDNA) genome. Interaction of HBx with the DDB1-CUL4-ROC1 (CRL4) E3 ligase is critical for this function. Using substrate-trapping proteomics, we identified the structural maintenance of chromosomes (SMC) complex proteins SMC5 and SMC6 as CRL4(HBx) substrates. HBx expression and HBV infection degraded the SMC5/6 complex in human hepatocytes in vitro and in humanized mice in vivo. HBx targets SMC5/6 for ubiquitylation by the CRL4(HBx) E3 ligase and subsequent degradation by the proteasome. Using a minicircle HBV (mcHBV) reporter system with HBx-dependent activity, we demonstrate that SMC5/6 knockdown, or inhibition with a dominant-negative SMC6, enhance HBx null mcHBV-Gluc gene expression. Furthermore, SMC5/6 knockdown rescued HBx-deficient HBV replication in human hepatocytes. These results indicate that a primary function of HBx is to degrade SMC5/6, which restricts HBV replication by inhibiting HBV gene expression. PMID:27626656

  5. FPGA accelerator for protein secondary structure prediction based on the GOR algorithm

    PubMed Central

    2011-01-01

    Background Protein is an important molecule that performs a wide range of functions in biological systems. Recently, the protein folding attracts much more attention since the function of protein can be generally derived from its molecular structure. The GOR algorithm is one of the most successful computational methods and has been widely used as an efficient analysis tool to predict secondary structure from protein sequence. However, the execution time is still intolerable with the steep growth in protein database. Recently, FPGA chips have emerged as one promising application accelerator to accelerate bioinformatics algorithms by exploiting fine-grained custom design. Results In this paper, we propose a complete fine-grained parallel hardware implementation on FPGA to accelerate the GOR-IV package for 2D protein structure prediction. To improve computing efficiency, we partition the parameter table into small segments and access them in parallel. We aggressively exploit data reuse schemes to minimize the need for loading data from external memory. The whole computation structure is carefully pipelined to overlap the sequence loading, computing and back-writing operations as much as possible. We implemented a complete GOR desktop system based on an FPGA chip XC5VLX330. Conclusions The experimental results show a speedup factor of more than 430x over the original GOR-IV version and 110x speedup over the optimized version with multi-thread SIMD implementation running on a PC platform with AMD Phenom 9650 Quad CPU for 2D protein structure prediction. However, the power consumption is only about 30% of that of current general-propose CPUs. PMID:21342582

  6. Diversity in growth and protein degradation by dairy relevant lactic acid bacteria species in reconstituted whey.

    PubMed

    Pescuma, Micaela; Hébert, Elvira M; Bru, Elena; Font de Valdez, Graciela; Mozzi, Fernanda

    2012-05-01

    The high nutritional value of whey makes it an interesting substrate for the development of fermented foods. The aim of this work was to evaluate the growth and proteolytic activity of sixty-four strains of lactic acid bacteria in whey to further formulate a starter culture for the development of fermented whey-based beverages. Fermentations were performed at 37 °C for 24 h in 10 and 16% (w/v) reconstituted whey powder. Cultivable populations, pH, and proteolytic activity (o-phthaldialdehyde test) were determined at 6 and 24 h incubation. Hydrolysis of whey proteins was analysed by Tricine SDS-PAGE. A principal component analysis (PCA) was applied to evaluate the behaviour of strains. Forty-six percent of the strains grew between 1 and 2 Δlog CFU/ml while 19% grew less than 0·9 Δlog CFU/ml in both reconstituted whey solutions. Regarding the proteolytic activity, most of the lactobacilli released amino acids and small peptides during the first 6 h incubation while streptococci consumed the amino acids initially present in whey to sustain growth. Whey proteins were degraded by the studied strains although to different extents. Special attention was paid to the main allergenic whey protein, β-lactoglobulin, which was degraded the most by Lactobacillus acidophilus CRL 636 and Lb. delbrueckii subsp. bulgaricus CRL 656. The strain variability observed and the PCA applied in this study allowed selecting appropriate strains able to improve the nutritional characteristics (through amino group release and protein degradation) and storage (decrease in pH) of whey. PMID:22559062

  7. Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

    PubMed

    Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. PMID:26505494

  8. Phenotypes on demand via switchable target protein degradation in multicellular organisms

    PubMed Central

    Faden, Frederik; Ramezani, Thomas; Mielke, Stefan; Almudi, Isabel; Nairz, Knud; Froehlich, Marceli S.; Höckendorff, Jörg; Brandt, Wolfgang; Hoehenwarter, Wolfgang; Dohmen, R. Jürgen; Schnittger, Arp; Dissmeyer, Nico

    2016-01-01

    Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation. PMID:27447739

  9. Phenotypes on demand via switchable target protein degradation in multicellular organisms.

    PubMed

    Faden, Frederik; Ramezani, Thomas; Mielke, Stefan; Almudi, Isabel; Nairz, Knud; Froehlich, Marceli S; Höckendorff, Jörg; Brandt, Wolfgang; Hoehenwarter, Wolfgang; Dohmen, R Jürgen; Schnittger, Arp; Dissmeyer, Nico

    2016-01-01

    Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation. PMID:27447739

  10. Acceleration through passive destabilization: protein folding in a weak hydrophobic environment

    NASA Astrophysics Data System (ADS)

    Jewett, Andrew; Baumketner, Andrij; Shea, Joan-Emma

    2004-03-01

    The GroEL chaperonin is a biomolecule which assists the folding of an extremely diverse range of proteins in Eubacteria. Some proteins undergo many rounds of ATP-regulated binding and dissociation from GroEL/ES before folding. It has been proposed that transient stress from ATP-regulated binding and release from GroEL/ES frees frustrated proteins from misfolded conformations. However recent evidence suggests that chaperonin-accelerated protein folding can take place entirely within a mutated GroEL+ES cavity that is unable to open and release the protein. Using molecular dynamics, we demonstrate that static confinement within a weakly hydrophobic (attractive) cavity (similar to the interior of the cavity formed by the GroEL+ES complex) is sufficient to significantly accelerate the folding of a highly frustrated protein-like heteropolymer. Our frustrated molecule benifits kinetically from a static hydrophobic environment that destabilizes misfolded conformations. This may shed light on the mechanisms used by other chaperones which do not depend on ATP.

  11. Results of a screening programme to identify plants or plant extracts that inhibit ruminal protein degradation.

    PubMed

    Selje, N; Hoffmann, E M; Muetzel, S; Ningrat, R; Wallace, R J; Becker, K

    2007-07-01

    One aim of the EC Framework V project, 'Rumen-up' (QLK5-CT-2001-00 992), was to find plants or plant extracts that would inhibit the nutritionally wasteful degradation of protein in the rumen. A total of 500 samples were screened in vitro using 14C-labelled casein in a 30-min incubation with ruminal digesta. Eight were selected for further investigation using a batch fermentation system and soya protein and bovine serum albumin as proteolysis substrates; proteolysis was monitored over 12 h by the disappearance of soluble protein and the production of branched SCFA and NH3. Freeze-dried, ground foliage of Peltiphyllum peltatum, Helianthemum canum, Arbutus unedo, Arctostaphylos uva-ursi and Knautia arvensis inhibited proteolysis (P < 0.05), while Daucus carota, Clematis vitalba and Erica arborea had little effect. Inhibition by the first four samples appeared to be caused by the formation of insoluble tannin-protein complexes. The samples were rich in phenolics and inhibition was reversed by polyethyleneglycol. In contrast, K. arvensis contained low concentrations of phenolics and no tannins, had no effect in the 30-min assay, yet inhibited the degradation rate of soluble protein (by 14 %, P < 0.0001) and the production of branched SCFA (by 17 %, P < 0.05) without precipitating protein in the 12-h batch fermentation. The effects showed some resemblance to those obtained in parallel incubations containing 3 mum-monensin, suggesting that K. arvensis may be a plant-derived feed additive that can suppress growth and activity of key proteolytic ruminal micro-organisms in a manner similar to that already well known for monensin. PMID:17445338

  12. A molecular nanodevice for targeted degradation of mRNA during protein synthesis

    PubMed Central

    Lee, Kyung-Ho; Min, Seung-Eui; Kim, Haseong; Lee, Seung-Goo; Kim, Dong-Myung

    2016-01-01

    RNase H is an endonuclease that catalyzes the cleavage of RNA. Because it only acts on RNA in RNA:DNA hybrids, RNase H can be used for targeted degradation of RNA when used in combination with antisense oligodeoxyribonucleotides (ASODNs) designed against a specific sequence of the target RNA. In this study, ASODN and RNase H were co-conjugated on magnetic nanoparticles. The resulting nanoparticles, having integrated functions of probing and processing target RNA, were able to remove target mRNA sequences more effectively than free ASODNs. The paramagnetic property of the nanoparticles also enabled timed engagement and disengagement of the RNA-degrading components in a given system, and these nanoparticles were able to be used for ON/OFF control of gene expression during cell-free protein synthesis reactions. PMID:26857021

  13. The role of local protein synthesis and degradation in axon regeneration

    PubMed Central

    Gumy, Laura F.; Tan, Chin Lik; Fawcett, James W.

    2010-01-01

    In axotomised regenerating axons, the first step toward successful regeneration is the formation of a growth cone. This requires a variety of dynamic morphological and biochemical changes in the axon, including the appearance of many new cytoskeletal, cell surface and signalling molecules. These changes suggest the activation of coordinated complex cellular processes. A recent development has been the demonstration that the regenerative ability of some axons depends on their capacity to locally synthesise new proteins and degrade others at the injury site autonomously from the cell body. There are also events involving the degradation of cytoskeletal and other molecules, and activation of signalling pathways, with axotomy-induced calcium changes probably being an initiating event. A future challenge will be to understand how this complex network of processes interacts in order to find therapeutic ways of promoting the regeneration of CNS axons. PMID:19520073

  14. mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy

    PubMed Central

    Zhao, Jinghui; Zhai, Bo; Gygi, Steven P.; Goldberg, Alfred Lewis

    2015-01-01

    Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440–443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth. PMID:26669439

  15. Conformational Changes in Acetylcholine Binding Protein Investigated by Temperature Accelerated Molecular Dynamics

    PubMed Central

    Mohammad Hosseini Naveh, Zeynab; Malliavin, Therese E.; Maragliano, Luca; Cottone, Grazia; Ciccotti, Giovanni

    2014-01-01

    Despite the large number of studies available on nicotinic acetylcholine receptors, a complete account of the mechanistic aspects of their gating transition in response to ligand binding still remains elusive. As a first step toward dissecting the transition mechanism by accelerated sampling techniques, we study the ligand-induced conformational changes of the acetylcholine binding protein (AChBP), a widely accepted model for the full receptor extracellular domain. Using unbiased Molecular Dynamics (MD) and Temperature Accelerated Molecular Dynamics (TAMD) simulations we investigate the AChBP transition between the apo and the agonist-bound state. In long standard MD simulations, both conformations of the native protein are stable, while the agonist-bound structure evolves toward the apo one if the orientation of few key sidechains in the orthosteric cavity is modified. Conversely, TAMD simulations initiated from the native conformations are able to produce the spontaneous transition. With respect to the modified conformations, TAMD accelerates the transition by at least a factor 10. The analysis of some specific residue-residue interactions points out that the transition mechanism is based on the disruption/formation of few key hydrogen bonds. Finally, while early events of ligand dissociation are observed already in standard MD, TAMD accelerates the ligand detachment and, at the highest TAMD effective temperature, it is able to produce a complete dissociation path in one AChBP subunit. PMID:24551117

  16. Stress-Regulated Translational Attenuation Adapts Mitochondrial Protein Import Through Tim17A Degradation

    PubMed Central

    Rainbolt, T. Kelly; Atanassova, Neli; Genereux, Joseph C.; Wiseman, R. Luke

    2014-01-01

    SUMMARY Stress-regulated signaling pathways protect mitochondrial proteostasis, and thus mitochondrial function, from pathologic insults. Despite the importance of stress-regulated signaling pathways in mitochondrial proteome maintenance, the molecular mechanisms by which these pathways maintain mitochondrial proteostasis remain largely unknown. Here, we identify Tim17A as a stress-regulated subunit of the Translocase of the Inner Membrane 23 (TIM23) mitochondrial protein import complex. We show that Tim17A protein levels are decreased downstream of stress-regulated translational attenuation induced by eIF2α phosphorylation through a mechanism dependent on the mitochondrial protease YME1L. Furthermore, we demonstrate that decreasing Tim17A protein levels attenuates TIM23-dependent protein import, promotes the induction of mitochondrial Unfolded Protein Response-associated proteostasis genes, and confers stress-resistance in C. elegans and mammalian cells. Thus, our results indicate that Tim17A degradation is a stress-responsive mechanism by which cells adapt mitochondrial protein import efficiency and promote mitochondrial proteostasis in response to the numerous pathologic insults that induce stress-regulated translation attenuation. PMID:24315374

  17. Staphylococcus epidermidis Esp Degrades Specific Proteins Associated with Staphylococcus aureus Biofilm Formation and Host-Pathogen Interaction

    PubMed Central

    Iwamoto, Takeo; Takada, Koji; Okuda, Ken-ichi; Tajima, Akiko; Iwase, Tadayuki

    2013-01-01

    Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (EspS235A) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction. PMID:23316041

  18. Environmental aging in polycrystalline-Si photovoltaic modules: comparison of chamber-based accelerated degradation studies with field-test data

    NASA Astrophysics Data System (ADS)

    Lai, T.; Biggie, R.; Brooks, A.; Potter, B. G.; Simmons-Potter, K.

    2015-09-01

    Lifecycle degradation testing of photovoltaic (PV) modules in accelerated-degradation chambers can enable the prediction both of PV performance lifetimes and of return-on-investment for installations of PV systems. With degradation results strongly dependent on chamber test parameters, the validity of such studies relative to fielded, installed PV systems must be determined. In the present work, accelerated aging of a 250 W polycrystalline silicon module is compared to real-time performance degradation in a similar polycrystalline-silicon, fielded, PV technology that has been operating since October 2013. Investigation of environmental aging effects are performed in a full-scale, industrial-standard environmental chamber equipped with single-sun irradiance capability providing illumination uniformity of 98% over a 2 x 1.6 m area. Time-dependent, photovoltaic performance (J-V) is evaluated over a recurring, compressed night-day cycle providing representative local daily solar insolation for the southwestern United States, followed by dark (night) cycling. This cycle is synchronized with thermal and humidity environmental variations that are designed to mimic, as closely as possible, test-yard conditions specific to a 12 month weather profile for a fielded system in Tucson, AZ. Results confirm the impact of environmental conditions on the module long-term performance. While the effects of temperature de-rating can be clearly seen in the data, removal of these effects enables the clear interpretation of module efficiency degradation with time and environmental exposure. With the temperature-dependent effect removed, the normalized efficiency is computed and compared to performance results from another panel of similar technology that has previously experienced identical climate changes in the test yard. Analysis of relative PV module efficiency degradation for the chamber-tested system shows good comparison to the field-tested system with ~2.5% degradation following

  19. Rumen Degradability and Small Intestinal Digestibility of the Amino Acids in Four Protein Supplements

    PubMed Central

    Wang, Y.; Jin, L.; Wen, Q. N.; Kopparapu, N. K.; Liu, J.; Liu, X. L.; Zhang, Y. G.

    2016-01-01

    The supplementation of livestock feed with animal protein is a present cause for public concern, and plant protein shortages have become increasingly prominent in China. This conflict may be resolved by fully utilizing currently available sources of plant protein. We estimated the rumen degradability and the small intestinal digestibility of the amino acids (AA) in rapeseed meal (RSM), soybean meal (SBM), sunflower seed meal (SFM) and sesame meal (SSM) using the mobile nylon bag method to determine the absorbable AA content of these protein supplements as a guide towards dietary formulations for the dairy industry. Overall, this study aimed to utilize protein supplements effectively to guide dietary formulations to increase milk yield and save plant protein resources. To this end, we studied four cows with a permanent rumen fistula and duodenal T-shape fistula in a 4×4 Latin square experimental design. The results showed that the total small intestine absorbable amino acids and small intestine absorbable essential amino acids were higher in the SBM (26.34% and 13.11% dry matter [DM], respectively) than in the SFM (13.97% and 6.89% DM, respectively). The small intestine absorbable Lys contents of the SFM, SSM, RSM and SBM were 0.86%, 0.88%, 1.43%, and 2.12% (DM basis), respectively, and the absorbable Met contents of these meals were 0.28%, 1.03%, 0.52%, and 0.47% (DM basis), respectively. Among the examined food sources, the milk protein score of the SBM (0.181) was highest followed by those of the RSM (0.136), SSM (0.108) and SFM (0.106). The absorbable amino acid contents of the protein supplements accurately reflected protein availability, which is an important indicator of the balance of feed formulation. Therefore, a database detailing the absorbable AA should be established. PMID:26732449

  20. Rumen Degradability and Small Intestinal Digestibility of the Amino Acids in Four Protein Supplements.

    PubMed

    Wang, Y; Jin, L; Wen, Q N; Kopparapu, N K; Liu, J; Liu, X L; Zhang, Y G

    2016-02-01

    The supplementation of livestock feed with animal protein is a present cause for public concern, and plant protein shortages have become increasingly prominent in China. This conflict may be resolved by fully utilizing currently available sources of plant protein. We estimated the rumen degradability and the small intestinal digestibility of the amino acids (AA) in rapeseed meal (RSM), soybean meal (SBM), sunflower seed meal (SFM) and sesame meal (SSM) using the mobile nylon bag method to determine the absorbable AA content of these protein supplements as a guide towards dietary formulations for the dairy industry. Overall, this study aimed to utilize protein supplements effectively to guide dietary formulations to increase milk yield and save plant protein resources. To this end, we studied four cows with a permanent rumen fistula and duodenal T-shape fistula in a 4×4 Latin square experimental design. The results showed that the total small intestine absorbable amino acids and small intestine absorbable essential amino acids were higher in the SBM (26.34% and 13.11% dry matter [DM], respectively) than in the SFM (13.97% and 6.89% DM, respectively). The small intestine absorbable Lys contents of the SFM, SSM, RSM and SBM were 0.86%, 0.88%, 1.43%, and 2.12% (DM basis), respectively, and the absorbable Met contents of these meals were 0.28%, 1.03%, 0.52%, and 0.47% (DM basis), respectively. Among the examined food sources, the milk protein score of the SBM (0.181) was highest followed by those of the RSM (0.136), SSM (0.108) and SFM (0.106). The absorbable amino acid contents of the protein supplements accurately reflected protein availability, which is an important indicator of the balance of feed formulation. Therefore, a database detailing the absorbable AA should be established. PMID:26732449

  1. Ruminal degradation and intestinal digestibility of protein and amino acids in high-protein feedstuffs commonly used in dairy diets.

    PubMed

    Paz, H A; Klopfenstein, T J; Hostetler, D; Fernando, S C; Castillo-Lopez, E; Kononoff, P J

    2014-10-01

    A study was conducted to determine the rumen degradation and intestinal digestibility of crude protein (CP) and AA, and AA composition of the rumen-undegradable protein (RUP) from 3 sources of blood meal (BM1, BM2, and BM3), canola meal (CM), low-fat distillers dried grains with solubles (LFDG), soybean meal (SBM), and expeller soybean meal (ESBM). Two Holstein cows fitted with ruminal and proximal duodenal cannulas were used for in situ incubation of 16h and for the mobile bag technique. To correct for bacterial contamination of the RUP, 2 methods were used: purines and DNA as bacterial markers. Ruminal degradations of CP were 85.3, 29.8, 40.7, 75.7, 76.9, 68.8, and 37.0 ± 3.93% for BM1, BM2, BM3, CM, LFDG, SBM, and ESBM, respectively. Ruminal degradation of both total essential AA and nonessential AA followed a similar pattern to that of CP across feedstuffs. Based on the ratio of AA concentration in the RUP to AA concentration in the original feedstuff, ruminal incubation decreased (ratio <1) the concentrations of His, Lys, and Trp, and increased (ratio >1) the concentrations of Ile and Met across feedstuffs. Compared with purines, the use of DNA as bacterial marker resulted in a higher estimate of bacterial CP contamination for CM and lower estimates for LFDG and ESBM. Intestinal digestibility of RUP could not be estimated for BM1, BM3, and SBM due to insufficient recovery of residue. For the remaining feedstuffs, intestinal digestibility of RUP was highest for ESBM, followed by BM2 and LFDG, and lowest for CM: 98.8, 87.9, 89.7, and 72.4 ± 1.40%, respectively. Intestinal absorbable dietary protein was higher for BM2 compared with CM and LFDG, at 61.7, 17.9, and 20.7 ± 2.73% CP, respectively. As prices fluctuate, intestinal absorbable protein or AA may be used as a tool to aid in the selection among feedstuffs with different protein quality. PMID:25108871

  2. Recycling of a regulatory protein by degradation of the RNA to which it binds.

    PubMed

    Deikus, Gintaras; Babitzke, Paul; Bechhofer, David H

    2004-03-01

    When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3'-to-5' exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase(-) strain showed accumulation of a stable, TRAP-protected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase(-) strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription. PMID:14976255

  3. Ectomycorrhizal Fungal Protein Degradation Ability Predicted by Soil Organic Nitrogen Availability.

    PubMed

    Rineau, Francois; Stas, Jelle; Nguyen, Nhu H; Kuyper, Thomas W; Carleer, Robert; Vangronsveld, Jaco; Colpaert, Jan V; Kennedy, Peter G

    2016-03-01

    In temperate and boreal forest ecosystems, nitrogen (N) limitation of tree metabolism is alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in soil switches from inorganic (NH4 (+) and NO3 (-)) to organic (mostly proteins). It has been hypothesized that ECM fungi adapt to the most common N source in their environment, which implies that fungi growing in older forests would have greater protein degradation abilities. Moreover, recent results for a model ECM fungal species suggest that organic N uptake requires a glucose supply. To test the generality of these hypotheses, we screened 55 strains of 13 Suillus species with different ecological preferences for their in vitro protein degradation abilities. Suillus species preferentially occurring in mature forests, where soil contains more organic matter, had significantly higher protease activity than those from young forests with low-organic-matter soils or species indifferent to forest age. Within species, the protease activities of ecotypes from soils with high or low soil organic N content did not differ significantly, suggesting resource partitioning between mineral and organic soil layers. The secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition had variable effects on secreted protease activity; in some species, it triggered activity, but in others, activity was repressed at high concentrations. Collectively, our results indicate that protease activity, a key ectomycorrhizal functional trait, is positively related to environmental N source availability but is also influenced by additional factors, such as carbon availability. PMID:26682855

  4. Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR{alpha} agonist WY14643 in rat hepatocytes

    SciTech Connect

    Wieneke, N.; Neuschaefer-Rube, F.; Bode, L.M.; Kuna, M.; Andres, J.; Carnevali, L.C.; Hirsch-Ernst, K.I.; Pueschel, G.P.

    2009-10-01

    Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feedback-controlled regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase II enzymes of xenobiotic metabolism. We have recently shown, that PPAR{alpha} agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR{alpha} agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPAR{alpha} agonist WY14643 to a larger extent than after induction with either compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UGT1A10. The PPAR{alpha}-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-induced reduction in energy expenditure by fatty acids as natural PPAR{alpha} ligands. The synergism of the PPAR{alpha} agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics.

  5. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling.

    PubMed

    Johnston, Christopher A; Taylor, J Philip; Gao, Yajun; Kimple, Adam J; Grigston, Jeffrey C; Chen, Jin-Gui; Siderovski, David P; Jones, Alan M; Willard, Francis S

    2007-10-30

    Heterotrimeric G protein signaling is important for cell-proliferative and glucose-sensing signal transduction pathways in the model plant organism Arabidopsis thaliana. AtRGS1 is a seven-transmembrane, RGS domain-containing protein that is a putative membrane receptor for d-glucose. Here we show, by using FRET, that d-glucose alters the interaction between the AtGPA1 and AtRGS1 in vivo. AtGPA1 is a unique heterotrimeric G protein alpha subunit that is constitutively GTP-bound given its high spontaneous nucleotide exchange coupled with slow GTP hydrolysis. Analysis of a point mutation in AtRGS1 that abrogates GTPase-accelerating activity demonstrates that the regulation of AtGPA1 GTP hydrolysis mediates sugar signal transduction during Arabidopsis development, in contrast to animals where nucleotide exchange is the limiting step in the heterotrimeric G protein nucleotide cycle. PMID:17951432

  6. Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice*

    PubMed Central

    Garyali, Punitee; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.

    2014-01-01

    Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a−/−, Epm2b−/−, and Epm2a−/− Epm2b−/− mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b−/− and Epm2a−/− Epm2b−/− cells) but not laforin (Epm2a−/− cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. PMID:24914213

  7. Two waves of proteasome-dependent protein degradation in the hippocampus are required for recognition memory consolidation.

    PubMed

    Figueiredo, Luciana S; Dornelles, Arethuza S; Petry, Fernanda S; Falavigna, Lucio; Dargél, Vinicius A; Köbe, Luiza M; Aguzzoli, Cristiano; Roesler, Rafael; Schröder, Nadja

    2015-04-01

    Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation. PMID:25687693

  8. Association of Bio-energy Processing-Induced Protein Molecular Structure Changes with CNCPS-Based Protein Degradation and Digestion of Co-products in Dairy Cows.

    PubMed

    Li, Xinxin; Zhang, Yonggen; Yu, Peiqiang

    2016-05-25

    The primary objective of this study was to develop a model to predict Cornell Net Carbohydrate Protein System (CNCPS) protein degradation and digestion based on protein molecular structure changes induced by bio-energy processing in different types of co-products (CoPR, CoPC, CoPS = co-products from bioprocessing of rapeseed, canola seed, and soybean, respectively). The results showed that the inherent structure changes induced by the processing had a close relationship with CNCPS predicted protein degradable, undegradable, and digestible contents. The amide I to II ratio and α-helix to β-sheet ratio could be used to predict total degradable protein (R(2) = 0.99, RSD = 0.84, P < 0.001). Total CNCPS intestinal digestible protein could be predicted by protein structure α-helix to β-sheet ratio (R(2) = 0.93, RSD = 0.33, P < 0.001). In conclusion, the processing-induced protein molecular structure changes were highly linked to protein nutritive value of the co-products and could be used as predictors for CNCPS protein degradation and digestion in dairy cattle. PMID:27112731

  9. The molecular components of the extracellular protein-degradation pathways of the ectomycorrhizal fungus Paxillus involutus

    PubMed Central

    Shah, Firoz; Rineau, Francois; Canbäck, Björn; Johansson, Tomas; Tunlid, Anders

    2013-01-01

    Proteins contribute to a major part of the organic nitrogen (N) in forest soils. This N is mobilized and becomes available to trees as a result of the depolymerizing activities of symbiotic ectomycorrhizal fungi. The mechanisms by which these fungi depolymerize proteins and assimilate the released N are poorly characterized. Biochemical analysis and transcriptome profiling were performed to examine the proteolytic machinery and the uptake system of the ectomycorrhizal basidiomycete Paxillus involutus during the assimilation of organic N from various protein sources and extracts of organic matter. All substrates induced secretion of peptidase activity with an acidic pH optimum, mostly contributed by aspartic peptidases. The peptidase activity was transiently repressed by ammonium. Transcriptional analysis revealed a large number of extracellular endo- and exopeptidases. The expression levels of these peptidases were regulated in parallel with transporters and enzymes involved in the assimilation and metabolism of the released peptides and amino acids. For the first time the molecular components of the protein degradation pathways of an ectomycorrhizal fungus are described. The data suggest that the transcripts encoding these components are regulated in response to the chemical properties and the availability of the protein substrates. PMID:23902518

  10. The kinesin KIF9 and reggie/flotillin proteins regulate matrix degradation by macrophage podosomes

    PubMed Central

    Cornfine, Susanne; Himmel, Mirko; Kopp, Petra; el Azzouzi, Karim; Wiesner, Christiane; Krüger, Marcus; Rudel, Thomas; Linder, Stefan

    2011-01-01

    Podosomes are actin-based matrix contacts in a variety of cell types, most notably monocytic cells, and are characterized by their ability to lyse extracellular matrix material. Besides their dependence on actin regulation, podosomes are also influenced by microtubules and microtubule-dependent transport processes. Here we describe a novel role for KIF9, a previously little-characterized member of the kinesin motor family, in the regulation of podosomes in primary human macrophages. We find that small interfering RNA (siRNA)/short-hairpin RNA–induced knockdown of KIF9 significantly affects both numbers and matrix degradation of podosomes. Overexpression and microinjection experiments reveal that the unique C-terminal region of KIF9 is crucial for these effects, presumably through binding of specific interactors. Indeed, we further identify reggie-1/flotillin-2, a signaling mediator between intracellular vesicles and the cell periphery, as an interactor of the KIF9 C-terminus. Reggie-1 dynamically colocalizes with KIF9 in living cells, and, consistent with KIF9-mediated effects, siRNA-induced knockdown of reggies/flotillins significantly impairs matrix degradation by podosomes. In sum, we identify the kinesin KIF9 and reggie/flotillin proteins as novel regulators of macrophage podosomes and show that their interaction is critical for the matrix-degrading ability of these structures. PMID:21119006

  11. ORMDL orosomucoid-like proteins are degraded by free-cholesterol-loading–induced autophagy

    PubMed Central

    Wang, Shuhui; Robinet, Peggy; Smith, Jonathan D.; Gulshan, Kailash

    2015-01-01

    Eukaryotic cells have evolved robust mechanisms to counter excess cholesterol including redistribution of lipids into different compartments and compensatory up-regulation of phospholipid biosynthesis. We demonstrate here that excess cellular cholesterol increased the activity of the endoplasmic reticulum (ER) enzyme serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme in sphingomyelin synthesis. This increased SPT activity was not due to altered levels of SPTLC1 or SPTLC2, the major subunits of SPT. Instead, cholesterol loading decreased the levels of ORMDL1, a negative regulator of SPT activity, due to its increased turnover. Several lines of evidence demonstrated that free-cholesterol–induced autophagy, which led to increased turnover of ORMDL1. Cholesterol loading induced ORMDL1 redistribution from the ER to cytoplasmic p62 positive autophagosomes. Coimmunoprecipitation analysis of cholesterol-loaded cells showed increased association between ORMDL1 and p62. The lysosomal inhibitor chloroquine or siRNA knockdown of Atg7 inhibited ORMDL1 degradation by cholesterol, whereas proteasome inhibitors showed no effect. ORMDL1 degradation was specific to free-cholesterol loading as autophagy induced by serum starvation or general ER stress did not lead to ORMDL1 degradation. ORMDL proteins are thus previously unidentified responders to excess cholesterol, exiting the ER to activate SPT and increase sphingomyelin biosynthesis, which may buffer excess cellular cholesterol. PMID:25775599

  12. Distinct protein degradation profiles are induced by different disuse models of skeletal muscle atrophy

    PubMed Central

    Bialek, Peter; Morris, Carl; Parkington, Jascha; St. Andre, Michael; Owens, Jane; Yaworsky, Paul; Seeherman, Howard

    2011-01-01

    Skeletal muscle atrophy can be a consequence of many diseases, environmental insults, inactivity, age, and injury. Atrophy is characterized by active degradation, removal of contractile proteins, and a reduction in muscle fiber size. Animal models have been extensively used to identify pathways that lead to atrophic conditions. We used genome-wide expression profiling analyses and quantitative PCR to identify the molecular changes that occur in two clinically relevant mouse models of muscle atrophy: hindlimb casting and Achilles tendon laceration (tenotomy). Gastrocnemius muscle samples were collected 2, 7, and 14 days after casting or injury. The total amount of muscle loss, as measured by wet weight and muscle fiber size, was equivalent between models on day 14, although tenotomy resulted in a more rapid induction of muscle atrophy. Furthermore, tenotomy resulted in the regulation of significantly more mRNA transcripts then did casting. Analysis of the regulated genes and pathways suggest that the mechanisms of atrophy are distinct between these models. The degradation following casting was ubiquitin-proteasome mediated, while degradation following tenotomy was lysosomal and matrix-metalloproteinase mediated, suggesting a possible role for autophagy. These data suggest that there are multiple mechanisms leading to muscle atrophy and that specific therapeutic agents may be necessary to combat atrophy resulting from different conditions. PMID:21791639

  13. Optical characterization of voltage-accelerated degradation in CH3NH3PbI3 perovskite solar cells.

    PubMed

    Handa, Taketo; Tex, David M; Shimazaki, Ai; Aharen, Tomoko; Wakamiya, Atsushi; Kanemitsu, Yoshihiko

    2016-05-16

    We investigate the performance degradation mechanism of CH3NH3PbI3 perovskite solar cells under bias voltage in air and nitrogen atmospheres using photoluminescence and electroluminescence techniques. When applying forward bias, the power conversion efficiency of the solar cells decreased significantly in air, but showed no degradation in nitrogen atmosphere. Time-resolved photoluminescence measurements on these devices revealed that the application of forward bias in air accelerates the generation of non-radiative recombination centers in the perovskite layer buried in the device. We found a negative correlation between the electroluminescence intensity and the injected current intensity in air. The irreversible change of the perovskite grain surface in air initiates the degradation of the perovskite solar cells. PMID:27409964

  14. Degradation of LIM domain-binding protein three during processing of Spanish dry-cured ham.

    PubMed

    Gallego, Marta; Mora, Leticia; Fraser, Paul D; Aristoy, María-Concepción; Toldrá, Fidel

    2014-04-15

    Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9 months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-90) providing evidence for the complexity and variability of proteolytic reactions throughout the whole process of dry-curing. Methionine oxidation has been observed in several peptides by the end of the process. The potential of some of the identified peptides to be used as biomarkers of dry-cured ham processing has also been considered. PMID:24295685

  15. Protein Transmission, Seeding and Degradation: Key Steps for α-Synuclein Prion-Like Propagation

    PubMed Central

    Ximerakis, Methodios; Vekrellis, Kostas

    2014-01-01

    Converging lines of evidence suggest that cell-to-cell transmission and the self-propagation of pathogenic amyloidogenic proteins play a central role in the initiation and the progression of several neurodegenerative disorders. This "prion-like" hypothesis has been recently reported for α-synuclein, a presynaptic protein implicated in the pathogenesis of Parkinson's disease (PD) and related disorders. This review summarizes recent findings on α-synuclein prion-like propagation, focusing on its transmission, seeding and degradation and discusses some key questions that remain to be explored. Understanding how α-synuclein exits cells and propagates from one brain region to another will lead to the development of new therapeutic strategies for the treatment of PD, aiming at slowing or stopping the disease progression. PMID:25548532

  16. Protein degradation by rumen microbes of heat-treated whole cottonseed.

    PubMed

    Tagari, H; Pena, F; Satter, L D

    1986-06-01

    In vitro and in situ rumen digestion trials were conducted to measure the effect of heat treatment on protein degradation by rumen microbes of cottonseed autoclaved and dry-heated (120, 140, 160 and 180 C) for different lengths of time (20, 40, 60, 90 and 120 min). Autoclaving (120 C, 1 kg/cm2 of pressure for 60 min) was effective in reducing ammonia nitrogen concentration in vitro, but dry heat at this temperature was without effect. Dry heating caused a linear decrease of in vitro ammonia concentration as temperature increased from 140 to 180 C and as exposure time increased from 20 to 120 min. The in situ results corroborated the in vitro findings. Crude protein disappearance from dacron bags decreased from 87 to 48% as temperature increased from 140 to 180 C after 20 min of heating. PMID:3755430

  17. Tyrosine-rich acidic matrix protein (TRAMP) accelerates collagen fibril formation in vitro.

    PubMed

    MacBeath, J R; Shackleton, D R; Hulmes, D J

    1993-09-15

    Tyrosine-rich acidic matrix protein (TRAMP) is a recently discovered protein that co-purifies with porcine skin lysyl oxidase and is equivalent to the M(r) 22,000 extracellular matrix protein from bovine skin that co-purifies with dermatan sulfate proteoglycans (Cronshaw, A. D., MacBeath, J. R. E., Shackleton, D. R., Collins, J. F., Fothergill-Gilmore, L. A., and Hulmes, D. J. S. (1993) Matrix 13, 255-266; Neame, P. J., Choi, H. U., and Rosenberg, L. C. (1989) J. Biol. Chem. 264, 5474-5479). The effect of TRAMP on collagen fibril formation was studied in vitro by reconstitution of fibrils from lathyritic rat skin collagen I. Fibril formation was initiated by the warm start procedure, in which acidic collagen solutions and double strength neutral buffer, both preincubated separately at 34 degrees C, were mixed. When monitored by turbidimetry, TRAMP was found to accelerate collagen fibril formation. Acceleration occurred at sub-stoichiometric molar ratios of TRAMP collagen, and the presence of TRAMP stabilized the fibrils against low temperature dissociation. It was confirmed by centrifugation that the amount of fibrillar collagen formed in the presence of TRAMP was greater than in its absence. By SDS-polyacrylamide gel electrophoresis and scanning densitometry, binding of TRAMP to collagen was detected that approached saturation with a molar ratio of TRAMP to collagen of approximately 1:2. Fibrils formed in the presence of TRAMP were normal when observed by electron microscopy, although fibril diameters were smaller than the controls. TRAMP was found to partially reverse the inhibitory effects of urea and increased ionic strength on the kinetics of fibril formation, although inhibition by glucose was unaffected. TRAMP also accelerated the assembly of pepsin-treated collagen, where the non-helical, telopeptide regions were partially removed. Acceleration of collagen fibril formation by TRAMP is discussed in the light of the known effects of other extracellular matrix

  18. Quercetin downregulates Mcl-1 by acting on mRNA stability and protein degradation

    PubMed Central

    Spagnuolo, C; Cerella, C; Russo, M; Chateauvieux, S; Diederich, M; Russo, G L

    2011-01-01

    Background: We recently demonstrated that quercetin, a flavonoid naturally present in food and beverages belonging to the large class of phytochemicals, was able to sensitise leukaemic cells isolated from patients with chronic lymphocytic leukaemia (CLL) when associated with recombinant tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) or anti-CD95. We also showed that quercetin potentiated the effect of fludarabine on resistant B cells from CLL patients. Resistance to therapy in CLL depends on the expression and activity of anti-apoptotic proteins of the Bcl-2 family. Among these, myeloid cell leukaemia-1 (Mcl-1) has been associated with apoptotic resistance in CLL. Therefore, we investigate here whether the sensitising activity of this flavonoid, which leads to increased apoptosis in both cell lines and CLL, could be related to Mcl-1 expression and stability. Results: B cells isolated from CLL patients showed different levels of Mcl-1 protein expression, resulting, in several cases, in increased sensitivity to fludarabine. Quercetin significantly enhanced the downregulation of Mcl-1 in B cells isolated from selected patients expressing detectable levels of Mcl-1. In U-937 cells, quercetin increased Mcl-1 mRNA instability in the presence of actinomycin D. When cells were treated with MG-132, a proteasome inhibitor, Mcl-1 protein level increased. However, quercetin, in the presence of Z-Vad-FMK, continued to lower Mcl-1 protein expression, indicating its independence from caspase-mediated degradation. In contrast, co-treatment of quercetin and MG-132 did not revert the effect of MG-132 mono-treatment, thus suggesting a possible interference of quercetin in regulating the proteasome-dependent degradation of Mcl-1. Gossypol, a small-molecule inhibitor of Bcl-2 family members, mimics the activity of quercetin by lowering Mcl-1 expression and sensitising U-937 cells to apoptosis induced by recombinant TRAIL and the Fas-ligand. Conclusion: This study

  19. Inhibition of PCSK9 Transcription by Berberine Involves Down-regulation of Hepatic HNF1α Protein Expression through the Ubiquitin-Proteasome Degradation Pathway*

    PubMed Central

    Dong, Bin; Li, Hai; Singh, Amar Bahadur; Cao, Aiqin; Liu, Jingwen

    2015-01-01

    Our previous in vitro studies have identified hepatocyte nuclear factor 1α (HNF1α) as an obligated trans-activator for PCSK9 gene expression and demonstrated its functional involvement in the suppression of PCSK9 expression by berberine (BBR), a natural cholesterol-lowering compound. In this study, we investigated the mechanism underlying the inhibitory effect of BBR on HNF1α-mediated PCSK9 transcription. Administration of BBR to hyperlipidemic mice and hamsters lowered circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting the gene expression of HNF1α. However, hepatic HNF1α protein levels were markedly reduced in BBR-treated animals as compared with the control. Using HepG2 cells as a model system, we obtained evidence that BBR treatment let to accelerated degradation of HNF1α protein. By applying inhibitors to selectively block the ubiquitin proteasome system (UPS) and autophagy-lysosomal pathway, we show that HNF1α protein content in HepG2 cells was not affected by bafilomycin A1 treatment, but it was dose-dependently increased by UPS inhibitors bortezomib and MG132. Bortezomib treatment elevated HNF1α and PCSK9 cellular levels with concomitant reductions of LDL receptor protein. Moreover, HNF1α protein displayed a multiubiquitination ladder pattern in cells treated with BBR or overexpressing ubiquitin. By expressing GFP-HNF1α fusion protein in cells, we observed that blocking UPS resulted in accumulation of GFP-HNF1α in cytoplasm. Importantly, we show that the BBR reducing effects on HNF1α protein and PCSK9 gene transcription can be eradicated by proteasome inhibitors. Altogether, our studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin-induced proteasomal degradation of HNF1α. PMID:25540198

  20. Tumor necrosis factor-α-induced apoptosis of gastric cancer MKN28 cells: accelerated degradation of the inhibitor of apoptosis family members.

    PubMed

    Kitagawa, Maki; Shiozaki, Atsushi; Ichikawa, Daisuke; Nakashima, Shingo; Kosuga, Toshiyuki; Konishi, Hirotaka; Komatsu, Shuhei; Fujiwara, Hitoshi; Okamoto, Kazuma; Otsuji, Eigo

    2015-01-15

    The role of the inhibitor of apoptosis (IAP) family members in tumor necrosis factor-α (TNF-α)-induced apoptosis of human gastric cancer MKN28 cells was explored. TNF-α induced up-regulation of cIAP2, whereas cycloheximide (CHX) induced down-regulation of XIAP and survivin. Degradation of cIAP1 and XIAP, but not survivin, was accelerated by co-treatment of cells with TNF-α and CHX, and TNF-α-induced up-regulation of cIAP2 was inhibited by BMS-345541 (NF-κB inhibitor). Treatment of MKN28 cells with TNF-α plus CHX induced degradation of survivin and activation of caspase-8 and -3, followed by degradation of cIAP1 and XIAP and apoptosis. Proteasome inhibitors (MG132 and epoxomicin) suppressed TNF-α plus CHX-induced degradation of survivin, cIAP1, and XIAP as well as apoptosis. A caspase inhibitor (z-VAD-fmk) suppressed TNF-α plus CHX-induced apoptosis, but allowed degradation of survivin, cIAP1 and XIAP. TNF-α receptor 1 and 2 were expressed on MKN28 cells. The magnitude of apoptosis induced by TNF-α plus BMS-345541 was much less than that induced by TNF-α plus CHX. These findings suggest that TNF-α plus CHX-induced apoptosis of gastric cancer MKN28 cells may be caused by accelerated degradation of the IAP family members (survivin, cIAP1, and XIAP), in addition to inhibition of NF-κB-dependent synthesis of anti-apoptotic molecules. PMID:25513960

  1. Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots*

    PubMed Central

    Li, Lei; Nelson, Clark J.; Solheim, Cory; Whelan, James; Millar, A. Harvey

    2012-01-01

    The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a 15N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (KS) and degradation (KD) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify KS and KD for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. KS and KD correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive 15N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability. PMID:22215636

  2. Amyloid-beta protein clearance and degradation (ABCD) pathways and their role in Alzheimer's disease.

    PubMed

    Baranello, Robert J; Bharani, Krishna L; Padmaraju, Vasudevaraju; Chopra, Nipun; Lahiri, Debomoy K; Greig, Nigel H; Pappolla, Miguel A; Sambamurti, Kumar

    2015-01-01

    Amyloid-β proteins (Aβ) of 42 (Aβ42) and 40 aa (Aβ40) accumulate as senile plaques (SP) and cerebrovascular amyloid protein deposits that are defining diagnostic features of Alzheimer's disease (AD). A number of rare mutations linked to familial AD (FAD) on the Aβ precursor protein (APP), Presenilin-1 (PS1), Presenilin- 2 (PS2), Adamalysin10, and other genetic risk factors for sporadic AD such as the ε4 allele of Apolipoprotein E (ApoE-ε4) foster the accumulation of Aβ and also induce the entire spectrum of pathology associated with the disease. Aβ accumulation is therefore a key pathological event and a prime target for the prevention and treatment of AD. APP is sequentially processed by β-site APP cleaving enzyme (BACE1) and γ-secretase, a multisubunit PS1/PS2-containing integral membrane protease, to generate Aβ. Although Aβ accumulates in all forms of AD, the only pathways known to be affected in FAD increase Aβ production by APP gene duplication or via base substitutions on APP and γ-secretase subunits PS1 and PS2 that either specifically increase the yield of the longer Aβ42 or both Aβ40 and Aβ42. However, the vast majority of AD patients accumulate Aβ without these known mutations. This led to proposals that impairment of Aβ degradation or clearance may play a key role in AD pathogenesis. Several candidate enzymes, including Insulin-degrading enzyme (IDE), Neprilysin (NEP), Endothelin-converting enzyme (ECE), Angiotensin converting enzyme (ACE), Plasmin, and Matrix metalloproteinases (MMPs) have been identified and some have even been successfully evaluated in animal models. Several studies also have demonstrated the capacity of γ-secretase inhibitors to paradoxically increase the yield of Aβ and we have recently established that the mechanism is by skirting Aβ degradation. This review outlines major cellular pathways of Aβ degradation to provide a basis for future efforts to fully characterize the panel of pathways responsible for A

  3. Automated Technologies and Novel Techniques to Accelerate Protein Crystallography for Structrual Genomics

    SciTech Connect

    Manjasetty,B.; Turnbull, A.; Panjikar, S.; Bussow, K.; Chance, M.

    2008-01-01

    The sequence infrastructure that has arisen through large-scale genomic projects dedicated to protein analysis, has provided a wealth of information and brought together scientists and institutions from all over the world. As a consequence, the development of novel technologies and methodologies in proteomics research is helping to unravel the biochemical and physiological mechanisms of complex multivariate diseases at both a functional and molecular level. In the late sixties, when X-ray crystallography had just been established, the idea of determining protein structure on an almost universal basis was akin to an impossible dream or a miracle. Yet only forty years after, automated protein structure determination platforms have been established. The widespread use of robotics in protein crystallography has had a huge impact at every stage of the pipeline from protein cloning, over-expression, purification, crystallization, data collection, structure solution, refinement, validation and data management- all of which have become more or less automated with minimal human intervention necessary. Here, recent advances in protein crystal structure analysis in the context of structural genomics will be discussed. In addition, this review aims to give an overview of recent developments in high throughput instrumentation, and technologies and strategies to accelerate protein structure/function analysis.

  4. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Lecker, S. H.; Goldberg, A. L.

    1998-01-01

    In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

  5. Estrogen inhibits phorbol ester-induced I kappa B alpha transcription and protein degradation.

    PubMed

    Sun, W H; Keller, E T; Stebler, B S; Ershler, W B

    1998-03-27

    Estrogen (E2) is known to prevent bone loss and the mechanism is, at least in part, mediated by inhibition of expression of cytokines such as interleukin-6 (IL-6). Expression of IL-6 is tightly regulated and the transcription factor NF kappa B can upregulate IL-6 gene expression by binding to its promoter region. NF kappa B is kept in an inactive state by associating with its cytoplasmic inhibitor I kappa B alpha. Upon mitogenic stimulation, I kappa B alpha becomes phosphorylated, followed by a rapid protein degradation. As a result, NF kappa B is released and translocate to the nucleus where DNA binding occurs. It has been shown that E2 treatment downregulates mitogen-induced IL-6 expression by inhibiting NF kappa B activity. Here, we sought to determine whether E2 regulates IL-6 gene expression by modulating the levels of I kappa B alpha. Our results show that E2 treatment almost completely inhibits phorbol ester-induced I kappa B alpha protein degradation. In addition, E2 inhibits phorbol ester-stimulated I kappa B alpha gene expression. Taken together, our results suggest that E2 maintains steady state levels of I kappa B alpha upon mitogen stimulation, resulting in inhibition of NF kappa B activation and IL-6 gene expression. This may explain the protective effect of E2 on bone loss. PMID:9535726

  6. Chitin Degradation Proteins Produced by the Marine Bacterium Vibrio harveyi Growing on Different Forms of Chitin

    PubMed Central

    Svitil, A. L.; Chadhain, S.; Moore, J. A.; Kirchman, D. L.

    1997-01-01

    Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on (beta)-chitin (isolated from squid pen) than on (alpha)-chitin (isolated from snow crab), probably because of the more open structure of (beta)-chitin. When exposed to different types of chitin, V. harveyi excreted several chitin-degrading proteins into the culture media. Some chitinases were present with all of the tested chitins, while others were unique to a particular chitin. We cloned and identified six separate chitinase genes from V. harveyi. These chitinases appear to be unique based on DNA restriction patterns, immunological data, and enzyme activity. This marine bacterium and probably others appear to synthesize separate chitinases for efficient utilization of different forms of chitin and chitin by-products. PMID:16535505

  7. LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor

    PubMed Central

    de Laat, Rian; Meabon, James S.; Wiley, Jesse C.; Hudson, Mark P.; Montine, Thomas J.; Bothwell, Mark

    2015-01-01

    Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP) by β-secretase and γ-secretase generate amyloid β (Aβ) peptides, which are thought to contribute to Alzheimer's disease (AD). Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD. PMID:25758563

  8. Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation

    PubMed Central

    Kaneko, Masayuki; Iwase, Ikuko; Yamasaki, Yuki; Takai, Tomoko; Wu, Yan; Kanemoto, Soshi; Matsuhisa, Koji; Asada, Rie; Okuma, Yasunobu; Watanabe, Takeshi; Imaizumi, Kazunori; Nomura, Yausyuki

    2016-01-01

    Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism by which unfolded proteins that accumulate in the ER are transported to the cytosol for ubiquitin–proteasome-mediated degradation. Ubiquitin ligases (E3s) are a group of enzymes responsible for substrate selectivity and ubiquitin chain formation. The purpose of this study was to identify novel E3s involved in ERAD. Thirty-seven candidate genes were selected by searches for proteins with RING-finger motifs and transmembrane regions, which are the major features of ERAD E3s. We performed gene expression profiling for the identified E3s in human and mouse tissues. Several genes were specifically or selectively expressed in both tissues; the expression of four genes (RNFT1, RNF185, CGRRF1 and RNF19B) was significantly upregulated by ER stress. To determine the involvement of the ER stress-responsive genes in ERAD, we investigated their ER localisation, in vitro autoubiquitination activity and ER stress resistance. All were partially localised to the ER, whereas CGRRF1 did not possess E3 activity. RNFT1 and RNF185, but not CGRRF1 and RNF19B, exhibited significant resistance to ER stressor in an E3 activity-dependent manner. Thus, these genes are possible candidates for ERAD E3s. PMID:27485036

  9. NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein.

    PubMed

    Maeda, Tomoji; Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu; Nakajima, Toshihiro; Komano, Hiroto

    2016-05-13

    Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. PMID:27084451

  10. An Armadillo Motif in Ufd3 Interacts with Cdc48 and is Involved in Ubiquitin Homeostasis and Protein Degradation

    SciTech Connect

    Zhao, G.; Li, G; Schindelin, H; Lennarz, W

    2009-01-01

    The yeast AAA-ATPase Cdc48 and the ubiquitin fusion degradation (UFD) proteins play important, evolutionarily conserved roles in ubiquitin dependent protein degradation. The N-terminal domain of Cdc48 interacts with substrate-recruiting cofactors, whereas the C terminus of Cdc48 binds to proteins such as Ufd3 that process substrates. Ufd3 is essential for efficient protein degradation and for maintaining cellular ubiquitin levels. This protein contains an N-terminal WD40 domain, a central ubiquitin-binding domain, and a C-terminal Cdc48-binding PUL domain. The crystal structure of the PUL domain reveals an Armadillo repeat with high structural similarity to importin-a, and the Cdc48-binding site could be mapped to the concave surface of the PUL domain by biochemical studies. Alterations of the Cdc48 binding site of Ufd3 by site-directed mutagenesis resulted in a depletion of cellular ubiquitin pools and reduced activity of the ubiquitin fusion degradation pathway. Therefore, our data provide direct evidence that the functions of Ufd3 in ubiquitin homeostasis and protein degradation depend on its interaction with the C terminus of Cdc48.

  11. A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation.

    PubMed

    Patel, Avinash; Lee, Hyun O; Jawerth, Louise; Maharana, Shovamayee; Jahnel, Marcus; Hein, Marco Y; Stoynov, Stoyno; Mahamid, Julia; Saha, Shambaditya; Franzmann, Titus M; Pozniakovski, Andrej; Poser, Ina; Maghelli, Nicola; Royer, Loic A; Weigert, Martin; Myers, Eugene W; Grill, Stephan; Drechsel, David; Hyman, Anthony A; Alberti, Simon

    2015-08-27

    Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases. PMID:26317470

  12. Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation

    PubMed Central

    Lee, Jae Ho; Lee, Gha Young; Jang, Hagoon; Choe, Sung Sik; Koo, Seung-Hoi; Kim, Jae Bum

    2014-01-01

    Sterol regulatory element binding protein1c (SREBP1c) is a key transcription factor for de novo lipogenesis during the postprandial state. During nutritional deprivation, hepatic SREBP1c is rapidly suppressed by fasting signals to prevent lipogenic pathways. However, the molecular mechanisms that control SREBP1c turnover in response to fasting status are not thoroughly understood. To elucidate which factors are involved in the inactivation of SREBP1c, we attempted to identify SREBP1c-interacting proteins by mass spectrometry analysis. Since we observed that ring finger protein20 (RNF20) ubiquitin ligase was identified as one of SREBP1c-interacting proteins, we hypothesized that fasting signaling would promote SREBP1c degradation in an RNF20-dependent manner. In this work, we demonstrate that RNF20 physically interacts with SREBP1c, leading to degradation of SREBP1c via ubiquitination. In accordance with these findings, RNF20 represses the transcriptional activity of SREBP1c and turns off the expression of lipogenic genes that are targets of SREBP1c. In contrast, knockdown of RNF20 stimulates the expression of SREBP1c and lipogenic genes and induces lipogenic activity in primary hepatocytes. Furthermore, activation of protein kinase A (PKA) with glucagon or forskolin enhances the expression of RNF20 and potentiates the ubiquitination of SREBP1c via RNF20. In wild-type and db/db mice, adenoviral overexpression of RNF20 markedly suppresses FASN promoter activity and reduces the level of hepatic triglycerides, accompanied by a decrease in the hepatic lipogenic program. Here, we reveal that RNF20-induced SREBP1c ubiquitination down-regulates hepatic lipogenic activity upon PKA activation. Conclusion: RNF20 acts as a negative regulator of hepatic fatty acid metabolism through degradation of SREBP1c upon PKA activation. Knowledge regarding this process enhances our understanding of how SREBP1c is able to turn off hepatic lipid metabolism during nutritional deprivation

  13. Targeted Protein Degradation by Salmonella under Phagosome-Mimicking Culture Conditions Investigated Using Comparative Peptidomics

    SciTech Connect

    Manes, Nathan P.; Gustin, Jean K.; Rue, Joanne; Mottaz, Heather M.; Purvine, Samuel O.; Norbeck, Angela D.; Monroe, Matthew E.; Zimmer, Jennifer S.; Metz, Thomas O.; Adkins, Joshua N.; Smith, Richard D.; Heffron, Fred

    2007-04-01

    The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Due to the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g., contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study Salmonella enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal compartment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified 5,163 distinct peptides originating from 682 proteins and the data clearly indicated that compared to cells cultured in the rich medium, Salmonella cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed by using bottom-up proteomics, and when the peptidomic and proteomic data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.

  14. Alteration of cardiac glycoside positive inotropic action by modulators of protein synthesis and degradation

    SciTech Connect

    Nosek, T.M.; Adams, R.J.

    1986-03-05

    Numerous membrane bound and cytoplasmic proteins participate in the cardiac expression of the positive inotropic action (PIA) of digitalis glycosides including the Na,K-ATPase (NKA). Exposure of the myocardium to an inhibitor of protein synthesis (cycloheximide, CYC) or of protein degradation (leupeptin, LEU) alters the PIA of ouabain in isolated, paced guinea pig papillary muscles (PM) in opposite ways. In vivo exposure to CYC for 3 hr resulted in a 30% depression of the in vitro PIA of ouabain at 1.7..mu..M compared to control. In vivo exposure to LEU for 1 hr resulted in a 47% enhancement of the in vitro PIA of 1.7..mu..M ouabain. Neither drug had an apparent effect on the ouabain PIA ED50. Neither CYC nor LEU exposure to PM in vitro affect resting or developed tension or the response of skinned PM to calcium. The mechanisms of the PIA alterations by CYC or LEU do not involve a direct effect on the digitalis receptor. Exposure of isolated cardiac sarcolemma enriched in NKA to 10-100..mu..M CYC or LEU did not affect NKA activity or /sup 3/H-ouabain binding. Although direct physicochemical effects of CYC or LEU may be involved in the alterations of the ouabain PIA, it is possible that modulation of the cellular levels or turnover rate of short-lived proteins may affect cardiac regulation of the digitalis PIA.

  15. Regulation of Endoplasmic Reticulum-Associated Protein Degradation (ERAD) by Ubiquitin

    PubMed Central

    Lemus, Leticia; Goder, Veit

    2014-01-01

    Quality control of protein folding inside the endoplasmic reticulum (ER) includes chaperone-mediated assistance in folding and the selective targeting of terminally misfolded species to a pathway called ER-associated protein degradation, or simply ERAD. Once selected for ERAD, substrates will be transported (back) into the cytosol, a step called retrotranslocation. Although still ill defined, retrotranslocation likely involves a protein conducting channel that is in part formed by specific membrane-embedded E3 ubiquitin ligases. Early during retrotranslocation, reversible self-ubiquitination of these ligases is thought to aid in initiation of substrate transfer across the membrane. Once being at least partially exposed to the cytosol, substrates will become ubiquitinated on the cytosolic side of the ER membrane by the same E3 ubiquitin ligases. Ubiquitin on substrates was originally thought to be a permanent modification that (1) promotes late steps of retrotranslocation by recruiting the energy-providing ATPase Cdc48p/p97 via binding to its associated adaptor proteins and that (2) serves to target substrates to the proteasome. Recently it became evident, however, that the poly-ubiquitin chains (PUCs) on ERAD substrates are often subject to extensive remodeling, or processing, at several stages during ERAD. This review recapitulates the current knowledge and recent findings about PUC processing on ERAD substrates and ubiquitination of ERAD machinery components and discusses their functional consequences. PMID:25100021

  16. Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators

    PubMed Central

    Liu, Yangfan P.; Tsai, I-Chun; Morleo, Manuela; Oh, Edwin C.; Leitch, Carmen C.; Massa, Filomena; Lee, Byung-Hoon; Parker, David S.; Finley, Daniel; Zaghloul, Norann A.; Franco, Brunella; Katsanis, Nicholas

    2014-01-01

    Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients. PMID:24691443

  17. Comparison of sludge digestion under aerobic and anaerobic conditions with a focus on the degradation of proteins at mesophilic temperature.

    PubMed

    Shao, Liming; Wang, Tianfeng; Li, Tianshui; Lü, Fan; He, Pinjing

    2013-07-01

    Aerobic and anaerobic digestion are popular methods for the treatment of waste activated sludge. However, the differences in degradation of sludge during aerobic and anaerobic digestion remain unclear. In this study, the sludge degradation during aerobic and anaerobic digestion was investigated at mesophilic temperature, focused on protein based on the degradation efficiency and degree of humification. The duration of aerobic and anaerobic digestion was about 90 days. The final degradation efficiency of volatile solid was 66.1 ± 1.6% and 66.4 ± 2.4% under aerobic and anaerobic conditions, respectively. The final degradation efficiency of protein was 67.5 ± 1.4% and 65.1 ± 2.6% under aerobic and anaerobic conditions, respectively. The degradation models of volatile solids were consistent with those of protein under both aerobic and anaerobic conditions. The solubility of protein under aerobic digestion was greater than that under anaerobic digestion. Moreover, the humification index of dissolved organic matter of aerobic digestion was greater than that during anaerobic digestion. PMID:23685650

  18. Evaluation of Plasma Fibrinogen Degradation Products and Total Serum Protein Concentration in Oral Submucous Fibrosis

    PubMed Central

    B.N.V.S., Satish; B., Maharudrappa; K.M., Prashant; Hugar, Deepa; Allad, Umesh; Prabhu, Prasanth S.

    2014-01-01

    Background: Oral submucous fibrosis (OSMF) is a potentially malignant disorder with a multifactorial etiology. Malnutrition is a major problem for the inhabitants of most countries where OSMF is prevalent. Recently, a new direction in the etiopathogenesis was provided by the identification of fibrinogen degradation products (FDP) in the plasma of OSMF patients. Aims and Objectives: To assess the role of FDP in the etiology of OSMF and to correlate with the nutritional status by evaluating the total serum protein level. The study also determines to evaluate the correlation between the levels of plasma FDP with respect to the staging and grading of OSMF. Correlation between the levels of Total Serum Protein (TSP) with respect to the staging and grading of OSMF was also evaluated. Materials and Methods: The study included 30 cases clinically and histopathologically diagnosed as oral submucous fibrosis. The FDP levels were assessed using both qualitative and semi quantitative method as supplied by ‘Tulip Diagnostics (P) Ltd. Total Serum Protein (TSP) estimation was done by Biuret method using Liquixx Protein kit by Erba, Manheim. Results: The study indicates that in qualitative assessment of FDP only 14 subjects showed the presence of FDP levels>200ng/ml. In semiquantitative assessment there is no significant association between varying clinical stages and histopathological grades and FDP levels. Total serum Protein level showed a marginal increase in all subjects. The study revealed a positive correlation between FDP and TSP in all OSMF subjects. Conclusion: A larger sample size which would be a better representation of the population and the use of different methods which have higher sensitivities and specificities to evaluate FDP level and detailed fractional analysis of protein along with immunoglobulin profiling would facilitate in attaining more conclusive results. PMID:24995245

  19. NITRIC OXIDE-DEPENDENT PROTEASOMAL DEGRADATION OF CYTOCHROME P450 2B PROTEINS*

    PubMed Central

    Lee, Choon-Myung; Kim, Bong-Yoon; Li, Lian; Morgan, Edward T.

    2007-01-01

    Exposure to inflammatory agents or cytokines causes the suppression of cytochrome P450 (CYP) enzyme activities and expression in liver and primary hepatocyte cultures. We showed previously that phenobarbital-induced CYP2B protein is down-regulated in primary cultures of rat hepatocytes following exposure to bacterial endotoxin (LPS) in a nitric oxide (NO)-dependent manner. In the present study, we found that CYP2B proteins in primary rat hepatocyte cultures were suppressed more than 60% after 6h treatment with interleukin-1β (IL-1). This effect was NO-dependent, and treatment of cells with the NO-donors (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione (GSNO), and S-nitroso, N-acetylpenicillamine (SNAP) also suppressed CYP2B proteins. However, the down-regulation by IL-1 was insensitive to inhibition of cGMP-dependent protein kinases. The down-regulation by IL-1 or NO donors was abolished by treatments with the proteasome inhibitors MG132 and lactacystin that did not affect NO production. The calpain inhibitor E64-d or the lysosomal protease inhibitors NH4Cl and chloroquine did not attenuate the down-regulation of CYP2B by IL-1. Treatment of HeLa cells expressing c-myc-tagged CYP2B1 with NOC-18 down-regulated its expression and enhanced its ubiquitination. Treatment of rat liver microsomes with GSNO caused S-nitrosylation of CYP2B protein, and enhanced the ubiquitination pattern of CYP2B compared to unmodified CYP2B in an in vitro ubiquitination assay. These data are consistent with the hypothesis that NO-dependent CYP2B ubiquitination and proteasomal degradation are dependent on protein modification by reactive nitrogen species. PMID:17993647

  20. C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

    PubMed

    Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

    2015-09-11

    In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. PMID:26231209

  1. Protein degradation by ubiquitin–proteasome system in formation and labilization of contextual conditioning memory

    PubMed Central

    Sol Fustiñana, María; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro

    2014-01-01

    The ubiquitin–proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates. PMID:25135196

  2. Protein degradation by ubiquitin-proteasome system in formation and labilization of contextual conditioning memory.

    PubMed

    Sol Fustiñana, María; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-09-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates. PMID:25135196

  3. Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation

    PubMed Central

    Gao, Caiji; Zhuang, Xiaohong; Cui, Yong; Fu, Xi; He, Yilin; Zhao, Qiong; Zeng, Yonglun; Shen, Jinbo; Luo, Ming; Jiang, Liwen

    2015-01-01

    Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process. PMID:25624505

  4. Anabolic therapy with growth hormone accelerates protein gain in surgical patients requiring nutritional rehabilitation.

    PubMed Central

    Byrne, T A; Morrissey, T B; Gatzen, C; Benfell, K; Nattakom, T V; Scheltinga, M R; LeBoff, M S; Ziegler, T R; Wilmore, D W

    1993-01-01

    .05). GH administration altered substrate oxidation (respiratory quotient = 0.94 +/- 0.02 GH vs. 1.17 +/- 0.05 STD, p < or = 0.0002) and the use of available energy, resulting in a 66% increase in the efficiency of protein deposition (13.37 +/- 0.8 g/1000 kcal vs. 8.04 g +/- 3.06 g/1000 kcal, p < or = 0.04). CONCLUSIONS: GH administration accelerated protein gain in stable adult patients receiving aggressive nutritional therapy without a significant increase in body fat or a disproportionate expansion of ECW. GH therapy accelerated nutritional repletion and, therefore, may shorten the convalescence of the malnourished patient requiring a major surgical procedure. PMID:8215633

  5. Degradation of the interferon-induced 68,000-M(r) protein kinase by poliovirus requires RNA.

    PubMed Central

    Black, T L; Barber, G N; Katze, M G

    1993-01-01

    Control of the interferon-induced double-stranded RNA (dsRNA) activated protein kinase (referred to as P68 because of its M(r) of 68,000 in human cells) by animal viruses is essential to avoid decreases in protein synthetic rates during infection. We have previously demonstrated that poliovirus establishes a unique way of regulating the protein kinase, namely by inducing the specific degradation of P68 during infection (T. L. Black, B. Safer, A. Hovanessian, and M. G. Katze, J. Virol. 63:2244-2251, 1989). In the present study we investigated the mechanisms by which P68 degradation occurred. To do this we used an in vitro degradation assay which faithfully reproduced the in vivo events. Although viral gene expression was required for P68 degradation, the major poliovirus proteases, 2A and 3C, were found not to be directly involved with P68 proteolysis. However, the protease responsible for P68 degradation required divalent cations for maximal activity and probably has both an RNA and a protein component since trypsin and ribonuclease abrogated the activity. Despite this requirement for divalent cations and RNA, activation of the kinase was not required for proteolysis since a catalytically inactive P68 was still degraded. Mapping of P68 protease-sensitive sites by using in vitro translated truncation and deletion mutants revealed that sites required for degradation resided in the amino terminus and colocalized to dsRNA-binding domains. Finally, we found that preincubation of cell extracts with the synthetic dsRNA poly(I-C) largely prevented P68 proteolysis, providing additional evidence for the critical role of RNA. On the basis of these data, we present a hypothetical model depicting possible mechanisms of P68 degradation in poliovirus-infected cells. Images PMID:7678306

  6. Novel durable bio-photocatalyst purifiers, a non-heterogeneous mechanism: accelerated entrapped dye degradation into structural polysiloxane-shield nano-reactors.

    PubMed

    Dastjerdi, Roya; Montazer, Majid; Shahsavan, Shadi; Böttcher, Horst; Moghadam, M B; Sarsour, Jamal

    2013-01-01

    This research has designed innovative Ag/TiO(2) polysiloxane-shield nano-reactors on the PET fabric to develop novel durable bio-photocatalyst purifiers. To create these very fine nano-reactors, oppositely surface charged multiple size nanoparticles have been applied accompanied with a crosslinkable amino-functionalized polysiloxane (XPs) emulsion. Investigation of photocatalytic dye decolorization efficiency revealed a non-heterogeneous mechanism including an accelerated degradation of entrapped dye molecules into the structural polysiloxane-shield nano-reactors. In fact, dye molecules can be adsorbed by both Ag and XPs due to their electrostatic interactions and/or even via forming a complex with them especially with silver NPs. The absorbed dye and active oxygen species generated by TiO(2) were entrapped by polysiloxane shelter and the presence of silver nanoparticles further attract the negative oxygen species closer to the adsorbed dye molecules. In this way, the dye molecules are in close contact with concentrated active oxygen species into the created nano-reactors. This provides an accelerated degradation of dye molecules. This non-heterogeneous mechanism has been detected on the sample containing all of the three components. Increasing the concentration of Ag and XPs accelerated the second step beginning with an enhanced rate. Further, the treated samples also showed an excellent antibacterial activity. PMID:23010055

  7. Conditioning nerve crush accelerates cytoskeletal protein transport in sprouts that form after a subsequent crush

    SciTech Connect

    McQuarrie, I.G.; Jacob, J.M. )

    1991-03-01

    To examine the relationship between axonal outgrowth and the delivery of cytoskeletal proteins to the growing axon tip, outgrowth was accelerated by using a conditioning nerve crush. Because slow component b (SCb) of axonal transport is the most rapid vehicle for carrying cytoskeletal proteins to the axon tip, the rate of SCb was measured in conditioned vs. sham-conditioned sprouts. In young Sprague-Dawley rats, the conditioning crush was made to sciatic nerve branches at the knee; 14 days later, the test crush was made where the L4 and L5 spinal nerves join to form the sciatic nerve in the flank. Newly synthesized proteins were labeled in motor neurons by injecting {sup 35}S-methionine into the lumbar spinal cord 7 days before the test crush. The wave of pulse-labeled SCb proteins reached the crush by the time it was made and subsequently entered sprouts. The nerve was removed and sectioned for SDS-PAGE and fluorography 4-12 days after the crush. Tubulins, neurofilament proteins, and representative 'cytomatrix' proteins (actin, calmodulin, and putative microtubule-associated proteins) were removed from gels for liquid scintillation counting. Labeled SCb proteins entered sprouts without first accumulating in parent axon stumps, presumably because sprouts begin to grow within hours after axotomy. The peak of SCb moved 11% faster in conditioned than in sham-conditioned sprouts: 3.0 vs. 2.7 mm/d (p less than 0.05). To confirm that sprouts elongate more rapidly when a test crush is preceded by a conditioning crush, outgrowth distances were measured in a separate group of rats by labeling fast axonal transport with {sup 3}H-proline 24 hours before nerve retrieval.

  8. Differential Expression in Phanerochaete chrysosporium of Membrane-Associated Proteins Relevant to Lignin Degradation

    SciTech Connect

    Shary, Semarjit; Kapich, Alexander N.; Panisko, Ellen A.; Magnuson, Jon K.; Cullen, Dan; Hammel, Ken

    2008-10-02

    Fungal lignin-degrading systems must include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, secretion of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Despite their importance, little is known about the nature or regulation of these membrane-associated components. We grew the white rot basidiomycete Phanerochaete chrysosporium on cellulose or glucose as the carbon source and monitored the mineralization of a 14C-labeled synthetic lignin by these cultures to assess their ligninolytic competence. The results showed that the cellulose-grown cultures were ligninolytic, whereas the glucose-grown ones were not. We isolated microsomal membrane fractions from both types of culture and analyzed tryptic digests of them by shotgun liquid chromatography/tandem mass spectrometry. Comparison of the results against the predicted P. chrysosporium proteome showed that a catalase (Joint Genome Institute P. chrysosporium protein I.D. 124398), an alcohol oxidase (126879), two transporters (137220 and 132234), and two cytochrome P450s (5011 and 8912) were up-regulated under ligninolytic conditions. Real time reverse transcription polymerase chain reaction assays showed that RNA transcripts encoding all of these proteins were also up-regulated in ligninolytic cultures. Catalase 124398, alcohol oxidase 126879, and transporter 137220 were found in a proteomic analysis of partially purified plasma membranes from ligninolytic P. chrysosporium, and are therefore most likely associated with the outer envelope of the fungus.

  9. Arabidopsis thaliana LSM proteins function in mRNA splicing and degradation

    PubMed Central

    Golisz, Anna; Sikorski, Pawel J.; Kruszka, Katarzyna; Kufel, Joanna

    2013-01-01

    Sm-like (Lsm) proteins have been identified in all organisms and are related to RNA metabolism. Here, we report that Arabidopsis nuclear AtLSM8 protein, as well as AtLSM5, which localizes to both the cytoplasm and nucleus, function in pre-mRNA splicing, while AtLSM5 and the exclusively cytoplasmic AtLSM1 contribute to 5′–3′ mRNA decay. In lsm8 and sad1/lsm5 mutants, U6 small nuclear RNA (snRNA) was reduced and unspliced mRNA precursors accumulated, whereas mRNA stability was mainly affected in plants lacking AtLSM1 and AtLSM5. Some of the mRNAs affected in lsm1a lsm1b and sad1/lsm5 plants were also substrates of the cytoplasmic 5′–3′ exonuclease AtXRN4 and of the decapping enzyme AtDCP2. Surprisingly, a subset of substrates was also stabilized in the mutant lacking AtLSM8, which supports the notion that plant mRNAs are actively degraded in the nucleus. Localization of LSM components, purification of LSM-interacting proteins as well as functional analyses strongly suggest that at least two LSM complexes with conserved activities in RNA metabolism, AtLSM1-7 and AtLSM2-8, exist also in plants. PMID:23620288

  10. Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris

    PubMed Central

    Haon, Mireille; Grisel, Sacha; Navarro, David; Gruet, Antoine; Berrin, Jean-Guy; Bignon, Christophe

    2015-01-01

    Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users’ community. PMID:26441929

  11. Tripartite motif protein 32 facilitates cell growth and migration via degradation of Abl-interactor 2.

    PubMed

    Kano, Satoshi; Miyajima, Naoto; Fukuda, Satoshi; Hatakeyama, Shigetsugu

    2008-07-15

    Tripartite motif protein 32 (TRIM32) mRNA has been reported to be highly expressed in human head and neck squamous cell carcinoma, but the involvement of TRIM32 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that TRIM32 binds to Abl-interactor 2 (Abi2), which is known as a tumor suppressor and a cell migration inhibitor, and we showed that TRIM32 mediates the ubiquitination of Abi2. Overexpression of TRIM32 promoted degradation of Abi2, resulting in enhancement of cell growth, transforming activity, and cell motility, whereas a dominant-negative mutant of TRIM32 lacking the RING domain inhibited the degradation of Abi2. In addition, we found that TRIM32 suppresses apoptosis induced by cis-diamminedichloroplatinum (II) in HEp2 cell lines. These findings suggest that TRIM32 is a novel oncogene that promotes tumor growth, metastasis, and resistance to anticancer drugs. PMID:18632609

  12. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer.

    PubMed

    Raina, Kanak; Lu, Jing; Qian, Yimin; Altieri, Martha; Gordon, Deborah; Rossi, Ann Marie K; Wang, Jing; Chen, Xin; Dong, Hanqing; Siu, Kam; Winkler, James D; Crew, Andrew P; Crews, Craig M; Coleman, Kevin G

    2016-06-28

    Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC. PMID:27274052

  13. Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

    PubMed

    Haon, Mireille; Grisel, Sacha; Navarro, David; Gruet, Antoine; Berrin, Jean-Guy; Bignon, Christophe

    2015-01-01

    Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community. PMID:26441929

  14. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer

    PubMed Central

    Raina, Kanak; Lu, Jing; Qian, Yimin; Altieri, Martha; Gordon, Deborah; Rossi, Ann Marie K.; Wang, Jing; Chen, Xin; Dong, Hanqing; Siu, Kam; Winkler, James D.; Crew, Andrew P.; Crews, Craig M.; Coleman, Kevin G.

    2016-01-01

    Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC. PMID:27274052

  15. Plasminogen activator/coagulase gene of Yersinia pestis is responsible for degradation of plasmid-encoded outer membrane proteins.

    PubMed Central

    Sodeinde, O A; Sample, A K; Brubaker, R R; Goguen, J D

    1988-01-01

    The related family of virulence plasmids found in the three major pathogens of the genus Yersinia all have the ability to encode a set of outer membrane proteins. In Y. enterocolitica and Y. pseudotuberculosis, these proteins are major constituents of the outer membrane when their synthesis is fully induced. In contrast, they have been difficult to detect in Y. pestis. It has recently been established that Y. pestis does synthesize these proteins, but that they are rapidly degraded due to some activity determined by the 9.5-kilobase plasmid commonly found in Y. pestis strains. We show that mutations in the pla gene of this plasmid, which encodes both the plasminogen activator and coagulase activities, blocked this degradation. A cloned 1.4-kilobase DNA fragment carrying pla was also sufficient to cause degradation in the absence of the 9.5-kilobase plasmid. Images PMID:2843471

  16. Return to quiescence of mouse neural stem cells by degradation of a proactivation protein.

    PubMed

    Urbán, Noelia; van den Berg, Debbie L C; Forget, Antoine; Andersen, Jimena; Demmers, Jeroen A A; Hunt, Charles; Ayrault, Olivier; Guillemot, François

    2016-07-15

    Quiescence is essential for long-term maintenance of adult stem cells. Niche signals regulate the transit of stem cells from dormant to activated states. Here, we show that the E3-ubiquitin ligase Huwe1 (HECT, UBA, and WWE domain-containing 1) is required for proliferating stem cells of the adult mouse hippocampus to return to quiescence. Huwe1 destabilizes proactivation protein Ascl1 (achaete-scute family bHLH transcription factor 1) in proliferating hippocampal stem cells, which prevents accumulation of cyclin Ds and promotes the return to a resting state. When stem cells fail to return to quiescence, the proliferative stem cell pool becomes depleted. Thus, long-term maintenance of hippocampal neurogenesis depends on the return of stem cells to a transient quiescent state through the rapid degradation of a key proactivation factor. PMID:27418510

  17. Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation

    SciTech Connect

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri; Su, Xiong; Liu, Jialiu; Sundaresan, Sinju; Stahl, Philip D.

    2013-05-03

    Highlights: •Hominoid-specific oncogene TBC1D3 is targeted to plasma membrane by palmitoylation. •TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. •TBC1D3 palmitoylation governs growth factors-induced TBC1D3 degradation. •Post-translational modifications may regulate oncogenic properties of TBC1D3. -- Abstract: Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis.

  18. Multiple sequence signals direct recognition and degradation of protein substrates by the AAA+ protease HslUV.

    PubMed

    Sundar, Shankar; McGinness, Kathleen E; Baker, Tania A; Sauer, Robert T

    2010-10-29

    Proteolysis is important for protein quality control and for the proper regulation of many intracellular processes in prokaryotes and eukaryotes. Discerning substrates from other cellular proteins is a key aspect of proteolytic function. The Escherichia coli HslUV protease is a member of a major family of ATP-dependent AAA+ degradation machines. HslU hexamers recognize and unfold native protein substrates and then translocate the polypeptide into the degradation chamber of the HslV peptidase. Although a wealth of structural information is available for this system, relatively little is known about mechanisms of substrate recognition. Here, we demonstrate that mutations in the unstructured N-terminal and C-terminal sequences of two model substrates alter HslUV recognition and degradation kinetics, including changes in V(max). By introducing N- or C-terminal sequences that serve as recognition sites for specific peptide-binding proteins, we show that blocking either terminus of the substrate interferes with HslUV degradation, with synergistic effects when both termini are obstructed. These results support a model in which one terminus of the substrate is tethered to the protease and the other terminus is engaged by the translocation/unfolding machinery in the HslU pore. Thus, degradation appears to consist of discrete steps, which involve the interaction of different terminal sequence signals in the substrate with different receptor sites in the HslUV protease. PMID:20837023

  19. Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine, and Ozone.

    PubMed

    Choe, Jong Kwon; Richards, David H; Wilson, Corey J; Mitch, William A

    2015-11-17

    Proteins are important targets of chemical disinfectants. To improve the understanding of disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is driven by their disinfectant rate constants rather than their geometrical arrangement. Across the model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs with its high disinfectant rate constants and supports its hypothesized role as a sacrificial antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine. These results concur with the prevalence of geometrical motifs featuring histidines or lysines near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer pathway, histidine, tyrosine, and lysine degradation followed their relative O3 rate constants. Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did not correlate with loss of particular amino acids, suggesting the need to characterize the oxidation of specific geometric motifs to understand structural degradation. PMID:26488608

  20. Taxol and tau overexpression induced calpain-dependent degradation of the microtubule-destabilizing protein SCG10.

    PubMed

    Vega, Irving E; Hamano, Tadanori; Propost, Josh A; Grenningloh, Gabriele; Yen, Shu-Hui

    2006-11-01

    Microtubule-stabilizing and -destabilizing proteins play a crucial role in regulating the dynamic instability of microtubules during neuronal development and synaptic transmission. The microtubule-destabilizing protein SCG10 is a neuron-specific protein implicated in neurite outgrowth. The SCG10 protein is significantly reduced in mature neurons, suggesting that its expression is developmentally regulated. In contrast, the microtubule-stabilizing protein tau is expressed in mature neurons and its function is essential for the maintenance of neuronal polarity and neuronal survival. Thus, the establishment and maintenance of neuronal polarity may down-regulate the protein level/function of SCG10. In this report, we show that treatment of PC12 cells and neuroblastoma cells with the microtubule-stabilizing drug Taxol induced a rapid degradation of the SCG10 protein. Consistently, overexpression of tau protein in neuroblastoma cells also induced a reduction in SCG10 protein levels. Calpain inhibitor MDL-28170, but not caspase inhibitors, blocked a significant decrease in SCG10 protein levels. Collectively, these results indicate that tau overexpression and Taxol treatment induced a calpain-dependent degradation of the microtubule-destabilizing protein SCG10. The results provide evidence for the existence of an intracellular mechanism involved in the regulation of SCG10 upon microtubule stabilization. PMID:16822511

  1. Network analysis and cross species comparison of protein-protein interaction networks of human, mouse and rat cytochrome P450 proteins that degrade xenobiotics.

    PubMed

    Karthikeyan, Bagavathy Shanmugam; Akbarsha, Mohammad Abdulkader; Parthasarathy, Subbiah

    2016-06-21

    Cytochrome P450 (CYP) enzymes that degrade xenobiotics play a critical role in the metabolism and biotransformation of drugs and xenobiotics in humans as well as experimental animal models such as mouse and rat. These proteins function as a network collectively as well as independently. Though there are several reports on the organization, regulation and functionality of various CYP enzymes at the molecular level, the understanding of organization and functionality of these proteins at the holistic level remain unclear. The objective of this study is to understand the organization and functionality of xenobiotic degrading CYP enzymes of human, mouse and rat using network theory approaches and to study species differences that exist among them at the holistic level. For our analysis, a protein-protein interaction (PPI) network for CYP enzymes of human, mouse and rat was constructed using the STRING database. Topology, centrality, modularity and robustness analyses were performed for our predicted CYP PPI networks that were then validated by comparison with randomly generated network models. Network centrality analyses of CYP PPI networks reveal the central/hub proteins in the network. Modular analysis of the CYP PPI networks of human, mouse and rat resulted in functional clusters. These clusters were subjected to ontology and pathway enrichment analysis. The analyses show that the cluster of the human CYP PPI network is enriched with pathways principally related to xenobiotic/drug metabolism. Endo-xenobiotic crosstalk dominated in mouse and rat CYP PPI networks, and they were highly enriched with endogenous metabolic and signaling pathways. Thus, cross-species comparisons and analyses of human, mouse and rat CYP PPI networks gave insights about species differences that existed at the holistic level. More investigations from both reductionist and holistic perspectives can help understand CYP metabolism and species extrapolation in a much better way. PMID:27194593

  2. Tuning calcite morphology and growth acceleration by a rational design of highly stable protein-mimetics

    NASA Astrophysics Data System (ADS)

    Chen, Chun-Long; Qi, Jiahui; Tao, Jinhui; Zuckermann, Ronald N.; Deyoreo, James J.

    2014-09-01

    In nature, proteins play a significant role in biomineral formation. One of the ultimate goals of bioinspired materials science is to develop highly stable synthetic molecules that mimic the function of these natural proteins by controlling crystal formation. Here, we demonstrate that both the morphology and the degree of acceleration or inhibition observed during growth of calcite in the presence of peptoids can be rationally tuned by balancing the electrostatic and hydrophobic interactions, with hydrophobic interactions playing the dominant role. While either strong electrostatic or hydrophobic interactions inhibit growth and reduces expression of the {104} faces, correlations between peptoid-crystal binding energies and observed changes in calcite growth indicate moderate electrostatic interactions allow peptoids to weakly adsorb while moderate hydrophobic interactions cause disruption of surface-adsorbed water layers, leading to growth acceleration with retained expression of the {104} faces. This study provides fundamental principles for designing peptoids as crystallization promoters, and offers a straightforward screening method based on macroscopic crystal morphology. Because peptoids are sequence-specific, highly stable, and easily synthesized, peptoid-enhanced crystallization offers a broad range of potential applications.

  3. Tuning calcite morphology and growth acceleration by a rational design of highly stable protein-mimetics

    SciTech Connect

    Chen, Chunlong; Qi, Jiahui; Tao, Jinhui; Zuckermann, Ronald; De Yoreo, James J.

    2014-09-05

    In nature, proteins play a significant role in biomineral formation. One of the ultimate goals of bioinspired materials science is to develop highly stable synthetic molecules that mimic the function of these natural proteins by controlling crystal formation. Here, we demonstrate that both the morphology and the degree of acceleration or inhibition observed during growth of calcite in the presence of peptoids can be rationally tuned by balancing the electrostatic interactions (EI) and hydrophobic interactions (HI), with HI playing the dominant role. While either strong EI or HI inhibit growth and suppress (104) face expression, correlations between peptoid-crystal binding energies and observed changes in calcite growth indicate moderate EI allow peptoids to weakly adsorb while moderate HI cause disruption of surface-adsorbed water layers, leading to growth acceleration with retained expression of (104) faces. This study provides fundamental principles for designing peptoids as crystallization promoters, and offers a straightforward screening method based on macroscopic crystal morphology. Because peptoids are sequence-specific, highly stable, and easily synthesized, peptoid-enhanced crystallization offers a broad range of potential applications.

  4. Degradation of IGF-binding protein-3 by proteases in cultured FRTL-5 rat thyroid cells.

    PubMed

    Wang, J F; Becks, G P; Hill, D J

    1997-02-01

    In this study, we have found that IGF-binding protein-3 (IGFBP-3) in calf serum added to tissue culture medium is degraded by cultured FRTL-5 cells and a major 31 kDa fragment of IGFBP-3 is produced. When FRTL-5 rat thyroid cells were cultured in 6H medium (modified F-12M medium containing TSH, insulin, hydrocortisone, somatostatin, transferrin, and glycyl-histidyl-lysine) containing 5% calf serum, both 44-46 and 31 kDa IGFBPs were found in conditioned medium by ligand blot analysis using 125I-labelled IGF-II. However, predominantly the 44-46 kDa IGFBP was detected in unconditioned 6H medium containing 5% calf serum. When calf serum in the media was replaced by human serum similar results were obtained, and the 44-46 kDa and 31 kDa IGFBPs were recognized using a human IGFBP-3 antibody following Western blot analysis. FRTL-5 cells secreted only small amounts of an endogenous 29 kDa IGFBP, thought to be IGFBP-5. To separate the 31 kDa fragment of IGFBP-3 from the endogenous IGFBP-5, culture media were fractionated by concanavalin-A-Sepharose chromatography and aliquots of both flow-through and eluate from the column were analyzed by ligand blotting. A 31 kDa IGFBP was found in the eluate fractions from concanavalin-A-Sepharose chromatography following the separation of conditioned 6H medium supplemented with calf serum, suggesting that this species was an N-linked glycoprotein and could be derived from the degradation of serum IGFBP-3 by FRTL-5 cells. Using a modified zymographic assay, we examined whether the degradation of IGFBP-3 could depend on the cell membrane. Confluent FRTL-5 cells were washed with PBS and overlaid with liquid agarose solution. After the agarose had solidified, unconditioned 6H medium containing 5% calf serum was incubated with the cells at 37 degrees C for 16 h. Both 44-46 and 31 kDa IGFBP species were found in the overlying, conditioned medium by ligand blot. However, the 31 kDa IGFBP was not found in medium in the absence of FRTL-5 cells

  5. Actin-associated protein palladin promotes tumor cell invasion by linking extracellular matrix degradation to cell cytoskeleton

    PubMed Central

    von Nandelstadh, Pernilla; Gucciardo, Erika; Lohi, Jouko; Li, Rui; Sugiyama, Nami; Carpen, Olli; Lehti, Kaisa

    2014-01-01

    Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP–negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain–containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion. PMID:24989798

  6. Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

    PubMed

    Sánchez-Lanzas, Raul; Alvarez-Castelao, Beatriz; Bermejo, Teresa; Ayuso, Teresa; Tuñón, Teresa; Castaño, José G

    2016-08-01

    Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway. PMID:27130438

  7. Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.

    PubMed

    Kolter, Thomas; Sandhoff, Konrad

    2005-01-01

    Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family. PMID:16212488

  8. The effects of cutting or of stretching skeletal muscle in vitro on the rates of protein synthesis and degradation

    NASA Technical Reports Server (NTRS)

    Seider, M. J.; Kapp, R.; Chen, C.-P.; Booth, F. W.

    1980-01-01

    Skeletal muscle preparations using cut muscle fibers have often been used in studies of protein metabolism. The present paper reports an investigation of the effect of muscle cutting or stretching in vitro on the rates of protein synthesis and/or degradation. Protein synthesis and content, and ATP and phosphocreatine levels were monitored in soleus and extensor digitorum longus muscles from the rat with various extents of muscle fiber cuts and following stretching to about 120% the resting length. Rates of protein synthesis are found to be significantly lower and protein degradation higher in the cut muscles than in uncut controls, while ATP and phosphocreatine concentrations decreased. Stretched intact muscles, on the other hand, are observed to have higher concentrations of high-energy phosphates than unstretched muscles, while rates of protein degradation were not affected. Results thus demonstrate that the cutting of skeletal muscle fibers alters many aspects of muscle metabolism, and that moderate decreases in ATP concentration do not alter rates of protein concentration in intact muscles in vitro.

  9. The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

    PubMed Central

    Mehnert, Martin; Sommermeyer, Franziska; Berger, Maren; Kumar Lakshmipathy, Sathish; Gauss, Robert; Aebi, Markus; Jarosch, Ernst; Sommer, Thomas

    2015-01-01

    Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1. PMID:25428985

  10. The senescence-accelerated prone mouse (SAMP8): a model of age-related cognitive decline with relevance to alterations of the gene expression and protein abnormalities in Alzheimer's disease.

    PubMed

    Butterfield, D Allan; Poon, H Fai

    2005-10-01

    The senescence-accelerated mouse (SAM) is an accelerated aging model that was established through phenotypic selection from a common genetic pool of AKR/J strain of mice. The SAM model was established in 1981, including nine major senescence-accelerated mouse prone (SAMP) substrains and three major senescence-accelerated mouse resistant (SAMR) substrains, each of which exhibits characteristic disorders. Recently, SAMP8 have drawn attention in gerontological research due to its characteristic learning and memory deficits at old age. Many recent reports provide insight into mechanisms of the cognitive impairment and pathological changes in SAMP8. Therefore, this mini review examines the recent findings of SAMP8 mice abnormalities at the gene and protein levels. The genes and proteins described in this review are functionally categorized into neuroprotection, signal transduction, protein folding/degradation, cytoskeleton/transport, immune response and reactive oxygen species (ROS) production. All of these processes are involved in learning and memory. Although these studies provide insight into the mechanisms that contribute to the learning and memory decline in aged SAMP8 mice, higher throughput techniques of proteomics and genomics are necessary to study the alterations of gene expression and protein abnormalities in SAMP8 mice brain in order to more completely understand the central nervous system dysfunction in this mouse model. The SAMP8 is a good animal model to investigate the fundamental mechanisms of age-related learning and memory deficits at the gene and protein levels. PMID:16026957

  11. Proteomic and SAGE profiling of murine melanoma progression indicates the reduction of proteins responsible for ROS degradation.

    PubMed

    de Souza, Gustavo A; Godoy, Lyris M F; Teixeira, Veronica R; Otake, Andreia H; Sabino, Adão; Rosa, José C; Dinarte, Anemari R; Pinheiro, Daniel G; Silva, Wilson A; Eberlin, Marcos N; Chammas, Roger; Greene, Lewis J

    2006-03-01

    Using 2-DE of total cell protein extracts, we compared soluble proteins from murine melanoma lines Tm1 and Tm5 with proteins from the nontumoral cell melan-a from which they were derived. Seventy-one of the 452 spots (average) detected with CBB were differentially accumulated, i.e., increased or decreased twofold. Forty-four spots were identified by PMF/MALDI-TOF, 15 with increased and 29 with decreased protein levels. SAGE showed that 17/34 (50%) of the differentially accumulated proteins, pI range 4-7, presented similar differences at the mRNA level. Major reductions in protein were observed in tumor cells of proteins that degrade reactive oxygen species (ROS). Decreases of > or = twofold in GST, superoxide dismutase, aldehyde dehydrogenase, thioredoxin, peroxiredoxin 2, and peroxiredoxin 6 protein were observed. SAGE indicated the reduction of other proteins involved in ROS degradation. As expected, the accumulation of exogenous peroxides was significantly higher in the tumor cells while the levels of glutathionylation were two times lower in the tumor cells compared to melan-a. The differential accumulation of proteins involved in oncogene/tumor suppressor pathways was observed. Melanoma cells can favor survival pathways activated by ROS by inhibiting p53 pathways and activation of Ras and c-myc pathways. PMID:16429458

  12. Mutant huntingtin represses CBP, but not p300, by binding and protein degradation.

    PubMed

    Cong, Shu-Yan; Pepers, Barry A; Evert, Bernd O; Rubinsztein, David C; Roos, Raymund A C; van Ommen, Gert-Jan B; Dorsman, Josephine C

    2005-12-01

    Huntington's disease can be used as a model to study neurodegenerative disorders caused by aggregation-prone proteins. It has been proposed that the entrapment of transcription factors in aggregates plays an important role in pathogenesis. We now report that the transcriptional activity of CBP is already repressed in the early time points by soluble mutant huntingtin, whereas the histone acetylase activity of CBP/p300 is gradually diminished over time. Mutant huntingtin bound much stronger to CBP than normal huntingtin, possibly contributing to repression. Especially at the later time points, CBP protein level was gradually reduced via the proteasome pathway. In sharp contrast, p300 was unaffected by mutant huntingtin. This selective degradation of CBP was absent in spinocerebellar ataxia 3. Thus, mutant huntingtin specifically affects CBP and not p300 both at the early and later time points, via multiple mechanisms. In addition to the reduction of CBP, also the altered ratio of these closely related histone acetyl transferases may affect chromatin structure and transcription and thus contribute to neurodegeneration. PMID:16456924

  13. Mutant huntingtin represses CBP, but not p300, by binding and protein degradation.

    PubMed

    Cong, Shu-Yan; Pepers, Barry A; Evert, Bernd O; Rubinsztein, David C; Roos, Raymund A C; van Ommen, Gert-Jan B; Dorsman, Josephine C

    2005-09-01

    Huntington's disease can be used as a model to study neurodegenerative disorders caused by aggregation-prone proteins. It has been proposed that the entrapment of transcription factors in aggregates plays an important role in pathogenesis. We now report that the transcriptional activity of CBP is already repressed in the early time points by soluble mutant huntingtin, whereas the histone acetylase activity of CBP/p300 is gradually diminished over time. Mutant huntingtin bound much stronger to CBP than normal huntingtin, possibly contributing to repression. Especially at the later time points, CBP protein level was gradually reduced via the proteasome pathway. In sharp contrast, p300 was unaffected by mutant huntingtin. This selective degradation of CBP was absent in spinocerebellar ataxia 3. Thus, mutant huntingtin specifically affects CBP and not p300 both at the early and later time points, via multiple mechanisms. In addition to the reduction of CBP, also the altered ratio of these closely related histone acetyltransferases may affect chromatin structure and transcription and thus contribute to neurodegeneration. PMID:15994095

  14. Effects of feed intake and protein degradability on ruminal characteristics and site of digestion in steers.

    PubMed

    Firkins, J L; Berger, L L; Merchen, N R; Fahey, G C; Nelson, D R

    1986-08-01

    Four multiple-fistulated Hereford steers were used in a 4 X 4 Latin square design with a 2 X 2 factorial arrangement of treatments [two intakes (9.1 and 6.1 kg dry matter/d) and two protein sources differing in ruminal degradability (dry distillers grains and dry corn gluten feed)]. Steers fed at the high intake had faster fluid dilution rates (7.63 versus 6.52%/h), higher ruminal fluid outflows (120.2 versus 91.7 L/d), lower apparent ruminal digestibilities of organic matter (41.3 versus 44.3%) and neutral detergent fiber (56.0 versus 60.2%), and lower total tract digestibilities of neutral detergent fiber (64.3 versus 68.7%) than when they were fed at the low intake. Steers fed dry corn gluten feed had higher apparent ruminal digestibilities of organic matter (45.5 versus 40.1%) and neutral detergent fiber (60.2 versus 56.0%) and lower duodenal flows of nonammonia-nonbacterial N (40.1 versus 52.2% of N intake) than when they were fed dry distillers grains. Efficiency of ruminal bacterial growth was higher when steers were fed at the high versus low intakes. Efficiency of ruminal bacterial growth and site and extent of fiber digestion, especially hemicellulose, but not ruminal escape of protein, can be readily altered by manipulation of feed intake of moderately high forage diets. PMID:3020102

  15. Application of Universal Stress Proteins in Probing the Dynamics of Potent Degraders in Complex Terephthalate Metagenome

    PubMed Central

    Mbah, Andreas N.; Isokpehi, Raphael D.

    2013-01-01

    The culture-independent strategies to study microbial diversity and function have led to a revolution in environmental genomics, enabling fundamental questions about the distribution of microbes and their influence on bioremediation to be addressed. In this research we used the expression of universal stress proteins as a probe to determine the changes in degrading microbial population from a highly toxic terephthalate wastewater to a less toxic activated sludge bioreactor. The impact of relative toxicities was significantly elaborated at the levels of genus and species. The results indicated that 23 similar prokaryotic phyla were represented in both metagenomes irrespective of their relative abundance. Furthermore, the following bacteria taxa Micromonosporaceae, Streptomyces, Cyanothece sp. PCC 7822, Alicyclobacillus acidocaldarius, Bacillus halodurans, Leuconostoc mesenteroides, Lactococcus garvieae, Brucellaceae, Ralstonia solanacearum, Verminephrobacter eiseniae, Azoarcus, Acidithiobacillus ferrooxidans, Francisella tularensis, Methanothermus fervidus, and Methanocorpusculum labreanum were represented only in the activated sludge bioreactor. These highly dynamic microbes could serve as taxonomic biomarkers for toxic thresholds related to terephthalate and its derivatives. This paper, highlights the application of universal stress proteins in metagenomics analysis. Dynamics of microbial consortium of this nature can have future in biotechnological applications in bioremediation of toxic chemicals and radionuclides. PMID:24151583

  16. Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients.

    PubMed

    Gruosso, Tina; Mieulet, Virginie; Cardon, Melissa; Bourachot, Brigitte; Kieffer, Yann; Devun, Flavien; Dubois, Thierry; Dutreix, Marie; Vincent-Salomon, Anne; Miller, Kyle Malcolm; Mechta-Grigoriou, Fatima

    2016-01-01

    Anti-cancer drugs often increase reactive oxygen species (ROS) and cause DNA damage. Here, we highlight a new cross talk between chronic oxidative stress and the histone variant H2AX, a key player in DNA repair. We observe that persistent accumulation of ROS, due to a deficient JunD-/Nrf2-antioxidant response, reduces H2AX protein levels. This effect is mediated by an enhanced interaction of H2AX with the E3 ubiquitin ligase RNF168, which is associated with H2AX poly-ubiquitination and promotes its degradation by the proteasome. ROS-mediated H2AX decrease plays a crucial role in chemosensitivity. Indeed, cycles of chemotherapy that sustainably increase ROS reduce H2AX protein levels in Triple-Negative breast cancer (TNBC) patients. H2AX decrease by such treatment is associated with an impaired NRF2-antioxidant response and is indicative of the therapeutic efficiency and survival of TNBC patients. Thus, our data describe a novel ROS-mediated regulation of H2AX turnover, which provides new insights into genetic instability and treatment efficacy in TNBC patients. PMID:27006338

  17. Acceleration of crossbridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function.

    PubMed

    Tong, Carl W; Stelzer, Julian E; Greaser, Marion L; Powers, Patricia A; Moss, Richard L

    2008-10-24

    Normal cardiac function requires dynamic modulation of contraction. beta1-adrenergic-induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non-PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, k(rel) (s-1), and delayed force development, k(df) (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline k(rel) and k(df) similar to WT myocardium, but, unlike WT, k(rel) and k(df) were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction. PMID:18802026

  18. Inhibition of ABCA1 protein degradation promotes HDL cholesterol efflux capacity and RCT and reduces atherosclerosis in mice.

    PubMed

    Huang, LinZhang; Fan, BaoYan; Ma, Ang; Shaul, Philip W; Zhu, HaiBo

    2015-05-01

    ABCA1 plays a key role in the initial lipidation of apoA-I, which generates circulating HDL cholesterol. Whereas it is known that the transcriptional upregulation of ABCA1 promotes HDL formation and reverse cholesterol transport (RCT), it is not known how the inhibition of ABCA1 protein degradation impacts HDL function. Employing the small molecule triacetyl-3-hydroxyphenyladenosine (IMM-H007), we determined how the attenuation of ABCA1 protein degradation affects HDL cholesterol efflux capacity, RCT, and atherosclerotic lesion formation. Pulse-chase analysis revealed that IMM-H007 inhibits ABCA1 degradation and facilitates its cell-surface localization in macrophages, and additional studies in macrophages showed that IMM-H007 thereby promotes cholesterol efflux. IMM-H007 treatment of Paigen diet-fed mice caused an increase in circulating HDL level, it increased the cholesterol efflux capacity of HDL, and it enhanced in vivo RCT from macrophages to the plasma, liver, and feces. Furthermore, ABCA1 degradation suppression by IMM-H007 reduced atherosclerotic plaque formation in apoE(-/-) mice. Thus, via effects on both ABCA1-expressing cells and circulating HDL function, the inhibition of ABCA1 protein degradation by IMM-H007 promotes HDL cholesterol efflux capacity and RCT and attenuates atherogenesis. IMM-H007 potentially represents a lead compound for the development of agents to augment HDL function. PMID:25761370

  19. A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

    PubMed Central

    Doherty, F J; Wassell, J A; Mayer, R J

    1987-01-01

    Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy. Images Fig. 1. Fig. 5. PMID:3593223

  20. A Doping Lattice of Aluminum and Copper with Accelerated Electron Transfer Process and Enhanced Reductive Degradation Performance

    PubMed Central

    Zhang, Lin; Gao, Xue; Zhang, Zhixuan; Zhang, Mingbo; Cheng, Yiqian; Su, Jixin

    2016-01-01

    Treatment of azo dye effluents has received increasing concerns over the years due to their potential harms to natural environment and human health. The present study described the degrading ability of the as-synthesized crystalline Al-Cu alloys for removal of high-concentration Acid Scarlet 3R in alkaline aqueous solutions and its degradation mechanism. Al-Cu alloy particles with Al/Cu ratios 19:1 were successfully synthesized by high-energy mechanical milling. Characterization results showed that 10 h mechanical alloying process could lead to the formation of crystalline Al(Cu) solid solution. Batch experiment results confirmed the excellent ability of Al-Cu alloy particles for the degradation of 3R in aqueous solution. Under a certain condition ([Al-Cu]0 = 2 g/L, [3R]0 = 200 mg/L, [NaCl]0 = 25 g/L, initial pH = 10.9), the 3R could be completely degraded within only 3 min. It was also found that the degradation reaction followed zero-order kinetics model with respect to the initial dye concentration. The intermediate compounds were identified by UV-vis, FT-IR and HPLC-MS, and a pathway was proposed. Additionally, post-treatment Al-Cu alloy particles were characterized by SEM and TEM, and the results showed that the degradation might be attributed to the corrosion effect of Al-Cu alloys. PMID:27535800

  1. A Doping Lattice of Aluminum and Copper with Accelerated Electron Transfer Process and Enhanced Reductive Degradation Performance.

    PubMed

    Zhang, Lin; Gao, Xue; Zhang, Zhixuan; Zhang, Mingbo; Cheng, Yiqian; Su, Jixin

    2016-01-01

    Treatment of azo dye effluents has received increasing concerns over the years due to their potential harms to natural environment and human health. The present study described the degrading ability of the as-synthesized crystalline Al-Cu alloys for removal of high-concentration Acid Scarlet 3R in alkaline aqueous solutions and its degradation mechanism. Al-Cu alloy particles with Al/Cu ratios 19:1 were successfully synthesized by high-energy mechanical milling. Characterization results showed that 10 h mechanical alloying process could lead to the formation of crystalline Al(Cu) solid solution. Batch experiment results confirmed the excellent ability of Al-Cu alloy particles for the degradation of 3R in aqueous solution. Under a certain condition ([Al-Cu]0 = 2 g/L, [3R]0 = 200 mg/L, [NaCl]0 = 25 g/L, initial pH = 10.9), the 3R could be completely degraded within only 3 min. It was also found that the degradation reaction followed zero-order kinetics model with respect to the initial dye concentration. The intermediate compounds were identified by UV-vis, FT-IR and HPLC-MS, and a pathway was proposed. Additionally, post-treatment Al-Cu alloy particles were characterized by SEM and TEM, and the results showed that the degradation might be attributed to the corrosion effect of Al-Cu alloys. PMID:27535800

  2. Crystal structure of phenylacetic acid degradation protein PaaG from Thermus thermophilus HB8.

    PubMed

    Kichise, Tomoyasu; Hisano, Tamao; Takeda, Kazuki; Miki, Kunio

    2009-09-01

    Microbial degradation of phenylacetic acid proceeds via the hybrid pathway that includes formation of a coenzyme A thioester, ring hydroxylation, non-oxygenolytic ring opening, and beta-oxidation-like reactions. A phenylacetic acid degradation protein PaaG is a member of the crotonase superfamily, and is a candidate non-oxygenolytic ring-opening enzyme. The crystal structure of PaaG from Thermus thermophilus HB8 was determined at a resolution of 1.85 A. PaaG consists of three identical subunits related by local three-fold symmetry. The monomer is comprised of a spiral and a helical domain with a fold characteristic of the crotonase superfamily. A putative active site residue, Asp136, is situated in an active site cavity and surrounded by several hydrophobic and hydrophilic residues. The active site cavity is sufficiently large to accommodate a ring substrate. Two conformations are observed for helix H2 located adjacent to the active site. Helix H2 is kinked at Asn81 in two subunits, whereas it is kinked at Leu77 in the other subunit, and the side chain of Tyr80 is closer to Asp136. This indicates that catalytic reaction of PaaG may proceed with large conformational changes at the active site. Asp136 is the only conserved polar residue in the active site. It is located at the same position as those of 4-chlorobenzoyl-CoA dehalogenase and peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase, indicating that PaaG may undergo isomerization or a ring-opening reaction via a Delta(3),Delta(2)-enoyl-CoA isomerase-like mechanism. PMID:19452559

  3. Maize toxin degrades peritrophic matrix proteins and stimulates compensatory transcriptome responses in fall armyworm midgut.

    PubMed

    Fescemyer, Howard W; Sandoya, Germán V; Gill, Torrence A; Ozkan, Seval; Marden, James H; Luthe, Dawn S

    2013-03-01

    Understanding the molecular mechanisms underlying insect compensatory responses to plant defenses could lead to improved plant resistance to herbivores. The Mp708 inbred line of maize produces the maize insect resistant 1-cysteine protease (Mir1-CP) toxin. Reduced feeding and growth of fall armyworm larvae fed on Mp708 was previously linked to impairment of nutrient utilization and degradation of the midgut (MG) peritrophic matrix (PM) by Mir1-CP. Here we examine the biochemical and transcriptional responses of fall armyworm larvae to Mir1-CP. Insect Intestinal Mucin (IIM) was severely depleted from pure PMs treated in vitro with recombinant Mir1-CP. Larvae fed on Mp708 midwhorls excrete frass largely depleted of IIM. Cracks, fissures and increased porosity previously observed in the PM of larvae fed on Mp708 midwhorls could ensue when Mir1-CP degrades the IIM that cross-links chitin fibrils in the PM. Both targeted and global transcriptome analyses were performed to determine how complete dissolution of the structure and function of the PM is prevented, enabling larvae to continue growing in the presence of Mir1-CP. The MGs from fall armyworm fed on Mp708 upregulate expression of genes encoding proteins involved in PM production as an apparent compensation to replace the disrupted PM structure and restore appropriate counter-current MG gradients. Also, several families of digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on diets lacking Mir1-CP (artificial diet, midwhorls from Tx601 or B73 maize). Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function. PMID:23306018

  4. Clenbuterol changes phosphorylated FOXO1 localization and decreases protein degradation in the sartorius muscle of neonatal chicks.

    PubMed

    Shimamoto, Saki; Ijiri, Daichi; Nakashima, Kazuki; Kawaguchi, Mana; Ishimaru, Yoshitaka; Furukawa, Airi; Ohtsuka, Akira

    2016-08-01

    To investigate the intracellular signaling mechanisms by which clenbuterol reduces muscle protein degradation, we examined the phosphorylation level and intracellular localization of FOXO1 in the sartorius muscle of neonatal chicks. One-day-old chicks were given a single intraperitoneal injection of clenbuterol (0.1 mg/kg body weight). Three hours after injection, AKT protein was phosphorylated in the sartorius muscle by clenbuterol injection. Coincidentally, clenbuterol increased cytosolic level of phosphorylated FOXO1 protein, while it decreased nuclear level of FOXO1 protein in the sartorius muscle. Furthermore, clenbuterol decreased the expression of mRNAs for muscle-specific ubiquitin ligases (atrogin-1/MAFbx and MuRF1) in the sartorius muscle accompanied by decreased plasma 3-methylhistidine concentration, an index of muscle protein degradation, at 3 h after injection. These results suggested that, in the sartorius muscle of the chicks, clenbuterol changed the intracellular localization of phosphorylated FOXO1, and consequently decreased protein degradation via suppressing the expression of genes encoding muscle-specific ubiquitin ligases. PMID:27055887

  5. Effect of Source of Rumen-Degraded Protein on Production and Ruminal Metabolism in Lactating Dairy Cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty-eight (8 with ruminal cannulas) lactating Holstein cows were assigned to a 16-wk, 4 x 4 Latin squares study to examine the effect on production and ruminal metabolism of feeding differing proportions of rumen-degraded protein (RDP) from soybean meal and urea. Diets contained dry matter [(DM) ...

  6. The interaction of harvesting time of day of switchgrass hay and ruminal degradability of supplemental protein to beef steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to interact AM ( 0600 ) vs PM ( 1800 ) harvest with ruminal degradability of a protein supplement (HI or LO ) to change voluntary intake, apparent digestibility or N retention by steers fed Alamo switchgrass (Panicum virgatum L.) hay. Black steers (255 ± 14kg BW) we...

  7. Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation

    PubMed Central

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri; Su, Xiong; Liu, Jialiu; Sundaresan, Sinju; Stahl, Philip D.

    2013-01-01

    Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in-vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis. PMID:23578663

  8. Estimating Rumen Degradable Protein in Forage Legume Hays and Silages by In Situ Disappearance Kinetics vs. Alternative Methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Currently, the in situ kinetic method is considered the “gold standard” for estimating rumen degradable protein (RDP) in forages. When such estimates are impractical (e.g. numerous samples, limited sample quantities, or lack of ruminally fistulated cattle) alternative RDP methods are used, but their...

  9. Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A

    PubMed Central

    Kim, Nari; Kim, Min-Jung; Sung, Pil Soo; Bae, Yong Chul; Shin, Eui-Cheol; Yoo, Joo-Yeon

    2016-01-01

    Hepatitis C virus (HCV) utilizes autophagy to promote its propagation. Here we show the autophagy-mediated suppression of HCV replication via the endoplasmic reticulum (ER) protein SCOTIN. SCOTIN overexpression inhibits HCV replication and infectious virion production in cells infected with cell culture-derived HCV. HCV nonstructural 5A (NS5A) protein, which is a critical factor for HCV RNA replication, interacts with the IFN-β-inducible protein SCOTIN, which transports NS5A to autophagosomes for degradation. Furthermore, the suppressive effect of SCOTIN on HCV replication is impaired in both ATG7-silenced cells and cells treated with autophagy or lysosomal inhibitors. SCOTIN does not affect the overall flow of autophagy; however, it is a substrate for autophagic degradation. The physical association between the transmembrane/proline-rich domain (TMPRD) of SCOTIN and Domain-II of NS5A is essential for autophagosomal trafficking and NS5A degradation. Altogether, our findings suggest that IFN-β-induced SCOTIN recruits the HCV NS5A protein to autophagosomes for degradation, thereby restricting HCV replication. PMID:26868272

  10. Degradation of oxidized proteins by the proteasome: Distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways.

    PubMed

    Raynes, Rachel; Pomatto, Laura C D; Davies, Kelvin J A

    2016-08-01

    The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known function of the proteasome is protein degradation through the 26S ubiquitin-proteasome system, responsible for the vast majority of protein degradation during homeostasis. However, the proteasome also plays an important role in adaptive immune responses and adaptation to oxidative stress. The unbound 20S proteasome, the core common to all proteasome conformations, is the main protease responsible for degrading oxidized proteins. During periods of acute stress, the 19S regulatory cap of the 26S proteasome disassociates from the proteolytic core, allowing for immediate ATP/ubiquitin-independent protein degradation by the 20S proteasome. Despite the abundance of unbound 20S proteasome compared to other proteasomal conformations, many publications fail to distinguish between the two proteolytic systems and often regard the 26S proteasome as the dominant protease. Further confounding the issue are the differential roles these two proteolytic systems have in adaptation and aging. In this review, we will summarize the increasing evidence that the 20S core proteasome constitutes the major conformation of the proteasome system and that it is far from a latent protease requiring activation by binding regulators. PMID:27155164

  11. Chondroitin Sulfate Accelerates Trans-Golgi-to-Surface Transport of Proteoglycan Amyloid Precursor Protein.

    PubMed

    Mihov, Deyan; Raja, Eva; Spiess, Martin

    2015-08-01

    The amyloid precursor protein (APP) is a membrane protein implicated in the pathogenesis of Alzheimer's disease. APP is a part-time proteoglycan, as splice variants lacking exon 15 are modified by a chondroitin sulfate glycosaminoglycan (GAG) chain. Investigating the effect of the GAG chain on the trafficking of APP in non-polarized cells, we found it to increase the steady-state surface-to-intracellular distribution, to reduce the rate of endocytosis and to accelerate transport kinetics from the trans-Golgi network (TGN) to the plasma membrane. Deletion of the cytosolic domain resulted in delayed surface arrival of GAG-free APP, but did not affect the rapid export kinetics of the proteoglycan form. Protein-free GAG chains showed the same TGN-to-cell surface transport kinetics as proteoglycan APP. Endosome ablation experiments were performed to distinguish between indirect endosomal and direct pathways to the cell surface. Surprisingly, TGN-to-cell surface transport of both GAG-free and proteoglycan APP was found to be indirect via transferrin-positive endosomes. Our results show that GAGs act as alternative sorting determinants in cellular APP transport that are dominant over cytoplasmic signals and involve distinct sorting mechanisms. PMID:25951880

  12. Mitochondrial Complex I Deficiency Increases Protein Acetylation and Accelerates Heart Failure

    PubMed Central

    Karamanlidis, Georgios; Lee, Chi Fung; Garcia-Menendez, Lorena; Kolwicz, Stephen C.; Suthammarak, Wichit; Gong, Guohua; Sedensky, Margaret M.; Morgan, Philip G.; Wang, Wang; Tian, Rong

    2013-01-01

    Summary Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases including heart failure but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by inactivation of the Ndufs4 gene, a protein critical for Complex I (C-I) assembly, in the mouse heart (cKO). While C-I supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD+/NADH ratio by C-I deficiency inhibited Sirt3 activity, leading to increase in protein acetylation, and sensitization of the permeability transition in mitochondria (mPTP). NAD+ precursor supplementation to cKO mice partially normalized the NAD+/NADH ratio, protein acetylation and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target. PMID:23931755

  13. Heat shock transcription factor δ³² is targeted for degradation via an ubiquitin-like protein ThiS in Escherichia coli.

    PubMed

    Xu, Xibing; Niu, Yulong; Liang, Ke; Wang, Jianmei; Li, Xufeng; Yang, Yi

    2015-04-01

    The posttranslational modification of proteins with ubiquitin and ubiquitin-like proteins (UBLs) plays an important role in eukaryote biology, through which substrate proteins are targeted for degradation by the proteasome. Prokaryotes have been thought to degrade proteins by an ubiquitin independent pathway. Here, we show that ThiS, an ubiquitin-like protein, is covalently attached to δ(32) and at least 27 other proteins, leading to their subsequent degradation by proteases, in a similar manner to the ubiquitin-proteasome system (UPS) in eukaryotes. Molecular biology and biochemical studies confirm that specific lysine sites in δ(32) can be modified by ThiS. The results presented here establish a new model for δ(32) degradation and show that Escherichia coli uses a small-protein modifier to control protein stability. PMID:25721662

  14. Lymphocytes accelerate epithelial tight junction assembly: role of AMP-activated protein kinase (AMPK).

    PubMed

    Tang, Xiao Xiao; Chen, Hao; Yu, Sidney; Zhang, Li; Caplan, Michael J; Chan, Hsiao Chang

    2010-01-01

    The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK. PMID:20808811

  15. Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

    SciTech Connect

    Song, Xiuming; Yao, Jing; Wang, Fei; Zhou, Mi; Zhou, Yuxin; Wang, Hu; Wei, Libin; Zhao, Li; Li, Zhiyu; Lu, Na Guo, Qinglong

    2013-09-01

    Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function.

  16. The requirements for rumen-degradable protein per unit of fermentable organic matter differ between fibrous feed sources

    PubMed Central

    Soliva, Carla R.; Amelchanka, Sergej L.; Kreuzer, Michael

    2015-01-01

    Ruminant feed evaluation systems use constant minimum requirements of rumen-degradable protein (RDP) and often relate this to apparently degradable organic matter (OM). However, studies with tropical forages indicate that RDP: apparently degraded OM might not be constant across high-fiber diets. This was tested with semi-continuous ruminal cultures (Rusitec) using dried contrasting low-protein fiber sources: brachiaria hay (high in fiber, medium lignified), apple pomace (medium in fiber, highly lignified), and sugar beet pulp (medium in fiber and lignification). Each feed was incubated at 14 g dry matter day−1 with 0, 0.85, 1.7, 3.4, 6.8, 13.6, or 27.2 mg g−1 urea. The amount of urea needed to reach a similar basal concentration of ammonia in the incubation fluid was tested for each feed in advance. Apparent fiber and OM degradability were determined after 48 h of incubation. Data was evaluated by regressions and analysis of variance. The response curve of incubation fluid ammonia to urea supplementation was similar in slope in all feeds. Plateaus in apparent OM degradability in relation to ammonia concentration were determined. The ammonia concentration where apparent OM and fiber degradability reached 95% of maximum was approached in the order of pomace < pulp < hay. With regard to fiber degradability, a plateau was reached at ≥ 80 g kg−1 crude protein only with hay and pomace, whilst a linear relationship existed between RDP and OM degradation for pulp. In hay the ratio RDP: OM degraded was equal to 1.6 but was only 1.0 in the other feeds. There was no obvious lack of branched short-chain fatty acids at low RDP. Thus, the hypothesis was confirmed but the demand for RDP seems even higher in tropical forage compared to food industrial byproducts. The efficiency of urea to promote apparent OM and fiber degradation was also variable. Thus, it seems that minimum thresholds of either RDP or ruminal ammonia concentration may not be reflected appropriately by

  17. Metal-assisted and microwave accelerated-evaporative crystallization: Application to lysozyme protein

    NASA Astrophysics Data System (ADS)

    Mauge-Lewis, Kevin

    In response to the growing need for new crystallization techniques that afford for rapid processing times along with control over crystal size and distribution, the Aslan Research Group has recently demonstrated the use of Metal-Assisted and Microwave-Accelerated Evaporative Crystallization MA-MAEC technique in conjunction with metal nanoparticles and nanostructures for the crystallization of amino acids and organic small molecules. In this study, we have employed the newly developed MA-MAEC technique to the accelerated crystallization of chicken egg-white lysozyme on circular crystallization platforms in order to demonstrate the proof-of-principle application of the method for protein crystallization. The circular crystallization platforms are constructed in-house from poly (methyl methacrylate) (PMMA) and silver nanoparticle films (SNFs), indium tin oxide (ITO) and iron nano-columns. In this study, we prove the MA-MAEC method to be a more effective technique in the rapid crystallization of macromolecules in comparison to other conventional methods. Furthermore, we demonstrate the use of the novel iCrystal system, which incorporates the use of continuous, low wattage heating to facilitate the rapid crystallization of the lysozyme while still retaining excellent crystal quality. With the incorporation of the iCrystal system, we observe crystallization times that are even shorter than those produced by the MA-MAEC technique using a conventional microwave oven in addition to significantly improved crystal quality.

  18. Strontium exerts dual effects on calcium phosphate cement: Accelerating the degradation and enhancing the osteoconductivity both in vitro and in vivo.

    PubMed

    Kuang, Guan-Ming; Yau, W P; Wu, Jun; Yeung, Kelvin W K; Pan, Haobo; Lam, W M; Lu, W W; Chiu, K Y

    2015-05-01

    Calcium phosphate cements (CPCs) have long been used as osteoconductive bone substitutes in the treatment of bone defects. However, the degradation rate of CPC is typically too slow to match the new bone growth rate. It is known that strontium increases the solubility of hydroxyapatite as well as exerts both anabolic and anticatabolic effects on bone. Therefore, we hypothesized that the incorporation of strontium would accelerate the degradation rate and enhance the osteoconductivity of CPC. In this study, Three groups, CPC (0% Sr-CPC), 5% Sr-CPC, and 10% Sr-CPC, were prepared, with the total molar ratio for Sr/(Sr+Ca) in the cement powder phase being 0, 5, and 10%, respectively. In the immersion test, less residual weight was observed in both 5% Sr-CPC and 10% Sr-CPC groups than CPC group. In addition, a higher osteoblastic cell proliferation rate and alkaline phosphatase activity were obtained in the strontium groups. In a rat femur bone defect model comparing CPC with 10% Sr-CPC, at 2 weeks postoperation, early endochondral ossification was found in the 10% Sr-CPC group, whereas only fibrous tissue was observed in control group; at 4-16 weeks postoperation, progressive osteoconduction toward the cement was observed in both groups. At 32 weeks, a higher peri-cement bone area and reduced cement area were noted in the 10% Sr-CPC group. In conclusion, in the 10% Sr-CPC group, strontium exerts dual effects on CPC: accelerating degradation rate and enhancing osteoconductivity, as shown here both in vitro and in vivo. PMID:25087971

  19. Replacing a single atom accelerates the folding of a protein and increases its thermostability.

    PubMed

    Arnold, Ulrich; Raines, Ronald T

    2016-07-12

    The conformational attributes of proline can have a substantial effect on the folding of polypeptide chains into a native structure and on the stability of that structure. Replacing the 4S hydrogen of a proline residue with fluorine is known to elicit stereoelectronic effects that favor a cis peptide bond. Here, semisynthesis is used to replace a cis-proline residue in ribonuclease A with (2S,4S)-4-fluoroproline. This subtle substitution accelerates the folding of the polypeptide chain into its three-dimensional structure and increases the thermostability of that structure without compromising its catalytic activity. Thus, an appropriately situated fluorine can serve as a prosthetic atom in the context of a protein. PMID:27336677

  20. Acceleration of protein folding by four orders of magnitude through a single amino acid substitution

    PubMed Central

    Roderer, Daniel J. A.; Schärer, Martin A.; Rubini, Marina; Glockshuber, Rudi

    2015-01-01

    Cis prolyl peptide bonds are conserved structural elements in numerous protein families, although their formation is energetically unfavorable, intrinsically slow and often rate-limiting for folding. Here we investigate the reasons underlying the conservation of the cis proline that is diagnostic for the fold of thioredoxin-like thiol-disulfide oxidoreductases. We show that replacement of the conserved cis proline in thioredoxin by alanine can accelerate spontaneous folding to the native, thermodynamically most stable state by more than four orders of magnitude. However, the resulting trans alanine bond leads to small structural rearrangements around the active site that impair the function of thioredoxin as catalyst of electron transfer reactions by more than 100-fold. Our data provide evidence for the absence of a strong evolutionary pressure to achieve intrinsically fast folding rates, which is most likely a consequence of proline isomerases and molecular chaperones that guarantee high in vivo folding rates and yields. PMID:26121966

  1. Acceleration of protein folding by four orders of magnitude through a single amino acid substitution.

    PubMed

    Roderer, Daniel J A; Schärer, Martin A; Rubini, Marina; Glockshuber, Rudi

    2015-01-01

    Cis prolyl peptide bonds are conserved structural elements in numerous protein families, although their formation is energetically unfavorable, intrinsically slow and often rate-limiting for folding. Here we investigate the reasons underlying the conservation of the cis proline that is diagnostic for the fold of thioredoxin-like thiol-disulfide oxidoreductases. We show that replacement of the conserved cis proline in thioredoxin by alanine can accelerate spontaneous folding to the native, thermodynamically most stable state by more than four orders of magnitude. However, the resulting trans alanine bond leads to small structural rearrangements around the active site that impair the function of thioredoxin as catalyst of electron transfer reactions by more than 100-fold. Our data provide evidence for the absence of a strong evolutionary pressure to achieve intrinsically fast folding rates, which is most likely a consequence of proline isomerases and molecular chaperones that guarantee high in vivo folding rates and yields. PMID:26121966

  2. In-situ Ruminal Protein, Fiber, and Dry Matter Degradability of Legume Silages and Hays as Influenced by Protein-binding Polyphenols and Conditioning Methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conditioning and conservation methods may alter polyphenol binding in forage legumes and the degradability of crude protein (CP), neutral detergent fiber (NDF) and dry matter (DM) in the rumen. In this study, alfalfa, birdsfoot trefoil with 6 or 15 g/kg condensed tannins (CT), and red clover with ~1...

  3. Protein Degradation Pathways Regulate the Functions of Helicases in the DNA Damage Response and Maintenance of Genomic Stability

    PubMed Central

    Sommers, Joshua A.; Suhasini, Avvaru N.; Brosh, Robert M.

    2015-01-01

    Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom’s syndrome helicase (BLM) provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA) interstrand cross-link (ICL) repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1) helps to balance translesion synthesis (TLS) and homologous recombination (HR) repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF), a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR) pathway. Stability of the Werner syndrome helicase-nuclease (WRN) involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB) implicated in transcription-coupled repair (TCR) is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis. PMID:25906194

  4. Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation

    PubMed Central

    Xiong, Hui; Wang, Danling; Chen, Linan; Choo, Yeun Su; Ma, Hong; Tang, Chengyuan; Xia, Kun; Jiang, Wei; Ronai, Ze’ev; Zhuang, Xiaoxi; Zhang, Zhuohua

    2009-01-01

    Mutations in PARKIN, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of Parkinson disease (PD). Although mutations in these genes lead to the same disease state, the functional relationships between them and how their respective disease-associated mutations cause PD are largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed PPD complex) to promote ubiquitination and degradation of Parkin substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human brain lysates. Genetic ablation of either Pink1 or Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well as decreased degradation and increased accumulation of aberrantly expressed Parkin substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat shock–induced misfolded protein. In contrast, PD-pathogenic Parkin and PINK1 mutations showed reduced ability to promote degradation of Parkin substrates. This study identified a functional ubiquitin E3 ligase complex consisting of PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of the complex, which may constitute a mechanism underlying PD pathogenesis. PMID:19229105

  5. Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris.

    PubMed

    Whyteside, Graham; Alcocer, Marcos J C; Kumita, Janet R; Dobson, Christopher M; Lazarou, Maria; Pleass, Richard J; Archer, David B

    2011-01-01

    We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general. PMID:21818368

  6. Native-State Stability Determines the Extent of Degradation Relative to Secretion of Protein Variants from Pichia pastoris

    PubMed Central

    Whyteside, Graham; Alcocer, Marcos J. C.; Kumita, Janet R.; Dobson, Christopher M.; Lazarou, Maria; Pleass, Richard J.; Archer, David B.

    2011-01-01

    We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general. PMID:21818368

  7. Induction of muscle protein degradation and weight loss by a tumor product.

    PubMed

    Todorov, P T; McDevitt, T M; Cariuk, P; Coles, B; Deacon, M; Tisdale, M J

    1996-03-15

    Splenocytes from mice bearing a cachexia-inducing tumor (MAC16) have been fused with mouse myeloma cells to produce hybridomas, which have been cloned to produce antibody reactive to a material which copurified with a lipid-mobilizing factor isolated from the same tumor. The monoclonal antibody has been used to investigate factors potentially involved in the development of cachexia. The major protein detectable by immunoprecipitation of a partially purified lipid-mobilizing factor was M(r) 69,000, whereas Western blotting showed two bands of M(r) 69,000 and M(r) 24,000. Although the monoclonal antibody did not neutralize lipid-mobilizing activity in an in vitro assay, it did neutralize a serum factor capable of protein degradation in isolated gastrocnemius muscle. Affinity purification of MAC16 tumor homogenates using the monoclonal antibody yielded two immunoreactive bands of M(r) 69,000 and M(r) 24,000, which were further fractionated on a hydrophobic column (C8). This material was capable of inducing tyrosine release from isolated gastrocnemius muscle, and the effect could be blocked with the monoclonal antibody. The two immunoreactive bands from the hydrophobic column were capable of inducing weight loss in mice, whereas nonimmunoreactive fractions had no effect on body weight. The M(r) 24,000 species had a unique amino acid sequence, whereas the M(r)