Presynaptic Protein Synthesis Is Required for Long-Term Plasticity of GABA Release.

PubMed

Younts, Thomas J; Monday, Hannah R; Dudok, Barna; Klein, Matthew E; Jordan, Bryen A; Katona, István; Castillo, Pablo E

2016-10-19

Long-term changes of neurotransmitter release are critical for proper brain function. However, the molecular mechanisms underlying these changes are poorly understood. While protein synthesis is crucial for the consolidation of postsynaptic plasticity, whether and how protein synthesis regulates presynaptic plasticity in the mature mammalian brain remain unclear. Here, using paired whole-cell recordings in rodent hippocampal slices, we report that presynaptic protein synthesis is required for long-term, but not short-term, plasticity of GABA release from type 1 cannabinoid receptor (CB 1 )-expressing axons. This long-term depression of inhibitory transmission (iLTD) involves cap-dependent protein synthesis in presynaptic interneuron axons, but not somata. Translation is required during the induction, but not maintenance, of iLTD. Mechanistically, CB 1 activation enhances protein synthesis via the mTOR pathway. Furthermore, using super-resolution STORM microscopy, we revealed eukaryotic ribosomes in CB 1 -expressing axon terminals. These findings suggest that presynaptic local protein synthesis controls neurotransmitter release during long-term plasticity in the mature mammalian brain. Copyright © 2016 Elsevier Inc. All rights reserved.

Endoplasmic reticulum stress inhibits expression of genes involved in thyroid hormone synthesis and their key transcriptional regulators in FRTL-5 thyrocytes

PubMed Central

Wen, Gaiping; Eder, Klaus

2017-01-01

Endoplasmic reticulum (ER) stress is characterized by the accumulation of misfolded proteins due to an impairment of ER quality control pathways leading to the activation of a defense system, called unfolded protein response (UPR). While thyrocytes are supposed to be highly susceptible to environmental conditions that cause ER stress due to the synthesis of large amounts of secretory proteins required for thyroid hormone synthesis, systematic investigations on the effect of ER stress on expression of key genes of thyroid hormone synthesis and their transcriptional regulators are lacking. Since the aim of the ER stress-induced UPR is to restore ER homeostasis and to facilitate cell survival through transient shutdown of ribosomal protein translation, we hypothesized that the expression of genes involved in thyroid hormone synthesis and their transcriptional regulators, all of which are not essential for cell survival, are down-regulated in thyrocytes during ER stress, while sterol regulatory element-binding proteins (SREBPs) are activated during ER stress in thyrocytes. Treatment of FRTL-5 thyrocytes with the ER stress inducer tunicamycin (TM) dose-dependently increased the mRNA and/or protein levels of known UPR target genes, stimulated phosphorylation of the ER stress sensor protein kinase RNA-like ER kinase (PERK) and of the PERK target protein eukaryotic initiation factor 2α (eIF2α) and caused splicing of the ER stress-sensitive transcription factor X-box binding protein (XBP-1) (P < 0.05). The mRNA levels and/or protein levels of genes involved in thyroid hormone synthesis, sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), their transcriptional regulators and thyrotropin (TSH) receptor and the uptake of Na125I were reduced at the highest concentration of TM tested (0.1 μg/mL; P < 0.05). Proteolytic activation of the SREBP-1c pathway was not observed in FRTL-5 cells treated with TM, whereas TM reduced proteolytic activation of the SREBP-2 pathway at 0.1 μg TM/mL (P < 0.05). In conclusion, the expression of key genes involved in thyroid hormone synthesis and their critical regulators and of the TSH receptor as well as the uptake of iodide is attenuated in thyrocytes during mild ER stress. Down-regulation of NIS, TPO and TG during ER stress is likely the consequence of impaired TSH/TSHR signaling in concert with reduced expression of critical transcriptional regulators of these genes. PMID:29095946

Endoplasmic reticulum stress inhibits expression of genes involved in thyroid hormone synthesis and their key transcriptional regulators in FRTL-5 thyrocytes.

PubMed

Wen, Gaiping; Ringseis, Robert; Eder, Klaus

2017-01-01

Endoplasmic reticulum (ER) stress is characterized by the accumulation of misfolded proteins due to an impairment of ER quality control pathways leading to the activation of a defense system, called unfolded protein response (UPR). While thyrocytes are supposed to be highly susceptible to environmental conditions that cause ER stress due to the synthesis of large amounts of secretory proteins required for thyroid hormone synthesis, systematic investigations on the effect of ER stress on expression of key genes of thyroid hormone synthesis and their transcriptional regulators are lacking. Since the aim of the ER stress-induced UPR is to restore ER homeostasis and to facilitate cell survival through transient shutdown of ribosomal protein translation, we hypothesized that the expression of genes involved in thyroid hormone synthesis and their transcriptional regulators, all of which are not essential for cell survival, are down-regulated in thyrocytes during ER stress, while sterol regulatory element-binding proteins (SREBPs) are activated during ER stress in thyrocytes. Treatment of FRTL-5 thyrocytes with the ER stress inducer tunicamycin (TM) dose-dependently increased the mRNA and/or protein levels of known UPR target genes, stimulated phosphorylation of the ER stress sensor protein kinase RNA-like ER kinase (PERK) and of the PERK target protein eukaryotic initiation factor 2α (eIF2α) and caused splicing of the ER stress-sensitive transcription factor X-box binding protein (XBP-1) (P < 0.05). The mRNA levels and/or protein levels of genes involved in thyroid hormone synthesis, sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), their transcriptional regulators and thyrotropin (TSH) receptor and the uptake of Na125I were reduced at the highest concentration of TM tested (0.1 μg/mL; P < 0.05). Proteolytic activation of the SREBP-1c pathway was not observed in FRTL-5 cells treated with TM, whereas TM reduced proteolytic activation of the SREBP-2 pathway at 0.1 μg TM/mL (P < 0.05). In conclusion, the expression of key genes involved in thyroid hormone synthesis and their critical regulators and of the TSH receptor as well as the uptake of iodide is attenuated in thyrocytes during mild ER stress. Down-regulation of NIS, TPO and TG during ER stress is likely the consequence of impaired TSH/TSHR signaling in concert with reduced expression of critical transcriptional regulators of these genes.

Higher insulin sensitivity in EDL muscle of rats fed a low-protein, high-carbohydrate diet inhibits the caspase-3 and ubiquitin-proteasome proteolytic systems but does not increase protein synthesis.

PubMed

Dos Santos, Maísa Pavani; Batistela, Emanuele; Pereira, Mayara Peron; Paula-Gomes, Silvia; Zanon, Neusa Maria; Kettelhut, Isis do Carmo; Karatzaferi, Christina; Andrade, Claudia Marlise Balbinotti; de França, Suélem Aparecida; Baviera, Amanda Martins; Kawashita, Nair Honda

2016-08-01

Compared with the extensor digitorum longus (EDL) muscle of control rats (C), the EDL muscle of rats fed a low-protein, high-carbohydrate diet (LPHC) showed a 36% reduction in mass. Muscle mass is determined by the balance between protein synthesis and proteolysis; thus, the aim of this work was to evaluate the components involved in these processes. Compared with the muscle from C rats, the EDL muscle from LPHC diet-fed rats showed a reduction (34%) in the in vitro basal protein synthesis and a 22% reduction in the in vitro basal proteolysis suggesting that the reduction in the mass can be associated with a change in the rate of the two processes. Soon after euthanasia, in the EDL muscles of the rats fed the LPHC diet for 15days, the activity of caspase-3 and that of components of the ubiquitin-proteasome system (atrogin-1 content and chymotrypsin-like activity) were decreased. The phosphorylation of p70(S6K) and 4E-BP1, proteins involved in protein synthesis, was also decreased. We observed an increase in the insulin-stimulated protein content of p-Akt. Thus, the higher insulin sensitivity in the EDL muscle of LPHC rats seemed to contribute to the lower proteolysis in LPHC rats. However, even with the higher insulin sensitivity, the reduction in p-E4-BP1 and p70(S6K) indicates a reduction in protein synthesis, showing that factors other than insulin can have a greater effect on the control of protein synthesis. Copyright © 2016 Elsevier Inc. All rights reserved.

A Tomato Peroxidase Involved in the Synthesis of Lignin and Suberin1

PubMed Central

Quiroga, Mónica; Guerrero, Consuelo; Botella, Miguel A.; Barceló, Araceli; Amaya, Iraida; Medina, María I.; Alonso, Francisco J.; de Forchetti, Silvia Milrad; Tigier, Horacio; Valpuesta, Victoriano

2000-01-01

The last step in the synthesis of lignin and suberin has been proposed to be catalyzed by peroxidases, although other proteins may also be involved. To determine which peroxidases are involved in the synthesis of lignin and suberin, five peroxidases from tomato (Lycopersicon esculentum) roots, representing the majority of the peroxidase activity in this organ, have been partially purified and characterized kinetically. The purified peroxidases with isoelectric point (pI) values of 3.6 and 9.6 showed the highest catalytic efficiency when the substrate used was syringaldazine, an analog of lignin monomer. Using a combination of transgenic expression and antibody recognition, we now show that the peroxidase pI 9.6 is probably encoded by TPX1, a tomato peroxidase gene we have previously isolated. In situ RNA hybridization revealed that TPX1 expression is restricted to cells undergoing synthesis of lignin and suberin. Salt stress has been reported to induce the synthesis of lignin and/or suberin. This stress applied to tomato caused changes in the expression pattern of TPX1 and induced the TPX1 protein. We propose that the TPX1 product is involved in the synthesis of lignin and suberin. PMID:10759507

Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

PubMed

Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

2012-12-27

L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

Protein synthesis in geostimulated root caps

NASA Technical Reports Server (NTRS)

Feldman, L. J.

1982-01-01

A study is presented of the processes occurring in the root cap of corn which are requisite for the formation of root cap inhibitor and which can be triggered or modulated by both light and gravity. The results of this study indicate the importance of protein synthesis for light-induced gravitropic bending in roots. Root caps in which protein synthesis is prevented are unable to induce downward bending. This suggests that light acts by stimulating proteins which are necessary for the translation of the gravitropic stimulus into a growth response (downward bending). The turnover of protein with time was also examined in order to determine whether light acts by stimulating the synthesis of unique proteins required for downward growth. It is found that auxin in combination with light allows for the translation of the gravitropic stimulus into a growth response at least in part through the modification of protein synthesis. It is concluded that unique proteins are stimulated by light and are involved in promoting the downward growth in roots which are responding to gravity.

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

USDA-ARS?s Scientific Manuscript database

Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. In...

Proteome Profile of Starch Granules Purified from Rice (Oryza sativa) Endosperm.

PubMed

Xing, Shihai; Meng, Xiaoxi; Zhou, Lihui; Mujahid, Hana; Zhao, Chunfang; Zhang, Yadong; Wang, Cailin; Peng, Zhaohua

2016-01-01

Starch is the most important food energy source in cereals. Many of the known enzymes involved in starch biosynthesis are partially or entirely granule-associated in the endosperm. Studying the proteome of rice starch granules is critical for us to further understand the mechanisms underlying starch biosynthesis and packaging of starch granules in rice amyloplasts, consequently for the improvement of rice grain quality. In this article, we developed a protocol to purify starch granules from mature rice endosperm and verified the quality of purified starch granules by microscopy observations, I2 staining, and Western blot analyses. In addition, we found the phenol extraction method was superior to Tris-HCl buffer extraction method with respect to the efficiency in recovery of starch granule associated proteins. LC-MS/MS analysis showed identification of already known starch granule associated proteins with high confidence. Several proteins reported to be involved in starch synthesis in prior genetic studies in plants were also shown to be enriched with starch granules, either directly or indirectly, in our studies. In addition, our results suggested that a few additional candidate proteins may also be involved in starch synthesis. Furthermore, our results indicated that some starch synthesis pathway proteins are subject to protein acetylation modification. GO analysis and KEGG pathway enrichment analysis showed that the identified proteins were mainly located in plastids and involved in carbohydrate metabolism. This study substantially advances the understanding of the starch granule associated proteome in rice and post translational regulation of some starch granule associated proteins.

Roles of Fragile X Mental Retardation Protein in Dopaminergic Stimulation-induced Synapse-associated Protein Synthesis and Subsequent α-Amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) Receptor Internalization*

PubMed Central

Wang, Hansen; Kim, Susan S.; Zhuo, Min

2010-01-01

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of α-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome. PMID:20457613

Roles of fragile X mental retardation protein in dopaminergic stimulation-induced synapse-associated protein synthesis and subsequent alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) receptor internalization.

PubMed

Wang, Hansen; Kim, Susan S; Zhuo, Min

2010-07-09

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.

Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet - A pilot study.

PubMed

Jourdan, Marion; Nair, K Sreekumaran; Carter, Rickey E; Schimke, Jill; Ford, G Charles; Marc, Julie; Aussel, Christian; Cynober, Luc

2015-06-01

Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 h. [ring-(13)C6] phenylalanine and [(15)N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; P = 0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake. Copyright © 2014. Published by Elsevier Ltd.

Therapeutic Interventions to Disrupt the Protein Synthetic Machinery in Melanoma

PubMed Central

Kardos, Gregory R.; Robertson, Gavin P.

2015-01-01

Control of the protein synthetic machinery is deregulated in many cancers, including melanoma, in order to increase protein production. Tumor suppressors and oncogenes play key roles in protein synthesis from the transcription of rRNA and ribosome biogenesis to mRNA translation initiation and protein synthesis. Major signaling pathways are altered in melanoma to modulate the protein synthetic machinery thereby promoting tumor development. However, despite the importance of this process in melanoma development, involvement of the protein synthetic machinery in this cancer type is an underdeveloped area of study. Here, we review the coupling of melanoma development to deregulation of the protein synthetic machinery. We examine existing knowledge regarding RNA Polymerase I inhibition and mRNA translation focusing on their inhibition for therapeutic applications in melanoma. Furthermore, the contribution of amino acid biosynthesis and involvement of ribosomal proteins are also reviewed as future therapeutic strategies to target deregulated protein production in melanoma. PMID:26139519

Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

PubMed

Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

2016-06-01

Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n TBA exhibit increased ( < 0.05) pAKT protein levels. These data indicate the TBA-mediated increases in protein synthesis likely involve GPCR, MMP2/9, hbEGF, EGFR, erbB2, and IGF-1R. However, the mechanism through which TBA mediates changes in protein degradation is different and appears to involve only the EGFR and erbB2. Furthermore, it appears the protein kinase B pathway is involved in TBA's effects on fused BSC cultures.

Early steps in protein synthesis and their regulation: a background study related to the biological effects of radiation. Progress report, July 1, 1975--June 30, 1976

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zamecnik, P.C.

This is a continuing study of protein synthesis, involving a search for the role of Ap/sub 4/A and other unusual nucleotides in growth regulation; studies of the mechanism of action of aminoacyl-tRNA ligases and the effect thereof on protein synthesis; a search for new regulators of the translation step, in cell-free systems; and an effort to improve the sensitivity and quantitation of chemical sequencing at the 3'-end of messenger RNA.

Whereas Short-Term Facilitation Is Presynaptic, Intermediate-Term Facilitation Involves Both Presynaptic and Postsynaptic Protein Kinases and Protein Synthesis

ERIC Educational Resources Information Center

Jin, Iksung; Kandel, Eric R.; Hawkins, Robert D.

2011-01-01

Whereas short-term plasticity involves covalent modifications that are generally restricted to either presynaptic or postsynaptic structures, long-term plasticity involves the growth of new synapses, which by its nature involves both pre- and postsynaptic alterations. In addition, an intermediate-term stage of plasticity has been identified that…

Using Simple Manipulatives to Improve Student Comprehension of a Complex Biological Process: Protein Synthesis

ERIC Educational Resources Information Center

Guzman, Karen; Bartlett, John

2012-01-01

Biological systems and living processes involve a complex interplay of biochemicals and macromolecular structures that can be challenging for undergraduate students to comprehend and, thus, misconceptions abound. Protein synthesis, or translation, is an example of a biological process for which students often hold many misconceptions. This article…

The Proteome and Lipidome of Thermococcus kodakarensis across the Stationary Phase

PubMed Central

Yoshinaga, Marcos Y.; Garcia Prado, Franka; Hinrichs, Kai-Uwe; Thomm, Michael

2016-01-01

The majority of cells in nature probably exist in a stationary-phase-like state, due to nutrient limitation in most environments. Studies on bacteria and yeast reveal morphological and physiological changes throughout the stationary phase, which lead to an increased ability to survive prolonged nutrient limitation. However, there is little information on archaeal stationary phase responses. We investigated protein- and lipid-level changes in Thermococcus kodakarensis with extended time in the stationary phase. Adaptations to time in stationary phase included increased proportion of membrane lipids with a tetraether backbone, synthesis of proteins that ensure translational fidelity, specific regulation of ABC transporters (upregulation of some, downregulation of others), and upregulation of proteins involved in coenzyme production. Given that the biological mechanism of tetraether synthesis is unknown, we also considered whether any of the protein-level changes in T. kodakarensis might shed light on the production of tetraether lipids across the same period. A putative carbon-nitrogen hydrolase, a TldE (a protease in Escherichia coli) homologue, and a membrane bound hydrogenase complex subunit were candidates for possible involvement in tetraether-related reactions, while upregulation of adenosylcobalamin synthesis proteins might lend support to a possible radical mechanism as a trigger for tetraether synthesis. PMID:27274708

The Proteome and Lipidome of Thermococcus kodakarensis across the Stationary Phase.

PubMed

Gagen, Emma J; Yoshinaga, Marcos Y; Garcia Prado, Franka; Hinrichs, Kai-Uwe; Thomm, Michael

2016-01-01

The majority of cells in nature probably exist in a stationary-phase-like state, due to nutrient limitation in most environments. Studies on bacteria and yeast reveal morphological and physiological changes throughout the stationary phase, which lead to an increased ability to survive prolonged nutrient limitation. However, there is little information on archaeal stationary phase responses. We investigated protein- and lipid-level changes in Thermococcus kodakarensis with extended time in the stationary phase. Adaptations to time in stationary phase included increased proportion of membrane lipids with a tetraether backbone, synthesis of proteins that ensure translational fidelity, specific regulation of ABC transporters (upregulation of some, downregulation of others), and upregulation of proteins involved in coenzyme production. Given that the biological mechanism of tetraether synthesis is unknown, we also considered whether any of the protein-level changes in T. kodakarensis might shed light on the production of tetraether lipids across the same period. A putative carbon-nitrogen hydrolase, a TldE (a protease in Escherichia coli) homologue, and a membrane bound hydrogenase complex subunit were candidates for possible involvement in tetraether-related reactions, while upregulation of adenosylcobalamin synthesis proteins might lend support to a possible radical mechanism as a trigger for tetraether synthesis.

Changes in the pattern of protein synthesis during zoospore germination in Blastocladiella emersonii.

PubMed Central

Silva, A M; Maia, J C; Juliani, M H

1987-01-01

Using two-dimensional gel electrophoresis, we analyzed the pattern of proteins synthesized during Blastocladiella emersonii zoospore germination in an inorganic solution, in both the presence and absence of actinomycin D. During the transition from zoospore to round cells (the first 25 min), essentially no qualitative differences were noticeable, indicating that the earliest stages of germination are entirely preprogrammed with stored RNA. Later in germination (after 25 min), however, changes in the pattern of protein synthesis were found. Some of these proteins (a total of 6 polypeptides) correspond possibly to a selective translation of stored messages, whereas the majority of the changed proteins (22 polypeptides) corresponds to newly synthesized mRNA. Thus, multiple levels of protein synthesis regulation seem to occur during zoospore germination, involving both transcriptional and translational controls. We also analyzed the pattern of protein synthesis during germination in a nutrient medium; synthesis of specific polypeptides occurred during late germination. During early germination posttranslational control was also observed, several labeled proteins from zoospores being specifically degraded or charge modified. Images PMID:3571161

Jatropha curcas Protein Concentrate Stimulates Insulin Signaling, Lipogenesis, Protein Synthesis and the PKCα Pathway in Rat Liver.

PubMed

León-López, Liliana; Márquez-Mota, Claudia C; Velázquez-Villegas, Laura A; Gálvez-Mariscal, Amanda; Arrieta-Báez, Daniel; Dávila-Ortiz, Gloria; Tovar, Armando R; Torres, Nimbe

2015-09-01

Jatropha curcas is an oil seed plant that belongs to the Euphorbiaceae family. Nontoxic genotypes have been reported in Mexico. The purpose of the present work was to evaluate the effect of a Mexican variety of J. curcas protein concentrate (JCP) on weight gain, biochemical parameters, and the expression of genes and proteins involved in insulin signaling, lipogenesis, cholesterol and protein synthesis in rats. The results demonstrated that short-term consumption of JCP increased serum glucose, insulin, triglycerides and cholesterol levels as well as the expression of transcription factors involved in lipogenesis and cholesterol synthesis (SREBP-1 and LXRα). Moreover, there was an increase in insulin signaling mediated by Akt phosphorylation and mTOR. JCP also increased PKCα protein abundance and the activation of downstream signaling pathway targets such as the AP1 and NF-κB transcription factors typically activated by phorbol esters. These results suggested that phorbol esters are present in JCP, and that they could be involved in the activation of PKC which may be responsible for the high insulin secretion and consequently the activation of insulin-dependent pathways. Our data suggest that this Mexican Jatropha variety contains toxic compounds that produce negative metabolic effects which require caution when using in the applications of Jatropha-based products in medicine and nutrition.

Amino acid starvation in Escherichia coli K-12: characteristics of the translation process.

PubMed Central

Subrahmanyam, C S; Das, H K

1976-01-01

Some characteristics of the translation process during amino acid starvation in Escherichia coli have been examined. Once starvation has been established, premature termination of polypeptides is negligible and complete proteins are formed. There is some preference for the synthesis of shorter proteins. The number of ribosomes involved in protein synthesis appears to decline to about half during amino acid-starvation. The assembly time of proteins during amino acid starvation is increased to only about fourfold, though protein synthesis maintained by turnover is reduced to 10%. To explain these observations, a model has been proposed for the course of events that possibly take place from the onset of starvation. PMID:780337

Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet – A pilot study

PubMed Central

Jourdan, Marion; Nair, K. Sreekumaran; Carter, Rickey E.; Schimke, Jill; Ford, G. Charles; Marc, Julie; Aussel, Christian; Cynober, Luc

2015-01-01

Background and Aims Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. Methods To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 hours. [ring-13C6] phenylalanine and [15N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. Results FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; p=0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. Conclusions Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake. PMID:24972455

Proteome Profile of Starch Granules Purified from Rice (Oryza sativa) Endosperm

PubMed Central

Xing, Shihai; Meng, Xiaoxi; Zhou, Lihui; Mujahid, Hana; Zhao, Chunfang; Zhang, Yadong; Wang, Cailin; Peng, Zhaohua

2016-01-01

Starch is the most important food energy source in cereals. Many of the known enzymes involved in starch biosynthesis are partially or entirely granule-associated in the endosperm. Studying the proteome of rice starch granules is critical for us to further understand the mechanisms underlying starch biosynthesis and packaging of starch granules in rice amyloplasts, consequently for the improvement of rice grain quality. In this article, we developed a protocol to purify starch granules from mature rice endosperm and verified the quality of purified starch granules by microscopy observations, I2 staining, and Western blot analyses. In addition, we found the phenol extraction method was superior to Tris-HCl buffer extraction method with respect to the efficiency in recovery of starch granule associated proteins. LC-MS/MS analysis showed identification of already known starch granule associated proteins with high confidence. Several proteins reported to be involved in starch synthesis in prior genetic studies in plants were also shown to be enriched with starch granules, either directly or indirectly, in our studies. In addition, our results suggested that a few additional candidate proteins may also be involved in starch synthesis. Furthermore, our results indicated that some starch synthesis pathway proteins are subject to protein acetylation modification. GO analysis and KEGG pathway enrichment analysis showed that the identified proteins were mainly located in plastids and involved in carbohydrate metabolism. This study substantially advances the understanding of the starch granule associated proteome in rice and post translational regulation of some starch granule associated proteins. PMID:27992503

[Molecular mechanisms of skeletal muscle hypertrophy].

PubMed

Astratenkova, I V; Rogozkin, V A

2014-06-01

Enzymes Akt, AMPK, mTOR, S6K and PGC-1a coactivator take part in skeletal muscles in the regulation of synthesis of proteins. The expression of these proteins is regulated by growth factors, hormones, nutrients, mechanical loading and leads to an increase in muscle mass and skeletal muscle hypertrophy. The review presents the results of studies published in the past four years, which expand knowledge on the effects of various factors on protein synthesis in skeletal muscle. The attention is focused on the achievements that reveal and clarify the signaling pathways involved in the regulation of protein synthesis in skeletal muscle. The central place is taken by mTOR enzyme which controls and regulates the main stages of the cascade of reactions of muscle proteins providing synthesis in the conditions of human life. coactivator PGC-1a.

Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis.

PubMed

Liu, Long; Tian, Jiao; Nan, Hao; Tian, Mengmeng; Li, Yuan; Xu, Xiaodong; Huang, Baicheng; Zhou, Enmin; Hiscox, Julian A; Chen, Hongying

2016-06-01

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

PubMed Central

Lizardi, Paul M.; Luck, David J. L.

1972-01-01

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes. PMID:4261038

Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells.

PubMed

Rivedal, Edgar; Leithe, Edward

2005-01-15

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces transient inhibition of gap junction intercellular communication (GJIC) in several cell types. The initial block in GJIC has been attributed to protein kinase C (PKC) mediated phosphorylation of connexin gap junction proteins, including connexin43 (Cx43). Restoration of GJIC, associated with normalization of the Cx43 phosphorylation status, has been ascribed to different events, including dephosphorylation of Cx43 and de novo synthesis of Cx43 or other, non-gap junctional, proteins. The data presented suggest that restoration of GJIC during continuous TPA exposure in normal and transformed rat liver epithelial cells is dependent on synthesis of Cx43 protein, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Reactivation of inactivated Cx43 by dephosphorylation does not appear to be involved in the recovery of GJIC. Both PKC and MAP kinase is involved in TPA-induced degradation of Cx43 and inhibition of GJIC. We show that coincubation of TPA with the protein synthesis inhibitor cycloheximide or the transcription inhibitor actinomycin D results in synergistic enhancement of the level of activated ERK1/2. Together, the present data highlight Cx43 degradation and synthesis as critical determinants in TPA-induced modifications of cell-cell communication via gap junctions.

A Proposed Model of Self-Generated Analogical Reasoning for the Concept of Translation in Protein Synthesis

ERIC Educational Resources Information Center

Salih, Maria

2008-01-01

This paper explored and described the analogical reasoning occurring in the minds of different science achievement groups for the concept of translation in protein synthesis. "What is the process of self-generated analogical reasoning?", "What types of matching was involved?" and "What are the consequences of the matching…

Oxygen Supplementation Improves Protein Milieu Supportive of Protein Synthesis and Antioxidant Function in the Cortex of Alzheimer's Disease Model Mice-a Quantitative Proteomic Study.

PubMed

Wang, Hao; Hong, Xiaoyu; Li, Shuiming; Wang, Yong

2017-10-01

Protein synthesis has been reported to be impaired in early-stage Alzheimer's disease (AD). Previously, we found that oxygen supplementation improved cognitive function and reduced mitochondrial damage in AD model mice. In the present study, we examined the effects of supplemental oxygen treatment on protein synthesis and oxidative damage. The synthesis of numerous proteins involved in mRNA splicing, transcription regulation, and translation was found to be significantly upregulated in cortex tissues of AD model mice given a supplemental oxygen treatment (OT group), relative to those of non-treated control AD model mice (Ctrl group), suggesting that impairment in protein synthesis may be alleviated by increased oxygen inhalation. Methionine oxidation and oxidation levels in general were similar between the OT and Ctrl groups, indicating that the oxygen supplementation treatment did not cause increases in peptide oxidation levels. On the contrary, the OT group exhibited upregulation of several proteins associated with antioxidant defense. These results support further exploration into the development of supplementary oxygen treatment as a potential therapy for AD.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2005-08-09

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2010-11-16

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

2009-07-14

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2006-10-31

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2007-08-28

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2007-07-03

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2010-11-02

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2007-05-15

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2007-02-27

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Shultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2007-04-03

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horst, M.N.

1990-12-01

Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated;more » a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine.« less

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella

PubMed Central

Solano, Cristina; García, Begoña; Latasa, Cristina; Toledo-Arana, Alejandro; Zorraquino, Violeta; Valle, Jaione; Casals, Joan; Pedroso, Enrique; Lasa, Iñigo

2009-01-01

Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3′-5′-cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di-GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEF-domain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP–dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable. PMID:19416883

Stimulation of skeletal muscle myofibrillar protein synthesis, p70 S6 kinase phosphorylation, and ribosomal protein S6 phosphorylation by inhibition of myostatin in mature mice.

PubMed

Welle, Stephen; Burgess, Kerri; Mehta, Sangeeta

2009-03-01

Knocking out myostatin activity during development increases the rate of muscle protein synthesis. The present study was done to determine whether postdevelopmental loss of myostatin activity stimulates myofibrillar protein synthesis and the phosphorylation of some of the proteins involved in regulation of protein synthesis rate. Myostatin activity was inhibited for 4 days, in 4- to 5-mo-old male mice, with injections of an anti-myostatin antibody (JA16). The mean myofibrillar synthesis rate increased 19% (P < 0.01) relative to the mean rate in saline-treated mice, as determined by incorporation of deuterium-labeled phenylalanine. JA16 increased phosphorylation of p70 S6 kinase (S6K) and ribosomal protein S6 (rpS6) 1.9-fold (P < 0.05). It did not affect phosphorylation of eukaryotic initiation factor 4E-binding protein-1 or Akt. Microarrays and real-time PCR analyses indicated that JA16 administration did not selectively enrich levels of mRNAs encoding myofibrillar proteins, ribosomal proteins, or translation initiation and elongation factors. Rapamycin treatment did not affect the rate of myofibrillar protein synthesis whether or not the mice received JA16 injections, although it eliminated the phosphorylation of S6K and rpS6. We conclude that the normal level of myostatin activity in mature muscle is sufficient to inhibit myofibrillar synthesis rate and phosphorylation of S6K and rpS6. Reversal of the inhibition of myofibrillar synthesis with an anti-myostatin antibody is not dependent on mTOR activation.

Skeletal-muscle growth and protein turnover.

PubMed Central

Millward, D J; Garlick, P J; Stewart, R J; Nnanyelugo, D O; Waterlow, J C

1975-01-01

Because of turnover, protein synthesis and breakdown can each be involved in the regulation of the growth of tissue protein. To investigate the regulation of skeletal-muscle-protein growth we measured rates of protein synthesis and breakdown in growing rats during development on a good diet, during development on a marginally low-protein diet and during rehabilitation on a good diet after a period of severe protein deficiency. Rates of protein synthesis were measured in vivo with a constant intravenous infusion of [14C]tyrosine. The growth rate of muscle protein was measured and the rate of breakdown calculated as breakdown rate=synthesis rate-growth rate. These measurements showed that during development on a good diet there was a fall with age in the rate of protein synthesis resulting from a fall in capacity (RNA concentration) and activity (synthesis rate per unit of RNA). There was a fall with age in the breakdown rate so that the rate was highest in the weaning rats, with a half-life of 3 days. There was a direct correlation between the fractional growth and breakdown rates. During rehabilitation on the good diet, rapid growth was also accompanied by high rates of protein breakdown. During growth on the inadequate diet protein synthesis rates were lesss than in controls, but growth occurred because of decreased rates of protein breakdown. This compression was not complete, however, since ultimate muscle size was only one-half that of controls. It is suggested that increased rates of protein breakdown are a necessary accompaniment to muscle growth and may result from the way in which myofibrils proliferate. PMID:1180916

The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimura, Akira; Kawahara, Nobuhiro; Takagi, Hiroshi, E-mail: hiro@bs.naist.jp

Highlights: Black-Right-Pointing-Pointer NO is produced from L-arginine in response to elevated temperature in yeast. Black-Right-Pointing-Pointer Tah18 was first identified as the yeast protein involved in NO synthesis. Black-Right-Pointing-Pointer Tah18-dependent NO synthesis confers tolerance to high-temperature on yeast cells. -- Abstract: Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. In the unicellular eukaryote yeast, NO may be involved in stress response pathways, but its role is poorly understood due to the lack of mammalian NO synthase (NOS) orthologues. Previously, we have proposed the oxidative stress-induced L-arginine synthesis and its physiologicalmore » role under stress conditions in yeast Saccharomyces cerevisiae. Here, our experimental results indicated that increased conversion of L-proline into L-arginine led to NO production in response to elevated temperature. We also showed that the flavoprotein Tah18, which was previously reported to transfer electrons to the Fe-S cluster protein Dre2, was involved in NO synthesis in yeast. Gene knockdown analysis demonstrated that Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. As it appears that such a unique cell protection mechanism is specific to yeasts and fungi, it represents a promising target for antifungal activity.« less

Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

PubMed Central

2011-01-01

Background Elucidation of molecular mechanism of silver nanoparticles (SNPs) biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution) of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro) and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro) SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver nanoparticles using C. reinhardtii as a model system. PMID:22152042

Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling*

PubMed Central

Liu, Rui; Kenney, Justin W.; Manousopoulou, Antigoni; Johnston, Harvey E.; Kamei, Makoto; Woelk, Christopher H.; Xie, Jianling; Schwarzer, Michael; Proud, Christopher G.

2016-01-01

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells. PMID:27512079

Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mTOR-independent process.

PubMed

Jeyapalan, Asumthia S; Orellana, Renan A; Suryawan, Agus; O'Connor, Pamela M J; Nguyen, Hanh V; Escobar, Jeffery; Frank, Jason W; Davis, Teresa A

2007-08-01

Skeletal muscle protein synthesis is elevated in neonates in part due to an enhanced response to the rise in insulin and amino acids after eating. In vitro studies suggest that glucose plays a role in protein synthesis regulation. To determine whether glucose, independently of insulin and amino acids, is involved in the postprandial rise in skeletal muscle protein synthesis, pancreatic-substrate clamps were performed in neonatal pigs. Insulin secretion was inhibited with somatostatin and insulin was infused to reproduce fasting or fed levels, while glucose and amino acids were clamped at fasting or fed levels. Fractional protein synthesis rates and translational control mechanisms were examined. Raising glucose alone increased protein synthesis in fast-twitch glycolytic muscles but not in other tissues. The response in muscle was associated with increased phosphorylation of protein kinase B (PKB) and enhanced formation of the active eIF4E.eIF4G complex but no change in phosphorylation of AMP-activated protein kinase (AMPK), tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mTOR), 4E-binding protein-1 (4E-BP1), ribosomal protein S6 kinase (S6K1), or eukaryotic elongation factor 2 (eEF2). Raising glucose, insulin, and amino acids increased protein synthesis in most tissues. The response in muscle was associated with phosphorylation of PKB, mTOR, S6K1, and 4E-BP1 and enhanced eIF4E.eIF4G formation. The results suggest that the postprandial rise in glucose, independently of insulin and amino acids, stimulates protein synthesis in neonates, and this response is specific to fast-twitch glycolytic muscle and occurs by AMPK- and mTOR-independent pathways.

No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

PubMed

Steinkellner, Hannes; Singh, Himanshu Narayan; Muckenthaler, Martina U; Goldenberg, Hans; Moganty, Rajeswari R; Scheiber-Mojdehkar, Barbara; Sturm, Brigitte

2017-07-20

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported. Copyright © 2017 Elsevier B.V. All rights reserved.

Mutation in the pssA gene involved in exopolysaccharide synthesis leads to several physiological and symbiotic defects in Rhizobium leguminosarum bv. trifolii.

PubMed

Janczarek, Monika; Rachwał, Kamila

2013-12-05

The symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii 24.2 secretes large amounts of acidic exopolysaccharide (EPS), which plays a crucial role in establishment of effective symbiosis with clover. The biosynthesis of this heteropolymer is conducted by a multi-enzymatic complex located in the bacterial inner membrane. PssA protein, responsible for the addition of glucose-1-phosphate to a polyprenyl phosphate carrier, is involved in the first step of EPS synthesis. In this work, we characterize R. leguminosarum bv. trifolii strain Rt270 containing a mini-Tn5 transposon insertion located in the 3'-end of the pssA gene. It has been established that a mutation in this gene causes a pleiotropic effect in rhizobial cells. This is confirmed by the phenotype of the mutant strain Rt270, which exhibits several physiological and symbiotic defects such as a deficiency in EPS synthesis, decreased motility and utilization of some nutrients, decreased sensitivity to several antibiotics, an altered extracellular protein profile, and failed host plant infection. The data of this study indicate that the protein product of the pssA gene is not only involved in EPS synthesis, but also required for proper functioning of Rhizobium leguminosarum bv. trifolii cells.

Insulinotropic and muscle protein synthetic effects of branched-chain amino acids: potential therapy for type 2 diabetes and sarcopenia.

PubMed

Manders, Ralph J; Little, Jonathan P; Forbes, Scott C; Candow, Darren G

2012-11-08

The loss of muscle mass and strength with aging (i.e., sarcopenia) has a negative effect on functional independence and overall quality of life. One main contributing factor to sarcopenia is the reduced ability to increase skeletal muscle protein synthesis in response to habitual feeding, possibly due to a reduction in postprandial insulin release and an increase in insulin resistance. Branched-chain amino acids (BCAA), primarily leucine, increases the activation of pathways involved in muscle protein synthesis through insulin-dependent and independent mechanisms, which may help counteract the "anabolic resistance" to feeding in older adults. Leucine exhibits strong insulinotropic characteristics, which may increase amino acid availability for muscle protein synthesis, reduce muscle protein breakdown, and enhance glucose disposal to help maintain blood glucose homeostasis.

Hypolipidemic effect of dietary pea proteins: Impact on genes regulating hepatic lipid metabolism.

PubMed

Rigamonti, Elena; Parolini, Cinzia; Marchesi, Marta; Diani, Erika; Brambilla, Stefano; Sirtori, Cesare R; Chiesa, Giulia

2010-05-01

Controversial data on the lipid-lowering effect of dietary pea proteins have been provided and the mechanisms behind this effect are not completely understood. The aim of the study was to evaluate a possible hypolipidemic activity of a pea protein isolate and to determine whether pea proteins could affect the hepatic lipid metabolism through regulation of genes involved in cholesterol and fatty acid homeostasis. Rats were fed Nath's hypercholesterolemic diets for 28 days, the protein sources being casein or a pea protein isolate from Pisum sativum. After 14 and 28 days of dietary treatment, rats fed pea proteins had markedly lower plasma cholesterol and triglyceride levels than rats fed casein (p<0.05). Pea protein-fed rats displayed higher hepatic mRNA levels of LDL receptor versus those fed casein (p<0.05). Hepatic mRNA concentration of genes involved in fatty acids synthesis, such as fatty acid synthase and stearoyl-CoA desaturase, was lower in pea protein-fed rats than in rats fed casein (p<0.05). In conclusion, the present study demonstrates a marked cholesterol and triglyceride-lowering activity of pea proteins in rats. Moreover, pea proteins appear to affect cellular lipid homeostasis by upregulating genes involved in hepatic cholesterol uptake and by downregulating fatty acid synthesis genes.

Proteomic and enzymatic response under Cr(VI) overload in yeast isolated from textile-dye industry effluent.

PubMed

Irazusta, Verónica; Bernal, Anahí Romina; Estévez, María Cristina; de Figueroa, Lucía I C

2018-02-01

Cyberlindnera jadinii M9 and Wickerhamomyces anomalus M10 isolated from textile-dye liquid effluents has shown capacity for chromium detoxification via Cr(VI) biological reduction. The aim of the study was to evaluate the effect of hexavalent chromium on synthesis of novel and/or specific proteins involved in chromium tolerance and reduction in response to chromium overload in two indigenous yeasts. A study was carried out following a proteomic approach with W. anomalus M10 and Cy. jadinii M9 strains. For this, proteins extracts belonging to total cell extracts, membranes and mitochondria were analyzed. When Cr(VI) was added to culture medium there was an over-synthesis of 39 proteins involved in different metabolic pathways. In both strains, chromium supplementation changed protein biosynthesis by upregulating proteins involved in stress response, methionine metabolism, energy production, protein degradation and novel oxide-reductase enzymes. Moreover, we observed that Cy. jadinii M9 and W. anomalus M10 displayed ability to activate superoxide dismutase, catalase and chromate reductase activity. Two enzymes from the total cell extracts, type II nitroreductase (Frm2) and flavoprotein wrbA (Ycp4), were identified as possibly responsible for inducing crude chromate-reductase activity in cytoplasm of W. anomalus M10 under chromium overload. In Cy.jadinii M9, mitochondrial Ferredoxine-NADP reductase (Yah1) and membrane FAD flavoprotein (Lpd1) were identified as probably involved in Cr(VI) reduction. To our knowledge, this is the first study proposing chromate reductase activity of these four enzymes in yeast and reporting a relationship between protein synthesis, enzymatic response and chromium biospeciation in Cy. jadinii and W. anomalus. Copyright © 2017 Elsevier Inc. All rights reserved.

Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

PubMed Central

Nakagawa, K.; Lokugamage, K.G.; Makino, S.

2017-01-01

Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

Experimental studies related to the origin of the genetic code and the process of protein synthesis - A review

NASA Technical Reports Server (NTRS)

Lacey, J. C., Jr.; Mullins, D. W., Jr.

1983-01-01

A survey is presented of the literature on the experimental evidence for the genetic code assignments and the chemical reactions involved in the process of protein synthesis. In view of the enormous number of theoretical models that have been advanced to explain the origin of the genetic code, attention is confined to experimental studies. Since genetic coding has significance only within the context of protein synthesis, it is believed that the problem of the origin of the code must be dealt with in terms of the origin of the process of protein synthesis. It is contended that the answers must lie in the nature of the molecules, amino acids and nucleotides, the affinities they might have for one another, and the effect that those affinities must have on the chemical reactions that are related to primitive protein synthesis. The survey establishes that for the bulk of amino acids, there is a direct and significant correlation between the hydrophobicity rank of the amino acids and the hydrophobicity rank of their anticodonic dinucleotides.

SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation

PubMed Central

Matsuda, Morihiro; Korn, Bobby S.; Hammer, Robert E.; Moon, Young-Ah; Komuro, Ryutaro; Horton, Jay D.; Goldstein, Joseph L.; Brown, Michael S.; Shimomura, Iichiro

2001-01-01

In liver, the synthesis of cholesterol and fatty acids increases in response to cholesterol deprivation and insulin elevation, respectively. This regulatory mechanism underlies the adaptation to cholesterol synthesis inhibitors (statins) and high calorie diets (insulin). In nonhepatic cells, lipid synthesis is controlled by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose active domains are released proteolytically to enter the nucleus and activate genes involved in the synthesis and uptake of cholesterol and fatty acids. SCAP (SREBP cleavage-activating protein) is a sterol-regulated escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of cleavage in the Golgi. Here, we produced a conditional deficiency of SCAP in mouse liver by genomic recombination mediated by inducible Cre recombinase. SCAP-deficient mice showed an 80% reduction in basal rates of cholesterol and fatty acid synthesis in liver, owing to decreases in mRNAs encoding multiple biosynthetic enzymes. Moreover, these mRNAs failed to increase normally in response to cholesterol deprivation produced by a cholesterol synthesis inhibitor and to insulin elevation produced by a fasting–refeeding protocol. These data provide in vivo evidence that SCAP and the SREBPs are required for hepatic lipid synthesis under basal and adaptive conditions. PMID:11358865

Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. II. Mitochondrial protein synthesis.

PubMed

Heude, M; Chanet, R

1975-04-01

The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) anc chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the (see article) genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) process for the (see article) induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressed or derepressed state of the mitochondria.

Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.

PubMed

Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

2016-08-01

The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

Identification of proteins associated with polyhydroxybutyrate granules from Herbaspirillum seropedicae SmR1--old partners, new players.

PubMed

Tirapelle, Evandro F; Müller-Santos, Marcelo; Tadra-Sfeir, Michelle Z; Kadowaki, Marco A S; Steffens, Maria B R; Monteiro, Rose A; Souza, Emanuel M; Pedrosa, Fabio O; Chubatsu, Leda S

2013-01-01

Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae.

Identification of Proteins Associated with Polyhydroxybutyrate Granules from Herbaspirillum seropedicae SmR1 - Old Partners, New Players

PubMed Central

Tirapelle, Evandro F.; Müller-Santos, Marcelo; Tadra-Sfeir, Michelle Z.; Kadowaki, Marco A. S.; Steffens, Maria B. R.; Monteiro, Rose A.; Souza, Emanuel M.; Pedrosa, Fabio O.; Chubatsu, Leda S.

2013-01-01

Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae. PMID:24086439

Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

PubMed

Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

2015-09-15

Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1. Copyright © 2015 the American Physiological Society.

Metaproteomics Provides Functional Insight into Activated Sludge Wastewater Treatment

PubMed Central

Wilmes, Paul; Wexler, Margaret; Bond, Philip L.

2008-01-01

Background Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR). Methodology/Principal Findings A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism “Candidatus Accumulibacter phosphatis”. When EBPR failed, the sludge was dominated by tetrad-forming α-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from “Candidatus Accumulibacter phosphatis” and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid β oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected. Conclusions/Significance Importantly, this study provides direct evidence linking the metabolic activities of “Accumulibacter” to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models. PMID:18392150

Analysis of soybean root proteins affected by gibberellic acid treatment under flooding stress.

PubMed

Oh, Myeong Won; Nanjo, Yohei; Komatsu, Setsuko

2014-01-01

Flooding is a serious abiotic stress for soybean because it restricts growth and reduces grain yields. To investigate the effect of gibberellic acid (GA) on soybean under flooding stress, root proteins were analyzed using a gel-free proteomic technique. Proteins were extracted from the roots of 4-days-old soybean seedlings exposed to flooding stress in the presence and absence of exogenous GA3 for 2 days. A total of 307, 324, and 250 proteins were identified from untreated, and flooding-treated soybean seedlings without or with GA3, respectively. Secondary metabolism- and cell-related proteins, and proteins involved in protein degradation/synthesis were decreased by flooding stress; however, the levels of these proteins were restored by GA3 supplementation under flooding. Fermentation- and cell wall-related proteins were not affected by GA3 supplementation. Furthermore, putative GA-responsive proteins, which were identified by the presence of a GA-responsive element in the promoter region, were less abundant by flooding stress; however, these proteins were more abundant by GA3 supplementation under flooding. Taken together, these results suggest that GA3 affects the abundance of proteins involved in secondary metabolism, cell cycle, and protein degradation/synthesis in soybeans under flooding stress.

Regulation of Staphylococcal Penicillinase Synthesis

PubMed Central

Imsande, John; Zyskind, Judith W.; Mile, Imre

1972-01-01

5-Methyl tryptophan was found to be an efficient inducer of penicillinase synthesis in Staphylococcus aureus. Addition of actinomycin D or tryptophan to the culture medium shuts off the 5-methyl tryptophan-induced synthesis of penicillinase with an apparent half-life of approximately 1 to 2 min, respectively. Hence, in the induction of penicillinase synthesis, 5-methyl tryptophan seems to function as a structural analogue of penicillin rather than by becoming incorporated in proteins and thereby creating faulty penicillinase repressor or antirepressor. This conclusion is supported by similarities in the structures of the two compounds as revealed by solid atomic models. The fact that S. aureus exposed to 14C-penicillin in the absence of protein synthesis failed to synthesize penicillinase at an increased level when cell growth was resumed strongly suggests that a protein involved in the regulation of penicillinase synthesis must be synthesized in the presence of the penicillinase inducer. In turn, this observation suggests that the penicillinase inducer promotes penicillinase synthesis by directing the penicillinase regulatory protein (i.e., the penicillinase antirepressor) to acquire a different conformation when it is synthesized in the presence of the penicillinase inducer. A working model for the regulation of penicillinase synthesis based on these and other data has been constructed and is presented. Images PMID:4333374

Mechanism of Protein Biosynthesis in Mammalian Mitochondria

PubMed Central

Christian, Brooke E.; Spremulli, Linda L.

2011-01-01

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. PMID:22172991

ROLE OF NEUROTRANSMITTERS AND PROTEIN SYNTHESIS IN SHORT- AND LONG-TERM MEMORY

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bennett, E.L.; Rosenzweig, M.R.; Flood, J.F.

1978-10-01

Anisomycin is an effective inhibitor of cerebral protein synthesis in mice and is also an effective amnestic agent for both passive and active behavioral tasks. From use of anisomycin in combination with a variety of stimulant and depressant drugs, we conclude that the level of arousal following acquisition plays an important role in determining the duration and the rate of the biosynthetic phase of memory formation. While we have interpreted the experiments with anisomycin as evidence for an essential role of protein in memory storage, others have suggested that side effects of inhibitors of protein synthesis on catecholamine metabolism aremore » the main cause of amnesia. Several experiments were therefore done to compare the effects of anisemycin and catecholamine inhibitors on memory. We conclude that anisomycin's principal amnestic mechanism does not involve inhibition of the catecholamine system. The results strengthen our conclusion that protein synthesis is an essential component for longterm memory trace formation. Also, it is suggested that proteins synthesized in the neuronal cell body are used, in conjunction with other molecules, to produce permanent and semi-permanent anatomical changes.« less

Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis.

PubMed

Tudor, Jennifer C; Davis, Emily J; Peixoto, Lucia; Wimmer, Mathieu E; van Tilborg, Erik; Park, Alan J; Poplawski, Shane G; Chung, Caroline W; Havekes, Robbert; Huang, Jiayan; Gatti, Evelina; Pierre, Philippe; Abel, Ted

2016-04-26

Sleep deprivation is a public health epidemic that causes wide-ranging deleterious consequences, including impaired memory and cognition. Protein synthesis in hippocampal neurons promotes memory and cognition. The kinase complex mammalian target of rapamycin complex 1 (mTORC1) stimulates protein synthesis by phosphorylating and inhibiting the eukaryotic translation initiation factor 4E-binding protein 2 (4EBP2). We investigated the involvement of the mTORC1-4EBP2 axis in the molecular mechanisms mediating the cognitive deficits caused by sleep deprivation in mice. Using an in vivo protein translation assay, we found that loss of sleep impaired protein synthesis in the hippocampus. Five hours of sleep loss attenuated both mTORC1-mediated phosphorylation of 4EBP2 and the interaction between eukaryotic initiation factor 4E (eIF4E) and eIF4G in the hippocampi of sleep-deprived mice. Increasing the abundance of 4EBP2 in hippocampal excitatory neurons before sleep deprivation increased the abundance of phosphorylated 4EBP2, restored the amount of eIF4E-eIF4G interaction and hippocampal protein synthesis to that seen in mice that were not sleep-deprived, and prevented the hippocampus-dependent memory deficits associated with sleep loss. These findings collectively demonstrate that 4EBP2-regulated protein synthesis is a critical mediator of the memory deficits caused by sleep deprivation. Copyright © 2016, American Association for the Advancement of Science.

L-Phenylalanine catabolism and 2-phenylethanol synthesis in Yarrowia lipolytica--mapping molecular identities through whole-proteome quantitative mass spectrometry analysis.

PubMed

Celińska, Ewelina; Olkowicz, Mariola; Grajek, Włodzimierz

2015-08-01

A world-wide effort is now being pursued towards the development of flavors and fragrances (F&F) production independently from traditional sources, as well as autonomously from depleting fossil fuel supplies. Biotechnological production of F&F by microbes has emerged as a vivid solution to the current market limitations. Amongst a wide variety of fragrant chemicals, 2-PE is of significant interest to both scientific and industrial community. Although the general overview of the 2-PE synthesis pathway is commonly known, involvement of particular molecular identities in this pathway has not been elucidated in Yarrowia lipolytica to date. The aim of this study was mapping molecular identities involved in 2-PE synthesis in Y. lipolytica. To acquire a comprehensive landscape of the proteins that are directly and indirectly involved in L-Phe degradation and 2-PE synthesis, we took advantage of comprehensibility and sensitivity of high-throughput LC-MS/MS-quantitative analysis. Amongst a number of proteins involved in amino acid turnover and the central carbon metabolism, enzymes involved in L-Phe conversion to 2-PE have been identified. Results on yeast-to-hyphae transition in relation to the character of the provided nitrogen source have been presented. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Anaphase Promoting Complex Is Required for Memory Function in Mice

ERIC Educational Resources Information Center

Kuczera, Tanja; Stilling, Roman Manuel; Hsia, Hung-En; Bahari-Javan, Sanaz; Irniger, Stefan; Nasmyth, Kim; Sananbenesi, Farahnaz; Fischer, Andre

2011-01-01

Learning and memory processes critically involve the orchestrated regulation of de novo protein synthesis. On the other hand it has become clear that regulated protein degradation also plays a major role in neuronal plasticity and learning behavior. One of the key pathways mediating protein degradation is proteosomal protein destruction. The…

Identification of the Mitochondrial Heme Metabolism Complex

PubMed Central

Medlock, Amy E.; Shiferaw, Mesafint T.; Marcero, Jason R.; Vashisht, Ajay A.; Wohlschlegel, James A.; Phillips, John D.; Dailey, Harry A.

2015-01-01

Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex. PMID:26287972

Regulation of myofibrillar accumulation in chick muscle cultures - Evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile proteins

NASA Technical Reports Server (NTRS)

Silver, Geri; Etlinger, Joseph D.

1985-01-01

The effects of calcium on the synthesis and the degradation of individual myofibrillar proteins were investigated using primary chick-leg skeletal muscle cultures labeled with S-35-methionine (for protein accumulation experiments) or Ca(2+)-45 (for calcium efflux experiments). It was found that the turnover of individual contractile proteins is regulated nonuniformly by a calcium-dependent mechanism involving lysosomes. The results also indicate that contractile proteins are released from the myofibril before their breakdown to amino acids.

Disrupting the Acyl Carrier Protein/SpoT Interaction In Vivo: Identification of ACP Residues Involved in the Interaction and Consequence on Growth

PubMed Central

Angelini, Sandra; My, Laetitia; Bouveret, Emmanuelle

2012-01-01

In bacteria, Acyl Carrier Protein (ACP) is the central cofactor for fatty acid biosynthesis. It carries the acyl chain in elongation and must therefore interact successively with all the enzymes of this pathway. Yet, ACP also interacts with proteins of diverse unrelated function. Among them, the interaction with SpoT has been proposed to be involved in regulating ppGpp levels in the cell in response to fatty acid synthesis inhibition. In order to better understand this mechanism, we screened for ACP mutants unable to interact with SpoT in vivo by bacterial two-hybrid, but still functional for fatty acid synthesis. The position of the selected mutations indicated that the helix II of ACP is responsible for the interaction with SpoT. This suggested a mechanism of recognition similar to one used for the enzymes of fatty acid synthesis. Consistently, the interactions tested by bacterial two-hybrid of ACP with fatty acid synthesis enzymes were also affected by the mutations that prevented the interaction with SpoT. Yet, interestingly, the corresponding mutant strains were viable, and the phenotypes of one mutant suggested a defect in growth regulation. PMID:22558350

The chemical basis for the origin of the genetic code and the process of protein synthesis

NASA Technical Reports Server (NTRS)

1981-01-01

The principles upon which the process of protein synthesis and the genetic code were established are elucidated. Extensive work on nuclear magnetic resonance studies of both monomermonomer and monoamino acid polynucleotide interactions is included. A new method of general utility for studying any amino acid interacting with any polynucleotide was developed. This system involves the use of methyl esters of amino acids interacting with polynucleotides.

Induction of heat-shock response and alterations of protein phosphorylation by a novel topoisomerase II inhibitor, withangulatin A, in 9L rat brain tumor cells.

PubMed

Lee, W C; Lin, K Y; Chen, C M; Chen, Z T; Liu, H J; Lai, Y K

1991-10-01

Withangulatin A is a newly identified in vitro topoisomerase II inhibitor isolated from the Chinese antitumor herb Physalis angulata. In vivo, it was found to be cytotoxic, capable of suppressing general protein synthesis and of inducing the synthesis of a small set of proteins including those generated by heat-shock treatment. The 70 kDa protein generated by withangulatin A was unequivocally identified as the heat-shock protein 70 (HSP70) since both proteins migrated to the same position on two-dimensional polyacrylamide gels, could be recognized by a monoclonal antibody to human HSP70, and exhibited identical peptide maps. The induction of protein synthesis by withangulatin A was regulated at the transcriptional level since it was aborted in cells pre-treated with actinomycin D. However, the initiation of this process did not require de novo protein synthesis since it was not affected by cycloheximide. Other cellular effect of withangulatin A was alterations of protein phosphorylation including an enhancement of phosphorylation of a 65 kDa protein which was also detected in the heat-shocked cells. Moreover, this process was observed within 7.5 min after the initial heat treatment which is much faster than the onset of HSP synthesis. Therefore, increased phosphorylation of the 65 kDa protein may represent one of the earliest signals generated by both heat-shock and withangluatin A and may be involved in the upstream regulation of heat-shock response in cells.

Evolution of Protein Synthesis from an RNA World

PubMed Central

Noller, Harry F.

2012-01-01

SUMMARY Because of the molecular complexity of the ribosome and protein synthesis, it is a challenge to imagine how translation could have evolved from a primitive RNA World. Two specific suggestions are made here to help to address this, involving separate evolution of the peptidyl transferase and decoding functions. First, it is proposed that translation originally arose not to synthesize functional proteins, but to provide simple (perhaps random) peptides that bound to RNA, increasing its available structure space, and therefore its functional capabilities. Second, it is proposed that the decoding site of the ribosome evolved from a mechanism for duplication of RNA. This process involved homodimeric “duplicator RNAs,” resembling the anticodon arms of tRNAs, which directed ligation of trinucleotides in response to an RNA template. PMID:20610545

NUCLEAR ENVELOPE-ASSOCIATED RESUMPTION OF RNA SYNTHESIS IN LATE MITOSIS OF HELA CELLS

PubMed Central

Simmons, T.; Heywood, P.; Hodge, L.

1973-01-01

The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G1) has been investigated in synchronized HeLa S3 cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G1 was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope. PMID:4752403

Erythrophagocytosis induces heat shock protein synthesis by human monocytes-macrophages.

PubMed

Clerget, M; Polla, B S

1990-02-01

Exposure of cells to elevated temperatures and other environmental stresses results in the expression of specific genes encoding the so-called heat shock proteins (HSPs). Since exogenous H2O2 induces in human monocytes the synthesis of HSPs, and previous induction of HSPs protects these cells from oxidative injury, we investigated whether HSP synthesis was also induced during generation of reactive oxygen species by the phagocyte itself during phagocytosis. As a model system, we analyzed the effects of erythrophagocytosis on protein synthesis by the human premonocytic line U937, in which phagocytosis is induced during differentiation with 1,25-dihydroxyvitamin D3. Exposure to whole erythrocytes, but not to erythrocyte ghosts, induced in the phagocytic cells only the synthesis of the 70- and 83- to 90-kDa HSPs and a 32-kDa oxidation-related stress protein identical by partial peptide mapping to heme oxygenase. The radioprotective aminothiol N-(2'-mercaptoethyl)-1,3-propanediamine (WR-1065), which can substitute for glutathione as hydrogen donor, prevented this induction. These results suggest that oxygen free radicals generated in the presence of hemoglobin-derived iron and consecutive glutathione depletion are involved in induction of stress protein synthesis during erythrophagocytosis. HSPs synthesized during phagocytosis may play a role in the phagocyte's defense mechanisms and in protective immunity.

Interactions between late acting proteins required for peptidoglycan synthesis during sporulation

PubMed Central

Fay, Allison; Meyer, Pablo; Dworkin, Jonathan

2010-01-01

The requirement of peptidoglycan synthesis for growth complicates the analysis of interactions between proteins involved in this pathway. In particular, the later steps that involve membrane-linked substrates have proven largely recalcitrant to in vivo analysis. Here we have taken advantage of the peptidoglycan synthesis that occurs during sporulation in Bacillus subtilis to examine the interactions between SpoVE, a non-essential, sporulation-specific homolog of the well-conserved and essential SEDS proteins, and SpoVD, a non-essential class B penicillin binding protein (PBP). We found that localization of SpoVD is dependent on SpoVE and that SpoVD protects SpoVE from in vivo proteolysis. Co-immunoprecipitations and Fluorescence Resonance Energy Transfer experiments indicated that SpoVE and SpoVD interact and co-affinity purification in E. coli demonstrated that this interaction is direct. Finally, we generated a functional protein consisting of a SpoVE-SpoVD fusion and found that a loss-of-function point mutation in either part of the fusion resulted in a loss of function of the entire fusion that was not complemented by a wild type protein. Thus, SpoVE has a direct and functional interaction with SpoVD and this conclusion will facilitate understanding the essential function SpoVE and related SEDS proteins such as FtsW and RodA play in bacterial growth and division. PMID:20417640

Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents.

PubMed

Manfredi, L H; Paula-Gomes, S; Zanon, N M; Kettelhut, I C

2017-10-19

Myostatin is a novel negative regulator of skeletal muscle mass. Myostatin expression is also found in heart in a much less extent, but it can be upregulated in pathological conditions, such as heart failure. Myostatin may be involved in inhibiting protein synthesis and/or increasing protein degradation in skeletal and cardiac muscles. Herein, we used cell cultures and isolated muscles from rats to determine protein degradation and synthesis. Muscles incubated with myostatin exhibited an increase in proteolysis with an increase of Atrogin-1, MuRF1 and LC3 genes. Extensor digitorum longus muscles and C2C12 myotubes exhibited a reduction in protein turnover. Cardiomyocytes showed an increase in proteolysis by activating autophagy and the ubiquitin proteasome system, and a decrease in protein synthesis by decreasing P70S6K. The effect of myostatin on protein metabolism is related to fiber type composition, which may be associated to the extent of atrophy mediated effect of myostatin on muscle.

Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents

PubMed Central

Manfredi, L.H.; Paula-Gomes, S.; Zanon, N.M.; Kettelhut, I.C.

2017-01-01

Myostatin is a novel negative regulator of skeletal muscle mass. Myostatin expression is also found in heart in a much less extent, but it can be upregulated in pathological conditions, such as heart failure. Myostatin may be involved in inhibiting protein synthesis and/or increasing protein degradation in skeletal and cardiac muscles. Herein, we used cell cultures and isolated muscles from rats to determine protein degradation and synthesis. Muscles incubated with myostatin exhibited an increase in proteolysis with an increase of Atrogin-1, MuRF1 and LC3 genes. Extensor digitorum longus muscles and C2C12 myotubes exhibited a reduction in protein turnover. Cardiomyocytes showed an increase in proteolysis by activating autophagy and the ubiquitin proteasome system, and a decrease in protein synthesis by decreasing P70S6K. The effect of myostatin on protein metabolism is related to fiber type composition, which may be associated to the extent of atrophy mediated effect of myostatin on muscle. PMID:29069231

Protein-protein interactions among enzymes of starch biosynthesis in high-amylose barley genotypes reveal differential roles of heteromeric enzyme complexes in the synthesis of A and B granules.

PubMed

Ahmed, Zaheer; Tetlow, Ian J; Ahmed, Regina; Morell, Matthew K; Emes, Michael J

2015-04-01

The present study investigated the role of protein phosphorylation, and protein complex formation between key enzymes of amylopectin synthesis, in barley genotypes exhibiting "high amylose" phenotypes. Starch branching enzyme (SBE) down-regulated lines (ΔSBEIIa and ΔSBEIIb), starch synthase (SS)IIa (ssiia(-), sex6) and SSIII (ssiii(-), amo1) mutants were compared to a reference genotype, OAC Baxter. Down-regulation of either SBEIIa or IIb caused pleiotropic effects on SSI and starch phosphorylase (SP) and resulted in formation of novel protein complexes in which the missing SBEII isoform was substituted by SBEI and SP. In the ΔSBEIIb down-regulated line, soluble SP activity was undetectable. Nonetheless, SP was incorporated into a heteromeric protein complex with SBEI and SBEIIa and was readily detected in starch granules. In amo1, unlike other mutants, the data suggest that both SBEIIa and SBEIIb are in a protein complex with SSI and SSIIa. In the sex6 mutant no protein complexes involving SBEIIa or SBEIIb were detected in amyloplasts. Studies with Pro-Q Diamond revealed that GBSS, SSI, SSIIa, SBEIIb and SP are phosphorylated in their granule bound state. Alteration in the granule proteome in ΔSBEIIa and ΔSBEIIb lines, suggests that different protein complexes are involved in the synthesis of A and B granules. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates.

PubMed

Zitterbart, Robert; Krumrey, Michael; Seitz, Oliver

2017-07-01

The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2 + 3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

RomA, A Periplasmic Protein Involved in the Synthesis of the Lipopolysaccharide, Tunes Down the Inflammatory Response Triggered by Brucella.

PubMed

Valguarnera, Ezequiel; Spera, Juan M; Czibener, Cecilia; Fulgenzi, Fabiana R; Casabuono, Adriana C; Altabe, Silvia G; Pasquevich, Karina A; Guaimas, Francisco; Cassataro, Juliana; Couto, Alicia S; Ugalde, Juan E

2018-03-28

Brucellaceae are stealthy pathogens with the ability to survive and replicate in the host in the context of a strong immune response. This capacity relies on several virulence factors that are able to modulate the immune system and in their structural components that have low proinflammatory activities. Lipopolysaccharide (LPS), the main component of the outer membrane, is a central virulence factor of Brucella, and it has been well established that it induces a low inflammatory response. We describe here the identification and characterization of a novel periplasmic protein (RomA) conserved in alpha-proteobacteria, which is involved in the homeostasis of the outer membrane. A mutant in this gene showed several phenotypes, such as membrane defects, altered LPS composition, reduced adhesion, and increased virulence and inflammation. We show that RomA is involved in the synthesis of LPS, probably coordinating part of the biosynthetic complex in the periplasm. Its absence alters the normal synthesis of this macromolecule and affects the homeostasis of the outer membrane, resulting in a strain with a hyperinflammatory phenotype. Our results suggest that the proper synthesis of LPS is central to maximize virulence and minimize inflammation.

Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

PubMed

Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

2018-01-01

DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

Protein synthesis in sperm: dialog between mitochondria and cytoplasm.

PubMed

Gur, Yael; Breitbart, Haim

2008-01-30

Ejaculated sperm are capable of using mRNAs transcripts for protein translation during the final maturation steps before fertilization. In a capacitation-dependent process, nuclear-encoded mRNAs are translated by mitochondrial-type ribosomes while the cytoplasmic translation machinery is not involved. Our findings suggest that new proteins are synthesized to replace degraded proteins while swimming and waiting in the female reproductive tract before fertilization, or produced due to the specific needs of the capacitating spermatozoa. In addition, a growing number of articles have reported evidence for the correlation of nuclear-encoded mRNA and protein synthesis in somatic mitochondria. It is known that all of the proteins necessary for the replication, transcription and translation of the genes encoded in mtDNA are now encoded in the nuclear genome. This genetic investment is far out of proportion to the number of proteins involved, as there have been multiple movements and duplications of genes. However, the evolutionary retention (or secondary uptake) of the mitochondrial machinery for translation of nuclear-encoded mRNAs may shed light on this paradox.

Nucleolin promotes in vitro translation of feline calicivirus genomic RNA.

PubMed

Hernández, Beatriz Alvarado; Sandoval-Jaime, Carlos; Sosnovtsev, Stanislav V; Green, Kim Y; Gutiérrez-Escolano, Ana Lorena

2016-02-01

Feline calicivirus depends on host-cell proteins for its replication. We previously showed that knockdown of nucleolin (NCL), a phosphoprotein involved in ribosome biogenesis, resulted in the reduction of FCV protein synthesis and virus yield. Here, we found that NCL may not be involved in FCV binding and entry into cells, but it binds to both ends of the FCV genomic RNA, and stimulates its translation in vitro. AGRO100, an aptamer that specifically binds and inactivates NCL, caused a strong reduction in FCV protein synthesis. This effect could be reversed by the addition of full-length NCL but not by a ΔrNCL, lacking the N-terminal domain. Consistent with this, FCV infection of CrFK cells stably expressing ΔrNCL led to a reduction in virus protein translation. These results suggest that NCL is part of the FCV RNA translational complex, and that the N-terminal part of the protein is required for efficient FCV replication. Copyright © 2015 Elsevier Inc. All rights reserved.

Nicotine shifts the temporal activation of hippocampal protein kinase A and extracellular signal-regulated kinase 1/2 to enhance long-term, but not short-term, hippocampus-dependent memory.

PubMed

Gould, Thomas J; Wilkinson, Derek S; Yildirim, Emre; Poole, Rachel L F; Leach, Prescott T; Simmons, Steven J

2014-03-01

Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 h but not 2 h post-training, delineating time points for STM (2 h) and LTM (4 h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 h but not 2 h post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning, and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories. Copyright © 2014 Elsevier Inc. All rights reserved.

Nicotine Shifts the Temporal Activation of Hippocampal Protein Kinase A and Extracellular Signal-Regulated Kinase 1/2 to Enhance Long-Term, but not Short-term, Hippocampus-Dependent Memory

PubMed Central

Gould, Thomas J.; Wilkinson, Derek S.; Yildirim, Emre; Poole, Rachel L. F.; Leach, Prescott T.; Simmons, Steven J.

2014-01-01

Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular regulated signaling kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 hours but not 2 hours post-training, delineating time points for STM (2 hours) and LTM (4 hours and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 hours but not 2 hours post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories. PMID:24457151

Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

PubMed

Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

2016-03-01

T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278.

In vivo synthesis of nano-selenium by Tetrahymena thermophila SB210.

PubMed

Cui, Yin-Hua; Li, Ling-Li; Zhou, Nan-Qing; Liu, Jing-Hua; Huang, Qing; Wang, Hui-Juan; Tian, Jie; Yu, Han-Qing

2016-12-01

Nano-selenium has a great potential to be used in chemical, biological, medical and environmental fields. Biological methods for nano-selenium synthesis have attracted wide interests, because they can be operated at ambient temperature and pressure without complicated equipments. In this work, a protozoa, Tetrahymena thermophila (T. thermophila) SB210, was used to in vivo synthesize nano-selenium. The biosynthesized nano-selenium was characterized using transmission electron microscopy, energy dispersive X-ray spectroscopy and Raman spectroscopy. The synthesized amorphous spherical selenium nanoparticles had diameters of 50-500nm with the coexistence of irregular nano-selenium. The expressions of glutathione (GSH) synthesis related gene glutathione synthase, cysteine-rich protein metallothionein related gene metallothionein-1 and [2Fe-2S] cluster-binding protein related gene were up-regulated in the nano-selenium producing group. Also, the subsequent GSH detection and in vitro synthesis experimental results suggest the three proteins were likely to be involved in the nano-selenium synthesis process. Copyright © 2016 Elsevier Inc. All rights reserved.

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB

PubMed Central

Fiume, Giuseppe; Rossi, Annalisa; de Laurentiis, Annamaria; Falcone, Cristina; Pisano, Antonio; Vecchio, Eleonora; Pontoriero, Marilena; Scala, Iris; Scialdone, Annarita; Masci, Francesca Fasanella; Mimmi, Selena; Palmieri, Camillo; Scala, Giuseppe; Quinto, Ileana

2013-01-01

Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control. PMID:23776612

How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction.

PubMed

Hameed, Dharjath S; Sapmaz, Aysegul; Ovaa, Huib

2017-03-15

Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.

A dual mechanism of cellulose deficiency in shv3svl1

PubMed Central

Yeats, Trevor H.; Somerville, Chris R.

2016-01-01

ABSTRACT SHAVEN3 (SHV3) and its homolog SHAVEN3-like 1 (SVL1) encode glycosylphosphatidylinositol (GPI)-anchored proteins (GAPs) that are involved in cellulose biosynthesis and hypocotyl elongation in Arabidopsis thaliana. In a recent report, we showed that the cellulose and hypocotyl elongation defects of the shv3svl1 double mutant are greatly enhanced by exogenous sucrose in the growth medium. Further investigation of this phenomenon showed that shv3svl1 exhibits a hyperpolarized plasma membrane (PM) proton gradient that is coupled with enhanced accumulation of sucrose via the PM sucrose/proton symporter SUC1. The resulting high intracellular sucrose concentration appears to favor starch synthesis at the expense of cellulose synthesis. Here, we describe our interpretation of these results in terms of 2 potential regulators of cellulose synthesis: intracellular sucrose concentration and a putative signaling pathway that involves SHV3-like proteins. PMID:27494413

Protein synthesis and its regulation: a background study related to the biological effects of radiation. Progress report, July 1, 1976--August 31, 1977

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zamecnik, P.C.

Results of studies are reported on delineation of the steps involved in protein synthesis and the role of transfer and messenger RNAs in the translation process. During the past year we have studied the mechanisms by which an oncogenic RNA virus modifies the growth process, and have begun to elucidate the role a novel dinucleotide, discovered in these laboratories, plays in rapidly growing cells in tissue culture.

Activity-dependent rapid local RhoA synthesis is required for hippocampal synaptic plasticity.

PubMed

Briz, Victor; Zhu, Guoqi; Wang, Yubin; Liu, Yan; Avetisyan, Mariam; Bi, Xiaoning; Baudry, Michel

2015-02-04

Dendritic protein synthesis and actin cytoskeleton reorganization are important events required for the consolidation of hippocampal LTP and memory. However, the temporal and spatial relationships between these two processes remain unclear. Here, we report that treatment of adult rat hippocampal slices with BDNF or with tetraethylammonium (TEA), which induces a chemical form of LTP, produces a rapid and transient increase in RhoA protein levels. Changes in RhoA were restricted to dendritic spines of CA3 and CA1 and require de novo protein synthesis regulated by mammalian target of rapamycin (mTOR). BDNF-mediated stimulation of RhoA activity, cofilin phosphorylation, and actin polymerization were completely suppressed by protein synthesis inhibitors. Furthermore, intrahippocampal injections of RhoA antisense oligodeoxynucleotides inhibited theta burst stimulation (TBS)-induced RhoA upregulation in dendritic spines and prevented LTP consolidation. Addition of calpain inhibitors after BDNF or TEA treatment maintained RhoA levels elevated and prolonged the effects of BDNF and TEA on actin polymerization. Finally, the use of isoform-selective calpain inhibitors revealed that calpain-2 was involved in RhoA synthesis, whereas calpain-1 mediated RhoA degradation. Overall, this mechanism provides a novel link between dendritic protein synthesis and reorganization of the actin cytoskeleton in hippocampal dendritic spines during LTP consolidation. Copyright © 2015 the authors 0270-6474/15/352269-14$15.00/0.

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

PubMed Central

Guedon, G; Sovia, D; Ebel, J P; Befort, N; Remy, P

1985-01-01

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:4092696

Metabotropic glutamate receptor I (mGluR1) antagonism impairs cocaine-induced conditioned place preference via inhibition of protein synthesis.

PubMed

Yu, Fei; Zhong, Peng; Liu, Xiaojie; Sun, Dalong; Gao, Hai-Qing; Liu, Qing-Song

2013-06-01

Antagonism of group I metabotropic glutamate receptors (mGluR1 and mGluR5) reduces behavioral effects of drugs of abuse, including cocaine. However, the underlying mechanisms remain poorly understood. Activation of mGluR5 increases protein synthesis at synapses. Although mGluR5-induced excessive protein synthesis has been implicated in the pathology of fragile X syndrome, it remains unknown whether group I mGluR-mediated protein synthesis is involved in any behavioral effects of drugs of abuse. We report that group I mGluR agonist DHPG induced more pronounced initial depression of inhibitory postsynaptic currents (IPSCs) followed by modest long-term depression (I-LTD) in dopamine neurons of rat ventral tegmental area (VTA) through the activation of mGluR1. The early component of DHPG-induced depression of IPSCs was mediated by the cannabinoid CB1 receptors, while DHPG-induced I-LTD was dependent on protein synthesis. Western blotting analysis indicates that mGluR1 was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation. We also show that cocaine conditioning activated translation machinery in the VTA via an mGluR1-dependent mechanism. Furthermore, intra-VTA microinjections of mGluR1 antagonist JNJ16259685 and protein synthesis inhibitor cycloheximide significantly attenuated or blocked the acquisition of cocaine-induced conditioned place preference (CPP) and activation of translation elongation factors. Taken together, these results suggest that mGluR1 antagonism inhibits de novo protein synthesis; this effect may block the formation of cocaine-cue associations and thus provide a mechanism for the reduction in CPP to cocaine.

Schistosome-derived omega-1 drives Th2 polarization by suppressing protein synthesis following internalization by the mannose receptor

PubMed Central

Everts, Bart; Hussaarts, Leonie; Driessen, Nicole N.; Meevissen, Moniek H.J.; Schramm, Gabriele; van der Ham, Alwin J.; van der Hoeven, Barbara; Scholzen, Thomas; Burgdorf, Sven; Mohrs, Markus; Pearce, Edward J.; Hokke, Cornelis H.; Haas, Helmut; Smits, Hermelijn H.

2012-01-01

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming. PMID:22966004

Bioinspired synthesis of magnetic nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

David, Anand

2009-01-01

The synthesis of magnetic nanoparticles has long been an area of active research. Magnetic nanoparticles can be used in a wide variety of applications such as magnetic inks, magnetic memory devices, drug delivery, magnetic resonance imaging (MRI) contrast agents, and pathogen detection in foods. In applications such as MRI, particle uniformity is particularly crucial, as is the magnetic response of the particles. Uniform magnetic particles with good magnetic properties are therefore required. One particularly effective technique for synthesizing nanoparticles involves biomineralization, which is a naturally occurring process that can produce highly complex nanostructures. Also, the technique involves mild conditions (ambientmore » temperature and close to neutral pH) that make this approach suitable for a wide variety of materials. The term 'bioinspired' is important because biomineralization research is inspired by the naturally occurring process, which occurs in certain microorganisms called 'magnetotactic bacteria'. Magnetotactic bacteria use biomineralization proteins to produce magnetite crystals having very good uniformity in size and morphology. The bacteria use these magnetic particles to navigate according to external magnetic fields. Because these bacteria synthesize high quality crystals, research has focused on imitating aspects of this biomineralization in vitro. In particular, a biomineralization iron-binding protein found in a certain species of magnetotactic bacteria, magnetospirillum magneticum, AMB-1, has been extracted and used for in vitro magnetite synthesis; Pluronic F127 gel was used to increase the viscosity of the reaction medium to better mimic the conditions in the bacteria. It was shown that the biomineralization protein mms6 was able to facilitate uniform magnetite synthesis. In addition, a similar biomineralization process using mms6 and a shorter version of this protein, C25, has been used to synthesize cobalt ferrite particles. The overall goal of this project is to understand the mechanism of magnetite particle synthesis in the presence of the biomineralization proteins, mms6 and C25. Previous work has hypothesized that the mms6 protein helps to template magnetite and cobalt ferrite particle synthesis and that the C25 protein templates cobalt ferrite formation. However, the effect of parameters such as the protein concentration on the particle formation is still unknown. It is expected that the protein concentration significantly affects the nucleation and growth of magnetite. Since the protein provides iron-binding sites, it is expected that magnetite crystals would nucleate at those sites. In addition, in the previous work, the reaction medium after completion of the reaction was in the solution phase, and magnetic particles had a tendency to fall to the bottom of the medium and aggregate. The research presented in this thesis involves solid Pluronic gel phase reactions, which can be studied readily using small-angle x-ray scattering, which is not possible for the solution phase experiments. In addition, the concentration effect of both of the proteins on magnetite crystal formation was studied.« less

Molecular cellular mechanisms of peptide regulation of melatonin synthesis in pinealocyte culture.

PubMed

Khavinson, V Kh; Linkova, N S; Kvetnoy, I M; Kvetnaia, T V; Polyakova, V O; Korf, H-W

2012-06-01

The effects of epithalone and vilone peptides on the synthesis of melatonin and factors involved in this process, arylalkylamine-N-acetyltransferase (AANAT) enzyme and pCREB transcription protein, were studied in rat pinealocyte culture. Epithalone stimulated AANAT and pCREB synthesis and increased melatonin level in culture medium. Simultaneous addition of norepinephrine and peptides into the culture potentiated the expression of AANAT and pCREB.

Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation.

PubMed

Alkozi, Hanan Awad; Perez de Lara, María J; Pintor, Jesús

2017-09-01

Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non-pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p < 0.05, n = 6) with a concomitant rise in N-acetyl serotonin and melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin-dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p < 0.001, n = 4). This mechanism was also established by measuring N-acetyl serotonin and melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca-calmodulin and the calmodulin-dependent protein kinase II are involved. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteins Exported via the PrsD-PrsE Type I Secretion System and the Acidic Exopolysaccharide Are Involved in Biofilm Formation by Rhizobium leguminosarum

PubMed Central

Russo, Daniela M.; Williams, Alan; Edwards, Anne; Posadas, Diana M.; Finnie, Christine; Dankert, Marcelo; Downie, J. Allan; Zorreguieta, Angeles

2006-01-01

The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria. PMID:16740954

Replication of tobacco mosaic virus RNA.

PubMed Central

Buck, K W

1999-01-01

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA. PMID:10212941

Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro.

PubMed

Muñoz-Félix, José M; Cuesta, Cristina; Perretta-Tejedor, Nuria; Subileau, Mariela; López-Hernández, Francisco J; López-Novoa, José M; Martínez-Salgado, Carlos

2016-09-01

Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Thermal Shock Induces Host Proteostasis Disruption and Endoplasmic Reticulum Stress in the Model Symbiotic Cnidarian Aiptasia.

PubMed

Oakley, Clinton A; Durand, Elysanne; Wilkinson, Shaun P; Peng, Lifeng; Weis, Virginia M; Grossman, Arthur R; Davy, Simon K

2017-06-02

Coral bleaching has devastating effects on coral survival and reef ecosystem function, but many of the fundamental cellular effects of thermal stress on cnidarian physiology are unclear. We used label-free liquid chromatography-tandem mass spectrometry to compare the effects of rapidly (33.5 °C, 24 h) and gradually (30 and 33.5 °C, 12 days) elevated temperatures on the proteome of the model symbiotic anemone Aiptasia. We identified 2133 proteins in Aiptasia, 136 of which were differentially abundant between treatments. Thermal shock, but not acclimation, resulted in significant abundance changes in 104 proteins, including those involved in protein folding and synthesis, redox homeostasis, and central metabolism. Nineteen abundant structural proteins showed particularly reduced abundance, demonstrating proteostasis disruption and potential protein synthesis inhibition. Heat shock induced antioxidant mechanisms and proteins involved in stabilizing nascent proteins, preventing protein aggregation and degrading damaged proteins, which is indicative of endoplasmic reticulum stress. Host proteostasis disruption occurred before either bleaching or symbiont photoinhibition was detected, suggesting host-derived reactive oxygen species production as the proximate cause of thermal damage. The pronounced abundance changes in endoplasmic reticulum proteins associated with proteostasis and protein turnover indicate that these processes are essential in the cellular response of symbiotic cnidarians to severe thermal stress.

Protein accumulation in leaves and roots associated with improved drought tolerance in creeping bentgrass expressing an ipt gene for cytokinin synthesis

PubMed Central

Merewitz, Emily B.; Gianfagna, Thomas; Huang, Bingru

2011-01-01

Cytokinins (CKs) may be involved in the regulation of plant adaptation to drought stress. The objectives of the study were to identify proteomic changes in leaves and roots in relation to improved drought tolerance in transgenic creeping bentgrass (Agrostis stolonifera) containing a senescence-activated promoter (SAG12) and the isopentyl transferase (ipt) transgene that increases endogenous CK content. Leaves of SAG12-ipt bentgrass exhibited less severe senescence under water stress, as demonstrated by maintaining lower electrolyte leakage and lipid peroxidation, and higher photochemical efficiency (Fv/Fm), compared with the null transformant (NT) plants. SAG12-ipt plants had higher root/shoot ratios and lower lipid peroxidation in leaves under water stress than the NT plants. The suppression of drought-induced leaf senescence and root dieback in the transgenic plants was associated with the maintenance of greater antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase). The SAG12-ipt and NT plants exhibited differential protein expression patterns under well-watered and drought conditions in both leaves and roots. Under equivalent leaf water deficit (47% relative water content), SAG12-ipt plants maintained higher abundance of proteins involved in (i) energy production within both photosynthesis and respiration [ribulose 1,5-bisphosphate carboxylase (RuBisCO) and glyceraldehyde phosphate dehydrogenase (GAPDH)]; (ii) amino acid synthesis (methionine and glutamine); (iii) protein synthesis and destination [chloroplastic elongation factor (EF-Tu) and protein disulphide isomerases (PDIs)]; and (iv) antioxidant defence system (catalase and peroxidase) than the NT plants. These results suggest that increased endogenous CKs under drought stress may directly or indirectly regulate protein abundance and enzymatic activities involved in the above-mentioned metabolic processes, thereby enhancing plant drought tolerance. PMID:21831843

Mechanism of protein biosynthesis in mammalian mitochondria.

PubMed

Christian, Brooke E; Spremulli, Linda L

2012-01-01

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression. Copyright © 2011 Elsevier B.V. All rights reserved.

Role of Oxidative Stress as Key Regulator of Muscle Wasting during Cachexia.

PubMed

Ábrigo, Johanna; Elorza, Alvaro A; Riedel, Claudia A; Vilos, Cristian; Simon, Felipe; Cabrera, Daniel; Estrada, Lisbell; Cabello-Verrugio, Claudio

2018-01-01

Skeletal muscle atrophy is a pathological condition mainly characterized by a loss of muscular mass and the contractile capacity of the skeletal muscle as a consequence of muscular weakness and decreased force generation. Cachexia is defined as a pathological condition secondary to illness characterized by the progressive loss of muscle mass with or without loss of fat mass and with concomitant diminution of muscle strength. The molecular mechanisms involved in cachexia include oxidative stress, protein synthesis/degradation imbalance, autophagy deregulation, increased myonuclear apoptosis, and mitochondrial dysfunction. Oxidative stress is one of the most common mechanisms of cachexia caused by different factors. It results in increased ROS levels, increased oxidation-dependent protein modification, and decreased antioxidant system functions. In this review, we will describe the importance of oxidative stress in skeletal muscles, its sources, and how it can regulate protein synthesis/degradation imbalance, autophagy deregulation, increased myonuclear apoptosis, and mitochondrial dysfunction involved in cachexia.

Comparative proteomic analysis of engineered Saccharomyces cerevisiae with enhanced free fatty acid accumulation.

PubMed

Chen, Liwei; Lee, Jaslyn Jie Lin; Zhang, Jianhua; Chen, Wei Ning

2016-02-01

The engineered Saccharomyces cerevisiae strain △faa1△faa4 [Acot5s] was demonstrated to accumulate more free fatty acids (FFA) previously. Here, comparative proteomic analysis was performed to get a global overview of metabolic regulation in the strain. Over 500 proteins were identified, and 82 of those proteins were found to change significantly in the engineered strains. Proteins involved in glycolysis, acetate metabolism, fatty acid synthesis, TCA cycle, glyoxylate cycle, the pentose phosphate pathway, respiration, transportation, and stress response were found to be upregulated in △faa1△faa4 [Acot5s] as compared to the wild type. On the other hand, proteins involved in glycerol, ethanol, ergosterol, and cell wall synthesis were downregulated. Taken together with our metabolite analysis, our results showed that the disruption of Faa1 and Faa4 and expression of Acot5s in the engineered strain △faa1△faa4 [Acot5s] not only relieved the feedback inhibition of fatty acyl-CoAs on fatty acid synthesis, but also caused a major metabolic rearrangement. The rearrangement redirected carbon flux toward the pathways which generate the essential substrates and cofactors for fatty acid synthesis, such as acetyl-CoA, ATP, and NADPH. Therefore, our results help shed light on the mechanism for the increased production of fatty acids in the engineered strains, which is useful in providing information for future studies in biofuel production.

Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta “Gold Standard” Leaves

PubMed Central

Yu, Juanjuan; Zhang, Jinzheng; Zhao, Qi; Liu, Yuelu; Chen, Sixue; Guo, Hongliang; Shi, Lei; Dai, Shaojun

2016-01-01

Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta “Gold Standard” is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta “Gold Standard”, as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta “Gold Standard”. For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves. PMID:27005614

Endothelin-1 (ET-1) stimulates carboxy terminal Smad2 phosphorylation in vascular endothelial cells by a mechanism dependent on ET receptors and de novo protein synthesis.

PubMed

Sharifat, Narges; Mohammad Zadeh, Ghorban; Ghaffari, Mohammad-Ali; Dayati, Parisa; Kamato, Danielle; Little, Peter J; Babaahmadi-Rezaei, Hossein

2017-01-01

G protein-coupled receptor (GPCR) agonists through their receptors can transactivate protein tyrosine kinase receptors such as epidermal growth factor receptor and serine/threonine kinase receptors most notably transforming growth factor (TGF)-β receptor (TβRI). This signalling mechanism represents a major expansion in the cellular outcomes attributable to GPCR signalling. This study addressed the role and mechanisms involved in GPCR agonist, endothelin-1 (ET-1)-mediated transactivation of the TβRI in bovine aortic endothelial cells (BAECs). The in-vitro model used BAECs. Signalling intermediate phospho-Smad2 in the carboxy terminal was detected and quantified by Western blotting. ET-1 treatment of BAECs resulted in a time and concentration-dependent increase in pSmad2C. Peak phosphorylation was evident with 100 nm treatment of ET-1 at 4-6 h. TβRI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ET A and ET B receptor antagonist ET-1-mediated pSmad2C levels were inhibited. The ET-mediated pSmad2C was blocked by the protein synthesis inhibitor, cycloheximide. In BAECs, ET-1 via the ETB receptor is involved in transactivation of the TβRI. The transactivation-dependent response is dependent upon de novo protein synthesis. © 2016 Royal Pharmaceutical Society.

Ribosome profiling reveals the what, when, where and how of protein synthesis.

PubMed

Brar, Gloria A; Weissman, Jonathan S

2015-11-01

Ribosome profiling, which involves the deep sequencing of ribosome-protected mRNA fragments, is a powerful tool for globally monitoring translation in vivo. The method has facilitated discovery of the regulation of gene expression underlying diverse and complex biological processes, of important aspects of the mechanism of protein synthesis, and even of new proteins, by providing a systematic approach for experimental annotation of coding regions. Here, we introduce the methodology of ribosome profiling and discuss examples in which this approach has been a key factor in guiding biological discovery, including its prominent role in identifying thousands of novel translated short open reading frames and alternative translation products.

Pulmonary inflammation-induced loss and subsequent recovery of skeletal muscle mass require functional poly-ubiquitin conjugation.

PubMed

Ceelen, Judith J M; Schols, Annemie M W J; Thielen, Nathalie G M; Haegens, Astrid; Gray, Douglas A; Kelders, Marco C J M; de Theije, Chiel C; Langen, Ramon C J

2018-05-02

Pulmonary inflammation in response to respiratory infections can evoke muscle wasting. Increased activity of the ubiquitin (Ub)-proteasome system (UPS) and the autophagy lysosome pathway (ALP) have been implicated in inflammation-induced muscle atrophy. Since poly-Ub conjugation is required for UPS-mediated proteolysis and has been implicated in the ALP, we assessed the effect of impaired ubiquitin conjugation on muscle atrophy and recovery following pulmonary inflammation, and compared activation and suppression of these proteolytic systems to protein synthesis regulation. Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS. Proteolysis (UPS and ALP) and synthesis signaling were examined in gastrocnemius muscle homogenates. Ub-conjugation-dependency of muscle atrophy and recovery was addressed using Ub-K48R (K48R) mice with attenuated poly-ubiquitin conjugation, and compared to UBWT control mice. Pulmonary inflammation caused a decrease in skeletal muscle mass which was accompanied by a rapid increase in expression of UPS and ALP constituents and reduction in protein synthesis signaling acutely after LPS. Muscle atrophy was attenuated in K48R mice, while ALP and protein synthesis signaling were not affected. Muscle mass recovery starting 72 h post LPS, correlated with reduced expression of UPS and ALP constituents and restoration of protein synthesis signaling. K48R mice however displayed impaired recovery of muscle mass. Pulmonary inflammation-induced muscle atrophy is in part attributable to UPS-mediated proteolysis, as activation of ALP- and suppression of protein synthesis signaling occur independently of poly-Ub conjugation during muscle atrophy. Recovery of muscle mass following pulmonary inflammation involves inverse regulation of proteolysis and protein synthesis signaling, and requires a functional poly-Ub conjugation.

Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

PubMed Central

Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

2013-01-01

Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05), and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates. PMID:23340676

The muscle fiber type–fiber size paradox: hypertrophy or oxidative metabolism?

PubMed Central

van Wessel, T.; de Haan, A.; van der Laarse, W. J.

2010-01-01

An inverse relationship exists between striated muscle fiber size and its oxidative capacity. This relationship implies that muscle fibers, which are triggered to simultaneously increase their mass/strength (hypertrophy) and fatigue resistance (oxidative capacity), increase these properties (strength or fatigue resistance) to a lesser extent compared to fibers increasing either of these alone. Muscle fiber size and oxidative capacity are determined by the balance between myofibrillar protein synthesis, mitochondrial biosynthesis and degradation. New experimental data and an inventory of critical stimuli and state of activation of the signaling pathways involved in regulating contractile and metabolic protein turnover reveal: (1) higher capacity for protein synthesis in high compared to low oxidative fibers; (2) competition between signaling pathways for synthesis of myofibrillar proteins and proteins associated with oxidative metabolism; i.e., increased mitochondrial biogenesis via AMP-activated protein kinase attenuates the rate of protein synthesis; (3) relatively higher expression levels of E3-ligases and proteasome-mediated protein degradation in high oxidative fibers. These observations could explain the fiber type–fiber size paradox that despite the high capacity for protein synthesis in high oxidative fibers, these fibers remain relatively small. However, it remains challenging to understand the mechanisms by which contractile activity, mechanical loading, cellular energy status and cellular oxygen tension affect regulation of fiber size. Therefore, one needs to know the relative contribution of the signaling pathways to protein turnover in high and low oxidative fibers. The outcome and ideas presented are relevant to optimizing treatment and training in the fields of sports, cardiology, oncology, pulmonology and rehabilitation medicine. Electronic supplementary material The online version of this article (doi:10.1007/s00421-010-1545-0) contains supplementary material, which is available to authorized users. PMID:20602111

Long-term sensitization training in Aplysia leads to an increase in the expression of BiP, the major protein chaperon of the ER.

PubMed

Kuhl, D; Kennedy, T E; Barzilai, A; Kandel, E R

1992-12-01

Long-term memory for sensitization of the gill- and siphon-withdrawal reflexes in Aplysia californica requires RNA and protein synthesis. These long-term behavioral changes are accompanied by long-term facilitation of the synaptic connections between the gill and siphon sensory and motor neurons, which are similarly dependent on transcription and translation. In addition to showing an increase in over-all protein synthesis, long-term facilitation is associated with changes in the expression of specific early, intermediate, and late proteins, and with the growth of new synaptic connections between the sensory and motor neurons of the reflex. We previously focused on early proteins and have identified four proteins as members of the immunoglobulin family of cell adhesion molecules related to NCAM and fasciclin II. We have now cloned the cDNA corresponding to one of the late proteins, and identified it as the Aplysia homolog of BiP, an ER resident protein involved in the folding and assembly of secretory and membrane proteins. Behavioral training increases the steady-state level of BiP mRNA in the sensory neurons. The increase in the synthesis of BiP protein is first detected 3 h after the onset of facilitation, when the increase in overall protein synthesis reaches its peak and the formation of new synaptic terminals becomes apparent. These findings suggest that the chaperon function of BiP might serve to fold proteins and assemble protein complexes necessary for the structural changes characteristic of long-term memory.

Skeletal Muscle Myofibrillar and Sarcoplasmic Protein Synthesis Rates Are Affected Differently by Altitude-Induced Hypoxia in Native Lowlanders

PubMed Central

Holm, Lars; Haslund, Mads Lyhne; Robach, Paul; van Hall, Gerrit; Calbet, Jose A. L.; Saltin, Bengt; Lundby, Carsten

2010-01-01

As a consequence to hypobaric hypoxic exposure skeletal muscle atrophy is often reported. The underlying mechanism has been suggested to involve a decrease in protein synthesis in order to conserve O2. With the aim to challenge this hypothesis, we applied a primed, constant infusion of 1-13C-leucine in nine healthy male subjects at sea level and subsequently at high-altitude (4559 m) after 7–9 days of acclimatization. Physical activity levels and food and energy intake were controlled prior to the two experimental conditions with the aim to standardize these confounding factors. Blood samples and expired breath samples were collected hourly during the 4 hour trial and vastus lateralis muscle biopsies obtained at 1 and 4 hours after tracer priming in the overnight fasted state. Myofibrillar protein synthesis rate was doubled; 0.041±0.018 at sea-level to 0.080±0.018%⋅hr−1 (p<0.05) when acclimatized to high altitude. The sarcoplasmic protein synthesis rate was in contrast unaffected by altitude exposure; 0.052±0.019 at sea-level to 0.059±0.010%⋅hr−1 (p>0.05). Trends to increments in whole body protein kinetics were seen: Degradation rate elevated from 2.51±0.21 at sea level to 2.73±0.13 µmol⋅kg−1⋅min−1 (p = 0.05) at high altitude and synthesis rate similar; 2.24±0.20 at sea level and 2.43±0.13 µmol⋅kg−1⋅min−1 (p>0.05) at altitude. We conclude that whole body amino acid flux is increased due to an elevated protein turnover rate. Resting skeletal muscle myocontractile protein synthesis rate was concomitantly elevated by high-altitude induced hypoxia, whereas the sarcoplasmic protein synthesis rate was unaffected by hypoxia. These changed responses may lead to divergent adaptation over the course of prolonged exposure. PMID:21187972

Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

PubMed

Sørensen, Brita Singers; Busk, Morten; Overgaard, Jens; Horsman, Michael R; Alsner, Jan

2015-01-01

The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis. Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein. Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined. We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo protein synthesis, depending on whether the two factors induced alone or overlapping, and as such it is important for in vivo studies to take this into account.

Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro

PubMed Central

Sørensen, Brita Singers; Busk, Morten; Overgaard, Jens; Horsman, Michael R.; Alsner, Jan

2015-01-01

Background The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis. Methods Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein. Results Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined. Conclusions We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo protein synthesis, depending on whether the two factors induced alone or overlapping, and as such it is important for in vivo studies to take this into account. PMID:26274822

Design, Synthesis and Affinity Properties of Biologically Active Peptide and Protein Conjugates of Cotton Cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, J. V.; Goheen, Steven C.

The formation of peptide and protein conjugates of cellulose on cotton fabrics provides promising leads for the development of wound healing, antibacterial, and decontaminating textiles. An approach to the design, synthesis, and analysis of bioconjugates containing cellulose peptide and protein conjugates includes: 1) computer graphic modeling for a rationally designed structure; 2) attachment of the peptide or protein to cotton cellulose through a linker amino acid, and 3) characterization of the resulting bioconjugate. Computer graphic simulation of protein and peptide cellulose conjugates gives a rationally designed biopolymer to target synthetic modifications to the cotton cellulose. Techniques for preparing these typesmore » of conjugates involve both sequential assembly of the peptide on the fabric and direct crosslinking of the peptide or protein as cellulose bound esters or carboxymethylcellulose amides.« less

Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure

PubMed Central

Létoquart, Juliette; van Tran, Nhan; Caroline, Vonny; Aleksandrov, Alexey; Lazar, Noureddine; van Tilbeurgh, Herman; Liger, Dominique; Graille, Marc

2015-01-01

Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm5U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity. PMID:26438534

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation.

PubMed

Schlimpert, Susan; Wasserstrom, Sebastian; Chandra, Govind; Bibb, Maureen J; Findlay, Kim C; Flärdh, Klas; Buttner, Mark J

2017-07-25

During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

Antisense RNA to the first N-glycosylation gene, ALG7, inhibits protein N-glycosylation and secretion by Xenopus oocytes.

PubMed

Kukuruzinska, M A; Apekin, V; Lamkin, M S; Hiltz, A; Rodriguez, A; Lin, C C; Paz, M A; Oppenheim, F G

1994-02-15

N-Glycosylation has been shown to affect the rate of glycoprotein transport through the secretory pathway. In order to identify the critical components in the N-glycosylation pathway that directly influence protein secretion, we have studied the effects of downregulation of the first gene in the dolichol pathway, ALG7, on the synthesis, glycosylation and secretion of native and heterologous proteins by Xenopus laevis oocytes. Our strategy involved the use of ALG7 antisense RNA (asRNA) to lower the effective abundance of the ALG7 protein in oocytes. The results showed that there was an inverse dose-response relationship between ALG7 asRNA and the amount of glycosylated and secreted proteins. These effects were also observed for heterologously expressed rat parotid amylase. Since ALG7 asRNA did not inhibit overall protein synthesis, we conclude that downregulation of ALG7 expression directly lowered protein export.

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

PubMed

Varley, C L; Royds, J A; Brown, B L; Dobson, P R

2001-01-01

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

Solid-phase synthesis and screening of N-acylated polyamine (NAPA) combinatorial libraries for protein binding.

PubMed

Iera, Jaclyn A; Jenkins, Lisa M Miller; Kajiyama, Hiroshi; Kopp, Jeffrey B; Appella, Daniel H

2010-11-15

Inhibitors for protein-protein interactions are challenging to design, in part due to the unique and complex architectures of each protein's interaction domain. Most approaches to develop inhibitors for these interactions rely on rational design, which requires prior structural knowledge of the target and its ligands. In the absence of structural information, a combinatorial approach may be the best alternative to finding inhibitors of a protein-protein interaction. Current chemical libraries, however, consist mostly of molecules designed to inhibit enzymes. In this manuscript, we report the synthesis and screening of a library based on an N-acylated polyamine (NAPA) scaffold that we designed to have specific molecular features necessary to inhibit protein-protein interactions. Screens of the library identified a member with favorable binding properties to the HIV viral protein R (Vpr), a regulatory protein from HIV, that is involved in numerous interactions with other proteins critical for viral replication. Published by Elsevier Ltd.

Overexpression of caveolin-3-enhanced protein synthesis rather than proteolysis inhibition in C2C12 myoblasts: relationship with myostatin activity.

PubMed

Hadj Sassi, Abdessattar; Monteil, Julien; Sauvant, Patrick; Atgié, Claude

2012-12-01

Caveolin-3 (cav-3), which is involved in the regulation of signal transduction and vesicular trafficking, could interact with activin receptor IIB to inhibit myostatin (MSTN) activity and may therefore play a role in muscle development and hypertrophy. MSTN is a member of the transforming growth factor-β family, identified as a negative regulator of skeletal muscle mass. The expression of MSTN is fiber-type specific and the greatest amount of MSTN is present in fiber, which is composed of myosin heavy chain (MHC) type IIb. MSTN acts through the activin receptor IIB to activate smad2/3 which leads to an increase in gene transcription involved in muscle atrophy. Muscle hypertrophy is a consequence of two mechanisms: (1) the inhibition of proteolysis such as the calcium-dependent proteolytic system calpains and calpastatin and (2) an increase in protein synthesis through the Akt/mTOR/p70s6K pathway. In order to determine which of the two processes predominates in inhibition of MSTN activity in a cav-3 context, we transfected a C2C12 cell line with plasmids containing mstn or cav-3 wild genes. The results reported in this study demonstrate that inhibition of MSTN activity by overexpression of cav-3 induces an activation of protein synthesis rather than an inhibition of proteolysis through the calcium proteolytic system. The inhibition of phosphorylation of smad-3 due to overexpression of cav-3 causes an increase in the phosphorylation of the ribosomal protein S6, promoting the synthesis of MHC type II, probably through activation of Akt/mTOR/p70s6K. These data highlight the role of protein synthesis as the predominant mechanism in muscle hypertrophy observed when the expression of MSTN is altered and confirm the value of studying the physiological role of MSTN in the growing processes of skeletal muscle.

Influence of omega-3 fatty acids on skeletal muscle protein metabolism and mitochondrial bioenergetics in older adults.

PubMed

Lalia, Antigoni Z; Dasari, Surendra; Robinson, Matthew M; Abid, Hinnah; Morse, Dawn M; Klaus, Katherine A; Lanza, Ian R

2017-04-01

Omega-3 polyunsaturated fatty acids (n3-PUFA) are recognized for their anti-inflammatory effects and may be beneficial in the context of sarcopenia. We determined the influence of n3-PUFA on muscle mitochondrial physiology and protein metabolism in older adults. Twelve young (18-35 years) and older (65-85 years) men and women were studied at baseline. Older adults were studied again following n3-PUFA supplementation (3.9g/day, 16 weeks). Muscle biopsies were used to evaluate respiratory capacity (high resolution respirometry) and oxidant emissions (spectrofluorometry) in isolated mitochondria. Maximal respiration was significantly lower in older compared to young. n3-PUFA did not change respiration, but significantly reduced oxidant emissions. Participants performed a single bout of resistance exercise, followed by biopsies at 15 and 18 hours post exercise. Several genes involved in muscle protein turnover were significantly altered in older adults at baseline and following exercise, yet muscle protein synthesis was similar between age groups under both conditions. Following n3-PUFA supplementation, mixed muscle, mitochondrial, and sarcoplasmic protein synthesis rates were increased in older adults before exercise. n3-PUFA increased post-exercise mitochondrial and myofibrillar protein synthesis in older adults. These results demonstrate that n3-PUFA reduce mitochondrial oxidant emissions, increase postabsorptive muscle protein synthesis, and enhance anabolic responses to exercise in older adults.

Influence of omega-3 fatty acids on skeletal muscle protein metabolism and mitochondrial bioenergetics in older adults

PubMed Central

Lalia, Antigoni Z.; Dasari, Surendra; Robinson, Matthew M.; Abid, Hinnah; Morse, Dawn M.; Klaus, Katherine A.; Lanza, Ian R.

2017-01-01

Omega-3 polyunsaturated fatty acids (n3-PUFA) are recognized for their anti-inflammatory effects and may be beneficial in the context of sarcopenia. We determined the influence of n3-PUFA on muscle mitochondrial physiology and protein metabolism in older adults. Twelve young (18-35 years) and older (65-85 years) men and women were studied at baseline. Older adults were studied again following n3-PUFA supplementation (3.9g/day, 16 weeks). Muscle biopsies were used to evaluate respiratory capacity (high resolution respirometry) and oxidant emissions (spectrofluorometry) in isolated mitochondria. Maximal respiration was significantly lower in older compared to young. n3-PUFA did not change respiration, but significantly reduced oxidant emissions. Participants performed a single bout of resistance exercise, followed by biopsies at 15 and 18 hours post exercise. Several genes involved in muscle protein turnover were significantly altered in older adults at baseline and following exercise, yet muscle protein synthesis was similar between age groups under both conditions. Following n3-PUFA supplementation, mixed muscle, mitochondrial, and sarcoplasmic protein synthesis rates were increased in older adults before exercise. n3-PUFA increased post-exercise mitochondrial and myofibrillar protein synthesis in older adults. These results demonstrate that n3-PUFA reduce mitochondrial oxidant emissions, increase postabsorptive muscle protein synthesis, and enhance anabolic responses to exercise in older adults. PMID:28379838

Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

PubMed

Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

2010-02-01

By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

Complementary transcriptome and proteome profiling in cabbage buds of a recessive male sterile mutant provides new insights into male reproductive development.

PubMed

Ji, Jialei; Yang, Limei; Fang, Zhiyuan; Zhuang, Mu; Zhang, Yangyong; Lv, Honghao; Liu, Yumei; Li, Zhansheng

2018-05-15

Plant male reproductive development is a very complex biological process that involves multiple metabolic pathways. To reveal novel insights into male reproductive development, we conducted an integrated profiling of gene activity in the developing buds of a cabbage recessive genetic male sterile mutant. Using RNA-Seq and label-free quantitative proteomics, 2881 transcripts and 1245 protein species were identified with significant differential abundance between the male sterile line 83121A and its isogenic maintainer line 83121B. Analyses of function annotations and correlations between transcriptome and proteome and protein interaction networks were also conducted, which suggested that the male sterility involves a complex regulatory pattern. Moreover, several key biological processes, such as fatty acid metabolism, tapetosome biosynthesis, amino acid metabolism and protein synthesis and degradation were identified as being of relevance to male reproductive development. A large number of protein species involved in sporopollenin synthesis, amino acid synthesis, ribosome assembly, protein processing in endoplasmic reticulum and lipid transfer were observed to be significantly down-accumulated in 83121A buds, indicating their potential roles in the regulation of cabbage microspore abortion. In summary, the conjoint analysis of the transcriptome and proteome provided a global picture regarding the molecular dynamics in male sterile buds of 83121A. Male sterile mutants are excellent materials for the study of plant male reproductive development. This study revealed the molecular dynamics of recessive male sterility in cabbage at the transcriptome and proteome levels, which deepens our understanding of the metabolic pathways involved in male development. Moreover, the male sterility-related genes identified in this study could provide a reference for the artificial regulation of cabbage fertility by using genetic engineering technology, which may result in potential applications in agriculture such as production of hybrid seeds using male sterility. Copyright © 2018 Elsevier B.V. All rights reserved.

Lower weight gain and hepatic lipid content in hamsters fed high fat diets supplemented with white rice protein, brown rice protein, soy protein, and their hydrolysates.

PubMed

Zhang, Huijuan; Bartley, Glenn E; Mitchell, Cheryl R; Zhang, Hui; Yokoyama, Wallace

2011-10-26

The physiological effects of the hydrolysates of white rice protein (WRP), brown rice protein (BRP), and soy protein (SP) hydrolyzed by the food grade enzyme, alcalase2.4 L, were compared to the original protein source. Male Syrian Golden hamsters were fed high-fat diets containing either 20% casein (control) or 20% extracted proteins or their hydrolysates as the protein source for 3 weeks. The brown rice protein hydrolysate (BRPH) diet group reduced weight gain 76% compared with the control. Animals fed the BRPH supplemented diet also had lower final body weight, liver weight, very low density lipoprotein cholesterol (VLDL-C), and liver cholesterol, and higher fecal fat and bile acid excretion than the control. Expression levels of hepatic genes for lipid oxidation, PPARα, ACOX1, and CPT1, were highest for hamsters fed the BRPH supplemented diet. Expression of CYP7A1, the gene regulating bile acid synthesis, was higher in all test groups. Expression of CYP51, a gene coding for an enzyme involved in cholesterol synthesis, was highest in the BRPH diet group. The results suggest that BRPH includes unique peptides that reduce weight gain and hepatic cholesterol synthesis.

The effect of G protein-coupled receptor kinase 2 (GRK2) on lactation and on proliferation of mammary epithelial cells from dairy cows.

PubMed

Hou, Xiaoming; Hu, Hongliu; Lin, Ye; Qu, Bo; Gao, Xuejun; Li, Qingzhang

2016-07-01

Milk protein is an important component of milk and a nutritional source for human consumption. To better understand the molecular events underlying synthesis of milk proteins, the global gene expression patterns in mammary glands of dairy cow with high-quality milk (>3% milk protein; >3.5% milk fat) and low-quality milk (<3% milk protein; <3.5% milk fat) were examined via digital gene expression study. A total of 139 upregulated and 66 downregulated genes were detected in the mammary tissues of lactating cows with high-quality milk compared with the tissues of cows with low-quality milk. A pathway enrichment study of these genes revealed that the top 5 pathways that were differentially affected in the tissues of cows with high- versus low-quality milk involved metabolic pathways, cancer, cytokine-cytokine receptor interactions, regulation of the actin cytoskeleton, and insulin signaling. We also found that the G protein-coupled receptor kinase 2 (GRK2) was one of the most highly upregulated genes in lactating mammary tissue with low-quality milk compared with tissue with high-quality milk. The knockdown of GRK2 in cultured bovine mammary epithelial cells enhanced CSN2 expression and activated signaling molecules related to translation, including protein kinase B, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase 1 (S6K1), whereas overexpression of GRK2 had the opposite effects. However, expression of genes involved in the mitogen-activated protein kinase pathway was positively regulated by GRK2. Therefore, GRK2 seems to act as a negative mediator of milk-protein synthesis via the protein kinase B-mammalian target of rapamycin signaling axis. Furthermore, GRK2 may negatively control milk-protein synthesis by activating the mitogen-activated protein kinase pathway in dairy cow mammary epithelial cells. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis.

PubMed

Pillai, Ramesh S; Artus, Caroline G; Filipowicz, Witold

2004-10-01

MicroRNAs (miRNAs) are approximately 21-nt-long RNAs involved in regulating development, differentiation, and other processes in eukaryotes. In metazoa, nearly all miRNAs control gene expression by imperfectly base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs and repressing protein synthesis by an unknown mechanism. It is also unknown whether miRNA-mRNA duplexes containing mismatches and bulges provide specific features that are recognized by factors mediating the repression. miRNAs form part of ribonucleoprotein complexes, miRNPs, that contain Argonaute (Ago) and other proteins. Here we demonstrate that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3'-UTR of a reporter mRNA. Inhibition of protein synthesis occurred without a change in the reporter mRNA level and was dependent on the number, but not the position, of the hairpins tethering hAgo2 to the 3'-UTR. These findings indicate that a primary function of miRNAs is to guide their associated proteins to the mRNA. Copyright 2004 RNA Society

Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency.

PubMed

Kemp, John P; Smith, Paul M; Pyle, Angela; Neeve, Vivienne C M; Tuppen, Helen A L; Schara, Ulrike; Talim, Beril; Topaloglu, Haluk; Holinski-Feder, Elke; Abicht, Angela; Czermin, Birgit; Lochmüller, Hanns; McFarland, Robert; Chinnery, Patrick F; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N; Taylor, Robert W; Horvath, Rita

2011-01-01

Mutations in several mitochondrial DNA and nuclear genes involved in mitochondrial protein synthesis have recently been reported in combined respiratory chain deficiency, indicating a generalized defect in mitochondrial translation. However, the number of patients with pathogenic mutations is small, implying that nuclear defects of mitochondrial translation are either underdiagnosed or intrauterine lethal. No comprehensive studies have been reported on large cohorts of patients with combined respiratory chain deficiency addressing the role of nuclear genes affecting mitochondrial protein synthesis to date. We investigated a cohort of 52 patients with combined respiratory chain deficiency without causative mitochondrial DNA mutations, rearrangements or depletion, to determine whether a defect in mitochondrial translation defines the pathomechanism of their clinical disease. We followed a combined approach of sequencing known nuclear genes involved in mitochondrial protein synthesis (EFG1, EFTu, EFTs, MRPS16, TRMU), as well as performing in vitro functional studies in 22 patient cell lines. The majority of our patients were children (<15 years), with an early onset of symptoms <1 year of age (65%). The most frequent clinical presentation was mitochondrial encephalomyopathy (63%); however, a number of patients showed cardiomyopathy (33%), isolated myopathy (15%) or hepatopathy (13%). Genomic sequencing revealed compound heterozygous mutations in the mitochondrial transfer ribonucleic acid modifying factor (TRMU) in a single patient only, presenting with early onset, reversible liver disease. No pathogenic mutation was detected in any of the remaining 51 patients in the other genes analysed. In vivo labelling of mitochondrial polypeptides in 22 patient cell lines showed overall (three patients) or selective (four patients) defects of mitochondrial translation. Immunoblotting for mitochondrial proteins revealed decreased steady state levels of proteins in some patients, but normal or increased levels in others, indicating a possible compensatory mechanism. In summary, candidate gene sequencing in this group of patients has a very low detection rate (1/52), although in vivo labelling of mitochondrial translation in 22 patient cell lines indicate that a nuclear defect affecting mitochondrial protein synthesis is responsible for about one-third of combined respiratory chain deficiencies (7/22). In the remaining patients, the impaired respiratory chain activity is most likely the consequence of several different events downstream of mitochondrial translation. Clinical classification of patients with biochemical analysis, genetic testing and, more importantly, in vivo labelling and immunoblotting of mitochondrial proteins show incoherent results, but a systematic review of these data in more patients may reveal underlying mechanisms, and facilitate the identification of novel factors involved in combined respiratory chain deficiency.

Gonadectomy-related adrenocortical tumors in ferrets demonstrate increased expression of androgen and estrogen synthesizing enzymes together with high inhibin expression.

PubMed

de Jong, M K; ten Asbroek, E E M; Sleiderink, A J; Conley, A J; Mol, J A; Schoemaker, N J

2014-07-01

The 2 objectives of this study were to (1) measure by quantitative polymerase chain reaction the expression of genes involved in steroid and inhibin synthesis in adrenocortical tumors of gonadectomized ferrets and (2) localize by immunohistochemistry several proteins that are key to adrenal steroidogenesis. Relative to the control adrenals, expression of the messenger RNAs encoding StAR (steroidogenic acute regulatory protein; P = 0.039), CYP11A (P = 0.019), CYP21 (P = 0.01), and 3β-HSD (P = 0.004), all involved in the synthesis of mineralocorticoids and glucocorticoids, were decreased in the adrenocortical tumors. In contrast, expression of cytochrome B5 (CytB5; P = 0.0001) and aromatase (P = 0.003), involved in androgen and estrogen synthesis, and both inhibin α-subunit (P = 0.002) and βB-subunit (P = 0.001) were upregulated. In tumors, immunostaining of CYP21 was low, whereas staining of Cyp17 and CytB5, necessary for androgen synthesis, was present. It is concluded that ferret adrenocortical tumors express genes for androgen production. In addition, the expression of aromatase and inhibin suggests an even more gonadal differentiation, which is reminiscent to the fact that both gonads and adrenals are derived from a common urogenital primordial cell. Copyright © 2014 Elsevier Inc. All rights reserved.

Total chemical synthesis of proteins without HPLC purification† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01883a Click here for additional data file.

PubMed Central

Loibl, S. F.; Harpaz, Z.; Zitterbart, R.

2016-01-01

The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2–6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8–33% overall yield with 90–98% purity despite the omission of HPLC purification. PMID:28451120

Urea synthesis in patients with chronic pancreatitis: relation to glucagon secretion and dietary protein intake.

PubMed

Hamberg, O; Andersen, V; Sonne, J; Larsen, S; Vilstrup, H

2001-12-01

Up-regulation of urea synthesis by amino acids and dietary protein intake may be impaired in patients with chronic pancreatitis (CP) due to the reduced glucagon secretion. Conversely, urea synthesis may be increased as a result of the chronic inflammation. The aims of the study were to determine urea synthesis kinetics in CP patients in relation to glucagon secretion (study I) and during an increase in protein intake (study II). In study I, urea synthesis rate, calculated as urinary excretion rate corrected for accumulation in total body water and intestinal loss, was measured during infusion of alanine in 7 CP patients and 5 control subjects on spontaneous protein intake. The functional hepatic nitrogen clearance (FHNC), i.e. urea synthesis expressed independent of changes in plasma amino acid concentration, was calculated as the slope of the linear relation between urea synthesis rate and plasma alpha -amino nitrogen concentration. In study II, 6 of the patients of study I had urea synthesis and FHNC determined before and after a period of 14 days of supplementation with a protein-enriched liquid (dietary sequence randomized). Study I: Alanine infusion increased urea synthesis rate by a factor of 10 in the control subjects, and by a factor of 5 in the CP patients (P<0.01). FHNC was 31.9+/-2.4 l/h in the control subjects and 16.5+/-2.0 l/h (P<0.05) in the CP patients. The glucagon response to alanine infusion (AUC) was reduced by 75 % in the CP patients. The reduction in FHNC paralleled the reduced glucagon response (r(2)=0.55, P<0.01). Study II: The spontaneous protein intake was 0.75+/-0.14 g/(kg x day) and increased during the high protein period to 1.77+/-0.12 g/(kg x day). This increased alanine stimulated urea synthesis by a factor of 1.3 (P<0.05), FHNC from 13.5+/-2.6 l/h to 19.4+/-3.1 l/h (P<0.01), and the glucagon response to alanine infusion (AUC) by a factor of 1.8 (P<0.05). Urea synthesis rate and FHNC are markedly reduced in CP patients. This is associated with, and probably a result of, impaired glucagon secretion, and predicts a lower than normal postprandial hepatic loss of amino nitrogen. An increase in dietary protein intake increases alanine stimulated urea synthesis and FHNC by a mechanism that involves an increase in glucagon. This indicates that the low FHNC during spontaneous protein intake included an adaptation to the low protein intake, effectuated by a further decrease in glucagon secretion. Copyright 2001 Harcourt Publishers Ltd.

Cannabinoid modulation of hippocampal long-term memory is mediated by mTOR signaling.

PubMed

Puighermanal, Emma; Marsicano, Giovanni; Busquets-Garcia, Arnau; Lutz, Beat; Maldonado, Rafael; Ozaita, Andrés

2009-09-01

Cognitive impairment is one of the most important negative consequences associated with cannabis consumption. We found that CB1 cannabinoid receptor (CB1R) activation transiently modulated the mammalian target of rapamycin (mTOR)/p70S6K pathway and the protein synthesis machinery in the mouse hippocampus, which correlated with the amnesic properties of delta9-tetrahydrocannabinol (THC). In addition, non-amnesic doses of either the mTOR blocker rapamycin or the protein synthesis inhibitor anisomycin abrogated the amnesic-like effects of THC, pointing to a mechanism involving new protein synthesis. Moreover, using pharmacological and genetic tools, we found that THC long-term memory deficits were mediated by CB1Rs expressed on GABAergic interneurons through a glutamatergic mechanism, as both the amnesic-like effects and p70S6K phosphorylation were reduced in GABA-CB1R knockout mice and by NMDA blockade.

Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin.

PubMed

Webert, Holger; Freibert, Sven-Andreas; Gallo, Angelo; Heidenreich, Torsten; Linne, Uwe; Amlacher, Stefan; Hurt, Ed; Mühlenhoff, Ulrich; Banci, Lucia; Lill, Roland

2014-10-31

Maturation of iron-sulphur (Fe/S) proteins involves complex biosynthetic machinery. In vivo synthesis of [2Fe-2S] clusters on the mitochondrial scaffold protein Isu1 requires the cysteine desulphurase complex Nfs1-Isd11, frataxin, ferredoxin Yah1 and its reductase Arh1. The roles of Yah1-Arh1 have remained enigmatic, because they are not required for in vitro Fe/S cluster assembly. Here, we reconstitute [2Fe-2S] cluster synthesis on Isu1 in a reaction depending on Nfs1-Isd11, frataxin, Yah1, Arh1 and NADPH. Unlike in the bacterial system, frataxin is an essential part of Fe/S cluster biosynthesis and is required simultaneously and stoichiometrically to Yah1. Reduced but not oxidized Yah1 tightly interacts with apo-Isu1 indicating a dynamic interaction between Yah1-apo-Isu1. Nuclear magnetic resonance structural studies identify the Yah1-apo-Isu1 interaction surface and suggest a pathway for electron flow from reduced ferredoxin to Isu1. Together, our study defines the molecular function of the ferredoxin Yah1 and its human orthologue FDX2 in mitochondrial Fe/S cluster synthesis.

Alkylation Damage by Lipid Electrophiles Targets Functional Protein Systems*

PubMed Central

Codreanu, Simona G.; Ullery, Jody C.; Zhu, Jing; Tallman, Keri A.; Beavers, William N.; Porter, Ned A.; Marnett, Lawrence J.; Zhang, Bing; Liebler, Daniel C.

2014-01-01

Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions. PMID:24429493

Microbial symbionts affect Pisum sativum proteome and metabolome under Didymella pinodes infection.

PubMed

Desalegn, G; Turetschek, R; Kaul, H-P; Wienkoop, S

2016-06-30

The long cultivation of field pea led to an enormous diversity which, however, seems to hold just little resistance against the ascochyta blight disease complex. The potential of below ground microbial symbiosis to prime the immune system of Pisum for an upcoming pathogen attack has hitherto received little attention. This study investigates the effect of beneficial microbes on the leaf proteome and metabolome as well as phenotype characteristics of plants in various symbiont interactions (mycorrhiza, rhizobia, co-inoculation, non-symbiotic) after infestation by Didymella pinodes. In healthy plants, mycorrhiza and rhizobia induced changes in RNA metabolism and protein synthesis. Furthermore, metal handling and ROS dampening was affected in all mycorrhiza treatments. The co-inoculation caused the synthesis of stress related proteins with concomitant adjustment of proteins involved in lipid biosynthesis. The plant's disease infection response included hormonal adjustment, ROS scavenging as well as synthesis of proteins related to secondary metabolism. The regulation of the TCA, amino acid and secondary metabolism including the pisatin pathway, was most pronounced in rhizobia associated plants which had the lowest infection rate and the slowest disease progression. A most comprehensive study of the Pisum sativum proteome and metabolome infection response to Didymella pinodes is provided. Several distinct patterns of microbial symbioses on the plant metabolism are presented for the first time. Upon D. pinodes infection, rhizobial symbiosis revealed induced systemic resistance e.g. by an enhanced level of proteins involved in pisatin biosynthesis. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Biochemical localization of a protein involved in Gluconacetobacter hansenii cellulose synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iyer, Prashanti R; Catchmark, Jeffrey M; Brown, Nicole Robitaille

2011-02-08

Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.

Mechanical Stimulation Induces mTOR Signaling via an ERK-Independent Mechanism: Implications for a Direct Activation of mTOR by Phosphatidic Acid

PubMed Central

You, Jae Sung; Frey, John W.; Hornberger, Troy A.

2012-01-01

Signaling by mTOR is a well-recognized component of the pathway through which mechanical signals regulate protein synthesis and muscle mass. However, the mechanisms involved in the mechanical regulation of mTOR signaling have not been defined. Nevertheless, recent studies suggest that a mechanically-induced increase in phosphatidic acid (PA) may be involved. There is also evidence which suggests that mechanical stimuli, and PA, utilize ERK to induce mTOR signaling. Hence, we reasoned that a mechanically-induced increase in PA might promote mTOR signaling via an ERK-dependent mechanism. To test this, we subjected mouse skeletal muscles to mechanical stimulation in the presence or absence of a MEK/ERK inhibitor, and then measured several commonly used markers of mTOR signaling. Transgenic mice expressing a rapamycin-resistant mutant of mTOR were also used to confirm the validity of these markers. The results demonstrated that mechanically-induced increases in p70s6k T389 and 4E-BP1 S64 phosphorylation, and unexpectedly, a loss in total 4E-BP1, were fully mTOR-dependent signaling events. Furthermore, we determined that mechanical stimulation induced these mTOR-dependent events, and protein synthesis, through an ERK-independent mechanism. Similar to mechanical stimulation, exogenous PA also induced mTOR-dependent signaling via an ERK-independent mechanism. Moreover, PA was able to directly activate mTOR signaling in vitro. Combined, these results demonstrate that mechanical stimulation induces mTOR signaling, and protein synthesis, via an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR. Furthermore, it appears that a decrease in total 4E-BP1 may be part of the mTOR-dependent mechanism through which mechanical stimuli activate protein synthesis. PMID:23077579

R-Baclofen Reverses a Social Behavior Deficit and Elevated Protein Synthesis in a Mouse Model of Fragile X Syndrome

PubMed Central

Qin, Mei; Huang, Tianjian; Kader, Michael; Krych, Leland; Xia, Zengyan; Burlin, Thomas; Zeidler, Zachary; Zhao, Tingrui

2015-01-01

Background: Fragile X syndrome (FXS) is the most common known inherited form of intellectual disability and the single genomic cause of autism spectrum disorders. It is caused by the absence of a fragile X mental retardation gene (Fmr1) product, FMRP, an RNA-binding translation suppressor. Elevated rates of protein synthesis in the brain and an imbalance between synaptic signaling via glutamate and γ-aminobutyric acid (GABA) are both considered important in the pathogenesis of FXS. In a mouse model of FXS (Fmr1 knockout [KO]), treatment with R-baclofen reversed some behavioral and biochemical phenotypes. A remaining crucial question is whether R-baclofen is also able to reverse increased brain protein synthesis rates. Methods: To answer this question, we measured regional rates of cerebral protein synthesis in vivo with the L-[1-14C]leucine method in vehicle- and R-baclofen–treated wildtype and Fmr1 KO mice. We further probed signaling pathways involved in the regulation of protein synthesis. Results: Acute R-baclofen administration corrected elevated protein synthesis and reduced deficits on a test of social behavior in adult Fmr1 KO mice. It also suppressed activity of the mammalian target of rapamycin pathway, particularly in synaptosome-enriched fractions, but it had no effect on extracellular-regulated kinase 1/2 activity. Ninety min after R-baclofen treatment, we observed an increase in metabotropic glutamate receptor 5 expression in the frontal cortex, a finding that may shed light on the tolerance observed in human studies with this drug. Conclusions: Our results suggest that treatment via activation of the GABA (GABA receptor subtype B) system warrants further study in patients with FXS. PMID:25820841

Trophic Mode-Dependent Proteomic Analysis Reveals Functional Significance of Light-Independent Chlorophyll Synthesis in Synechocystis sp. PCC 6803.

PubMed

Fang, Longfa; Ge, Haitao; Huang, Xiahe; Liu, Ye; Lu, Min; Wang, Jinlong; Chen, Weiyang; Xu, Wu; Wang, Yingchun

2017-01-09

The photosynthetic model organism Synechocystis sp. PCC 6803 can grow in different trophic modes, depending on the availability of light and exogenous organic carbon source. However, how the protein profile changes to facilitate the cells differentially propagate in different modes has not been comprehensively investigated. Using isobaric labeling-based quantitative proteomics, we simultaneously identified and quantified 45% Synechocystis proteome across four different trophic modes, i.e., autotrophic, heterotrophic, photoheterotrophic, and mixotrophic modes. Among the 155 proteins that are differentially expressed across four trophic modes, proteins involved in nitrogen assimilation and light-independent chlorophyll synthesis are dramatically upregulated in the mixotrophic mode, concomitant with a dramatic increase of P II phosphorylation that senses carbon and nitrogen assimilation status. Moreover, functional study using a mutant defective in light-independent chlorophyll synthesis revealed that this pathway is important for chlorophyll accumulation under a cycled light/dark illumination regime, a condition mimicking day/night cycles in certain natural habitats. Collectively, these results provide the most comprehensive information on trophic mode-dependent protein expression in cyanobacterium, and reveal the functional significance of light-independent chlorophyll synthesis in trophic growth. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Role of G protein-coupled estrogen receptor-1 in estradiol 17β-induced alterations in protein synthesis and protein degradation rates in fused bovine satellite cell cultures.

PubMed

Kamanga-Sollo, E; Thornton, K J; White, M E; Dayton, W R

2017-01-01

In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters in several countries. Treatment with E2 stimulates protein synthesis rate and suppresses protein degradation rate in fused bovine satellite cell (BSC) cultures; however, the mechanisms involved in these effects are not known with certainty. Although the genomic effects of E2 mediated through the classical estrogen receptors have been characterized, recent studies indicate that binding of E2 to the G protein-coupled estrogen receptor (GPER)-1 mediates nongenomic effects of E2 on cellular function. Our current data show that inhibition of GPER-1, matrix metalloproteinases 2 and 9 (MMP2/9), or heparin binding epidermal growth factor-like growth factor (hbEGF) suppresses E2 stimulate protein synthesis rate in cultured BSCs (P < 0.001) suggesting that all of these are required in order for E2 to stimulate protein synthesis in these cultures. In contrast, inhibition of GPER-1, MMP2/9, or hbEGF has no effect on the ability of E2 to suppress protein degradation rates in fused BSC cultures indicating that these factors are not required in order for E2 to suppress protein degradation rate in these cells. Furthermore, treatment of fused BSC cultures with E2 increased (P < 0.05) pAKT levels indicating that the pAKT pathway may play a role in E2-stimulated effects on cultured BSC. In summary, our current data show that active GPER-1, MMP2/9, and hbEGF are necessary for E2-stimulated protein synthesis but not for E2-simulated suppression of protein degradation in cultured BSC. In addition, E2 treatment increases pAKT levels in cultured BSC. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5

PubMed Central

Yang, Yang; Zengel, James; Sun, Minghao; Sleeman, Katrina; Timani, Khalid Amine; Aligo, Jason; Rota, Paul

2015-01-01

ABSTRACT Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of vaccines and potential antiviral drugs. PMID:26378167

Structural approaches to the study of oligosaccharides in glycoprotein quality control.

PubMed

Ito, Yukishige; Hagihara, Shinya; Matsuo, Ichiro; Totani, Kiichiro

2005-10-01

High-mannose-type oligosaccharides have been shown to play important roles in protein quality control. Several intracellular proteins, such as lectins, chaperones and glycan-processing enzymes, are involved in this process. These include calnexin/calreticulin, UDP-glucose:glycoprotein glucosyltransferase (UGGT), cargo receptors (such as VIP36 and ERGIC-53), mannosidase-like proteins (e.g. EDEM and Htm1p) and ubiquitin ligase (Fbs). They are thought to recognize high-mannose-type glycans with subtly different structures, although the precise specificities are yet to be clarified. In order to gain a clear understanding of these protein-carbohydrate interactions, comprehensive synthesis of high-mannose-type glycans was conducted. In addition, two approaches to the synthesis of artificial glycoproteins with homogeneous oligosaccharides were investigated. Furthermore, a novel substrate of UGGT was discovered.

Characterization of proteins in soybean roots under flooding and drought stresses.

PubMed

Oh, MyeongWon; Komatsu, Setsuko

2015-01-30

Flooding and drought affect soybean growth because soybean is a stress-sensitive crop. In 2-day-old plants exposed to 2-day flooding or drought, the fresh weight of roots was markedly suppressed, although the root morphology clearly differed between two conditions. To understand the response mechanisms of soybean to flooding and drought stresses, a gel-free proteomic technique was used. A total of 97 and 48 proteins were significantly changed in response to flooding and drought stresses, respectively. Proteins involved in protein synthesis were decreased by flooding stress and increased by drought. Glycolysis-related proteins were increased in roots by both flooding and drought stresses. Fermentation, stress, and cell wall-related proteins were increased in response to flooding stress, whereas cell organization and redox-related proteins were increased under drought stress. Among the identified proteins, three S-adenosylmethionine synthetases were commonly decreased and increased in response to flooding and drought stresses, respectively. The mRNA expression levels of S-adenosylmethionine synthetase genes displayed a similar tendency to the changes in protein abundance. These results suggest that S-adenosylmethionine synthetase is involved in the regulation of stress response because it was changed in response to flooding and drought stresses. This study reported on the response mechanisms of soybean to flooding and drought stresses using the gel-free proteomic technique. Proteins involved in protein synthesis were decreased by flooding stress and increased by drought. Glycolysis-related proteins were increased in roots by both flooding and drought stresses. Fermentation, stress, and cell wall-related proteins were increased in response to flooding stress, whereas cell organization and redox-related proteins were increased under drought stress. Among the identified proteins, three S-adenosylmethionine synthetases were commonly decreased and increased in response to flooding and drought stresses, respectively. The mRNA expression levels of S-adenosylmethionine synthetase genes displayed a similar tendency to the changes in protein abundance. These results suggest that S-adenosylmethionine synthetase is involved in the regulation of stress response because it was changed in response to flooding and drought stresses. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein turnover, nitrogen balance and rehabilitation.

PubMed

Fern, E B; Waterlow, J C

1983-01-01

Not many studies have been done on protein turnover during recovery from malnutrition. Some relevant information can, however, be obtained from measurements on normal growing animals, since rehabilitation and normal growth have in common a rapid rate of net protein synthesis. The key question is the extent to which net gain in protein results from an increase in synthesis or a decrease in breakdown or both. Different studies have used different methods, and all methods for measuring protein turnover have some disadvantages and sources of error. It is important to bear this in mind in evaluating the results. Consequently, part of this paper will be devoted to questions of methodology. Whole body protein turnover has been measured in children recovering from severe malnutrition. During the phase of rapid catch-up growth the rate of protein synthesis is increased. As might be expected, it increases linearly with the rate of weight gain. At the same time there is a smaller increase in the rate of protein breakdown. The resultant of these two processes is that, over and above the basal rate of protein synthesis, 1.4 grams of protein have to be synthesized for 1 gram to be laid down. Very similar results have been obtained in rapidly growing young pigs. Experimental studies on muscle growth in general confirm the conclusion that, at least in muscle, rapid growth is associated with rapid rates of protein breakdown as well as of synthesis. This has been shown in muscles of young growing rats, as well as in muscles in which hypertrophy has been induced by stretch or other stimuli. In contrast, the evidence suggests that rapid growth involves a fall in the rate of protein degradation. The magnitude of the nitrogen balance under any conditions is determined by the difference between synthesis and breakdown. In the absence of any storage of amino acids, this must be the same as the difference between intake and excretion (S - B = I - E). A question of great interest is whether, at a given intake, the extent of N balance is determined primarily by regulation of synthesis and breakdown or by regulation of amino acid oxidation. Clearly, a reduction in amino acid degradation is equivalent to an increase in amino acid intake. An interesting subject for future research is the extent to which the amino acid degrading enzymes adapt to the requirements imposed by growth and rehabilitation.

Metabolic reprogramming in cancer cells: glycolysis, glutaminolysis, and Bcl-2 proteins as novel therapeutic targets for cancer.

PubMed

Li, Chunxia; Zhang, Guifeng; Zhao, Lei; Ma, Zhijun; Chen, Hongbing

2016-01-20

Nearly a century ago, Otto Warburg made the ground-breaking observation that cancer cells, unlike normal cells, prefer a seemingly inefficient mechanism of glucose metabolism: aerobic glycolysis, a phenomenon now referred to as the Warburg effect. The finding that rapidly proliferating cancer cells favors incomplete metabolism of glucose, producing large amounts of lactate as opposed to synthesizing ATP to sustain cell growth, has confounded scientists for years. Further investigation into the metabolic phenotype of cancer has expanded our understanding of this puzzling conundrum, and has opened new avenues for the development of anti-cancer therapies. Enhanced glycolytic flux is now known to allow for increased synthesis of intermediates for sustaining anabolic pathways critical for cancer cell growth. Alongside the increase in glycolysis, cancer cells transform their mitochondria into synthesis machines supported by augmented glutaminolysis, supplying lipid production, amino acid synthesis, and the pentose phosphate pathways. Inhibition of several of the key enzymes involved in these pathways has been demonstrated to effectively obstruct cancer cell growth and multiplication, sensitizing them to apoptosis. The modulation of various regulatory proteins involved in metabolic processes is central to cancerous reprogramming of metabolism. The finding that members of one of the major protein families involved in cell death regulation also aberrantly regulated in cancers, the Bcl-2 family of proteins, are also critical mediators of metabolic pathways, provides strong evidence for the importance of the metabolic shift to cancer cell survival. Targeting the anti-apoptotic members of the Bcl-2 family of proteins is proving to be a successful way to selectively target cancer cells and induce apoptosis. Further understanding of how cancer cells modify metabolic regulation to increase channeling of substrates into biosynthesis will allow for the discovery of novel drug targets to treat cancer. In the present review, we focused on the recent developments in therapeutic targeting of different steps in glycolysis, glutaminolysis and on the metabolic regulatory role of Bcl-2 family proteins.

Ndrg2 is a PGC-1α/ERRα target gene that controls protein synthesis and expression of contractile-type genes in C2C12 myotubes.

PubMed

Foletta, Victoria C; Brown, Erin L; Cho, Yoshitake; Snow, Rod J; Kralli, Anastasia; Russell, Aaron P

2013-12-01

The stress-responsive, tumor suppressor N-myc downstream-regulated gene 2 (Ndrg2) is highly expressed in striated muscle. In response to anabolic and catabolic signals, Ndrg2 is suppressed and induced, respectively, in mouse C2C12 myotubes. However, little is known about the mechanisms regulating Ndrg2 expression in muscle, as well as the biological role for Ndrg2 in differentiated myotubes. Here, we show that Ndrg2 is a target of a peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and estrogen-related receptor alpha (ERRα) transcriptional program and is induced in response to endurance exercise, a physiological stress known also to increase PGC-1α/ERRα activity. Analyses of global gene and protein expression profiles in C2C12 myotubes with reduced levels of NDRG2, suggest that NDRG2 affects muscle growth, contractile properties, MAPK signaling, ion and vesicle transport and oxidative phosphorylation. Indeed, suppression of NDRG2 in myotubes increased protein synthesis and the expression of fast glycolytic myosin heavy chain isoforms, while reducing the expression of embryonic myosin Myh3, other contractile-associated genes and the MAPK p90 RSK1. Conversely, enhanced expression of NDRG2 reduced protein synthesis, and furthermore, partially blocked the increased protein synthesis rates elicited by a constitutively active form of ERRα. In contrast, suppressing or increasing levels of NDRG2 did not affect mRNA expression of genes involved in mitochondrial biogenesis that are regulated by PGC-1α or ERRα. This study shows that in C2C12 myotubes Ndrg2 is a novel PGC-1α/ERRα transcriptional target, which influences protein turnover and the regulation of genes involved in muscle contraction and function. © 2013 Elsevier B.V. All rights reserved.

Interaction Network and Localization of Brucella abortus Membrane Proteins Involved in the Synthesis, Transport, and Succinylation of Cyclic β-1,2-Glucans

PubMed Central

Guidolin, Leticia S.; Morrone Seijo, Susana M.; Guaimas, Francisco F.

2015-01-01

ABSTRACT Cyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that play an important role in the virulence and interaction of Brucella with the host. Once synthesized in the cytoplasm by the CβG synthase (Cgs), CβG are transported to the periplasm by the CβG transporter (Cgt) and succinylated by the CβG modifier enzyme (Cgm). Here, we used a bacterial two-hybrid system and coimmunoprecipitation techniques to study the interaction network between these three integral inner membrane proteins. Our results indicate that Cgs, Cgt, and Cgm can form both homotypic and heterotypic interactions. Analyses carried out with Cgs mutants revealed that the N-terminal region of the protein (Cgs region 1 to 418) is required to sustain the interactions with Cgt and Cgm as well as with itself. We demonstrated by single-cell fluorescence analysis that in Brucella, Cgs and Cgt are focally distributed in the membrane, particularly at the cell poles, whereas Cgm is mostly distributed throughout the membrane with a slight accumulation at the poles colocalizing with the other partners. In summary, our results demonstrate that Cgs, Cgt, and Cgm form a membrane-associated biosynthetic complex. We propose that the formation of a membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating their synthesis with the transport and modification. IMPORTANCE In this study, we analyzed the interaction and localization of the proteins involved in the synthesis, transport, and modification of Brucella abortus cyclic β-1,2-glucans (CβG), which play an important role in the virulence and interaction of Brucella with the host. We demonstrate that these proteins interact, forming a complex located mainly at the cell poles; this is the first experimental evidence of the existence of a multienzymatic complex involved in the metabolism of osmoregulated periplasmic glucans in bacteria and argues for another example of pole differentiation in Brucella. We propose that the formation of this membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating synthesis with the transport and modification. PMID:25733613

Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms

PubMed Central

Adams, Nancy E.; Maurelli, Anthony T.

2015-01-01

Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB–RB transitions. PMID:25998263

Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms.

PubMed

Fisher, Derek J; Adams, Nancy E; Maurelli, Anthony T

2015-08-01

Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB-RB transitions.

Binding domain-driven intracellular trafficking of sterols for synthesis of steroid hormones, bile acids and oxysterols.

PubMed

Midzak, Andrew; Papadopoulos, Vassilios

2014-09-01

Steroid hormones, bioactive oxysterols and bile acids are all derived from the biological metabolism of lipid cholesterol. The enzymatic pathways generating these compounds have been an area of intense research for almost a century, as cholesterol and its metabolites have substantial impacts on human health. Owing to its high degree of hydrophobicity and the chemical properties that it confers to biological membranes, the distribution of cholesterol in cells is tightly controlled, with subcellular organelles exhibiting highly divergent levels of cholesterol. The manners in which cells maintain such sterol distributions are of great interest in the study of steroid and bile acid synthesis, as limiting cholesterol substrate to the enzymatic pathways is the principal mechanism by which production of steroids and bile acids is regulated. The mechanisms by which cholesterol moves within cells, however, remain poorly understood. In this review, we examine the subcellular machinery involved in cholesterol metabolism to steroid hormones and bile acid, relating it to both lipid- and protein-based mechanisms facilitating intracellular and intraorganellar cholesterol movement and delivery to these pathways. In particular, we examine evidence for the involvement of specific protein domains involved in cholesterol binding, which impact cholesterol movement and metabolism in steroidogenesis and bile acid synthesis. A better understanding of the physical mechanisms by which these protein- and lipid-based systems function is of fundamental importance to understanding physiological homeostasis and its perturbation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The Consolidation of Object and Context Recognition Memory Involve Different Regions of the Temporal Lobe

ERIC Educational Resources Information Center

Balderas, Israela; Rodriguez-Ortiz, Carlos J.; Salgado-Tonda, Paloma; Chavez-Hurtado, Julio; McGaugh, James L.; Bermudez-Rattoni, Federico

2008-01-01

These experiments investigated the involvement of several temporal lobe regions in consolidation of recognition memory. Anisomycin, a protein synthesis inhibitor, was infused into the hippocampus, perirhinal cortex, insular cortex, or basolateral amygdala of rats immediately after the sample phase of object or object-in-context recognition memory…

Proteomic analysis of Arabidopsis thaliana leaves in response to acute boron deficiency and toxicity reveals effects on photosynthesis, carbohydrate metabolism, and protein synthesis.

PubMed

Chen, Mei; Mishra, Sasmita; Heckathorn, Scott A; Frantz, Jonathan M; Krause, Charles

2014-02-15

Boron (B) stress (deficiency and toxicity) is common in plants, but as the functions of this essential micronutrient are incompletely understood, so too are the effects of B stress. To investigate mechanisms underlying B stress, we examined protein profiles in leaves of Arabidopsis thaliana plants grown under normal B (30 μM), compared to plants transferred for 60 and 84 h (i.e., before and after initial visible symptoms) in deficient (0 μM) or toxic (3 mM) levels of B. B-responsive polypeptides were sequenced by mass spectrometry, following 2D gel electrophoresis, and 1D gels and immunoblotting were used to confirm the B-responsiveness of some of these proteins. Fourteen B-responsive proteins were identified, including: 9 chloroplast proteins, 6 proteins of photosynthetic/carbohydrate metabolism (rubisco activase, OEC23, photosystem I reaction center subunit II-1, ATPase δ-subunit, glycolate oxidase, fructose bisphosphate aldolase), 6 stress proteins, and 3 proteins involved in protein synthesis (note that the 14 proteins may fall into multiple categories). Most (8) of the B-responsive proteins decreased under both B deficiency and toxicity; only 3 increased with B stress. Boron stress decreased, or had no effect on, 3 of 4 oxidative stress proteins examined, and did not affect total protein. Hence, our results indicate relatively early specific effects of B stress on chloroplasts and protein synthesis. Copyright © 2013 Elsevier GmbH. All rights reserved.

Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis

PubMed Central

Alamgir, Md; Eroukova, Veronika; Jessulat, Matthew; Xu, Jianhua; Golshani, Ashkan

2008-01-01

Background Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s) for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, ~4700 strains) for increased sensitivity to paromomycin, which targets the process of protein synthesis. Results As expected, our analysis indicated that the majority of deletion strains (134) with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45), cellular component biogenesis and organization (28), DNA maintenance (21), transport (20), others (38) and unknown (39). These may represent minor cellular target sites (side-effects) for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis. Conclusion We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s). PMID:19055778

Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis.

PubMed

Alamgir, Md; Eroukova, Veronika; Jessulat, Matthew; Xu, Jianhua; Golshani, Ashkan

2008-12-03

Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s) for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, approximately 4700 strains) for increased sensitivity to paromomycin, which targets the process of protein synthesis. As expected, our analysis indicated that the majority of deletion strains (134) with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45), cellular component biogenesis and organization (28), DNA maintenance (21), transport (20), others (38) and unknown (39). These may represent minor cellular target sites (side-effects) for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis. We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s).

Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure.

PubMed

Létoquart, Juliette; van Tran, Nhan; Caroline, Vonny; Aleksandrov, Alexey; Lazar, Noureddine; van Tilbeurgh, Herman; Liger, Dominique; Graille, Marc

2015-12-15

Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm(5)U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

PubMed Central

Jacinto-Loeza, Eva; Vivanco-Domínguez, Serafín; Guarneros, Gabriel; Hernández-Sánchez, Javier

2008-01-01

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 μg/μl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA. PMID:18583364

Soya protein stimulates bile acid excretion by the liver and intestine through direct and indirect pathways influenced by the presence of dietary cholesterol.

PubMed

Arellano-Martínez, Gloria Leticia; Granados, Omar; Palacios-González, Berenice; Torres, Nimbe; Medina-Vera, Isabel; Tovar, Armando R

2014-06-28

Several studies using different animal models have demonstrated that the consumption of soya protein (SP) reduces serum cholesterol concentrations by increasing the excretion of bile acids (BA). However, the mechanism by which SP enhances BA excretion is not fully understood. Therefore, the aim of the present study was to determine whether the consumption of SP regulates the expression of key enzymes involved in hepatic BA synthesis and the transporters involved in reverse cholesterol transport (RCT) via fibroblast growth factor 15 (FGF15) and/or small heterodimer protein (SHP) in rats. To achieve this aim, four groups of rats were fed experimental diets containing 20 % casein (C) or SP with or without the addition of 0·2 % cholesterol and the expression of hepatic genes involved in BA synthesis and the ileal and hepatic RCT was measured. Rats fed the SP diet had higher concentrations of ileal FGF15 and hepatic FGF15 receptor (FGFR4) and increased expression of SHP and liver receptor homolog 1 (LRH1) than those fed the C diet; as a result, the excretion of faecal BA was greater. The addition of cholesterol to the diet repressed the protein abundance of FGF15 and FGFR4; however, SP increased the expression of SHP and LRH1 to a lesser extent. Nonetheless, the expression of ABCG5/8 was increased in the intestine of rats fed the SP diet, and the effect was enhanced by the addition of cholesterol to the diet. In conclusion, SP in the presence of cholesterol increases BA synthesis via the repressions of FGF15 and SHP and accelerates BA excretion to prevent cholesterol overload in the enterocytes by increasing RCT.

Heat-induced accumulation of protein synthesis elongation factor 1A indicates an important role in heat tolerance in potato

USDA-ARS?s Scientific Manuscript database

Heat stress substantially reduces crop productivity worldwide, and will become more severe due to global warming. Identification of proteins involved in heat stress response may help develop varieties for heat tolerance. Eukaryotic elongation factor 1A (eEF1A) is a cytosolic, multifunctional protei...

The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates.

PubMed

Matts, R L; Hurst, R

1992-09-05

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.

Microarray Analyses of Gene Expression during Adventitious Root Development in Pinus contorta1[w

PubMed Central

Brinker, Monika; van Zyl, Leonel; Liu, Wenbin; Craig, Deborah; Sederoff, Ronald R.; Clapham, David H.; von Arnold, Sara

2004-01-01

In order to investigate the gene expression pattern during adventitious root development, RNA of Pinus contorta hypocotyls, pulse-treated with the auxin indole-3-butyric acid and harvested at distinct developmental time points of root development, was hybridized to microarrays containing 2,178 cDNAs from Pinus taeda. Over the period of observation of root development, the transcript levels of 220 genes changed significantly. During the root initiation phase, genes involved in cell replication and cell wall weakening and a transcript encoding a PINHEAD/ZWILLE-like protein were up-regulated, while genes related to auxin transport, photosynthesis, and cell wall synthesis were down-regulated. In addition, there were changes in transcript abundance of genes related to water stress. During the root meristem formation phase the transcript abundances of genes involved in auxin transport, auxin responsive transcription, and cell wall synthesis, and of a gene encoding a B-box zinc finger-like protein, increased, while those encoding proteins involved in cell wall weakening decreased. Changes of transcript abundance of genes related to water stress during the root meristem formation and root formation phase indicate that the plant roots had become functional in water transport. Simultaneously, genes involved in auxin transport were up-regulated, while genes related to cell wall modification were down-regulated. Finally, during the root elongation phase down-regulation of transcripts encoding proteins involved in cell replication and stress occurred. Based on the observed changes in transcript abundances, we suggest hypotheses about the relative importance of various physiological processes during the auxin-induced development of roots in P. contorta. PMID:15247392

Hydrogen sulfide ameliorates aging-associated changes in the kidney.

PubMed

Lee, Hak Joo; Feliers, Denis; Barnes, Jeffrey L; Oh, Sae; Choudhury, Goutam Ghosh; Diaz, Vivian; Galvan, Veronica; Strong, Randy; Nelson, James; Salmon, Adam; Kevil, Christopher G; Kasinath, Balakuntalam S

2018-04-01

Aging is associated with replacement of normal kidney parenchyma by fibrosis. Because hydrogen sulfide (H 2 S) ameliorates kidney fibrosis in disease models, we examined its status in the aging kidney. In the first study, we examined kidney cortical H 2 S metabolism and signaling pathways related to synthesis of proteins including matrix proteins in young and old male C57BL/6 mice. In old mice, increase in renal cortical content of matrix protein involved in fibrosis was associated with decreased H 2 S generation and AMPK activity, and activation of insulin receptor (IR)/IRS-2-Akt-mTORC1-mRNA translation signaling axis that can lead to increase in protein synthesis. In the second study, we randomized 18-19 month-old male C57BL/6 mice to receive 30 μmol/L sodium hydrosulfide (NaHS) in drinking water vs. water alone (control) for 5 months. Administration of NaHS increased plasma free sulfide levels. NaHS inhibited the increase in kidney cortical content of matrix proteins involved in fibrosis and ameliorated glomerulosclerosis. NaHS restored AMPK activity and inhibited activation of IR/IRS-2-Akt-mTORC1-mRNA translation axis. NaHS inhibited age-related increase in kidney cortical content of p21, IL-1β, and IL-6, components of the senescence-associated secretory phenotype. NaHS abolished increase in urinary albumin excretion seen in control mice and reduced serum cystatin C levels suggesting improved glomerular clearance function. We conclude that aging-induced changes in the kidney are associated with H 2 S deficiency. Administration of H 2 S ameliorates aging-induced kidney changes probably by inhibiting signaling pathways leading to matrix protein synthesis.

Dendritic spine density and EphrinB2 levels of hippocampal and anterior cingulate cortex neurons increase sequentially during formation of recent and remote fear memory in the mouse.

PubMed

Abate, Georgia; Colazingari, Sandra; Accoto, Alessandra; Conversi, David; Bevilacqua, Arturo

2018-05-15

Memory consolidation is a dynamic process that involves a sequential remodeling of hippocampal-cortical circuits. Although synaptic events underlying memory consolidation are well assessed, fine molecular events controlling this process deserve further characterization. To this aim, we challenged male C57BL/6N mice in a contextual fear conditioning (CFC) paradigm and tested their memory 24 h, 7 days or 36 days later. Mice displayed a strong fear response at all time points with an increase in dendritic spine density and protein levels of the cell adhesion factor EphrinB2 in CA1 hippocampal neurons 24 h and 7 days post conditioning (p.c.), and in anterior cingulate cortex (ACC) neurons 36 days p.c. We then investigated whether the formation of remote memory and neuronal modifications in the ACC would depend on p.c. protein synthesis in hippocampal neurons. Bilateral intrahippocampal infusions with the protein synthesis inhibitor anisomycin administered immediately p.c. decreased fear response, neuronal spine growth and EphrinB2 protein levels of hippocampal and ACC neurons 24 h and 36 days p.c., respectively. Anisomycin infusion 24 h p.c. had no effects on fear response, increase in spine density and in EphrinB2 protein levels in ACC neurons 36 days p.c. Our results thus confirm that early but not late p.c. hippocampal protein synthesis is necessary for the formation of remote memory and provide the first evidence of a possible involvement of EphrinB2 in neuronal plasticity in the ACC. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of Fragile X Syndrome-Specific Molecular Markers in Human Fibroblasts: A Useful Model to Test the Efficacy of Therapeutic Drugs

PubMed Central

Kumari, Daman; Bhattacharya, Aditi; Nadel, Jeffrey; Moulton, Kristen; Zeak, Nicole M.; Glicksman, Anne; Dobkin, Carl; Brick, David J.; Schwartz, Philip H.; Smith, Carolyn B.; Klann, Eric; Usdin, Karen

2014-01-01

Fragile X Syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, FMRP, an RNA-binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout (KO) mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated extracellular signal regulated kinase 1/2 (p-ERK 1/2) and phosphorylated p70 ribosomal S6 kinase 1 (p-S6K1). Treatment with small molecules that inhibit S6K1, and a known FMRP target, phosphoinositide 3-kinase (PI3K) catalytic subunit p110β, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches. PMID:25224527

Nucleic acids encoding plant glutamine phenylpyruvate transaminase (GPT) and uses thereof

DOEpatents

Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

2016-03-29

Glutamine phenylpyruvate transaminase (GPT) proteins, nucleic acid molecules encoding GPT proteins, and uses thereof are disclosed. Provided herein are various GPT proteins and GPT gene coding sequences isolated from a number of plant species. As disclosed herein, GPT proteins share remarkable structural similarity within plant species, and are active in catalyzing the synthesis of 2-hydroxy-5-oxoproline (2-oxoglutaramate), a powerful signal metabolite which regulates the function of a large number of genes involved in the photosynthesis apparatus, carbon fixation and nitrogen metabolism.

Ribosomal Protein S6 Phosphorylation Is Involved in Novelty-Induced Locomotion, Synaptic Plasticity and mRNA Translation

PubMed Central

Puighermanal, Emma; Biever, Anne; Pascoli, Vincent; Melser, Su; Pratlong, Marine; Cutando, Laura; Rialle, Stephanie; Severac, Dany; Boubaker-Vitre, Jihane; Meyuhas, Oded; Marsicano, Giovanni; Lüscher, Christian; Valjent, Emmanuel

2017-01-01

The phosphorylation of the ribosomal protein S6 (rpS6) is widely used to track neuronal activity. Although it is generally assumed that rpS6 phosphorylation has a stimulatory effect on global protein synthesis in neurons, its exact biological function remains unknown. By using a phospho-deficient rpS6 knockin mouse model, we directly tested the role of phospho-rpS6 in mRNA translation, plasticity and behavior. The analysis of multiple brain areas shows for the first time that, in neurons, phospho-rpS6 is dispensable for overall protein synthesis. Instead, we found that phospho-rpS6 controls the translation of a subset of mRNAs in a specific brain region, the nucleus accumbens (Acb), but not in the dorsal striatum. We further show that rpS6 phospho-mutant mice display altered long-term potentiation (LTP) in the Acb and enhanced novelty-induced locomotion. Collectively, our findings suggest a previously unappreciated role of phospho-rpS6 in the physiology of the Acb, through the translation of a selective subclass of mRNAs, rather than the regulation of general protein synthesis. PMID:29311811

Synthesis of chlorophyll b: Localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit

PubMed Central

Eggink, Laura L; LoBrutto, Russell; Brune, Daniel C; Brusslan, Judy; Yamasato, Akihiro; Tanaka, Ayumi; Hoober, J Kenneth

2004-01-01

Background Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly. Results Fluorescence of a CAO-GFP fusion protein, transiently expressed in young pea leaves, was found at the periphery of mature chloroplasts and on thylakoid membranes by confocal fluorescence microscopy. However, when membranes from partially degreened cells of Chlamydomonas reinhardtii cw15 were resolved on sucrose gradients, full-length CAO was detected by immunoblot analysis only on the chloroplast envelope inner membrane. The electron paramagnetic resonance spectrum of CAO included a resonance at g = 4.3, assigned to the predicted mononuclear iron center. Instead of a spectrum of the predicted Rieske iron-sulfur center, a nearly symmetrical, approximately 100 Gauss peak-to-trough signal was observed at g = 2.057, with a sensitivity to temperature characteristic of an iron-sulfur center. A remarkably stable radical in the protein was revealed by an isotropic, 9 Gauss peak-to-trough signal at g = 2.0042. Fragmentation of the protein after incorporation of 125I- identified a conserved tyrosine residue (Tyr-422 in Chlamydomonas and Tyr-518 in Arabidopsis) as the radical species. The radical was quenched by chlorophyll a, an indication that it may be involved in the enzymatic reaction. Conclusion CAO was found on the chloroplast envelope and thylakoid membranes in mature chloroplasts but only on the envelope inner membrane in dark-grown C. reinhardtii cells. Such localization provides further support for the envelope membranes as the initial site of Chl b synthesis and assembly of LHCs during chloroplast development. Identification of a tyrosine radical in the protein provides insight into the mechanism of Chl b synthesis. PMID:15086960

mTORC1 controls long-term memory retrieval.

PubMed

Pereyra, Magdalena; Katche, Cynthia; de Landeta, Ana Belén; Medina, Jorge H

2018-06-08

Understanding how stored information emerges is a main question in the neurobiology of memory that is now increasingly gaining attention. However, molecular events underlying this memory stage, including involvement of protein synthesis, are not well defined. Mammalian target of rapamycin complex 1 (mTORC1), a central regulator of protein synthesis, has been implicated in synaptic plasticity and is required for memory formation. Using inhibitory avoidance (IA), we evaluated the role of mTORC1 in memory retrieval. Infusion of a selective mTORC1 inhibitor, rapamycin, into the dorsal hippocampus 15 or 40 min but not 3 h before testing at 24 h reversibly disrupted memory expression even in animals that had already expressed IA memory. Emetine, a general protein synthesis inhibitor, provoked a similar impairment. mTORC1 inhibition did not interfere with short-term memory retrieval. When infused before test at 7 or 14 but not at 28 days after training, rapamycin impaired memory expression. mTORC1 blockade in retrosplenial cortex, another structure required for IA memory, also impaired memory retention. In addition, pretest intrahippocampal rapamycin infusion impaired object location memory retrieval. Our results support the idea that ongoing protein synthesis mediated by activation of mTORC1 pathway is necessary for long but not for short term memory.

Identification of a Hyphantria cunea nucleopolyhedrovirus (NPV) gene that is involved in global protein synthesis shutdown and restricted Bombyx mori NPV multiplication in a B. mori cell line.

PubMed

Shirata, Noriko; Ikeda, Motoko; Kobayashi, Michihiro

2010-03-15

We previously demonstrated that Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication is restricted in permissive BmN-4 cells upon coinfection with Hyphantria cunea NPV (HycuNPV). Here, we show that HycuNPV-encoded hycu-ep32 gene is responsible for the restricted BmNPV multiplication in HycuNPV-coinfected BmN-4 cells. The only homologue for hycu-ep32 is in Orgyia pseudotsugata NPV. hycu-ep32 could encode a polypeptide of 312 amino acids, and it contains no characteristic domains or motifs to suggest its possible functions. hycu-ep32 is an early gene, and Hycu-EP32 expression reaches a maximum by 6 h postinfection. hycu-ep32-defective HycuNPV, vHycuDeltaep32, was generated, indicating that hycu-ep32 is nonessential in permissive SpIm cells. In BmN-4 cells, HycuNPV infection resulted in a severe global protein synthesis shutdown, while vHycuDeltaep32 did not cause any specific protein synthesis shutdown. These results indicate that the restriction of BmNPV multiplication by HycuNPV is caused by a global protein synthesis shutdown induced by hycu-ep32 upon coinfection with HycuNPV. Copyright 2009 Elsevier Inc. All rights reserved.

Concentration dependent requirement for local protein synthesis in motor neuron subtype specific response to axon guidance cues

PubMed Central

Nedelec, Stephane; Peljto, Mirza; Shi, Peng; Amoroso, Mackenzie W.; Kam, Lance C.; Wichterle, Hynek

2012-01-01

Formation of functional motor circuits relies on the ability of distinct spinal motor neuron subtypes to project their axons with high precision to appropriate muscle targets. While guidance cues contributing to motor axon pathfinding have been identified, the intracellular pathways underlying subtype specific responses to these cues remain poorly understood. In particular, it remains controversial whether responses to axon guidance cues depend on axonal protein synthesis. Using a growth cone collapse assay, we demonstrate that mouse embryonic stem cell (ESC) derived spinal motor neurons (ES-MNs) respond to ephrin-A5, Sema3f and Sema3a in a concentration dependent manner. At low doses, ES-MNs exhibit segmental or subtype specific responses, while this selectivity is lost at higher concentrations. Response to high doses of semaphorins and to all doses of ephrin-A5 is protein synthesis independent. In contrast, using microfluidic devices and stripe assays, we show that growth cone collapse and guidance at low concentrations of semaphorins relies on local protein synthesis in the axonal compartment. Similar bimodal response to low and high concentrations of guidance cues is observed in human ES-MNs, pointing to a general mechanism by which neurons increase their repertoire of responses to the limited set of guidance cues involved in neural circuit formation. PMID:22279234

pelo Is Required for High Efficiency Viral Replication

PubMed Central

Wu, Xiurong; He, Wan-Ting; Tian, Shuye; Meng, Dan; Li, Yuanyue; Chen, Wanze; Li, Lisheng; Tian, Lili; Zhong, Chuan-Qi; Han, Felicia; Chen, Jianming; Han, Jiahuai

2014-01-01

Viruses hijack host factors for their high speed protein synthesis, but information about these factors is largely unknown. In searching for genes that are involved in viral replication, we carried out a forward genetic screen for Drosophila mutants that are more resistant or sensitive to Drosophila C virus (DCV) infection-caused death, and found a virus-resistant line in which the expression of pelo gene was deficient. Our mechanistic studies excluded the viral resistance of pelo deficient flies resulting from the known Drosophila anti-viral pathways, and revealed that pelo deficiency limits the high level synthesis of the DCV capsid proteins but has no or very little effect on the expression of some other viral proteins, bulk cellular proteins, and transfected exogenous genes. The restriction of replication of other types of viruses in pelo deficient flies was also observed, suggesting pelo is required for high level production of capsids of all kinds of viruses. We show that both pelo deficiency and high level DCV protein synthesis increase aberrant 80S ribosomes, and propose that the preferential requirement of pelo for high level synthesis of viral capsids is at least partly due to the role of pelo in dissociation of stalled 80S ribosomes and clearance of aberrant viral RNA and proteins. Our data demonstrated that pelo is a host factor that is required for high efficiency translation of viral capsids and targeting pelo could be a strategy for general inhibition of viral infection. PMID:24722736

Post-retrieval late process contributes to persistence of reactivated fear memory.

PubMed

Nakayama, Daisuke; Yamasaki, Yoshiko; Matsuki, Norio; Nomura, Hiroshi

2013-05-16

Several studies have demonstrated the mechanisms involved in memory persistence after learning. However, little is known about memory persistence after retrieval. In this study, a protein synthesis inhibitor, anisomycin, was infused into the basolateral amygdala of mice 9.5 h after retrieval of contextual conditioned fear. Anisomycin attenuated fear memory after 7 d, but not after 2 d. In contrast, infusion of anisomycin 5- or 24-h post-retrieval was ineffective. These findings indicate that anisomycin attenuates the persistence of reactivated fear memory in a time-dependent manner. We propose that late protein synthesis is required for memory persistence after retrieval.

Oxime-based linker libraries as a general approach for the rapid generation and screening of multidentate inhibitors

PubMed Central

Bahta, Medhanit; Liu, Fa; Kim, Sung-Eun; Stephen, Andrew G.; Fisher, Robert J.; Burke, Terrence R.

2013-01-01

The described oxime-based library protocol provides detailed procedures for the linkage of aminooxy functionality with aldehyde building blocks that result in the generation of libraries of multidentate inhibitors. Synthesis of inhibitors for protein tyrosine phosphatases (PTPs) and antagonists directed against the human tumor susceptibility gene 101 (Tsg101) are shown as examples. Three steps are involved: a) the design and synthesis of aminooxy platforms; b) tethering with aldehydes to form oxime-based linkages with sufficient purity; and c) direct in vitro biological evaluation of oxime products without purification. Each coupling reaction is a) performed in capped microtubes at room temperature; b) diluted for inhibitory evaluation and c) screened with targets in microplates to provide IC50 or Kd values. The synthesis of the aminooxy platforms takes 3–5 days; tethering with the aldehydes takes 24 h; and inhibition assay of enzymes and protein-protein interactions (PPIs) takes 30 min and 2 h respectively. PMID:22422315

Proteomic analysis of common bean stem under drought stress using in-gel stable isotope labeling.

PubMed

Zadražnik, Tanja; Egge-Jacobsen, Wolfgang; Meglič, Vladimir; Šuštar-Vozlič, Jelka

2017-02-01

Drought is an abiotic stress that strongly influences plant growth, development and productivity. Proteome changes in the stem of the drought-tolerant common bean (Phaseolus vulgaris L.) cultivar Tiber have were when the plants were exposed to drought. Five-week-old plants were subjected to water deficit by withholding irrigation for 7, 12 and 17days, whereas control plants were regularly irrigated. Relative water content (RWC) of leaves, as an indicator of the degree of cell and tissue hydration, showed the highest statistically significant differences between control and drought-stressed plants after 17days of treatment, where RWC remained at 90% for control and declined to 45% for stressed plants. Plants exposed to drought for 17days and control plants at the same developmental stage were included in quantitative proteomic analysis using in-gel stable isotope labeling of proteins in combination with mass spectrometry. The quantified proteins were grouped into several functional groups, mainly into energy metabolism, photosynthesis, proteolysis, protein synthesis and proteins related to defense and stress. 70kDa heat shock protein showed the greatest increase in abundance under drought of all the proteins, suggesting its role in protecting plants against stress by re-establishing normal protein conformations and thus cellular homeostasis. The abundance of proteins involved in protein synthesis also increased under drought stress, important for recovery of damaged proteins involved in the plant cell's metabolic activities. Other important proteins in this study were related to proteolysis and folding, which are necessary for maintaining proper cellular protein homeostasis. Taken together, these results reveal the complexity of pathways involved in the drought stress response in common bean stems and enable comparison with the results of proteomic analysis of leaves, thus providing important information to further understand the biochemical and molecular mechanisms of drought response in this important legume. Copyright © 2016 Elsevier GmbH. All rights reserved.

Glucocorticoids Enhance Muscle Proteolysis through a Myostatin-Dependent Pathway at the Early Stage.

PubMed

Wang, Ruxia; Jiao, Hongchao; Zhao, Jingpeng; Wang, Xiaojuan; Lin, Hai

2016-01-01

Myostatin, a member of the TGF-β superfamily of secreted proteins, is expressed primarily in skeletal muscle. It negatively regulates muscle mass and is associated with glucocorticoid-induced muscle atrophy. However, it remains unclear whether myostatin is involved in glucocorticoid-induced muscle protein turnover. The aim of the present study was to investigate the role of myostatin in protein metabolism during dexamethasone (DEX) treatment. Protein synthesis rates and the expression of the genes for myostatin, ubiquitin-proteasome atrogin-1, MuRF1, FoxO1/3a and mTOR/p70S6K were determined. The results show that DEX decreased (P<0.05) protein synthesis rates while increasing the abundance of myostatin. DEX increased (P<0.05) the level of phospho-FoxO1/3a (Thr 24/32) and the expression of MuRF1. In contrast, DEX treatment had no detectable effect on atrogin-1 protein levels (P>0.05). The phosphorylation levels of mTOR and p70S6K were decreased by DEX treatment (P<0.05). Follistatin treatment inhibited the DEX-induced increase in myostatin (P<0.05) and the activation of phosphor-FoxO1/3a (Thr 24/32) (P< 0.05) and MuRF1 (P<0.05). Follistatin treatment had no influence on the protein synthesis rate or on the phosphorylation levels of mTOR (Ser 2448) and p70S6K (Thr 389) (P> 0.05). In conclusion, the present study suggests that the myostatin signalling pathway is associated with glucocorticoid-induced muscle protein catabolism at the beginning of exposure. Myostatin is not a main pathway associated with the suppression of muscle protein synthesis by glucocorticoids.

Glucocorticoids Enhance Muscle Proteolysis through a Myostatin-Dependent Pathway at the Early Stage

PubMed Central

Wang, Ruxia; Jiao, Hongchao; Zhao, Jingpeng; Wang, Xiaojuan; Lin, Hai

2016-01-01

Myostatin, a member of the TGF-β superfamily of secreted proteins, is expressed primarily in skeletal muscle. It negatively regulates muscle mass and is associated with glucocorticoid-induced muscle atrophy. However, it remains unclear whether myostatin is involved in glucocorticoid-induced muscle protein turnover. The aim of the present study was to investigate the role of myostatin in protein metabolism during dexamethasone (DEX) treatment. Protein synthesis rates and the expression of the genes for myostatin, ubiquitin-proteasome atrogin-1, MuRF1, FoxO1/3a and mTOR/p70S6K were determined. The results show that DEX decreased (P<0.05) protein synthesis rates while increasing the abundance of myostatin. DEX increased (P<0.05) the level of phospho-FoxO1/3a (Thr 24/32) and the expression of MuRF1. In contrast, DEX treatment had no detectable effect on atrogin-1 protein levels (P>0.05). The phosphorylation levels of mTOR and p70S6K were decreased by DEX treatment (P<0.05). Follistatin treatment inhibited the DEX-induced increase in myostatin (P<0.05) and the activation of phosphor-FoxO1/3a (Thr 24/32) (P< 0.05) and MuRF1 (P<0.05). Follistatin treatment had no influence on the protein synthesis rate or on the phosphorylation levels of mTOR (Ser 2448) and p70S6K (Thr 389) (P> 0.05). In conclusion, the present study suggests that the myostatin signalling pathway is associated with glucocorticoid-induced muscle protein catabolism at the beginning of exposure. Myostatin is not a main pathway associated with the suppression of muscle protein synthesis by glucocorticoids. PMID:27227776

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor.

PubMed

Mikulík, K; Janda, I; Weiser, J; Stastná, J; Jiránová, A

1984-12-03

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.

NSs protein of Schmallenberg virus counteracts the antiviral response of the cell by inhibiting its transcriptional machinery.

PubMed

Barry, Gerald; Varela, Mariana; Ratinier, Maxime; Blomström, Anne-Lie; Caporale, Marco; Seehusen, Frauke; Hahn, Kerstin; Schnettler, Esther; Baumgärtner, Wolfgang; Kohl, Alain; Palmarini, Massimo

2014-08-01

Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways. © 2014 The Authors.

Response of Escherichia coli to Prolonged Berberine Exposure.

PubMed

Budeyri Gokgoz, Nilay; Avci, Fatma Gizem; Yoneten, Kubra Karaosmanoglu; Alaybeyoglu, Begum; Ozkirimli, Elif; Sayar, Nihat Alpagu; Kazan, Dilek; Sariyar Akbulut, Berna

2017-07-01

Berberine is a plant-derived alkaloid possessing antimicrobial activity; unfortunately, its efflux through multidrug resistance pumps reduces its efficacy. Cellular life span of Escherichia coli is generally shorter with prolonged berberine exposure; nevertheless, about 30% of the cells still remain robust following this treatment. To elucidate its mechanism of action and to identify proteins that could be involved in development of antimicrobial resistance, protein profiles of E. coli cells treated with berberine for 4.5 and 8 hours were compared with control cells. A total of 42 proteins were differentially expressed in cells treated with berberine for 8 hours when compared to control cells. In both 4.5 and 8 hours of berberine-treated cells, carbohydrate and peptide uptake regimens remained unchanged, although amino acid maintenance regimen switched from transport to synthesis. Defect in cell division persisted and this condition was confirmed by images obtained from scanning electron microscopy. Universal stress proteins were not involved in stress response. The significant increase in the abundance of elongation factors could suggest the involvement of these proteins in protection by exhibiting chaperone activities. Furthermore, the involvement of the outer membrane protein OmpW could receive special attention as a protein involved in response to antimicrobial agents, since the expression of only this porin protein was upregulated after 8 hours of exposure.

Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans

PubMed Central

Childress, Catherine; Feuerbacher, Leigh A.; Phillips, Linda; Burgum, Alex

2013-01-01

Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cyclic AMP (cAMP) receptor protein (CRP) indirectly increases ltxA expression, but the intermediary regulator is unknown. Integration host factor (IHF) binds to and represses the leukotoxin promoter, but neither CRP nor IHF is responsible for the anaerobic induction of ltxA RNA synthesis. Thus, we have undertaken studies to identify other regulators of leukotoxin transcription and to demonstrate how these proteins work together to modulate leukotoxin synthesis. First, analyses of ltxA RNA expression from defined leukotoxin promoter mutations in the chromosome identify positions −69 to −35 as the key control region and indicate that an activator protein modulates leukotoxin transcription. We show that Mlc, which is a repressor in Escherichia coli, functions as a direct transcriptional activator in A. actinomycetemcomitans; an mlc deletion mutant reduces leukotoxin RNA synthesis, and recombinant Mlc protein binds specifically at the −68 to −40 region of the leukotoxin promoter. Furthermore, we show that CRP activates ltxA expression indirectly by increasing the levels of Mlc. Analyses of Δmlc, Δihf, and Δihf Δmlc strains demonstrate that Mlc can increase RNA polymerase (RNAP) activity directly and that IHF represses ltxA RNA synthesis mainly by blocking Mlc binding. Finally, a Δihf Δmlc mutant still induces ltxA during anaerobic growth, indicating that there are additional factors involved in leukotoxin transcriptional regulation. A model for the coordinated regulation of leukotoxin transcription is presented. PMID:23475968

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes

PubMed Central

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M.; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H.; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-01-01

Aims Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein–protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. Methods and results We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. Conclusions This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing. PMID:27095734

Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

PubMed Central

Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

2016-01-01

Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

Glutamate transporter-dependent mTOR phosphorylation in Müller glia cells

PubMed Central

María López-Colomé, Ana; Martínez-Lozada, Zila; Guillem, Alain M; López, Edith; Ortega, Arturo

2012-01-01

Glu (glutamate), the excitatory transmitter at the main signalling pathway in the retina, is critically involved in changes in the protein repertoire through the activation of signalling cascades, which regulate protein synthesis at transcriptional and translational levels. Activity-dependent differential gene expression by Glu is related to the activation of ionotropic and metabotropic Glu receptors; however, recent findings suggest the involvement of Na+-dependent Glu transporters in this process. Within the retina, Glu uptake is aimed at the replenishment of the releasable pool, and for the prevention of excitotoxicity and is carried mainly by the GLAST/EAAT-1 (Na+-dependent glutamate/aspartate transporter/excitatory amino acids transporter-1) located in Müller radial glia. Based on the previous work showing the alteration of GLAST expression induced by Glu, the present work investigates the involvement of GLAST signalling in the regulation of protein synthesis in Müller cells. To this end, we explored the effect of D-Asp (D-aspartate) on Ser-2448 mTOR (mammalian target of rapamycin) phosphorylation in primary cultures of chick Müller glia. The results showed that D-Asp transport induces the time- and dose-dependent phosphorylation of mTOR, mimicked by the transportable GLAST inhibitor THA (threo-β-hydroxyaspartate). Signalling leading to mTOR phosphorylation includes Ca2+ influx, the activation of p60src, phosphatidylinositol 3-kinase, protein kinase B, mTOR and p70S6K. Interestingly, GLAST activity promoted AP-1 (activator protein-1) binding to DNA, supporting a function for transporter signalling in retinal long-term responses. These results add a novel receptor-independent pathway for Glu signalling in Müller glia, and further strengthen the critical involvement of these cells in the regulation of glutamatergic transmission in the retina. PMID:22817638

Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles

NASA Astrophysics Data System (ADS)

Ballottin, Daniela; Fulaz, Stephanie; Souza, Michele L.; Corio, Paola; Rodrigues, Alexandre G.; Souza, Ana O.; Gaspari, Priscyla M.; Gomes, Alexandre F.; Gozzo, Fábio; Tasic, Ljubica

2016-06-01

Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis—isolated as an endophytic fungus from Rizophora mangle—were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon their physical interaction with the AgNPs or when covalently bonded to them. Eight proteins in the AgNP dispersion were identified by mass spectrometry analyses. All these proteins are involved in metabolic pathways of the fungus and are important for carbon, phosphorous and nitrogen uptake, and for the fungal growth. Thereby, important proteins for fungi are also involved in the formation and stabilization of the biogenic AgNPs.

R-Baclofen Reverses a Social Behavior Deficit and Elevated Protein Synthesis in a Mouse Model of Fragile X Syndrome.

PubMed

Qin, Mei; Huang, Tianjian; Kader, Michael; Krych, Leland; Xia, Zengyan; Burlin, Thomas; Zeidler, Zachary; Zhao, Tingrui; Smith, Carolyn B

2015-03-28

Fragile X syndrome (FXS) is the most common known inherited form of intellectual disability and the single genomic cause of autism spectrum disorders. It is caused by the absence of a fragile X mental retardation gene (Fmr1) product, FMRP, an RNA-binding translation suppressor. Elevated rates of protein synthesis in the brain and an imbalance between synaptic signaling via glutamate and γ-aminobutyric acid (GABA) are both considered important in the pathogenesis of FXS. In a mouse model of FXS (Fmr1 knockout [KO]), treatment with R-baclofen reversed some behavioral and biochemical phenotypes. A remaining crucial question is whether R-baclofen is also able to reverse increased brain protein synthesis rates. To answer this question, we measured regional rates of cerebral protein synthesis in vivo with the L-[1-(14)C]leucine method in vehicle- and R-baclofen-treated wildtype and Fmr1 KO mice. We further probed signaling pathways involved in the regulation of protein synthesis. Acute R-baclofen administration corrected elevated protein synthesis and reduced deficits on a test of social behavior in adult Fmr1 KO mice. It also suppressed activity of the mammalian target of rapamycin pathway, particularly in synaptosome-enriched fractions, but it had no effect on extracellular-regulated kinase 1/2 activity. Ninety min after R-baclofen treatment, we observed an increase in metabotropic glutamate receptor 5 expression in the frontal cortex, a finding that may shed light on the tolerance observed in human studies with this drug. Our results suggest that treatment via activation of the GABA (GABA receptor subtype B) system warrants further study in patients with FXS. Published by Oxford University Press on behalf of CINP 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

RNA-binding proteins in plants: the tip of an iceberg?

NASA Technical Reports Server (NTRS)

Fedoroff, Nina V.; Federoff, N. V. (Principal Investigator)

2002-01-01

RNA-binding proteins, which are involved in the synthesis, processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellular regulatory proteins. Although relatively few RNA-binding proteins have been studied in plants, they are being identified with increasing frequency, both genetically and biochemically. RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have been elegantly analyzed in Clamydomonas reinhardtii have counterparts with similar functions in higher plants. Several recent reports describe mutations in genes encoding RNA-binding proteins that affect plant development and hormone signaling.

Probing Functional Heteromeric Chemokine Protein-Protein Interactions through Conformation-Assisted Oxime Ligation.

PubMed

Agten, Stijn M; Koenen, Rory R; Ippel, Hans; Eckardt, Veit; von Hundelshausen, Philipp; Mayo, Kevin H; Weber, Christian; Hackeng, Tilman M

2016-11-21

Protein-protein interactions (PPIs) govern most processes in living cells. Current drug development strategies are aimed at disrupting or stabilizing PPIs, which require a thorough understanding of PPI mechanisms. Examples of such PPIs are heteromeric chemokine interactions that are potentially involved in pathological disorders such as cancer, atherosclerosis, and HIV. It remains unclear whether this functional modulation is mediated by heterodimer formation or by the additive effects of mixed chemokines on their respective receptors. To address this issue, we report the synthesis of a covalent RANTES-PF4 heterodimer (termed OPRAH) by total chemical synthesis and oxime ligation, with an acceleration of the final ligation step driven by PPIs between RANTES and PF4. Compared to mixed separate chemokines, OPRAH exhibited increased biological activity, thus providing evidence that physical formation of the heterodimer indeed mediates enhanced function. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

DNA Microarray Analysis of the Expression Profile of Escherichia coli in Response to Treatment with 4,5-Dihydroxy-2-Cyclopenten-1-One

PubMed Central

Phadtare, Sangita; Kato, Ikunoshin; Inouye, Masayori

2002-01-01

We carried out DNA microarray-based global transcript profiling of Escherichia coli in response to 4,5-dihydroxy-2-cyclopenten-1-one to explore the manifestation of its antibacterial activity. We show that it has widespread effects in E. coli affecting genes encoding proteins involved in cell metabolism and membrane synthesis and functions. Genes belonging to the regulon involved in synthesis of Cys are upregulated. In addition, rpoS and RpoS-regulated genes responding to various stresses and a number of genes responding to oxidative stress are upregulated. PMID:12426362

Syndecan-2 regulates melanin synthesis via protein kinase C βII-mediated tyrosinase activation.

PubMed

Jung, Hyejung; Chung, Heesung; Chang, Sung Eun; Choi, Sora; Han, Inn-Oc; Kang, Duk-Hee; Oh, Eok-Soo

2014-05-01

Syndecan-2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan-2 in melanogenesis. Syndecan-2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA-mediated knockdown of syndecan-2 was associated with reduced melanin synthesis, whereas overexpression of syndecan-2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan-2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan-2 expression, and this up-regulation of syndecan-2 was required for UVB-induced melanin synthesis. Taken together, these data suggest that syndecan-2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin-associated diseases. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

PubMed Central

Woodard, John; Rasch, Ellen; Swift, Hewson

1961-01-01

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C14 and uridine-H3 were used as ribonucleic acid (RNA) precursors, thymidine-H3 as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C14 as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. PMID:13786522

No Escaping the Rat Race: Simulated Night Shift Work Alters the Time-of-Day Variation in BMAL1 Translational Activity in the Prefrontal Cortex.

PubMed

Marti, Andrea R; Patil, Sudarshan; Mrdalj, Jelena; Meerlo, Peter; Skrede, Silje; Pallesen, Ståle; Pedersen, Torhild T; Bramham, Clive R; Grønli, Janne

2017-01-01

Millions of people worldwide work during the night, resulting in disturbed circadian rhythms and sleep loss. This may cause deficits in cognitive functions, impaired alertness and increased risk of errors and accidents. Disturbed circadian rhythmicity resulting from night shift work could impair brain function and cognition through disrupted synthesis of proteins involved in synaptic plasticity and neuronal function. Recently, the circadian transcription factor brain-and-muscle arnt-like protein 1 (BMAL1) has been identified as a promoter of mRNA translation initiation, the most highly regulated step in protein synthesis, through binding to the mRNA "cap". In this study we investigated the effects of simulated shift work on protein synthesis markers. Male rats ( n = 40) were exposed to forced activity, either in their rest phase (simulated night shift work) or in their active phase (simulated day shift work) for 3 days. Following the third work shift, experimental animals and time-matched undisturbed controls were euthanized (rest work at ZT12; active work at ZT0). Tissue lysates from two brain regions (prefrontal cortex, PFC and hippocampus) implicated in cognition and sleep loss, were analyzed with m 7 GTP (cap) pull-down to examine time-of-day variation and effects of simulated shift work on cap-bound protein translation. The results show time-of-day variation of protein synthesis markers in PFC, with increased protein synthesis at ZT12. In the hippocampus there was little difference between ZT0 and ZT12. Active phase work did not induce statistically significant changes in protein synthesis markers at ZT0 compared to time-matched undisturbed controls. Rest work, however, resulted in distinct brain-region specific changes of protein synthesis markers compared to time-matched controls at ZT12. While no changes were observed in the hippocampus, phosphorylation of cap-bound BMAL1 and its regulator S6 kinase beta-1 (S6K1) was significantly reduced in the PFC, together with significant reduction in the synaptic plasticity associated protein activity-regulatedcytoskeleton-associated protein (Arc). Our results indicate considerable time-of-day and brain-region specific variation in cap-dependent translation initiation. We concludethat simulated night shift work in rats disrupts the pathways regulating the circadian component of the translation of mRNA in the PFC, and that this may partly explain impaired waking function during night shift work.

Copper-induced overexpression of genes encoding antioxidant system enzymes and metallothioneins involve the activation of CaMs, CDPKs and MEK1/2 in the marine alga Ulva compressa.

PubMed

Laporte, Daniel; Valdés, Natalia; González, Alberto; Sáez, Claudio A; Zúñiga, Antonio; Navarrete, Axel; Meneses, Claudio; Moenne, Alejandra

2016-08-01

Transcriptomic analyses were performed in the green macroalga Ulva compressa cultivated with 10μM copper for 24h. Nucleotide sequences encoding antioxidant enzymes, ascorbate peroxidase (ap), dehydroascorbate reductase (dhar) and glutathione reductase (gr), enzymes involved in ascorbate (ASC) synthesis l-galactose dehydrogenase (l-gdh) and l-galactono lactone dehydrogenase (l-gldh), in glutathione (GSH) synthesis, γ-glutamate-cysteine ligase (γ-gcl) and glutathione synthase (gs), and metal-chelating proteins metallothioneins (mt) were identified. Amino acid sequences encoded by transcripts identified in U. compressa corresponding to antioxidant system enzymes showed homology mainly to plant and green alga enzymes but those corresponding to MTs displayed homology to animal and plant MTs. Level of transcripts encoding the latter proteins were quantified in the alga cultivated with 10μM copper for 0-12 days. Transcripts encoding enzymes of the antioxidant system increased with maximal levels at day 7, 9 or 12, and for MTs at day 3, 7 or 12. In addition, the involvement of calmodulins (CaMs), calcium-dependent protein kinases (CDPKs), and the mitogen-activated protein kinase kinase (MEK1/2) in the increase of the level of the latter transcripts was analyzed using inhibitors. Transcript levels decreased with inhibitors of CaMs, CDPKs and MEK1/2. Thus, copper induces overexpression of genes encoding antioxidant enzymes, enzymes involved in ASC and GSH syntheses and MTs. The increase in transcript levels may involve the activation of CaMs, CDPKs and MEK1/2 in U. compressa. Copyright © 2016 Elsevier B.V. All rights reserved.

Liver protein synthesis stays elevated after chemotherapy in tumour-bearing mice.

PubMed

Samuels, Sue E; McLaren, Teresa A; Knowles, Andrew L; Stewart, Sarah A; Madelmont, Jean-Claude; Attaix, Didier

2006-07-28

We studied the effect of chemotherapy on liver protein synthesis in mice bearing colon 26 adenocarcinoma (C26). Liver protein mass decreased (-32%; P<0.05) in cachectic mice, but protein synthesis increased (20-35%; P<0.05) in cachectic mice, which is consistent with increased export protein synthesis. Increased protein synthesis in tumour-bearing mice was primarily mediated by increasing ( approximately 15%; P<0.05) the RNA concentration, i.e. the capacity for protein synthesis (Cs; mg RNA/g protein). Cystemustine, a nitrosourea chemotherapy that cures C26 with 100% efficacy, rapidly restored liver protein mass; protein synthesis however stayed higher than in healthy mice ( approximately 15%) throughout the initial and later stages of recovery. Chemotherapy had no significant effect on liver protein mass and synthesis in healthy mice. Reduced food intake was not a factor in this model. These data suggest a high priority for liver protein synthesis during cancer cachexia and recovery.

The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss).

PubMed

Pannevis, M C; Houlihan, D F

1992-01-01

To establish the energetic cost of protein synthesis, isolated trout hepatocytes were used to measure protein synthesis and respiration simultaneously at a variety of temperatures. The presence of bovine serum albumin was essential for the viability of isolated hepatocytes during isolation, but, in order to measure protein synthesis rates, oxygen consumption rates and RNA-to-protein ratios, BSA had to be washed from the cells. Isolated hepatocytes were found to be capable of protein synthesis and oxygen consumption at constant rates over a wide range of oxygen tension. Cycloheximide was used to inhibit protein synthesis. Isolated hepatocytes used on average 79.7 +/- 9.5% of their total oxygen consumption on cycloheximide-sensitive protein synthesis and 2.8 +/- 2.8% on maintaining ouabain-sensitive Na+/K(+)-ATPase activity. The energetic cost of protein synthesis in terms of moles of adenosine triphosphate per gram of protein synthesis decreased with increasing rates of protein synthesis at higher temperatures. It is suggested that the energetic cost consists of a fixed (independent of synthesis rate) and a variable component (dependent on synthesis rate).

Transcriptional profiling of Haemophilus parasuis SH0165 response to tilmicosin.

PubMed

Liu, Yingyu; Chen, Pin; Wang, Yang; Li, Wentao; Cheng, Shuang; Wang, Chunmei; Zhang, Anding; He, Qigai

2012-12-01

The Haemophilus parasuis respiratory tract pathogen poses a severe threat to the swine industry despite available antimicrobial therapies. To gain a more detailed understanding of the molecular mechanisms underlying H. parasuis response to tilmicosin treatment, microarray technology was applied to analyze the variation in gene expression of isolated H. parasuis SH0165 treated in vitro with subinhibitory (0.25 μg/ml) and inhibitory (8 μg/ml) concentrations. Tilmicosin treatment induced differential expression of 405 genes, the encoded products of which are mainly involved in the heat shock response, protein synthesis, and intracellular transportation. The subinhibitory and inhibitory concentrations of tilmicosin induced distinctive gene expression profiles of shared and unique changes, respectively. These changes included 302 genes mainly involved in protein export and the phosphotransferase system to sustain cell growth, and 198 genes mainly related to RNA polymerase, recombination, and repair to inhibit cell growth. In silico analysis of functions related to the differentially expressed genes suggested that adaptation of H. parasuis SH0165 to tilmicosin involves modulation of protein synthesis and membrane transport. Collectively, the genes comprising each transcriptional profile of H. parasuis response to tilmicosin provide novel insights into the physiological functions of this economically significant bacterium and may represent targets of future molecular therapeutic strategies.

Comprehensive analysis of gene expression patterns in Friedreich's ataxia fibroblasts by RNA sequencing reveals altered levels of protein synthesis factors and solute carriers

PubMed Central

Li, Yanjie; Lu, Yue; Lin, Kevin; Hauser, Lauren A.; Lynch, David R.

2017-01-01

ABSTRACT Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease usually caused by large homozygous expansions of GAA repeat sequences in intron 1 of the frataxin (FXN) gene. FRDA patients homozygous for GAA expansions have low FXN mRNA and protein levels when compared with heterozygous carriers or healthy controls. Frataxin is a mitochondrial protein involved in iron–sulfur cluster synthesis, and many FRDA phenotypes result from deficiencies in cellular metabolism due to lowered expression of FXN. Presently, there is no effective treatment for FRDA, and biomarkers to measure therapeutic trial outcomes and/or to gauge disease progression are lacking. Peripheral tissues, including blood cells, buccal cells and skin fibroblasts, can readily be isolated from FRDA patients and used to define molecular hallmarks of disease pathogenesis. For instance, FXN mRNA and protein levels as well as FXN GAA-repeat tract lengths are routinely determined using all of these cell types. However, because these tissues are not directly involved in disease pathogenesis, their relevance as models of the molecular aspects of the disease is yet to be decided. Herein, we conducted unbiased RNA sequencing to profile the transcriptomes of fibroblast cell lines derived from 18 FRDA patients and 17 unaffected control individuals. Bioinformatic analyses revealed significantly upregulated expression of genes encoding plasma membrane solute carrier proteins in FRDA fibroblasts. Conversely, the expression of genes encoding accessory factors and enzymes involved in cytoplasmic and mitochondrial protein synthesis was consistently decreased in FRDA fibroblasts. Finally, comparison of genes differentially expressed in FRDA fibroblasts to three previously published gene expression signatures defined for FRDA blood cells showed substantial overlap between the independent datasets, including correspondingly deficient expression of antioxidant defense genes. Together, these results indicate that gene expression profiling of cells derived from peripheral tissues can, in fact, consistently reveal novel molecular pathways of the disease. When performed on statistically meaningful sample group sizes, unbiased global profiling analyses utilizing peripheral tissues are critical for the discovery and validation of FRDA disease biomarkers. PMID:29125828

Strategies for investigating nuclear-cytoplasmic tRNA dynamics in yeast and mammalian cells.

PubMed

Pierce, Jacqueline B; Chafe, Shawn C; Eswara, Manoja B K; van der Merwe, George; Mangroo, Dev

2014-01-01

Nuclear-cytoplasmic tRNA transport involves multiple pathways that are segregated by the involvement of distinct proteins. The tRNA export process begins in the nucleolus, where the functionality of newly produced tRNAs are tested by aminoacylation, and ends with the delivery of the exported aminoacyl tRNAs to the eukaryotic elongation factor eEF-1A for utilization in protein synthesis in the cytoplasm. Recent studies have identified a number of proteins that participate in nuclear tRNA export in both yeast and mammals. However, genetic and biochemical evidence suggest that additional components, which have yet to be identified, also participate in nuclear-cytoplasmic tRNA trafficking. Here we review key strategies that have led to the identification and characterization of proteins that are involved in the nuclear tRNA export process in yeasts and mammals. The approaches described will greatly facilitate the identification and delineation of the roles of new proteins involved in nuclear export of tRNAs to the cytoplasm. Copyright © 2014 Elsevier Inc. All rights reserved.

Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: alterations in the activities and protein expression of glutathione-related enzymes.

PubMed

Gu, Feng; Chauhan, Ved; Chauhan, Abha

2013-12-01

Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence in autism suggests a deficit in glutathione (GSH), a major endogenous antioxidant. It is not known whether the synthesis, consumption, and/or regeneration of GSH is affected in autism. In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (P<0.05). Although there was no significant difference in GR activity between autism and control groups, 40% of autistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired activities in the cerebellum in autism, and lower GCL activity in autism may be related to decreased protein expression of GCLM. Copyright © 2013 Elsevier Inc. All rights reserved.

Proteomic analysis of early phase of conidia germination in Aspergillus nidulans.

PubMed

Oh, Young Taek; Ahn, Chun-Seob; Kim, Jeong Geun; Ro, Hyeon-Su; Lee, Chang-Won; Kim, Jae Won

2010-03-01

In order to investigate proteins involved in early phase of conidia germination, proteomic analysis was performed using two-dimensional gel electrophoresis (2D-GE) in conjunction with MALDI-TOF mass spectrometry (MS). The expression levels of 241 proteins varied quantitatively with statistical significance (P<0.05) at the early phase of the germination stage. Out of these 57 were identified by MALDI-TOF MS. Through classification of physiological functions from Conserved Domain Database analysis, among the identified proteins, 21, 13, and 6 proteins were associated with energy metabolism, protein synthesis, and protein folding process, respectively. Interestingly, eight proteins, which are involved in detoxification of reactive oxygen species (ROS) including catalase A, thioredoxin reductase, and mitochondrial peroxiredoxin, were also identified. The expression levels of the genes were further confirmed using Northern blot and reverse transcriptase (RT)-PCR analyses. This study represents the first proteomic analysis of early phase of conidia germination and will contribute to a better understanding of the molecular events involved in conidia germination process. Copyright (c) 2009 Elsevier Inc. All rights reserved.

Two-dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus.

PubMed

Vödisch, Martin; Albrecht, Daniela; Lessing, Franziska; Schmidt, André D; Winkler, Robert; Guthke, Reinhard; Brakhage, Axel A; Kniemeyer, Olaf

2009-03-01

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. We established a 2-D reference map for A. fumigatus. Using MALDI-TOF-MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.

Structure-based design, synthesis and crystallization of 2-arylquinazolines as lipid pocket ligands of p38α MAPK

PubMed Central

Bührmann, Mike; Wiedemann, Bianca M.; Müller, Matthias P.; Hardick, Julia; Ecke, Maria

2017-01-01

In protein kinase research, identifying and addressing small molecule binding sites other than the highly conserved ATP-pocket are of intense interest because this line of investigation extends our understanding of kinase function beyond the catalytic phosphotransfer. Such alternative binding sites may be involved in altering the activation state through subtle conformational changes, control cellular enzyme localization, or in mediating and disrupting protein-protein interactions. Small organic molecules that target these less conserved regions might serve as tools for chemical biology research and to probe alternative strategies in targeting protein kinases in disease settings. Here, we present the structure-based design and synthesis of a focused library of 2-arylquinazoline derivatives to target the lipophilic C-terminal binding pocket in p38α MAPK, for which a clear biological function has yet to be identified. The interactions of the ligands with p38α MAPK was analyzed by SPR measurements and validated by protein X-ray crystallography. PMID:28892510

The RNA binding and transport proteins staufen and fragile X mental retardation protein are expressed by rat primary afferent neurons and localize to peripheral and central axons.

PubMed

Price, T J; Flores, C M; Cervero, F; Hargreaves, K M

2006-09-15

Neuronal proteins have been traditionally viewed as being derived solely from the soma; however, accumulating evidence indicates that dendritic and axonal sites are capable of a more autonomous role in terms of new protein synthesis. Such extra-somal translation allows for more rapid, on-demand regulation of neuronal structure and function than would otherwise be possible. While mechanisms of dendritic RNA transport have been elucidated, it remains unclear how RNA is trafficked into the axon for this purpose. Primary afferent neurons of the dorsal root (DRG) and trigeminal (TG) ganglia have among the longest axons in the neuraxis and such axonal protein synthesis would be advantageous, given the greater time involved for protein trafficking to occur via axonal transport. Therefore, we hypothesized that these primary sensory neurons might express proteins involved in RNA transport. Rat DRG and TG neurons expressed staufen (stau) 1 and 2 (detected at the mRNA level) and stau2 and fragile x mental retardation protein (FMRP; detected at the protein level). Stau2 mRNA was also detected in human TG neurons. Stau2 and FMRP protein were localized to the sciatic nerve and dorsal roots by immunohistochemistry and to dorsal roots by Western blot. Stau2 and FMRP immunoreactivities colocalized with transient receptor potential channel type 1 immunoreactivity in sensory axons of the sciatic nerve and dorsal root, suggesting that these proteins are being transported into the peripheral and central terminals of nociceptive sensory axons. Based on these findings, we propose that stau2 and FMRP proteins are attractive candidates to subserve RNA transport in sensory neurons, linking somal transcriptional events to axonal translation.

THE RNA BINDING AND TRANSPORT PROTEINS STAUFEN AND FRAGILE X MENTAL RETARDATION PROTEIN ARE EXPRESSED BY RAT PRIMARY AFFERENT NEURONS AND LOCALIZE TO PERIPHERAL AND CENTRAL AXONS

PubMed Central

PRICE, T. J.; FLORES, C. M.; CERVERO, F.; HARGREAVES, K. M.

2007-01-01

Neuronal proteins have been traditionally viewed as being derived solely from the soma; however, accumulating evidence indicates that dendritic and axonal sites are capable of a more autonomous role in terms of new protein synthesis. Such extra-somal translation allows for more rapid, on-demand regulation of neuronal structure and function than would otherwise be possible. While mechanisms of dendritic RNA transport have been elucidated, it remains unclear how RNA is trafficked into the axon for this purpose. Primary afferent neurons of the dorsal root (DRG) and trigeminal (TG) ganglia have among the longest axons in the neuraxis and such axonal protein synthesis would be advantageous, given the greater time involved for protein trafficking to occur via axonal transport. Therefore, we hypothesized that these primary sensory neurons might express proteins involved in RNA transport. Rat DRG and TG neurons expressed staufen (stau) 1 and 2 (detected at the mRNA level) and stau2 and fragile × mental retardation protein (FMRP; detected at the protein level). Stau2 mRNA was also detected in human TG neurons. Stau2 and FMRP protein were localized to the sciatic nerve and dorsal roots by immunohistochemistry and to dorsal roots by Western blot. Stau2 and FMRP immunoreactivities colocalized with transient receptor potential channel type 1 immunoreactivity in sensory axons of the sciatic nerve and dorsal root, suggesting that these proteins are being transported into the peripheral and central terminals of nociceptive sensory axons. Based on these findings, we propose that stau2 and FMRP proteins are attractive candidates to subserve RNA transport in sensory neurons, linking somal transcriptional events to axonal translation. PMID:16809002

Dynamic changes in proteins during apple (Malus x domestica) fruit ripening and storage

PubMed Central

Shi, Yun; Jiang, Li; Zhang, Li; Kang, Ruoyi; Yu, Zhifang

2014-01-01

A proteomic study, using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight, was conducted in apple fruit (cv. ‘Golden Delicious’) starting at 10 days prior to harvest through 50 days in storage. Total protein was extracted using a phenol/sodium dodecyl sulfate protocol. More than 400 protein spots were detected in each gel and 55 differentially expressed proteins (p<0.05) were subjected to matrix-assisted laser desorption/ionization time-of-flight/time-of-flight analysis. Fifty-three of these proteins were finally identified using an apple expressed sequence tag database downloaded from Genome Database for Rosaceae and placed into six categories. The categories and the percentage of proteins placed in each category were stress response and defense (49.0%), energy and metabolism (34.0%), fruit ripening and senescence (5.6%), signal transduction (3.8%), cell structure (3.8%) and protein synthesis (3.8%). Proteins involved in several multiple metabolic pathways, including glycolysis, pentose–phosphate pathway, anti-oxidative systems, photosynthesis and cell wall synthesis, were downregulated, especially during the climacteric burst in respiration and during the senescent stages of fruit development. Proteins classified as allergens or involved in cell wall degradation were upregulated during the ripening process. Some protein spots exhibited a mixed pattern (increasing to maximal abundance followed by a decrease), such as 1-aminocyclopropane-1-carboxylate oxidase, L-ascorbate peroxidase and abscisic acid response proteins. The identification of differentially expressed proteins associated with physiological processes identified in the current study provides a baseline of information for understanding the metabolic processes and regulatory mechanisms that occur in climacteric apple fruit during ripening and senescence. PMID:26504530

Transcriptome survey of the lipid metabolic pathways involved in energy production and ecdysteroid synthesis in the salmon louse Caligus rogercresseyi (Crustacea: Copepoda).

PubMed

Gonçalves, Ana Teresa; Farlora, Rodolfo; Gallardo-Escárate, Cristian

2014-10-01

The goal of this study was to identify and analyze the lipid metabolic pathways involved in energy production and ecdysteroid synthesis in the ectoparasite copepod Caligus rogercresseyi. Massive transcriptome sequencing analysis was performed during the infectious copepodid larval stage, during the attached chalimus larval stage, and also in female and male adults. Thirty genes were selected for describing the pathways, and these were annotated for proteins or enzymes involved in lipid digestion, absorption, and transport; fatty acid degradation; the synthesis and degradation of ketone bodies; and steroid and ecdysteroid syntheses. Differential expression of these genes was analyzed by ontogenic stage and discussed considering each stage's feeding habits and energetic needs. Copepodids showed a low expression of fatty acid digestion genes, reflected by a non-feeding behavior, and the upregulation of genes involved in steroid biosynthesis, which was consistent with a pathway for cholesterol synthesis during ecdysis. The chalimus stage showed an upregulation of genes related to fatty acid digestion, absorption, and transport, as well as to fatty acid degradation and the synthesis of ketone bodies, therefore suggesting that lipids ingested from the mucus and skin of the host fish are metabolized as important sources of energy. Adult females also showed a pattern of high lipid metabolism for energy supply and mobilization in relation to reproduction and vitellogenesis. Adult females and males revealed different lipid metabolism patterns that reflected different energetic needs. This study reports for the first time the probable lipid metabolic pathways involved in the energy production and ecdysteroid synthesis of C. rogercresseyi. Copyright © 2014 Elsevier Inc. All rights reserved.

iTRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.

PubMed

Geng, Xiaofang; Wang, Gaiping; Qin, Yanli; Zang, Xiayan; Li, Pengfei; Geng, Zhi; Xue, Deming; Dong, Zimei; Ma, Kexue; Chen, Guangwen; Xu, Cunshuan

2015-01-01

The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR) remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.

Chirality and protein biosynthesis.

PubMed

Banik, Sindrila Dutta; Nandi, Nilashis

2013-01-01

Chirality is present at all levels of structural hierarchy of protein and plays a significant role in protein biosynthesis. The macromolecules involved in protein biosynthesis such as aminoacyl tRNA synthetase and ribosome have chiral subunits. Despite the omnipresence of chirality in the biosynthetic pathway, its origin, role in current pathway, and importance is far from understood. In this review we first present an introduction to biochirality and its relevance to protein biosynthesis. Major propositions about the prebiotic origin of biomolecules are presented with particular reference to proteins and nucleic acids. The problem of the origin of homochirality is unresolved at present. The chiral discrimination by enzymes involved in protein synthesis is essential for keeping the life process going. However, questions remained pertaining to the mechanism of chiral discrimination and concomitant retention of biochirality. We discuss the experimental evidence which shows that it is virtually impossible to incorporate D-amino acids in protein structures in present biosynthetic pathways via any of the two major steps of protein synthesis, namely aminoacylation and peptide bond formation reactions. Molecular level explanations of the stringent chiral specificity in each step are extended based on computational analysis. A detailed account of the current state of understanding of the mechanism of chiral discrimination during aminoacylation in the active site of aminoacyl tRNA synthetase and peptide bond formation in ribosomal peptidyl transferase center is presented. Finally, it is pointed out that the understanding of the mechanism of retention of enantiopurity has implications in developing novel enzyme mimetic systems and biocatalysts and might be useful in chiral drug design.

Functional expansion of human tRNA synthetases achieved by structural inventions

PubMed Central

Guo, Min; Schimmel, Paul; Yang, Xiang-Lei

2010-01-01

Known as an essential component of the translational apparatus, the aminoacyl-tRNA synthetase family catalyzes the first step reaction in protein synthesis, that is, to specifically attach each amino acid to its cognate tRNA. While preserving this essential role, tRNA synthetases developed other roles during evolution. Human tRNA synthetases, in particular, have diverse functions in different pathways involving angiogenesis, inflammation and apoptosis. The functional diversity is further illustrated in the association with various diseases through genetic mutations that do not affect aminoacylation or protein synthesis. Here we review the accumulated knowledge on how human tRNA synthetases used structural inventions to achieve functional expansions. PMID:19932696

[Antifungals cellular targets and mechanisms of resistance].

PubMed

Accoceberry, Isabelle; Noël, Thierry

2006-01-01

Antifungals of systemic use for the treatment of invasive fungal infections belong to four main chemical families which have globally three cellular targets in fungal cells: fluorinated pyrimidines act on deoxyribonucleic acid (DNA) replication and protein synthesis; polyenes and azoles are toxic for ergosterol and its biosynthetic pathway; lipopeptides inhibit the synthesis of cell wall beta glucans. The resistance mechanisms that are developed by some fungi begin to be well understood particularly in Candida yeasts. The underlying bases of these mechanisms are either mutations that modify the antifungal target, or that block access to the target, and, on the other hand, the overexpression of genes encoding the target, or some membrane proteins involved in the active efflux of antifungal drugs.

Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation

PubMed Central

Nollen, Ellen A. A.; Garcia, Susana M.; van Haaften, Gijs; Kim, Soojin; Chavez, Alejandro; Morimoto, Richard I.; Plasterk, Ronald H. A.

2004-01-01

Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation. PMID:15084750

Proteins Differentially Expressed in the Pancreas of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice Fed Ethanol For 3 Months.

PubMed

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-07-01

The aim of this study was to identify differentially expressed proteins in the pancreatic tissue of hepatic alcohol dehydrogenase-deficient deer mice fed ethanol to understand metabolic basis and mechanism of alcoholic chronic pancreatitis. Mice were fed liquid diet containing 3.5 g% ethanol daily for 3 months, and differentially expressed pancreatic proteins were identified by protein separation using 2-dimensional gel electrophoresis and identification by mass spectrometry. Nineteen differentially expressed proteins were identified by applying criteria established for protein identification in proteomics. An increased abundance was found for ribosome-binding protein 1, 60S ribosomal protein L31-like isoform 1, histone 4, calcium, and adenosine triphosphate (ATP) binding proteins and the proteins involved in antiapoptotic processes and endoplasmic reticulum function, stress, and/or homeostasis. Low abundance was found for endoA cytokeratin, 40S ribosomal protein SA, amylase 2b isoform precursor, serum albumin, and ATP synthase subunit β and the proteins involved in cell motility, structure, and conformation. Chronic ethanol feeding in alcohol dehydrogenase-deficient deer mice differentially expresses pancreatic functional and structural proteins, which can be used to develop biomarker(s) of alcoholic chronic pancreatitis, particularly amylase 2b precursor, and 60 kDa heat shock protein and those involved in ATP synthesis and blood osmotic pressure.

Modulation of gene expression in heart and liver of hibernating black bears (Ursus americanus)

PubMed Central

2011-01-01

Background Hibernation is an adaptive strategy to survive in highly seasonal or unpredictable environments. The molecular and genetic basis of hibernation physiology in mammals has only recently been studied using large scale genomic approaches. We analyzed gene expression in the American black bear, Ursus americanus, using a custom 12,800 cDNA probe microarray to detect differences in expression that occur in heart and liver during winter hibernation in comparison to summer active animals. Results We identified 245 genes in heart and 319 genes in liver that were differentially expressed between winter and summer. The expression of 24 genes was significantly elevated during hibernation in both heart and liver. These genes are mostly involved in lipid catabolism and protein biosynthesis and include RNA binding protein motif 3 (Rbm3), which enhances protein synthesis at mildly hypothermic temperatures. Elevated expression of protein biosynthesis genes suggests induction of translation that may be related to adaptive mechanisms reducing cardiac and muscle atrophies over extended periods of low metabolism and immobility during hibernation in bears. Coordinated reduction of transcription of genes involved in amino acid catabolism suggests redirection of amino acids from catabolic pathways to protein biosynthesis. We identify common for black bears and small mammalian hibernators transcriptional changes in the liver that include induction of genes responsible for fatty acid β oxidation and carbohydrate synthesis and depression of genes involved in lipid biosynthesis, carbohydrate catabolism, cellular respiration and detoxification pathways. Conclusions Our findings show that modulation of gene expression during winter hibernation represents molecular mechanism of adaptation to extreme environments. PMID:21453527

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

PubMed

Iwamoto, T; Grove, A; Montal, M O; Montal, M; Tomich, J M

1994-06-01

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA.

PubMed Central

de la Cruz, J; Vioque, A

2001-01-01

tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3' end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (deltassrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of deltassrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the deltassrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics. PMID:11780628

Negative and Translation Termination-Dependent Positive Control of FLI-1 Protein Synthesis by Conserved Overlapping 5′ Upstream Open Reading Frames in Fli-1 mRNA

PubMed Central

Sarrazin, Sandrine; Starck, Joëlle; Gonnet, Colette; Doubeikovski, Alexandre; Melet, Fabrice; Morle, François

2000-01-01

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5′ upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG −41 and GUG −37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5′ uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5′ uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5′ uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons. PMID:10757781

West nile virus infections suppress early viral RNA synthesis and avoid inducing the cell stress granule response.

PubMed

Courtney, S C; Scherbik, S V; Stockman, B M; Brinton, M A

2012-04-01

West Nile virus (WNV) recently became endemic in the United States and is a significant cause of human morbidity and mortality. Natural WNV strain infections do not induce stress granules (SGs), while W956IC (a lineage 2/1 chimeric WNV infectious clone) virus infections produce high levels of early viral RNA and efficiently induce SGs through protein kinase R (PKR) activation. Additional WNV chimeric viruses made by replacing one or more W956IC genes with the lineage 1 Eg101 equivalent in the W956IC backbone were analyzed. The Eg-NS4b+5, Eg-NS1+3+4a, and Eg-NS1+4b+5 chimeras produced low levels of viral RNA at early times of infection and inefficiently induced SGs, suggesting the possibility that interactions between viral nonstructural proteins and/or between viral nonstructural proteins and cell proteins are involved in suppressing early viral RNA synthesis and membrane remodeling during natural WNV strain infections. Detection of exposed viral double-stranded RNA (dsRNA) in W956IC-infected cells suggested that the enhanced early viral RNA synthesis surpassed the available virus-induced membrane protection and allowed viral dsRNA to activate PKR.

Global changes in the proteome of Cupriavidus necator H16 during poly-(3-hydroxybutyrate) synthesis from various biodiesel by-product substrates.

PubMed

Sharma, Parveen K; Fu, Jilagamazhi; Spicer, Victor; Krokhin, Oleg V; Cicek, Nazim; Sparling, Richard; Levin, David B

2016-12-01

Synthesis of poly-[3-hydroxybutyrate] (PHB) by Cupriavidus necator H16 in batch cultures was evaluated using three biodiesel-derived by-products as the sole carbon sources: waste glycerol (REG-80, refined to 80 % purity with negligible free fatty acids); glycerol bottom (REG-GB, with up to 65 % glycerol and 35 % free fatty acids), and free fatty acids (REG-FFA, with up to 75 % FFA and no glycerol). All the three substrates supported growth and PHB production by C. necator, with polymer accumulation ranging from 9 to 84 % cell dry weight (cdw), depending on the carbon source. To help understand these differences, proteomic analysis indicated that although C. necator H16 was able to accumulate PHB during growth on all three biodiesel by-products, no changes in the levels of PHB synthesis enzymes were observed. However, significant changes in the levels of expression were observed for two Phasin proteins involved with PHB accumulation, and for a number of gene products in the fatty acid β-oxidation pathway, the Glyoxylate Shunt, and the hydrogen (H2) synthesis pathways in C. necator cells cultured with different substrates. The glycerol transport protein (GlpF) was induced in REG-GB and REG-80 glycerol cultures only. Cupriavidus necator cells cultured with REG-GB and REG-FFA showed up-regulation of β-oxidation and Glyoxylate Shunt pathways proteins at 24 h pi, but H2 synthesis pathways enzymes were significantly down-regulated, compared with cells cultured with waste glycerol. Our data confirmed earlier observations of constitutive expression of PHB synthesis proteins, but further suggested that C. necator H16 cells growing on biodiesel-derived glycerol were under oxidative stress.

Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor

PubMed Central

Krall, Abigail S.; Xu, Shili; Graeber, Thomas G.; Braas, Daniel; Christofk, Heather R.

2016-01-01

Cellular amino acid uptake is critical for mTOR complex 1 (mTORC1) activation and cell proliferation. However, the regulation of amino acid uptake is not well-understood. Here we describe a role for asparagine as an amino acid exchange factor: intracellular asparagine exchanges with extracellular amino acids. Through asparagine synthetase knockdown and altering of media asparagine concentrations, we show that intracellular asparagine levels regulate uptake of amino acids, especially serine, arginine and histidine. Through its exchange factor role, asparagine regulates mTORC1 activity and protein synthesis. In addition, we show that asparagine regulation of serine uptake influences serine metabolism and nucleotide synthesis, suggesting that asparagine is involved in coordinating protein and nucleotide synthesis. Finally, we show that maintenance of intracellular asparagine levels is critical for cancer cell growth. Collectively, our results indicate that asparagine is an important regulator of cancer cell amino acid homeostasis, anabolic metabolism and proliferation. PMID:27126896

Powerful signals for weak muscles.

PubMed

Saini, Amarjit; Faulkner, Steve; Al-Shanti, Nasser; Stewart, Claire

2009-10-01

The aim of the present review is to summarise, evaluate and critique the different mechanisms involved in anabolic growth of skeletal muscle and the catabolic processes involved in cancer cachexia and sarcopenia of ageing. This is highly relevant, since they represent targets for future promising clinical investigations. Sarcopenia is an inevitable process associated with a gradual reduction in muscle mass and strength, associated with a reduction in motor unit number and atrophy of muscle fibres, especially the fast type IIa fibres. The loss of muscle mass with ageing is clinically important because it leads to diminished functional ability and associated complications. Cachexia is widely recognised as severe and rapid wasting accompanying disease states such as cancer or immunodeficiency disease. One of the main characteristics of cancer cachexia is asthenia or lack of strength, which is directly related to the muscle loss. Indeed, apart from the speed of loss, muscle wasting during cancer and ageing share many common metabolic pathways and mediators. In healthy young individuals, muscles maintain their mass and function because of a balance between protein synthesis and protein degradation associated with rates of anabolic and catabolic processes, respectively. Muscles grow (hypertrophy) when protein synthesis exceeds protein degradation. Conversely, muscles shrink (atrophy) when protein degradation dominates. These processes are not occurring independently of each other, but are finely coordinated by a web of intricate signalling networks. Such signalling networks are in charge of executing environmental and cellular cues that ultimately determine whether muscle proteins are synthesised or degraded. Increasing our understanding for the pathways involved in hypertrophy and atrophy and particularly the interaction of these pathways is essential in designing therapeutic strategies for both prevention and treatment of muscle wasting conditions with age and with disease.

Early changes in tissue amino acid metabolism and nutrient routing in rats fed a high-fat diet: evidence from natural isotope abundances of nitrogen and carbon in tissue proteins.

PubMed

Mantha, Olivier L; Polakof, Sergio; Huneau, Jean-François; Mariotti, François; Poupin, Nathalie; Zalko, Daniel; Fouillet, Helene

2018-05-01

Little is known about how diet-induced obesity and insulin resistance affect protein and amino acid (AA) metabolism in tissues. The natural relative abundances of the heavy stable isotopes of C (δ 13C) and N (δ 15N) in tissue proteins offer novel and promising biomarkers of AA metabolism. They, respectively, reflect the use of dietary macronutrients for tissue AA synthesis and the relative metabolic use of tissue AA for oxidation v. protein synthesis. In this study, δ 13C and δ 15N were measured in the proteins of various tissues in young adult rats exposed perinatally and/or fed after weaning with a normal- or a high-fat (HF) diet, the aim being to characterise HF-induced tissue-specific changes in AA metabolism. HF feeding was shown to increase the routing of dietary fat to all tissue proteins via non-indispensable AA synthesis, but did not affect AA allocation between catabolic and anabolic processes in most tissues. However, the proportion of AA directed towards oxidation rather than protein synthesis was increased in the small intestine and decreased in the tibialis anterior muscle and adipose tissue. In adipose tissue, the AA reallocation was observed in the case of perinatal or post-weaning exposure to HF, whereas in the small intestine and tibialis anterior muscle the AA reallocation was only observed after HF exposure that covered both the perinatal and post-weaning periods. In conclusion, HF exposure induced an early reorganisation of AA metabolism involving tissue-specific effects, and in particular a decrease in the relative allocation of AA to oxidation in several peripheral tissues.

Skeletal muscle plasticity induced by seasonal acclimatization involves IGF1 signaling: implications in ribosomal biogenesis and protein synthesis.

PubMed

Fuentes, Eduardo N; Zuloaga, Rodrigo; Valdes, Juan Antonio; Molina, Alfredo; Alvarez, Marco

2014-10-01

One of the most fundamental biological processes in living organisms that are affected by environmental fluctuations is growth. In fish, skeletal muscle accounts for the largest proportion of body mass, and the growth of this tissue is mainly controlled by the insulin-like growth factor (IGF) system. By using the carp (Cyprinus carpio), a fish that inhabits extreme conditions during winter and summer, we assessed the skeletal muscle plasticity induced by seasonal acclimatization and the relation of IGF signaling with protein synthesis and ribosomal biogenesis. The expression of igf1 in muscle decreased during winter in comparison with summer, whereas the expression for both paralogues of igf2 did not change significantly between seasons. The expression of igf1 receptor a (igf1ra), but not of igf1rb, was down-regulated in muscle during the winter as compared to the summer. A decrease in protein contents and protein phosphorylation for IGF signaling molecules in muscle was observed in winter-acclimatized carp. This was related with a decreased expression in muscle for markers of myogenesis (myoblast determination factor (myod), myogenic factor 5 (myf5), and myogenin (myog)); protein synthesis (myosin heavy chain (mhc) and myosin light chain (mlc3 and mlc1b)); and ribosomal biogenesis (pre-rRNA and ribosomal proteins). IGF signaling, and key markers of ribosomal biogenesis, protein synthesis, and myogenesis were affected by seasonal acclimatization, with differential regulation in gene expression and signaling pathway activation observed in muscle between both seasons. This suggests that these molecules are responsible for the muscle plasticity induced by seasonal acclimatization in carp. Copyright © 2014 Elsevier Inc. All rights reserved.

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

PubMed Central

Wang, Jigang; Zhang, Jianbin; Lee, Yew-Mun; Koh, Pin-Lang; Ng, Shukie; Bao, Feichao; Lin, Qingsong; Shen, Han-Ming

2016-01-01

ABSTRACT Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQTM). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy. PMID:27463841

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy.

PubMed

Wang, Jigang; Zhang, Jianbin; Lee, Yew-Mun; Koh, Pin-Lang; Ng, Shukie; Bao, Feichao; Lin, Qingsong; Shen, Han-Ming

2016-10-02

Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ TM ). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.

Reproductive adverse outcome pathways for chemical inhibitors of steroid synthesis in fish

EPA Science Inventory

A key physiological process controlling reproductive success of oviparous vertebrates (fish, amphibian, reptiles, birds) involves production of the egg yolk protein precursor vitellogenin (VTG). VTG production is an estrogen receptor (ER)-mediated process that, in females, is con...

Bringing Life Processes to Life

ERIC Educational Resources Information Center

Souders, Hannah

2018-01-01

Protein synthesis, involving such abstract concepts as "DNA," "RNA," "transcription," and "translation," is one of many biological processes too small to be seen by the naked eye. This combination of scientific jargon and sub-microscopic elements can make biology seem overwhelming to any student--but…

Recent advances in the identification of the host factors involved in dengue virus replication.

PubMed

Wang, Yi; Zhang, Ping

2017-02-01

Dengue virus (DENV) belongs to the genus Flavivirus of the family Flaviviridae and it is primarily transmitted via Aedes aegypti and Aedes albopictus mosquitoes. The life cycle of DENV includes attachment, endocytosis, protein translation, RNA synthesis, assembly, egress, and maturation. Recent researches have indicated that a variety of host factors, including cellular proteins and microRNAs, positively or negatively regulate the DENV replication process. This review summarizes the latest findings (from 2014 to 2016) in the identification of the host factors involved in the DENV life cycle and Dengue infection.

Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates

PubMed Central

Hackett, Jeremiah D.; Wisecaver, Jennifer H.; Brosnahan, Michael L.; Kulis, David M.; Anderson, Donald M.; Bhattacharya, Debashish; Plumley, F. Gerald; Erdner, Deana L.

2013-01-01

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand. PMID:22628533

Evolution of saxitoxin synthesis in cyanobacteria and dinoflagellates.

PubMed

Hackett, Jeremiah D; Wisecaver, Jennifer H; Brosnahan, Michael L; Kulis, David M; Anderson, Donald M; Bhattacharya, Debashish; Plumley, F Gerald; Erdner, Deana L

2013-01-01

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

Schisandrae Fructus Supplementation Ameliorates Sciatic Neurectomy-Induced Muscle Atrophy in Mice

PubMed Central

Kim, Joo Wan; Ku, Sae-Kwang; Kim, Ki Young; Kim, Sung Goo; Han, Min Ho; Kim, Gi-Young; Hwang, Hye Jin; Kim, Byung Woo; Kim, Cheol Min

2015-01-01

The objective of this study was to assess the possible beneficial skeletal muscle preserving effects of ethanol extract of Schisandrae Fructus (EESF) on sciatic neurectomy- (NTX-) induced hindlimb muscle atrophy in mice. Here, calf muscle atrophy was induced by unilateral right sciatic NTX. In order to investigate whether administration of EESF prevents or improves sciatic NTX-induced muscle atrophy, EESF was administered orally. Our results indicated that EESF dose-dependently diminished the decreases in markers of muscle mass and activity levels, and the increases in markers of muscle damage and fibrosis, inflammatory cell infiltration, cytokines, and apoptotic events in the gastrocnemius muscle bundles are induced by NTX. Additionally, destruction of gastrocnemius antioxidant defense systems after NTX was dose-dependently protected by treatment with EESF. EESF also upregulated muscle-specific mRNAs involved in muscle protein synthesis but downregulated those involved in protein degradation. The overall effects of 500 mg/kg EESF were similar to those of 50 mg/kg oxymetholone, but it showed more favorable antioxidant effects. The present results suggested that EESF exerts a favorable ameliorating effect on muscle atrophy induced by NTX, through anti-inflammatory and antioxidant effects related to muscle fiber protective effects and via an increase in protein synthesis and a decrease in protein degradation. PMID:26064425

Error-prone repair and translesion synthesis III: the activation of UmuD (or less is more).

PubMed

Bridges, Bryn A

2005-08-15

Following DNA damage to Escherichia coli bacteria, RecA protein is activated by binding to single stranded DNA and cleaves its own gene repressor (LexA protein). Two papers from Graham Walker's laboratory showed that several bacterial genes in addition to RecA are repressed by the LexA repressor and are inducible following DNA damage [C.J. Keyon, G.C. Walker, DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli, in: Proceedings of the National Academy of Sciences of the United States of America 77, 1980, pp. 2819--2823] and predicted that one of them (UmuD) might itself be subject to activation by a further cleavage reaction involving activated RecA protein [K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, G.C. Walker, umuD,C and mucA,B operans whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology, in: Proceedings of the National Academy of Sciences of the United States of America 82, 1985, pp. 4331--4335]. The processed form of UmuD, termed UmuD', later proved to be a subunit of DNA polymerase V, a key enzyme involved in translesion synthesis.

Understanding resistant germplasm-induced virulence variation through analysis of proteomics and suppression subtractive hybridization in a maize pathogen Curvularia lunata.

PubMed

Gao, Shigang; Liu, Tong; Li, Yingying; Wu, Qiong; Fu, Kehe; Chen, Jie

2012-12-01

Curvularia lunata is an important pathogen causing Curvularia leaf spot in maize. Significant pathogenic variation has been found in C. lunata. To better understand the mechanism of this phenomenon, we consecutively put the selective pressures of resistant maize population on C. lunata strain WS18 (low virulence) artificially. As a result, the virulence of this strain was significantly enhanced. Using 2DE, 12 up-regulated and four down-regulated proteins were identified in virulence-increased strain compared to WS18. Our analysis revealed that melanin synthesis-related proteins (Brn1, Brn2, and scytalone dehydratase) and stress tolerance-related proteins (HSP 70) directly involved in the potential virulence growth as crucial markers or factors in C. lunata. To validate 2DE results and screen differential genes at mRNA level, we constructed a subtracted cDNA library (tester: virulence-increased strain; driver: WS18). A total of 188 unigenes were obtained this way, of which 14 were indicators for the evolution of pathogen virulence. Brn1 and hsp genes exhibited similar expression patterns corresponding to proteins detected by 2DE. Overall, our results indicated that differential proteins or genes, being involved with melanin synthesis or tolerance response to stress, could be considered as hallmarks of virulence increase in C. lunata. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

PubMed Central

Uluisik, Irem; Kaya, Alaattin; Fomenko, Dmitri E.; Karakaya, Huseyin C.; Carlson, Bradley A.; Gladyshev, Vadim N.; Koc, Ahmet

2011-01-01

Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance. PMID:22114689

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-06-01

Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing. Published by Oxford University Press on behalf of the European Society of Cardiology 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soranzo, Thomas; Martin, Donald K.; Lenormand, Jean -Luc

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for bothmore » direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.« less

Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

DOE PAGES

Soranzo, Thomas; Martin, Donald K.; Lenormand, Jean -Luc; ...

2017-06-13

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for bothmore » direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.« less

VBNC Legionella pneumophila cells are still able to produce virulence proteins.

PubMed

Alleron, Laëtitia; Khemiri, Arbia; Koubar, Mohamad; Lacombe, Christian; Coquet, Laurent; Cosette, Pascal; Jouenne, Thierry; Frere, Jacques

2013-11-01

Legionella pneumophila is the agent responsible for legionellosis. Numerous bacteria, including L. pneumophila, can enter into a viable but not culturable (VBNC) state under unfavorable environmental conditions. In this state, cells are unable to form colonies on standard medium but are still alive. Here we show that VBNC L. pneumophila cells, obtained by monochloramine treatment, were still able to synthesize proteins, some of which are involved in virulence. Protein synthesis was measured using (35)S-labeling and the proteomes of VBNC and culturable cells then compared. This analysis allowed the identification of nine proteins that were accumulated in the VBNC state. Among them, four were involved in virulence, i.e., the macrophage infectivity potentiator protein, the hypothetical protein lpl2247, the ClpP protease proteolytic subunit and the 27 kDa outer membrane protein. Others, i.e., the enoyl reductase, the electron transfer flavoprotein (alpha and beta subunits), the 50S ribosomal proteins (L1 and L25) are involved in metabolic and energy production pathways. However, resuscitation experiments performed with Acanthamoeba castellanii failed, suggesting that the accumulation of virulence factors by VBNC cells is not sufficient to maintain their virulence. Copyright © 2013 Elsevier Ltd. All rights reserved.

Modulation of GDP-fucose level for generating proteins with reduced rate of fucosylation (WO2010141855).

PubMed

Taupin, Philippe

2011-09-01

The application (WO2010141855) is in the field of glycobiology, and involves the control of the rate of fucosylation of proteins by exogenous factors. It aims at controlling the rate of protein fucosylation with inhibitors (drugs or nucleic acid antagonists) of enzymes involved in the synthesis of GDP-fucose. Mammalian cell lines were cultured in the presence of inhibitors, for example, siRNA. The rates of GDP-fucose in cells and during protein fucosylation were characterized. The level of protein fucosylation decreases rapidly in response to a decrease in GDP-fucose level. The relationship between the rate of fucosylation of proteins and the level of GDP-fucose in a cell is non-linear. Reduction in the rate of protein fucosylation can be achieved with a minimal reduction of the level of GDP-fucose in cells. The paradigm may be used to synthesize proteins and antibodies, with a reduced rate of fucosylation. The application claims that the use of drugs or nucleic acid antagonists that inhibit the enzymes involved in GDP-fucose biosynthesis optimizes the level of GDP-fucose present in cells, and reduces the rate of fucosylation of glycoproteins.

Changes in transcription during recovery from heat injury in Salmonella typhimurium and effects of BCAA on recovery.

PubMed

Hsu-Ming, Wen; Naito, Kimitaka; Kinoshita, Yoshimasa; Kobayashi, Hiroshi; Honjoh, Ken-ichi; Tashiro, Kousuke; Miyamoto, Takahisa

2012-06-01

Mechanisms of recovery from heat injury in Salmonella typhimurium were elucidated. Recovery of the heat-injured S. typhimurium cells in TSB resulted in full recovery after 3 h of incubation at 37°C. The DNA microarray analysis of 30- and 60-min recovering cells resulted in an increase in transcription of 89 and 141 genes, respectively. Among them, 15 genes, with known function, seemed to be somewhat involved in recovery. They encoded proteins involved in branched-chain amino acid (BCAA) transport (livJ, livH), cell envelope integrity (ddg), heat-shock response (cpxP, rrmJ), phage shock protein (pspA), ribosome modulation factor (rmf), virulence (sseB) transcriptional regulation (rpoE, rpoH, rseA, rseB, rseC) and ArcB signal transduction (sixA) and cytoplasmic membrane protein (fxsA). Among them, the effects of BCAA supplementation on recovery from heat injury were studied to confirm the importance of the BCAA transport liv genes during recovery. It was found that supplementation of TSB with 0.1% BCAA resulted in an enhanced recovery of injured cells in comparison to those recovered in TSB without BCAA. Supplementation of BCAA at 0.1% resulted in a cell count increase 4.4-fold greater than that of the control after 1 h incubation. It seems that BCAA promoted the recovery by promoting protein synthesis either directly through their use in translation or indirectly through stimulation of protein synthesis by activation of the Lrp protein.

Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response.

PubMed

Van Cutsem, Emmanuel; Simonart, Géraldine; Degand, Hervé; Faber, Anne-Marie; Morsomme, Pierre; Boutry, Marc

2011-02-01

Nicotiana tabacum leaves are covered by trichomes involved in the secretion of large amounts of secondary metabolites, some of which play a major role in plant defense. However, little is known about the metabolic pathways that operate in these structures. We undertook a proteomic analysis of N. tabacum trichomes in order to identify their protein complement. Efficient trichome isolation was obtained by abrading frozen leaves. After homogenization, soluble proteins and a microsomal fraction were prepared by centrifugation. Gel-based and gel-free proteomic analyses were then performed. 2-DE analysis of soluble proteins led to the identification of 1373 protein spots, which were digested and analyzed by MS/MS, leading to 680 unique identifications. Both soluble proteins and microsomal fraction were analyzed by LC MALDI-MS/MS after trypsin digestion, leading to 858 identifications, many of which had not been identified after 2-DE, indicating that the two methods complement each other. Many enzymes putatively involved in secondary metabolism were identified, including enzymes involved in the synthesis of terpenoid precursors and in acyl sugar production. Several transporters were also identified, some of which might be involved in secondary metabolite transport. Various (a)biotic stress response proteins were also detected, supporting the role of trichomes in plant defense. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

PubMed

Paiva, Ana L S; Oliveira, Jose T A; de Souza, Gustavo A; Vasconcelos, Ilka M

2016-12-02

Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

An assembly of proteins and lipid domains regulates transport of phosphatidylserine to phosphatidylserine decarboxylase 2 in Saccharomyces cerevisiae.

PubMed

Riekhof, Wayne R; Wu, Wen-I; Jones, Jennifer L; Nikrad, Mrinalini; Chan, Mallory M; Loewen, Christopher J R; Voelker, Dennis R

2014-02-28

Saccharomyces cerevisiae uses multiple biosynthetic pathways for the synthesis of phosphatidylethanolamine. One route involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), the transport of this lipid to endosomes, and decarboxylation by PtdSer decarboxylase 2 (Psd2p) to produce phosphatidylethanolamine. Several proteins and protein motifs are known to be required for PtdSer transport to occur, namely the Sec14p homolog PstB2p/Pdr17p; a PtdIns 4-kinase, Stt4p; and a C2 domain of Psd2p. The focus of this work is on defining the protein-protein and protein-lipid interactions of these components. PstB2p interacts with a protein encoded by the uncharacterized gene YPL272C, which we name Pbi1p (PstB2p-interacting 1). PstB2p, Psd2, and Pbi1p were shown to be lipid-binding proteins specific for phosphatidic acid. Pbi1p also interacts with the ER-localized Scs2p, a binding determinant for several peripheral ER proteins. A complex between Psd2p and PstB2p was also detected, and this interaction was facilitated by a cryptic C2 domain at the extreme N terminus of Psd2p (C2-1) as well the previously characterized C2 domain of Psd2p (C2-2). The predicted N-terminal helical region of PstB2p was necessary and sufficient for promoting the interaction with both Psd2p and Pbi1p. Taken together, these results support a model for PtdSer transport involving the docking of a PtdSer donor membrane with an acceptor via specific protein-protein and protein-lipid interactions. Specifically, our model predicts that this process involves an acceptor membrane complex containing the C2 domains of Psd2p, PstB2p, and Pbi1p that ligate to Scs2p and phosphatidic acid present in the donor membrane, forming a zone of apposition that facilitates PtdSer transfer.

Assembly of Lipopolysaccharide in Escherichia coli Requires the Essential LapB Heat Shock Protein*

PubMed Central

Klein, Gracjana; Kobylak, Natalia; Lindner, Buko; Stupak, Anna; Raina, Satish

2014-01-01

Here, we describe two new heat shock proteins involved in the assembly of LPS in Escherichia coli, LapA and LapB (lipopolysaccharide assembly protein A and B). lapB mutants were identified based on an increased envelope stress response. Envelope stress-responsive pathways control key steps in LPS biogenesis and respond to defects in the LPS assembly. Accordingly, the LPS content in ΔlapB or Δ(lapA lapB) mutants was elevated, with an enrichment of LPS derivatives with truncations in the core region, some of which were pentaacylated and exhibited carbon chain polymorphism. Further, the levels of LpxC, the enzyme that catalyzes the first committed step of lipid A synthesis, were highly elevated in the Δ(lapA lapB) mutant. Δ(lapA lapB) mutant accumulated extragenic suppressors that mapped either to lpxC, waaC, and gmhA, or to the waaQ operon (LPS biosynthesis) and lpp (Braun's lipoprotein). Increased synthesis of either FabZ (3-R-hydroxymyristoyl acyl carrier protein dehydratase), slrA (novel RpoE-regulated non-coding sRNA), lipoprotein YceK, toxin HicA, or MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) suppressed some of the Δ(lapA lapB) defects. LapB contains six tetratricopeptide repeats and, at the C-terminal end, a rubredoxin-like domain that was found to be essential for its activity. In pull-down experiments, LapA and LapB co-purified with LPS, Lpt proteins, FtsH (protease), DnaK, and DnaJ (chaperones). A specific interaction was also observed between WaaC and LapB. Our data suggest that LapB coordinates assembly of proteins involved in LPS synthesis at the plasma membrane and regulates turnover of LpxC, thereby ensuring balanced biosynthesis of LPS and phospholipids consistent with its essentiality. PMID:24722986

Metabolic consequences of physical inactivity.

PubMed

Biolo, Gianni; Ciocchi, Beniamino; Stulle, Manuela; Piccoli, Arianna; Lorenzon, Stefania; Dal Mas, Viviana; Barazzoni, Rocco; Zanetti, Michela; Guarnieri, Gianfranco

2005-01-01

Physical inactivity is associated with alteration of normal physiologic processes leading to muscle atrophy, reduced exercise capacity, insulin resistance, and altered energy balance. Bed rest studies in human beings using stable isotopes of amino acids indicate that muscle unloading decreases the turnover rates of muscle and whole-body proteins, with a prevailing inhibition of protein synthesis. In the fasting state, muscle and whole-body nitrogen loss was not accelerated during bed rest. In experimental postprandial states, the amino acid-mediated stimulation of protein synthesis was impaired, whereas the ability of combined insulin and glucose infusion to decrease whole-body proteolysis was not affected by muscle inactivity. Thus, an impaired ability of protein/amino acid feeding to stimulate body protein synthesis is the major catabolic mechanism for the effect of bed rest on protein metabolism. This suggests that a protein intake level greater than normal could be required to achieve the same postprandial anabolic effect during muscle inactivity. Metabolic adaptation to muscle inactivity also involves development of resistance to the glucoregulatory action of insulin, decreased energy requirements, and increased insulin and leptin secretion. These alterations may lead to the development of the metabolic syndrome that is defined as the association of hyperinsulinemia, dyslipidemia, hypertension, hyperglycemia, and abdominal obesity. This cluster of metabolic abnormalities is a risk factor for coronary artery disease and stroke. Evidence indicates that exercise training programs may counteract all of these abnormalities both in healthy sedentary subjects and in patients affected by a variety of chronic disease states.

Unilateral hindlimb casting induced a delayed generalized muscle atrophy during rehabilitation that is prevented by a whey or a high protein diet but not a free leucine-enriched diet.

PubMed

Magne, Hugues; Savary-Auzeloux, Isabelle; Migné, Carole; Peyron, Marie-Agnès; Combaret, Lydie; Rémond, Didier; Dardevet, Dominique

2013-01-01

Sarcopenia is the general muscle mass and strength loss associated with ageing. Muscle atrophy could be made worse by exposure to acute periods of immobilization, because muscle disuse by itself is a stimulus for atrophy. Using a model of unilateral hindlimb casting in old adult rats, we have already demonstrated that the primary effect of immobilization was atrophy in the casted leg, but was also surprisingly associated with a retarded atrophy in the non-casted leg during rehabilitation. In search of mechanisms involved in this generalized atrophy, we demonstrated in the present study that contrary to pair-fed non-immobilized control animals, muscle protein synthesis in the non-immobilized limb was unable to adapt and to respond positively to food intake. Because pair-fed control rats did not lose muscle mass, this defect in muscle protein synthesis may represent one of the explanation for the muscle mass loss observed in the non-immobilized rats. Nevertheless, in order to stimulate protein turn over and generate a positive nitrogen balance required to maintain the whole muscle mass in immobilized rats, we tested a dietary free leucine supplementation (an amino acid known for its stimulatory effect on protein metabolism) during the rehabilitation period. Leucine supplementation was able to overcome the anabolic resistance in the non-immobilized limb. A greater muscle protein synthesis up-regulation associated with a stimulation of the mTOR signalling pathway was indeed recorded but it remained inefficient to prevent the loss of muscle in the non-immobilized limb. By contrast, we demonstrated here that whey protein or high protein diets were able to prevent the muscle mass loss of the non-immobilized limb by sustaining muscle protein synthesis during the entire rehabilitation period.

Unilateral Hindlimb Casting Induced a Delayed Generalized Muscle Atrophy during Rehabilitation that Is Prevented by a Whey or a High Protein Diet but Not a Free Leucine-Enriched Diet

PubMed Central

Magne, Hugues; Savary-Auzeloux, Isabelle; Migné, Carole; Peyron, Marie-Agnès; Combaret, Lydie; Rémond, Didier; Dardevet, Dominique

2013-01-01

Sarcopenia is the general muscle mass and strength loss associated with ageing. Muscle atrophy could be made worse by exposure to acute periods of immobilization, because muscle disuse by itself is a stimulus for atrophy. Using a model of unilateral hindlimb casting in old adult rats, we have already demonstrated that the primary effect of immobilization was atrophy in the casted leg, but was also surprisingly associated with a retarded atrophy in the non-casted leg during rehabilitation. In search of mechanisms involved in this generalized atrophy, we demonstrated in the present study that contrary to pair-fed non-immobilized control animals, muscle protein synthesis in the non-immobilized limb was unable to adapt and to respond positively to food intake. Because pair-fed control rats did not lose muscle mass, this defect in muscle protein synthesis may represent one of the explanation for the muscle mass loss observed in the non-immobilized rats. Nevertheless, in order to stimulate protein turn over and generate a positive nitrogen balance required to maintain the whole muscle mass in immobilized rats, we tested a dietary free leucine supplementation (an amino acid known for its stimulatory effect on protein metabolism) during the rehabilitation period. Leucine supplementation was able to overcome the anabolic resistance in the non-immobilized limb. A greater muscle protein synthesis up-regulation associated with a stimulation of the mTOR signalling pathway was indeed recorded but it remained inefficient to prevent the loss of muscle in the non-immobilized limb. By contrast, we demonstrated here that whey protein or high protein diets were able to prevent the muscle mass loss of the non-immobilized limb by sustaining muscle protein synthesis during the entire rehabilitation period. PMID:24015173

Effects of beta-hydroxy-beta-methylbutyrate (HMB) on the expression of ubiquitin ligases, protein synthesis pathways and contractile function in extensor digitorum longus (EDL) of fed and fasting rats.

PubMed

Gerlinger-Romero, Frederico; Guimarães-Ferreira, Lucas; Yonamine, Caio Yogi; Salgueiro, Rafael Barrera; Nunes, Maria Tereza

2018-03-01

Beta-hydroxy-beta-methylbutyrate (HMB), a leucine metabolite, enhances the gain of skeletal muscle mass by increasing protein synthesis or attenuating protein degradation or both. The aims of this study were to investigate the effect of HMB on molecular factors controlling skeletal muscle protein synthesis and degradation, as well as muscle contractile function, in fed and fasted conditions. Wistar rats were supplied daily with HMB (320 mg/kg body weight diluted in NaCl-0.9%) or vehicle only (control) by gavage for 28 days. After this period, some of the animals were subjected to a 24-h fasting, while others remained in the fed condition. The EDL muscle was then removed, weighed and used to evaluate the genes and proteins involved in protein synthesis (AKT/4E-BP1/S6) and degradation (Fbxo32 and Trim63). A sub-set of rats were used to measure in vivo muscle contractile function. HMB supplementation increased AKT phosphorylation during fasting (three-fold). In the fed condition, no differences were detected in atrogenes expression between control and HMB supplemented group; however, HMB supplementation did attenuate the fasting-induced increase in their expression levels. Fasting animals receiving HMB showed improved sustained tetanic contraction times (one-fold) and an increased muscle to tibia length ratio (1.3-fold), without any cross-sectional area changes. These results suggest that HMB supplementation under fasting conditions increases AKT phosphorylation and attenuates the increased of atrogenes expression, followed by a functional improvement and gain of skeletal muscle weight, suggesting that HMB protects skeletal muscle against the deleterious effects of fasting.

A Dietary Supplementation with Leucine and Antioxidants Is Capable to Accelerate Muscle Mass Recovery after Immobilization in Adult Rats

PubMed Central

Savary-Auzeloux, Isabelle; Magne, Hugues; Migné, Carole; Oberli, Marion; Breuillé, Denis; Faure, Magali; Vidal, Karine; Perrot, Marie; Rémond, Didier; Combaret, Lydie; Dardevet, Dominique

2013-01-01

Prolonged inactivity induces muscle loss due to an activation of proteolysis and decreased protein synthesis; the latter is also involved in the recovery of muscle mass. The aim of the present work was to explore the evolution of muscle mass and protein metabolism during immobilization and recovery and assess the effect of a nutritional strategy for counteracting muscle loss and facilitating recovery. Adult rats (6–8 months) were subjected to unilateral hindlimb casting for 8 days (I0–I8) and then permitted to recover for 10 to 40 days (R10–R40). They were fed a Control or Experimental diet supplemented with antioxidants/polyphenols (AOX) (I0 to I8), AOX and leucine (AOX + LEU) (I8 to R15) and LEU alone (R15 to R40). Muscle mass, absolute protein synthesis rate and proteasome activities were measured in gastrocnemius muscle in casted and non-casted legs in post prandial (PP) and post absorptive (PA) states at each time point. Immobilized gastrocnemius protein content was similarly reduced (-37%) in both diets compared to the non-casted leg. Muscle mass recovery was accelerated by the AOX and LEU supplementation (+6% AOX+LEU vs. Control, P<0.05 at R40) due to a higher protein synthesis both in PA and PP states (+23% and 31% respectively, Experimental vs. Control diets, P<0.05, R40) without difference in trypsin- and chymotrypsin-like activities between diets. Thus, this nutritional supplementation accelerated the recovery of muscle mass via a stimulation of protein synthesis throughout the entire day (in the PP and PA states) and could be a promising strategy to be tested during recovery from bed rest in humans. PMID:24312309

Live vaccines for human metapneumovirus designed by reverse genetics.

PubMed

Buchholz, Ursula J; Nagashima, Kunio; Murphy, Brian R; Collins, Peter L

2006-10-01

Human metapneumovirus (HMPV) was first described in 2001 and has quickly become recognized as an important cause of respiratory tract disease worldwide, especially in the pediatric population. A vaccine against HMPV is required to prevent severe disease associated with infection in infancy. The primary strategy is to develop a live-attenuated virus for intranasal immunization, which is particularly well suited against a respiratory virus. Reverse genetics provides a means of developing highly characterized 'designer' attenuated vaccine candidates. To date, several promising vaccine candidates have been developed, each using a different mode of attenuation. One candidate involves deletion of the G glycoprotein, providing attenuation that is probably based on reduced efficiency of attachment. A second candidate involves deletion of the M2-2 protein, which participates in regulating RNA synthesis and whose deletion has the advantageous property of upregulating transcription and increasing antigen synthesis. A third candidate involves replacing the P protein gene of HMPV with its counterpart from the related avian metapneumovirus, thereby introducing attenuation owing to its chimeric nature and host range restriction. Another live vaccine strategy involves using an attenuated parainfluenza virus as a vector to express HMPV protective antigens, providing a bivalent pediatric vaccine. Additional modifications to provide improved vaccines will also be discussed.

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation.

PubMed

Orellana, Renán A; Jeyapalan, Asumthia; Escobar, Jeffery; Frank, Jason W; Nguyen, Hanh V; Suryawan, Agus; Davis, Teresa A

2007-11-01

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

Desensitization of the growth hormone-induced Janus kinase 2 (Jak 2)/signal transducer and activator of transcription 5 (Stat5)-signaling pathway requires protein synthesis and phospholipase C.

PubMed

Fernández, L; Flores-Morales, A; Lahuna, O; Sliva, D; Norstedt, G; Haldosén, L A; Mode, A; Gustafsson, J A

1998-04-01

Signal transducers and activators of transcription (Stat) proteins are latent cytoplasmic transcription factors that are tyrosine phosphorylated by Janus kinases (Jak) in response to GH and other cytokines. GH activates Stat5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation. However, the mechanisms that turn off the GH-activated Jak2/Stat5 pathway are unknown. Continuous exposure to GH of BRL-4 cells, a rat hepatoma cell line stably transfected with rat GH receptor, induces a rapid but transient activation of Jak2 and Stat5. GH-induced Stat5 DNA-binding activity was detected after 2 min and reached a maximum at 10 min. Continued exposure to GH resulted in a desensitization characterized by 1) a rapid decrease in Stat5 DNA-binding activity. The rate of decrease of activity was rapid up to 1 h of GH treatment, and the remaining activity declined slowly thereafter. The activity of Stat5 present after 5 h is still higher than the control levels and almost 10-20% with respect to maximal activity at 10 min; and 2) the inability of further GH treatment to reinduce activation of Stat5. In contrast, with transient exposures of BRL-4 cells to GH, Stat5 DNA-binding activity could repeatedly be induced. GH-induced Jak2 and Stat5 activities were independent of ongoing protein synthesis. However, Jak2 tyrosine phosphorylation and Stat5 DNA-binding activity were prolonged for at least 4 h in the presence of cycloheximide, which suggests that the maintenance of desensitization requires ongoing protein synthesis. Furthermore, inhibition of protein synthesis potentiated GH-induced transcriptional activity in BRL-4 cells transiently transfected with SPIGLE1CAT, a reporter plasmid activated by Stat5. GH-induced Jak2 and Stat5 activation were not affected by D609 or mepacrine, both inhibitors of phospholipase C. However, in the presence of D609 and mepacrine, GH maintained prolonged Jak2 and Stat5 activation. Transactivation of SPIGLE1 by GH was potentiated by mepacrine and D609 but not by the phospholipase A2 inhibitor AACOCF3. Thus, a regulatory circuit of GH-induced transcription through the Jak2/Stat5-signaling pathway includes a prompt GH-induced activation of Jak2/Stat5 followed by a negative regulatory response; ongoing protein synthesis and intracellular signaling pathways, where phospholipase C activity is involved, play a critical role to desensitize the GH-activated Jak2/Stat5-signaling pathway.

Genetics Home Reference: TRNT1 deficiency

MedlinePlus

... in the production (synthesis) of other proteins. During protein synthesis, a molecule called transfer RNA (tRNA) helps assemble ... thought to be less able to participate in protein synthesis. Researchers suspect that protein synthesis in cellular structures ...

Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development.

PubMed

Dezfulian, Mohammad H; Foreman, Curtis; Jalili, Espanta; Pal, Mrinal; Dhaliwal, Rajdeep K; Roberto, Don Karl A; Imre, Kathleen M; Kohalmi, Susanne E; Crosby, William L

2017-04-07

Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking. To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised. Taken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.

Identification of endoplasmic reticulum proteins involved in glycan assembly: synthesis and characterization of P3-(4-azidoanilido)uridine 5'-triphosphate, a membrane-topological photoaffinity probe for uridine diphosphate-sugar binding proteins.

PubMed Central

Rancour, D M; Menon, A K

1998-01-01

Much of the enzymic machinery required for the assembly of cell surface carbohydrates is located in the endoplasmic reticulum (ER) of eukaryotic cells. Structural information on these proteins is limited and the identity of the active polypeptide(s) is generally unknown. This paper describes the synthesis and characteristics of a photoaffinity reagent that can be used to identify and analyse members of the ER glycan assembly apparatus, specifically those glycosyltransferases, nucleotide phosphatases and nucleotide-sugar transporters that recognize uridine nucleotides or UDP-sugars. The photoaffinity reagent, P3-(4-azidoanilido)uridine 5'-triphosphate (AAUTP), was synthesized easily from commercially available precursors. AAUTP inhibited the activity of ER glycosyltransferases that utilize UDP-GlcNAc and UDP-Glc, indicating that it is recognized by UDP-sugar-binding proteins. In preliminary tests AAUTP[alpha-32P] labelled bovine milk galactosyltransferase, a model UDP-sugar-utilizing enzyme, in a UV-light-dependent, competitive and saturable manner. When incubated with rat liver ER vesicles, AAUTP[alpha-32P] labelled a discrete subset of ER proteins; labelling was light-dependent and metal ion-specific. Photolabelling of intact ER vesicles with AAUTP[alpha-32P] caused selective incorporation of radioactivity into proteins with cytoplasmically disposed binding sites; UDP-Glc:glycoprotein glucosyltransferase, a lumenal protein, was labelled only when the vesicle membrane was disrupted. These data indicate that AAUTP is a membrane topological probe of catalytic sites in target proteins. Strategies for using AAUTP to identify and study novel ER proteins involved in glycan assembly are discussed. PMID:9677326

New Molecular Targets of Anticancer Therapy - Current Status and Perspectives.

PubMed

Zajac, Marianna; Muszalska, Izabela; Jelinska, Anna

2016-01-01

Molecularly targeted anticancer therapy involves the use of drugs or other substances affecting specific molecular targets that play a part in the development, progression and spread of a given neoplasm. By contrast, the majority of classical chemotherapeutics act on all rapidly proliferating cells, both healthy and cancerous ones. Target anticancer drugs are designed to achieve a particular aim and they usually act cytostatically, not cytotoxically like classical chemotherapeutics. At present, more than 300 biological molecular targets have been identified. The proteins involved in cellular metabolism include (among others) receptor proteins, signal transduction proteins, mRNA thread matrix synthesis proteins participating in neoplastic transformation, cell cycle control proteins, functional and structural proteins. The receptor proteins that are targeted by currently used anticancer drugs comprise the epithelial growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor(VEGFR). Target anticancer drugs may affect extracellular receptor domains (antibodies) or intracellular receptor domains (tyrosine kinase inhibitors). The blocking of the mRNA thread containing information about the structure of oncogenes (signal transduction proteins) is another molecular target of anticancer drugs. That type of treatment, referred to as antisense therapy, is in clinical trials. When the synthesis of genetic material is disturbed, in most cases the passage to the next cycle phase is blocked. The key proteins responsible for the blockage are cyclines and cycline- dependent kinases (CDK). Clinical trials are focused on natural and synthetic substances capable of blocking various CDKs. The paper discusses the molecular targets and chemical structure of target anticancer drugs that have been approved for and currently applied in antineoplastic therapy together with indications and contraindications for their application.

Proteomic analysis of proteins related to rice grain chalkiness using iTRAQ and a novel comparison system based on a notched-belly mutant with white-belly

PubMed Central

2014-01-01

Background Grain chalkiness is a complex trait adversely affecting appearance and milling quality, and therefore has been one of principal targets for rice improvement. Eliminating chalkiness from rice has been a daunting task due to the complex interaction between genotype and environment and the lack of molecular markers. In addition, the molecular mechanisms underlying grain chalkiness formation are still imperfectly understood. Results We identified a notched-belly mutant (DY1102) with high percentage of white-belly, which only occurs in the bottom part proximal to the embryo. Using this mutant, a novel comparison system that can minimize the effect of genetic background and growing environment was developed. An iTRAQ-based comparative display of the proteins between the bottom chalky part and the upper translucent part of grains of DY1102 was performed. A total of 113 proteins responsible for chalkiness formation was identified. Among them, 70 proteins are up-regulated and 43 down-regulated. Approximately half of these differentially expressed proteins involved in central metabolic or regulatory pathways including carbohydrate metabolism (especially cell wall synthesis) and protein synthesis, folding and degradation, providing proteomic confirmation of the notion that chalkiness formation involves diverse but delicately regulated pathways. Protein metabolism was the most abundant category, accounting for 27.4% of the total differentially expressed proteins. In addition, down regulation of PDIL 2–3 and BiP was detected in the chalky tissue, indicating the important role of protein metabolism in grain chalkiness formation. Conclusions Using this novel comparison system, our comprehensive survey of endosperm proteomics in the notched-belly mutant provides a valuable proteomic resource for the characterization of pathways contributing to chalkiness formation at molecular and biochemical levels. PMID:24924297

Bio-Based Approaches to Inorganic Material Synthesis (Preprint)

DTIC Science & Technology

2007-03-01

involves the fungus and plant pathogen Fusarium oxysporum acting on amorphous silica in rice husks to transform it into crystalline silica...nanoparticles after 24 hrs at room temperature [3]. Specific cationic proteins from F. oxysporum were found to be associated with the crystalline silica 2 after

A Plant 5S Ribosomal RNA Mimic Regulates Alternative Splicing of Transcription Factor IIIA Pre-mRNAs

PubMed Central

Hammond, Ming C.; Wachter, Andreas; Breaker, Ronald R.

2009-01-01

Transcription factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with striking resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs (pre-mRNAs) from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. Since the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing. PMID:19377483

A plant 5S ribosomal RNA mimic regulates alternative splicing of transcription factor IIIA pre-mRNAs.

PubMed

Hammond, Ming C; Wachter, Andreas; Breaker, Ronald R

2009-05-01

Transcription factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with clear resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. As the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing.

The growing impact of lyophilized cell-free protein expression systems

PubMed Central

Hunt, J. Porter; Yang, Seung Ook; Wilding, Kristen M.; Bundy, Bradley C.

2017-01-01

ABSTRACT Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis. PMID:27791452

HIV replication enhances production of free fatty acids, low density lipoproteins and many key proteins involved in lipid metabolism: a proteomics study.

PubMed

Rasheed, Suraiya; Yan, Jasper S; Lau, Alex; Chan, Arvan S

2008-08-20

HIV-infected patients develop multiple metabolic abnormalities including insulin resistance, lipodystrophy and dyslipidemia. Although progression of these disorders has been associated with the use of various protease inhibitors and other antiretroviral drugs, HIV-infected individuals who have not received these treatments also develop lipid abnormalities albeit to a lesser extent. How HIV alters lipid metabolism in an infected cell and what molecular changes are affected through protein interaction pathways are not well-understood. Since many genetic, epigenetic, dietary and other factors influence lipid metabolism in vivo, we have chosen to study genome-wide changes in the proteomes of a human T-cell line before and after HIV infection in order to circumvent computational problems associated with multiple variables. Four separate experiments were conducted including one that compared 14 different time points over a period of >3 months. By subtractive analyses of protein profiles overtime, several hundred differentially expressed proteins were identified in HIV-infected cells by mass spectrometry and each protein was scrutinized for its biological functions by using various bioinformatics programs. Herein, we report 18 HIV-modulated proteins and their interaction pathways that enhance fatty acid synthesis, increase low density lipoproteins (triglycerides), dysregulate lipid transport, oxidize lipids, and alter cellular lipid metabolism. We conclude that HIV replication alone (i.e. without any influence of antiviral drugs, or other human genetic factors), can induce novel cellular enzymes and proteins that are significantly associated with biologically relevant processes involved in lipid synthesis, transport and metabolism (p = <0.0002-0.01). Translational and clinical studies on the newly discovered proteins may now shed light on how some of these proteins may be useful for early diagnosis of individuals who might be at high risk for developing lipid-related disorders. The target proteins could then be used for future studies in the development of inhibitors for preventing lipid-metabolic anomalies. This is the first direct evidence that HIV-modulates production of proteins that are significantly involved in disrupting the normal lipid-metabolic pathways.

From The Cover: Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation

NASA Astrophysics Data System (ADS)

Nollen, Ellen A. A.; Garcia, Susana M.; van Haaften, Gijs; Kim, Soojin; Chavez, Alejandro; Morimoto, Richard I.; Plasterk, Ronald H. A.

2004-04-01

Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation. protein misfolding | neurodegenerative diseases

Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

PubMed

Gao, Song; Carson, James A

2016-01-01

Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. Copyright © 2016 the American Physiological Society.

IBT-based quantitative proteomics identifies potential regulatory proteins involved in pigmentation of purple sea cucumber, Apostichopus japonicus.

PubMed

Xing, Lili; Sun, Lina; Liu, Shilin; Li, Xiaoni; Zhang, Libin; Yang, Hongsheng

2017-09-01

Sea cucumbers are an important economic species and exhibit high yield value among aquaculture animals. Purple sea cucumbers are very rare and beautiful and have stable hereditary patterns. In this study, isobaric tags (IBT) were first used to reveal the molecular mechanism of pigmentation in the body wall of the purple sea cucumber. We analyzed the proteomes of purple sea cucumber in early pigmentation stage (Pa), mid pigmentation stage (Pb) and late pigmentation stage (Pc), resulting in the identification of 5580 proteins, including 1099 differentially expressed proteins in Pb: Pa and 339 differentially expressed proteins in Pc: Pb. GO and KEGG analyses revealed possible differentially expressed proteins, including"melanogenesis", "melanosome", "melanoma", "pigment-biosynthetic process", "Epidermis development", "Ras-signaling pathway", "Wnt-signaling pathway", "response to UV light", and "tyrosine metabolism", involved in pigment synthesis and regulation in purple sea cucumbers. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying pigmentation in sea cucumbers. Furthermore, these results may also provide the base for further identification of proteins involved in resistance mechanisms against melanoma, albinism, UV damage, and other diseases in sea cucumbers. Copyright © 2017. Published by Elsevier Inc.

Systematic identification of light-regulated cold-responsive proteome in a model cyanobacterium.

PubMed

Chen, Weiyang; Fang, Longfa; Huang, Xiahe; Ge, Haitao; Wang, Jinlong; Wang, Xiaorong; Zhang, Yuanya; Sui, Na; Xu, Wu; Wang, Yingchun

2018-05-15

Differential expression of cold-responsive proteins is necessary for cyanobacteria to acclimate to cold stress frequently occurring in their natural habitats. Accumulating evidence indicates that cold-induced expression of certain proteins is dependent on light illumination, but a systematic identification of light-dependent and/or light-independent cold-responsive proteins in cyanobacteria is still lacking. Herein, we comprehensively identified cold-responsive proteins in a model cyanobacterium Synechocystis sp. PCC 6803 (Hereafter Synechocystis) that was cold-stressed in light or in dark. In total, 72 proteins were identified as cold-responsive, including 19 and 17 proteins whose cold-responsiveness are light-dependent and light-independent, respectively. Bioinformatic analysis revealed that outer membrane proteins, proteins involved in translation, and proteins involved in divergent types of stress responses were highly enriched in the cold-responsive proteins. Moreover, a protein network responsible for nitrogen assimilation and amino acid biosynthesis, transcription, and translation were upregulated in response to the cold stress. The network contains both light-dependent and light-independent cold-responsive proteins, probably for fine tuning its activity to endow Synechocystis the flexibility necessary for cold adaptation in their natural habitats, where days and nights are alternating. Together, our results should serve as an important resource for future study toward understanding the mechanism of cold acclimation in cyanobacteria. Photosynthetic cyanobacteria need to acclimate to frequently occurring abiotic stresses such as cold in their natural habitats, and the acclimation process has to be coordinated with photosynthesis, the light-dependent process that provides carbon and energy for propagation of cyanobacteria. It is conceivable that cold-induced differential protein expression can also be regulated by light. Hence it is important to systematically identify cold responsive proteins that are regulated or not regulated by light to better understand the mechanism of cold acclimation in cyanobacteria. In this manuscript, we identified a network involved in protein synthesis that were upregulated by cold. The network contains both light-dependent and light-independent cold-inducible proteins, presumably for fine tuning the activity of the network in natural habitats of cyanobacteria where days and nights are alternating. This finding underscores the significance of proteome reprograming toward enhancing protein synthesis in cold adaptation. Copyright © 2018 Elsevier B.V. All rights reserved.

Proteomic Profiling of De Novo Protein Synthesis in Starvation-Induced Autophagy Using Bioorthogonal Noncanonical Amino Acid Tagging.

PubMed

Zhang, J; Wang, J; Lee, Y-M; Lim, T-K; Lin, Q; Shen, H-M

2017-01-01

Autophagy is an intracellular degradation process activated by stress factors such as nutrient starvation to maintain cellular homeostasis. There is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, up-to-date characterizing of these de novo proteins is technically difficult. In this chapter, we describe a novel method to identify newly synthesized proteins during starvation-mediated autophagy by bioorthogonal noncanonical amino acid tagging (BONCAT), in conjunction with isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics. l-azidohomoalanine (AHA) is an analog of methionine, and it can be readily incorporated into the newly synthesized proteins. The AHA-containing proteins can be enriched with avidin beads after a "click" reaction between alkyne-bearing biotin and the azide moiety of AHA. The enriched proteins are then subjected to iTRAQ™ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By using this technique, we have successfully profiled more than 700 proteins that are synthesized during starvation-induced autophagy. We believe that this approach is effective in identification of newly synthesized proteins in the process of autophagy and provides useful insights to the molecular mechanisms and biological functions of autophagy. © 2017 Elsevier Inc. All rights reserved.

Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation

PubMed Central

Totsuka, Akane; Okamoto, Emi; Miyahara, Taira; Kouno, Takanobu; Cano, Emilio A.; Sasaki, Nobuhiro; Watanabe, Aiko; Tasaki, Keisuke; Nishihara, Masahiro; Ozeki, Yoshihiro

2018-01-01

In a previous study, two genes responsible for white flower phenotypes in carnation were identified. These genes encoded enzymes involved in anthocyanin synthesis, namely, flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR), and showed reduced expression in the white flower phenotypes. Here, we identify another candidate gene for white phenotype in carnation flowers using an RNA-seq analysis followed by RT-PCR. This candidate gene encodes a transcriptional regulatory factor of the basic helix-loop-helix (bHLH) type. In the cultivar examined here, both F3H and DFR genes produced active enzyme proteins; however, expression of DFR and of genes for enzymes involved in the downstream anthocyanin synthetic pathway from DFR was repressed in the absence of bHLH expression. Occasionally, flowers of the white flowered cultivar used here have red speckles and stripes on the white petals. We found that expression of bHLH occurred in these red petal segments and induced expression of DFR and the following downstream enzymes. Our results indicate that a member of the bHLH superfamily is another gene involved in anthocyanin synthesis in addition to structural genes encoding enzymes. PMID:29681756

In vitro synthesis of the iron–molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins

PubMed Central

Curatti, Leonardo; Hernandez, Jose A.; Igarashi, Robert Y.; Soboh, Basem; Zhao, Dehua; Rubio, Luis M.

2007-01-01

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron–molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe2+, S2−, MoO42−, and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe2+, S2−, MoO42−, R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed. PMID:17978192

Immediate Translation of Formin DIAPH1 mRNA after Its Exiting the Nucleus Is Required for Its Perinuclear Localization in Fibroblasts

PubMed Central

Liao, Guoning; Liu, Gang

2013-01-01

DIAPH1 is a formin protein which promotes actin polymerization, stabilizes microtubules and consequently is involved in cytoskeleton dynamics, cell migration and differentiation. In contrast to the relatively well-understood signaling cascades that regulate DIAPH1 activity, its spatial regulation of biogenesis is not understood. A recent report showed that synthesis of DIAPH1 is confined in the perinuclear ER compartment through translation-dependent mRNA targeting. However, the underlying mechanism of DIAPH1 local synthesis is yet to be elucidated. Here, we provide evidence to demonstrate that the 5′-cap-mediated immediate translation of DIAPH1 mRNA upon exiting nucleus is required for localizing the mRNA in the perinuclear ER compartment. This is supported by data: 1) Delayed translation of DIAPH1 mRNA resulted in loss of perinuclear localization of the mRNA; 2) Once delocalized, DIAPH1 mRNA could not be retargeted to the perinuclear region; and 3) The translation of DIAPH1 mRNA is 5′-cap dependent. These results provide new insights into the novel mechanism of DIAPH1 local synthesis. In addition, these findings have led to the development of new approaches for manipulating DIAPH1 mRNA localization and local protein synthesis in cells for functional studies. Furthermore, a correlation of DIAPH1 mRNA and DIAPH1 protein localization has been demonstrated using a new method to quantify the intracellular distribution of protein. PMID:23840831

Amino acid homeostasis and signalling in mammalian cells and organisms

PubMed Central

Bröer, Angelika

2017-01-01

Cells have a constant turnover of proteins that recycle most amino acids over time. Net loss is mainly due to amino acid oxidation. Homeostasis is achieved through exchange of essential amino acids with non-essential amino acids and the transfer of amino groups from oxidised amino acids to amino acid biosynthesis. This homeostatic condition is maintained through an active mTORC1 complex. Under amino acid depletion, mTORC1 is inactivated. This increases the breakdown of cellular proteins through autophagy and reduces protein biosynthesis. The general control non-derepressable 2/ATF4 pathway may be activated in addition, resulting in transcription of genes involved in amino acid transport and biosynthesis of non-essential amino acids. Metabolism is autoregulated to minimise oxidation of amino acids. Systemic amino acid levels are also tightly regulated. Food intake briefly increases plasma amino acid levels, which stimulates insulin release and mTOR-dependent protein synthesis in muscle. Excess amino acids are oxidised, resulting in increased urea production. Short-term fasting does not result in depletion of plasma amino acids due to reduced protein synthesis and the onset of autophagy. Owing to the fact that half of all amino acids are essential, reduction in protein synthesis and amino acid oxidation are the only two measures to reduce amino acid demand. Long-term malnutrition causes depletion of plasma amino acids. The CNS appears to generate a protein-specific response upon amino acid depletion, resulting in avoidance of an inadequate diet. High protein levels, in contrast, contribute together with other nutrients to a reduction in food intake. PMID:28546457

Organization of chlorophyll biosynthesis and insertion of chlorophyll into the chlorophyll-binding proteins in chloroplasts.

PubMed

Wang, Peng; Grimm, Bernhard

2015-12-01

Oxygenic photosynthesis requires chlorophyll (Chl) for the absorption of light energy, and charge separation in the reaction center of photosystem I and II, to feed electrons into the photosynthetic electron transfer chain. Chl is bound to different Chl-binding proteins assembled in the core complexes of the two photosystems and their peripheral light-harvesting antenna complexes. The structure of the photosynthetic protein complexes has been elucidated, but mechanisms of their biogenesis are in most instances unknown. These processes involve not only the assembly of interacting proteins, but also the functional integration of pigments and other cofactors. As a precondition for the association of Chl with the Chl-binding proteins in both photosystems, the synthesis of the apoproteins is synchronized with Chl biosynthesis. This review aims to summarize the present knowledge on the posttranslational organization of Chl biosynthesis and current attempts to envision the proceedings of the successive synthesis and integration of Chl into Chl-binding proteins in the thylakoid membrane. Potential auxiliary factors, contributing to the control and organization of Chl biosynthesis and the association of Chl with the Chl-binding proteins during their integration into photosynthetic complexes, are discussed in this review.

Mitochondrial Protein Synthesis, Import, and Assembly

PubMed Central

Fox, Thomas D.

2012-01-01

The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

Synthesis of guanidinoacetate and creatine from amino acids by rat pancreas.

PubMed

da Silva, Robin P; Clow, Kathy; Brosnan, John T; Brosnan, Margaret E

2014-02-01

Creatine is an important molecule involved in cellular energy metabolism. Creatine is spontaneously converted to creatinine at a rate of 1·7% per d; creatinine is lost in the urine. Creatine can be obtained from the diet or synthesised from endogenous amino acids via the enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT). The liver has high GAMT activity and the kidney has high AGAT activity. Although the pancreas has both AGAT and GAMT activities, its possible role in creatine synthesis has not been characterised. In the present study, we examined the enzymes involved in creatine synthesis in the pancreas as well as the synthesis of guanidinoacetate (GAA) and creatine by isolated pancreatic acini. We found significant AGAT activity and somewhat lower GAMT activity in the pancreas and that pancreatic acini had measurable activities of both AGAT and GAMT and the capacity to synthesise GAA and creatine from amino acids. Creatine supplementation led to a decrease in AGAT activity in the pancreas, though it did not affect its mRNA or protein abundance. This was in contrast with the reduction of AGAT activity and mRNA and protein abundance in the kidney, suggesting that the regulatory mechanisms that control the expression of this enzyme in the pancreas are different from those in the kidney. Dietary creatine increased the concentrations of GAA, creatine and phosphocreatine in the pancreas. Unexpectedly, creatine supplementation decreased the concentrations of S-adenosylmethionine, while those of S-adenosylhomocysteine were not altered significantly.

Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet.

PubMed

Zhou, Zhong-Hua; Yang, Hui-Juan; Chen, Ming; Lou, Cheng-Fu; Zhang, Yao-Zhou; Chen, Ke-Ping; Wang, Yong; Yu, Mei-Lan; Yu, Fang; Li, Jian-Ying; Zhong, Bo-Xiong

2008-12-01

To gain an insight into the effects of different diets on growth and development of the domesticated silkworm at protein level, we employed comparative proteomic approach to investigate the proteomic differences of midgut, hemolymph, fat body and posterior silk gland of the silkworms reared on fresh mulberry leaves and on artificial diet. Seventy-six differentially expressed proteins were identified by MALDI TOF/TOF MS, and among them, 41 proteins were up-regulated, and 35 proteins were downregulated. Database searches, combined with GO analysis and KEGG pathway analysis revealed that some hemolymph proteins such as Nuecin, Gloverin-like proteins, PGRP, P50 and beta/-N-acetylglucosamidase were related to innate immunity of the silkworm, and some proteins identified in silkworm midgut including Myosin 1 light chain, Tropomyosin 1, Profilin, Serpin-2 and GSH-Px were involved in digestion and nutrition absorption. Moreover, two up-regulated enzymes in fat body of larvae reared on artificial diet were identified as V-ATPase subunit B and Arginine kinase which participate in energy metabolism. Furthermore, 6 down-regulated proteins identified in posterior silk gland of silkworm larvae reared on artificial diet including Ribosomal protein SA, EF-2, EF-1gamma, AspAT, ERp57 and PHB were related to silk synthesis. Our results suggested that the different diets could alter the expression of proteins related to immune system, digestion and absorption of nutrient, energy metabolism and silk synthesis poor nutrition and absorption of nutrition in silkworm. The results also confirmed that the poor nutrient absorption, weakened innate immunity, decreased energy metabolism and reduced silk synthesis are the main reasons for low cocoons yield, inferior filament quality, low survival rate of young larvae and insufficient resistance against specific pathogens in the silkworms fed on artificial diet.

Light aerobic physical exercise in combination with leucine and/or glutamine-rich diet can improve the body composition and muscle protein metabolism in young tumor-bearing rats.

PubMed

Salomão, Emilianne Miguel; Gomes-Marcondes, Maria Cristina Cintra

2012-12-01

Nutritional supplementation with some amino acids may influence host's responses and also certain mechanism involved in tumor progression. It is known that exercise influences body weight and muscle composition. Previous findings from our group have shown that leucine has beneficial effects on protein composition in cachectic rat model as the Walker 256 tumor. The main purpose of this study was to analyze the effects of light exercise and leucine and/or glutamine-rich diet in body composition and skeletal muscle protein synthesis and degradation in young tumor-bearing rats. Walker tumor-bearing rats were subjected to light aerobic exercise (swimming 30 min/day) and fed a leucine-rich (3%) and/or glutamine-rich (4%) diet for 10 days and compared to healthy young rats. The carcasses were analyzed as total water and fat body content and lean body mass. The gastrocnemious muscles were isolated and used for determination of total protein synthesis and degradation. The chemical body composition changed with tumor growth, increasing body water and reducing body fat content and total body nitrogen. After tumor growth, the muscle protein metabolism was impaired, showing that the muscle protein synthesis was also reduced and the protein degradation process was increased in the gastrocnemius muscle of exercised rats. Although short-term exercise (10 days) alone did not produce beneficial effects that would reduce tumor damage, host protein metabolism was improved when exercise was combined with a leucine-rich diet. Only total carcass nitrogen and protein were recovered by a glutamine-rich diet. Exercise, in combination with an amino acid-rich diet, in particular, leucine, had effects beyond reducing tumoral weight such as improving protein turnover and carcass nitrogen content in the tumor-bearing host.

Myasthenia gravis: past, present, and future

PubMed Central

Conti-Fine, Bianca M.; Milani, Monica; Kaminski, Henry J.

2006-01-01

Myasthenia gravis (MG) is an autoimmune syndrome caused by the failure of neuromuscular transmission, which results from the binding of autoantibodies to proteins involved in signaling at the neuromuscular junction (NMJ). These proteins include the nicotinic AChR or, less frequently, a muscle-specific tyrosine kinase (MuSK) involved in AChR clustering. Much is known about the mechanisms that maintain self tolerance and modulate anti-AChR Ab synthesis, AChR clustering, and AChR function as well as those that cause neuromuscular transmission failure upon Ab binding. This insight has led to the development of improved diagnostic methods and to the design of specific immunosuppressive or immunomodulatory treatments. PMID:17080188

Fragile X Mental Retardation Protein Is Required to Maintain Visual Conditioning-Induced Behavioral Plasticity by Limiting Local Protein Synthesis

PubMed Central

Liu, Han-Hsuan

2016-01-01

Fragile X mental retardation protein (FMRP) is thought to regulate neuronal plasticity by limiting dendritic protein synthesis, but direct demonstration of a requirement for FMRP control of local protein synthesis during behavioral plasticity is lacking. Here we tested whether FMRP knockdown in Xenopus optic tectum affects local protein synthesis in vivo and whether FMRP knockdown affects protein synthesis-dependent visual avoidance behavioral plasticity. We tagged newly synthesized proteins by incorporation of the noncanonical amino acid azidohomoalanine and visualized them with fluorescent noncanonical amino acid tagging (FUNCAT). Visual conditioning and FMRP knockdown produce similar increases in FUNCAT in tectal neuropil. Induction of visual conditioning-dependent behavioral plasticity occurs normally in FMRP knockdown animals, but plasticity degrades over 24 h. These results indicate that FMRP affects visual conditioning-induced local protein synthesis and is required to maintain the visual conditioning-induced behavioral plasticity. SIGNIFICANCE STATEMENT Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Exaggerated dendritic protein synthesis resulting from loss of fragile X mental retardation protein (FMRP) is thought to underlie cognitive deficits in FXS, but no direct evidence has demonstrated that FMRP-regulated dendritic protein synthesis affects behavioral plasticity in intact animals. Xenopus tadpoles exhibit a visual avoidance behavior that improves with visual conditioning in a protein synthesis-dependent manner. We showed that FMRP knockdown and visual conditioning dramatically increase protein synthesis in neuronal processes. Furthermore, induction of visual conditioning-dependent behavioral plasticity occurs normally after FMRP knockdown, but performance rapidly deteriorated in the absence of FMRP. These studies show that FMRP negatively regulates local protein synthesis and is required to maintain visual conditioning-induced behavioral plasticity in vivo. PMID:27383604

Fragile X Mental Retardation Protein Is Required to Maintain Visual Conditioning-Induced Behavioral Plasticity by Limiting Local Protein Synthesis.

PubMed

Liu, Han-Hsuan; Cline, Hollis T

2016-07-06

Fragile X mental retardation protein (FMRP) is thought to regulate neuronal plasticity by limiting dendritic protein synthesis, but direct demonstration of a requirement for FMRP control of local protein synthesis during behavioral plasticity is lacking. Here we tested whether FMRP knockdown in Xenopus optic tectum affects local protein synthesis in vivo and whether FMRP knockdown affects protein synthesis-dependent visual avoidance behavioral plasticity. We tagged newly synthesized proteins by incorporation of the noncanonical amino acid azidohomoalanine and visualized them with fluorescent noncanonical amino acid tagging (FUNCAT). Visual conditioning and FMRP knockdown produce similar increases in FUNCAT in tectal neuropil. Induction of visual conditioning-dependent behavioral plasticity occurs normally in FMRP knockdown animals, but plasticity degrades over 24 h. These results indicate that FMRP affects visual conditioning-induced local protein synthesis and is required to maintain the visual conditioning-induced behavioral plasticity. Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Exaggerated dendritic protein synthesis resulting from loss of fragile X mental retardation protein (FMRP) is thought to underlie cognitive deficits in FXS, but no direct evidence has demonstrated that FMRP-regulated dendritic protein synthesis affects behavioral plasticity in intact animals. Xenopus tadpoles exhibit a visual avoidance behavior that improves with visual conditioning in a protein synthesis-dependent manner. We showed that FMRP knockdown and visual conditioning dramatically increase protein synthesis in neuronal processes. Furthermore, induction of visual conditioning-dependent behavioral plasticity occurs normally after FMRP knockdown, but performance rapidly deteriorated in the absence of FMRP. These studies show that FMRP negatively regulates local protein synthesis and is required to maintain visual conditioning-induced behavioral plasticity in vivo. Copyright © 2016 the authors 0270-6474/16/367325-15$15.00/0.

Exploring the Denitrification Proteome of Paracoccus denitrificans PD1222

PubMed Central

Olaya-Abril, Alfonso; Hidalgo-Carrillo, Jesús; Luque-Almagro, Víctor M.; Fuentes-Almagro, Carlos; Urbano, Francisco J.; Moreno-Vivián, Conrado; Richardson, David J.; Roldán, María D.

2018-01-01

Denitrification is a respiratory process that produces nitrous oxide as an intermediate, which may escape to the atmosphere before its reduction to dinitrogen through the nitrous oxide reductase NosZ. In this work, the denitrification process carried out by Paracoccus denitrificans PD1222 has been explored through a quantitative proteomic analysis. Under anaerobic conditions, with nitrate as sole nitrogen source, the synthesis of all the enzymes involved in denitrification, the respiratory nitrate, nitrite, nitric oxide, and nitrous oxide reductases, was increased. However, the periplasmic and assimilatory nitrate reductases decreased. Synthesis of transporters for alcohols, D-methionine, sulfate and copper, most of the enzymes involved in the tricarboxylic acid cycle, and proteins involved in other metabolic processes like lysine catabolism, fatty acids degradation and acetyl-CoA synthesis, was increased during denitrification in P. denitrificans PD1222. As consequence, an enhanced production of the central metabolite acetyl-CoA was observed. After establishing the key features of the denitrification proteome, its changes by the influence of a competitive electron acceptor, oxygen, or competitive nitrogen source, ammonium, were evaluated. PMID:29896187

Exploring the Denitrification Proteome of Paracoccus denitrificans PD1222.

PubMed

Olaya-Abril, Alfonso; Hidalgo-Carrillo, Jesús; Luque-Almagro, Víctor M; Fuentes-Almagro, Carlos; Urbano, Francisco J; Moreno-Vivián, Conrado; Richardson, David J; Roldán, María D

2018-01-01

Denitrification is a respiratory process that produces nitrous oxide as an intermediate, which may escape to the atmosphere before its reduction to dinitrogen through the nitrous oxide reductase NosZ. In this work, the denitrification process carried out by Paracoccus denitrificans PD1222 has been explored through a quantitative proteomic analysis. Under anaerobic conditions, with nitrate as sole nitrogen source, the synthesis of all the enzymes involved in denitrification, the respiratory nitrate, nitrite, nitric oxide, and nitrous oxide reductases, was increased. However, the periplasmic and assimilatory nitrate reductases decreased. Synthesis of transporters for alcohols, D-methionine, sulfate and copper, most of the enzymes involved in the tricarboxylic acid cycle, and proteins involved in other metabolic processes like lysine catabolism, fatty acids degradation and acetyl-CoA synthesis, was increased during denitrification in P. denitrificans PD1222. As consequence, an enhanced production of the central metabolite acetyl-CoA was observed. After establishing the key features of the denitrification proteome, its changes by the influence of a competitive electron acceptor, oxygen, or competitive nitrogen source, ammonium, were evaluated.

An Amylase-Like Protein, AmyD, Is the Major Negative Regulator for α-Glucan Synthesis in Aspergillus nidulans during the Asexual Life Cycle.

PubMed

He, Xiaoxiao; Li, Shengnan; Kaminskyj, Susan

2017-03-27

α-Glucan affects fungal cell-cell interactions and is important for the virulence of pathogenic fungi. Interfering with production of α-glucan could help to prevent fungal infection. In our previous study, we reported that an amylase-like protein, AmyD, could repress α-glucan accumulation in Aspergillus nidulans . However, the underlying molecular mechanism was not clear. Here, we examined the localization of AmyD and found it was a membrane-associated protein. We studied AmyD function in α-glucan degradation, as well as with other predicted amylase-like proteins and three annotated α-glucanases. AmyC and AmyE share a substantial sequence identity with AmyD, however, neither affects α-glucan synthesis. In contrast, AgnB and MutA (but not AgnE) are functional α-glucanases that also repress α-glucan accumulation. Nevertheless, the functions of AmyD and these glucanases were independent from each other. The dynamics of α-glucan accumulation showed different patterns between the AmyD overexpression strain and the α-glucanase overexpression strains, suggesting AmyD may not be involved in the α-glucan degradation process. These results suggest the function of AmyD is to directly suppress α-glucan synthesis, but not to facilitate its degradation.

A Simple Protein Synthesis Model for the PURE System Operation.

PubMed

Mavelli, Fabio; Marangoni, Roberto; Stano, Pasquale

2015-06-01

The encapsulation of transcription-translation (TX-TL) cell-free machinery inside lipid vesicles (liposomes) is a key element in synthetic cell technology. The PURE system is a TX-TL kit composed of well-characterized parts, whose concentrations are fine tunable, which works according to a modular architecture. For these reasons, the PURE system perfectly fulfils the requirements of synthetic biology and is widely used for constructing synthetic cells. In this work, we present a simplified mathematical model to simulate the PURE system operations. Based on Michaelis-Menten kinetics and differential equations, the model describes protein synthesis dynamics by using 9 chemical species, 6 reactions and 16 kinetic parameters. The model correctly predicts the time course for messenger RNA and protein production and allows quantitative predictions. By means of this model, it is possible to foresee how the PURE system species affect the mechanism of proteins synthesis and therefore help in understanding scenarios where the concentration of the PURE system components has been modified purposely or as a result of stochastic fluctuations (for example after random encapsulation inside vesicles). The model also makes the determination of response coefficients for all species involved in the TX-TL mechanism possible and allows for scrutiny on how chemical energy is consumed by the three PURE system modules (transcription, translation and aminoacylation).

Proteomic Analysis of Differentially Expressed Proteins Involved in Peel Senescence in Harvested Mandarin Fruit

PubMed Central

Li, Taotao; Zhang, Jingying; Zhu, Hong; Qu, Hongxia; You, Shulin; Duan, Xuewu; Jiang, Yueming

2016-01-01

Mandarin (Citrus reticulata), a non-climacteric fruit, is an economically important fruit worldwide. The mechanism underlying senescence of non-climacteric fruit is poorly understood. In this study, a gel-based proteomic study followed by LC-ESI-MS/MS analysis was carried out to investigate the proteomic changes involved in peel senescence in harvested mandarin “Shatangju” fruit stored for 18 days. Over the course of the storage period, the fruit gradually senesced, accompanied by a decreased respiration rate and increased chlorophyll degradation and disruption of membrane integrity. Sixty-three proteins spots that showed significant differences in abundance were identified. The up-regulated proteins were mainly associated with cell wall degradation, lipid degradation, protein degradation, senescence-related transcription factors, and transcription-related proteins. In contrast, most proteins associated with ATP synthesis and scavenging of reactive oxygen species were significantly down-regulated during peel senescence. Three thioredoxin proteins and three Ca2+ signaling-related proteins were significantly up-regulated during peel senescence. It is suggested that mandarin peel senescence is associated with energy supply efficiency, decreased antioxidant capability, and increased protein and lipid degradation. In addition, activation of Ca2+ signaling and transcription factors might be involved in cell wall degradation and primary or secondary metabolism. PMID:27303420

Toxin-Antitoxin Systems as Multilevel Interaction Systems

PubMed Central

Goeders, Nathalie; Van Melderen, Laurence

2014-01-01

Toxin-antitoxin (TA) systems are small genetic modules usually composed of a toxin and an antitoxin counteracting the activity of the toxic protein. These systems are widely spread in bacterial and archaeal genomes. TA systems have been assigned many functions, ranging from persistence to DNA stabilization or protection against mobile genetic elements. They are classified in five types, depending on the nature and mode of action of the antitoxin. In type I and III, antitoxins are RNAs that either inhibit the synthesis of the toxin or sequester it. In type II, IV and V, antitoxins are proteins that either sequester, counterbalance toxin activity or inhibit toxin synthesis. In addition to these interactions between the antitoxin and toxin components (RNA-RNA, protein-protein, RNA-protein), TA systems interact with a variety of cellular factors, e.g., toxins target essential cellular components, antitoxins are degraded by RNAses or ATP-dependent proteases. Hence, TA systems have the capacity to interact with each other at different levels. In this review, we will discuss the different interactions in which TA systems are involved and their implications in TA system functions and evolution. PMID:24434905

Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

PubMed

Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

2017-12-09

Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

New insights into sulfur amino acids function in gut health and disease

USDA-ARS?s Scientific Manuscript database

The gastrointestinal tract (GIT) is a metabolically significant site of sulfur amino acids (SAAs) metabolism in the body. Aside from their role in protein synthesis, methionine and cysteine are involved in many biological functions and diseases. Methionine (MET) is an indispensable amino acid and is...

Post-Retrieval Late Process Contributes to Persistence of Reactivated Fear Memory

ERIC Educational Resources Information Center

Nakayama, Daisuke; Yamasaki, Yoshiko; Matsuki, Norio; Nomura, Hiroshi

2013-01-01

Several studies have demonstrated the mechanisms involved in memory persistence after learning. However, little is known about memory persistence after retrieval. In this study, a protein synthesis inhibitor, anisomycin, was infused into the basolateral amygdala of mice 9.5 h after retrieval of contextual conditioned fear. Anisomycin attenuated…

New insights into sulfur amino acid function in gut health and disease

USDA-ARS?s Scientific Manuscript database

The gastrointestinal tract (GIT) is a metabolically significant site of sulfur amino acids (SAA) metabolism in the body. Aside from their role in protein synthesis, methionine and cysteine are involved in many biological functions and diseases. Methionine (MET) is an indispensable AA and is transmet...

Quantitative Proteomic Analysis of Wheat Seeds during Artificial Ageing and Priming Using the Isobaric Tandem Mass Tag Labeling

PubMed Central

Lv, Yangyong; Zhang, Shuaibing; Wang, Jinshui; Hu, Yuansen

2016-01-01

Wheat (Triticum aestivum L.) is an important crop worldwide. The physiological deterioration of seeds during storage and seed priming is closely associated with germination, and thus contributes to plant growth and subsequent grain yields. In this study, wheat seeds during different stages of artificial ageing (45°C; 50% relative humidity; 98%, 50%, 20%, and 1% Germination rates) and priming (hydro-priming treatment) were subjected to proteomics analysis through a proteomic approach based on the isobaric tandem mass tag labeling. A total of 162 differentially expressed proteins (DEPs) mainly involved in metabolism, energy supply, and defense/stress responses, were identified during artificial ageing and thus validated previous physiological and biochemical studies. These DEPs indicated that the inability to protect against ageing leads to the incremental decomposition of the stored substance, impairment of metabolism and energy supply, and ultimately resulted in seed deterioration. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the up-regulated proteins involved in seed ageing were mainly enriched in ribosome, whereas the down-regulated proteins were mainly accumulated in energy supply (starch and sucrose metabolism) and stress defense (ascorbate and aldarate metabolism). Proteins, including hemoglobin 1, oleosin, agglutinin, and non-specific lipid-transfer proteins, were first identified in aged seeds and might be regarded as new markers of seed deterioration. Of the identified proteins, 531 DEPs were recognized during seed priming compared with unprimed seeds. In contrast to the up-regulated DEPs in seed ageing, several up-regulated DEPs in priming were involved in energy supply (tricarboxylic acid cycle, glycolysis, and fatty acid oxidation), anabolism (amino acids, and fatty acid synthesis), and cell growth/division. KEGG and protein-protein interaction analysis indicated that the up-regulated proteins in seed priming were mainly enriched in amino acid synthesis, stress defense (plant-pathogen interactions, and ascorbate and aldarate metabolism), and energy supply (oxidative phosphorylation and carbon metabolism). Therefore, DEPs associated with seed ageing and priming can be used to characterize seed vigor and optimize germination enhancement treatments. This work reveals new proteomic insights into protein changes that occur during seed deterioration and priming. PMID:27632285

Proteolytic activity during senescence of plants

NASA Technical Reports Server (NTRS)

Huffaker, R. C.

1990-01-01

Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolytic activities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein degradation. This field of study has great practical implications, e.g. maintaining photosynthesis during seed-fill in order to obtain greater crop yields. The current use of stay green' variants in the populations of several crop plants to produce increased yields shows the potential for future development. The near future should see exciting discoveries in these areas of research that will have far reaching effects on the construction of transgenic plants for future research accomplishments and agricultural use.

Quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside suppresses melanin synthesis by augmenting p38 MAPK and CREB signaling pathways and subsequent cAMP down-regulation in murine melanoma cells

PubMed Central

Jung, Hyun Gug; Kim, Han Hyuk; Paul, Souren; Jang, Jae Yoon; Cho, Yong Hun; Kim, Hyeon Jeong; Yu, Jae Myo; Lee, Eun Su; An, Bong Jeun; Kang, Sun Chul; Bang, Byung Ho

2015-01-01

In this study, the effect of purified quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosid (QCGG) on melanogenesis was investigated. QCGG was isolated from the calyx of a traditional Korean medicinal herb, Persimmon (Diospyros kaki). The hypopigmentation effects of QCGG were determined by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay, and Western blotting of α-MSH-stimulated B16F10 mouse melanoma cells. Our results showed that QCGG inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner as well as significantly reduced the expression of melanogenic proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1, tyrosinase-related protein-2, and tyrosinase. Moreover, QCGG inhibited intracellular cAMP levels, cAMP response element-binding protein (CREB), and p38 MAPK expression in α-MSH-stimulated B16F10 cells. Taken together, the suppressive effects of QCGG on melanogenesis may involve down-regulation of MITF and its downstream signaling pathway via phosphorylation of p38 MAPK and CREB along with reduced cAMP levels. These results indicate that QCGG reduced melanin synthesis by reducing expression of tyrosine and tyrosine-related proteins via extracellular signal-related protein kinase (ERK) activation, followed by down-regulation of CREB, p38, and MITF. PMID:26586997

Quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside suppresses melanin synthesis by augmenting p38 MAPK and CREB signaling pathways and subsequent cAMP down-regulation in murine melanoma cells.

PubMed

Jung, Hyun Gug; Kim, Han Hyuk; Paul, Souren; Jang, Jae Yoon; Cho, Yong Hun; Kim, Hyeon Jeong; Yu, Jae Myo; Lee, Eun Su; An, Bong Jeun; Kang, Sun Chul; Bang, Byung Ho

2015-11-01

In this study, the effect of purified quercetin-3-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosid (QCGG) on melanogenesis was investigated. QCGG was isolated from the calyx of a traditional Korean medicinal herb, Persimmon (Diospyros kaki). The hypopigmentation effects of QCGG were determined by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay, and Western blotting of α-MSH-stimulated B16F10 mouse melanoma cells. Our results showed that QCGG inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner as well as significantly reduced the expression of melanogenic proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1, tyrosinase-related protein-2, and tyrosinase. Moreover, QCGG inhibited intracellular cAMP levels, cAMP response element-binding protein (CREB), and p38 MAPK expression in α-MSH-stimulated B16F10 cells. Taken together, the suppressive effects of QCGG on melanogenesis may involve down-regulation of MITF and its downstream signaling pathway via phosphorylation of p38 MAPK and CREB along with reduced cAMP levels. These results indicate that QCGG reduced melanin synthesis by reducing expression of tyrosine and tyrosine-related proteins via extracellular signal-related protein kinase (ERK) activation, followed by down-regulation of CREB, p38, and MITF.

Altered Machinery of Protein Synthesis in Alzheimer's: From the Nucleolus to the Ribosome.

PubMed

Hernández-Ortega, Karina; Garcia-Esparcia, Paula; Gil, Laura; Lucas, José J; Ferrer, Isidre

2016-09-01

Ribosomes and protein synthesis have been reported to be altered in the cerebral cortex at advanced stages of Alzheimer's disease (AD). Modifications in the hippocampus with disease progression have not been assessed. Sixty-seven cases including middle-aged (MA) and AD stages I-VI were analyzed. Nucleolar chaperones nucleolin, nucleophosmin and nucleoplasmin 3, and upstream binding transcription factor RNA polymerase I gene (UBTF) mRNAs are abnormally regulated and their protein levels reduced in AD. Histone modifications dimethylated histone H3K9 (H3K9me2) and acetylated histone H3K12 (H3K12ac) are decreased in CA1. Nuclear tau declines in CA1 and dentate gyrus (DG), and practically disappears in neurons with neurofibrillary tangles. Subunit 28 ribosomal RNA (28S rRNA) expression is altered in CA1 and DG in AD. Several genes encoding ribosomal proteins are abnormally regulated and protein levels of translation initiation factors eIF2α, eIF3η and eIF5, and elongation factor eEF2, are altered in the CA1 region in AD. These findings show alterations in the protein synthesis machinery in AD involving the nucleolus, nucleus and ribosomes in the hippocampus in AD some of them starting at first stages (I-II) preceding neuron loss. These changes may lie behind reduced numbers of dendritic branches and reduced synapses of CA1 and DG neurons which cause hippocampal atrophy. © 2015 International Society of Neuropathology.

Gibberellin-Stimulation of Rhizome Elongation and Differential GA-Responsive Proteomic Changes in Two Grass Species

PubMed Central

Ma, Xiqing; Huang, Bingru

2016-01-01

Rapid and extensive rhizome development is a desirable trait for perennial grass growth and adaptation to environmental stresses. The objective of this study was to determine proteomic changes and associated metabolic pathways of gibberellin (GA) -regulation of rhizome elongation in two perennial grass species differing in rhizome development. Plants of a short-rhizome bunch-type tall fescue (TF; Festuca arundinacea; ‘BR’) and an extensive rhizomatous Kentucky bluegrass (KB; Poa pratensis; ‘Baron’) were treated with 10 μM GA3 in hydroponic culture in growth chambers. The average rhizome length in KB was significantly longer than that in TF regardless of GA3 treatment, and increased significantly with GA3 treatment, to a greater extent than that in TF. Comparative proteomic analysis using two-dimensional electrophoresis and mass spectrometry was performed to further investigate proteins and associated metabolic pathways imparting increased rhizome elongation by GA. A total of 37 and 38 differentially expressed proteins in response to GA3 treatment were identified in TF and KB plants, respectively, which were mainly involved in photosynthesis, energy and amino acid metabolism, protein synthesis, defense and cell development processes. Accelerated rhizome elongation in KB by GA could be mainly associated with the increased abundance of proteins involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and ATP synthase), amino acid metabolism (S-adenosylmethionine and adenosylhomocysteinase), protein synthesis (HSP90, elongation factor Tu and eukaryotic translation initiation factor 5A), cell-wall development (cell dividion cycle protein, alpha tubulin-2A and actin), and signal transduction (calreticulin). These proteins could be used as candidate proteins for further analysis of molecular mechanisms controlling rhizome growth. PMID:27446135

Stable isotope labeling tandem mass spectrometry (SILT) to quantify protein production and clearance rates

PubMed Central

Bateman, Randall J.; Munsell, Ling Y.; Chen, Xianghong; Holtzman, David M.; Yarasheski, Kevin E.

2007-01-01

In all biological systems, protein amount is a function of the rate of production and clearance. The speed of a response to a disturbance in protein homeostasis is determined by turnover rate. Quantifying alterations in protein synthesis and clearance rates is vital to understanding disease pathogenesis (e.g., aging, inflammation). No methods exist for quantifying production and clearance rates of low abundance (femtomole) proteins in vivo. We describe a novel, mass spectrometry-based method for quantitating low abundance protein synthesis and clearance rates in vitro and in vivo in animals and humans. The utility of this method is demonstrated with amyloid-beta (Aß), an important low abundance protein involved in Alzheimer's disease pathogenesis. We used in vivo stable isotope labeling, immunoprecipitation of Aß from cerebrospinal fluid, and quantitative liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-tandem MS) to quantify human Aß protein production and clearance rates. The method is sensitive and specific for stable isotope labeled amino acid incorporation into CNS (± 1% accuracy). This in vivo method can be used to identify pathophysiologic changes in protein metabolism; and may serve as a biomarker for monitoring disease risk, progression, or response to novel therapeutic agents. The technique is adaptable to other macromolecules, such as carbohydrates or lipids. PMID:17383190

Physiological and molecular alterations promoted by Schizotetranychus oryzae mite infestation in rice leaves

PubMed Central

Buffon, Giseli; Blasi, Édina A. R.; Adamski, Janete M.; Ferla, Noeli J.; Berger, Markus; Santi, Lucélia; Lavallée-Adam, Mathieu; Yates, John R.; Beys-da-Silva, Walter O.; Sperotto, Raul A.

2016-01-01

Infestation of phytophagous mite Schizotetranychus oryzae in rice causes critical yield losses. To better understand this interaction, we employed Multidimensional Protein Identification Technology (MudPIT) approach to identify differentially expressed proteins. We detected 18 unique proteins in control and 872 in infested leaves, respectively, along with 32 proteins more abundant in control leaves. S. oryzae infestation caused decreased abundance of proteins related to photosynthesis (mostly photosystem II-related), carbon assimilation and energy production, chloroplast detoxification, defense, fatty acid and gibberellin synthesis. On the other hand, infestation caused increased abundance of proteins involved in protein modification and degradation, gene expression at the translation level, protein partitioning to different organelles, lipid metabolism, actin cytoskeleton remodeling, and synthesis of jasmonate, amino acid and molecular chaperones. Our results also suggest that S. oryzae infestation promotes cell wall remodeling and interferes with ethylene biosynthesis in rice leaves. Proteomic data were positively correlated with enzymatic assays and RT-qPCR analysis. Our findings describe the protein expression patterns of infested rice leaves, and suggest that the acceptor side of PSII is probably the major damaged target in the photosynthetic apparatus. These data will be useful in future biotechnological approaches aiming to induce phytophagous mite resistance in rice. PMID:26667653

Regulation of peroxisome proliferator-activated receptor gamma on milk fat synthesis in dairy cow mammary epithelial cells.

PubMed

Liu, Lili; Lin, Ye; Liu, Lixin; Wang, Lina; Bian, Yanjie; Gao, Xuejun; Li, Qingzhang

2016-12-01

Peroxisome proliferator-activated receptor gamma (PPARγ) participates in lipogenesis in rats, goats, and humans. However, the exact mechanism of PPARγ regulation on milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) remains largely unexplored. The aim of this study was to investigate the role of PPARγ regarding milk fat synthesis in DCMECs and to ascertain whether milk fat precursor acetic acid and palmitic acid could interact with PPARγ signaling to regulate milk fat synthesis. For this study, we examined the effects of PPARγ overexpression and gene silencing on cell growth, triacylglycerol synthesis, and the messenger RNA (mRNA) and protein expression levels of genes involved in milk fat synthesis in DCMECs. In addition, we investigated the influences of acetic acid and palmitic acid on the mRNA and protein levels of milk lipogenic genes and triacylglycerol synthesis in DCMECs transfected with PPARγ small interfering RNA (siRNA) and PPARγ expression vector. The results showed that when PPARγ was silenced, cell viability, proliferation, and triacylglycerol secretion were obviously reduced. Gene silencing of PPARγ significantly downregulated the expression levels of milk fat synthesis-related genes in DCMECs. PPARγ overexpression improved cell viability, proliferation, and triacylglycerol secretion. The expression levels of milk lipogenic genes were significantly increased when PPARγ was overexpressed. Acetic acid and palmitic acid could markedly improve triacylglycerol synthesis and upregulate the expression levels of PPARγ and other lipogenic genes in DCMECs. These results suggest that PPARγ is a positive regulator of milk fat synthesis in DCMECs and that acetic acid and palmitic acid could partly regulate milk fat synthesis in DCMECs via PPARγ signaling.

N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

PubMed

Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

2010-07-01

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

Mechanism of plant-mediated synthesis of silver nanoparticles - A review on biomolecules involved, characterisation and antibacterial activity.

PubMed

Rajeshkumar, S; Bharath, L V

2017-08-01

Engineering a reliable and eco-accommodating methodology for the synthesis of metal nanoparticles is a crucial step in the field of nanotechnology. Plant-mediated synthesis of metal nanoparticles has been developed as a substitute to defeat the limitations of conventional synthesis approaches such as physical and chemical methods. Biomolecules, such as proteins, amino acids, enzymes, flavonoids, and terpenoids from several plant extracts have been used as a stabilising and reducing agents for the synthesis of AgNPs. Regardless of an extensive range of biomolecules assistance in the synthesis procedure, researchers are facing a significant challenge to synthesise stable and geometrically controlled AgNPs. In the past decade, several efforts were made to develop Plant-mediated synthesis methods to produce stable, cost effective and eco-friendly AgNPs. More than hundred different plants extract sources for synthesising AgNPs were described in the last decade by several researchers. Most of the reviews were focused on various plant sources for synthesis, various characterization techniques for characteristic analysis, and antibacterial activity against bacterial. There are many reviews are available for the plant-mediated synthesis of AgNPs as well as antibacterial activity of AgNPs but this is the first review article mainly focused on biomolecules of plants and its various parts and operating conditions involved in the synthesis. Apart from, this review includes the characterisation of AgNPs and antibacterial activity of such nanoparticles with size, shape and method used for this study. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics and Yield in Rice (Oryza Sativa)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Zhaohua PEng; Ronald, Palmela; Wang, Guo-Liang

This project aims to identify the regulatory genes of rice cell wall synthesis pathways using a cell wall removal and regeneration system. We completed the gene expression profiling studies following the time course from cell wall removal to cell wall regeneration in rice suspension cells. We also completed, total proteome, nuclear subproteome and histone modification studies following the course from cell wall removal and cell wall regeneration process. A large number of differentially expressed regulatory genes and proteins were identified. Meanwhile, we generated RNAi and over-expression transgenic rice for 45 genes with at least 10 independent transgenic lines for eachmore » gene. In addition, we ordered T-DNA and transposon insertion mutants for 60 genes from Korea, Japan, and France and characterized the mutants. Overall, we have mutants and transgenic lines for over 90 genes, exceeded our proposed goal of generating mutants for 50 genes. Interesting Discoveries a) Cell wall re-synthesis in protoplasts may involve a novel cell wall synthesis mechanism. The synthesis of the primary cell wall is initiated in late cytokinesis with further modification during cell expansion. Phragmoplast plays an essential role in cell wall synthesis. It services as a scaffold for building the cell plate and formation of a new cell wall. Only one phragmoplast and one new cell wall is produced for each dividing cell. When the cell wall was removed enzymatically, we found that cell wall re-synthesis started from multiple locations simultaneously, suggesting that a novel mechanism is involved in cell wall re-synthesis. This observation raised many interesting questions, such as how the starting sites of cell wall synthesis are determined, whether phragmoplast and cell plate like structures are involved in cell wall re-synthesis, and more importantly whether the same set of enzymes and apparatus are used in cell wall re-synthesis as during cytokinesis. Given that many known cell wall synthesis pathway genes are induced by removal of cell wall, some cell wall synthesis apparatus must be shared in both cases. The cell wall re-synthesis mechanism may have broad application because our preliminary assay indicates that the cell wall characteristics are highly different from those produced during cytokinesis. A thorough understanding on the regulation of cell wall re-synthesis may lead to improvement of cell wall characteristics. b) Removal of cell wall results in chromatin decondensation Another interesting observation was that removal of cell wall was associated with substantial chromatin change. Our DNA DAPI stain, chromatin MNase digestion, histone modification proteomics, protein differential expression analysis, and DNA oligo array studies all supported that substantial chromatin change was associated with removal of cell wall treatment. It is still under investigation if the chromatin change is associated with activation of cell wall synthesis genes, in which chromatin remodeling is required. Another possibility is that the cell wall is required for stabilizing the chromatin structure in plant cells. Given that spindle fiber is directly connected with both chromatin structure and cell wall synthesis, it is possible that there is an intrinsic connection between cell wall and chromatin.« less

Primaquine: Modes of Action and Mechanisms of Drug Resistance.

DTIC Science & Technology

1975-06-30

on in vitro protein synthesis, nucleic acid synthesis in vitro and in isolated nuclei, in vitro lipid synthesis, andmembrane transport and permeability...vitro protein synthesis, nucleic acid synthesis in vitro and in isolated nuclei, in vitro lipid synthesis, and membrane transport and permeability. In...protein synthesis. 7 III. The effects of primaquine on nucleic acid synthesis in isolated nuclei. 7 IV. The effects of primaquine on DNA and RNA syntheses

Characterization of Cellulose Synthesis in Plant Cells

PubMed Central

Maleki, Samaneh Sadat; Mohammadi, Kourosh; Ji, Kong-shu

2016-01-01

Cellulose is the most significant structural component of plant cell wall. Cellulose, polysaccharide containing repeated unbranched β (1-4) D-glucose units, is synthesized at the plasma membrane by the cellulose synthase complex (CSC) from bacteria to plants. The CSC is involved in biosynthesis of cellulose microfibrils containing 18 cellulose synthase (CesA) proteins. Macrofibrils can be formed with side by side arrangement of microfibrils. In addition, beside CesA, various proteins like the KORRIGAN, sucrose synthase, cytoskeletal components, and COBRA-like proteins have been involved in cellulose biosynthesis. Understanding the mechanisms of cellulose biosynthesis is of great importance not only for improving wood production in economically important forest trees to mankind but also for plant development. This review article covers the current knowledge about the cellulose biosynthesis-related gene family. PMID:27314060

Comparative Proteomic Analyses Provide New Insights into Low Phosphorus Stress Responses in Maize Leaves

PubMed Central

Zhang, Kewei; Liu, Hanhan; Tao, Peilin; Chen, Huan

2014-01-01

Phosphorus deficiency limits plant growth and development. To better understand the mechanisms behind how maize responds to phosphate stress, we compared the proteome analysis results of two groups of maize leaves that were treated separately with 1,000 µM (control, +P) and 5 µM of KH2PO4 (intervention group, −P) for 25 days. In total, 1,342 protein spots were detected on 2-DE maps and 15.43% had changed (P<0.05; ≥1.5-fold) significantly in quantity between the +P and −P groups. These proteins are involved in several major metabolic pathways, including photosynthesis, carbohydrate metabolism, energy metabolism, secondary metabolism, signal transduction, protein synthesis, cell rescue and cell defense and virulence. The results showed that the reduction in photosynthesis under low phosphorus treatment was due to the down-regulation of the proteins involved in CO2 enrichment, the Calvin cycle and the electron transport system. Electron transport and photosynthesis restrictions resulted in a large accumulation of peroxides. Maize has developed many different reactive oxygen species (ROS) scavenging mechanisms to cope with low phosphorus stress, including up-regulating its antioxidant content and antioxidase activity. After being subjected to phosphorus stress over a long period, maize may increase its internal phosphorus utilization efficiency by altering photorespiration, starch synthesis and lipid composition. These results provide important information about how maize responds to low phosphorus stress. PMID:24858307

Choline pathways during normal and stimulated renal growth in rats.

PubMed Central

Bean, G H; Lowenstein, L M

1978-01-01

Cellular membrane synthesis occurs during normal and stimulated renal growth. Choline in the kidney is utilized as a precursor for membrane synthesis via the choline kinase reaction. We investigated choline phosphorylation during normal and stimulated renal growth. Rapidly growing neonatal rat kidneys contained relatively high levels of choline kinase activity (61 pmol phosphorylcholine/min per mg protein). Choline kinase activity and phosphorylcholine production then fell gradually over the 1st mo of life; by 1 mo phosphorylcholine production was 34 pmol phosphorylcholine/min per mg protein. Choline kinase activity increased by 27% (P less than 0.001) in 28-day-old rats when renal growth was stimulated by contralateral nephrectomy; the increase occurred within 2 h after surgery. Thus, changes in the activity of this important enzyme in the initiation of membrane synthesis is associated both with normal renal development and with adaptation to nephron loss. The findings further suggest that the cell membrane may be involved in the initiation of compensatory renal growth. PMID:659614

A murC gene from coryneform bacteria.

PubMed

Wachi, M; Wijayarathna, C D; Teraoka, H; Nagai, K

1999-02-01

The upstream flanking region of the ftsQ and ftsZ genes of Brevibacterium flavum MJ233, which belongs to the coryneform bacteria, was amplified by the inverse polymerase chain reaction method and cloned in Escherichia coli. Complementation analysis of E. coli mutant with a defective cell-wall synthesis mechanism with the cloned fragment and its DNA sequencing indicated the presence of the murC gene, encoding UDP-N-acetylmuramate:L-alanine ligase involved in peptidoglycan synthesis, just upstream from the ftsQ gene. The B. flavum murC gene could encode a protein of 486 amino acid residues with a calculated molecular mass of 51 198 Da. A 50-kDa protein was synthesized by the B. flavum murC gene in an in vitro transcription/translation system using E. coli S30 lysate. These results indicate that the genes responsible for cell-wall synthesis and cell division are located as a cluster in B. flavum similar to the E. coli mra region.

Automated Fmoc-based solid-phase synthesis of peptide thioesters with self-purification effect and application in the construction of immobilized SH3 domains.

PubMed

Mende, Franziska; Beisswenger, Michael; Seitz, Oliver

2010-08-18

Peptide thioesters are important building blocks in the total synthesis of proteins and protein domains via fragment ligation. However, synthetic access of peptide thioesters still is a bottleneck of this powerful ligation chemistry. The commonly used methods for the Fmoc-based synthesis of peptide thioesters involve nonautomated solution steps that have to be performed after the solid-phase assembly of the peptide. Usually, HPLC purification is required. Herein, a method that enables crude peptides to be used in divergent native chemical ligations reactions is described. We present an Fmoc-based solid-phase synthesis of peptide thioesters with self-purification which facilitates access to these important building blocks, since the often cumbersome HPLC purification can be avoided. Fmoc-protected amino acids are coupled on a safety catch sulfonamide resin. The self-purifying effect is achieved through the combination of (a) N-terminal coupling of a cleavable cyclization linker and subsequent backbone-to-side chain cyclization, (b) activation of the sulfonamide linkage by alkylation, (c) thiolysis for the selective detachment of truncation products, and (d) TFA cleavage for the liberation of the desired peptide thioester in unprotected form. We have previously shown a method wherein cyclization was performed after carboxymethylation of the sulfonamide. However, the automation of this method was difficult and side reactions at methionine residues hampered the general applicability. The new design involves peptide synthesis on a modified carboxy-functionalized sulfonamide linker, a substantially milder activation of the sulfonamide bond and the use of monomethoxytrityl as well as 2-phenyl-isopropyl protecting groups. This approach solved the problems with methionine containing peptides and enabled the complete automation of the self-purifying synthesis of peptide thioesters. The study also addressed problems in the synthesis of difficult peptides. Aggregated truncation products can resist extraction and contaminate full-length thioesters obtained after TFA cleavage. It is shown that significant enhancements of the purity were achieved when mild acidic extractions were included in the wash protocols after thiolysis. The potential of the method was demonstrated in the parallel synthesis of 20-40 amino acid long peptide thioesters, which were obtained in excellent purities. The thioesters and cysteinyl peptides were used without purification in the assembly of immobilized SH3 protein domains of SHO1 in yeast. A cysteine scan by native chemical ligation suggested single amino acid to cysteine substitutions that (a) confer useful ligation yields, (b) support correct folding, and (c) sustain the function of the folded protein domain. The chemical synthesis of the SH3-domain of SHO1 succeeded in highest yields when cysteine placements at positions S23, F24, and E36 were avoided. The synthetic SH3 mutants were examined in a binding assay, which indicated that N27C, L30C, and D34C mutations provide functional SH3-domain.

GPER-1 and estrogen receptor-β ligands modulate aldosterone synthesis.

PubMed

Caroccia, Brasilina; Seccia, Teresa M; Campos, Abril Gonzalez; Gioco, Francesca; Kuppusamy, Maniselvan; Ceolotto, Giulio; Guerzoni, Eugenia; Simonato, Francesca; Mareso, Sara; Lenzini, Livia; Fassina, Ambrogio; Rossi, Gian Paolo

2014-11-01

Fertile women have lower blood pressure and cardiovascular risk than age-matched men, which suggests that estrogens exert cardiovascular protective effects. However, whether 17 β-estradiol (E2) blunts aldosterone secretion, and thereby affects the gender dimorphism of blood pressure, is unknown. We therefore sought for the estrogen receptor (ER) subtypes in human adrenocortical tissues ex vivo by performing gene and protein expression studies. We also investigated the effect of E2 on aldosterone synthesis and the involved receptors through in vitro functional experiments in the adrenocortical cells HAC15. We found that in the human adrenal cortex and aldosterone-producing adenoma cells, the most expressed ERs were the ERβ and the G protein-coupled receptor-1 (GPER-1), respectively. After selective ERβ blockade, E2 (10 nmol/L) markedly increased both the expression of aldosterone synthase and the production of aldosterone (+5- to 7-fold vs baseline, P < .001). Under the same condition, the GPER-1 receptor agonist 1-[4-(6-bromo-benzo (1, 3)dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinolin-8-yl]-ethanone (G-1) (10 nmol/L) mimicked this effect, which was abrogated by cotreatment with either the GPER-1 receptor antagonist (3aS*,4R*,9bR*)-4-(6-Bro-mo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G-15), or a selective protein kinase A inhibitor 8-Bromo-2-monobutyryladenosine-3,5-cyclic mono-phosphorothioate, Rp-isomer. Silencing of the ERβ significantly raised aldosterone synthase expression and aldosterone production. Conversely, silencing of the GPER-1 lowered aldosterone synthase gene and protein expression. Moreover, it blunted the stimulatory effect of E2 on aldosterone synthase that was seen during ERβ blockade. These results support the conclusion that in humans, E2 inhibits aldosterone synthesis by acting via ERβ. Pharmacologic disinhibition of ERβ unmasks a potent secretagogue effect of E2 that involves GPER-1 and protein kinase A signaling.

Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons.

PubMed

Lebeau, Geneviève; Miller, Linda C; Tartas, Maylis; McAdam, Robyn; Laplante, Isabel; Badeaux, Frédérique; DesGroseillers, Luc; Sossin, Wayne S; Lacaille, Jean-Claude

2011-01-01

The two members of the Staufen family of RNA-binding proteins, Stau1 and Stau2, are present in distinct ribonucleoprotein complexes and associate with different mRNAs. Stau1 is required for protein synthesis-dependent long-term potentiation (L-LTP) in hippocampal pyramidal cells. However, the role of Stau2 in synaptic plasticity remains unexplored. We found that unlike Stau1, Stau2 is not required for L-LTP. In contrast, Stau2, but not Stau1, is necessary for DHPG-induced protein synthesis-dependent long-term depression (mGluR-LTD). While Stau2 is involved in early development of spines, its down-regulation does not alter spine morphology or spontaneous miniature synaptic activity in older cultures where LTD occurs. In addition, Stau2, but not Stau1, knockdown reduces the dendritic localization of Map1b mRNA, a specific transcript involved in mGluR-LTD. Moreover, mGluR stimulation with DHPG induces Map1b, but not Map2, mRNA dissociation from mRNA granules containing Stau2 and the ribosomal protein P0. This dissociation was not observed in cells in which Stau2 was depleted. Finally, Stau2 knockdown reduces basal Map1b protein expression in dendrites and prevents DHPG-induced increases in dendritic Map1b protein level. We suggest a role for Stau2 in the generation and regulation of Map1b mRNA containing granules that are required for mGluR-LTD.

Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function. [UV radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minor, P.D.; Dimmock, N.J.

1977-05-15

Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and ..cap alpha..-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at highmore » concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent.« less

Identification and Characterization of Major Lipid Particle Proteins of the Yeast Saccharomyces cerevisiae

PubMed Central

Athenstaedt, Karin; Zweytick, Dagmar; Jandrositz, Anita; Kohlwein, Sepp Dieter; Daum, Günther

1999-01-01

Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism. PMID:10515935

A review on biogenic synthesis of ZnO nanoparticles using plant extracts and microbes: A prospect towards green chemistry.

PubMed

Ahmed, Shakeel; Annu; Chaudhry, Saif Ali; Ikram, Saiqa

2017-01-01

Nanotechnology is emerging as an important area of research with its tremendous applications in all fields of science, engineering, medicine, pharmacy, etc. It involves the materials and their applications having one dimension in the range of 1-100nm. Generally, various techniques are used for syntheses of nanoparticles (NPs) viz. laser ablation, chemical reduction, milling, sputtering, etc. These conventional techniques e.g. chemical reduction method, in which various hazardous chemicals are used for the synthesis of NPs later become liable for innumerable health risks due to their toxicity and endangering serious concerns for environment, while other approaches are expensive, need high energy for the synthesis of NPs. However, biogenic synthesis method to produce NPs is eco-friendly and free of chemical contaminants for biological applications where purity is of concerns. In biological method, different biological entities such as extract, enzymes or proteins of a natural product are used to reduce and stabilised formation of NPs. The nature of these biological entities also influence the structure, shape, size and morphology of synthesized NPs. In this review, biogenic synthesis of zinc oxide (ZnO) NPs, procedures of syntheses, mechanism of formation and their various applications have been discussed. Various entities such as proteins, enzymes, phytochemicals, etc. available in the natural reductants are responsible for synthesis of ZnO NPs. Copyright © 2016 Elsevier B.V. All rights reserved.

Peripheral Injection of SB203580 Inhibits the Inflammatory-Dependent Synthesis of Proinflammatory Cytokines in the Hypothalamus

PubMed Central

Herman, Andrzej P.; Krawczyńska, Agata; Antushevich, Hanna

2014-01-01

The study was designed to determine the effects of peripheral injection of SB203580 on the synthesis of interleukin- (IL-) 1β, IL-6, and tumor necrosis factor (TNF) α in the hypothalamus of ewes during prolonged inflammation. Inflammation was induced by the administration of lipopolysaccharide (LPS) (400 ng/kg) over 7 days. SB203580 is a selective ATP-competitive inhibitor of the p38 mitogen-activated protein kinase (MAPK), which is involved in the regulation of proinflammatory cytokines IL-1β, IL-6 and TNFα synthesis. Intravenous injection of SB203580 successfully inhibited (P < 0.01) synthesis of IL-1β and reduced (P < 0.01) the production of IL-6 in the hypothalamus. The p38 MAPK inhibitor decreased (P < 0.01) gene expression of TNFα but its effect was not observed at the level of TNFα protein synthesis. SB203580 also reduced (P < 0.01) LPS-stimulated IL-1 receptor type 1 gene expression. The conclusion that inhibition of p38 MAPK blocks LPS-induced proinflammatory cytokine synthesis seems to initiate new perspectives in the treatment of chronic inflammatory diseases also within the central nervous system. However, potential proinflammatory effects of SB203580 treatment suggest that all therapies using p38 MAPK inhibitors should be introduced very carefully with analysis of all expected and unexpected consequences of treatment. PMID:24995301

Inhibition of Cholesterol Synthesis in HepG2 Cells by GINST-Decreasing HMG-CoA Reductase Expression Via AMP-Activated Protein Kinase.

PubMed

Han, Joon-Seung; Sung, Jong Hwan; Lee, Seung Kwon

2017-11-01

GINST, a hydrolyzed ginseng extract, has been reported to have antidiabetic effects and to reduce hyperglycemia and hyperlipidemia. Hypercholesterolemia is caused by diet or genetic factors and can lead to atherosclerosis and coronary heart disease. Thus, the purpose of this study is to determine whether GINST and the ginsenoside metabolite, IH-901 (compound K), reduce cholesterol synthesis in HepG2 cells and the signal transduction pathways involved. Concentrations of cholesterol were measured by using an enzymatic method. Expression levels of sterol regulatory element-binding protein 2 (SREBP2), HMG-CoA reductase (HMGCR), peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins α (C/EBPα), GAPDH, and phosphorylation of AMP-activated protein kinase α (AMPKα), protein kinase B (PKB, also known as Akt), and mechanistic target of rapamycin complex 1 (mTORC1) were measured using western blot. Total cholesterol concentration decreased after GINST treatment for 24 and 48 h. Expression of HMGCR decreased more with GINST than with the inhibitors, U18666A and atorvastatin, after 48 h in a dose-dependent manner. Phosphorylation of AMPKα increased 2.5x by GINST after 360 min of treatment, and phosphorylation of Akt decreased after 120 and 360 min. We separated compound K from GINST extracts flash chromatography. Compound K decreased cholesterol synthesis in HepG2 cells at 24 and 48 h. Therefore, we conclude that GINST inhibits cholesterol synthesis in HepG2 cells by decreasing HMGCR expression via AMPKα activation. GINST, a hydrolyzed ginseng extract, can inhibit cholesterol synthesis in liver cells via activation of AMPKα. IH-901 (compound K), which is the main component with bioactivity in GINST, also has anticholesterol effects. Thus, we suggest that GINST can be used to reduce hypercholesterolemia. © 2017 Institute of Food Technologists®.

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors

PubMed Central

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios

2017-01-01

Abstract Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. PMID:28973457

The Crc protein inhibits the production of polyhydroxyalkanoates in Pseudomonas putida under balanced carbon/nitrogen growth conditions.

PubMed

La Rosa, Ruggero; de la Peña, Fernando; Prieto, María Axiliadora; Rojo, Fernando

2014-01-01

Pseudomonas putida synthesizes polyhydroxyalkanoates (PHAs) as storage compounds. PHA synthesis is more active when the carbon source is in excess and the nitrogen source is limiting, but can also occur at a lower rate under balanced carbon/nitrogen ratios. This work shows that PHA synthesis is controlled by the Crc global regulator, a protein that optimizes carbon metabolism by inhibiting the expression of genes involved in the use of non-preferred carbon sources. Crc acts post-transcriptionally. The mRNAs of target genes contain characteristic catabolite activity (CA) motifs near the ribosome binding site. Sequences resembling CA motifs can be predicted for the phaC1 gene, which codes for a PHA polymerase, and for phaI and phaF, which encode proteins associated to PHA granules. Our results show that Crc inhibits the translation of phaC1 mRNA, but not that of phaI or phaF, reducing the amount of PHA accumulated in the cell. Crc inhibited PHA synthesis during exponential growth in media containing a balanced carbon/nitrogen ratio. No inhibition was seen when the carbon/nitrogen ratio was imbalanced. This extends the role of Crc beyond that of controlling the hierarchical utilization of carbon sources and provides a link between PHA synthesis and the global regulatory networks controlling carbon flow. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

PubMed

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

2017-09-29

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein1[OPEN

PubMed Central

Fernie, Alisdair R.; Stitt, Mark

2015-01-01

Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied 13CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%–4% d−1), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. PMID:25810096

Quantifying protein synthesis and degradation in Arabidopsis by dynamic 13CO2 labeling and analysis of enrichment in individual amino acids in their free pools and in protein.

PubMed

Ishihara, Hirofumi; Obata, Toshihiro; Sulpice, Ronan; Fernie, Alisdair R; Stitt, Mark

2015-05-01

Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied (13)CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d(-1)), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. © 2015 American Society of Plant Biologists. All Rights Reserved.

Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments.

PubMed

Lee, Kyung-Ho; Kim, Dong-Myung

2013-11-01

Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential regulation of protein synthesis by amino acids and insulin in peripheral and visceral tissues of neonatal pigs

PubMed Central

Suryawan, Agus; O’Connor, Pamela M. J.; Bush, Jill A.; Nguyen, Hanh V.

2009-01-01

The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. In the current study, we examined the individual roles of amino acids and insulin in the regulation of protein synthesis in peripheral and visceral tissues of the neonate by performing pancreatic glucose–amino acid clamps in overnight-fasted 7-day-old pigs. We infused pigs (n = 8–12/group) with insulin at 0, 10, 22, and 110 ng kg−0.66 min−1 to achieve ~0, 2, 6 and 30 μU ml−1 insulin so as to simulate below fasting, fasting, intermediate, and fed insulin levels, respectively. At each insulin dose, amino acids were maintained at the fasting or fed level. In conjunction with the highest insulin dose, amino acids were also allowed to fall below the fasting level. Tissue protein synthesis was measured using a flooding dose of L-[4-3H] phenylalanine. Both insulin and amino acids increased fractional rates of protein synthesis in longissimus dorsi, gastrocnemius, masseter, and diaphragm muscles. Insulin, but not amino acids, increased protein synthesis in the skin. Amino acids, but not insulin, increased protein synthesis in the liver, pancreas, spleen, and lung and tended to increase protein synthesis in the jejunum and kidney. Neither insulin nor amino acids altered protein synthesis in the stomach. The results suggest that the stimulation of protein synthesis by feeding in most tissues of the neonate is regulated by the post-prandial rise in amino acids. However, the feeding-induced stimulation of protein synthesis in skeletal muscles is independently mediated by insulin as well as amino acids. PMID:18683020

Directed evolution of an extremely stable fluorescent protein.

PubMed

Kiss, Csaba; Temirov, Jamshid; Chasteen, Leslie; Waldo, Geoffrey S; Bradbury, Andrew R M

2009-05-01

In this paper we describe the evolution of eCGP123, an extremely stable green fluorescent protein based on a previously described fluorescent protein created by consensus engineering (CGP: consensus green protein). eCGP123 could not be denatured by a standard thermal melt, preserved almost full fluorescence after overnight incubation at 80 degrees C and possessed a free energy of denaturation of 12.4 kcal/mol. It was created from CGP by a recursive process involving the sequential introduction of three destabilizing heterologous inserts, evolution to overcome the destabilization and finally 'removal' of the destabilizing insert by gene synthesis. We believe that this approach may be generally applicable to the stabilization of other proteins.

Enhancement of RNA Synthesis, Protein Synthesis, and Abscission by Ethylene

PubMed Central

Abeles, F. B.; Holm, R. E.

1966-01-01

Ethylene stimulated RNA and protein synthesis in bean (Phaseolus vulgaris L. var. Red Kidney) abscission zone explants prior to abscission. The effect of ethylene on RNA synthesis and abscission was blocked by actinomycin D. Carbon dioxide, which inhibits the effect of ethylene on abscission, also inhibited the influence of ethylene on protein synthesis. An aging period appears to be essential before bean explants respond to ethylene. Stimulation of protein synthesis by ethylene occurred only in receptive or senescent explants. Treatment of juvenile explants with ethylene, which has no effect on abscission also has no effect on protein synthesis. Evidence in favor of a hormonal role for ethylene during abscission is discussed. PMID:16656405

Floral reversion mechanism in longan (Dimocarpus longan Lour.) revealed by proteomic and anatomic analyses.

PubMed

You, Xiangrong; Wang, Lingxia; Liang, Wenyu; Gai, Yonghong; Wang, Xiaoyan; Chen, Wei

2012-02-02

Two-dimensional gel electrophoresis (2-DE) was used to analyze the proteins related to floral reversion in Dimocarpus longan Lour. Proteins were extracted from buds undergoing the normal process of flowering and from those undergoing floral reversion in three developing stages in D. longan. Differentially expressed proteins were identified from the gels after 2-DE analysis, which were confirmed using matrix-assisted laser desorption/ionization-time of flying-mass spectroscopy and protein database search. A total of 39 proteins, including 18 up-regulated and 21 down-regulated proteins, were classified into different categories, such as energy and substance metabolism, protein translation, secondary metabolism, phytohormone, cytoskeleton structure, regulation, and stress tolerance. Among these, the largest functional class was associated with primary metabolism. Down-regulated proteins were involved in photosynthesis, transcription, and translation, whereas up-regulated proteins were involved in respiration. Decreased flavonoid synthesis and up-regulated GA20ox might be involved in the floral reversion process. Up-regulated 14-3-3 proteins played a role in the regulation of floral reversion in D. longan by responding to abiotic stress. Observations via transmission electron microscopy revealed the ultrastructure changes in shedding buds undergoing floral reversion. Overall, the results provided insights into the molecular basis for the floral reversion mechanism in D. longan. Copyright © 2011 Elsevier B.V. All rights reserved.

Minced beef is more rapidly digested and absorbed than beef steak, resulting in greater postprandial protein retention in older men.

PubMed

Pennings, Bart; Groen, Bart B L; van Dijk, Jan-Willem; de Lange, Anneke; Kiskini, Alexandra; Kuklinski, Marjan; Senden, Joan M G; van Loon, Luc J C

2013-07-01

Older individuals generally experience a reduced food-chewing efficiency. As a consequence, food texture may represent an important factor that modulates dietary protein digestion and absorption kinetics and the subsequent postprandial protein balance. We assessed the effect of meat texture on the dietary protein digestion rate, amino acid availability, and subsequent postprandial protein balance in vivo in older men. Ten older men (mean ± SEM age: 74 ± 2 y) were randomly assigned to a crossover experiment that involved 2 treatments in which they consumed 135 g of specifically produced intrinsically L-[1-(13)C]phenylalanine-labeled beef, which was provided as beef steak or minced beef. Meat consumption was combined with continuous intravenous L-[ring-(2)H5]phenylalanine and L-[ring-(2)H2]tyrosine infusion to assess beef protein digestion and absorption kinetics as well as whole-body protein balance and skeletal muscle protein synthesis rates. Meat protein-derived phenylalanine appeared more rapidly in the circulation after minced beef than after beef steak consumption (P < 0.05). Also, its availability in the circulation during the 6-h postprandial period was greater after minced beef than after beef steak consumption (61 ± 3% compared with 49 ± 3%, respectively; P < 0.01). The whole-body protein balance was more positive after minced beef than after beef steak consumption (29 ± 2 compared with 19 ± 3 μmol phenylalanine/kg, respectively; P < 0.01). Skeletal muscle protein synthesis rates did not differ between treatments when assessed over a 6-h postprandial period. Minced beef is more rapidly digested and absorbed than beef steak, which results in increased amino acid availability and greater postprandial protein retention. However, this does not result in greater postprandial muscle protein synthesis rates. This trial was registered at clinicaltrials.gov as NCT01145131.

Involvement of neuron-derived orphan receptor-1 (NOR-1) in LDL-induced mitogenic stimulus in vascular smooth muscle cells: role of CREB.

PubMed

Rius, Jordi; Martínez-González, José; Crespo, Javier; Badimon, Lina

2004-04-01

Low density lipoproteins (LDLs) modulate the expression of key genes involved in atherogenesis. Recently, we have shown that the transcription factor neuron-derived orphan receptor-1 (NOR-1) is involved in vascular smooth muscle cell (VSMC) proliferation. Our aim was to analyze whether NOR-1 is involved in LDL-induced mitogenic effects in VSMC. LDL induced NOR-1 expression in a time- and dose-dependent manner. Antisense oligonucleotides against NOR-1 inhibit DNA synthesis induced by LDL in VSMCs as efficiently as antisense against the protooncogene c-fos. The upregulation of NOR-1 mRNA levels by LDL involves pertusis-sensitive G protein-coupled receptors, Ca2+ mobilization, protein kinases A (PKA) and C (PKC) activation, and mitogen-activated protein kinase pathways (MAPK) (p44/p42 and p38). LDL promotes cAMP response element binding protein (CREB) activation (phosphorylation in Ser133). In transfection assays a dominant-negative of CREB inhibits NOR-1 promoter activity, while mutation of specific (cAMP response element) CRE sites in the NOR-1 promoter abolishes LDL-induced NOR-1 promoter activity. In VSMCs, LDL-induced mitogenesis involves NOR-1 upregulation through a CREB-dependent mechanism. CREB could play a role in the modulation by LDL of key genes (containing CRE sites) involved in atherogenesis.

Cell-free protein synthesis: the state of the art.

PubMed

Whittaker, James W

2013-02-01

Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology. Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids.

Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

PubMed

Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

2017-06-01

Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

Biochemical Foundations of Health and Energy Conservation in Hibernating Free-ranging Subadult Brown Bear Ursus arctos*

PubMed Central

Hansen, Rasmus; Sønderkær, Mads; Arinell, Karin; Swenson, Jon E.; Revsbech, Inge G.

2016-01-01

Brown bears (Ursus arctos) hibernate for 5–7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general. PMID:27609515

Enhanced skeletal muscle protein synthesis rates in pigs treated with somatotropin requires fed amino acids levels

USDA-ARS?s Scientific Manuscript database

Chronic somatotropin (pST) treatment in pigs increases skeletal muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin alone could not account for the pST-induced increase in protein synthesis. This study...

Metal insertion into NiFe-hydrogenases.

PubMed

Blokesch, M; Paschos, A; Theodoratou, E; Bauer, A; Hube, M; Huth, S; Böck, A

2002-08-01

The synthesis and the insertion of the metallocentre of NiFe-hydrogenases is a complex process, in which seven maturation enzymes plus ATP, GTP and carbamoyl phosphate are involved. The review summarizes what is known about the properties and activities of these auxiliary proteins, and postulates a pathway along which maturation may take place.

Arabidopsis glutaredoxin s17 contributes to vegetative growth, mineral accumulation, and redox balance during iron deficiency

USDA-ARS?s Scientific Manuscript database

Iron (Fe) is an essential mineral nutrient and a metal cofactor required for many proteins and enzymes involved in the processes of DNA synthesis, respiration, and photosynthesis. Iron limitation can have detrimental effects on plant growth and development. Such effects are mediated, at least in par...

Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum

USDA-ARS?s Scientific Manuscript database

Aspartate 1-decarboxylase (ADC) and dopa decarboxylase (DDC) provide b–alanine and dopamine used in insect cuticle tanning. Beta-alanine is conjugated with dopamine to yield N-b-alanyldopamine (NBAD), a substrate for the phenoloxidase laccase that catalyzes the synthesis of cuticle protein cross-li...

How-to-Do-It: A Physical Model Illustrating Protein Synthesis on the Ribosome.

ERIC Educational Resources Information Center

Rogerson, Allen C.; Cheney, Richard W., Jr.

1989-01-01

Describes a way to help students grasp intermediate steps in the movement and relationships of the various components involved in the addition of an amino acid to a nascent peptide chain. Includes drawings of the model in operation, construction details, and suggested shapes and labeling of components. (RT)

Gene expression microarray analysis encompassing metamorphosis and the onset of calcification in the scleractinian coral Montastraea faveolata.

PubMed

Reyes-Bermudez, Alejandro; Desalvo, Michael K; Voolstra, Christian R; Sunagawa, Shinichi; Szmant, Alina M; Iglesias-Prieto, Roberto; Medina, Mónica

2009-01-01

Similar to many marine invertebrates, scleractinian corals experience a dramatic morphological transformation, as well as a habitat switch, upon settlement and metamorphosis. At this time, planula larvae transform from non-calcifying, demersal, motile organisms into sessile, calcifying, benthic juvenile polyps. We performed gene expression microarray analyses between planulae, aposymbiotic primary polyps, and symbiotic adult tissue to elucidate the molecular mechanisms underlying coral metamorphosis and early stages of calcification in the Robust/Short clade scleractinian coral Montastraea faveolata. Among the annotated genes, the most abundant upregulated transcripts in the planula stage are involved in protein synthesis, chromatin assembly and mitochondrial metabolism; the polyp stage, morphogenesis, protein catabolism and organic matrix synthesis; and the adult stage, sexual reproduction, stress response and symbiosis. We also present evidence showing that the planula and adult transcriptomes are more similar to each other than to the polyp transcriptome. Our results also point to a large number of uncharacterized adult coral-specific genes likely involved in coral-specific functions such as symbiosis and calcification.

Biosynthesis of coenzyme Q in eukaryotes.

PubMed

Kawamukai, Makoto

2016-01-01

Coenzyme Q (CoQ) is a component of the electron transport chain that participates in aerobic cellular respiration to produce ATP. In addition, CoQ acts as an electron acceptor in several enzymatic reactions involving oxidation-reduction. Biosynthesis of CoQ has been investigated mainly in Escherichia coli and Saccharomyces cerevisiae, and the findings have been extended to various higher organisms, including plants and humans. Analyses in yeast have contributed greatly to current understanding of human diseases related to CoQ biosynthesis. To date, human genetic disorders related to mutations in eight COQ biosynthetic genes have been reported. In addition, the crystal structures of a number of proteins involved in CoQ synthesis have been solved, including those of IspB, UbiA, UbiD, UbiX, UbiI, Alr8543 (Coq4 homolog), Coq5, ADCK3, and COQ9. Over the last decade, knowledge of CoQ biosynthesis has accumulated, and striking advances in related human genetic disorders and the crystal structure of proteins required for CoQ synthesis have been made. This review focuses on the biosynthesis of CoQ in eukaryotes, with some comparisons to the process in prokaryotes.

N-acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis.

PubMed

Yi, Dan; Hou, Yongqing; Wang, Lei; Long, Minhui; Hu, Shengdi; Mei, Huimin; Yan, Liqiong; Hu, Chien-An Andy; Wu, Guoyao

2016-02-01

Dietary supplementation with N-acetylcysteine (NAC) has been reported to improve intestinal health and treat gastrointestinal diseases. However, the underlying mechanisms are not fully understood. According to previous reports, NAC was thought to exert its effect through glutathione synthesis. This study tested the hypothesis that NAC enhances enterocyte growth and protein synthesis independently of cellular glutathione synthesis. Intestinal porcine epithelial cells were cultured for 3 days in Dulbecco's modified Eagle medium containing 0 or 100 μM NAC. To determine a possible role for GSH (the reduced form of glutathione) in mediating the effect of NAC on cell growth and protein synthesis, additional experiments were conducted using culture medium containing 100 μM GSH, 100 μM GSH ethyl ester (GSHee), diethylmaleate (a GSH-depletion agent; 10 μM), or a GSH-synthesis inhibitor (buthionine sulfoximine, BSO; 20 μM). NAC increased cell proliferation, GSH concentration, and protein synthesis, while inhibiting proteolysis. GSHee enhanced cell proliferation and GSH concentration without affecting protein synthesis but inhibited proteolysis. Conversely, BSO or diethylmaleate reduced cell proliferation and GSH concentration without affecting protein synthesis, while promoting protein degradation. At the signaling level, NAC augmented the protein abundance of total mTOR, phosphorylated mTOR, and phosphorylated 70S6 kinase as well as mRNA levels for mTOR and p70S6 kinase in IPEC-1 cells. Collectively, these results indicate that NAC upregulates expression of mTOR signaling proteins to stimulate protein synthesis in enterocytes independently of GSH generation. Our findings provide a hitherto unrecognized biochemical mechanism for beneficial effects of NAC in intestinal cells.

A potential role for zinc transporter 7 in testosterone synthesis in mouse Leydig tumor cells.

PubMed

Chu, Qingqing; Chi, Zhi-Hong; Zhang, Xiuli; Liang, Dan; Wang, Xuemei; Zhao, Yue; Zhang, Li; Zhang, Ping

2016-06-01

Previous studies have demonstrated that zinc (Zn) is an essential trace element which is involved in male reproduction. The zinc transporter (ZnT) family, SLC30a, is involved in the maintenance of Zn homeostasis and in mediating intracellular signaling events; however, relatively little is known regarding the effect of ZnTs on testosterone synthesis. Thus, in the present study, we aimed to determine the effect of Zn transporter 7 (ZnT7) on testosterone synthesis in male CD-1 mice and mouse Leydig cells. The findings of the present study revealed that the concentrations of Zn in the testes and Leydig cells were significantly lower in mice fed a Zn-deficient diet compared with the control mice fed a Zn-adequate diet. In addition, ZnT7 was principally expressed and colocalized with steroidogenic acute regulatory protein (StAR) in the Leydig cells of male CD-1 mice. ZnT7 expression was downregulated in the mice fed a Zn-deficient diet, which led to decreases in the expression of the enzymes involved in testosterone synthesis namely cholesterol side‑chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/D5-D4 isomerase (3β-HSD) as well as decreased serum testosterone levels. These results suggested that Znt7 may be involved in testosterone synthesis in the mouse testes. To examine this hypothesis, we used the mouse Leydig tumor cell line (MLTC-1 cell line) in which the ZnT7 gene had been silenced, in order to gauge the impact of changes in ZnT7 expression on testosterone secretion and the enzymes involved in testosterone synthesis. The results demonstrated that ZnT7 gene silencing downregulated the expression of StAR, P450scc and 3β-HSD as well as progesterone concentrations in the human chorionic gonadotrophin (hCG)-stimulated MLTC-1 cells. Taken together, these findings reveal that ZnT7 may play an important role in the regulation of testosterone synthesis by modulating steroidogenic enzymes, and may represent a therapeutic target in testosterone deficiency.

Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.

PubMed

Milles, J; Krämer, J; Prange, A

2007-12-01

In general, proteome studies compare different states of metabolism to investigate external or internal influences on protein expression. In the context of mycotoxin production the method could open another view on this complex and could be helpful to gain knowledge about proteins which are involved in metabolism (enzymes, transporters). In this short technical report, we describe a new protocol suitable for protein preparation for whole proteome analysis ofFusarium graminearum. Cell lysis was performed by grinding the mycelium with liquid nitrogen. Proteins were extracted with TCA/acetone and then cleaned; the isolated proteins were separated in a 2D-gel electrophoresis system (BioRad) using different pH gradients. The protocol established seems also generally applicable for other mycotoxin producing fungi.

ABCE1 is essential for S phase progression in human cells

PubMed Central

Toompuu, Marina; Kärblane, Kairi; Pata, Pille; Truve, Erkki; Sarmiento, Cecilia

2016-01-01

ABSTRACT ABCE1 is a highly conserved protein universally present in eukaryotes and archaea, which is crucial for the viability of different organisms. First identified as RNase L inhibitor, ABCE1 is currently recognized as an essential translation factor involved in several stages of eukaryotic translation and ribosome biogenesis. The nature of vital functions of ABCE1, however, remains unexplained. Here, we study the role of ABCE1 in human cell proliferation and its possible connection to translation. We show that ABCE1 depletion by siRNA results in a decreased rate of cell growth due to accumulation of cells in S phase, which is accompanied by inefficient DNA synthesis and reduced histone mRNA and protein levels. We infer that in addition to the role in general translation, ABCE1 is involved in histone biosynthesis and DNA replication and therefore is essential for normal S phase progression. In addition, we analyze whether ABCE1 is implicated in transcript-specific translation via its association with the eIF3 complex subunits known to control the synthesis of cell proliferation-related proteins. The expression levels of a few such targets regulated by eIF3A, however, were not consistently affected by ABCE1 depletion. PMID:26985706

Some pharmacological aspects of drug dependence.

PubMed

Chesher, G B

1975-12-06

The self-administration of drugs to achieve altered states of consciousness is recognized as normal human behaviour. Community attitudes towards drug use vary according to the drug and often bear little relationship to the known pharmacological and toxicological effects of the drug. For an objective assessment of the potential dangers associated with drug use, a distinction is made between drug use and drug abuse. It is stressed that the progression from drug use to drug abuse involves social and psychological factors in addition to the pharmacological factors which are outlined in this paper. The sequential development of drug dependency is described under the headings: Induction; continued consumption; compulsive consumption; withdrawal; abstinence; reinduction. Man uses psychotropic drugs because he finds the effects rewarding. Some experimental models to explore the neurophysiological basis of the reward are described. Experiments employing inhibitors of protein synthesis suggest that the phenomena of tolerance and physical dependence involve the synthesis of new protein. It has been suggested that the new protein might be new receptor molecules for the drug or neurotransmitter substances. These new receptors might constitute a "drug memory" and provide a possible explanation for high relapse rate of drug dependent subjects. A pharmacological basis for the methadone maintenance programme of management of narcotic dependent subjects is briefly outlined.

Involvement of Dynein and Spectrin with Early Melanosome Transport and Melanosomal Protein Trafficking

PubMed Central

Watabe, Hidenori; Valencia, Julio C.; Le Pape, Elodie; Yamaguchi, Yuji; Nakamura, Masayuki; Rouzaud, François; Hoashi, Toshihiko; Kawa, Yoko; Mizoguchi, Masako; Hearing, Vincent J.

2007-01-01

Melanosomes are unique membrane-bound organelles specialized for the synthesis and distribution of melanin. Mechanisms involved in the trafficking of proteins to melanosomes and in the transport of mature pigmented melanosomes to the dendrites of melanocytic cells are being characterized but details about those processes during early stages of melanosome maturation are not well understood. Early melanosomes must remain in the perinuclear area until critical components are assembled. In this study, we characterized the processing of two distinct melanosomal proteins, TYR and Pmel17, to elucidate protein processing in early or late steps of the secretory pathway, respectively, and to determine mechanisms underlying the subcellular localization and transport of early melanosomes. We used immunological, biochemical and molecular approaches to demonstrate that the movement of early melanosomes in the perinuclear area depends primarily on microtubules but not on actin filaments. In contrast, the trafficking of TYR and Pmel17 depends on cytoplasmic dynein and its interaction with the spectrin/ankyrin system which is involved with the sorting of cargo from the plasma membrane. These results provide important clues towards understanding the processes involved with early events in melanosome formation and transport. PMID:17687388

Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

PubMed

Liu, Ying; Li, Yueqin; Zhang, Di; Liu, Jiali; Gou, Kemian; Cui, Sheng

2015-05-01

The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism.

Heparan sulfate proteoglycans undergo differential expression alterations in right sided colorectal cancer, depending on their metastatic character.

PubMed

Fernández-Vega, Iván; García-Suárez, Olivia; García, Beatriz; Crespo, Ainara; Astudillo, Aurora; Quirós, Luis M

2015-10-20

Heparan sulfate proteoglycans (HSPGs) are complex molecules involved in the growth, invasion and metastatic properties of cancerous cells. This study analyses the alterations in the expression patterns of these molecules in right sided colorectal cancer (CRC), both metastatic and non-metastatic. Twenty right sided CRCs were studied. A transcriptomic approach was used, employing qPCR to analyze both the expression of the enzymes involved in heparan sulfate (HS) chains biosynthesis, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate (CS) chains, we include the study of the genes involved in the biosynthesis of these glycosaminoglycans. Immunohistochemical techniques were also used to analyze tissue expression of particular genes showing significant expression differences, of potential interest. Changes in proteoglycan core proteins differ depending on their location; those located intracellularly or in the extracellular matrix show very similar alteration patterns, while those located on the cell surface vary greatly depending on the nature of the tumor: glypicans 1, 3, 6 and betaglycan are affected in the non-metastatic tumors, whereas in the metastatic, only glypican-1 and syndecan-1 are modified, the latter showing opposing alterations in levels of RNA and of protein, suggesting post-transcriptional regulation in these tumors. Furthermore, in non-metastatic tumors, polymerization of glycosaminoglycan chains is modified, particularly affecting the synthesis of the tetrasaccharide linker and the initiation and elongation of CS chains, HS chains being less affected. Regarding the enzymes responsible for the modificaton of the HS chains, alterations were only found in non-metastatic tumors, affecting N-sulfation and the isoforms HS6ST1, HS3ST3B and HS3ST5. In contrast, synthesis of the CS chains suggests changes in epimerization and sulfation of the C4 and C2 in both types of tumor. Right sided CRCs show alterations in the expression of HSPGs, including the expression of the cell surface core proteins, many glycosiltransferases and some enzymes that modify the HS chains depending on the metastatic nature of the tumor, resulting more affected in non-metastatic ones. However, matrix proteoglycans and enzymes involved in CS fine structure synthesis are extensively modified independetly of the presence of lymph node metastasis.

Genetics Home Reference: HSD10 disease

MedlinePlus

... in the production (synthesis) of proteins . While most protein synthesis occurs in the fluid surrounding the nucleus (cytoplasm), ... few proteins are synthesized in the mitochondria. During protein synthesis, in either the mitochondria or the cytoplasm, molecules ...

Non-encapsidation Activities of the Capsid Proteins of Positive-strand RNA Viruses

PubMed Central

Ni, Peng; Kao, C. Cheng

2013-01-01

Viral capsid proteins (CPs) are characterized by their role in forming protective shells around viral genomes. However, CPs have additional and important roles in the virus infection cycles and in the cellular response to infection. These activities involve CP binding to RNAs in both sequence-specific and nonspecific manners as well as association with other proteins. This review focuses on CPs of both plant and animal-infecting viruses with positive-strand RNA genomes. We summarize the structural features of CPs and describe their modulatory roles in viral translation, RNA-dependent RNA synthesis, and host defense responses. PMID:24074574

Chromophore-assisted light inactivation of pKi-67 leads to inhibition of ribosomal RNA synthesis.

PubMed

Rahmanzadeh, R; Hüttmann, G; Gerdes, J; Scholzen, T

2007-06-01

Expression of the nuclear Ki-67 protein (pKi-67) is strongly associated with cell proliferation. For this reason, antibodies against this protein are widely used as prognostic tools for the assessment of cell proliferation in biopsies from cancer patients. Despite this broad application in histopathology, functional evidence for the physiological role of pKi-67 is still missing. Recently, we proposed a function of pKi-67 in the early steps of ribosomal RNA (rRNA) synthesis. Here, we have examined the involvement of pKi-67 in this process by photochemical inhibition using chromophore-assisted light inactivation (CALI). Anti-pKi-67 antibodies were labelled with the fluorochrome fluorescein 5(6)-isothiocyanate and were irradiated after binding to their target protein. Performing CALI in vitro on cell lysates led to specific cross-linking of pKi-67. Moreover, the upstream binding factor (UBF) necessary for rRNA transcription was also partly subjected to cross-link formation, indicating a close spatial proximity of UBF and pKi-67. CALI in living cells, using micro-injected antibody, caused a striking relocalization of UBF from foci within the nucleoli to spots located at the nucleolar rim or within the nucleoplasm. pKi-67-CALI resulted in dramatic inhibition of RNA polymerase I-dependent nucleolar rRNA synthesis, whereas RNA polymerase II-dependent nucleoplasmic RNA synthesis remained almost unaltered. Our data presented here argue for a crucial role of pKi-67 in RNA polymerase I-dependent nucleolar rRNA synthesis.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

Energetic costs of protein synthesis do not differ between red- and white-blooded Antarctic notothenioid fishes.

PubMed

Lewis, Johanne M; Grove, Theresa J; O'Brien, Kristin M

2015-09-01

Antarctic icefishes (Family Channichthyidae) within the suborder Notothenioidei lack the oxygen-binding protein hemoglobin (Hb), and six of the 16 species of icefishes lack myoglobin (Mb) in heart ventricle. As iron-centered proteins, Hb and Mb can promote the formation of reactive oxygen species (ROS) that damage biological macromolecules. Consistent with this, our previous studies have shown that icefishes have lower levels of oxidized proteins and lipids in oxidative muscle compared to red-blooded notothenioids. Because oxidized proteins are usually degraded by the 20S proteasome and must be resynthesized, we hypothesized that rates of protein synthesis would be lower in icefishes compared to red-blooded notothenioids, thereby reducing the energetic costs of protein synthesis and conferring a benefit to the loss of Hb and Mb. Rates of protein synthesis were quantified in hearts, and the fraction of oxygen consumption devoted to protein synthesis was measured in isolated hepatocytes and cardiomyocytes of notothenioids differing in the expression of Hb and cardiac Mb. Neither rates of protein synthesis nor the energetic costs of protein synthesis differed among species, suggesting that red-blooded species do not degrade and replace oxidatively modified proteins at a higher rate compared to icefishes but rather, persist with higher levels of oxidized proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation

PubMed Central

Shih, Yu-Tzu; Hsueh, Yi-Ping

2016-01-01

Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders. PMID:26984393

VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

PubMed

Shih, Yu-Tzu; Hsueh, Yi-Ping

2016-03-17

Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

Atrogin-1 affects muscle protein synthesis and degradation when energy metabolism is impaired by the antidiabetes drug berberine.

PubMed

Wang, Huiling; Liu, Dajun; Cao, Peirang; Lecker, Stewart; Hu, Zhaoyong

2010-08-01

Defects in insulin/IGF-1 signaling stimulate muscle protein loss by suppressing protein synthesis and increasing protein degradation. Since an herbal compound, berberine, lowers blood levels of glucose and lipids, we proposed that it would improve insulin/IGF-1 signaling, blocking muscle protein losses. We evaluated whether berberine ameliorates muscle atrophy in db/db mice, a model of type 2 diabetes, by measuring protein synthesis and degradation in muscles of normal and db/db mice treated with or without berberine. We also examined mechanisms for berberine-induced changes in muscle protein metabolism. Berberine administration decreased protein synthesis and increased degradation in muscles of normal and db/db mice. The protein catabolic mechanism depended on berberine-stimulated expression of the E3 ubiquitin ligase, atrogin-1. Atrogin-1 not only increased proteolysis but also reduced protein synthesis by mechanisms that were independent of decreased phosphorylation of Akt or forkhead transcription factors. Impaired protein synthesis was dependent on a reduction in eIF3-f, an essential regulator of protein synthesis. Berberine impaired energy metabolism, activating AMP-activated protein kinase and providing an alternative mechanism for the stimulation of atrogin-1 expression. When we increased mitochondrial biogenesis by expressing peroxisome proliferator-activated receptor gamma coactivator-1alpha, berberine-induced changes in muscle protein metabolism were prevented. Berberine impairs muscle metabolism by two novel mechanisms. It impairs mitochonidrial function stimulating the expression of atrogin-1 without affecting phosphorylation of forkhead transcription factors. The increase in atrogin-1 not only stimulated protein degradation but also suppressed protein synthesis, causing muscle atrophy.

Cellulose Synthesis in Agrobacterium tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alan R. White; Ann G. Matthysse

2004-07-31

We have cloned the celC gene and its homologue from E. coli, yhjM, in an expression vector and expressed the both genes in E. coli; we have determined that the YhjM protein is able to complement in vitro cellulose synthesis by extracts of A. tumefaciens celC mutants, we have purified the YhjM protein product and are currently examining its enzymatic activity; we have examined whole cell extracts of CelC and various other cellulose mutants and wild type bacteria for the presence of cellulose oligomers and cellulose; we have examined the ability of extracts of wild type and cellulose mutants includingmore » CelC to incorporate UDP-14C-glucose into cellulose and into water-soluble, ethanol-insoluble oligosaccharides; we have made mutants which synthesize greater amounts of cellulose than the wild type; and we have examined the role of cellulose in the formation of biofilms by A. tumefaciens. In addition we have examined the ability of a putative cellulose synthase gene from the tunicate Ciona savignyi to complement an A. tumefaciens celA mutant. The greatest difference between our knowledge of bacterial cellulose synthesis when we started this project and current knowledge is that in 1999 when we wrote the original grant very few bacteria were known to synthesize cellulose and genes involved in this synthesis were sequenced only from Acetobacter species, A. tumefaciens and Rhizobium leguminosarum. Currently many bacteria are known to synthesize cellulose and genes that may be involved have been sequenced from more than 10 species of bacteria. This additional information has raised the possibility of attempting to use genes from one bacterium to complement mutants in another bacterium. This will enable us to examine the question of which genes are responsible for the three dimensional structure of cellulose (since this differs among bacterial species) and also to examine the interactions between the various proteins required for cellulose synthesis. We have carried out one preliminary experiment of this type and have successfully complemented an A. tumefaciens CelC mutant with the homologous gene (yhjM) from E. coli.« less

Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

USDA-ARS?s Scientific Manuscript database

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were...

Do rice suspension-cultured cells treated with abscisic acid mimic developing seeds?

PubMed

Matsuno, Koya; Fujimura, Tatsuhito

2015-08-01

Starch synthesis is activated in the endosperm during seed development and also in rice suspension cells cultured with abscisic acid. In the anticipation that the mechanisms of starch synthesis are similar between the endosperm and the suspension cells cultured with abscisic acid, expression of genes involved in starch synthesis was evaluated in the suspension cells after abscisic acid treatment. However, it was found that the regulatory mechanism of starch synthesis in the suspension cells cultured with abscisic acid was different from that in developing seeds. Expression analyses of genes involved in oil bodies, which accumulate in the embryo and aleurone layer, and seed storage proteins, which accumulate mainly in the endosperm, showed that the former were activated in the suspension cells cultured with abscisic acid, but the latter were not. Master regulators for embryogenesis, OsVP1 (homologue of AtABI3) and OsLFL1 (homologue of AtFUS3 or AtLFL2), were expressed in the suspension cells at levels comparable to those in the embryo. From these results, it is suggested that interactions between regulators and abscisic acid control the synthesis of phytic acid and oil bodies in the cultured cells and embryo. We suggest that the system of suspension cells cultured with abscisic acid helps to reveal the mechanisms of phytic acid and oil body synthesis in embryo.

Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis

PubMed Central

Zhang, Junxiang; Yuan, Hui; Yang, Yong; Fish, Tara; Lyi, Sangbom M.; Thannhauser, Theodore W; Zhang, Lugang; Li, Li

2016-01-01

Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development. PMID:27006483

Microtubule antagonists activate programmed cell death (apoptosis) in cultured rat hepatocytes.

PubMed Central

Tsukidate, K.; Yamamoto, K.; Snyder, J. W.; Farber, J. L.

1993-01-01

We investigated the mechanism of lethal injury following the disruption of microtubules in cultured hepatocytes treated with vinblastine (VBL) or colchicine (COL). These agents kill hepatocytes by a process readily distinguished from two well-known pathways that lead to a loss of viability, namely, oxidative stress and inhibition of mitochondrial electron transport. Cell killing with VBL and COL was accompanied by fragmentation of DNA. Both the loss of viability and the fragmentation of DNA were prevented by the inhibition of protein synthesis within 6 hours following exposure to VBL or COL. Cell death and the fragmentation of DNA were also prevented when Ca2+ was removed from the culture medium. By contrast, the inhibition of protein kinase C prevented cell killing by VBL or COL, but did not alter the extent of DNA fragmentation. The requirements here for protein synthesis, extracellular Ca2+, and protein kinase C activity define a model of apoptosis, or programmed cell death, that seems to involve mechanisms that can be dissociated from the fragmentation of DNA. Images Figure 2 PMID:8362985

Using Denatured Egg White as a Macroscopic Model for Teaching Protein Structure and Introducing Protein Synthesis for High School Students

NASA Astrophysics Data System (ADS)

Correia, Paulo R. M.; Torres, Bayardo B.

2007-12-01

The success of teaching molecular and atomic phenomena depends on the didactical strategy and the media selection adopted, in consideration of the level of abstraction of the subject to be taught and the students' capability to deal with abstract operations. Dale's cone of experience was employed to plan three 50-minute classes to discuss protein denaturation from a chemical point of view. Only low abstraction level activities were selected: (i) two demonstrations showing the denaturation of albumin by heating and by changing the solvent, (ii) the assembly of a macroscopic model representing the protein molecule, and (iii) a role-play for simulating glucagon synthesis. A student-centered approach and collaborative learning were used throughout the classes. The use of macroscopic models is a powerful didactical strategy to represent molecular and atomic events. They can convert microscopic entities into touchable objects, reducing the abstraction level required to discuss chemistry with high school students. Thus, interesting topics involving molecules and their behavior can take place efficiently when mediated by concrete experiences.

Modulation of protein synthesis by polyamines.

PubMed

Igarashi, Kazuei; Kashiwagi, Keiko

2015-03-01

Polyamines are ubiquitous small basic molecules that play important roles in cell growth and viability. Since polyamines mainly exist as a polyamine-RNA complex, we looked for proteins whose synthesis is preferentially stimulated by polyamines at the level of translation, and thus far identified 17 proteins in Escherichia coli and 6 proteins in eukaryotes. The mechanisms of polyamine stimulation of synthesis of these proteins were investigated. In addition, the role of eIF5A, containing hypusine formed from spermidine, on protein synthesis is described. These results clearly indicate that polyamines and eIF5A contribute to cell growth and viability through modulation of protein synthesis. © 2015 International Union of Biochemistry and Molecular Biology.

Biotin and Lipoic Acid: Synthesis, Attachment and Regulation

PubMed Central

Cronan, John E.

2014-01-01

Summary Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well-described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized and thus there is no transcriptional control of the synthetic genes. In contrast transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA a dual function protein that both represses biotin operon transcription and ligates biotin to its cognate protein. PMID:26442940

Mitogenic signaling pathways of growth factors can be distinguished by the involvement of pertussis toxin-sensitive guanosine triphosphate-binding protein and of protein kinase C.

PubMed Central

Nishizawa, N; Okano, Y; Chatani, Y; Amano, F; Tanaka, E; Nomoto, H; Nozawa, Y; Kohno, M

1990-01-01

We have examined the possible involvements of pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)-binding protein (Gp) and protein kinase C (PKC) in the mitogenic signaling pathways of various growth factors by the use of PT-pretreated and/or 12-O-tetradecanoyl phorbol-13-acetate (TPA)-pretreated mouse fibroblasts. Effects of PT pretreatment (inactivation of PT-sensitive Gp) and TPA pretreatment (depletion of PKC) on mitogen-induced DNA synthesis varied significantly and systematically in response to growth factors: mitogenic responses of cells to thrombin, bombesin, and bradykinin were almost completely abolished both in PT- and TPA-pretreated cells; responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vanadate were reduced to approximately 50% both in PT- and TPA-pretreated cells compared with native cells; response to basic fibroblast growth factor (bFGF) was not affected in PT-pretreated cells but was inhibited to some extent in TPA-pretreated cells. Thus, growth factors examined have been classified into three groups with regard to the involvements of PT-sensitive Gp and PKC in their signal transduction pathways. Binding of each growth factor to its receptor was not affected significantly by pretreatment of cells with PT or TPA. Inhibitory effects of PT and TPA pretreatment on each mitogen-induced DNA synthesis were not additive, suggesting that the functions of PT-sensitive Gp and PKC lie on an identical signal transduction pathway. Although all three groups of mitogens activated PKC, signaling of each growth factor depends to a varying extent on the function of PKC. Our results indicate that a single peptide growth factor such as EGF, PDGF, or bFGF acts through multiple signaling pathways to induce cell proliferation. Images PMID:2129194

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

PubMed Central

Aspesi, Anna; Pavesi, Elisa; Robotti, Elisa; Crescitelli, Rossella; Boria, Ilenia; Avondo, Federica; Moniz, Hélène; Da Costa, Lydie; Mohandas, Narla; Roncaglia, Paola; Ramenghi, Ugo; Ronchi, Antonella; Gustincich, Stefano; Merlin, Simone; Marengo, Emilio; Ellis, Steven R.; Follenzi, Antonia; Santoro, Claudio; Dianzani, Irma

2014-01-01

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA. PMID:24835311

Transcription profile of brewery yeast under fermentation conditions.

PubMed

James, T C; Campbell, S; Donnelly, D; Bond, U

2003-01-01

Yeast strains, used in the brewing industry, experience distinctive physiological conditions. During a brewing fermentation, yeast are exposed to anaerobic conditions, high pressure, high specific gravity and low temperatures. The purpose of this study was to examine the global gene expression profile of yeast subjected to brewing stress. We have carried out a microarray analysis of a typical brewer's yeast during the course of an 8-day fermentation in 15 degrees P wort. We used the probes derived from Saccharomyces cerevisiae genomic DNA on the chip and RNA isolated from three stages of brewing. This analysis shows a high level of expression of genes involved in fatty acid and ergosterol biosynthesis early in fermentation. Furthermore, genes involved in respiration and mitochondrial protein synthesis also show higher levels of expression. Surprisingly, we observed a complete repression of many stress response genes and genes involved in protein synthesis throughout the 8-day period compared with that at the start of fermentation. This microarray data set provides an analysis of gene expression under brewing fermentation conditions. The data provide an insight into the various metabolic processes altered or activated by brewing conditions of growth. This study leads to future experiments whereby selective alterations in brewing conditions could be introduced to take advantage of the changing transcript profile to improve the quality of the brew.

Structure-based drug design, synthesis and biological assays of P. falciparum Atg3-Atg8 protein-protein interaction inhibitors

NASA Astrophysics Data System (ADS)

Villa, Stefania; Legnani, Laura; Colombo, Diego; Gelain, Arianna; Lammi, Carmen; Bongiorno, Daniele; Ilboudo, Denise P.; McGee, Kellen E.; Bosch, Jürgen; Grazioso, Giovanni

2018-03-01

The proteins involved in the autophagy (Atg) pathway have recently been considered promising targets for the development of new antimalarial drugs. In particular, inhibitors of the protein-protein interaction (PPI) between Atg3 and Atg8 of Plasmodium falciparum retarded the blood- and liver-stages of parasite growth. In this paper, we used computational techniques to design a new class of peptidomimetics mimicking the Atg3 interaction motif, which were then synthesized by click-chemistry. Surface plasmon resonance has been employed to measure the ability of these compounds to inhibit the Atg3-Atg8 reciprocal protein-protein interaction. Moreover, P. falciparum growth inhibition in red blood cell cultures was evaluated as well as the cyto-toxicity of the compounds.

Comparative proteomics analysis of the rice roots colonized by Herbaspirillum seropedicae strain SmR1 reveals induction of the methionine recycling in the plant host.

PubMed

Alberton, Dayane; Müller-Santos, Marcelo; Brusamarello-Santos, Liziane Cristina Campos; Valdameri, Glaucio; Cordeiro, Fabio Aparecido; Yates, Marshall Geoffrey; de Oliveira Pedrosa, Fabio; de Souza, Emanuel Maltempi

2013-11-01

Although the use of plant growth-promoting bacteria in agriculture is a reality, the molecular basis of plant-bacterial interaction is still poorly understood. We used a proteomic approach to study the mechanisms of interaction of Herbaspirillum seropedicae SmR1 with rice. Root proteins of rice seedlings inoculated or noninoculated with H. seropedicae were separated by 2-D electrophoresis. Differentially expressed proteins were identified by MALDI-TOF/TOF and MASCOT program. Among the identified proteins of H. seropedicae, the dinitrogenase reductase NifH and glutamine synthetase GlnA, which participate in nitrogen fixation and ammonium assimilation, respectively, were the most abundant. The rice proteins up-regulated included the S-adenosylmethionine synthetase, methylthioribose kinase, and acireductone dioxygenase 1, all of which are involved in the methionine recycling. S-Adenosylmethionine synthetase catalyzes the synthesis of S-adenosylmethionine, an intermediate used in transmethylation reactions and in ethylene, polyamine, and phytosiderophore biosynthesis. RT-qPCR analysis also confirmed that the methionine recycling and phytosiderophore biosynthesis genes were up-regulated, while ACC oxidase mRNA level was down-regulated in rice roots colonized by bacteria. In agreement with these results, ethylene production was reduced approximately three-fold in rice roots colonized by H. seropedicae. The results suggest that H. seropedicae stimulates methionine recycling and phytosiderophore synthesis and diminishes ethylene synthesis in rice roots.

Rapid Reversion from Monomer to Dimer Regenerates the Ultraviolet-B Photoreceptor UV RESISTANCE LOCUS8 in Intact Arabidopsis Plants1[W][OA

PubMed Central

Heilmann, Monika; Jenkins, Gareth I.

2013-01-01

Arabidopsis (Arabidopsis thaliana) UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor that specifically mediates photomorphogenic responses to ultraviolet (UV)-B in plants. UV-B photoreception induces the conversion of the UVR8 dimer into a monomer that interacts with the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) protein to regulate gene expression. However, it is not known how the dimeric photoreceptor is regenerated in plants. Here, we show, by using inhibitors of protein synthesis and degradation via the proteasome, that the UVR8 dimer is not regenerated by rapid de novo synthesis following destruction of the monomer. Rather, regeneration occurs by reversion from the monomer to the dimer. However, regeneration of dimeric UVR8 in darkness following UV-B exposure occurs much more rapidly in vivo than in vitro with illuminated plant extracts or purified UVR8, indicating that rapid regeneration requires intact cells. Rapid dimer regeneration in vivo requires protein synthesis, the presence of a carboxyl-terminal 27-amino acid region of UVR8, and the presence of COP1, which is known to interact with the carboxyl-terminal region. However, none of these factors can account fully for the difference in regeneration kinetics in vivo and in vitro, indicating that additional proteins or processes are involved in UVR8 dimer regeneration in vivo. PMID:23129206

Identification and subsequent phosphorylation of sequestered partially processed caseins in the lactating guinea-pig mammary gland.

PubMed Central

Boulton, A P; Pascall, J C; Craig, R K

1984-01-01

Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway, and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labelled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and alpha-lactalbumin sequestered within membrane-bound vesicles. After a 30 min labelling period, higher-Mr caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent approx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6477529

The Rules and Functions of Nucleocytoplasmic Shuttling Proteins.

PubMed

Fu, Xuekun; Liang, Chao; Li, Fangfei; Wang, Luyao; Wu, Xiaoqiu; Lu, Aiping; Xiao, Guozhi; Zhang, Ge

2018-05-12

Biological macromolecules are the basis of life activities. There is a separation of spatial dimension between DNA replication and RNA biogenesis, and protein synthesis, which is an interesting phenomenon. The former occurs in the cell nucleus, while the latter in the cytoplasm. The separation requires protein to transport across the nuclear envelope to realize a variety of biological functions. Nucleocytoplasmic transport of protein including import to the nucleus and export to the cytoplasm is a complicated process that requires involvement and interaction of many proteins. In recent years, many studies have found that proteins constantly shuttle between the cytoplasm and the nucleus. These shuttling proteins play a crucial role as transport carriers and signal transduction regulators within cells. In this review, we describe the mechanism of nucleocytoplasmic transport of shuttling proteins and summarize some important diseases related shuttling proteins.

Morphological and proteomic analysis of biofilms from the Antarctic archaeon, Halorubrum lacusprofundi.

PubMed

Liao, Y; Williams, T J; Ye, J; Charlesworth, J; Burns, B P; Poljak, A; Raftery, M J; Cavicchioli, R

2016-11-22

Biofilms enhance rates of gene exchange, access to specific nutrients, and cell survivability. Haloarchaea in Deep Lake, Antarctica, are characterized by high rates of intergenera gene exchange, metabolic specialization that promotes niche adaptation, and are exposed to high levels of UV-irradiation in summer. Halorubrum lacusprofundi from Deep Lake has previously been reported to form biofilms. Here we defined growth conditions that promoted the formation of biofilms and used microscopy and enzymatic digestion of extracellular material to characterize biofilm structures. Extracellular DNA was found to be critical to biofilms, with cell surface proteins and quorum sensing also implicated in biofilm formation. Quantitative proteomics was used to define pathways and cellular processes involved in forming biofilms; these included enhanced purine synthesis and specific cell surface proteins involved in DNA metabolism; post-translational modification of cell surface proteins; specific pathways of carbon metabolism involving acetyl-CoA; and specific responses to oxidative stress. The study provides a new level of understanding about the molecular mechanisms involved in biofilm formation of this important member of the Deep Lake community.

Morphological and proteomic analysis of biofilms from the Antarctic archaeon, Halorubrum lacusprofundi

PubMed Central

Liao, Y.; Williams, T. J.; Ye, J.; Charlesworth, J.; Burns, B. P.; Poljak, A.; Raftery, M. J.; Cavicchioli, R.

2016-01-01

Biofilms enhance rates of gene exchange, access to specific nutrients, and cell survivability. Haloarchaea in Deep Lake, Antarctica, are characterized by high rates of intergenera gene exchange, metabolic specialization that promotes niche adaptation, and are exposed to high levels of UV-irradiation in summer. Halorubrum lacusprofundi from Deep Lake has previously been reported to form biofilms. Here we defined growth conditions that promoted the formation of biofilms and used microscopy and enzymatic digestion of extracellular material to characterize biofilm structures. Extracellular DNA was found to be critical to biofilms, with cell surface proteins and quorum sensing also implicated in biofilm formation. Quantitative proteomics was used to define pathways and cellular processes involved in forming biofilms; these included enhanced purine synthesis and specific cell surface proteins involved in DNA metabolism; post-translational modification of cell surface proteins; specific pathways of carbon metabolism involving acetyl-CoA; and specific responses to oxidative stress. The study provides a new level of understanding about the molecular mechanisms involved in biofilm formation of this important member of the Deep Lake community. PMID:27874045

Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumins on the Synthesis and Stability of Gold Nanostructures

PubMed Central

Miranda, Érica G. A.; Tofanello, Aryane; Brito, Adrianne M. M.; Lopes, David M.; Albuquerque, Lindomar J. C.; de Castro, Carlos E.; Costa, Fanny N.; Giacomelli, Fernando C.; Ferreira, Fabio F.; Araújo-Chaves, Juliana C.; Nantes, Iseli L.

2016-01-01

The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3–12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from 15 days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After 2 months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with the gold surface. Therefore, the cysteine side chain of albumins is important for the colloidal stabilization of GNPs rather than as the reducing agent for the synthesis. Despite the presence of more reactive gold species at more acidic pH values, i.e., below 6.0, in these conditions the loss of native albumin structure impaired GNP synthesis. Alkaline pH values (9–12) combined the unfavorable conditions of denaturated protein structure with less reactive gold species. Therefore, an optimal condition for the synthesis of GNPs using serum albumins involves more reactive gold salt species combined with a reducing and negatively charged form of the protein, all favored at pH 6–7. PMID:27066476

Translation initiation mediated by nuclear cap-binding protein complex.

PubMed

Ryu, Incheol; Kim, Yoon Ki

2017-04-01

In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

The antiviral protein Viperin suppresses T7 promoter dependent RNA synthesis-possible implications for its antiviral activity.

PubMed

Dukhovny, Anna; Shlomai, Amir; Sklan, Ella H

2018-05-25

Viperin is a multifunctional interferon-inducible broad-spectrum antiviral protein. Viperin belongs to the S-Adenosylmethionine (SAM) superfamily of enzymes known to catalyze a wide variety of radical-mediated reactions. However, the exact mechanism by which viperin exerts its functions is still unclear. Interestingly, for many RNA viruses viperin was shown to inhibit viral RNA accumulation by interacting with different viral non-structural proteins. Here, we show that viperin inhibits RNA synthesis by bacteriophage T7 polymerase in mammalian cells. This inhibition is specific and occurs at the RNA level. Viperin expression significantly reduced T7-mediated cytoplasmic RNA levels. The data showing that viperin inhibits the bacteriophage T7 polymerase supports the conservation of viperin's antiviral activity between species. These results highlight the possibility that viperin might utilize a broader mechanism of inhibition. Accordingly, our results suggest a novel mechanism involving polymerase inhibition and provides a tractable system for future mechanistic studies of viperin.

Fe2+ chelator proferrorosamine A: a gene cluster of Erwinia rhapontici P45 involved in its synthesis and its impact on growth of Erwinia amylovora CFBP1430.

PubMed

Born, Yannick; Remus-Emsermann, Mitja N P; Bieri, Marco; Kamber, Tim; Piel, Jörn; Pelludat, Cosima

2016-02-01

Proferrorosamine A (proFRA) is an iron (Fe2+) chelator produced by the opportunistic plant pathogen Erwinia rhapontici P45. To identify genes involved in proFRA synthesis, transposon mutagenesis was performed. The identified 9.3 kb gene cluster, comprising seven genes, designated rosA-rosG, encodes proteins that are involved in proFRA synthesis. Based on gene homologies, a biosynthetic pathway model for proFRA is proposed. To obtain a better understanding of the effect of proFRA on non-proFRA producing bacteria, E. rhapontici P45 was co-cultured with Erwinia amylovora CFBP1430, a fire-blight-causing plant pathogen. E. rhapontici P45, but not corresponding proFRA-negative mutants, led to a pink coloration of E. amylovora CFBP1430 colonies on King's B agar, indicating accumulation of the proFRA-iron complex ferrorosamine, and growth inhibition in vitro. By saturating proFRA-containing extracts with Fe2+, the inhibitory effect was neutralized, suggesting that the iron-chelating capability of proFRA is responsible for the growth inhibition of E. amylovora CFBP1430.

A Role for Iron-Sulfur Clusters in the Regulation of Transcription Factor Yap5-dependent High Iron Transcriptional Responses in Yeast*

PubMed Central

Li, Liangtao; Miao, Ren; Bertram, Sophie; Jia, Xuan; Ward, Diane M.; Kaplan, Jerry

2012-01-01

Yeast respond to increased cytosolic iron by activating the transcription factor Yap5 increasing transcription of CCC1, which encodes a vacuolar iron importer. Using a genetic screen to identify genes involved in Yap5 iron sensing, we discovered that a mutation in SSQ1, which encodes a mitochondrial chaperone involved in iron-sulfur cluster synthesis, prevented expression of Yap5 target genes. We demonstrated that mutation or reduced expression of other genes involved in mitochondrial iron-sulfur cluster synthesis (YFH1, ISU1) prevented induction of the Yap5 response. We took advantage of the iron-dependent catalytic activity of Pseudaminobacter salicylatoxidans gentisate 1,2-dioxygenase expressed in yeast to measure changes in cytosolic iron. We determined that reductions in iron-sulfur cluster synthesis did not affect the activity of cytosolic gentisate 1,2-dioxygenase. We show that loss of activity of the cytosolic iron-sulfur cluster assembly complex proteins or deletion of cytosolic glutaredoxins did not reduce expression of Yap5 target genes. These results suggest that the high iron transcriptional response, as well as the low iron transcriptional response, senses iron-sulfur clusters. PMID:22915593

Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes.

PubMed

Heinz, Stefan; Braspenning, Joris

2015-01-01

An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulation factors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulation factor deficiencies.

Assessment of protein synthesis in highly aerobic canine species at the onset and during exercise training.

PubMed

Miller, Benjamin F; Ehrlicher, Sarah E; Drake, Joshua C; Peelor, Frederick F; Biela, Laurie M; Pratt-Phillips, Shannon; Davis, Michael; Hamilton, Karyn L

2015-04-01

Canis lupus familiaris, the domesticated dog, is capable of extreme endurance performance. The ability to perform sustained aerobic exercise is dependent on a well-developed mitochondrial reticulum. In this study we examined the cumulative muscle protein and DNA synthesis in groups of athletic dogs at the onset of an exercise training program and following a strenuous exercise training program. We hypothesized that both at the onset and during an exercise training program there would be greater mitochondrial protein synthesis rates compared with sedentary control with no difference in mixed or cytoplasmic protein synthesis rates. Protein synthetic rates of three protein fractions and DNA synthesis were determined over 1 wk using (2)H2O in competitive Alaskan Huskies and Labrador Retrievers trained for explosive device detection. Both groups of dogs had very high rates of skeletal muscle protein synthesis in the sedentary state [Alaskan Huskies: Mixed = 2.28 ± 0.12, cytoplasmic (Cyto) = 2.91 ± 0.10, and mitochondrial (Mito) = 2.62 ± 0.07; Labrador Retrievers: Mixed = 3.88 ± 0.37, Cyto = 3.85 ± 0.06, and Mito = 2.92 ± 0.20%/day]. Mitochondrial (Mito) protein synthesis rates did not increase at the onset of an exercise training program. Exercise-trained dogs maintained Mito protein synthesis during exercise training when mixed (Mixed) and cytosolic (Cyto) fractions decreased, and this coincided with a decrease in p-RpS6 but also a decrease in p-ACC signaling. Contrary to our hypothesis, canines did not have large increases in mitochondrial protein synthesis at the onset or during an exercise training program. However, dogs have a high rate of protein synthesis compared with humans that perhaps does not necessitate an extra increase in protein synthesis at the onset of aerobic exercise training. Copyright © 2015 the American Physiological Society.

Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells.

PubMed

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J; Méndez, Ernesto; Arias, Carlos F

2015-10-01

Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

PubMed Central

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

2015-01-01

ABSTRACT Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. PMID:26246569

Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. I. Nuclear-directed protein synthesis.

PubMed

Heude, M; Chanet, R; Moustacchi, E

1975-04-01

The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stationary phage haploid yeast cells was examined. This was carried out using cycloheximide (CH), a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis is required for the increase in survival seen after the liquid holding of cells at both stages, as well as for the "petite" recovery seen after the liquid holding of exponential phase cells. The characteristic negative liquid holding effect observed for the UV induction of "petites" in stationary phase cells (increase of the frequency of "petites" during storage) remained following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of a synthesis of proteins.

MECHANISMS IN ENDOCRINOLOGY: Exogenous insulin does not increase muscle protein synthesis rate when administered systemically: a systematic review.

PubMed

Trommelen, Jorn; Groen, Bart B L; Hamer, Henrike M; de Groot, Lisette C P G M; van Loon, Luc J C

2015-07-01

Though it is well appreciated that insulin plays an important role in the regulation of muscle protein metabolism, there is much discrepancy in the literature on the capacity of exogenous insulin administration to increase muscle protein synthesis rates in vivo in humans. To assess whether exogenous insulin administration increases muscle protein synthesis rates in young and older adults. A systematic review of clinical trials was performed and the presence or absence of an increase in muscle protein synthesis rate was reported for each individual study arm. In a stepwise manner, multiple models were constructed that excluded study arms based on the following conditions: model 1, concurrent hyperaminoacidemia; model 2, insulin-induced hypoaminoacidemia; model 3, supraphysiological insulin concentrations; and model 4, older, more insulin resistant, subjects. From the presented data in the current systematic review, we conclude that: i) exogenous insulin and amino acid administration effectively increase muscle protein synthesis, but this effect is attributed to the hyperaminoacidemia; ii) exogenous insulin administered systemically induces hypoaminoacidemia which obviates any insulin-stimulatory effect on muscle protein synthesis; iii) exogenous insulin resulting in supraphysiological insulin levels exceeding 50, 000  pmol/l may effectively augment muscle protein synthesis; iv) exogenous insulin may have a diminished effect on muscle protein synthesis in older adults due to age-related anabolic resistance; and v) exogenous insulin administered systemically does not increase muscle protein synthesis in healthy, young adults. © 2015 European Society of Endocrinology.

Chronic ethanol feeding modulates the synthesis of digestive enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ponnappa, B.C.; Hoek, J.B.; Rubin, E.

1987-05-01

The effects of chronic ethanol feeding on pancreatic protein synthesis were investigated. Protein synthesis was assessed by studying the rate of incorporation of /sup 3/H-leucine into TCA-precipitable proteins in isolated pancreatic acini from rats. Chronic ethanol ingestion increased the rate of pancreatic protein synthesis by 2-4 fold. The onset of the increase in protein synthesis was detectable two days after ethanol feeding, reached a maximum after 7 days and remained unchanged after 4 months on the ethanol-containing diet. The rate of synthesis of individual digestive enzymes was studied by SDS-PAGE on extracts obtained from purified zymogen granules. Ethanol feeding inducedmore » an increase in the rate of synthesis of most of the digestive enzymes; chymotrypsinogen, trypsinogen and an unidentified protein were increased to a greater extent than other digestive enzymes. By contrast, the synthesis of amylase was selectively decreased after ethanol feeding. These results suggest that chronic ethanol ingestion has specific effects on the rate of synthesis of individual digestive enzymes in the exocrine pancreas.« less

Chronological protein synthesis in regenerating rat liver.

PubMed

He, Jinjun; Hao, Shuai; Zhang, Hao; Guo, Fuzheng; Huang, Lingyun; Xiao, Xueyuan; He, Dacheng

2015-07-01

Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, (35) S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through (35) S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5-5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacterial cellulose synthesis mechanism of facultative anaerobe Enterobacter sp. FY-07.

PubMed

Ji, Kaihua; Wang, Wei; Zeng, Bing; Chen, Sibin; Zhao, Qianqian; Chen, Yueqing; Li, Guoqiang; Ma, Ting

2016-02-25

Enterobacter sp. FY-07 can produce bacterial cellulose (BC) under aerobic and anaerobic conditions. Three potential BC synthesis gene clusters (bcsI, bcsII and bcsIII) of Enterobacter sp. FY-07 have been predicted using genome sequencing and comparative genome analysis, in which bcsIII was confirmed as the main contributor to BC synthesis by gene knockout and functional reconstitution methods. Protein homology, gene arrangement and gene constitution analysis indicated that bcsIII had high identity to the bcsI operon of Enterobacter sp. 638; however, its arrangement and composition were same as those of BC synthesizing operon of G. xylinum ATCC53582 except for the flanking sequences. According to the BC biosynthesizing process, oxygen is not directly involved in the reactions of BC synthesis, however, energy is required to activate intermediate metabolites and synthesize the activator, c-di-GMP. Comparative transcriptome and metabolite quantitative analysis demonstrated that under anaerobic conditions genes involved in the TCA cycle were downregulated, however, genes in the nitrate reduction and gluconeogenesis pathways were upregulated, especially, genes in three pyruvate metabolism pathways. These results suggested that Enterobacter sp. FY-07 could produce energy efficiently under anaerobic conditions to meet the requirement of BC biosynthesis.

Bacterial cellulose synthesis mechanism of facultative anaerobe Enterobacter sp. FY-07

PubMed Central

Ji, Kaihua; Wang, Wei; Zeng, Bing; Chen, Sibin; Zhao, Qianqian; Chen, Yueqing; Li, Guoqiang; Ma, Ting

2016-01-01

Enterobacter sp. FY-07 can produce bacterial cellulose (BC) under aerobic and anaerobic conditions. Three potential BC synthesis gene clusters (bcsI, bcsII and bcsIII) of Enterobacter sp. FY-07 have been predicted using genome sequencing and comparative genome analysis, in which bcsIII was confirmed as the main contributor to BC synthesis by gene knockout and functional reconstitution methods. Protein homology, gene arrangement and gene constitution analysis indicated that bcsIII had high identity to the bcsI operon of Enterobacter sp. 638; however, its arrangement and composition were same as those of BC synthesizing operon of G. xylinum ATCC53582 except for the flanking sequences. According to the BC biosynthesizing process, oxygen is not directly involved in the reactions of BC synthesis, however, energy is required to activate intermediate metabolites and synthesize the activator, c-di-GMP. Comparative transcriptome and metabolite quantitative analysis demonstrated that under anaerobic conditions genes involved in the TCA cycle were downregulated, however, genes in the nitrate reduction and gluconeogenesis pathways were upregulated, especially, genes in three pyruvate metabolism pathways. These results suggested that Enterobacter sp. FY-07 could produce energy efficiently under anaerobic conditions to meet the requirement of BC biosynthesis. PMID:26911736

Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice.

PubMed

Marini, Juan C; Didelija, Inka Cajo

2015-01-01

Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L), and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.

RINT-1 interacts with MSP58 within nucleoli and plays a role in ribosomal gene transcription.

PubMed

Yang, Chuan-Pin; Kuo, Yu-Liang; Lee, Yi-Chao; Lee, Kuen-Haur; Chiang, Chi-Wu; Wang, Ju-Ming; Hsu, Che-Chia; Chang, Wen-Chang; Lin, Ding-Yen

2016-09-16

The nucleolus is the cellular site of ribosomal (r)DNA transcription and ribosome biogenesis. The 58-kDa microspherule protein (MSP58) is a nucleolar protein involved in rDNA transcription and cell proliferation. However, regulation of MSP58-mediated rDNA transcription remains unknown. Using a yeast two-hybrid system with MSP58 as bait, we isolated complementary (c)DNA encoding Rad50-interacting protein 1 (RINT-1), as a MSP58-binding protein. RINT-1 was implicated in the cell cycle checkpoint, membrane trafficking, Golgi apparatus and centrosome dynamic integrity, and telomere length control. Both in vitro and in vivo interaction assays showed that MSP58 directly interacts with RINT-1. Interestingly, microscopic studies revealed the co-localization of MSP58, RINT-1, and the upstream binding factor (UBF), a rRNA transcription factor, in the nucleolus. We showed that ectopic expression of MSP58 or RINT-1 resulted in decreased rRNA expression and rDNA promoter activity, whereas knockdown of MSP58 or RINT-1 by siRNA exerted the opposite effect. Coexpression of MSP58 and RINT-1 robustly decreased rRNA synthesis compared to overexpression of either protein alone, whereas depletion of RINT-1 from MSP58-transfected cells enhanced rRNA synthesis. We also found that MSP58, RINT-1, and the UBF were associated with the rDNA promoter using a chromatin immunoprecipitation assay. Because aberrant ribosome biogenesis contributes to neoplastic transformation, our results revealed a novel protein complex involved in the regulation of rRNA gene expression, suggesting a role for MSP58 and RINT-1 in cancer development. Copyright © 2016 Elsevier Inc. All rights reserved.

Growth, oxygen consumption, and protein and RNA synthesis rates in the yolk sac larvae of the African catfish (Clarias gariepinus).

PubMed

Smith, Richard W; Ottema, Colin

2006-03-01

Rapidly growing African catfish yolk sac larvae were investigated during the first 22 h after hatching. Body compartment protein concentration increased fourfold yet oxygen consumption remained constant (mean=21.3 +/- 3.2 nmol O2 mg(-1) protein h(-1)), suggesting fast growth results mainly from yolk sac protein absorption. The protein synthesis rates at 1-2 and 5-6 h also equaled the highest conceivable rates of muscle protein synthesis; 11.6-11.9% and 7.4-7.9% day(-1), respectively. Therefore the corresponding energetic costs of protein synthesis were almost the theoretical minimum; 13.0 +/- 1.7-16.3 +/- 2.8 micromol O2 mg(-1) protein synthesised. Total protein synthesis expenditure (74.5-77.7 micromol O2 g(-1) protein h(-1)) was also less than other yolk sac larvae. These protein synthesis rates resulted from high RNA concentrations (113.2 +/- 3.4 microg RNA mg(-1) protein) and were also correlated with RNA translational efficiency. High translational efficiency (1 h; 1.2+/-0.1 mg protein synthesised microg(-1) RNA day(-1)) equaled high synthesis rate (36.8 +/- 5.4 microg RNA microg(-1) DNA day(-1)) and both declined over 22 h. This investigation suggests rapid growth combines growth efficiency and compensatory energy partitioning. This accommodates the ontogenetic and phylogenetic standpoints imposed by energy budget limitations.

Insight into the SEA amide thioester equilibrium. Application to the synthesis of thioesters at neutral pH.

PubMed

Pira, S L; El Mahdi, O; Raibaut, L; Drobecq, H; Dheur, J; Boll, E; Melnyk, O

2016-07-26

The bis(2-sulfanylethyl)amide (SEA) N,S-acyl shift thioester surrogate has found a variety of useful applications in the field of protein total synthesis. Here we present novel insights into the SEA amide/thioester equilibrium in water which is an essential step in any reaction involving the thioester surrogate properties of the SEA group. We also show that the SEA amide thioester equilibrium can be efficiently displaced at neutral pH for accessing peptide alkylthioesters, i.e. the key components of the native chemical ligation (NCL) reaction.

Solid-phase synthesis and structure-activity relationships of novel biarylethers as melanin-concentrating hormone receptor-1 antagonists.

PubMed

Ma, Vu; Bannon, Anthony W; Baumgartner, Jamie; Hale, Clarence; Hsieh, Faye; Hulme, Christopher; Rorrer, Kirk; Salon, John; van Staden, Carlo; Tempest, Paul

2006-10-01

Melanin-concentrating hormone (MCH) is a cyclic 19 amino acid orexigenic neuropeptide. The action of MCH on feeding is thought to involve the activation of its respective G protein-coupled receptor MCH-R1. Consequently, antagonists that block MCH regulated MCH-R1 activity may provide a viable approach to the treatment of diet-induced obesity. This communication reports the discovery of a novel MCH-R1 receptor antagonist, the biarylether 7, identified through high throughput screening. The solid-phase synthesis and structure-activity relationship of related analogs is described.

Abscisic Acid Transport and Homeostasis in the Context of Stomatal Regulation.

PubMed

Merilo, Ebe; Jalakas, Pirko; Laanemets, Kristiina; Mohammadi, Omid; Hõrak, Hanna; Kollist, Hannes; Brosché, Mikael

2015-09-01

The discovery of cytosolic ABA receptors is an important breakthrough in stomatal research; signaling via these receptors is involved in determining the basal stomatal conductance and stomatal responsiveness. However, the source of ABA in guard cells is still not fully understood. The level of ABA increases in guard cells by de novo synthesis, recycling from inactive conjugates via β-glucosidases BG1 and BG2 and by import, whereas it decreases by hydroxylation, conjugation, and export. ABA importers include the NRT1/PTR family protein AIT1, ATP-binding cassette protein ABCG40, and possibly ABCG22, whereas the DTX family member DTX50 and ABCG25 function as ABA exporters. Here, we review the proteins involved in ABA transport and homeostasis and their physiological role in stomatal regulation. Recent experiments suggest that functional redundancy probably exists among ABA transporters between vasculature and guard cells and ABA recycling proteins, as stomatal functioning remained intact in abcg22, abcg25, abcg40, ait1, and bg1bg2 mutants. Only the initial response to reduced air humidity was significantly delayed in abcg22. Considering the reports showing autonomous ABA synthesis in guard cells, we discuss that rapid stomatal responses to atmospheric factors might depend primarily on guard cell-synthesized ABA, whereas in the case of long-term soil water deficit, ABA synthesized in the vasculature might have a significant role. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells.

PubMed

Chen, Lisa S; Redkar, Sanjeev; Bearss, David; Wierda, William G; Gandhi, Varsha

2009-11-05

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-X(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction.

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells

PubMed Central

Chen, Lisa S.; Redkar, Sanjeev; Bearss, David; Wierda, William G.

2009-01-01

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-XL, XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction. PMID:19734450

Role of host protein Ebp1 in influenza virus growth: intracellular localization of Ebp1 in virus-infected and uninfected cells.

PubMed

Honda, Ayae

2008-01-20

The cellular protein Ebp1 was identified to interact with PB1 protein of influenza virus RNA polymerase, and inhibit both RNA synthesis in vitro and influenza virus replication in vivo [Honda, A., Okamoto, T., Ishihama, A., 2007. Host factor Ebp1: selective inhibitor of influenza virus transcriptase. Genes Cells 12, 133-142]. The intracellular localization of Ebp1 that is involved in cell proliferation control was analyzed by direct immunostaining of cells before and after influenza virus infection. Ebp1 was found to localize in the nuclear membrane of uninfected cells, and to form nuclear aggregates with viral P proteins in virus-infected cells.

Echinococcus granulosus fatty acid binding proteins subcellular localization.

PubMed

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

PubMed

Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

2015-08-01

Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system.

PubMed

Kazuta, Yasuaki; Matsuura, Tomoaki; Ichihashi, Norikazu; Yomo, Tetsuya

2014-11-01

In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and β-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells.

PubMed

Stech, Marlitt; Quast, Robert B; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A; Kubick, Stefan

2014-01-01

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

PubMed Central

Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

2014-01-01

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Hwang, Jung-Shan; Yamada, Kazunori; Honda, Ayae; Nakade, Kohji; Ishihama, Akira

2000-01-01

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni2+-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris. PMID:10756019

Partial Support Ventilation and Mitochondrial-Targeted Antioxidants Protect against Ventilator-Induced Decreases in Diaphragm Muscle Protein Synthesis.

PubMed

Hudson, Matthew B; Smuder, Ashley J; Nelson, W Bradley; Wiggs, Michael P; Shimkus, Kevin L; Fluckey, James D; Szeto, Hazel H; Powers, Scott K

2015-01-01

Mechanical ventilation (MV) is a life-saving intervention in patients in respiratory failure. Unfortunately, prolonged MV results in the rapid development of diaphragm atrophy and weakness. MV-induced diaphragmatic weakness is significant because inspiratory muscle dysfunction is a risk factor for problematic weaning from MV. Therefore, developing a clinical intervention to prevent MV-induced diaphragm atrophy is important. In this regard, MV-induced diaphragmatic atrophy occurs due to both increased proteolysis and decreased protein synthesis. While efforts to impede MV-induced increased proteolysis in the diaphragm are well-documented, only one study has investigated methods of preserving diaphragmatic protein synthesis during prolonged MV. Therefore, we evaluated the efficacy of two therapeutic interventions that, conceptually, have the potential to sustain protein synthesis in the rat diaphragm during prolonged MV. Specifically, these experiments were designed to: 1) determine if partial-support MV will protect against the decrease in diaphragmatic protein synthesis that occurs during prolonged full-support MV; and 2) establish if treatment with a mitochondrial-targeted antioxidant will maintain diaphragm protein synthesis during full-support MV. Compared to spontaneously breathing animals, full support MV resulted in a significant decline in diaphragmatic protein synthesis during 12 hours of MV. In contrast, diaphragm protein synthesis rates were maintained during partial support MV at levels comparable to spontaneous breathing animals. Further, treatment of animals with a mitochondrial-targeted antioxidant prevented oxidative stress during full support MV and maintained diaphragm protein synthesis at the level of spontaneous breathing animals. We conclude that treatment with mitochondrial-targeted antioxidants or the use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during prolonged MV.

Association of CAD, a multifunctional protein involved in pyrimidine synthesis, with mLST8, a component of the mTOR complexes

PubMed Central

2013-01-01

Background mTOR is a genetically conserved serine/threonine protein kinase, which controls cell growth, proliferation, and survival. A multifunctional protein CAD, catalyzing the initial three steps in de novo pyrimidine synthesis, is regulated by the phosphorylation reaction with different protein kinases, but the relationship with mTOR protein kinase has not been known. Results CAD was recovered as a binding protein with mLST8, a component of the mTOR complexes, from HEK293 cells transfected with the FLAG-mLST8 vector. Association of these two proteins was confirmed by the co-immuoprecipitaiton followed by immunoblot analysis of transfected myc-CAD and FLAG-mLST8 as well as that of the endogenous proteins in the cells. Analysis using mutant constructs suggested that CAD has more than one region for the binding with mLST8, and that mLST8 recognizes CAD and mTOR in distinct ways. The CAD enzymatic activity decreased in the cells depleted of amino acids and serum, in which the mTOR activity is suppressed. Conclusion The results obtained indicate that mLST8 bridges between CAD and mTOR, and plays a role in the signaling mechanism where CAD is regulated in the mTOR pathway through the association with mLST8. PMID:23594158

Long-term social recognition memory is mediated by oxytocin-dependent synaptic plasticity in the medial amygdala.

PubMed

Gur, Rotem; Tendler, Alex; Wagner, Shlomo

2014-09-01

Recognition of specific individuals is fundamental to mammalian social behavior and is mediated in most mammals by the main and accessory olfactory systems. Both these systems innervate the medial amygdala (MeA), where activity of the neuropeptide oxytocin is thought to mediate social recognition memory (SRM). The specific contribution of the MeA to SRM formation and the specific actions of oxytocin in the MeA are unknown. We used the social discrimination test to evaluate short-term and long-term SRM in adult Sprague-Dawley male rats (n = 38). The role of protein synthesis in the MeA was investigated by local application of the protein synthesis blocker anisomycin (n = 11). Synaptic plasticity was assessed in vivo by recording the MeA evoked field potential responses to stimulation of the main (n = 21) and accessory (n = 56) olfactory bulbs before and after theta burst stimulation. Intracerebroventricular administration of saline, oxytocin, or oxytocin receptor antagonist was used to measure the effect of oxytocin on synaptic plasticity. Anisomycin application to the MeA prevented the formation of long-term SRM. In addition, the responses of MeA neurons underwent long-term depression (LTD) after theta burst stimulation of the accessory olfactory bulb, but not the main accessory bulb, in an oxytocin-dependent manner. No LTD was found in socially isolated rats, which are known to lack long-term SRM. Finally, accessory olfactory bulb stimulation before SRM acquisition blocked long-term SRM, supporting the involvement of LTD in the MeA in formation of long-term SRM. Our results indicate that long-term SRM in rats involves protein synthesis and oxytocin-dependent LTD in the MeA. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Convergent synthesis of proteins by kinetically controlled ligation

DOEpatents

Kent, Stephen; Pentelute, Brad; Bang, Duhee; Johnson, Erik; Durek, Thomas

2010-03-09

The present invention concerns methods and compositions for synthesizing a polypeptide using kinetically controlled reactions involving fragments of the polypeptide for a fully convergent process. In more specific embodiments, a ligation involves reacting a first peptide having a protected cysteyl group at its N-terminal and a phenylthioester at its C-terminal with a second peptide having a cysteine residue at its N-termini and a thioester at its C-termini to form a ligation product. Subsequent reactions may involve deprotecting the cysteyl group of the resulting ligation product and/or converting the thioester into a thiophenylester.

Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration.

PubMed

Chaki, Mounira; Álvarez de Morales, Paz; Ruiz, Carmelo; Begara-Morales, Juan C; Barroso, Juan B; Corpas, Francisco J; Palma, José M

2015-09-01

Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper. The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis. Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit. The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H2O2, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration

PubMed Central

Chaki, Mounira; Álvarez de Morales, Paz; Ruiz, Carmelo; Begara-Morales, Juan C.; Barroso, Juan B.; Corpas, Francisco J.; Palma, José M.

2015-01-01

Background and Aims Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper. Methods The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis. Key Results Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit. Conclusions The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H2O2, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit. PMID:25814060

Proteomic Analysis of the Endosperm Ontogeny of Jatropha curcas L. Seeds.

PubMed

Shah, Mohibullah; Soares, Emanoella L; Carvalho, Paulo C; Soares, Arlete A; Domont, Gilberto B; Nogueira, Fábio C S; Campos, Francisco A P

2015-06-05

Seeds of Jatropha curcas L. represent a potential source of raw material for the production of biodiesel. However, this use is hampered by the lack of basic information on the biosynthetic pathways associated with synthesis of toxic diterpenes, fatty acids, and triacylglycerols, as well as the pattern of deposition of storage proteins during seed development. In this study, we performed an in-depth proteome analysis of the endosperm isolated from five developmental stages which resulted in the identification of 1517, 1256, 1033, 752, and 307 proteins, respectively, summing up 1760 different proteins. Proteins with similar label free quantitation expression pattern were grouped into five clusters. The biological significance of these identifications is discussed with special focus on the analysis of seed storage proteins, proteins involved in the metabolism of fatty acids, carbohydrates, toxic components and proteolytic processing. Although several enzymes belonging to the biosynthesis of diterpenoid precursors were identified, we were unable to find any terpene synthase/cyclase, indicating that the synthesis of phorbol esters, the main toxic diterpenes, does not occur in seeds. The strategy used enabled us to provide a first in depth proteome analysis of the developing endosperm of this biodiesel plant, providing an important glimpse into the enzymatic machinery devoted to the production of C and N sources to sustain seed development.

Role for malonyl coenzyme A:acyl carrier protein transacylase (MCAT) in the growth-inhibitory effect of the calmodulin antagonist trifluoperazine in Mycobacterium bovis BCG.

PubMed

Sinha, Indrajit; Dick, Thomas

2004-06-01

To determine whether the fatty acid synthesis enzyme malonyl coenzyme A:acyl carrier protein transacylase (MCAT) is involved in the growth-inhibitory effect of trifluoperazine in the tubercle bacillus Mycobacterium bovis BCG. BCG was grown in liquid culture with various concentrations of trifluoperazine and growth was monitored by OD measurement. To determine the effect of trifluoperazine on MCAT protein level, total protein was extracted from BCG cultures and was analysed by 2D gel electrophoresis and western blot. To confirm trifluoperazine-dependent reduction in the MCAT protein level, two BCG strains overexpressing MCAT at a low and high constitutive level were similarly tested. The synergic effect of trifluoperazine and isoniazid was tested at sub-MIC levels in liquid cultures. Trifluoperazine inhibition of growth correlates with reduction in the steady-state level of MCAT protein. Overexpression of MCAT confers resistance to trifluoperazine. Trifluoperazine acts synergically (albeit weakly) with isoniazid and no resistance towards isoniazid alone was observed due to overexpression of MCAT. This suggests MCAT to be a specific target of trifluoperazine. These results indicate MCAT as a target of trifluoperazine and provide an explanation for the inhibitory effect of trifluoperazine on mycobacterial lipid synthesis observed earlier. This makes MCAT a potential target for new antimycobacterials.

The plasma membrane proteome of maize roots grown under low and high iron conditions.

PubMed

Hopff, David; Wienkoop, Stefanie; Lüthje, Sabine

2013-10-08

Iron (Fe) homeostasis is essential for life and has been intensively investigated for dicots, while our knowledge for species in the Poaceae is fragmentary. This study presents the first proteome analysis (LC-MS/MS) of plasma membranes isolated from roots of 18-day old maize (Zea mays L.). Plants were grown under low and high Fe conditions in hydroponic culture. In total, 227 proteins were identified in control plants, whereas 204 proteins were identified in Fe deficient plants and 251 proteins in plants grown under high Fe conditions. Proteins were sorted by functional classes, and most of the identified proteins were classified as signaling proteins. A significant number of PM-bound redox proteins could be identified including quinone reductases, heme and copper-containing proteins. Most of these components were constitutive, and others could hint at an involvement of redox signaling and redox homeostasis by change in abundance. Energy metabolism and translation seem to be crucial in Fe homeostasis. The response to Fe deficiency includes proteins involved in development, whereas membrane remodeling and assembly and/or repair of Fe-S clusters is discussed for Fe toxicity. The general stress response appears to involve proteins related to oxidative stress, growth regulation, an increased rigidity and synthesis of cell walls and adaption of nutrient uptake and/or translocation. This article is part of a Special Issue entitled: Plant Proteomics in Europe. Copyright © 2013 Elsevier B.V. All rights reserved.

GosB Inhibits Triacylglycerol Synthesis and Promotes Cell Survival in Mouse Mammary Epithelial Cells.

PubMed

Xu, Gaoxiao; Duan, Saixing; Hou, Jianye; Wei, Zhongxin; Zhao, Guangwei

2017-01-01

It has been demonstrated that the activator protein related transcription factor Finkel-Biskis-Jinkins murine osteosarcoma B (GosB) is involved in preadipocyte differentiation and triacylglycerol synthesis. However, the role of GosB in regulating the synthesis of milk fatty acid in mouse mammary glands remains unclear. This research uncovered potentially new roles of GosB in suppressing milk fatty acid synthesis. Results revealed that GosB had the highest expression in lung tissue and showed a higher expression level during nonlactation than during lactation. GosB inhibited the expression of fatty acid synthase (FASN) , stearoyl-CoA desaturase (SCD) , fatty acid binding protein 4 (FABP4) , diacylglycerol acyltransferase 1 (DGAT1) , perilipin 2 (PLIN2) , perilipin 3 (PLIN3) , and C/EBPα in mouse mammary gland epithelial cells (MEC). In addition, GosB reduced cellular triglyceride content and the accumulation of lipid droplets; in particular, GosB enhanced saturated fatty acid concentration (C16:0 and C18:0). The PPAR γ agonist, rosiglitazone (ROSI), promoted apoptosis and inhibited cell proliferation. GosB increased the expression of Bcl-2 and protected MEC from ROSI-induced apoptosis. Furthermore, MECs were protected from apoptosis through the GosB regulation of intracellular calcium concentrations. These findings suggest that GosB may regulate mammary epithelial cells milk fat synthesis and apoptosis via PPAR γ in mouse mammary glands.

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

PubMed

Sachse, Rita; Dondapati, Srujan K; Fenz, Susanne F; Schmidt, Thomas; Kubick, Stefan

2014-08-25

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Chemical Synthesis of Proteins

PubMed Central

Nilsson, Bradley L.; Soellner, Matthew B.; Raines, Ronald T.

2010-01-01

Proteins have become accessible targets for chemical synthesis. The basic strategy is to use native chemical ligation, Staudinger ligation, or other orthogonal chemical reactions to couple synthetic peptides. The ligation reactions are compatible with a variety of solvents and proceed in solution or on a solid support. Chemical synthesis enables a level of control on protein composition that greatly exceeds that attainable with ribosome-mediated biosynthesis. Accordingly, the chemical synthesis of proteins is providing previously unattainable insight into the structure and function of proteins. PMID:15869385

Quantitative proteomic analysis of post-flooding recovery in soybean root exposed to aluminum oxide nanoparticles.

PubMed

Yasmeen, Farhat; Raja, Naveed Iqbal; Mustafa, Ghazala; Sakata, Katsumi; Komatsu, Setsuko

2016-06-30

Aluminum oxide nanoparticles (Al2O3 NPs) are used in various commercial and agricultural products. Soybean exhibits severe reduction in growth under flooding condition. To examine the effects of Al2O3 NPs on the recovery of soybean from flooding, proteomic analysis was performed. Survival percentage and weight/length of root including hypocotyl were improved after 2 and 4days of flooding with 50ppm Al2O3 NPs leading to recovery as compared to flooding. A total of 211 common proteins were changed in abundance during the recovery period after treatment without or with Al2O3 NPs. These proteins were related to protein synthesis, stress, cell wall, and signaling. Among the identified stress-related proteins, S-adenosyl-l-methionine dependent methyltransferases were recovered from flooding with Al2O3 NPs. Hierarchical clustering divided the identified proteins into three clusters. Cluster II exhibited the greatest change in proteins related to protein synthesis, transport, and development during the recovery from flooding with Al2O3 NPs. However, activity of enolase remained unchanged during flooding leading to subsequent recovery with Al2O3 NPs. These results suggest that S-adenosyl-l-methionine dependent methyltransferases and enolase might be involved in mediating recovery responses by Al2O3 NPs. This study highlighted the role of Al2O3 NPs in recovery of soybean seedlings from flooding stress using gel-free proteomic technique. The key findings of this study are as follows: (i) survival percentage was enhanced at 50ppm Al2O3 NPs during the recovery stage; (ii) seedling weight and weight/length of root including hypocotyl improved at 50ppm Al2O3 NPs during the period of recovery; (iii) protein synthesis and stress related proteins were increased on recovery after flooding without or with Al2O3 NPs; (iv) the abundance of S-adenosyl-l-methionine dependent methyltransferases recovered from flooding with Al2O3 NPs; (v) glycolysis related proteins amplified under flooding with Al2O3 NPs; (vi) enolase enzyme remained unchanged during flooding leading to subsequent recovery from flooding with Al2O3 NPs. Collectively, these results suggest that S-adenosyl-l-methionine dependent methyltransferases and enolase are involved in response to flooding with Al2O3 NPs and might be helpful in recovery from flooding stress. Copyright © 2016 Elsevier B.V. All rights reserved.

RNA turnover and protein synthesis in fish cells.

PubMed

Smith, R W; Palmer, R M; Houlihan, D F

2000-03-01

Protein synthesis in fish has been previously correlated with RNA content. The present study investigates whether protein and RNA synthesis rates are similarly related. Protein and RNA synthesis rates were determined from 3H-phenylalanine and 3H-uridine incorporation, respectively, and expressed as % x day(-1) and half-lives, respectively. Three fibroblast cell lines were used: BF-2, RTP, CHSE 214, which are derived from the bluegill, rainbow trout and Chinook salmon, respectively. These cells contained similar RNA concentrations (approximately 175 microg RNA x mg(-1) cell protein). Therefore differences in protein synthesis rates, BF-2 (31.3 +/- 1.8)>RTP (25.1 +/- 1.7)>CHSE 214 (17.6 +/-1.1), were attributable to RNA translational efficiency. The most translationally efficient RNA (BF-2 cells), 1.8 mg protein synthesised x microg(-1) RNA x day(-1), corresponded to the lowest RNA half-life, 75.4 +/- 6.4 h. Translationally efficient RNA was also energetically efficient with BF-2 cells exploiting the least costly route of nucleotide supply (i.e. exogenous salvage) 3.5-6.0 times more than the least translationally efficient RNA (CHSE 214 cells). These data suggest that differential nucleotide supply, between intracellular synthesis and exogenous salvage, constitutes the area of pre-translational flexibility exploited to maintain RNA synthesis as a fixed energetic cost component of protein synthesis.

The limits to biocatalysis: pushing the envelope.

PubMed

Sheldon, Roger A; Brady, Dean

2018-06-12

In the period 1985 to 1995 applications of biocatalysis, driven by the need for more sustainable manufacture of chemicals and catalytic, (enantio)selective methods for the synthesis of pharmaceutical intermediates, largely involved the available hydrolases. This was followed, in the next two decades, by revolutionary developments in protein engineering and directed evolution for the optimisation of enzyme function and performance that totally changed the biocatalysis landscape. In the same period, metabolic engineering and synthetic biology revolutionised the use of whole cell biocatalysis in the synthesis of commodity chemicals by fermentation. In particular, developments in the enzymatic enantioselective synthesis of chiral alcohols and amines are highlighted. Progress in enzyme immobilisation facilitated applications under harsh industrial conditions, such as in organic solvents. The emergence of biocatalytic or chemoenzymatic cascade processes, often with co-immobilised enzymes, has enabled telescoping of multi-step processes. Discovering and inventing new biocatalytic processes, based on (meta)genomic sequencing, evolving enzyme promiscuity, chemomimetic biocatalysis, artificial metalloenzymes, and the introduction of non-canonical amino acids into proteins, are pushing back the limits of biocatalysis function. Finally, the integral role of biocatalysis in developing a biobased carbon-neutral economy is discussed.

"Dark matter" worlds of unstable RNA and protein.

PubMed

Baboo, Sabyasachi; Cook, Peter R

2014-01-01

Astrophysicists use the term "dark matter" to describe the majority of the matter and/or energy in the universe that is hidden from view, and biologists now apply it to the new families of RNA they are uncovering. We review evidence for an analogous hidden world containing peptides. The critical experiments involved pulse-labeling human cells with tagged amino acids for periods as short as five seconds. Results are extraordinary in two respects: both nucleus and cytoplasm become labeled, and most signals disappear with a half-life of less than one minute. Just as the synthesis of each mature mRNA is regulated by the abortive production of hundreds of shorter transcripts that are quickly degraded, it seems that the synthesis of each full-length protein in the stable proteome is regulated by an apparently wasteful production and degradation of shorter peptides. Some of the nuclear synthesis is probably a byproduct of nuclear ribosomes proofreading newly-made RNA for inappropriately-placed termination codons (a process that triggers "nonsense-mediated decay"). We speculate that some "dark-matter" peptides will play other important roles in the cell.

“Dark matter” worlds of unstable RNA and protein

PubMed Central

Baboo, Sabyasachi; Cook, Peter R

2014-01-01

Astrophysicists use the term “dark matter” to describe the majority of the matter and/or energy in the universe that is hidden from view, and biologists now apply it to the new families of RNA they are uncovering. We review evidence for an analogous hidden world containing peptides. The critical experiments involved pulse-labeling human cells with tagged amino acids for periods as short as five seconds. Results are extraordinary in two respects: both nucleus and cytoplasm become labeled, and most signals disappear with a half-life of less than one minute. Just as the synthesis of each mature mRNA is regulated by the abortive production of hundreds of shorter transcripts that are quickly degraded, it seems that the synthesis of each full-length protein in the stable proteome is regulated by an apparently wasteful production and degradation of shorter peptides. Some of the nuclear synthesis is probably a byproduct of nuclear ribosomes proofreading newly-made RNA for inappropriately-placed termination codons (a process that triggers “nonsense-mediated decay”). We speculate that some “dark-matter” peptides will play other important roles in the cell. PMID:25482115

Mechanical stimulation of skeletal muscle increases prostaglandin F2(alpha) synthesis and cyclooxygenase activity by a pertussis toxin sensitive mechanism

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman H.; Shansky, Janet; Solerssi, Rosa; Chromiak, Joseph

1992-01-01

Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increases the production of prostaglandin F(sub 2(alpha)), an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical activity, the activity of cyclooxygenase, a regulatory enzyme in prostaglandin synthesis, was increased 82% (P is less than .005), and this increase was maintained for at least 24 h. Kinetic analysis of the stretch-activated cyclooxygenase indicated a two to three-fold decrease in the enzyme's K(sub m) with no change in V(sub max). The stretch-induced increase in enzymatic activity was not inhibited by cycloheximide, was independent of cellular electrical activity (tetrodotoxin-insensitive), but was prevented by the G protein inhibitor pertussis toxin. Pertussis toxin also inhibited the stretch-induced increases in PGF(sub 2(alpha)) production, and cell growth. It is concluded that stretch of skeletal muscle increases the synthesis of the anabolic modulator PGF(sub 2(alpha)) by a G protein-dependent process which involves activation of cyclooxygenase by a posttranslational mechanism.

[beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose.

PubMed Central

Li, L.; Drake, R. R.; Clement, S.; Brown, R. M.

1993-01-01

Using differential product entrapment and photolabeling under specifying conditions, we identifIed a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Gossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring [beta]-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of [beta]-1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring [beta]-1,3-glucan synthesis, is coincident with [beta]-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of [beta]-1,3-glucan is discussed. PMID:12231766

Synaptic Plasticity and Translation Initiation

ERIC Educational Resources Information Center

Klann, Eric; Antion, Marcia D.; Banko, Jessica L.; Hou, Lingfei

2004-01-01

It is widely accepted that protein synthesis, including local protein synthesis at synapses, is required for several forms of synaptic plasticity. Local protein synthesis enables synapses to control synaptic strength independent of the cell body via rapid protein production from pre-existing mRNA. Therefore, regulation of translation initiation is…

Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

PubMed

Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

2010-01-01

Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation

PubMed Central

Epperson, L. Elaine; Rose, James C.; Carey, Hannah V.

2010-01-01

Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast. PMID:19923364

Chloramphenicol decreases CB1 receptor expression in the nucleus accumbens and prefrontal cortex and prevents amphetamine-induced conditioned place preference in rats.

PubMed

Amancio-Belmont, Octavio; Pérez-Vázquez, Diego; Ruiz-Contreras, Alejandra E; Pérez de la Mora, Miguel; Rueda-Orozco, Pavel E; Méndez-Díaz, Mónica; Prospero-Garcia, Oscar E

2017-08-01

Drug dependence seems to involve a learning and memory process. Since learning and memory depend on protein synthesis, drug dependence may depend on protein synthesis, too. Drug-induced reward is a crucial effect for the development of drug-dependence. We used chloramphenicol (CAP, a protein synthesis inhibitor), to evaluate its effects on amphetamine (amph)-seeking behavior, on CB1R expression and on protein synthesis in general, in specific areas of the brain. Two groups of Wistar adult male rats were subjected to amph-induced conditioned place preference (CPP). Rats in group 1 received amph and were kept in the chamber for 30min. Once this period elapsed, they received a subcutaneous injection of saline (veh) and were returned to their home-cage. Rats in group 2 were also treated with amph but received CAP (150mg/kgsc) instead of saline. Once CPP was evaluated rats were sacrificed and the prefrontal cortex (PFC), the nucleus accumbens (NAcc) and the hippocampus (Hipp) were isolated and prepared for CB1R Western blot analysis. A vivarium reared group of rats was added as a non-experimentally manipulated control group. Results indicate that group 1 developed CPP while increasing CB1R expression in the NAcc. Group 2 did not develop CPP, had lower CB1R expression in the PFC and lacked the CB1R increase in the NAcc observed in the amph+veh group. These results support the notion that among the underlying mechanisms for amph-seeking reward is an increase in CB1R, further supporting an interaction between dopamine/endocannabinoids in CPP learning. Copyright © 2017. Published by Elsevier Inc.

15d-PGJ2 upregulates synthesis of IL-8 in endothelial cells through induction of oxidative stress.

PubMed

Jozkowicz, Alicja; Was, Halina; Taha, Hevidar; Kotlinowski, Jerzy; Mleczko, Katarzyna; Cisowski, Jaroslaw; Weigel, Guenter; Dulak, Jozef

2008-12-01

15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) is a cyclopentenone prostaglandin regarded as antiinflammatory mediator, which can act through peroxisome proliferator-activated receptor-gamma (PPARgamma) or through G protein-coupled surface receptors. It has been demonstrated that 15d-PGJ(2) potently increases the generation of interleukin-8 (IL-8) in human microvascular endothelial cells (HMEC-1s); however, the mechanism of this induction is not known. The aim of the study was to find the pathway involved in 15d-PGJ(2)-mediated IL-8 stimulation. Our data confirmed that the effect of 15d-PGJ(2) is independent of PPARgamma. For the first time, we excluded the activation of G proteins and the contribution of G protein-coupled surface receptors in endothelial cells treated with 15d-PGJ(2). Instead, we demonstrated that stimulation of IL-8 involved induction of oxidative stress, activation of p38 kinases, and increase in stability of IL-8 mRNA. Upregulation of IL-8 promoter, although measurable, seemed to play a less-pronounced role. Additionally, our results indicate the involvement of cAMP elevation and may suggest a role for ATF2 transcription factor. Concomitant induction of heme oxygenase-1 in HMEC-1s did not influence the synthesis of IL-8. In summary, we showed that 15d-PGJ(2), acting through oxidative stress, may exert proinflammatory effects. The upregulation of IL-8 is mostly associated with p38-mediated stabilization of mRNA.

How does yeast respond to pressure?

PubMed

Fernandes, P M B

2005-08-01

The brewing and baking yeast Saccharomyces cerevisiae has been used as a model for stress response studies of eukaryotic cells. In this review we focus on the effect of high hydrostatic pressure (HHP) on S. cerevisiae. HHP exerts a broad effect on yeast cells characteristic of common stresses, mainly associated with protein alteration and lipid bilayer phase transition. Like most stresses, pressure induces cell cycle arrest. Below 50 MPa (500 atm) yeast cell morphology is unaffected whereas above 220 MPa wild-type cells are killed. S. cerevisiae cells can acquire barotolerance if they are pretreated with a sublethal stress due to temperature, ethanol, hydrogen peroxide, or pressure. Nevertheless, pressure only leads to protection against severe stress if, after pressure pretreatment, the cells are also re-incubated at room pressure. We attribute this effect to the inhibition of the protein synthesis apparatus under HHP. The global genome expression analysis of S. cerevisiae cells submitted to HHP revealed a stress response profile. The majority of the up-regulated genes are involved in stress defense and carbohydrate metabolism while most repressed genes belong to the cell cycle progression and protein synthesis categories. However, the signaling pathway involved in the pressure response is still to be elucidated. Nitric oxide, a signaling molecule involved in the regulation of a large number of cellular functions, confers baroprotection. Furthermore, S. cerevisiae cells in the early exponential phase submitted to 50-MPa pressure show induction of the expression level of the nitric oxide synthase inducible isoform. As pressure becomes an important biotechnological tool, studies concerning this kind of stress in microorganisms are imperative.

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

PubMed

Semino, C E; Specht, C A; Raimondi, A; Robbins, P W

1996-05-14

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

The effect of NGATHA altered activity on auxin signaling pathways within the Arabidopsis gynoecium

PubMed Central

Martínez-Fernández, Irene; Sanchís, Sofía; Marini, Naciele; Balanzá, Vicente; Ballester, Patricia; Navarrete-Gómez, Marisa; Oliveira, Antonio C.; Colombo, Lucia; Ferrándiz, Cristina

2014-01-01

The four NGATHA genes (NGA) form a small subfamily within the large family of B3-domain transcription factors of Arabidopsis thaliana. NGA genes act redundantly to direct the development of the apical tissues of the gynoecium, the style, and the stigma. Previous studies indicate that NGA genes could exert this function at least partially by directing the synthesis of auxin at the distal end of the developing gynoecium through the upregulation of two different YUCCA genes, which encode flavin monooxygenases involved in auxin biosynthesis. We have compared three developing pistil transcriptome data sets from wildtype, nga quadruple mutants, and a 35S::NGA3 line. The differentially expressed genes showed a significant enrichment for auxin-related genes, supporting the idea of NGA genes as major regulators of auxin accumulation and distribution within the developing gynoecium. We have introduced reporter lines for several of these differentially expressed genes involved in synthesis, transport and response to auxin in NGA gain- and loss-of-function backgrounds. We present here a detailed map of the response of these reporters to NGA misregulation that could help to clarify the role of NGA in auxin-mediated gynoecium morphogenesis. Our data point to a very reduced auxin synthesis in the developing apical gynoecium of nga mutants, likely responsible for the lack of DR5rev::GFP reporter activity observed in these mutants. In addition, NGA altered activity affects the expression of protein kinases that regulate the cellular localization of auxin efflux regulators, and thus likely impact auxin transport. Finally, protein accumulation in pistils of several ARFs was differentially affected by nga mutations or NGA overexpression, suggesting that these accumulation patterns depend not only on auxin distribution but could be also regulated by transcriptional networks involving NGA factors. PMID:24904608

Influence of Nrf2 activators on subcellular skeletal muscle protein and DNA synthesis rates after 6 weeks of milk protein feeding in older adults.

PubMed

Konopka, Adam R; Laurin, Jaime L; Musci, Robert V; Wolff, Christopher A; Reid, Justin J; Biela, Laurie M; Zhang, Qian; Peelor, Fredrick F; Melby, Christopher L; Hamilton, Karyn L; Miller, Benjamin F

2017-04-01

In older adults, chronic oxidative and inflammatory stresses are associated with an impaired increase in skeletal muscle protein synthesis after acute anabolic stimuli. Conjugated linoleic acid (CLA) and Protandim have been shown to activate nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor for the antioxidant response element and anti-inflammatory pathways. This study tested the hypothesis that compared to a placebo control (CON), CLA and Protandim would increase skeletal muscle subcellular protein (myofibrillar, mitochondrial, cytoplasmic) and DNA synthesis in older adults after 6 weeks of milk protein feeding. CLA decreased oxidative stress and skeletal muscle oxidative damage with a trend to increase messenger RNA (mRNA) expression of a Nrf2 target, NAD(P)H dehydrogenase quinone 1 (NQO1). However, CLA did not influence other Nrf2 targets (heme oxygenase-1 (HO-1), glutathione peroxidase 1 (Gpx1)) or protein or DNA synthesis. Conversely, Protandim increased HO-1 protein content but not the mRNA expression of downstream Nrf2 targets, oxidative stress, or skeletal muscle oxidative damage. Rates of myofibrillar protein synthesis were maintained despite lower mitochondrial and cytoplasmic protein syntheses after Protandim versus CON. Similarly, DNA synthesis was non-significantly lower after Protandim compared to CON. After Protandim, the ratio of protein to DNA synthesis tended to be greater in the myofibrillar fraction and maintained in the mitochondrial and cytoplasmic fractions, emphasizing the importance of measuring both protein and DNA synthesis to gain insight into proteostasis. Overall, these data suggest that Protandim may enhance proteostatic mechanisms of skeletal muscle contractile proteins after 6 weeks of milk protein feeding in older adults.

Cell signaling pathways in the adrenal cortex: Links to stem/progenitor biology and neoplasia.

PubMed

Penny, Morgan K; Finco, Isabella; Hammer, Gary D

2017-04-15

The adrenal cortex is a dynamic tissue responsible for the synthesis of steroid hormones, including mineralocorticoids, glucocorticoids, and androgens in humans. Advances have been made in understanding the role of adrenocortical stem/progenitor cell populations in cortex homeostasis and self-renewal. Recently, large molecular profiling studies of adrenocortical carcinoma (ACC) have given insights into proteins and signaling pathways involved in normal tissue homeostasis that become dysregulated in cancer. These data provide an impetus to examine the cellular pathways implicated in adrenocortical disease and study connections, or lack thereof, between adrenal homeostasis and tumorigenesis, with a particular focus on stem and progenitor cell pathways. In this review, we discuss evidence for stem/progenitor cells in the adrenal cortex, proteins and signaling pathways that may regulate these cells, and the role these proteins play in pathologic and neoplastic conditions. In turn, we also examine common perturbations in adrenocortical tumors (ACT) and how these proteins and pathways may be involved in adrenal homeostasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

TOR complex 1 regulates the yeast plasma membrane proton pump and pH and potassium homeostasis.

PubMed

Mahmoud, Shima; Planes, María Dolores; Cabedo, Marc; Trujillo, Cristina; Rienzo, Alessandro; Caballero-Molada, Marcos; Sharma, Sukesh C; Montesinos, Consuelo; Mulet, José Miguel; Serrano, Ramón

2017-07-01

We have identified in yeast a connection between two master regulators of cell growth: a biochemical connection involving the TORC1 protein kinase (which activates protein synthesis, nutrient uptake, and anabolism) and a biophysical connection involving the plasma membrane proton-pumping H + -ATPase Pma1 (which drives nutrient and K + uptake and regulates pH homeostasis). Raising the temperature to nonpermissive values in a TOR thermosensitive mutant decreases Pma1 activity. Rapamycin, a TORC1 inhibitor, inhibits Pma1 dependent on its receptor Fpr1 and on the protein phosphatase Sit4, a TORC1 effector. Mutation of either Sit4 or Tco89, a nonessential subunit of TORC1, decreases proton efflux, K + uptake, intracellular pH, cell growth, and tolerance to weak organic acids. Tco89 does not affect Pma1 activity but activates K + transport. © 2017 Federation of European Biochemical Societies.

Identification of the recA (tif) gene product of Escherichia coli

PubMed Central

Gudas, Lorraine J.; Mount, David W.

1977-01-01

Treatments that inhibit DNA synthesis in recA+lexA+Escherichia coli stimulate synthesis of a 40,000 molecular weight protein species (protein X). The protein X molecules produced by wild-type and mutant E. coli strains have been compared by two-dimensional gel electrophoresis. One recA mutant (DM1415 spr recA1) produced a protein X with a more acidic isoelectric point than protein X from the wild type, demonstrating that protein X is probably the product of the recA gene. Additional mutants carrying the recA-linked tif-1 mutation yielded a protein X that was more basic than the wild-type protein, indicating that the tif-1 mutation also alters the recA protein. Protein X molecules from the above mutants and wild-type E. coli have been shown to yield similar partial products upon limited proteolysis in sodium dodecyl sulfate, indicating they are the same protein species. These and additional studies suggest that (i) the tif-1 mutation alters a site on the recA protein that is sensitive to DNA synthesis inhibition, (ii) synthesis of recA protein is self-regulated, and (iii) synthesis of recA protein is also regulated by the lexA product with lexA-suppressor mutations such as spr resulting in constitutive synthesis of recA protein. Images PMID:341152

Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins

NASA Astrophysics Data System (ADS)

Bondalapati, Somasekhar; Jbara, Muhammad; Brik, Ashraf

2016-05-01

Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.

PI3K is required for both basal and LPA-induced DNA synthesis in oral carcinoma cells.

PubMed

Aasrum, Monica; Tjomsland, Vegard; Thoresen, G Hege; De Angelis, Paula M; Christoffersen, Thoralf; Brusevold, Ingvild J

2016-07-01

The glycerophospholipid lysophosphatidic acid (LPA), which is present in most tissues and in high concentrations in saliva, may exert profound effects on oral cancer cells. We have investigated mitogenic signalling induced by LPA in the two oral carcinoma cell lines, D2 and E10, focusing on the role of EGFR transactivation and downstream pathways. Two oral squamous carcinoma cell lines, D2 and E10, were analysed for effects of LPA on signalling pathways and induction of DNA synthesis. Pathway activation was investigated by examining phosphorylation of signalling proteins and by the use of specific pathway inhibitors. The D2 cells had higher levels of activated signalling proteins and higher DNA synthesis activity in the basal condition than E10 cells. EGF did not induce proliferation in D2 cells, whereas LPA induced proliferation in both cell lines, by mechanisms depending on EGFR transactivation. Release of EGFR ligands was involved in basal and LPA-induced proliferation in both D2 and E10 cells. The proliferation in D2 cells was dependent on the PI3K/Akt pathway, but not the MEK/ERK pathway. In E10 cells, the PI3K/Akt, MEK/ERK and p38 pathways were all involved in the proliferation. Transactivation of EGFR is required for LPA-induced DNA synthesis in D2 and E10 cells. Our results also show that although proliferation of oral carcinoma cells is regulated by several pathways, and differentially in E10 and D2 cells, the PI3K pathway has a crucial role in both cell lines. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Maternal-fetal cholesterol transport in the second half of mouse pregnancy does not involve LDL receptor-related protein 2.

PubMed

Zwier, M V; Baardman, M E; van Dijk, T H; Jurdzinski, A; Wisse, L J; Bloks, V W; Berger, R M F; DeRuiter, M C; Groen, A K; Plösch, T

2017-08-01

LDL receptor-related protein type 2 (LRP2) is highly expressed on both yolk sac and placenta. Mutations in the corresponding gene are associated with severe birth defects in humans, known as Donnai-Barrow syndrome. We here characterized the contribution of LRP2 and maternal plasma cholesterol availability to maternal-fetal cholesterol transport and fetal cholesterol levels in utero in mice. Lrp2 +/- mice were mated heterozygously to yield fetuses of all three genotypes. Half of the dams received a 0.5% probucol-enriched diet during gestation to decrease maternal HDL cholesterol. At E13.5, the dams received an injection of D7-labelled cholesterol and were provided with 1- 13 C acetate-supplemented drinking water. At E16.5, fetal tissues were collected and maternal cholesterol transport and fetal synthesis quantified by isotope enrichments in fetal tissues by GC-MS. The Lrp2 genotype did not influence maternal-fetal cholesterol transport and fetal cholesterol. However, lowering of maternal plasma cholesterol levels by probucol significantly reduced maternal-fetal cholesterol transport. In the fetal liver, this was associated with increased cholesterol synthesis rates. No indications were found for an interaction between the Lrp2 genotype and maternal probucol treatment. Maternal-fetal cholesterol transport and endogenous fetal cholesterol synthesis depend on maternal cholesterol concentrations but do not involve LRP2 in the second half of murine pregnancy. Our results suggest that the mouse fetus can compensate for decreased maternal cholesterol levels. It remains a relevant question how the delicate system of cholesterol transport and synthesis is regulated in the human fetus and placenta. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

[Rhythm of protein synthesis in cultures of hepatocytes from rats of different ages. Norm and effect of the peptide livagen].

PubMed

Brodskiĭ, V Ia; Khavinson, V Kh; Zolotarev, Iu A; Nechaeva, N V; Malinin, V V; Novikova, T E; Gvazava, I G; Fateeva, V I

2001-01-01

The circumhoralian rhythm of protein synthesis was determined in a monolayer culture of hepatocytes from rats at the age of 1 to 24 months and weighing from 45 to 480 g, respectively. The peptide lyvagen (Lys-Glu-Asp-Ala) obtained by directed chemical synthesis on the basis of amino acid analysis of the liver polypeptide preparations increased the level of protein synthesis in the hepatocytes from rats of different ages; the highest effect was observed in the cells of old animals. In old rats, lyvagen increased the amplitude of protein synthesis fluctuations. The peptide epitalon (Ala-Glu-Asp-Gly) constructed on the basis of analysis of the epiphysis peptides did not change the intensity of protein synthesis in the cultured hepatocytes.

The identification of high-affinity G protein-coupled receptor ligands from large combinatorial libraries using multicolor quantum dot-labeled cell-based screening

PubMed Central

Fu, Junjie; Lee, Timothy; Qi, Xin

2014-01-01

G protein-coupled receptors (GPCRs), which are involved in virtually every biological process, constitute the largest family of transmembrane receptors. Many top-selling and newly approved drugs target GPCRs. In this review, we aim to recapitulate efforts and progress in combinatorial library-assisted GPCR ligand discovery, particularly focusing on one-bead-one-compound library synthesis and quantum dot-labeled cell-based assays, which both effectively enhance the rapid identification of GPCR ligands with higher affinity and specificity. PMID:24941874

The mammalian iris-ciliary complex affects organization and synthesis of cytoskeletal proteins of organ and tissue cultured lens epithelial cells.

PubMed

Banerjee, A; Emanuel, K; Parafina, J; Bagchi, M

1992-10-01

A water soluble growth inhibitor was isolated from the mammalian ocular iris-ciliary complex. The molecular weight of this protein is 10 kD or lower as determined by ultrafiltration fractionation. The iris-ciliary (IC) complex water soluble protein(s) significantly inhibits synthesis of lower molecular weight proteins of the epithelial cells of the organ cultured mammalian ocular lens. It was also found that this inhibitory effect of IC is mediated via the structural organization of the lens. Monolayer cultures of the lens epithelial cells exposed to IC did not manifest any inhibition of their protein synthesis. Moreover, these tissue cultured lens epithelial (TCLE) cells showed a significant increase in their protein synthetic activities in response to the presence of IC factors in the culture medium. It is postulated that the IC activity is modulated via either the lens capsule, an extracellular matrix, or due to the specific organization of the intact lens. The specific effects of IC on the cytoskeletal organization and synthesis in the organ cultured lens epithelial (OCLE) and TCLE cells were also examined. Both groups, treated with IC factors, manifested significant alterations in their protein synthetic activities and cytoskeletal architecture. The 3H-leucine incorporation experiments showed that alpha-actin and alpha-tubulin synthesis is partially inhibited by IC factors in OCLE cells but vimentin synthesis is not, whereas in TCLE cells all of them showed increased synthesis in response to IC factors. Turnover rates of these proteins in both OCLE and TCLE cells were also computed. The immunofluorescence and microscopic evaluation of OCLE and TCLE cells exposed to IC factors illustrated significant alteration in the cytoarchitecture of the filaments. We demonstrate that an inhibitor(s) molecule of 10 kD or lower size isolated from IC inhibited protein synthesis of OCLE cells and stimulated protein synthesis in TCLE cells. The IC factor also affects the synthesis and organization of cytoskeletal filaments of both the OCLE and TCLE cells.

Association of protein kinase FA/GSK-3alpha (a proline-directed kinase and a regulator of protooncogenes) with human cervical carcinoma dedifferentiation/progression.

PubMed

Yang, S D; Yu, J S; Lee, T T; Ni, M H; Yang, C C; Ho, Y S; Tsen, T Z

1995-10-01

Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in poorly differentiated cervical carcinoma (82.8 +/- 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 +/- 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 +/- 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 +/- 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3alpha in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3alpha may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3alpha may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3alpha may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression.

Drought tolerance and proteomics studies of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene.

PubMed

Qin, Na; Xu, Weigang; Hu, Lin; Li, Yan; Wang, Huiwei; Qi, Xueli; Fang, Yuhui; Hua, Xia

2016-11-01

Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.

Differential expression of steroidogenic factors 1 and 2, cytochrome p450scc, and steroidogenic acute regulatory protein in human pancreas.

PubMed

Morales, Angélica; Vilchis, Felipe; Chávez, Bertha; Morimoto, Sumiko; Chan, Carlos; Robles-Díaz, Guillermo; Díaz-Sánchez, Vicente

2008-08-01

The aim of this study was to investigate the expression of the 4 gene transcripts, steroidogenic factors 1 (SF-1) and 2 (SF-2), steroidogenic acute regulatory (StAR), and cytochrome P450 11A1, involved in the synthesis of steroid hormones in normal human pancreas. Total RNA was extracted from normal male (n = 5) and female (n = 5) samples, obtained from the organ donor program. The expression levels of SF-1, SF-2, StAR protein, and P450scc were assessed by reverse transcription-polymerase chain reaction and complemented with immunohistochemistry analysis. Polymerase chain reaction products amplification for all genes was present in both male and female samples, although differential expression was observed. The signals detected were much more evident in male than in female messenger RNA isolates for SF-1, SF-2, and StAR protein. The expression for P450scc was more intense in female samples. A similar pattern was observed in the immunohistochemical studies. Normal human pancreas expresses 4 gene transcripts involved in steroid synthesis similarly to steroidogenic organs. A distinctive characteristic is the sexually dimorphic expression of these factors. These data provide further evidence to support that the pancreas is a truly steroidogenic tissue, highlighting the presence of sex- and location-related differences in the expression of steroidogenic factors.

Proteome analysis reveals differential expression of proteins involved in triacylglycerol accumulation by Rhodococcus jostii RHA1 after addition of methyl viologen.

PubMed

Dávila Costa, José Sebastián; Silva, Roxana A; Leichert, Lars; Alvarez, Héctor M

2017-03-01

Rhodococcus jostii RHA1 is able to degrade toxic compounds and accumulate high amounts of triacylglycerols (TAG) upon nitrogen starvation. These NADPH-dependent processes are essential for the adaptation of rhodococci to fluctuating environmental conditions. In this study, we used an MS-based, label-free and quantitative proteomic approach to better understand the integral response of R. jostii RHA1 to the presence of methyl viologen (MV) in relation to the synthesis and accumulation of TAG. The addition of MV promoted a decrease of TAG accumulation in comparison to cells cultivated under nitrogen-limiting conditions in the absence of this pro-oxidant. Proteomic analyses revealed that the abundance of key proteins of fatty acid biosynthesis, the Kennedy pathway, glyceroneogenesis and methylmalonyl-CoA pathway, among others, decreased in the presence of MV. In contrast, some proteins involved in lipolysis and β-oxidation of fatty acids were upregulated. Some metabolic pathways linked to the synthesis of NADPH remained activated during oxidative stress as well as under nitrogen starvation conditions. Additionally, exposure to MV resulted in the activation of complete antioxidant machinery comprising superoxide dismutases, catalases, mycothiol biosynthesis, mycothione reductase and alkyl hydroperoxide reductases, among others. Our study suggests that oxidative stress response affects TAG accumulation under nitrogen-limiting conditions through programmed molecular mechanisms when both stresses occur simultaneously.

Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of cell integrity of yeast Saccharomyces cerevisiae.

PubMed

Morimoto, Yuji; Tani, Motohiro

2015-02-01

Complex sphingolipids play important roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened yeast mutant strains showing a synthetic lethal interaction with loss of mannosylinositol phosphorylceramide (MIPC) synthesis and found that a specific group of glycosyltransferases involved in the synthesis of mannan-type N-glycans is essential for the growth of cells lacking MIPC synthases (Sur1 and Csh1). The genetic interaction was also confirmed by repression of MNN2, which encodes alpha-1,2-mannosyltransferase that synthesizes mannan-type N-glycans, by a tetracycline-regulatable system. MNN2-repressed sur1Δ csh1Δ cells exhibited high sensitivity to zymolyase treatment, and caffeine and sodium dodecyl sulfate (SDS) strongly inhibited the growth of sur1Δ csh1Δ cells, suggesting impairment of cell integrity due to the loss of MIPC synthesis. The phosphorylated form of Slt2, a mitogen-activated protein (MAP) kinase activated by impaired cell integrity, increased in sur1Δ csh1Δ cells, and this increase was dramatically enhanced by the repression of Mnn2. Moreover, the growth defect of MNN2-repressed sur1Δ csh1Δ cells was enhanced by the deletion of SLT2 or RLM1 encoding a downstream target of Slt2. These results indicated that loss of MIPC synthesis causes impairment of cell integrity, and this effect is enhanced by impaired synthesis of mannan-type N-glycans. © 2014 John Wiley & Sons Ltd.

Suppression of muscle wasting by the plant‐derived compound ursolic acid in a model of chronic kidney disease

PubMed Central

Yu, Rizhen; Chen, Ji‐an; Xu, Jing; Cao, Jin; Wang, Yanlin; Thomas, Sandhya S.

2016-01-01

Abstract Background Muscle wasting in chronic kidney disease (CKD) and other catabolic disorders contributes to morbidity and mortality, and there are no therapeutic interventions that regularly and safely block losses of muscle mass. We have obtained evidence that impaired IGF‐1/insulin signalling and increases in glucocorticoids, myostatin and/or inflammatory cytokines that contribute to the development of muscle wasting in catabolic disorders by activating protein degradation. Methods Using in vitro and in vivo models of muscle wasting associated with CKD or dexamethasone administration, we measured protein synthesis and degradation and examined mechanisms by which ursolic acid, derived from plants, could block the loss of muscle mass stimulated by CKD or excessive levels of dexamethasone. Results Using cultured C2C12 myotubes to study muscle wasting, we found that exposure to glucocorticoids cause loss of cell proteins plus an increase in myostatin; both responses are significantly suppressed by ursolic acid. Results from promoter and ChIP assays demonstrated a mechanism involving ursolic acid blockade of myostatin promoter activity that is related to CEBP/δ expression. In mouse models of CKD‐induced or dexamethasone‐induced muscle wasting, we found that ursolic acid blocked the loss of muscle mass by stimulating protein synthesis and decreasing protein degradation. These beneficial responses included decreased expression of myostatin and inflammatory cytokines (e.g. TGF‐β, IL‐6 and TNFα), which are initiators of muscle‐specific ubiquitin‐E3 ligases (e.g. Atrogin‐1, MuRF‐1 and MUSA1). Conclusions Ursolic acid improves CKD‐induced muscle mass by suppressing the expression of myostatin and inflammatory cytokines via increasing protein synthesis and reducing proteolysis. PMID:27897418

Comparative proteomic analysis of early somatic and zygotic embryogenesis in Theobroma cacao L.

PubMed

Noah, Alexandre Mboene; Niemenak, Nicolas; Sunderhaus, Stephanie; Haase, Christin; Omokolo, Denis Ndoumou; Winkelmann, Traud; Braun, Hans-Peter

2013-01-14

Somatic embryogenesis can efficiently foster the propagation of Theobroma cacao, but the poor quality of resulted plantlet hinders the use of this technique in the commercial scale. The current study has been initiated to systematically compare the physiological mechanisms underlying somatic and zygotic embryogenesis in T. cacao on the proteome level. About 1000 protein spots per fraction could be separated by two-dimensional isoelectric focusing/SDS PAGE. More than 50 of the protein spots clearly differed in abundance between zygotic and somatic embryos: 33 proteins spots were at least 3-fold higher in abundance in zygotic embryos and 20 in somatic embryos. Analyses of these protein spots differing in volume by mass spectrometry resulted in the identification of 68 distinct proteins. Many of the identified proteins are involved in genetic information processing (21 proteins), carbohydrate metabolism (11 proteins) and stress response (7 proteins). Somatic embryos especially displayed many stress related proteins, few enzymes involved in storage compound synthesis and an exceptional high abundance of endopeptidase inhibitors. Phosphoenolpyruvate carboxylase, which was accumulated more than 3-fold higher in zygotic embryos, represents a prominent enzyme in the storage compound metabolism in cacao seeds. Implications on the improvement of somatic embryogenesis in cacao are discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

Regulation of protein turnover by heat shock proteins.

PubMed

Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

2014-12-01

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system. Copyright © 2014 Elsevier Inc. All rights reserved.

Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

PubMed Central

Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

2011-01-01

It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes. PMID:21960964

miR-27a controls triacylglycerol synthesis in bovine mammary epithelial cells by targeting peroxisome proliferator-activated receptor gamma.

PubMed

Tang, K Q; Wang, Y N; Zan, L S; Yang, W C

2017-05-01

Growing evidence has revealed that microRNA are central elements in milk fat synthesis in mammary epithelial cells. A negative regulator of adipocyte fat synthesis, miR-27a has been reported to be involved in the regulation of milk fat synthesis in goat mammary epithelial cells; however, the regulatory role of miR-27a in bovine milk fat synthesis remains unclear. In the present study, primary bovine mammary epithelial cells (BMEC) were harvested from mid-lactation cows and cultured in Dulbecco's modified Eagle's medium/F-12 medium with 10% fetal bovine serum, 5 μg/mL of insulin, 1 μg/mL of hydrocortisone, 2 μg/mL of prolactin, 1 μg/mL of progesterone, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. We found that the overexpression of miR-27a significantly suppressed lipid droplet formation and decreased the cellular triacylglycerol (TAG) levels, whereas inhibition of miR-27a resulted in a greater lipid droplet formation and TAG accumulation in BMEC. Meanwhile, overexpression of miR-27a inhibited mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein beta (C/EBPβ), perilipin 2 (PLIN2), and fatty acid binding protein 3 (FABP3), whereas miR-27a downregulation increased PPARG, C/EBPβ, FABP3, and CCAAT enhancer binding protein alpha (C/EBPα) mRNA expression. Furthermore, Western blot analysis revealed the protein level of PPARG in miR-27a mimic and inhibitor transfection groups to be consistent with the mRNA expression response. Moreover, luciferase reporter assays verified that PPARG was the direct target of miR-27a. In summary, these results indicate that miR-27a has the ability to control TAG synthesis in BMEC via targeting PPARG, suggesting that miR-27a could potentially be used to improve beneficial milk components in dairy cows. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cellular Energetic Status Supervises the Synthesis of Bis-Diphosphoinositol Tetrakisphosphate Independently of AMP-Activated Protein Kinase

PubMed Central

Choi, Kuicheon; Mollapour, Elahe; Choi, Jae H.; Shears, Stephen B.

2009-01-01

Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP]2-InsP4 synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP]2-InsP4 synthesis without affecting other inositol phosphates. In addition, we report that the short-term increases in [PP]2-InsP4 levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP]2-InsP4 synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2′-amino-3′-methoxyflavone (PD98059), had similar off-target effects. We conclude that the levels and hence the signaling strength of [PP]2-InsP4 is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks. PMID:18460607

Simultaneous infusion of glutamine and branched-chain amino acids (BCAA) to septic rats does not have more favorable effect on protein synthesis in muscle, liver, and small intestine than separate infusions.

PubMed

Holecek, Milan; Muthny, Tomas; Kovarik, Miroslav; Sispera, Ludek

2006-01-01

Glutamine and branched-chain amino acids (BCAA; valine, leucine, and isoleucine) are used as nutrition supplements in the treatment of proteocatabolic illness. We hypothesized that simultaneous administration of BCAA and glutamine affects protein metabolism more significantly than separate administration. In the present study, we evaluated their effect on protein synthesis in skeletal muscle, liver, and jejunum of septic rats. Twenty-four hours after induction of sepsis by subcutaneous injection of turpentine, the rats were infused for 6 hours with 5 mL of 1.75% glutamine, 1.75% BCAA, 1.75% glutamine+BCAA, or saline solution. The control group consisted of intact rats infused with saline. Protein synthesis was measured at the end of infusion by a "flooding method" with [3,4,5-(3)H]phenylalanine. In turpentine-treated animals, we observed a decrease in glutamine concentration in blood plasma and skeletal muscle, a decrease in BCAA concentration in liver and jejunum, and a decrease in protein synthesis in all tissues. Glutamine or glutamine+BCAA infusion increased glutamine concentration in plasma and muscle and stimulated protein synthesis in the liver. The BCAA infusion enhanced concentrations of BCAA in plasma and tissues, but the effect of BCAA on protein synthesis was insignificant. Synergistic effect of simultaneous infusion of glutamine and BCAA on protein synthesis was not observed. We conclude that glutamine infusion to rats with septic injury may significantly improve impaired protein synthesis in the liver and that there is no synergistic effect of glutamine and BCAA infusion on protein synthesis in skeletal muscle, liver, and jejunum.

DECOUPLING OF PROTEIN AND RNA SYNTHESIS DURING DEUTERIUM PARTHENOGENESIS IN SEA URCHIN EGGS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gross, P.R.; Spindel, W.; Cousineau, G.H.

1963-10-29

The parthenogenetic activation of cell division and suppression of nucleic acid synthesis by deuterium in eggs of sea urchins was investigated. D/ sub 2/O treatment was found to evoke a high rate of protein synthesis in the eggs that was maintained for several hours. However, eggs whose protein synthesis was activated and that were making labeled cytasters showed no increment in RNA synthesis over controls. (P.C.H.)

A Working Model of Protein Synthesis Using Lego(TM) Building Blocks.

ERIC Educational Resources Information Center

Templin, Mark A.; Fetters, Marcia K.

2002-01-01

Uses Lego building blocks to improve the effectiveness of teaching about protein synthesis. Provides diagrams and pictures for a 2-3 day student activity. Discusses mRNA, transfer RNA, and a protein synthesis model. (MVL)

Protein synthesis is defended in the mitochondrial fraction of gill but not heart in cunner (Tautogolabrus adspersus) exposed to acute hypoxia and hypothermia.

PubMed

Lewis, Johanne M; Driedzic, William R

2010-02-01

The cunner, Tautogolabrus adspersus, is a north-temperate teleost which relies upon metabolic depression to survive the extreme low water temperatures of its habitat during the winter. Previous study has demonstrated a decrease in protein synthesis accompanies the metabolic depression observed at the whole animal level during seasonal low temperature exposure. As such, the objective of the current study was to determine: (i) if the response of decreased protein synthesis is conserved across environmental stressors and (ii) if the response of metabolic depression is conserved across levels of cellular organization. This was accomplished through the measurement of in vivo protein synthesis rates in the whole tissue, cytosolic and mitochondrial protein pools (reflective of nuclear encoded proteins imported into mitochondria) of heart and gill in cunner exposed to either acute low temperature (8-4 degrees C) or acute hypoxia (10% O(2) saturation). In both heart and gill, rates of protein synthesis in the whole tissue and cytosolic protein pools were substantially depressed by 80% in response to acute hypothermia. In hypoxic heart, protein synthesis was significantly decreased by 50-60% in the whole tissue, cytosolic and mitochondrial pools; however, in gill there was no significant difference in rates of protein synthesis in any cellular fraction between normoxic and hypoxic groups. Most strikingly the rate of new protein accumulation in the mitochondrial fraction of gill did not change in response to either a decrease in temperature or hypoxia. The defense of protein synthesis in the gill is most likely associated with the importance of maintaining ionic regulation and the oxidative capacity in this front line organ for gas and ion exchange.

Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sampson, D.A.; Jansen, G.R.

1985-04-01

Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of (3-/sup 3/H)phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary glandmore » protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis.« less

Synthesis and pH-dependent stability of purine-6-sulfenic acid, a putative reactive metabolite of 6-thiopurine.

PubMed

Abraham, R T; Benson, L M; Jardine, I

1983-10-01

Previous studies have shown that 6-thiopurine is metabolically activated by hepatic cytochrome P-450 to an intermediate capable of binding to proteins by a mixed disulfide linkage. The identity of the active metabolite was postulated to be purine-6-sulfenic acid. In the present report, we describe the synthesis of the sulfenic acid derivatives of 6-thiopurine and two structurally similar compounds, 9-methyl-6-thiopurine and 4-mercapto-1H-pyrazolo[3,4-d]-pyrimidine. The unusual pH-dependent stability profiles of these compounds in buffered aqueous media are presented and explained on the basis of a disproportionation mechanism of sulfenic acid decomposition. Studies with radiolabeled purine-6-sulfenic acid demonstrate that this species binds directly to hepatic microsomal protein. These results support the proposed involvement of purine-6-sulfenic acid in the metabolic activation and tissue binding of 6-thiopurine.

Depletion of microglia and inhibition of exosome synthesis halt tau propagation

PubMed Central

Asai, Hirohide; Ikezu, Seiko; Tsunoda, Satoshi; Medalla, Maria; Luebke, Jennifer; Haydar, Tarik; Wolozin, Benjamin; Butovsky, Oleg; Kügler, Sebastian; Ikezu, Tsuneya

2015-01-01

Accumulation of pathological tau protein is a major hallmark of Alzheimer’s disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus–based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target. PMID:26436904

Overview of Cell Synchronization.

PubMed

Banfalvi, Gaspar

2017-01-01

The widespread interest in cell synchronization is maintained by the studies of control mechanism involved in cell cycle regulation. During the synchronization distinct subpopulations of cells are obtained representing different stages of the cell cycle. These subpopulations are then used to study regulatory mechanisms of the cycle at the level of macromolecular biosynthesis (DNA synthesis, gene expression, protein synthesis), protein phosphorylation, development of new drugs, etc. Although several synchronization methods have been described, it is of general interest that scientists get a compilation and an updated view of these synchronization techniques. This introductory chapter summarizes: (1) the basic concepts and principal criteria of cell cycle synchronizations, (2) the most frequently used synchronization methods, such as physical fractionation (flow cytometry, dielectrophoresis, cytofluorometric purification), chemical blockade, (3) synchronization of embryonic cells, (4) synchronization at low temperature, (5) comparison of cell synchrony techniques, (6) synchronization of unicellular organisms, and (7) the effect of synchronization on transfection.

Using simple manipulatives to improve student comprehension of a complex biological process: protein synthesis.

PubMed

Guzman, Karen; Bartlett, John

2012-01-01

Biological systems and living processes involve a complex interplay of biochemicals and macromolecular structures that can be challenging for undergraduate students to comprehend and, thus, misconceptions abound. Protein synthesis, or translation, is an example of a biological process for which students often hold many misconceptions. This article describes an exercise that was developed to illustrate the process of translation using simple objects to represent complex molecules. Animations, 3D physical models, computer simulations, laboratory experiments and classroom lectures are also used to reinforce the students' understanding of translation, but by focusing on the simple manipulatives in this exercise, students are better able to visualize concepts that can elude them when using the other methods. The translation exercise is described along with suggestions for background material, questions used to evaluate student comprehension and tips for using the manipulatives to identify common misconceptions. Copyright © 2012 Wiley Periodicals, Inc.

Probing Functional Heteromeric Chemokine Protein–Protein Interactions through Conformation‐Assisted Oxime Ligation

PubMed Central

Agten, Stijn M.; Koenen, Rory R.; Ippel, Hans; Eckardt, Veit; von Hundelshausen, Philipp; Mayo, Kevin H.; Weber, Christian

2016-01-01

Abstract Protein–protein interactions (PPIs) govern most processes in living cells. Current drug development strategies are aimed at disrupting or stabilizing PPIs, which require a thorough understanding of PPI mechanisms. Examples of such PPIs are heteromeric chemokine interactions that are potentially involved in pathological disorders such as cancer, atherosclerosis, and HIV. It remains unclear whether this functional modulation is mediated by heterodimer formation or by the additive effects of mixed chemokines on their respective receptors. To address this issue, we report the synthesis of a covalent RANTES‐PF4 heterodimer (termed OPRAH) by total chemical synthesis and oxime ligation, with an acceleration of the final ligation step driven by PPIs between RANTES and PF4. Compared to mixed separate chemokines, OPRAH exhibited increased biological activity, thus providing evidence that physical formation of the heterodimer indeed mediates enhanced function. PMID:27785869

A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry.

PubMed

Chen, Guo-Qiang

2009-08-01

Biopolyesters polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer experts and medical researchers. PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines and biofuels have been developed and are covered in this critical review. Companies have been established or involved in PHA related R&D as well as large scale production. Recently, bacterial PHA synthesis has been found to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ or PhaC have been found to be useful for achieving protein purification and even specific drug targeting. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (142 references).

Role of eIF2α Kinases in Translational Control and Adaptation to Cellular Stress.

PubMed

Wek, Ronald C

2018-02-12

A central mechanism regulating translation initiation in response to environmental stress involves phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Phosphorylation of eIF2α causes inhibition of global translation, which conserves energy and facilitates reprogramming of gene expression and signaling pathways that help to restore protein homeostasis. Coincident with repression of protein synthesis, many gene transcripts involved in the stress response are not affected or are even preferentially translated in response to increased eIF2α phosphorylation by mechanisms involving upstream open reading frames (uORFs). This review highlights the mechanisms regulating eIF2α kinases, the role that uORFs play in translational control, and the impact that alteration of eIF2α phosphorylation by gene mutations or small molecule inhibitors can have on health and disease. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Soluble and filamentous proteins in Arabidopsis sieve elements.

PubMed

Batailler, Brigitte; Lemaître, Thomas; Vilaine, Françoise; Sanchez, Christian; Renard, Denis; Cayla, Thibaud; Beneteau, Julie; Dinant, Sylvie

2012-07-01

Phloem sieve elements are highly differentiated cells involved in the long-distance transport of photoassimilates. These cells contain both aggregated phloem-proteins (P-proteins) and soluble proteins, which are also translocated by mass flow. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to carry out a proteomic survey of the phloem exudate of Arabidopsis thaliana, collected by the ethylenediaminetetraacetic acid (EDTA)-facilitated method. We identified 287 proteins, a large proportion of which were enzymes involved in the metabolic precursor generation and amino acid synthesis, suggesting that sieve tubes display high levels of metabolic activity. RNA-binding proteins, defence proteins and lectins were also found. No putative P-proteins were detected in the EDTA-exudate fraction, indicating a lack of long-distance translocation of such proteins in Arabidopsis. In parallel, we investigated the organization of P-proteins, by high-resolution transmission electron microscopy, and the localization of the phloem lectin PP2, a putative P-protein component, by immunolocalization with antibodies against PP2-A1. Transmission electron microscopy observations of P-proteins revealed bundles of filaments resembling strings of beads. PP2-A1 was found weakly associated with these structures in the sieve elements and bound to plastids. These observations suggest that PP2-A1 is anchored to P-proteins and organelles rather than being a structural component of P-proteins. © 2012 Blackwell Publishing Ltd.

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis

PubMed Central

Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia

2017-01-01

Abstract Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an ‘all-or-none’ pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. PMID:28520982

Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting

PubMed Central

Fu, Suneng; Fan, Jason; Blanco, Joshua; Gimenez-Cassina, Alfredo; Danial, Nika N.; Watkins, Steve M.; Hotamisligil, Gökhan S.

2012-01-01

Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)–associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity. PMID:22927828

Polysome profiling in liver identifies dynamic regulation of endoplasmic reticulum translatome by obesity and fasting.

PubMed

Fu, Suneng; Fan, Jason; Blanco, Joshua; Gimenez-Cassina, Alfredo; Danial, Nika N; Watkins, Steve M; Hotamisligil, Gökhan S

2012-08-01

Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)-associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity.

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC).

PubMed

Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A; Schoenhard, Grant; Zemelman, Boris V; Mechref, Yehia; Raab-Graham, Kimberly F

2016-02-01

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder-neurological disorders that exhibit elevated mTORC1 activity. Through a protein-protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential phosphorylation of ribosomal proteins in Arabidopsis thaliana plants during day and night.

PubMed

Turkina, Maria V; Klang Årstrand, Hanna; Vener, Alexander V

2011-01-01

Protein synthesis in plants is characterized by increase in the translation rates for numerous proteins and central metabolic enzymes during the day phase of the photoperiod. The detailed molecular mechanisms of this diurnal regulation are unknown, while eukaryotic protein translation is mainly controlled at the level of ribosomal initiation complexes, which also involves multiple events of protein phosphorylation. We characterized the extent of protein phosphorylation in cytosolic ribosomes isolated from leaves of the model plant Arabidopsis thaliana harvested during day or night. Proteomic analyses of preparations corresponding to both phases of the photoperiod detected phosphorylation at eight serine residues in the C-termini of six ribosomal proteins: S2-3, S6-1, S6-2, P0-2, P1 and L29-1. This included previously unknown phosphorylation of the 40S ribosomal protein S6 at Ser-231. Relative quantification of the phosphorylated peptides using stable isotope labeling and mass spectrometry revealed a 2.2 times increase in the day/night phosphorylation ratio at this site. Phosphorylation of the S6-1 and S6-2 variants of the same protein at Ser-240 increased by the factors of 4.2 and 1.8, respectively. The 1.6 increase in phosphorylation during the day was also found at Ser-58 of the 60S ribosomal protein L29-1. It is suggested that differential phosphorylation of the ribosomal proteins S6-1, S6-2 and L29-1 may contribute to modulation of the diurnal protein synthesis in plants.

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

PubMed

Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

2017-09-06

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

Haloarchaea and the Formation of Gas Vesicles

PubMed Central

Pfeifer, Felicitas

2015-01-01

Halophilic Archaea (Haloarchaea) thrive in salterns containing sodium chloride concentrations up to saturation. Many Haloarchaea possess genes encoding gas vesicles, but only a few species, such as Halobacterium salinarum and Haloferax mediterranei, produce these gas-filled, proteinaceous nanocompartments. Gas vesicles increase the buoyancy of cells and enable them to migrate vertically in the water body to regions with optimal conditions. Their synthesis depends on environmental factors, such as light, oxygen supply, temperature and salt concentration. Fourteen gas vesicle protein (gvp) genes are involved in their formation, and regulation of gvp gene expression occurs at the level of transcription, including the two regulatory proteins, GvpD and GvpE, but also at the level of translation. The gas vesicle wall is solely formed of proteins with the two major components, GvpA and GvpC, and seven additional accessory proteins are also involved. Except for GvpI and GvpH, all of these are required to form the gas permeable wall. The applications of gas vesicles include their use as an antigen presenter for viral or pathogen proteins, but also as a stable ultrasonic reporter for biomedical purposes. PMID:25648404

Insights into the Melipona scutellaris (Hymenoptera, Apidae, Meliponini) fat body transcriptome.

PubMed

de Sousa, Cristina Soares; Serrão, José Eduardo; Bonetti, Ana Maria; Amaral, Isabel Marques Rodrigues; Kerr, Warwick Estevam; Maranhão, Andréa Queiroz; Ueira-Vieira, Carlos

2013-07-01

The insect fat body is a multifunctional organ analogous to the vertebrate liver. The fat body is involved in the metabolism of juvenile hormone, regulation of environmental stress, production of immunity regulator-like proteins in cells and protein storage. However, very little is known about the molecular mechanisms involved in fat body physiology in stingless bees. In this study, we analyzed the transcriptome of the fat body from the stingless bee Melipona scutellaris. In silico analysis of a set of cDNA library sequences yielded 1728 expressed sequence tags (ESTs) and 997 high-quality sequences that were assembled into 29 contigs and 117 singlets. The BLAST X tool showed that 86% of the ESTs shared similarity with Apis mellifera (honeybee) genes. The M. scutellaris fat body ESTs encoded proteins with roles in numerous physiological processes, including anti-oxidation, phosphorylation, metabolism, detoxification, transmembrane transport, intracellular transport, cell proliferation, protein hydrolysis and protein synthesis. This is the first report to describe a transcriptomic analysis of specific organs of M. scutellaris. Our findings provide new insights into the physiological role of the fat body in stingless bees.

Insights into the Melipona scutellaris (Hymenoptera, Apidae, Meliponini) fat body transcriptome

PubMed Central

de Sousa, Cristina Soares; Serrão, José Eduardo; Bonetti, Ana Maria; Amaral, Isabel Marques Rodrigues; Kerr, Warwick Estevam; Maranhão, Andréa Queiroz; Ueira-Vieira, Carlos

2013-01-01

The insect fat body is a multifunctional organ analogous to the vertebrate liver. The fat body is involved in the metabolism of juvenile hormone, regulation of environmental stress, production of immunity regulator-like proteins in cells and protein storage. However, very little is known about the molecular mechanisms involved in fat body physiology in stingless bees. In this study, we analyzed the transcriptome of the fat body from the stingless bee Melipona scutellaris. In silico analysis of a set of cDNA library sequences yielded 1728 expressed sequence tags (ESTs) and 997 high-quality sequences that were assembled into 29 contigs and 117 singlets. The BLAST X tool showed that 86% of the ESTs shared similarity with Apis mellifera (honeybee) genes. The M. scutellaris fat body ESTs encoded proteins with roles in numerous physiological processes, including anti-oxidation, phosphorylation, metabolism, detoxification, transmembrane transport, intracellular transport, cell proliferation, protein hydrolysis and protein synthesis. This is the first report to describe a transcriptomic analysis of specific organs of M. scutellaris. Our findings provide new insights into the physiological role of the fat body in stingless bees. PMID:23885214

Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cell Suspensions: A Proteomic Analysis.

PubMed

Gas-Pascual, Elisabet; Simonovik, Biljana; Heintz, Dimitri; Bergdoll, Marc; Schaller, Hubert; Bach, Thomas J

2015-08-01

The effect of an inhibitor of cycloartenol synthase (CAS, EC 5.4.99.8) on the proteome of tobacco BY-2 cells has been examined. CAS catalyzes the first committed step in phytosterol synthesis in plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Proteins were separated by two-dimensional electrophoresis and spots, that clearly looked differentially accumulated after visual inspection, were cut, in-gel trypsin digested, and peptides were analyzed by nano-HPLC-MS/MS. Distinct peptides were compared to sequences in the data banks and attributed to corresponding proteins and genes. Inhibition of CAS induced proteins that appear to mitigate the negative effects of the chemical exposure. However, as all enzymes that are directly involved in phytosterol biosynthesis are low-abundant proteins, significant changes in their levels could not be observed. Differences could be seen with enzymes involved in primary metabolism (glycolysis, pentose phosphate pathway etc.), in proteins of the chaperonin family, and those, like actin, that participate in formation and strengthening of the cytoskeleton and have some impact on cell growth and division.

Chronic leucine supplementation of a low protein diet increases protein synthesis in skeletal muscle and visceral tissues of neonatal pigs through mTOR signaling

USDA-ARS?s Scientific Manuscript database

Leucine acutely stimulates protein synthesis by activating the mammalian target of rapamycin (mTOR) signaling pathway. We hypothesized that leucine supplementation of a low protein diet will enhance protein synthesis and mTOR signaling in the neonate for prolonged periods. Fasted 5-d-old pigs (n=6–8...

Protein engineering in designing tailored enzymes and microorganisms for biofuels production

PubMed Central

Wen, Fei; Nair, Nikhil U; Zhao, Huimin

2009-01-01

Summary Lignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. However, the recalcitrant nature of lignocellulose poses technical hurdles to an economically viable biorefinery. Low enzymatic hydrolysis efficiency and low productivity, yield, and titer of biofuels are among the top cost contributors. Protein engineering has been used to improve the performances of lignocellulose-degrading enzymes, as well as proteins involved in biofuel synthesis pathways. Unlike its great success seen in other industrial applications, protein engineering has achieved only modest results in improving the lignocellulose-to-biofuels efficiency. This review will discuss the unique challenges that protein engineering faces in the process of converting lignocellulose to biofuels and how they are addressed by recent advances in this field. PMID:19660930

Heat shock proteins (Hsp 70) response is not systematic to cell stress: case of the mycotoxin ochratoxin A.

PubMed

Hassen, Wafa; Ayed-Boussema, Imen; Bouslimi, Amel; Bacha, Hassen

2007-12-05

Ochratoxin A (OTA) is a mycotoxin routinely detected in improperly stored animal and human food supplies as well as in human sera worldwide. OTA has multiple toxic effects; however, the most prominent is nephrotoxicity. Thus, OTA is involved in the pathogenesis of human nephropathy in Balkan areas. In this study, we address the question of the appropriate functioning of the basal cellular defense mechanisms, after exposure to OTA, which, up to now, has not been investigated satisfactorily. In this context, we have monitored the effect of OTA on (i) the inhibition of cell viability, (ii) the oxidative damage using the GSH depletion, (iii) the inhibition of protein synthesis through the incorporation of [(3)H] Leucine and (iv) the induction of Hsp 70 gene expression as a parameter of cytotoxicity, oxidative damage and particularly as a protective and adaptative response. This study was conducted using the Human Hep G2 hepatocytes and monkey kidney Vero cells under exposure conditions ranging from non-cytotoxic to sub-lethal. Our results clearly showed that OTA inhibits cell proliferation, strongly reduces protein synthesis and induces the decrease of GSH in concentration-dependent manner in both Hep G2 and Vero cells. However, although cytotoxicity and oxidative damage (main inducers of Hsp expression) occur, no change was observed in Hsp 70 level under the multiple tested conditions. Inhibition of protein synthesis could not explain the absence of Hsp 70 response since concentrations, which did not influence protein synthesis, also failed to display the expected Hsp 70 response. Our data are consistent with recently published reports where considerable differences were noticed in the ability of relevant toxicants to induce Hsp. These results raised doubt about the universal character of Hsp induction which seems to be more complex than originally envisioned. It could be concluded that Hsp 70 induction is not systematic to cell stress.

Calcineurin inhibition blocks within-, but not between-session fear extinction in mice

PubMed Central

Moulin, Thiago C.; Carneiro, Clarissa F. D.; Gonçalves, Marina M. C.; Junqueira, Lara S.; Amaral, Olavo B.

2015-01-01

Memory extinction involves the formation of a new associative memory that inhibits a previously conditioned association. Nonetheless, it could also depend on weakening of the original memory trace if extinction is assumed to have multiple components. The phosphatase calcineurin (CaN) has been described as being involved in extinction but not in the initial consolidation of fear learning. With this in mind, we set to study whether CaN could have different roles in distinct components of extinction. Systemic treatment with the CaN inhibitors cyclosporin A (CsA) or FK-506, as well as i.c.v. administration of CsA, blocked within-session, but not between-session extinction or initial learning of contextual fear conditioning. Similar effects were found in multiple-session extinction of contextual fear conditioning and in auditory fear conditioning, indicating that CaN is involved in different types of short-term extinction. Meanwhile, inhibition of protein synthesis by cycloheximide (CHX) treatment did not affect within-session extinction, but disrupted fear acquisition and slightly impaired between-session extinction. Our results point to a dissociation of within- and between-session extinction of fear conditioning, with the former being more dependent on CaN activity and the latter on protein synthesis. Moreover, the modulation of within-session extinction did not affect between-session extinction, suggesting that these components are at least partially independent. PMID:25691516

Fatty acid-binding protein 5 (FABP5) promotes lipolysis of lipid droplets, de novo fatty acid (FA) synthesis and activation of nuclear factor-kappa B (NF-κB) signaling in cancer cells.

PubMed

Senga, Shogo; Kobayashi, Narumi; Kawaguchi, Koichiro; Ando, Akira; Fujii, Hiroshi

2018-06-12

Fatty acid-binding proteins (FABPs) are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting them to the appropriate compartments in the cell. Epidermal fatty acid-binding protein (FABP5) is an intracellular lipid-binding protein that is abundantly expressed in adipocytes and macrophages. Previous studies have revealed that the FABP5 expression level is closely related to malignancy in various types of cancer. However, its precise functions in the metabolisms of cancer cells remain unclear. Here, we revealed that FABP5 knockdown significantly induced downregulation of the genes expression, such as hormone-sensitive lipase (HSL), monoacylglycerol lipase (MAGL), elongation of long-chain fatty acid member 6 (Elovl6), and acyl-CoA synthetase long-chain family member 1 (ACSL1), which are involved in altered lipid metabolism, lipolysis, and de novo FA synthesis in highly aggressive prostate and breast cancer cells. Moreover, we demonstrated that FABP5 induced inflammation and cytokine production through the nuclear factor-kappa B signaling pathway activated by reactive oxygen species and protein kinase C in PC-3 and MDA-MB-231 cells. Thus, FABP5 might regulate lipid quality and/or quantity to promote aggressiveness such as cell growth, invasiveness, survival, and inflammation in prostate and breast cancer cells. In the present study, we have revealed for the first time that high expression of FABP5 plays a critical role in alterations of lipid metabolism, leading to cancer development and metastasis in highly aggressive prostate and breast cancer cells. Copyright © 2018. Published by Elsevier B.V.

Effects of anaerobiosis on in vivo protein synthesis in the roots of a marine angiosperm zostera marina

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, R.D.; Alberte, R.S.

1989-04-01

The roots of the temperate seagrass Zostera marina undergo daily periods of anaerobiosis at night. These diurnal periods of anoxia alter many metabolic processes in the roots including carbon and nitrogen metabolism, amino acid synthesis, and synthesis and levels of ATP, ADP and AMP. To further characterize the effects of anaerobiosis, we determined in vivo rates of protein synthesis by measuring the relative incorporation of {sup 35}S-MET in TCA precipitated protein samples. Results from these studies show that in vivo protein synthesis decreases continuously during 12 h of anaerobiosis and correlates with changes in ATP levels under similar conditions. Furthermore,more » polypeptide patterns obtained by SDS-PAGE and 2D-SDSPAGE indicate that anaerobiosis leads to differential protein synthesis in the roots.« less