共查询到20条相似文献,搜索用时 30 毫秒
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Background
Misfolded proteins accumulating outside the bacterial cytoplasmic membrane can interfere with the secretory machinery, hence the existence of quality factors to eliminate these misfolded proteins is of capital importance in bacteria that are efficient producers of secretory proteins. These bacteria normally use a specific two-component system to respond to the stress produced by the accumulation of the misfolded proteins, by activating the expression of HtrA-like proteases to specifically eliminate the incorrectly folded proteins.Methodology/Principal Findings
Overproduction of alpha-amylase in S. lividans causing secretion stress permitted the identification of a two-component system (SCO4156-SCO4155) that regulates three HtrA-like proteases which appear to be involved in secretion stress response. Mutants in each of the genes forming part of the two-genes operon that encodes the sensor and regulator protein components accumulated misfolded proteins outside the cell, strongly suggesting the involvement of this two-component system in the S. lividans secretion stress response.Conclusions/Significance
To our knowledge this is the first time that a specific secretion stress response two-component system is found to control the expression of three HtrA-like protease genes in S. lividans, a bacterium that has been repeatedly used as a host for the synthesis of homologous and heterologous secretory proteins of industrial application. 相似文献5.
Naoshi Ohta Masato Sato Kiminori Ushida Mami Kokubo Takayuki Baba Kayoko Taniguchi Makoto Urai Koji Kihira Joji Mochida 《BMC biotechnology》2009,9(1):1-11
Background
Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles.Results
Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies.Conclusion
Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells. 相似文献6.
Background
Insulin degrading enzyme (IDE) is a major protease of amyloid beta peptide (Aβ), a prominent toxic protein in Alzheimer’s disease (AD) pathogenesis. Previous studies suggested that statins promote IDE secretion; however, the underlying mechanism is unknown, as IDE has no signal sequence.Results
In this study, we found that simvastatin (0.2 μM for 12 h) induced the degradation of extracellular Aβ40, which depended on IDE secretion from primary astrocytes. In addition, simvastatin increased IDE secretion from astrocytes in a time- and dose-dependent manner. Moreover, simvastatin-mediated IDE secretion was mediated by an autophagy-based unconventional secretory pathway, and autophagic flux regulated simvastatin-mediated IDE secretion. Finally, simvastatin activated autophagy via the LKB1-AMPK-mTOR signaling pathway in astrocytes.Conclusions
These results demonstrate a novel pathway for statin-mediated IDE secretion from astrocytes. Modulation of this pathway could provide a potential therapeutic target for treatment of Aβ pathology by enhancing extracellular clearance of Aβ.7.
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Mette Koefoed Claus M. Larsen Mirjam V. Faulenbach Allan Vaag Jan A. Ehses Marc Y. Donath James Norton McGuire Flemming Pociot Thomas Mandrup-Poulsen 《Clinical proteomics》2010,6(4):153-161
Introduction
High glucose concentrations induce the production of IL-1β in human pancreatic beta cells leading to impaired insulin secretion, decreased cell proliferation and apoptosis. Blockade of IL-1 signalling with the recombinant human IL-1 receptor antagonist anakinra reduces HbA1c in patients with type 2 diabetes. The aims of the present study were to identify: (1) candidate surrogates for improved glycemia in type 2 diabetic patients following treatment with anakinra, (2) proteins that change serum concentration because of anakinra treatment and (3) candidate biomarkers that may predict improved glycemia in type 2 diabetic subjects treated with anakinra.Methods
Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry was used to analyse serum from 67 type 2 diabetic patients who had received either placebo or anakinra for 13 weeks. Immunodepletion with magnetic protein G bead-coupled antibodies were used to identify three proteins and Western blotting confirmed the biomarker concentration pattern of four proteins.Results
Twelve proteins, including transthyretin (TTR) and transferrin (Tf), were identified as candidate surrogates for improved glycemia. Six proteins, including retinol-binding protein 4 (RPB4) and a protein tentatively identified as modified apolipoprotein-A1 (apo-AI), increased expression as a consequence of anakinra treatment and four proteins were candidate biomarkers that may predict improved glycemia following anakinra treatment. Furthermore, we found increased RBP4 to be associated with improved beta cell secretory function and increased TTR, RBP4 and modified apo-AI (peak at 28,601 Da) to be associated with decreased inflammation.Conclusions
Anakinra-induced changes in the serum proteome pool associated with a decreased cardiovascular disease risk, reduced inflammation and improved beta cell secretory function. 相似文献9.
Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment 总被引:1,自引:0,他引:1
Xochitl C Morgan Timothy L Tickle Harry Sokol Dirk Gevers Kathryn L Devaney Doyle V Ward Joshua A Reyes Samir A Shah Neal LeLeiko Scott B Snapper Athos Bousvaros Joshua Korzenik Bruce E Sands Ramnik J Xavier Curtis Huttenhower 《Genome biology》2012,13(9):1-18
Background
The inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis result from alterations in intestinal microbes and the immune system. However, the precise dysfunctions of microbial metabolism in the gastrointestinal microbiome during IBD remain unclear. We analyzed the microbiota of intestinal biopsies and stool samples from 231 IBD and healthy subjects by 16S gene pyrosequencing and followed up a subset using shotgun metagenomics. Gene and pathway composition were assessed, based on 16S data from phylogenetically-related reference genomes, and associated using sparse multivariate linear modeling with medications, environmental factors, and IBD status.Results
Firmicutes and Enterobacteriaceae abundances were associated with disease status as expected, but also with treatment and subject characteristics. Microbial function, though, was more consistently perturbed than composition, with 12% of analyzed pathways changed compared with 2% of genera. We identified major shifts in oxidative stress pathways, as well as decreased carbohydrate metabolism and amino acid biosynthesis in favor of nutrient transport and uptake. The microbiome of ileal Crohn's disease was notable for increases in virulence and secretion pathways.Conclusions
This inferred functional metagenomic information provides the first insights into community-wide microbial processes and pathways that underpin IBD pathogenesis. 相似文献10.
Huahua Wang Junjun Huang Weihong Liang Xiaolei Liang Yurong Bi 《Plant and Soil》2013,366(1-2):479-490
Background and Aims
Polyamines and nitric oxide (NO) are two important molecules modulating numerous environment stresses in plants. This study was to investigate the roles of polyamines and NO in aluminum (Al) tolerance in red kidney bean.Methods
The interaction between putrescine (Put) and NO under Al stress was examined. NO donor and scavenger were used to further examine the role of NO in Al-induced citrate secretion from roots by high performance liquid chromatography.Results
Al stress caused increase of endogenous free Put, and exogenous Put alleviated Al-induced inhibition of root elongation and Al accumulation. In addition, Put induced NO production and nitrate reductase (NR) activity under Al stress. Al- and Put-induced NO production could be reversed by NR inhibitor. Furthermore, Al stress stimulated citrate secretion from roots, and this response was stimulated by NO donor, whereas NO scavenger inhibited Al-induced citrate secretion from roots. Concomitantly, NO donor reduced Al accumulation in root apexes, while NO scavenger further enhanced Al accumulation. Al-induced inhibition of root growth was significantly improved by exogenous citrate treatment.Conclusions
Put and NO enhanced Al tolerance by modulating citrate secretion from roots, and NO may act downstream of Put in red kidney bean under Al stress. 相似文献11.
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Mohamed Belal Hamed Spyridoula Karamanou Solveig Ólafsdottir Joana Sofia Martins Basílio Kenneth Simoens Kostantinos C. Tsolis Lieve Van Mellaert Eik Elísabet Guðmundsdóttir Gudmundur Oli Hreggvidsson Jozef Anné Kristel Bernaerts Olafur H. Fridjonsson Anastassios Economou 《Microbial cell factories》2017,16(1):232
Background
The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces.Results
CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498–507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50–90 mg/L or 100–120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans.Conclusion
This study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.14.
Buz Barstow Christina M Agapakis Patrick M Boyle Gerald Grandl Pamela A Silver Edwin H Wintermute 《Journal of biological engineering》2011,5(1):1-15
Background
FeFe-hydrogenases are the most active class of H2-producing enzymes known in nature and may have important applications in clean H2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O2.Results
We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O2 levels eliminate growth. This pathway forms the basis for a genetic selection for O2 tolerance. Genetically selected hydrogenases did not show improved stability in O2 and in many cases had lost H2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.Conclusions
Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H2 metabolism. Our results also indicate a H2-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H2-activating catalysts. 相似文献15.
ABA signaling in stress-response and seed development 总被引:5,自引:0,他引:5
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Fabíola C Toledo Juliana E Perobelli Flávia PC Pedrosa Janete A Anselmo-Franci Wilma DG Kempinas 《Reproductive biology and endocrinology : RB&E》2011,9(1):1-9
Background
Hyperglycemia can impair the male reproductive system in experimental animals and in men during reproductive age. Studies have shown that vitamin C has some good effects on male reproductive system, and therefore vitamin C treatment could attenuate the dysfunctions in this system caused by hyperglycemia. Thus, the objective of this work was to evaluate whether vitamin C treatment could attenuate reproductive dysfunctions in hyperglycemic male rats.Methods
Adult male rats were divided into 3 groups: a normoglycemic (n = 10) and two hyperglycemic (that received a single dose of streptozotocin - 40 mg/kg BW). The two last groups (n = 10 per group) were divided into: hyperglycemic control (Hy) and hyperglycemic + 150 mg of vitamin C (HyC), by gavage during 30 consecutive days. The normoglycemic and hyperglycemic control groups received the vehicle (water). The first day after the treatment, the rats were anesthetized and killed to evaluate oxidative stress biomarkers (TBARS, SOD, GSHt and GSH-Px) in the erythrocytes, body and reproductive organ weights, sperm parameters, plasma hormone levels (FSH, LH and testosterone), testicular and epididymal histo-morphometry and histopathology.Results
Compared with the normoglycemic animals, hyperglycemic control rats showed reduced weight of the body and reproductive organ but testis weight was maintained. It was also observed reduction of testosterone and LH levels, seminiferous tubular diameter, sperm motility and sperm counts in the epididymis. In addition, there was an increase in morphological abnormalities on spermatozoa as well as in oxidative stress level. Vitamin C reduced the oxidative stress level, diminished the number of abnormal sperm, and increased testosterone and LH levels and seminiferous tubular diameter but did not show improvement of sperm motility in relation to the hyperglycemic control group. Hyperglycemia caused a rearrangement in the epididymal tissue components (stroma, ephitelium and lumen) as demonstrated by the stereological analysis results. However, this alteration was partially prevented by vitamin C treatment.Conclusions
We conclude that vitamin C partially attenuated some male reproductive system dysfunctions in hyperglycemic rats. 相似文献17.
Tomáš Helikar Bryan Kowal Sean McClenathan Mitchell Bruckner Thaine Rowley Alex Madrahimov Ben Wicks Manish Shrestha Kahani Limbu Jim A Rogers 《BMC systems biology》2012,6(1):1-14
Background
The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression.Results
In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU.Conclusions
The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology. 相似文献18.
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Background
In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation.Results
To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER.Conclusions
This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. 相似文献20.
Han Bin Pek Pei Yu Lim Chengcheng Liu Dong-Yup Lee Xuezhi Bi Fong Tian Wong Dave Siak-Wei Ow 《Biotechnology letters》2017,39(5):759-765