Fe-S Clusters Emerging as Targets of Therapeutic Drugs

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Laurence Vernis

2017-01-01

Full Text Available Fe-S centers exhibit strong electronic plasticity, which is of importance for insuring fine redox tuning of protein biological properties. In accordance, Fe-S clusters are also highly sensitive to oxidation and can be very easily altered in vivo by different drugs, either directly or indirectly due to catabolic by-products, such as nitric oxide species (NOS or reactive oxygen species (ROS. In case of metal ions, Fe-S cluster alteration might be the result of metal liganding to the coordinating sulfur atoms, as suggested for copper. Several drugs presented through this review are either capable of direct interaction with Fe-S clusters or of secondary Fe-S clusters alteration following ROS or NOS production. Reactions leading to Fe-S cluster disruption are also reported. Due to the recent interest and progress in Fe-S biology, it is very likely that an increasing number of drugs already used in clinics will emerge as molecules interfering with Fe-S centers in the near future. Targeting Fe-S centers could also become a promising strategy for drug development.

Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis.

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Valeria R Turowski

Full Text Available Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR and ferredoxin (Fd, two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.

fps/fes knockout mice display a lactation defect and the fps/fes tyrosine kinase is a component of E-cadherin-based adherens junctions in breast epithelial cells during lactation.

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Truesdell, Peter F; Zirngibl, Ralph A; Francis, Sarah; Sangrar, Waheed; Greer, Peter A

2009-10-15

The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase implicated in vesicular trafficking and cytokine and growth factor signaling in hematopoietic, neuronal, vascular endothelial and epithelial lineages. Genetic evidence has suggested a tumor suppressor role for Fps/Fes in breast and colon. Here we used fps/fes knockout mice to investigate potential roles for this kinase in development and function of the mammary gland. Fps/Fes expression was induced during pregnancy and lactation, and its kinase activity was dramatically enhanced. Milk protein and fat composition from nursing fps/fes-null mothers was normal; however, pups reared by them gained weight more slowly than pups reared by wild-type mothers. Fps/Fes displayed a predominantly dispersed punctate intracellular distribution which was consistent with vesicles within the luminal epithelial cells of lactating breast, while a small fraction co-localized with beta-catenin and E-cadherin on their basolateral surfaces. Fps/Fes was found to be a component of the E-cadherin adherens junction (AJ) complex; however, the phosphotyrosine status of beta-catenin and core AJ components in fps/fes-null breast tissue was unaltered, and epithelial cell AJs and gland morphology were intact. We conclude that Fps/Fes is not essential for the maintenance of epithelial cell AJs in the lactating breast but may instead play important roles in vesicular trafficking and milk secretion.

A missed Fe-S cluster handoff causes a metabolic shakeup.

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Berteau, Olivier

2018-05-25

The general framework of pathways by which iron-sulfur (Fe-S) clusters are assembled in cells is well-known, but the cellular consequences of disruptions to that framework are not fully understood. Crooks et al. report a novel cellular system that creates an acute Fe-S cluster deficiency, using mutants of ISCU, the main scaffold protein for Fe-S cluster assembly. Surprisingly, the resultant metabolic reprogramming leads to the accumulation of lipid droplets, a situation encountered in many poorly understood pathological conditions, highlighting unanticipated links between Fe-S assembly machinery and human disease. © 2018 Berteau.

Defining the Architecture of the Core Machinery for the Assembly of Fe-S Clusters in Human Mitochondria.

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Gakh, Oleksandr; Ranatunga, Wasantha; Galeano, Belinda K; Smith, Douglas S; Thompson, James R; Isaya, Grazia

2017-01-01

Although Fe-S clusters may assemble spontaneously from elemental iron and sulfur in protein-free systems, the potential toxicity of free Fe 2+ , Fe 3+ , and S 2- ions in aerobic environments underscores the requirement for specialized proteins to oversee the safe assembly of Fe-S clusters in living cells. Prokaryotes first developed multiprotein systems for Fe-S cluster assembly, from which mitochondria later derived their own system and became the main Fe-S cluster suppliers for eukaryotic cells. Early studies in yeast and human mitochondria indicated that Fe-S cluster assembly in eukaryotes is centered around highly conserved Fe-S proteins (human ISCU) that serve as scaffolds upon which new Fe-S clusters are assembled from (i) elemental sulfur, provided by a pyridoxal phosphate-dependent cysteine desulfurase (human NFS1) and its stabilizing-binding partner (human ISD11), and (ii) elemental iron, provided by an iron-binding protein of the frataxin family (human FXN). Further studies revealed that all of these proteins could form stable complexes that could reach molecular masses of megadaltons. However, the protein-protein interaction surfaces, catalytic mechanisms, and overall architecture of these macromolecular machines remained undefined for quite some time. The delay was due to difficulties inherent in reconstituting these very large multiprotein complexes in vitro or isolating them from cells in sufficient quantities to enable biochemical and structural studies. Here, we describe approaches we developed to reconstitute the human Fe-S cluster assembly machinery in Escherichia coli and to define its remarkable architecture. © 2017 Elsevier Inc. All rights reserved.

Activated Fps/Fes tyrosine kinase regulates erythroid differentiation and survival.

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Sangrar, Waheed; Gao, Yan; Bates, Barbara; Zirngibl, Ralph; Greer, Peter A

2004-10-01

A substantial body of evidence implicates the cytoplasmic protein tyrosine kinase Fps/Fes in regulation of myeloid differentiation and survival. In this study we wished to determine if Fps/Fes also plays a role in the regulation of erythropoiesis. Mice tissue-specifically expressing a "gain-of-function" mutant fps/fes transgene (fps(MF)) encoding an activated variant of Fps/Fes (MFps), were used to explore the in vivo biological role of Fps/Fes. Erythropoiesis in these mice was assessed by hematological analysis, lineage marker analysis, bone-marrow colony assays, and biochemical approaches. fps(MF) mice displayed reductions in peripheral red cell counts. However, there was an accumulation of immature erythroid precursors, which displayed increased survival. Fps/Fes and the related Fer kinase were both detected in early erythroid progenitors/blasts and in mature red cells. Fps/Fes was also activated in response to erythropoietin (EPO) and stem cell factor (SCF), two critical factors in erythroid development. In addition, increased Stat5A/B activation and reduced Erk1/2 phosphorylation was observed in fps(MF) primary erythroid cells in response to EPO or SCF, respectively. These data support a role for Fps/Fes in regulating the survival and differentiation of erythroid cells through modulation of Stat5A/B and Erk kinase pathways induced by EPO and SCF. The increased numbers and survival of erythroid progenitors from fps(MF) mice, and their differential responsiveness to SCF and EPO, implicates Fps/Fes in the commitment of multilineage progenitors to the erythroid lineage. The anemic phenotype in fps(MF) mice suggests that downregulation of Fps/Fes activity might be required for terminal erythroid differentiation.

The fps/fes proto-oncogene regulates hematopoietic lineage output.

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Sangrar, Waheed; Gao, Yan; Zirngibl, Ralph A; Scott, Michelle L; Greer, Peter A

2003-12-01

The fps/fes proto-oncogene is abundantly expressed in myeloid cells, and the Fps/Fes cytoplasmic protein-tyrosine kinase is implicated in signaling downstream from hematopoietic cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO). Studies using leukemic cell lines have previously suggested that Fps/Fes contributes to granulomonocytic differentiation, and that it might play a more selective role in promoting survival and differentiation along the monocytic pathway. In this study we have used a genetic approach to explore the role of Fps/Fes in hematopoiesis. We used transgenic mice that tissue-specifically express a mutant human fps/fes transgene (fps(MF)) that was engineered to encode Fps/Fes kinase that is activated through N-terminal myristoylation (MFps). Hematopoietic function was assessed using lineage analysis, hematopoietic progenitor cell colony-forming assays, and biochemical approaches. fps(MF) transgenic mice displayed a skewed hematopoietic output reflected by increased numbers of circulating granulocytic and monocytic cells and a corresponding decrease in lymphoid cells. Bone marrow colony assays of progenitor cells revealed a significant increase in the number of both granulomonocytic and multi-lineage progenitors. A molecular analysis of signaling in mature monocytic cells showed that MFps promoted GM-CSF-induced STAT3, STAT5, and ERK1/2 activation. These observations support a role for Fps/Fes in signaling pathways that contribute to lineage determination at the level of multi-lineage hematopoietic progenitors as well as the more committed granulomonocytic progenitors.

Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe-S cluster assembly centers.

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Galeano, B K; Ranatunga, W; Gakh, O; Smith, D Y; Thompson, J R; Isaya, G

2017-06-21

Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast and human systems elucidated a conserved mechanism for sulfur donation but did not provide any conclusive insights into the mechanism for iron delivery from the iron donor, frataxin, to the scaffold. We previously showed that oligomerization is a mechanism by which yeast frataxin (Yfh1) can promote assembly of the core machinery for Fe-S cluster synthesis both in vitro and in cells, in such a manner that the scaffold protein, Isu1, can bind to Yfh1 independent of the presence of the cysteine desulfurase, Nfs1. Here, in the absence of Yfh1, Isu1 was found to exist in two forms, one mostly monomeric with limited tendency to dimerize, and one with a strong propensity to oligomerize. Whereas the monomeric form is stabilized by zinc, the loss of zinc promotes formation of dimer and higher order oligomers. However, upon binding to oligomeric Yfh1, both forms take on a similar symmetrical trimeric configuration that places the Fe-S cluster coordinating residues of Isu1 in close proximity of iron-binding residues of Yfh1. This configuration is suitable for docking of Nfs1 in a manner that provides a structural context for coordinate iron and sulfur donation to the scaffold. Moreover, distinct structural features suggest that in physiological conditions the zinc-regulated abundance of monomeric vs. oligomeric Isu1 yields [Yfh1]·[Isu1] complexes with different Isu1 configurations that afford unique functional properties for Fe-S cluster assembly and delivery.

Protein kinase substrate identification on functional protein arrays

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Zhou Fang

2008-02-01

Full Text Available Abstract Background Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. Results To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Conclusion Functional protein microarrays are an

Analysis of blood clearance and labeled metabolites for the estrogen receptor tracer [F-18]-16α-fluorestradiol (FES)

International Nuclear Information System (INIS)

Mankoff, David A.; Tewson, Timothy J.; Eary, Janet F.

1997-01-01

[F-18] 16α-Fluoroestradiol (FES) has been shown to be a tracer of estrogen receptor content in breast tumors; however, quantitative analysis of FES images is complicated by the rapid metabolism of the tracer in vivo. To optimize FES PET imaging studies and to provide an input function for the quantitative analysis of the tracer FES uptake in breast tumors, we studied the clearance and metabolism of FES in 15 breast cancer patients. FES clearance, protein binding, and metabolite production and limited assays to determine the identity of labeled metabolites were performed. These studies show that FES was rapidly cleared from the blood and metabolized; at 20 min only 20% of the circulating radioactivity was unmetabolized FES, and much of this was protein bound. The detectable metabolites in either blood or urine are conjugation products, largely the glucuronide and the sulfate of FES, and these are excreted through the kidneys at a rate comparable to their introduction into the circulation. After 20 min postinjection the blood levels of radioactivity remain fairly constant. Our results, the first report on human metabolites, are in close agreement with previous animal studies of FES metabolism. These studies show that because FES clearance is rapid and metabolite background is nearly constant, imaging starting at 20 to 30 min after injection may provide good visualization of estrogen-containing tissues. Labeled metabolites need to be accounted for in quantifying FES uptake

The Fps/Fes kinase regulates leucocyte recruitment and extravasation during inflammation.

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Parsons, Sean A; Mewburn, Jeffrey D; Truesdell, Peter; Greer, Peter A

2007-12-01

Fps/Fes and Fer comprise a distinct subfamily of cytoplasmic protein-tyrosine kinases, and have both been implicated in the regulation of innate immunity. Previous studies showed that Fps/Fes-knockout mice were hypersensitive to systemic lipopolysaccharide (LPS) challenge, and Fer-deficient mice displayed enhanced recruitment of leucocytes in response to localized LPS challenge. We show here for the first time, a role for Fps in the regulation of leucocyte recruitment to areas of inflammation. Using the cremaster muscle intravital microscopy model, we observed increased leucocyte adherence to venules, and increased rates and degrees of transendothelial migration in Fps/Fes-knockout mice relative to wild-type animals subsequent to localized LPS challenge. There was also a decreased vessel wall shear rate in the post-capillary venules of LPS-challenged Fps/Fes-knockout mice, and an increase in neutrophil migration into the peritoneal cavity subsequent to thioglycollate challenge. Using flow cytometry to quantify the expression of surface molecules, we observed prolonged expression of the selectin ligand PSGL-1 on peripheral blood neutrophils from Fps/Fes-knockout mice stimulated ex vivo with LPS. These observations provide important insights into the observed in vivo behaviour of leucocytes in LPS-challenged Fps/Fes-knockout mice and provide evidence that the Fps/Fes kinase plays an important role in the innate immune response.

Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensible in vivo and conserved in evolution

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Ciesielski, Szymon; Schilke, Brenda; Osipiuk, Jerzy; Bigelow, Lance; Mulligan, Rory; Majewska, Julia; Joachimiak, Andrzej; Marszalek, Jaroslaw; Craig, Elizabeth A.; Dutkiewicz, Rafal

2012-01-01

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70’s ATPase activity. However, in apparent contradiction, we previously reported that an 8 fold decrease in Jac1’s affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, a total of eight surface exposed residues were determined to play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype and a reduction in the activity of Fe-S cluster containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensible role in the essential process of mitochondrial Fe-S cluster biogenesis. PMID:22306468

Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensable in vivo and conserved in evolution.

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Ciesielski, Szymon J; Schilke, Brenda A; Osipiuk, Jerzy; Bigelow, Lance; Mulligan, Rory; Majewska, Julia; Joachimiak, Andrzej; Marszalek, Jaroslaw; Craig, Elizabeth A; Dutkiewicz, Rafal

2012-03-16

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70's ATPase activity. However, in apparent contradiction, we previously reported that an 8-fold decrease in Jac1's affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here, we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, we determined that a total of eight surface-exposed residues play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype, and a reduction in the activity of Fe-S cluster-containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensable role in the essential process of mitochondrial Fe-S cluster biogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

The end for FES?

International Nuclear Information System (INIS)

Koedijk, K.

2009-01-01

In 1993, the Dutch Economic Structure Enhancement Fund (FES) was implemented. One of its objectives was to use some of the returns from gas extraction for important national investments that strengthen the economic structure. Up till 2007 the fund financed investments to the amount of 22 bln euro. Keeping FES in its current shape will require some adjustments. A frequently mentioned alternative is to turn FES into a financial investment fund (Sovereign Wealth Fund of SWF), thus allowing the fund to serve future generation. [nl

[Nitrogen oxide is involved in the regulation of the Fe-S cluster assembly in proteins and the formation of biofilms by Escherichia coli cells].

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Vasil'eva, S V; Streltsova, D A; Starostina, I A; Sanina, N A

2013-01-01

The functions of nitrogen oxide (NO) in the regulation of the reversible processes of Fe-S cluster assembly in proteins and the formation of Escherichia coli biofilms have been investigated. S-nitrosoglutathione (GSNO) and crystalline nitrosyl complexes of iron with sulfur-containing aliphatic ligands cisaconite (CisA) and penaconite have been used as NO donors for the first time. Wild-type E. coli cells of the strain MC4100, mutants deltaiscA and deltasufA, and the double paralog mutant deltaiscA/sufA with deletions in the alternative pathways of Fe2+ supply for cluster assembly (all derived from the above-named strain) were used in this study. Plankton growth of bacterial cultures, the mass of mature biofilms, and the expression of the SoxRS[2Fe-2S] regulon have been investigated and shown to depend on strain genotype, the process of Fe-S cluster assembly in iron-sulfur proteins, NO donor structure, and the presence of Fe2+ chelator ferene in the incubation medium. The antibiotic ciprofloxacine (CF) was used as an inhibitor of E. coli biofilm formation in the positive control. NO donors regulating Fe-S cluster assembly in E. coli have been shown to control plankton growth of the cultures and the process of mature biofilm formation; toxic doses of NO caused a dramatic (3- to 4-fold) stimulation of cell entry into biofilms as a response to nitrosative stress; NO donors CisA and GSNO in physiological concentrations suppressed the formation of mature biofilms, and the activity of these compounds was comparable to that of CE Regulation of both Fe-S cluster assembly in iron-sulfur proteins and biofilm formation by NO is indicative of the connection between these processes in E. coli.

TLR-activated repression of Fe-S cluster biogenesis drives a metabolic shift and alters histone and tubulin acetylation.

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Tong, Wing-Hang; Maio, Nunziata; Zhang, De-Liang; Palmieri, Erika M; Ollivierre, Hayden; Ghosh, Manik C; McVicar, Daniel W; Rouault, Tracey A

2018-05-22

Given the essential roles of iron-sulfur (Fe-S) cofactors in mediating electron transfer in the mitochondrial respiratory chain and supporting heme biosynthesis, mitochondrial dysfunction is a common feature in a growing list of human Fe-S cluster biogenesis disorders, including Friedreich ataxia and GLRX5-related sideroblastic anemia. Here, our studies showed that restriction of Fe-S cluster biogenesis not only compromised mitochondrial oxidative metabolism but also resulted in decreased overall histone acetylation and increased H3K9me3 levels in the nucleus and increased acetylation of α-tubulin in the cytosol by decreasing the lipoylation of the pyruvate dehydrogenase complex, decreasing levels of succinate dehydrogenase and the histone acetyltransferase ELP3, and increasing levels of the tubulin acetyltransferase MEC17. Previous studies have shown that the metabolic shift in Toll-like receptor (TLR)-activated myeloid cells involves rapid activation of glycolysis and subsequent mitochondrial respiratory failure due to nitric oxide (NO)-mediated damage to Fe-S proteins. Our studies indicated that TLR activation also actively suppresses many components of the Fe-S cluster biogenesis machinery, which exacerbates NO-mediated damage to Fe-S proteins by interfering with cluster recovery. These results reveal new regulatory pathways and novel roles of the Fe-S cluster biogenesis machinery in modifying the epigenome and acetylome and provide new insights into the etiology of Fe-S cluster biogenesis disorders.

Enhanced endotoxin sensitivity in fps/fes-null mice with minimal defects in hematopoietic homeostasis.

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Zirngibl, Ralph A; Senis, Yotis; Greer, Peter A

2002-04-01

The fps/fes proto-oncogene encodes a cytoplasmic protein tyrosine kinase implicated in growth factor and cytokine receptor signaling and thought to be essential for the survival and terminal differentiation of myeloid progenitors. Fps/Fes-null mice were healthy and fertile, displayed slightly reduced numbers of bone marrow myeloid progenitors and circulating mature myeloid cells, and were more sensitive to lipopolysaccharide (LPS). These phenotypes were rescued using a fps/fes transgene. This confirmed that Fps/Fes is involved in, but not required for, myelopoiesis and that it plays a role in regulating the innate immune response. Bone marrow-derived Fps/Fes-null macrophages showed no defects in granulocyte-macrophage colony-stimulating factor-, interleukin 6 (IL-6)-, or IL-3-induced activation of signal transducer and activator of transcription 3 (Stat3) and Stat5A or LPS-induced degradation of I kappa B or activation of p38, Jnk, Erk, or Akt.

Emerging critical roles of Fe-S clusters in DNA replication and repair

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Fuss, Jill O.; Tsai, Chi-Lin; Ishida, Justin P.; Tainer, John A.

2015-01-01

Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life – the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair. PMID:25655665

Fe/S protein biogenesis in trypanosomes — A review

Czech Academy of Sciences Publication Activity Database

Lukeš, Julius; Basu, Somsuvro

2015-01-01

Roč. 1853, č. 6 (2015), s. 1481-1492 ISSN 0167-4889 R&D Projects: GA ČR GAP305/12/2261; GA MŠk(CZ) EE2.3.30.0032; GA ČR(CZ) GA14-23986S EU Projects: European Commission(XE) 316304 Institutional support: RVO:60077344 Keywords : Fe/S cluster * Trypanosoma brucei * protists * Kinetoplastida Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.128, year: 2015

The E. coli monothiol glutaredoxin GrxD forms homodimeric and heterodimeric FeS cluster containing complexes.

Science.gov (United States)

Yeung, N; Gold, B; Liu, N L; Prathapam, R; Sterling, H J; Willams, E R; Butland, G

2011-10-18

Monothiol glutaredoxins (mono-Grx) represent a highly evolutionarily conserved class of proteins present in organisms ranging from prokaryotes to humans. Mono-Grxs have been implicated in iron sulfur (FeS) cluster biosynthesis as potential scaffold proteins and in iron homeostasis via an FeS-containing complex with Fra2p (homologue of E. coli BolA) in yeast and are linked to signal transduction in mammalian systems. However, the function of the mono-Grx in prokaryotes and the nature of an interaction with BolA-like proteins have not been established. Recent genome-wide screens for E. coli genetic interactions reported the synthetic lethality (combination of mutations leading to cell death; mutation of only one of these genes does not) of a grxD mutation when combined with strains defective in FeS cluster biosynthesis (isc operon) functions [Butland, G., et al. (2008) Nature Methods 5, 789-795]. These data connected the only E. coli mono-Grx, GrxD to a potential role in FeS cluster biosynthesis. We investigated GrxD to uncover the molecular basis of this synthetic lethality and observed that GrxD can form FeS-bound homodimeric and BolA containing heterodimeric complexes. These complexes display substantially different spectroscopic and functional properties, including the ability to act as scaffold proteins for intact FeS cluster transfer to the model [2Fe-2S] acceptor protein E. coli apo-ferredoxin (Fdx), with the homodimer being significantly more efficient. In this work, we functionally dissect the potential cellular roles of GrxD as a component of both homodimeric and heterodimeric complexes to ultimately uncover if either of these complexes performs functions linked to FeS cluster biosynthesis. © 2011 American Chemical Society

Probing protein phosphatase substrate binding

DEFF Research Database (Denmark)

Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

2012-01-01

Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein Prenylation

Energy Technology Data Exchange (ETDEWEB)

DeGraw, Amanda J.; Hast, Michael A.; Xu, Juhua; Mullen, Daniel; Beese, Lorena S.; Barany, George; Distefano, Mark D. (Duke); (UMM)

2010-11-15

Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications.

Activated Fps/Fes partially rescues the in vivo developmental potential of Flk1-deficient vascular progenitor cells.

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Haigh, Jody J; Ema, Masatsugu; Haigh, Katharina; Gertsenstein, Marina; Greer, Peter; Rossant, Janet; Nagy, Andras; Wagner, Erwin F

2004-02-01

Relatively little is known about the modulators of the vascular endothelial growth factor A (VEGF-A)/Flk1 signaling cascade. To functionally characterize this pathway, VEGF-A stimulation of endothelial cells was performed. VEGF-A-mediated Flk1 activation resulted in increased translocation of the endogenous Fps/Fes cytoplasmic tyrosine kinase to the plasma membrane and increased tyrosine phosphorylation, suggesting a role for Fps/Fes in VEGF-A/Flk1 signaling events. Addition of a myristoylation consensus sequence to Fps/Fes resulted in VEGF-A-independent membrane localization of Fps/Fes in endothelial cells. Expression of the activated Fps/Fes protein in Flk1-deficient embryonic stem (ES) cells rescued their contribution to the developing vascular endothelium in vivo by using ES cell-derived chimeras. Activated Fps/Fes contributed to this rescue event by restoring the migratory potential to Flk1 null progenitors, which is required for movement of hemangioblasts from the primitive streak region into the yolk sac proper. Activated Fps/Fes in the presence of Flk1 increased the number of hemangioblast colonies in vitro and increased the number of mesodermal progenitors in vivo. These results suggest that Fps/Fes may act synergistically with Flk1 to modulate hemangioblast differentiation into the endothelium. We have also demonstrated that activated Fps/Fes causes hemangioma formation in vivo, independently of Flk1, as a result of increasing vascular progenitor density.

Fe-S cluster coordination of the chromokinesin KIF4A alters its sub-cellular localization during mitosis.

Science.gov (United States)

Ben-Shimon, Lilach; Paul, Viktoria D; David-Kadoch, Galit; Volpe, Marina; Stümpfig, Martin; Bill, Eckhard; Mühlenhoff, Ulrich; Lill, Roland; Ben-Aroya, Shay

2018-05-30

Fe-S clusters act as co-factors of proteins with diverse functions, e.g. in DNA repair. Down-regulation of the cytosolic iron-sulfur protein assembly (CIA) machinery promotes genomic instability by the inactivation of multiple DNA repair pathways. Furthermore, CIA deficiencies are associated with so far unexplained mitotic defects. Here, we show that CIA2B and MMS19, constituents of the CIA targeting complex involved in facilitating Fe-S cluster insertion into cytosolic and nuclear target proteins, co-localize with components of the mitotic machinery. Down-regulation of CIA2B and MMS19 impairs the mitotic cycle. We identify the chromokinesin KIF4A as a mitotic component involved in these effects. KIF4A binds a Fe-S cluster in vitro through its conserved cysteine-rich domain. We demonstrate in vivo that this domain is required for the mitosis-related KIF4A localization and for the mitotic defects associated with KIF4A knockout. KIF4A is the first identified mitotic component carrying such a post-translational modification. These findings suggest that the lack of Fe-S clusters in KIF4A upon down-regulation of the CIA targeting complex contributes to the mitotic defects. © 2018. Published by The Company of Biologists Ltd.

A structural classification of substrate-binding proteins

NARCIS (Netherlands)

Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

2010-01-01

Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

Kinome signaling through regulated protein-protein interactions in normal and cancer cells.

Science.gov (United States)

Pawson, Tony; Kofler, Michael

2009-04-01

The flow of molecular information through normal and oncogenic signaling pathways frequently depends on protein phosphorylation, mediated by specific kinases, and the selective binding of the resulting phosphorylation sites to interaction domains present on downstream targets. This physical and functional interplay of catalytic and interaction domains can be clearly seen in cytoplasmic tyrosine kinases such as Src, Abl, Fes, and ZAP-70. Although the kinase and SH2 domains of these proteins possess similar intrinsic properties of phosphorylating tyrosine residues or binding phosphotyrosine sites, they also undergo intramolecular interactions when linked together, in a fashion that varies from protein to protein. These cooperative interactions can have diverse effects on substrate recognition and kinase activity, and provide a variety of mechanisms to link the stimulation of catalytic activity to substrate recognition. Taken together, these data have suggested how protein kinases, and the signaling pathways in which they are embedded, can evolve complex properties through the stepwise linkage of domains within single polypeptides or multi-protein assemblies.

Human-FES cooperative control for wrist movement: a preliminary study

Directory of Open Access Journals (Sweden)

Kai Gui

2016-07-01

Full Text Available Functional electrical stimulation (FES sometimes applies to patients with partial paralysis, so human voluntary control and FES control both exist. Our study aims to build a cooperative controller to achieve human-FES cooperation. This cooperative controller is formed by a classical FES controller and an impedance controller. The FES controller consists of a back propagation (BP neural network-based feedforward controller and a PID-based feedback controller. The function of impedance controller is to convert volitional force/torque, which is estimated from a three-stage filter based on EMG, into additional angle. The additional angle can reduce the FES intensity in our cooperative controller, comparing to that in classical FES controller. Some assessment experiments are designed to test the performance of the cooperative controller.

Regulation of human Nfu activity in Fe-S cluster delivery-characterization of the interaction between Nfu and the HSPA9/Hsc20 chaperone complex.

Science.gov (United States)

Wachnowsky, Christine; Liu, Yushi; Yoon, Taejin; Cowan, J A

2018-01-01

Iron-sulfur cluster biogenesis is a complex, but highly regulated process that involves de novo cluster formation from iron and sulfide ions on a scaffold protein, and subsequent delivery to final targets via a series of Fe-S cluster-binding carrier proteins. The process of cluster release from the scaffold/carrier for transfer to the target proteins may be mediated by a dedicated Fe-S cluster chaperone system. In human cells, the chaperones include heat shock protein HSPA9 and the J-type chaperone Hsc20. While the role of chaperones has been somewhat clarified in yeast and bacterial systems, many questions remain over their functional roles in cluster delivery and interactions with a variety of human Fe-S cluster proteins. One such protein, Nfu, has recently been recognized as a potential interaction partner of the chaperone complex. Herein, we examined the ability of human Nfu to function as a carrier by interacting with the human chaperone complex. Human Nfu is shown to bind to both chaperone proteins with binding affinities similar to those observed for IscU binding to the homologous HSPA9 and Hsc20, while Nfu can also stimulate the ATPase activity of HSPA9. Additionally, the chaperone complex was able to promote Nfu function by enhancing the second-order rate constants for Fe-S cluster transfer to target proteins and providing directionality in cluster transfer from Nfu by eliminating promiscuous transfer reactions. Together, these data support a hypothesis in which Nfu can serve as an alternative carrier protein for chaperone-mediated cluster release and delivery in Fe-S cluster biogenesis and trafficking. © 2017 Federation of European Biochemical Societies.

The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines

Energy Technology Data Exchange (ETDEWEB)

Kim, Hyo-Jin; Lee, Kang-Lok; Kim, Kyoung-Dong; Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr

2016-09-09

Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1. - Highlights: • Fep1, a prototype fungal iron uptake regulator, was isolated stably from Schizosaccharomyces pombe. • Fep1 exhibits UV–visible absorption spectrum, characteristic of [Fe-S] proteins. • The iron and sulfide contents in purified or reconstituted Fep1 also support [Fe-S]. • The conserved cysteines are critical for [Fe-S]-binding. • EPR spectra at 5 K and 123 K suggest a mixed population of [Fe-S].

Immobilization of aqueous Hg(II) by mackinawite (FeS)

International Nuclear Information System (INIS)

Liu Jianrong; Valsaraj, Kalliat T.; Devai, Istvan; DeLaune, R.D.

2008-01-01

As one of the major constituents of acid volatile sulfide (AVS) in anoxic sediments, mackinawite (FeS) is known for its ability to scavenge trace metals. The interaction between aqueous Hg(II) (added as HgCl 2 ) and synthetic FeS was studied via batch sorption experiments conducted under anaerobic conditions. Due to the release of H + during formation of hydrolyzed Hg(II) species which is more reactive than Hg 2+ in surface adsorption, the equilibrium pH decreased with the increase in Hg(II)/FeS molar ratio. Counteracting the loss of FeS solids at lower pH, the maximum capacity for FeS to remove aqueous Hg(II) was approximately 0.75 mol Hg(II) (mol FeS) -1 . The comparison of X-ray power diffraction (XRPD) patterns of synthetic FeS sorbent before and after sorption showed that the major products formed from the interaction between FeS and the aqueous Hg(II) were metacinnabar, cinnabar, and mercury iron sulfides. With the addition of FeS at 0.4 g L -1 to a 1 mM Hg(II) solution with an initial pH of 5.6, Fe 2+ release was approximately 0.77 mol Fe 2+ per mol Hg(II) removed, suggesting that 77% of Hg(II) was removed via precipitation reaction under these conditions, with 23% of Hg(II) removed by adsorption. Aeration does not cause significant release of Hg(II) into the water phase

Synthesis and electrical conductivity of nanocrystalline tetragonal FeS

International Nuclear Information System (INIS)

Zeng Shu-Lin; Wang Hui-Xian; Dong Cheng

2014-01-01

A convenient method for synthesis of tetragonal FeS using iron powder as iron source, is reported. Nanocrystalline tetragonal FeS samples were successfully synthesized by reacting metallic iron powder with sodium sulfide in acetate buffer solution. The obtained sample is single-phase tetragonal FeS with lattice parameters a = 0.3767 nm and c = 0.5037 nm, as revealed by X-ray diffraction. The sample consists of flat nanosheets with lateral dimensions from 20 nm up to 200 nm and average thickness of about 20 nm. We found that tetragonal FeS is a fairly good conductor from the electrical resistivity measurement on a pellet of the nanosheets. The temperature dependence of conductivity of the pellet was well fitted using an empirical equation wherein the effect of different grain boundaries was taken into consideration. This study provides a convenient, economic way to synthesize tetragonal FeS in a large scale and reports the first electrical conductivity data for tetragonal FeS down to liquid helium temperature. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

FES in Europe and beyond: Current Translational Research

Directory of Open Access Journals (Sweden)

Christine Azevedo Coste

2016-12-01

Full Text Available Capacity of adult neural and muscle tissues to respond to external Electrical Stimulation (ES is the biological basis for the development and implementation of mobility impairment physiotherapy protocols and of related assistive technologies, e.g, Functional Electrical Stimulation (FES. All body tissues, however, respond to electrical stimulation and, indeed, the most successful application of FES is electrical stimulation of the heart to revert or limit effects of arrhythmias (Pace-makers and Defibrillators. Here, we list and discuss results of FES current research activities, in particular those presented at 2016 Meetings: the PaduaMuscleDays, the Italian Institute of Myology Meeting, the 20th International Functional Electrical Stimulation Society (IFESS conference held in Montpellier and the Vienna Workshop on FES. Several papers were recently e-published in the European Journal of Translational Myology as reports of meeting presentations. All the events and publications clearly show that FES research in Europe and beyond is alive and promisses translation of results into clinical management of a very large population of persons with deficiencies.

Anti-friction performance of FeS nanoparticle synthesized by biological method

Energy Technology Data Exchange (ETDEWEB)

Zhou, Lu Hai, E-mail: lhzhou@t.shu.edu.cn [School of Materials Science and Engineering, Shanghai University, Shanghai 200444 (China); Wei, Xi Cheng [School of Materials Science and Engineering, Shanghai University, Shanghai 200444 (China); Ma, Zi Jian [Pipe and Bar Division of Baoshan Iron & Steel Co., Ltd., Shanghai 200941 (China); Mei, Bin [Shanghai Medical Instrumentation College, Shanghai 200093 (China)

2017-06-15

Highlights: • FeS nanoparticles were successfully prepared by a biological method. • The anti-friction performance of prepared nanoparticle under oil lubricating and dry condition were analyzed. • The anti-friction mechanism of FeS nanoparticle was discussed. - Abstract: FeS nanoparticle is prepared by a biological method. The size, morphology and structure of the FeS nanoparticle are characterized by the means of X-ray diffraction and transmission electron microscopy. The anti-friction behavior of the FeS nanoparticle as a lubricating oil additive is evaluated in the engine oil by using a face-to-face contact mode. The worn surface is characterized by using the scanning electron microscopy and secondary ion mass spectroscopy in order to find the reasons resulting in the reduction of friction coefficient due to the addition of the FeS nanoparticle. The anti-friction mechanism of the FeS nanoparticle is elucidated based on the experimental results.

Automated synthesis of the estrogen receptors imaging agent 18F-FES

International Nuclear Information System (INIS)

Guo Shen; Chen Guobao; Dai Hongfeng; Lin Meifu; Chen Wenxin

2011-01-01

Objective: 18 F-16α-17β-fluoroestradiol ( 18 F-FES), an estrogen receptors imaging agent, is synthesized with Tracerlab FX FN system. Methods: 18 F-FES is obtained by two steps reactions, including the nucleophilic displacement reaction of no-carrier-added 18 F-fluoride with 3-O-methoxymethyl-16, 17-O-sulfuryl-16-epiesteriol, then the intermediate is evaporated and hydrolyzed with HCI and finally gives 18 F-FES. Results: The synthesis of 18 F-FES can be completed in about 80 min.The radiochemical yield and radio-chemical purity are about 10% and 95% respectively. Conclusion: The procedure of synthesis is simple and automatical. 18 F-FES has an extremely low toxicity, which suggests that 18 F-FES may be a safe, a nd effective estrogen receptors imaging agent. (authors)

BCI-FES system for neuro-rehabilitation of stroke patients

Science.gov (United States)

Jure, Fabricio A.; Carrere, Lucía C.; Gentiletti, Gerardo G.; Tabernig, Carolina B.

2016-04-01

Nowadays, strokes are a growing cause of mortality and many people remain with motor sequelae and troubles in the daily activities. To treat this sequelae, alternative rehabilitation techniques are needed. In this article a Brain Computer Interface (BCI) system to control a Functional Electrical Stimulation (FES) system is presented. It can be used as a novel tool in easy setup clinical routines, to improve the rehabilitation process by mean of detecting patient´s motor intention, performing it by FES and finally receiving appropriate feedback The BCI-FES system presented here, consists of three blocks: the first one decodes the patient´s intention and it is composed by the patient, the acquisition hardware and the processing software (Emotiv EPOC®). The second block, based on Arduino’s technology, transforms the information into a valid command signal. The last one excites the patient´s neuromuscular system by means of a FES device. In order to evaluate the cerebral activity sensed by the device, topographic maps were obtained. The BCI-FES system was able to detect the patient´s motor intention and control the FES device. At the time of this publication, the system it’s being employing in a rehabilitation program with patients post stroke.

Transfer of sulfur from IscS to IscU during Fe/S cluster assembly.

Science.gov (United States)

Urbina, H D; Silberg, J J; Hoff, K G; Vickery, L E

2001-11-30

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [(35)S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of (35)S from [(35)S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.

Protein degradation: recognition of ubiquitinylated substrates

DEFF Research Database (Denmark)

Hartmann-Petersen, Rasmus; Gordon, Colin

2004-01-01

A cell-free system has been developed in budding yeast that provides direct evidence that the Dsk2/Dph1, Rad23/Rhp23 and Rpn10/Pus1 multi-ubiquitin-binding proteins, long implicated in substrate recognition and presentation to the 26S proteasome, actually fulfil such a role.......A cell-free system has been developed in budding yeast that provides direct evidence that the Dsk2/Dph1, Rad23/Rhp23 and Rpn10/Pus1 multi-ubiquitin-binding proteins, long implicated in substrate recognition and presentation to the 26S proteasome, actually fulfil such a role....

The Fps/Fes kinase regulates the inflammatory response to endotoxin through down-regulation of TLR4, NF-kappaB activation, and TNF-alpha secretion in macrophages.

Science.gov (United States)

Parsons, Sean A; Greer, Peter A

2006-12-01

Fps/Fes and Fer are members of a distinct subfamily of cytoplasmic protein tyrosine kinases that have recently been implicated in the regulation of innate immunity. Previous studies showed that mice lacking Fps/Fes are hypersensitive to systemic LPS challenge, and Fer-deficient mice displayed enhanced recruitment of leukocytes in response to local LPS challenge. This study identifies physiological, cellular, and molecular defects that contribute to the hyperinflammatory phenotype in Fps/Fes null mice. Plasma TNF-alpha levels were elevated in LPS challenged Fps/Fes null mice as compared with wild-type mice and cultured Fps/Fes null peritoneal macrophages treated with LPS showed increased TNF-alpha production. Cultured Fps/Fes null macrophages also displayed prolonged LPS-induced degradation of IkappaB-alpha, increased phosphorylation of the p65 subunit of NF-kappaB, and defective TLR4 internalization, compared with wild-type macrophages. Together, these observations provide a likely mechanistic basis for elevated proinflammatory cytokine secretion by Fps/Fes null macrophages and the increased sensitivity of Fps/Fes null mice to endotoxin. We posit that Fps/Fes modulates the innate immune response of macrophages to LPS, in part, by regulating internalization and down-regulation of the TLR4 receptor complex.

Can FES-rowing mediate bone mineral density in SCI: a pilot study.

Science.gov (United States)

Gibbons, R S; McCarthy, I D; Gall, A; Stock, C G; Shippen, J; Andrews, B J

2014-11-01

A single case study. To compare proximal tibia trabecular bone mineral density (BMD) of a participant with complete spinal cord injury (SCI), long-termed functional electrical stimulation-rowing (FES-R) trained, with previously reported SCI and non-SCI group norms. To estimate lower limb joint contact forces (JCFs) in the FES-R trained participant. UK University and orthopaedic hospital research centre. Bilateral proximal tibial trabecular BMD of the FES-R trained participant was measured using peripheral quantitative computerised tomography, and the data were compared with SCI and non-SCI groups. An instrumented four-channel FES-R system was used to measure the lower limb JCFs in the FES-R trained participant. Structurally, proximal tibial trabecular BMD was higher in the FES-R trained participant compared with the SCI group, but was less than the non-SCI group. Furthermore, left (184.7 mg cm(-3)) and right (160.7 mg cm(-3)) BMD were well above the threshold associated with non-traumatic fracture. The knee JCFs were above the threshold known to mediate BMD in SCI, but below threshold at the hip and ankle. As pathological fractures predominate in the distal femur and proximal tibia in chronic SCI patients, the fact that the FES-R trained participant's knee JCFs were above those known to partially prevent bone loss, suggests that FES-R training may provide therapeutic benefit. Although the elevated bilateral proximal tibial BMD of the FES-R participant provides circumstantial evidence of osteogenesis, this single case precludes any statement on the clinical significance. Further investigations are required involving larger numbers and additional channels of FES to increase loading at the hip and ankle.

Charge density study of two FeS2 polymorphs

DEFF Research Database (Denmark)

Schmøkel, Mette Stokkebro; Jørgensen, Mads Ry Vogel; Bjerg, Lasse

Experimental charge density studies of inorganic solids have proven to be a difficult task due to systematic errors related to data collection such as absorption and extinction; however, the use of synchrotron radiation has the potential to minimize these problems. [1] One of the pioneering...... experimental electron density studies of an inorganic solid containing a transition metal was presented by Stevens et al. [2] who investigated the effect of crystal-field splitting of the partially filled iron d-orbitals in the pyrite structure of FeS2. Other studies of various FeS2 structures, including...... pyrite, has been performed by Gibbs et al. [3], however, these are all based on theoretical calculations rather than experiment. In the current study we revisit FeS2 through an experimental charge density study of the two low-spin iron FeS2 structures, pyrite and marcasite. High-quality, low...

Comparison of FeS, FeS + S and solid superacid catalytic properties for coal hydro-liquefaction

Energy Technology Data Exchange (ETDEWEB)

Zhicai Wang; Hengfu Shui; Dexiang Zhang; Jinsheng Gao [East China University of Science and Technology, Shanghai (China). College of Resource and Environment Engineering

2007-03-15

Catalyst plays an important role in direct coal liquefaction. This paper focuses on the catalytic behavior of a novel SO{sub 4}{sup 2-}/ZrO{sub 2} superacid catalyst in coal hydro-liquefaction. A series of hydro-liquefaction experiments were conducted under mild conditions - 400{sup o}C, 30 min and H{sub 2} initial pressure 4 MPa in a batch autoclave with a volume of 100 ml. The catalytic property of SO{sub 4}{sup 2-}/ZrO{sub 2} was compared with FeS and FeS + S by Shenhua coal. The liquefaction products catalyzed by different catalysts were analyzed by FTIR spectrum, {sup 1}H NMR spectrum and element analysis. In addition, the SO{sub 4}{sup 2-}/ZrO{sub 2} solid superacid was characterized. The results indicated that the SO{sub 4}{sup 2-}/ZrO{sub 2} solid superacid shows outstanding catalytic property for direct liquefaction of coal and gives the highest coal conversion and gas + oil yield compared to other two catalysts. The THF conversion and the extraction yield of CS{sub 2}/NMP mixed solvent of liquefied coal catalyzed with SO{sub 4}{sup 2-}/ZrO{sub 2} are 76.3%, daf and 81.2%, daf respectively, and the yield of gas + oil is 62.5%, daf under the condition used in this study. The pyrolysis of coal macromolecular clusters can be promoted by catalysts such as FeS, FeS + S and SO{sub 4}{sup 2-}/ZrO{sub 2}. There may be only the pyrolysis of volatile matter and the relaxation of the structure of coal macromolecular clusters in non-catalytic liquefaction at 400{sup o}C. Added sulfur in FeS can improve the catalytic activity of hydrogenation. SO{sub 4}{sup 2-}/ZrO{sub 2} is a notable catalyst in the study of coal direct liquefaction because it shows excellent catalytic activities for the pyrolysis and the hydrogenation. In addition, it has been found that the C-O bond is the most stable group in coal liquefaction reaction except for the covalent bond between carbon and carbon. 34 refs., 6 figs., 6 tabs.

sEMG Signal Acquisition Strategy towards Hand FES Control

Directory of Open Access Journals (Sweden)

Cinthya Lourdes Toledo-Peral

2018-01-01

Full Text Available Due to damage of the nervous system, patients experience impediments in their daily life: severe fatigue, tremor or impaired hand dexterity, hemiparesis, or hemiplegia. Surface electromyography (sEMG signal analysis is used to identify motion; however, standardization of electrode placement and classification of sEMG patterns are major challenges. This paper describes a technique used to acquire sEMG signals for five hand motion patterns from six able-bodied subjects using an array of recording and stimulation electrodes placed on the forearm and its effects over functional electrical stimulation (FES and volitional sEMG combinations, in order to eventually control a sEMG-driven FES neuroprosthesis for upper limb rehabilitation. A two-part protocol was performed. First, personalized templates to place eight sEMG bipolar channels were designed; with these data, a universal template, called forearm electrode set (FELT, was built. Second, volitional and evoked movements were recorded during FES application. 95% classification accuracy was achieved using two sessions per movement. With the FELT, it was possible to perform FES and sEMG recordings simultaneously. Also, it was possible to extract the volitional and evoked sEMG from the raw signal, which is highly important for closed-loop FES control.

Interactions of iron-bound frataxin with ISCU and ferredoxin on the cysteine desulfurase complex leading to Fe-S cluster assembly.

Science.gov (United States)

Cai, Kai; Frederick, Ronnie O; Tonelli, Marco; Markley, John L

2018-06-01

Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron‑sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe 2+ but not Fe 3+ . While FXN (with or without bound Fe 2+ ) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the cysteine desulfurase complex ([NFS1] 2 :[ISD11] 2 :[Acp] 2 ), abbreviated as (NIA) 2 , where "N" represents the cysteine desulfurase (NFS1), "I" represents the accessory protein (ISD11), and "A" represents acyl carrier protein (Acp). FXN binds (NIA) 2 weakly in the absence of ISCU but more strongly in its presence. Fe 2+ -FXN binds to the (NIA) 2 -ISCU 2 complex without release of iron. However, upon the addition of both l-cysteine and a reductant (either reduced FDX2 or DTT), Fe 2+ is released from FXN as consistent with Fe 2+ -FXN being the proximal source of iron for Fe-S cluster assembly. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells

DEFF Research Database (Denmark)

Grankowski, N; Gasior, E; Issinger, O G

1993-01-01

Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential...... phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein...

Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates

Directory of Open Access Journals (Sweden)

Amy eCurran

2011-08-01

Full Text Available The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs. While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16 and 34. Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ~70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites. Of these, 74 (27% were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Resilient carbon encapsulation of iron pyrite (FeS2) cathodes in lithium ion batteries

Science.gov (United States)

Yoder, Tara S.; Tussing, Matthew; Cloud, Jacqueline E.; Yang, Yongan

2015-01-01

Converting iron pyrite (FeS2) from a non-cyclable to a cyclable cathode material for lithium ion batteries has been an ongoing challenge in recent years. Herein we report a promising mitigation strategy: wet-chemistry based conformal encapsulation of synthetic FeS2 nanocrystals in a resilient carbon (RC) matrix (FeS2@RC). The FeS2@RC composite was fabricated by dispersing autoclave-synthesized FeS2 nanocrystals in an aqueous glucose solution, polymerizing the glucose in a hydrothermal reactor, and finally heating the polymer/FeS2 composite in a tube furnace to partially carbonize the polymer. The FeS2@RC electrodes showed superior cyclability compared with the FeS2 electrodes, that is, 25% versus 1% of retention at the 20th cycle. Based on electrochemical analysis, XRD study, and SEM characterization, the performance enhancement was attributed to RC's ability to accommodate volume fluctuation, enhance charge transfer, alleviate detrimental side reactions, and suppress loss of the active material. Furthermore, the remaining issues associated with the current system were identified and future research directions were proposed.

Sliding mode closed-Loop control of FES: controlling the shank movement

NARCIS (Netherlands)

Jezernik, Saso; Wassink, R.G.V.; Keller, Thierry

2004-01-01

Functional electrical stimulation (FES) enables restoration of movement in individuals with spinal cord injury. FES-based devices use electric current pulses to stimulate and excite the intact peripheral nerves. They produce muscle contractions, generate joint torques, and thus, joint movements.

Fps/Fes and Fer protein-tyrosinekinases play redundant roles in regulating hematopoiesis.

Science.gov (United States)

Senis, Yotis A; Craig, Andrew W B; Greer, Peter A

2003-08-01

The highly related protein-tyrosine kinases Fps (also called Fes) and Fer are sole members of a subfamily of kinases. In this study, knock-in mice harboring kinase-inactivating mutations in both fps and fer alleles were used to assess functional redundancy between Fps and Fer kinases in regulating hematopoiesis. Mice harboring kinase-inactivating mutations in fps and fer alleles were generated previously. Compound homozygous mice were bred that lack both Fps and Fer kinase activities and progeny were analyzed for potential defects in viability and fertility. Potential differences in hematopoiesis were analyzed by lineage analysis of bone marrow cells, peripheral blood counts, and hematopoietic progenitor cell colony-forming assays. Mice devoid of both Fps and Fer kinase activities were viable and displayed reduced fertility. Circulating levels of neutrophils, erythrocytes, and platelets were elevated in compound mutant mice compared to wild-type controls, suggesting that hematopoiesis is deregulated in the absence of Fps and Fer kinases. Compound mutant mice also showed reduced overall bone marrow cellularity, and lineage analysis revealed elevated CD11b(hi)Ly-6G(lo) myeloid cells, which may reflect increased granulocyte progenitors. Although no differences in the overall number of granulocyte/monocyte colony-forming progenitors were observed, qualitative differences in myeloid colonies from compound mutant mice suggested a role for Fps and Fer kinases in regulating cell-cell adhesion or a skewing in cellularity of colonies. Mice lacking both Fps and Fer kinase activities develop normally, show reduced fertility, and display defects in hematopoiesis, thus providing evidence for functional redundancy between Fps and Fer kinases in regulating hematopoiesis.

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

Directory of Open Access Journals (Sweden)

Weatherford Wendy

2005-05-01

Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

Science.gov (United States)

Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

Preparation of FeS2 nanotube arrays based on layer-by-layer assembly and their photoelectrochemical properties

International Nuclear Information System (INIS)

Wang, Mudan; Xue, Dongpeng; Qin, Haiying; Zhang, Lei; Ling, Guoping; Liu, Jiabin; Fang, Youtong; Meng, Liang

2016-01-01

Graphical abstract: - Highlights: • Amorphous Fe 2 O 3 nanotube arrays are prepared via layer-by-layer assembly. • Pyrite FeS 2 nanotube arrays are obtained by sulfurizing Fe 2 O 3 nanotube arrays. • Various electrochemical properties are characterized. • A comparison between FeS 2 nanotube and nanoparticle films is conducted. • Nanotube arrays show enhanced corrosion resistance and photoresponse. - Abstract: Well-aligned one-dimensional iron pyrite FeS 2 nanotube arrays have been fabricated via layer-by-layer assembly technique on ZnO nanorod arrays in combination with subsequent sulfurization. The as-prepared products were confirmed to be pure phase pyrite FeS 2 with Fe/S ratio approaching 1/2. Typical nanotube structure was observed for the FeS 2 with average outer diameter of 150 ± 20 nm and wall thickness of 50 ± 5 nm. Comparisons of photoelectrochemical properties between FeS 2 nanotubes and FeS 2 nanoparticles were conducted. Tafel polarization curves and electrochemical impedance spectroscopy indicate that FeS 2 nanotubes possess high corrosion resistance and electrochemical stability. The J–V curves show that the photocurrent at 1.0 V for FeS 2 nanotubes is more than five times larger than that of FeS 2 nanoparticles, indicating enhanced photoresponse and rapid charge transfer performances of 1-D nanotube structure. The enhanced photoelectrochemical properties mainly benefit from the unique architecture features of nanotube array structure.

Hybrid FES-robot cooperative control of ambulatory gait rehabilitation exoskeleton.

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del-Ama, Antonio J; Gil-Agudo, Angel; Pons, José L; Moreno, Juan C

2014-03-04

Robotic and functional electrical stimulation (FES) approaches are used for rehabilitation of walking impairment of spinal cord injured individuals. Although devices are commercially available, there are still issues that remain to be solved. Control of hybrid exoskeletons aims at blending robotic exoskeletons and electrical stimulation to overcome the drawbacks of each approach while preserving their advantages. Hybrid actuation and control have a considerable potential for walking rehabilitation but there is a need of novel control strategies of hybrid systems that adequately manage the balance between FES and robotic controllers. Combination of FES and robotic control is a challenging issue, due to the non-linear behavior of muscle under stimulation and the lack of developments in the field of hybrid control. In this article, a cooperative control strategy of a hybrid exoskeleton is presented. This strategy is designed to overcome the main disadvantages of muscular stimulation: electromechanical delay and change in muscle performance over time, and to balance muscular and robotic actuation during walking.Experimental results in healthy subjects show the ability of the hybrid FES-robot cooperative control to balance power contribution between exoskeleton and muscle stimulation. The robotic exoskeleton decreases assistance while adequate knee kinematics are guaranteed. A new technique to monitor muscle performance is employed, which allows to estimate muscle fatigue and implement muscle fatigue management strategies. Kinesis is therefore the first ambulatory hybrid exoskeleton that can effectively balance robotic and FES actuation during walking. This represents a new opportunity to implement new rehabilitation interventions to induce locomotor activity in patients with paraplegia.Acronym list: 10 mWT: ten meters walking test; 6 MWT: six minutes walking test; FSM: finite-state machine; t-FSM: time-domain FSM; c-FSM: cycle-domain FSM; FES: functional electrical

Altered sterol metabolism in budding yeast affects mitochondrial iron-sulfur (Fe-S) cluster synthesis.

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Ward, Diane M; Chen, Opal S; Li, Liangtao; Kaplan, Jerry; Bhuiyan, Shah Alam; Natarajan, Selvamuthu K; Bard, Martin; Cox, James E

2018-05-17

Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho- or Rho0) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron-sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial Fe metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29. Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increase mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

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Mutsuki Amano

Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

Substrate-Bound Protein Gradients to Study Haptotaxis

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Sebastien G. Ricoult

2015-03-01

Full Text Available Cells navigate in response to inhomogeneous distributions of extracellular guidance cues. The cellular and molecular mechanisms underlying migration in response to gradients of chemical cues have been investigated for over a century. Following the introduction of micropipettes and more recently microfluidics for gradient generation, much attention and effort was devoted to study cellular chemotaxis, which is defined as guidance by gradients of chemical cues in solution. Haptotaxis, directional migration in response to gradients of substrate-bound cues, has received comparatively less attention; however it is increasingly clear that in vivo many physiologically relevant guidance proteins – including many secreted cues – are bound to cellular surfaces or incorporated into extracellular matrix and likely function via a haptotactic mechanism. Here, we review the history of haptotaxis. We examine the importance of the reference surface, the surface in contact with the cell that is not covered by the cue, which forms a gradient opposing the gradient of the protein cue and must be considered in experimental designs and interpretation of results. We review and compare microfluidics, contact-printing, light patterning and 3D fabrication to pattern substrate-bound protein gradients in vitro, and focus on their application to study axon guidance. The range of methods to create substrate-bound gradients discussed herein make possible systematic analyses of haptotactic mechanisms. Furthermore, understanding the fundamental mechanisms underlying cell motility will inform bioengineering approaches to program cell navigation and recover lost function.

Interpretation of the photoelectron spectra of FeS(2)(-) by a multiconfiguration computational approach.

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Clima, Sergiu; Hendrickx, Marc F A

2007-11-01

The ground states of FeS(2) and FeS(2)(-), and several low-lying excited electronic states of FeS(2) that are responsible for the FeS(2)(-) photoelectron spectrum, are calculated. At the B3LYP level an open, quasi-linear [SFeS](-) conformation is found as the most stable structure, which is confirmed at the ab initio CASPT2 computational level. Both the neutral and the anionic unsaturated complexes possess high-spin electronic ground states. For the first time a complete assignment of the photoelectron spectrum of FeS(2)(-) is proposed. The lowest energy band in this spectrum is ascribed to an electron detachment from the two highest-lying 3dpi antibonding orbitals (with respect to the iron-sulfur bonding) of iron. The next-lowest experimental band corresponds to an electron removal from nonbonding, nearly pure sulfur orbitals. The two highest bands in the spectra are assigned as electron detachments from pi and sigma bonding mainly sulfur orbitals.

Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU.

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Hoff, Kevin G; Ta, Dennis T; Tapley, Tim L; Silberg, Jonathan J; Vickery, Larry E

2002-07-26

Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.

Prediction of membrane transport proteins and their substrate specificities using primary sequence information.

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Nitish K Mishra

Full Text Available Membrane transport proteins (transporters move hydrophilic substrates across hydrophobic membranes and play vital roles in most cellular functions. Transporters represent a diverse group of proteins that differ in topology, energy coupling mechanism, and substrate specificity as well as sequence similarity. Among the functional annotations of transporters, information about their transporting substrates is especially important. The experimental identification and characterization of transporters is currently costly and time-consuming. The development of robust bioinformatics-based methods for the prediction of membrane transport proteins and their substrate specificities is therefore an important and urgent task.Support vector machine (SVM-based computational models, which comprehensively utilize integrative protein sequence features such as amino acid composition, dipeptide composition, physico-chemical composition, biochemical composition, and position-specific scoring matrices (PSSM, were developed to predict the substrate specificity of seven transporter classes: amino acid, anion, cation, electron, protein/mRNA, sugar, and other transporters. An additional model to differentiate transporters from non-transporters was also developed. Among the developed models, the biochemical composition and PSSM hybrid model outperformed other models and achieved an overall average prediction accuracy of 76.69% with a Mathews correlation coefficient (MCC of 0.49 and a receiver operating characteristic area under the curve (AUC of 0.833 on our main dataset. This model also achieved an overall average prediction accuracy of 78.88% and MCC of 0.41 on an independent dataset.Our analyses suggest that evolutionary information (i.e., the PSSM and the AAIndex are key features for the substrate specificity prediction of transport proteins. In comparison, similarity-based methods such as BLAST, PSI-BLAST, and hidden Markov models do not provide accurate predictions

In vivo fluorescent detection of Fe-S clusters coordinated by human GRX2.

Science.gov (United States)

Hoff, Kevin G; Culler, Stephanie J; Nguyen, Peter Q; McGuire, Ryan M; Silberg, Jonathan J; Smolke, Christina D

2009-12-24

A major challenge to studying Fe-S cluster biosynthesis in higher eukaryotes is the lack of simple tools for imaging metallocluster binding to proteins. We describe the first fluorescent approach for in vivo detection of 2Fe2S clusters that is based upon the complementation of Venus fluorescent protein fragments via human glutaredoxin 2 (GRX2) coordination of a 2Fe2S cluster. We show that Escherichia coli and mammalian cells expressing Venus fragments fused to GRX2 exhibit greater fluorescence than cells expressing fragments fused to a C37A mutant that cannot coordinate a metallocluster. In addition, we find that maximal fluorescence in the cytosol of mammalian cells requires the iron-sulfur cluster assembly proteins ISCU and NFS1. These findings provide evidence that glutaredoxins can dimerize within mammalian cells through coordination of a 2Fe2S cluster as observed with purified recombinant proteins. Copyright 2009 Elsevier Ltd. All rights reserved.

An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates.

Science.gov (United States)

Molle, Virginie; Leiba, Jade; Zanella-Cléon, Isabelle; Becchi, Michel; Kremer, Laurent

2010-11-01

Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.

Beyond Histones: New Substrate Proteins of Lysine Deacetylases in Arabidopsis Nuclei

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Magdalena Füßl

2018-04-01

Full Text Available The reversible acetylation of lysine residues is catalyzed by the antagonistic action of lysine acetyltransferases and deacetylases, which can be considered as master regulators of their substrate proteins. Lysine deacetylases, historically referred to as histone deacetylases, have profound functions in regulating stress defenses and development in plants. Lysine acetylation of the N-terminal histone tails promotes gene transcription and decondensation of chromatin, rendering the DNA more accessible to the transcription machinery. In plants, the classical lysine deacetylases from the RPD3/HDA1-family have thus far mainly been studied in the context of their deacetylating activities on histones, and their versatility in molecular activities is still largely unexplored. Here we discuss the potential impact of lysine acetylation on the recently identified nuclear substrate proteins of lysine deacetylases from the Arabidopsis RPD3/HDA1-family. Among the deacetylase substrate proteins, many interesting candidates involved in nuclear protein import, transcriptional regulation, and chromatin remodeling have been identified. These candidate proteins represent key starting points for unraveling new molecular functions of the Arabidopsis lysine deacetylases. Site-directed engineering of lysine acetylation sites on these target proteins might even represent a new approach for optimizing plant growth under climate change conditions.

PID Controller Design for FES Applied to Ankle Muscles in Neuroprosthesis for Standing Balance

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Hossein Rouhani

2017-06-01

Full Text Available Closed-loop controlled functional electrical stimulation (FES applied to the lower limb muscles can be used as a neuroprosthesis for standing balance in neurologically impaired individuals. The objective of this study was to propose a methodology for designing a proportional-integral-derivative (PID controller for FES applied to the ankle muscles toward maintaining standing balance for several minutes and in the presence of perturbations. First, a model of the physiological control strategy for standing balance was developed. Second, the parameters of a PID controller that mimicked the physiological balance control strategy were determined to stabilize the human body when modeled as an inverted pendulum. Third, this PID controller was implemented using a custom-made Inverted Pendulum Standing Apparatus that eliminated the effect of visual and vestibular sensory information on voluntary balance control. Using this setup, the individual-specific FES controllers were tested in able-bodied individuals and compared with disrupted voluntary control conditions in four experimental paradigms: (i quiet-standing; (ii sudden change of targeted pendulum angle (step response; (iii balance perturbations that simulate arm movements; and (iv sudden change of targeted angle of a pendulum with individual-specific body-weight (step response. In paradigms (i to (iii, a standard 39.5-kg pendulum was used, and 12 subjects were involved. In paradigm (iv 9 subjects were involved. Across the different experimental paradigms and subjects, the FES-controlled and disrupted voluntarily-controlled pendulum angle showed root mean square errors of <1.2 and 2.3 deg, respectively. The root mean square error (all paradigms, rise time, settle time, and overshoot [paradigms (ii and (iv] in FES-controlled balance were significantly smaller or tended to be smaller than those observed with voluntarily-controlled balance, implying improved steady-state and transient responses of FES

Arsenic sorption to nanoparticulate mackinawite (FeS): An examination of phosphate competition.

Science.gov (United States)

Niazi, Nabeel Khan; Burton, Edward D

2016-11-01

Nanoparticulate mackinawite (FeS) can be an important host-phase for arsenic (As) in sulfidic, subsurface environments. Although not previously investigated, phosphate (PO 4 3- ) may compete with As for available sorption sites on FeS, thereby enhancing As mobility in FeS-bearing soils, sediments and groundwater systems. In this study, we examine the effect of PO 4 3- on sorption of arsenate (As(V)) and arsenite (As(III)) to nanoparticulate FeS at pH 6, 7 and 9. Results show that PO 4 3- (at 0.01-1.0 mM P) did not significantly affect sorption of either As(V) or As(III) to nanoparticulate FeS at initial aqueous As concentrations ranging from 0.01 to 1.0 mM. At pH 9 and 7, sorption of both As(III) and As(V) to nanoparticulate FeS was similar, with distribution coefficient (K d ) values spanning 0.76-15 L g -1 (which corresponds to removal of 87-98% of initial aqueous As(III) and As(V) concentrations). Conversely, at pH 6, the sorption of As(III) was characterized by substantially higher K d values (6.3-93.4 L g -1 ) than those for As(V) (K d  = 0.21-0.96 L g -1 ). Arsenic K-edge X-ray absorption near edge structure (XANES) spectroscopy indicated that up to 52% of the added As(V) was reduced to As(III) in As(V) sorption experiments, as well as the formation of minor amounts of an As 2 S 3 -like species. In As(III) sorption experiments, XANES spectroscopy also demonstrated the formation of an As 2 S 3 -like species and the partial oxidation of As(III) to As(V) (despite the strictly O 2 -free experimental conditions). Overall, the XANES data indicate that As sorption to nanoparticulate FeS involves several redox transformations and various sorbed species, which display a complex dependency on pH and As loading but that are not influenced by the co-occurrence of PO 4 3- . This study shows that nanoparticulate FeS can help to immobilize As(III) and As(V) in sulfidic subsurface environments where As co-exists with PO 4 3- . Copyright © 2016 Elsevier Ltd. All

Exploring hierarchical FeS2/C composite nanotubes arrays as advanced cathode for lithium ion batteries

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Pan, G. X.; Cao, F.; Xia, X. H.; Zhang, Y. J.

2016-11-01

Rational construction of advanced FeS2 cathode is one of research hotspots, and of great importance for developing high-performance lithium ion batteries (LIBs). Herein we report a facile hydrolysis-sulfurization method for fabrication of FeS2/C nanotubes arrays with the help of sacrificial Co2(OH)2CO3 nanowires template and glucose carbonization. Self-supported FeS2/C nanotubes consist of interconnected nanoburrs of 5-20 nm, and show hierarchical porous structure. The FeS2/C nanotubes arrays are demonstrated with enhanced cycling life and noticeable high-rate capability with capacities ranging from 735 mAh g-1 at 0.25 C to 482 mAh g-1 at 1.5 C, superior to those FeS2 counterparts in the literature. The composite nanotubes arrays architecture plays positive roles in the electrochemical enhancement due to combined advantages of large electrode-electrolyte contact area, good strain accommodation, improved electrical conductivity, and enhanced structural stability.

Hydroxyapatite coating on the titanium substrate modulated by a recombinant collagen-like protein

International Nuclear Information System (INIS)

Pan Mingli; Kong Xiangdong; Cai Yurong; Yao Juming

2011-01-01

Research highlights: → Hydroxyapatite was deposited on alkali-heat treated Ti substrate by immersing in 1.5 x SBF solution containing the recombinant collagen-like protein. → The recombinant collagen-like protein accelerated the preferential nucleation and growth of hydroxyapatite along c axis on the Ti substrate. → Hydroxyapatite-collagen composite on the Ti substrate promoted the attachment, subsequently proliferation and differentiation of MG-63 cells. - Abstract: Plenty of techniques have been developed to modify the surface character of titanium (Ti) and its alloys in order to realize their biological bond to natural bone. In this work, a biomimetic process was employed to form a hydroxyapatite (HAp) coating on the alkali-heat treated Ti substrate in 1.5 times simulated body fluid (1.5 x SBF) with the addition of a recombinant collagen-like protein. The coating was characterized using SEM-EDX, FESEM, and XRD. Results showed that the recombinant collagen-like protein could accelerate the preferential nucleation and directional growth along c axis of HAp on the pretreated Ti substrates. The investigation of in vitro cell cultivation showed that the existence of recombinant collagen-like protein in coating could improve the initial cell adhesion, proliferation and differentiation of MG-63 cells, which implied the materials possessed excellent biocompatibility and had a wide potential in biomedical application.

Hydroxyapatite coating on the titanium substrate modulated by a recombinant collagen-like protein

Energy Technology Data Exchange (ETDEWEB)

Pan Mingli [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Kong Xiangdong [College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Cai Yurong [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Yao Juming, E-mail: yaoj@zstu.edu.cn [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China)

2011-04-15

Research highlights: {yields} Hydroxyapatite was deposited on alkali-heat treated Ti substrate by immersing in 1.5 x SBF solution containing the recombinant collagen-like protein. {yields} The recombinant collagen-like protein accelerated the preferential nucleation and growth of hydroxyapatite along c axis on the Ti substrate. {yields} Hydroxyapatite-collagen composite on the Ti substrate promoted the attachment, subsequently proliferation and differentiation of MG-63 cells. - Abstract: Plenty of techniques have been developed to modify the surface character of titanium (Ti) and its alloys in order to realize their biological bond to natural bone. In this work, a biomimetic process was employed to form a hydroxyapatite (HAp) coating on the alkali-heat treated Ti substrate in 1.5 times simulated body fluid (1.5 x SBF) with the addition of a recombinant collagen-like protein. The coating was characterized using SEM-EDX, FESEM, and XRD. Results showed that the recombinant collagen-like protein could accelerate the preferential nucleation and directional growth along c axis of HAp on the pretreated Ti substrates. The investigation of in vitro cell cultivation showed that the existence of recombinant collagen-like protein in coating could improve the initial cell adhesion, proliferation and differentiation of MG-63 cells, which implied the materials possessed excellent biocompatibility and had a wide potential in biomedical application.

Vascular defects in gain-of-function fps/fes transgenic mice correlate with PDGF- and VEGF-induced activation of mutant Fps/Fes kinase in endothelial cells.

Science.gov (United States)

Sangrar, W; Mewburn, J D; Vincent, S G; Fisher, J T; Greer, P A

2004-05-01

Fps/Fes is a cytoplasmic tyrosine kinase that is abundantly expressed in the myeloid, endothelial, epithelial, neuronal and platelet lineages. Genetic manipulation in mice has uncovered potential roles for this kinase in hematopoiesis, innate immunity, inflammation and angiogenesis. We have utilized a genetic approach to explore the role of Fps/Fes in angiogenesis. A hypervascular line of mice generated by expression of a 'gain-of-function' human fps/fes transgene (fps(MF)) encoding a myristoylated variant of Fps (MFps) was used in these studies. The hypervascular phenotype of this line was extensively characterized by intravital microscopy and biochemical approaches. fps(MF) mice exhibited 1.6-1.7-fold increases in vascularity which was attributable to increases in the number of secondary vessels. Vessels were larger, exhibited varicosities and disorganized patterning, and were found to have defects in histamine-induced permeability. Biochemical characterization of endothelial cell (EC) lines derived from fps(MF) mice revealed that MFps was hypersensitive to activation by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). MFps mediates enhanced sensitization to VEGF and PDGF signaling in ECs. We propose that this hypersensitization contributes to excessive angiogenic signaling and that this underlies the observed hypervascular phenotype of fps(MF) mice. These phenotypes recapitulate important aspects of the vascular defects observed in both VEGF and angiopoietin-1 transgenic mice. The fps/fes proto-oncogene product therefore represents a novel player in the regulation of angiogenesis, and the fps(MF) line of mice constitutes a unique new murine model for the study of this process.

Nueva era de CIESPAL: Proyecto CIESPAL-FES

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Fausto Jaramillo

2015-01-01

Full Text Available El artículo presenta una breve descripción del Proyecto -CIESPAL-FES- sus objetivos y un resumen de la investigación que dio lugar al desarrollo del pensum para los becarios que habían de capacitarse en la televisión.

PID Controller Design for FES Applied to Ankle Muscles in Neuroprosthesis for Standing Balance.

Science.gov (United States)

Rouhani, Hossein; Same, Michael; Masani, Kei; Li, Ya Qi; Popovic, Milos R

2017-01-01

Closed-loop controlled functional electrical stimulation (FES) applied to the lower limb muscles can be used as a neuroprosthesis for standing balance in neurologically impaired individuals. The objective of this study was to propose a methodology for designing a proportional-integral-derivative (PID) controller for FES applied to the ankle muscles toward maintaining standing balance for several minutes and in the presence of perturbations. First, a model of the physiological control strategy for standing balance was developed. Second, the parameters of a PID controller that mimicked the physiological balance control strategy were determined to stabilize the human body when modeled as an inverted pendulum. Third, this PID controller was implemented using a custom-made Inverted Pendulum Standing Apparatus that eliminated the effect of visual and vestibular sensory information on voluntary balance control. Using this setup, the individual-specific FES controllers were tested in able-bodied individuals and compared with disrupted voluntary control conditions in four experimental paradigms: (i) quiet-standing; (ii) sudden change of targeted pendulum angle (step response); (iii) balance perturbations that simulate arm movements; and (iv) sudden change of targeted angle of a pendulum with individual-specific body-weight (step response). In paradigms (i) to (iii), a standard 39.5-kg pendulum was used, and 12 subjects were involved. In paradigm (iv) 9 subjects were involved. Across the different experimental paradigms and subjects, the FES-controlled and disrupted voluntarily-controlled pendulum angle showed root mean square errors of controlled balance were significantly smaller or tended to be smaller than those observed with voluntarily-controlled balance, implying improved steady-state and transient responses of FES-controlled balance. At the same time, the FES-controlled balance required similar torque levels (no significant difference) as voluntarily

Protein adsorption on tailored substrates: long-range forces and conformational changes

Energy Technology Data Exchange (ETDEWEB)

Bellion, M; Santen, L [Department of Theoretical Physics, Saarland University, 66041 Saarbruecken (Germany); Mantz, H; Haehl, H; Quinn, A; Nagel, A; Gilow, C; Weitenberg, C; Schmitt, Y; Jacobs, K [Department of Experimental Physics, Saarland University, 66041 Saarbruecken (Germany)], E-mail: k.jacobs@physik.uni-saarland.de

2008-10-08

Adsorption of proteins onto solid surfaces is an everyday phenomenon that is not yet fully understood. To further the current understanding, we have performed in situ ellipsometry studies to reveal the adsorption kinetics of three different proteins, lysozyme, {alpha}-amylase and bovine serum albumin. As substrates we offer Si wafers with a controlled Si oxide layer thickness and a hydrophilic or hydrophobic surface functionalization, allowing the tailoring of the influence of short- and long-range interactions. Our studies show that not only the surface chemistry determines the properties of an adsorbed protein layer but also the van der Waals contributions of a composite substrate. We compare the experimental findings to results of a colloidal Monte Carlo approach that includes conformational changes of the adsorbed proteins induced by density fluctuations.

A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

Science.gov (United States)

Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

2018-01-01

In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.

Directory of Open Access Journals (Sweden)

Wenhan Zhu

2011-03-01

Full Text Available A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter.

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

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Neil Arvin Bretaña

Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

Science.gov (United States)

Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

2012-01-01

Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

Muscle pathology in lower motor neuron paraplegia and h-b FES

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Ugo Carraro

2010-03-01

Full Text Available After complete Spinal Cord Injury (SCI, causing complete disconnection between the muscle fibers and the nervous system, the denervated muscles become unexcitable with commercial electrical stimulators within several months and undergo severe atrophy and disorganization of contractile apparatus after 1-3 years. Years after the injury the surviving and regenerated myofibers are substituted with adipocytes and collagen. To counteract the progressive changes transforming muscle into an unexcitable tissue, we developed a novel therapy concept for paraplegic patients with complete lower motor neuron (LMN denervation of the lower extremities. The new stimulators for home-based functional electrical stimulation (h-b FES have been designed to reverse longstanding and severe atrophy of LMN denervated muscles by delivering high-intensity (up to 2,4 J and long-duration impulses (up to 150 ms able to elicit contractions of denervated skeletal muscle fibers in absence of nerve. Concurrent to the development of the stimulation equipment, specific clinical assessments and training strategies were developed at the Wilhelminenspital Wien, Austria. Main results of our clinical study on 20 patients, which completed a 2 years h-b FES program are: 1. significant +33% increase of muscle size and +75% of the mean diameter of muscle fibers, with striking improvements of the ultra-structural organization of contractile material; 2. recovery of the tetanic contractility with significant increase in muscle force output during electrical stimulation; 3. five subjects performed FES-assisted stand-up and stepping-in-place exercises;. 4. data from ultrastructural analyses indicating that the shorter the time span between SCI and the beginning of h-b FES, the larger were the number and the size of recovered fibers. The study demonstrates that h-b FES of permanent LMN denervated muscle is an effective home therapy that results in rescue of muscle mass, function and perfusion

Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

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Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

2016-02-15

In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Psychometric properties of the original and short versions of the Falls Efficacy Scale-International (FES-I) in people with Parkinson's disease.

Science.gov (United States)

Jonasson, Stina B; Nilsson, Maria H; Lexell, Jan

2017-05-31

Fear of falling is common in people with Parkinson's disease (PD) and is associated with an increased risk for future falls, activity limitations and a reduced quality of life. The Falls Efficacy Scale-International (FES-I) assesses fear of falling conceptualized as concerns about falling. The original FES-I has good psychometric properties in people with PD, but whether this applies also for the short version of FES-I remains to be shown. The aim of the present study was to evaluate the psychometric properties of the short FES-I and to compare these with the original FES-I in the same sample of people with PD. The investigated psychometric properties included known groups validity, data completeness, scaling assumptions, targeting and reliability. A postal survey, which included the original, full-length FES-I, was distributed to 174 people with PD. Responders received a second survey after two weeks. From these data, short FES-I total scores were calculated by extracting the items that are included in the short version of the scale. Median age and PD duration of the 101 responders (43% women) were 73 and 5 years, respectively. The original as well as the short FES-I scores were able to discriminate (p falling, activity avoidance, falls, near falls, and with various self-rated PD severity, respectively. Both versions of FES-I had a high level of data completeness (0.7 to 0.9% missing item responses). Scaling assumptions were acceptable for the original as well as the short FES-I. While the short FES-I had 19% floor effect, the original version was better targeted. Both versions were reliable and obtained high values for internal consistency (Cronbach's alpha >0.8) and test-retest reliability (Intraclass Correlation Coefficient > 0.9). Both the original and short FES-I revealed generally good psychometric properties in people with PD, although the original scale was better targeted. Due to the higher floor effect in the short FES-I, the present findings favors

Impact of FeS Mineralogy on TCE Degradation

Science.gov (United States)

Iron- and sulfate-reducing conditions are often encountered in permeable reactive barrier (PRB) systems that are constructed to remove TCE from groundwater, which usually leads to the accumulation of FeS mineral phases in the matrix of the PRB. Poorly crystalline mackinawite (Fe...

Strategies for Rapid Muscle Fatigue Reduction during FES Exercise in Individuals with Spinal Cord Injury: A Systematic Review.

Science.gov (United States)

Ibitoye, Morufu Olusola; Hamzaid, Nur Azah; Hasnan, Nazirah; Abdul Wahab, Ahmad Khairi; Davis, Glen M

2016-01-01

Rapid muscle fatigue during functional electrical stimulation (FES)-evoked muscle contractions in individuals with spinal cord injury (SCI) is a significant limitation to attaining health benefits of FES-exercise. Delaying the onset of muscle fatigue is often cited as an important goal linked to FES clinical efficacy. Although the basic concept of fatigue-resistance has a long history, recent advances in biomedical engineering, physiotherapy and clinical exercise science have achieved improved clinical benefits, especially for reducing muscle fatigue during FES-exercise. This review evaluated the methodological quality of strategies underlying muscle fatigue-resistance that have been used to optimize FES therapeutic approaches. The review also sought to synthesize the effectiveness of these strategies for persons with SCI in order to establish their functional impacts and clinical relevance. Published scientific literature pertaining to the reduction of FES-induced muscle fatigue was identified through searches of the following databases: Science Direct, Medline, IEEE Xplore, SpringerLink, PubMed and Nature, from the earliest returned record until June 2015. Titles and abstracts were screened to obtain 35 studies that met the inclusion criteria for this systematic review. Following the evaluation of methodological quality (mean (SD), 50 (6) %) of the reviewed studies using the Downs and Black scale, the largest treatment effects reported to reduce muscle fatigue mainly investigated isometric contractions of limited functional and clinical relevance (n = 28). Some investigations (n = 13) lacked randomisation, while others were characterised by small sample sizes with low statistical power. Nevertheless, the clinical significance of emerging trends to improve fatigue-resistance during FES included (i) optimizing electrode positioning, (ii) fine-tuning of stimulation patterns and other FES parameters, (iii) adjustments to the mode and frequency of exercise training

Strategies for Rapid Muscle Fatigue Reduction during FES Exercise in Individuals with Spinal Cord Injury: A Systematic Review.

Directory of Open Access Journals (Sweden)

Morufu Olusola Ibitoye

Full Text Available Rapid muscle fatigue during functional electrical stimulation (FES-evoked muscle contractions in individuals with spinal cord injury (SCI is a significant limitation to attaining health benefits of FES-exercise. Delaying the onset of muscle fatigue is often cited as an important goal linked to FES clinical efficacy. Although the basic concept of fatigue-resistance has a long history, recent advances in biomedical engineering, physiotherapy and clinical exercise science have achieved improved clinical benefits, especially for reducing muscle fatigue during FES-exercise. This review evaluated the methodological quality of strategies underlying muscle fatigue-resistance that have been used to optimize FES therapeutic approaches. The review also sought to synthesize the effectiveness of these strategies for persons with SCI in order to establish their functional impacts and clinical relevance.Published scientific literature pertaining to the reduction of FES-induced muscle fatigue was identified through searches of the following databases: Science Direct, Medline, IEEE Xplore, SpringerLink, PubMed and Nature, from the earliest returned record until June 2015. Titles and abstracts were screened to obtain 35 studies that met the inclusion criteria for this systematic review.Following the evaluation of methodological quality (mean (SD, 50 (6 % of the reviewed studies using the Downs and Black scale, the largest treatment effects reported to reduce muscle fatigue mainly investigated isometric contractions of limited functional and clinical relevance (n = 28. Some investigations (n = 13 lacked randomisation, while others were characterised by small sample sizes with low statistical power. Nevertheless, the clinical significance of emerging trends to improve fatigue-resistance during FES included (i optimizing electrode positioning, (ii fine-tuning of stimulation patterns and other FES parameters, (iii adjustments to the mode and frequency of exercise

Strategies for Rapid Muscle Fatigue Reduction during FES Exercise in Individuals with Spinal Cord Injury: A Systematic Review

Science.gov (United States)

Ibitoye, Morufu Olusola; Hamzaid, Nur Azah; Hasnan, Nazirah; Abdul Wahab, Ahmad Khairi; Davis, Glen M.

2016-01-01

Background Rapid muscle fatigue during functional electrical stimulation (FES)-evoked muscle contractions in individuals with spinal cord injury (SCI) is a significant limitation to attaining health benefits of FES-exercise. Delaying the onset of muscle fatigue is often cited as an important goal linked to FES clinical efficacy. Although the basic concept of fatigue-resistance has a long history, recent advances in biomedical engineering, physiotherapy and clinical exercise science have achieved improved clinical benefits, especially for reducing muscle fatigue during FES-exercise. This review evaluated the methodological quality of strategies underlying muscle fatigue-resistance that have been used to optimize FES therapeutic approaches. The review also sought to synthesize the effectiveness of these strategies for persons with SCI in order to establish their functional impacts and clinical relevance. Methods Published scientific literature pertaining to the reduction of FES-induced muscle fatigue was identified through searches of the following databases: Science Direct, Medline, IEEE Xplore, SpringerLink, PubMed and Nature, from the earliest returned record until June 2015. Titles and abstracts were screened to obtain 35 studies that met the inclusion criteria for this systematic review. Results Following the evaluation of methodological quality (mean (SD), 50 (6) %) of the reviewed studies using the Downs and Black scale, the largest treatment effects reported to reduce muscle fatigue mainly investigated isometric contractions of limited functional and clinical relevance (n = 28). Some investigations (n = 13) lacked randomisation, while others were characterised by small sample sizes with low statistical power. Nevertheless, the clinical significance of emerging trends to improve fatigue-resistance during FES included (i) optimizing electrode positioning, (ii) fine-tuning of stimulation patterns and other FES parameters, (iii) adjustments to the mode and

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

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Sophie A Comyn

2016-07-01

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

Reduced Graphene Oxide-Wrapped FeS2 Composite as Anode for High-Performance Sodium-Ion Batteries

Science.gov (United States)

Wang, Qinghong; Guo, Can; Zhu, Yuxuan; He, Jiapeng; Wang, Hongqiang

2018-06-01

Iron disulfide is considered to be a potential anode material for sodium-ion batteries due to its high theoretical capacity. However, its applications are seriously limited by the weak conductivity and large volume change, which results in low reversible capacity and poor cycling stability. Herein, reduced graphene oxide-wrapped FeS2 (FeS2/rGO) composite was fabricated to achieve excellent electrochemical performance via a facile two-step method. The introduction of rGO effectively improved the conductivity, BET surface area, and structural stability of the FeS2 active material, thus endowing it with high specific capacity, good rate capability, as well as excellent cycling stability. Electrochemical measurements show that the FeS2/rGO composite had a high initial discharge capacity of 1263.2 mAh g-1 at 100 mA g-1 and a high discharge capacity of 344 mAh g-1 at 10 A g-1, demonstrating superior rate performance. After 100 cycles at 100 mA g-1, the discharge capacity remained at 609.5 mAh g-1, indicating the excellent cycling stability of the FeS2/rGO electrode.

Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

Science.gov (United States)

Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

2014-01-01

Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

Molecular dynamics simulations of protein-tyrosine phosphatase 1B: II. Substrate-enzyme interactions and dynamics

DEFF Research Database (Denmark)

Peters, Günther H.j.; Frimurer, T. M.; Andersen, J. N.

2000-01-01

Molecular dynamics simulations of protein tyrosine phosphatase 1B (PTP1B) complexed with the phosphorylated peptide substrate DADEpYL and the free substrate have been conducted to investigate 1) the physical forces involved in substrate-protein interactions, 2) the importance of enzyme...... to substrate binding. Based on essential dynamics analysis of the PTP1B/DADEpYL trajectory, it is shown that internal motions in the binding pocket occur in a subspace of only a few degrees of freedom. in particular, relatively large flexibilities are observed along several eigenvectors in the segments: Arg(24...... for catalysis. Analysis of the individual enzyme-substrate interaction energies revealed that mainly electrostatic forces contribute to binding. Indeed, calculation of the electrostatic field of the enzyme reveals that only the field surrounding the binding pocket is positive, while the remaining protein...

A gait stability investigation into FES-assisted paraplegic walking based on the walker tipping index.

Science.gov (United States)

Ming, Dong; Bai, Yanru; Liu, Xiuyun; Qi, Hongzhi; Cheng, Longlong; Wan, Baikun; Hu, Yong; Wong, Yatwa; Luk, Keith D K; Leong, John C Y

2009-12-01

The gait outcome measures used in clinical trials of paraplegic locomotor training determine the effectiveness of improved walking function assisted by the functional electrical stimulation (FES) system. Focused on kinematic, kinetic or physiological changes of paraplegic patients, traditional methods cannot quantify the walking stability or identify the unstable factors of gait in real time. Up until now, the published studies on dynamic gait stability for the effective use of FES have been limited. In this paper, the walker tipping index (WTI) was used to analyze and process gait stability in FES-assisted paraplegic walking. The main instrument was a specialized walker dynamometer system based on a multi-channel strain-gauge bridge network fixed on the frame of the walker. This system collected force information for the handle reaction vector between the patient's upper extremities and the walker during the walking process; the information was then converted into walker tipping index data, which is an evaluation indicator of the patient's walking stability. To demonstrate the potential usefulness of WTI in gait analysis, a preliminary clinical trial was conducted with seven paraplegic patients who were undergoing FES-assisted walking training and seven normal control subjects. The gait stability levels were quantified for these patients under different stimulation patterns and controls under normal walking with knee-immobilization through WTI analysis. The results showed that the walking stability in the FES-assisted paraplegic group was worse than that in the control subject group, with the primary concern being in the anterior-posterior plane. This new technique is practical for distinguishing useful gait information from the viewpoint of stability, and may be further applied in FES-assisted paraplegic walking rehabilitation.

A gait stability investigation into FES-assisted paraplegic walking based on the walker tipping index

Science.gov (United States)

Ming, Dong; Bai, Yanru; Liu, Xiuyun; Qi, Hongzhi; Cheng, Longlong; Wan, Baikun; Hu, Yong; Wong, Yatwa; Luk, Keith D. K.; Leong, John C. Y.

2009-12-01

The gait outcome measures used in clinical trials of paraplegic locomotor training determine the effectiveness of improved walking function assisted by the functional electrical stimulation (FES) system. Focused on kinematic, kinetic or physiological changes of paraplegic patients, traditional methods cannot quantify the walking stability or identify the unstable factors of gait in real time. Up until now, the published studies on dynamic gait stability for the effective use of FES have been limited. In this paper, the walker tipping index (WTI) was used to analyze and process gait stability in FES-assisted paraplegic walking. The main instrument was a specialized walker dynamometer system based on a multi-channel strain-gauge bridge network fixed on the frame of the walker. This system collected force information for the handle reaction vector between the patient's upper extremities and the walker during the walking process; the information was then converted into walker tipping index data, which is an evaluation indicator of the patient's walking stability. To demonstrate the potential usefulness of WTI in gait analysis, a preliminary clinical trial was conducted with seven paraplegic patients who were undergoing FES-assisted walking training and seven normal control subjects. The gait stability levels were quantified for these patients under different stimulation patterns and controls under normal walking with knee-immobilization through WTI analysis. The results showed that the walking stability in the FES-assisted paraplegic group was worse than that in the control subject group, with the primary concern being in the anterior-posterior plane. This new technique is practical for distinguishing useful gait information from the viewpoint of stability, and may be further applied in FES-assisted paraplegic walking rehabilitation.

Cross-cultural adaptation and measurement properties testing of the Iconographical Falls Efficacy Scale (Icon-FES).

Science.gov (United States)

Franco, Marcia Rodrigues; Pinto, Rafael Zambelli; Delbaere, Kim; Eto, Bianca Yumie; Faria, Maíra Sgobbi; Aoyagi, Giovana Ayumi; Steffens, Daniel; Pastre, Carlos Marcelo

2018-02-14

The Iconographical Falls Efficacy Scale (Icon-FES) is an innovative tool to assess concern of falling that uses pictures as visual cues to provide more complete environmental contexts. Advantages of Icon-FES over previous scales include the addition of more demanding balance-related activities, ability to assess concern about falling in highly functioning older people, and its normal distribution. To perform a cross-cultural adaptation and to assess the measurement properties of the 30-item and 10-item Icon-FES in a community-dwelling Brazilian older population. The cross-cultural adaptation followed the recommendations of international guidelines. We evaluated the measurement properties (i.e. internal consistency, test-retest reproducibility, standard error of the measurement, minimal detectable change, construct validity, ceiling/floor effect, data distribution and discriminative validity), in 100 community-dwelling people aged ≥60 years. The 30-item and 10-item Icon-FES-Brazil showed good internal consistency (alpha and omega >0.70) and excellent intra-rater reproducibility (ICC 2,1 =0.96 and 0.93, respectively). According to the standard error of the measurement and minimal detectable change, the magnitude of change needed to exceed the measurement error and variability were 7.2 and 3.4 points for the 30-item and 10-item Icon-FES, respectively. We observed an excellent correlation between both versions of the Icon-FES and Falls Efficacy Scale - International (rho=0.83, pmeasurement properties to evaluate concern about falling among the community-dwelling older population. Copyright © 2018 Associação Brasileira de Pesquisa e Pós-Graduação em Fisioterapia. Publicado por Elsevier Editora Ltda. All rights reserved.

Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling.

Science.gov (United States)

Schumacher, Dominik; Lemke, Oliver; Helma, Jonas; Gerszonowicz, Lena; Waller, Verena; Stoschek, Tina; Durkin, Patrick M; Budisa, Nediljko; Leonhardt, Heinrich; Keller, Bettina G; Hackenberger, Christian P R

2017-05-01

The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.

Cross-cultural validation of the Falls Efficacy Scale-International (FES-I) in Portuguese community-dwelling older adults.

Science.gov (United States)

Figueiredo, Daniela; Santos, Sónia

The Falls Efficacy Scale-International (FES-I) is a highly reliable instrument to assess fear of falling among older population. This study aimed to develop a European Portuguese version of the FES-I (FES-I (P) ) and analyse its psychometric properties in terms of internal consistency, test-retest reliability, concurrent and convergent validity. A cross-sectional study was conducted. Data collection integrated a socio-demographic questionnaire which included falls history and presence/absence of fear of falling, the Activities-specific Balance Confidence Scale (ABC), the Hospital Anxiety and Depression Scale (HADS), the Timed Up and Go (TUG) and the Five Times Sit to Stand Test (FTSST). Descriptive and inferential statistical analyses were performed. A total of 100 Portuguese community-dwelling older people (74.27±8.7years old) have participated in the study. From these, 82 have participated in the reliability study. The FES-I (P) had excellent internal consistency (α=0,978) and test-retest reliability (ICC 2,1 =0,999). A significant negative correlation was found between the FES-I (P) and the ABC (r s =-0.85; pPortuguese community-living older people. Future studies should explore the FES-I (P) responsiveness to change over time and analyse its psychometric properties in samples of both non-community-dwelling and community-dwelling older adults with different health conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

Directory of Open Access Journals (Sweden)

Johanna M Mattsson

Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

Identification of secreted proteins of Aspergillus oryzae associated with growth on solid cereal substrates

NARCIS (Netherlands)

Biesebeke, R. te; Boussier, A.; Biezen, N. van; Hondel, C.A.M.J.J. van den; Punt, P.J.

2006-01-01

Filamentous growth of Aspergillus oryzae on solid cereal substrates involves secretion of substrate converting enzymes and a solid substrate specific polarised hyphal growth phenotype. To identify proteins produced under these specific conditions, the extracts of A. oryzae grown on wheat-based media

The intermembrane space protein Erv1 of Trypanosoma brucei is essential for mitochondrial Fe-S cluster assembly and operates alone

Czech Academy of Sciences Publication Activity Database

Haindrich, Alexander C.; Boudova, M.; Vancová, Marie; Peña-Diaz, Priscila; Horáková, Eva; Lukeš, Julius

2017-01-01

Roč. 214, JUN (2017), s. 47-51 ISSN 0166-6851 R&D Projects: GA ČR GA15-21974S; GA ČR(CZ) GA16-18699S Institutional support: RVO:60077344 Keywords : Trypanosoma * Erv1 * Fe-S cluster assembly * mitochondrion Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 2.536, year: 2016

A Regulatory Circuit Composed of a Transcription Factor, IscR, and a Regulatory RNA, RyhB, Controls Fe-S Cluster Delivery

Directory of Open Access Journals (Sweden)

Pierre Mandin

2016-09-01

Full Text Available Fe-S clusters are cofactors conserved through all domains of life. Once assembled by dedicated ISC and/or SUF scaffolds, Fe-S clusters are conveyed to their apo-targets via A-type carrier proteins (ATCs. Escherichia coli possesses four such ATCs. ErpA is the only ATC essential under aerobiosis. Recent studies reported a possible regulation of the erpA mRNA by the small RNA (sRNA RyhB, which controls the expression of many genes under iron starvation. Surprisingly, erpA has not been identified in recent transcriptomic analysis of the iron starvation response, thus bringing into question the actual physiological significance of the putative regulation of erpA by RyhB. Using an sRNA library, we show that among 26 sRNAs, only RyhB represses the expression of an erpA-lacZ translational fusion. We further demonstrate that this repression occurs during iron starvation. Using mutational analysis, we show that RyhB base pairs to the erpA mRNA, inducing its disappearance. In addition, IscR, the master regulator of Fe-S homeostasis, represses expression of erpA at the transcriptional level when iron is abundant, but depleting iron from the medium alleviates this repression. The conjunction of transcriptional derepression by IscR and posttranscriptional repression by RyhB under Fe-limiting conditions is best described as an incoherent regulatory circuit. This double regulation allows full expression of erpA at iron concentrations for which Fe-S biogenesis switches from the ISC to the SUF system. We further provide evidence that this regulatory circuit coordinates ATC usage to iron availability.

EMG based FES for post-stroke rehabilitation

Science.gov (United States)

Piyus, Ceethal K.; Anjaly Cherian, V.; Nageswaran, Sharmila

2017-11-01

Annually, 15 million in world population experiences stroke. Nearly 9 million stroke survivors every year experience mild to severe disability. The loss of upper extremity function in stroke survivors still remains a major rehabilitation challenge. The proposed EMG Abstract—Annually, 15 million in world population experiences stroke. Nearly 9 million stroke survivors every year experience mild to severe disability. The loss of upper extremity function in stroke survivors still remains a major rehabilitation challenge. The proposed EMG based FES system can be used for effective upper limb motor re-education in post stroke upper limb rehabilitation. The governing feature of the designed system is its synchronous activation, in which the FES stimulation is dependent on the amplitude of the EMG signal acquired from the unaffected upper limb muscle of the hemiplegic patient. This proportionate operation eliminates the undesirable damage to the patient’s skin by generating stimulus in proportion to voluntary EMG signals. This feature overcomes the disadvantages of currently available manual motor re-education systems. This model can be used in home-based post stroke rehabilitation, to effectively improve the upper limb functions.

The Impact of FeS Mineralogy on TCE Degradation

Science.gov (United States)

Iron- and sulfate-reducing conditions are often encountered in permeable reactive barrier (PRB) systems that are constructed to remove TCE from groundwater, which usually leads to the accumulation of FeS mineral phases in the matrix of the PRB. Poorly crystalline mackinawite (Fe...

Structure and property correlations in FeS

Energy Technology Data Exchange (ETDEWEB)

Kuhn, S.J. [Materials Science & Technology Division, Oak Ridge National Laboratory , Oak Ridge , TN 37831 (United States); Department of Physics , University of Notre Dame , Notre Dame , IN 46556 (United States); Kidder, M.K. [Chemical Sciences Division, Oak Ridge National Laboratory , Oak Ridge , TN 37831 (United States); Parker, D.S. [Materials Science & Technology Division, Oak Ridge National Laboratory , Oak Ridge , TN 37831 (United States); Cruz, C. dela [Quantum Condensed Matter Division, Oak Ridge National Laboratory , Oak Ridge , TN 37831 (United States); McGuire, M.A.; Chance, W.M.; Li, Li [Materials Science & Technology Division, Oak Ridge National Laboratory , Oak Ridge , TN 37831 (United States); Debeer-Schmitt, L. [Chemical and Engineering Materials Division, Oak Ridge National Laboratory , Oak Ridge , TN 37831 (United States); Ermentrout, J. [Materials Science & Technology Division, Oak Ridge National Laboratory , Oak Ridge , TN 37831 (United States); Littrell, K.C. [Chemical and Engineering Materials Division, Oak Ridge National Laboratory , Oak Ridge , TN 37831 (United States); Eskildsen, M.R. [Department of Physics , University of Notre Dame , Notre Dame , IN 46556 (United States); Sefat, A.S. [Materials Science & Technology Division, Oak Ridge National Laboratory , Oak Ridge , TN 37831 (United States)

2017-03-15

Highlights: • Similar to other iron chalcogenides of FeSe and FeTe, the structure and composition of FeS is highly correlated to its superconductivity. For iron-sulfide (FeS), we report the correlation between the structural details with its magnetic and superconducting properties. • While our FeS with a = 3.6772(7) Å is a filamentary superconductor coexisting with an antiferromagnetic phase, previously reported samples with a > 3.68 Å are bulk superconductors with no magnetism, and those with a ≈ 3.674 Å show magnetic properties. The a lattice of ≥3.68 Å seem to be crucial for causing bulk superconductivity in the tetragonal phase, which is relevant to iron stoichiometry and sulfur height from the iron plane. • For Fe{sub 0.93}S, we report evidence for the coexistence of antiferromagnetism at T{sub N} = 116 and filamentary superconductivity below T{sub c} = 4 K. While temperature neutron diffraction data reveals antiferromagnetic commensurate ordering with wave vector k{sub m} = (0.25,0.25,0), our magnetization results shows shielding and diamagnetism. - Abstract: For iron-sulfide (FeS), we investigate the correlation between the structural details, including its dimensionality and composition, with its magnetic and superconducting properties. We compare, theoretically and experimentally, the two-dimensional (2D) layered tetragonal (“t-FeS”) phase with the 3D hexagonal ('h-FeS') phase. X-ray diffraction reveals iron-deficient chemical compositions of t-Fe{sub 0.93(1)}S and h-Fe{sub 0.84(1)}S that show no low-temperature structural transitions. First-principles calculations reveal a high sensitivity of the 2D structure to the electronic and magnetic properties, predicting marginal antiferromagnetic instability for our compound (sulfur height of z{sub S} = 0.252) with an ordering energy of about 11 meV/Fe, while the 3D phase is magnetically stable. Experimentally, h-Fe{sub 0.84}S orders magnetically well above room

Bacillus subtilis BY-kinase PtkA controls enzyme activity and localization of its protein substrates

DEFF Research Database (Denmark)

Jers, Carsten; Pedersen, Malene Mejer; Paspaliari, Dafni Katerina

2010-01-01

-phosphorylated proteins in B. subtilis. We found that the majority of these proteins could be phosphorylated by PtkA in vitro. Among these new substrates, single-stranded DNA exonuclease YorK, and aspartate semialdehyde dehydrogenase Asd were activated by PtkA-dependent phosphorylation. Because enzyme activity......A was dramatically altered in Delta ptkA background. Our results confirm that PtkA can control enzyme activity of its substrates in some cases, but also reveal a new mode of action for PtkA, namely ensuring correct cellular localization of its targets.......P>Bacillus subtilis BY-kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. Our recent phosphoproteome study identified nine new tyrosine...

The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.

Science.gov (United States)

Lill, Roland; Hoffmann, Bastian; Molik, Sabine; Pierik, Antonio J; Rietzschel, Nicole; Stehling, Oliver; Uzarska, Marta A; Webert, Holger; Wilbrecht, Claudia; Mühlenhoff, Ulrich

2012-09-01

Mitochondria play a key role in iron metabolism in that they synthesize heme, assemble iron-sulfur (Fe/S) proteins, and participate in cellular iron regulation. Here, we review the latter two topics and their intimate connection. The mitochondrial Fe/S cluster (ISC) assembly machinery consists of 17 proteins that operate in three major steps of the maturation process. First, the cysteine desulfurase complex Nfs1-Isd11 as the sulfur donor cooperates with ferredoxin-ferredoxin reductase acting as an electron transfer chain, and frataxin to synthesize an [2Fe-2S] cluster on the scaffold protein Isu1. Second, the cluster is released from Isu1 and transferred toward apoproteins with the help of a dedicated Hsp70 chaperone system and the glutaredoxin Grx5. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Functional defects of the core ISC assembly machinery are signaled to cytosolic or nuclear iron regulatory systems resulting in increased cellular iron acquisition and mitochondrial iron accumulation. In fungi, regulation is achieved by iron-responsive transcription factors controlling the expression of genes involved in iron uptake and intracellular distribution. They are assisted by cytosolic multidomain glutaredoxins which use a bound Fe/S cluster as iron sensor and additionally perform an essential role in intracellular iron delivery to target metalloproteins. In mammalian cells, the iron regulatory proteins IRP1, an Fe/S protein, and IRP2 act in a post-transcriptional fashion to adjust the cellular needs for iron. Thus, Fe/S protein biogenesis and cellular iron metabolism are tightly linked to coordinate iron supply and utilization. This article is part of a Special Issue entitled: Cell Biology of Metals. Copyright © 2012 Elsevier B.V. All rights reserved.

Optimization of FES-assisted rising motion in individuals with paraplegia

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Charles Fattal

2011-12-01

Full Text Available The objective of this paper is to investigate what would be the optimal strategy for voluntary trunk movement, which would minimize hip, knee and ankle torques demanding as well minimal upper limb participation during Functional Electrical Stimulation (FES-assisted sit to stand motion in person suffering from Spinal cord injury. Our results suggest that paraplegic patients should bend their body forward in order to use linear momentum of the trunk in sit off phase, i.e. they should generate the motion similar to the one of healthy subjects. Those results have been experimentally tested using a closed-loop controller for FES-assisted standing-up. The controller should automatically trigger leg stimulation in optimal moment with respect to the trunk motion in order to decrease arm participation during rising phase of the motion.

Correction to: Fe-S cluster assembly in the supergroup Excavata.

Science.gov (United States)

Peña-Diaz, Priscila; Lukeš, Julius

2018-05-29

The article "Fe-S cluster assembly in the supergroup Excavata", written by Priscila Peña‑Diaz, Julius Lukeš was originally published electronically on the publisher's internet portal (currently SpringerLink) without open access.

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

International Nuclear Information System (INIS)

Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro; Yamaguchi, Kazuo; Garcia, Andres J

2011-01-01

The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG 7 ). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni 2+ -ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG 7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII 7-10 ) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII 7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

Energy Technology Data Exchange (ETDEWEB)

Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro [World Premier International (WPI) Research Center Initiative, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science - NIMS (Japan); Yamaguchi, Kazuo [Department of Chemistry, Faculty of Science and Research Institute for Photofunctionalized Materials, Kanagawa University (Japan); Garcia, Andres J, E-mail: NAKANISHI.Jun@nims.go.jp [Institute for Bioengineering and Bioscience, Woodruff School of Mechanical Engineering, Georgia Institute of Technology (United States)

2011-08-15

The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG{sub 7}). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni{sup 2+}-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG{sub 7} underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII{sub 7-10}) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII{sub 7-10} was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

FES Bike Race preparation to Cybathlon 2016 by EMA team: a short case report

Directory of Open Access Journals (Sweden)

Juliana Araujo Guimarães

2017-12-01

Full Text Available FES-assisted cycling has been recommended to people struggling to emerge from a disability to more functioning life after spinal cord injury. Recommendations issued by a gowing number of scientific papershas promised toimprove body composition and physical activity levels, as well as to controlinvoluntary muscle response; favoring activity and participation which break new grounds in expanding locomotion, leisure and occupational options for people with paraplegia and tetraplegia. In this report we described our experience to select and prepare a pilot to compete in the FES Bike Race modality at Cybathlon 2016 in Kloten (Zurick. He was a man, 38 years old, with a complete spinal cord injury, level T9, three years of injury. He took part in a two preparation phases lasting respectively 18 and 12 weeks each: (1st pre-FES-cycling and a (2nd FES-cycling. The 1st phase aimed to explore electrical stimulation response in the quadricps, hamstrings and gluteus muscles; searching for a standard muscular recruitment enable to propel the pedals of a trike. Following, in the 2nd phase, stationary to mobile FES-cycling was performed at the same time the development of the automation and control systems were being incorporated in the trike. We adapted a commercial tadpole trycicle anda pilot controlled system. Although we had planned a three session by week protocol, for reasons of term and time to finish the trike development and be prepared to compete, in the last two weeks before the Cybatlhon an intense level of exercise was maintained. After the race, we noticedinflammatory signs on the left knee which later revealed a patella fracture. The video footage analysis confirmed ithappened during the race’s first lap.

RanBP3 influences interactions between CRM1 and its nuclear protein export substrates

OpenAIRE

Englmeier, Ludwig; Fornerod, Maarten; Bischoff, F. Ralf; Petosa, Carlo; Mattaj, Iain W.; Kutay, Ulrike

2001-01-01

We investigated the role of RanBP3, a nuclear member of the Ran-binding protein 1 family, in CRM1-mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1–export substrate interaction. Nuclear RanBP3 stimulates CRM1-dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mec...

Advertising Discourse Analysis of FES stores: Killing Love, Cowards Show

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Cristian Venegas Ahumada

2013-08-01

Full Text Available The objective is to analyze the structural and photographic discourse of the Autumn-Winter campaign 2008 of FES stores for young people. This was done by a semiotic theory and a critical-structural methodology of discourse. An analysis of 4 advertising photographs was done, and at once an analysis of the discourse “FES says no to violence against Women”, which explains the campaign’s target. The result is: The discourse was subjected to production condition (society of control and makes advertising a way to homogenize subjectivity of masses to consume. Recognition conditions demonstrate that this advertising discourse of symbolic violence means a type of violation of Men and Women Rights. An action like this requires commitment of Psychology in order to promote the social humanizing change, by means of university teaching and professional tasks.

Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

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Stabel Silvia

2002-04-01

Full Text Available Abstract Background The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro. Results We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B, alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues. Conclusion The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

Adaptação transcultural e avaliação das propriedades psicométricas da Falls Efficacy Scale - International em idosos Brasileiros (FES-I-BRASIL Cross-cultural adaptation and evaluation of the psychometric properties of the Falls Efficacy Scale - International Among Elderly Brazilians (FES-I-BRAZIL

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Flávia F. O. Camargos

2010-06-01

Full Text Available OBJETIVOS: Adaptar culturalmente a Falls Efficacy Scale-International (FES-I e avaliar suas propriedades psicométricas em uma amostra de idosos brasileiros da comunidade. MÉTODOS: Conforme recomendações da Rede Européia de prevenção às quedas, o instrumento foi traduzido para o português do Brasil e adaptado culturalmente para a população brasileira (FES-I-Brasil. A FES-I-Brasil foi aplicada em 163 idosos (73,44±5,51 anos, e foram coletados dados demográficos e relacionados à história de quedas. Dentre esses idosos, 58 foram distribuídos aleatoriamente para avaliação da confiabilidade. A confiabilidade foi analisada pelo Índice de Correlação Intraclasse (ICC e a consistência interna pelo α de Cronbach. A estrutura interna da FES-I-Brasil foi avaliada pela análise fatorial exploratória. O modelo de regressão logística foi utilizado para identificar quais tarefas da escala eram mais relevantes para discriminar quedas. Para análise de sensibilidade e especificidade da FES-I-Brasil, empregou-se a curva Receiving Operator Characteristic (ROC. RESULTADOS: A consistência interna da FES-I-Brasil foi α=0,93, e a confiabilidade foi ICC=0,84 e 0,91 (intra e interexaminadores, respectivamente. A análise fatorial sugeriu dois fatores que verificavam preocupação em cair durante atividades de socialização e de vida diária (básicas e instrumentais e tarefas relacionadas ao controle postural. Uma pontuação >23 pontos na FES-I-Brasil sugeriu associação com histórico de queda esporádica, ao passo que uma pontuação >31 pontos ensejou uma associação com queda recorrente. CONCLUSÕES: A FES-I-Brasil apresentou-se semântica, linguística e psicometricamente adequada para avaliar o medo de cair na população de idosos brasileiros da comunidade.OBJECTIVES: To culturally adapt the Falls Efficacy Scale - International (FES-I and assess its psychometric properties in a sample of community-dwelling elderly Brazilians. METHODS

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

Directory of Open Access Journals (Sweden)

Jun Nakanishi, Hidekazu Nakayama, Kazuo Yamaguchi, Andres J Garcia and Yasuhiro Horiike

2011-01-01

Full Text Available The development of methods for the off–on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs of three disulfide compounds containing (i a photocleavable poly(ethylene glycol (PEG, (ii nitrilotriacetic acid (NTA and (iii hepta(ethylene glycol (EG7. Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7–10 to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

FES-assisted Cycling Improves Aerobic Capacity and Locomotor Function Postcerebrovascular Accident.

Science.gov (United States)

Aaron, Stacey E; Vanderwerker, Catherine J; Embry, Aaron E; Newton, Jennifer H; Lee, Samuel C K; Gregory, Chris M

2018-03-01

After a cerebrovascular accident (CVA) aerobic deconditioning contributes to diminished physical function. Functional electrical stimulation (FES)-assisted cycling is a promising exercise paradigm designed to target both aerobic capacity and locomotor function. This pilot study aimed to evaluate the effects of an FES-assisted cycling intervention on aerobic capacity and locomotor function in individuals post-CVA. Eleven individuals with chronic (>6 months) post-CVA hemiparesis completed an 8-wk (three times per week; 24 sessions) progressive FES-assisted cycling intervention. V˙O2peak, self-selected, and fastest comfortable walking speeds, gait, and pedaling symmetry, 6-min walk test (6MWT), balance, dynamic gait movements, and health status were measured at baseline and posttraining. Functional electrical stimulation-assisted cycling significantly improved V˙O2peak (12%, P = 0.006), self-selected walking speed (SSWS, 0.05 ± 0.1 m·s, P = 0.04), Activities-specific Balance Confidence scale score (12.75 ± 17.4, P = 0.04), Berg Balance Scale score (3.91 ± 4.2, P = 0.016), Dynamic Gait Index score (1.64 ± 1.4, P = 0.016), and Stroke Impact Scale participation/role domain score (12.74 ± 16.7, P = 0.027). Additionally, pedal symmetry, represented by the paretic limb contribution to pedaling (paretic pedaling ratio [PPR]) significantly improved (10.09% ± 9.0%, P = 0.016). Although step length symmetry (paretic step ratio [PSR]) did improve, these changes were not statistically significant (-0.05% ± 0.1%, P = 0.09). Exploratory correlations showed moderate association between change in SSWS and 6-min walk test (r = 0.74), and moderate/strong negative association between change in PPR and PSR. These results support FES-assisted cycling as a means to improve both aerobic capacity and locomotor function. Improvements in SSWS, balance, dynamic walking movements, and participation in familial and societal roles are important targets for rehabilitation of individuals

Upper Limb Rehabilitation Robot Powered by PAMs Cooperates with FES Arrays to Realize Reach-to-Grasp Trainings

Science.gov (United States)

Su, Chen; Jiang, Xiaobo

2017-01-01

The reach-to-grasp activities play an important role in our daily lives. The developed RUPERT for stroke patients with high stiffness in arm flexor muscles is a low-cost lightweight portable exoskeleton rehabilitation robot whose joints are unidirectionally actuated by pneumatic artificial muscles (PAMs). In order to expand the useful range of RUPERT especially for patients with flaccid paralysis, functional electrical stimulation (FES) is taken to activate paralyzed arm muscles. As both the exoskeleton robot driven by PAMs and the neuromuscular skeletal system under FES possess the highly nonlinear and time-varying characteristics, iterative learning control (ILC) is studied and is taken to control this newly designed hybrid rehabilitation system for reaching trainings. Hand function rehabilitation refers to grasping. Because of tiny finger muscles, grasping and releasing are realized by FES array electrodes and matrix scan method. By using the surface electromyography (EMG) technique, the subject's active intent is identified. The upper limb rehabilitation robot powered by PAMs cooperates with FES arrays to realize active reach-to-grasp trainings, which was verified through experiments. PMID:29065566

Upper Limb Rehabilitation Robot Powered by PAMs Cooperates with FES Arrays to Realize Reach-to-Grasp Trainings

Directory of Open Access Journals (Sweden)

Xikai Tu

2017-01-01

Full Text Available The reach-to-grasp activities play an important role in our daily lives. The developed RUPERT for stroke patients with high stiffness in arm flexor muscles is a low-cost lightweight portable exoskeleton rehabilitation robot whose joints are unidirectionally actuated by pneumatic artificial muscles (PAMs. In order to expand the useful range of RUPERT especially for patients with flaccid paralysis, functional electrical stimulation (FES is taken to activate paralyzed arm muscles. As both the exoskeleton robot driven by PAMs and the neuromuscular skeletal system under FES possess the highly nonlinear and time-varying characteristics, iterative learning control (ILC is studied and is taken to control this newly designed hybrid rehabilitation system for reaching trainings. Hand function rehabilitation refers to grasping. Because of tiny finger muscles, grasping and releasing are realized by FES array electrodes and matrix scan method. By using the surface electromyography (EMG technique, the subject’s active intent is identified. The upper limb rehabilitation robot powered by PAMs cooperates with FES arrays to realize active reach-to-grasp trainings, which was verified through experiments.

Growth-promoting effect on iron-sulfur proteins on axenic cultures of Entamoeba dispar

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Khalifa S.A.M.

2006-03-01

Full Text Available A growth-promoting factor (GPF that promotes the growth of Entamoeba dispar under axenic culture conditions was found in fractions of mitochondria (Mt, hydrogenosomes (Hg and chloroplasts (Cp obtained from cells of six different protozoan, mammalian and plant species. We were able to extract the GPF from the Cp-rich leaf cells of a plant (spiderwort: Commelina communis L. in an acetone-soluble fraction as a complex of chlorophyll with low molecular weight proteins (molecular weight [MW] approximately 4,600. We also found that on treatment with 0.6 % complexes of 2-mercapthoethanol (2ME, complexes of chlorophyll-a with iron-sulphur (Fe-S proteins (e.g., ferredoxins [Fd] from spinach and Clostridium pasteurianum and noncomplex rubredoxin (Rd from C. pasteurianum have a growth-promoting effect on E. dispar. These findings suggest that E. dispar may lack a sufficient quantity of some essential components of Fe-S proteins, such as Fe-S center.

Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis.

Science.gov (United States)

Xue, Qiao; Cui, Ying-Lu; Zheng, Qing-Chuan; Zhang, Hong-Xing

2016-05-01

BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure-function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.

Biomass carbon composited FeS2 as cathode materials for high-rate rechargeable lithium-ion battery

Science.gov (United States)

Xu, Xin; Meng, Zhen; Zhu, Xueling; Zhang, Shunlong; Han, Wei-Qiang

2018-03-01

Pyrite FeS2 has long been used as commercial primary lithium batteries at room temperature. To achieve rechargeable FeS2 battery, biomass-carbon@FeS2 composites are prepared using green and renewable auricularia auricula as carbon source through the process of carbonization and sulfuration. The auricularia auricula has strong swelling characteristics to absorb aqueous solution which can effectively absorb Fe ions into its body. FeS2 homogeneously distributed in biomass carbon matrix performs high electronic and ionic conductivity. The specific capacity of biomass-carbon@FeS2 composites remains 850 mAh g-1 after 80 cycles at 0.5C and 700 mAh g-1 at the rate of 2C after 150 cycles. Biomass-carbon@FeS2 composites exhibit high-rate capacity in lithium-ion battery.

Restoration of Movement using FES: An Introductory Study I

Science.gov (United States)

Ahmed, M.; Huq, M. S.; Ibrahim, B. S. K. K.

2018-03-01

FES has been applied for movement restoration, rehabilitation and therapy in spinal cord injury and nervous system failure subjects, whose number rise worldwide every year. Despite the increase, assist devices that are expected to aid healthcare delivery are not in abundance and could be as a result of standards imposed due to the sensitive condition of the subjects. Although closed loop control systems are expected to positively aid in that regards, the delicacy of the plant was a big constraint. An existing model from the works Ferrarin and Pedotti was elaborated from the knee swinging to the sit-to-stand movements and from the two mathematical models it can be inferred that even though similar higher level of excitation would be required for sit-to-stand manoeuvre. As part of research to improve on closed approach for the FES system, an appraisal was successfully done on the neuromuscular model together with fatigue models from the works on Lynch. The remodelled fatigue models to suit the research were presented.

Analysis of NFU-1 metallocofactor binding-site substitutions-impacts on iron-sulfur cluster coordination and protein structure and function.

Science.gov (United States)

Wesley, Nathaniel A; Wachnowsky, Christine; Fidai, Insiya; Cowan, J A

2017-11-01

Iron-sulfur (Fe/S) clusters are ancient prosthetic groups found in numerous metalloproteins and are conserved across all kingdoms of life due to their diverse, yet essential functional roles. Genetic mutations to a specific subset of mitochondrial Fe/S cluster delivery proteins are broadly categorized as disease-related under multiple mitochondrial dysfunction syndrome (MMDS), with symptoms indicative of a general failure of the metabolic system. Multiple mitochondrial dysfunction syndrome 1 (MMDS1) arises as a result of the missense mutation in NFU1, an Fe/S cluster scaffold protein, which substitutes a glycine near the Fe/S cluster-binding pocket to a cysteine (p.Gly208Cys). This substitution has been shown to promote protein dimerization such that cluster delivery to NFU1 is blocked, preventing downstream cluster trafficking. However, the possibility of this additional cysteine, located adjacent to the cluster-binding site, serving as an Fe/S cluster ligand has not yet been explored. To fully understand the consequences of this Gly208Cys replacement, complementary substitutions at the Fe/S cluster-binding pocket for native and Gly208Cys NFU1 were made, along with six other variants. Herein, we report the results of an investigation on the effect of these substitutions on both cluster coordination and NFU1 structure and function. The data suggest that the G208C substitution does not contribute to cluster binding. Rather, replacement of the glycine at position 208 changes the oligomerization state as a result of global structural alterations that result in the downstream effects manifest as MMDS1, but does not perturb the coordination chemistry of the Fe-S cluster. © 2017 Federation of European Biochemical Societies.

Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements.

Science.gov (United States)

Rachel, Natalie M; Quaglia, Daniela; Lévesque, Éric; Charette, André B; Pelletier, Joelle N

2017-11-01

Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side-chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site-specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100-fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5-cd]indolizine-type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α-helical, β-sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q-GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG-reactive glutamines can be readily introduced into a protein domain for fluorescent labeling. © 2017 The Protein Society.

Fully automatic control of paraplegic FES pedaling using higher-order sliding mode and fuzzy logic control.

Science.gov (United States)

Farhoud, Aidin; Erfanian, Abbas

2014-05-01

In this paper, a fully automatic robust control strategy is proposed for control of paraplegic pedaling using functional electrical stimulation (FES). The method is based on higher-order sliding mode (HOSM) control and fuzzy logic control. In FES, the strength of muscle contraction can be altered either by varying the pulse width (PW) or by the pulse amplitude (PA) of the stimulation signal. The proposed control strategy regulates simultaneously both PA and PW (i.e., PA/PW modulation). A HOSM controller is designed for regulating the PW and a fuzzy logic controller for the PA. The proposed control scheme is free-model and does not require any offline training phase and subject-specific information. Simulation studies on a virtual patient and experiments on three paraplegic subjects demonstrate good tracking performance and robustness of the proposed control strategy against muscle fatigue and external disturbances during FES-induced pedaling. The results of simulation studies show that the power and cadence tracking errors are 5.4% and 4.8%, respectively. The experimental results indicate that the proposed controller can improve pedaling system efficacy and increase the endurance of FES pedaling. The average of power tracking error over three paraplegic subjects is 7.4±1.4% using PA/PW modulation, while the tracking error is 10.2±1.2% when PW modulation is used. The subjects could pedal for 15 min with about 4.1% power loss at the end of experiment using proposed control strategy, while the power loss is 14.3% using PW modulation. The controller could adjust the stimulation intensity to compensate the muscle fatigue during long period of FES pedaling.

An Automatic Identification Procedure to Promote the use of FES-Cycling Training for Hemiparetic Patients

Directory of Open Access Journals (Sweden)

Emilia Ambrosini

2014-01-01

Full Text Available Cycling induced by Functional Electrical Stimulation (FES training currently requires a manual setting of different parameters, which is a time-consuming and scarcely repeatable procedure. We proposed an automatic procedure for setting session-specific parameters optimized for hemiparetic patients. This procedure consisted of the identification of the stimulation strategy as the angular ranges during which FES drove the motion, the comparison between the identified strategy and the physiological muscular activation strategy, and the setting of the pulse amplitude and duration of each stimulated muscle. Preliminary trials on 10 healthy volunteers helped define the procedure. Feasibility tests on 8 hemiparetic patients (5 stroke, 3 traumatic brain injury were performed. The procedure maximized the motor output within the tolerance constraint, identified a biomimetic strategy in 6 patients, and always lasted less than 5 minutes. Its reasonable duration and automatic nature make the procedure usable at the beginning of every training session, potentially enhancing the performance of FES-cycling training.

ErpA, an iron sulfur (Fe S) protein of the A-type essential for respiratory metabolism in E.coli.

NARCIS (Netherlands)

Loiseau, L.; Gerez, C.; Bekker, M.; Ollagnier-de Choudens, S.; Py, B.; Sanakis, Y.; Teixeira De Mattos, M.J.; Fontecave, M.; Barras, F.

2007-01-01

Understanding the biogenesis of iron-sulfur (Fe-S) proteins is relevant to many fields, including bioenergetics, gene regulation, and cancer research. Several multiprotein complexes assisting Fe-S assembly have been identified in both prokaryotes and eukaryotes. Here, we identify in Escherichia coli

In vivo imaging of estrogen receptor concentration in the endometrium and myometrium using FES PET - influence of menstrual cycle and endogenous estrogen level

Energy Technology Data Exchange (ETDEWEB)

Tsuchida, Tatsuro [Department of Radiology, Faculty of Medical Sciences, University of Fukui, Yoshida-gun, Fukui 910-1193 (Japan)]. E-mail: tsucchy@fmsrsa.fukui-med.ac.jp; Okazawa, Hidehiko [Biomedical Imaging Research Center, University of Fukui, Yoshida-gun, Fukui 910-1193 (Japan); Mori, Tetsuya [Biomedical Imaging Research Center, University of Fukui, Yoshida-gun, Fukui 910-1193 (Japan); Kobayashi, Masato [Biomedical Imaging Research Center, University of Fukui, Yoshida-gun, Fukui 910-1193 (Japan); Yoshida, Yoshio [Department of Obstetrics and Gynecology, Faculty of Medical Sciences, University of Fukui, Yoshida-gun, Fukui 910-1193 (Japan); Fujibayashi, Yasuhisa [Biomedical Imaging Research Center, University of Fukui, Yoshida-gun, Fukui 910-1193 (Japan); Itoh, Harumi [Department of Radiology, Faculty of Medical Sciences, University of Fukui, Yoshida-gun, Fukui 910-1193 (Japan)

2007-02-15

Purpose: The goals of this study were to measure estrogen receptor (ER) concentration in the endometrium and myometrium using 16{alpha}-[{sup 18}F]fluoro-17{beta}-estradiol (FES) positron emission tomography (PET) and to investigate the relationship between changes in these parameters with the menstrual cycle and endogenous estrogen levels. Methods: Sixteen female healthy volunteers were included in this study. After blood sampling to measure endogenous estrogen level, FES PET image was acquired 60 min postinjection of FES. After whole-body imaging of FES PET, averaged standardized uptake values (SUVs) in the endometrium and myometrium were measured, and the relationship between FES uptake and menstrual cycle or endogenous estrogen level was evaluated. Results: Endometrial SUV was significantly higher in the proliferative phase than in the secretory phase (6.03{+-}1.05 vs. 3.97{+-}1.29, P=.022). In contrast, there was no significant difference in myometrial SUV when the proliferative and secretory phases were compared (P=.23). Further, there was no correlation between SUV and endogenous estrogen level in the proliferative phase. Conclusions: The change of ER concentration relative to menstrual cycle as characterized by FES PET was consistent with those from previous reports that used an immunohistochemical technique. These data suggest that FES PET is a feasible, noninvasive method for characterizing changes in ER concentration.

In vivo imaging of estrogen receptor concentration in the endometrium and myometrium using FES PET - influence of menstrual cycle and endogenous estrogen level

International Nuclear Information System (INIS)

Tsuchida, Tatsuro; Okazawa, Hidehiko; Mori, Tetsuya; Kobayashi, Masato; Yoshida, Yoshio; Fujibayashi, Yasuhisa; Itoh, Harumi

2007-01-01

Purpose: The goals of this study were to measure estrogen receptor (ER) concentration in the endometrium and myometrium using 16α-[ 18 F]fluoro-17β-estradiol (FES) positron emission tomography (PET) and to investigate the relationship between changes in these parameters with the menstrual cycle and endogenous estrogen levels. Methods: Sixteen female healthy volunteers were included in this study. After blood sampling to measure endogenous estrogen level, FES PET image was acquired 60 min postinjection of FES. After whole-body imaging of FES PET, averaged standardized uptake values (SUVs) in the endometrium and myometrium were measured, and the relationship between FES uptake and menstrual cycle or endogenous estrogen level was evaluated. Results: Endometrial SUV was significantly higher in the proliferative phase than in the secretory phase (6.03±1.05 vs. 3.97±1.29, P=.022). In contrast, there was no significant difference in myometrial SUV when the proliferative and secretory phases were compared (P=.23). Further, there was no correlation between SUV and endogenous estrogen level in the proliferative phase. Conclusions: The change of ER concentration relative to menstrual cycle as characterized by FES PET was consistent with those from previous reports that used an immunohistochemical technique. These data suggest that FES PET is a feasible, noninvasive method for characterizing changes in ER concentration

Effectiveness of functional electrical stimulation (fes) versus conventional electrical stimulation in gait rehabilitation of patients with stroke

International Nuclear Information System (INIS)

Sharif, F.; Ghulam, S.; Malik, A.N.

2017-01-01

To compare the effectiveness of functional electrical stimulation (FES) versus conventional electrical stimulation in gait rehabilitation of patients with stroke for finding the most appropriate problem-oriented treatment for foot drop patients in a shorter time period. Study Design: Randomized controlled trial. Place and Duration of Study:Armed Forces Institute of Rehabilitation Medicine, Rawalpindi, from July to December 2016. Methodology: Subjects with foot drop due to stroke were allotted randomly into 1 of 2 groups receiving standard rehabilitation with Functional Electrical Stimulation (FES) or Electrical Muscle Stimulation (EMS). FES was applied on tibialis anterior 30 minutes/day, five days/week for six weeks. EMS was also applied on the tibialis anterior five days/week for six weeks. Outcome measures included Fugl-Meyer Assessment Scale, Modified Ashworth Scale, Berg Balance Scale (BBS), Time Up and Go Test (TUG) and Gait Dynamic Index (GDI). They were recorded at baseline, after 3 and 6 weeks. Pre- and post-treatment scores were analyzed between two groups on SPSS-20. Results: After six weeks of intervention, significant improvement was recorded in Fugl-Meyer Assessment score (p<0.001), modified Ashworth Scale score (p=0.027), Berg Balance Scale score (p<0.001), Time Up and Go Test (p<0.001) and Gait Dynamic Index (p=0.012) of the group subjected to FES. Conclusion: Gait training with FES is more effective than EMS in improving mobility, balance, gait performance and reducing spasticity in stroke patients. The research will help clinicians to select appropriate treatment of foot drop in stroke patients. (author)

Microbial proteins produced from cannery wastes. II. Different cellulosic wastes used as substrate

Energy Technology Data Exchange (ETDEWEB)

Lee, H.C.; Chen, S.H.; Chang, J.S.; Lee, S.C.

1980-01-01

The production of protein by the cultivation of Cellulomonas species 34 on cellulosic wastes was studied. Maximum protein production on bamboo shoot husks was 4.77 g/L in 48 h when aeration was 1 vol./min, stirring rate was 1000 rpm, and substrate concentration was 3%. The chemical compounds of asparagus peel and pineapple peel and the conditions for their treatment with NaOH for protein fermentation are given.

Preparation and development of FeS2 Quantum Dots on SiO2 nanostructures immobilized in biopolymers and synthetic polymers as nanoparticles and nanofibers catalyst for antibiotic degradation.

Science.gov (United States)

Gao, Wei; Razavi, Razieh; Fakhri, Ali

2018-03-22

The FeS 2 Quantum Dots (QDs) decorated SiO 2 nanostructure were prepared by hydrothermal synthesis method. Chitosan and polypyrrole as polymers were used for the immobilization process. The characteristic structure of prepared samples was analyzed using several techniques such as X-ray diffraction, scanning and transmittance electron microscopy, photoluminescence and UV-vis spectroscopy. The mean crystallite sizes of FeS 2 QDs/SiO 2 nanocomposites, FeS 2 QDs/SiO 2 -chitosan nanocomposites and FeS 2 QDs/SiO 2 -polypyrrole nanohybrids are 56.12, 76.38, and 83.24nm, respectively. The band gap energy of FeS 2 QDs/SiO 2 nanocomposites, FeS 2 QDs/SiO 2 -chitosan nanocomposites and FeS 2 QDs/SiO 2 -polypyrrole nanohybrids were found out to be 3.0, 2.8, and 2.7eV, respectively. The photocatalysis properties were investigated by degradation of ampicillin under UV light illumination. The effect of experimental variables, such as, pH and time, on photo-degradation efficiency was studied. The results show that the three prepared samples nanopowders under UV light was in pH3 at 60min. As it could be seen that the amount of ampicillin degradation was increased with the loading of FeS 2 QDs on SiO 2 and FeS 2 QDs/SiO 2 on chitosan nanoparticles and polypyrrole nanofiber. The antibacterial experiment was investigated under visible light illumination and the FeS 2 QDs/SiO 2 -chitosan nanocomposites and FeS 2 QDs/SiO 2 -polypyrrole nanohybrids demonstrate good antibacterial compared to FeS 2 QDs/SiO 2 nanocomposites. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of different substrates on the yield and protein content of ...

African Journals Online (AJOL)

The effects of seven substrates for the cultivation, yield and protein content of the mushroom, Pleurotus tuberregium (Fries) Singer were investigated. The experimental design used was completely randomized design (CRD) of 7 treatments and 10 replicates. The highest fresh weight yield was obtained from mushrooms ...

DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

Science.gov (United States)

Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

2013-01-01

Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

Setting the pace: insights and advancements gained while preparing for an FES bike race.

Science.gov (United States)

McDaniel, John; Lombardo, Lisa M; Foglyano, Kevin M; Marasco, Paul D; Triolo, Ronald J

2017-11-17

The reduction in physical activity following a spinal cord injury often leads to a decline in mental and physical health. Developing an exercise program that is effective and enjoyable is paramount for this population. Although functional electrical stimulation (FES) stationary cycling has been utilized in rehabilitation settings, implementing an overground cycling program for those with spinal cord injuries has greater technical challenges. Recently our laboratory team focused on training five individuals with compete spinal cord injuries utilizing an implanted pulse generator for an overground FES bike race in CYBATHLON 2016 held in Zurich, Switzerland. The advancements in muscle strength and endurance and ultimately cycling power our pilots made during this training period not only helped propel our competing pilot to win gold at the CYBATHLON 2016, but allowed our pilots to ride their bikes outside within their communities. Such a positive outcome has encouraged us to put effort into developing more widespread use of FES overground cycling as a rehabilitative tool for those with spinal cord injuries. This commentary will describe our approach to the CYBATHLON 2016 including technological advancements, bike design and the training program.

Photoelectrochemical energy conversion obtained with ultrathin organo-metallic-chemical-vapor-deposition layer of FeS2 (pyrite) on TiO2

International Nuclear Information System (INIS)

Ennaoui, A.; Fiechter, S.; Tributsch, H.; Giersig, M.; Vogel, R.; Weller, H.

1992-01-01

Ultrathin (10 to 20 nm thick), polycrystalline films of FeS 2 (pyrite) were grown on TiO 2 (anatase) by chemical vapor deposition. The FeS 2 films were characterized using optical absorption and high-resolution electron microscopy. Photoelectrochemical solar cells, using TiO 2 (anatase) coated with FeS 2 ultrathin films, generated high open-circuit photo-voltages, of up to 600 mV, compared with a single crystal of pyrite electrode (200 mV). The photoelectrochemical behavior shows a strong dependence of photovoltage and photocurrent on the pH of the solution. This paper reports that it is explained by electron injection from the conduction band of FeS 2 to the conduction band of TiO 2 . Regeneration of holes is taking place by electron transfer from the redox system in the electrolyte

Participation to the first Cybathlon: an overview of the FREEWHEELS team FES-cycling solution

Directory of Open Access Journals (Sweden)

Benoît Sijobert

2017-12-01

Full Text Available This article is a contribution to a special issue aiming at collecting data and documenting the different specificities of the teams which participated into Cybathlon 2016 FES-bike discipline. Our team prepared one paraplegic pilot over one year and developed a FES-cycling device based on existing commercial products. Our pilot (47 y.o, spinal cord lesion T3 AIS A since year 1995 was qualified for the final race and finished in 6th position over 12 participants in the discipline, covering a total distance of 750m at an average speed of 5.71km/h, propelled by his own quadriceps and hamstrings muscles.

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and[2Fe-2S

NARCIS (Netherlands)

Berg, van den W.A.M.; Hagen, W.R.; Dongen, van W.M.A.M.

2000-01-01

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state

Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation.

Science.gov (United States)

Lessmann, Eva; Ngo, Mike; Leitges, Michael; Minguet, Susana; Ridgway, Neale D; Huber, Michael

2007-02-01

The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.

Hydrolysis of protein and model dipeptide substrated by attached and nonattached marine Pseudomonas sp. strain NCIMB 2021

International Nuclear Information System (INIS)

Griffith, P.C.; Fletcher, M.

1991-01-01

Rates of substrate hydrolysis by nonattached bacteria and by bacteria attached to particles derived from marine diatom frustules were estimated by using two substrates, a dipeptide analog and a protein. Adsorption of the two substrates onto the particles was also evaluated. Methyl-coumarinyl-amide-leucine (MCA-leucine) was used to estimate hydrolysis of dipeptides by measuring an increase in fluorescence as MCA-leucine was hydrolyzed to leucine and the fluorochrome methylcoumarin. To examine hydrolysis of a larger molecule, was prepared a radiolabeled protein by 14 C-methylation of bovine serum albumin. The rate of protein hydrolysis in samples of particle-attached or nonattached bacteria was estimated by precipitating all nonhydrolyzed protein with cold trichloroacetic acid and then determining the trichloroacetic acid-soluble radiolabeled material, which represented methyl- 14 C-peptides and -amino acids. About 25% of the MCA-leucine adsorbed to the particles. MCA-leucine was hydrolyzed faster by nonattached than attached bacteria, which was probably related to its tendency to remain dissolved in the liquid phase. In contrast, almost 100% of the labeled protein adsorbed to the particles. Accordingly, protein was much less available to nonattached bacteria but was rapidly hydrolyzed by attached bacteria

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

Science.gov (United States)

Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K; Smith, Douglas Y; Söderberg, Christopher A G; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

2016-05-06

The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Regulatory Circuit Composed of a Transcription Factor, IscR, and a Regulatory RNA, RyhB, Controls Fe-S Cluster Delivery.

Science.gov (United States)

Mandin, Pierre; Chareyre, Sylvia; Barras, Frédéric

2016-09-20

Fe-S clusters are cofactors conserved through all domains of life. Once assembled by dedicated ISC and/or SUF scaffolds, Fe-S clusters are conveyed to their apo-targets via A-type carrier proteins (ATCs). Escherichia coli possesses four such ATCs. ErpA is the only ATC essential under aerobiosis. Recent studies reported a possible regulation of the erpA mRNA by the small RNA (sRNA) RyhB, which controls the expression of many genes under iron starvation. Surprisingly, erpA has not been identified in recent transcriptomic analysis of the iron starvation response, thus bringing into question the actual physiological significance of the putative regulation of erpA by RyhB. Using an sRNA library, we show that among 26 sRNAs, only RyhB represses the expression of an erpA-lacZ translational fusion. We further demonstrate that this repression occurs during iron starvation. Using mutational analysis, we show that RyhB base pairs to the erpA mRNA, inducing its disappearance. In addition, IscR, the master regulator of Fe-S homeostasis, represses expression of erpA at the transcriptional level when iron is abundant, but depleting iron from the medium alleviates this repression. The conjunction of transcriptional derepression by IscR and posttranscriptional repression by RyhB under Fe-limiting conditions is best described as an incoherent regulatory circuit. This double regulation allows full expression of erpA at iron concentrations for which Fe-S biogenesis switches from the ISC to the SUF system. We further provide evidence that this regulatory circuit coordinates ATC usage to iron availability. Regulatory small RNAs (sRNAs) have emerged as major actors in the control of gene expression in the last few decades. Relatively little is known about how these regulators interact with classical transcription factors to coordinate genetic responses. We show here how an sRNA, RyhB, and a transcription factor, IscR, regulate expression of an essential gene, erpA, in the bacterium E

Controlled expression of nif and isc iron-sulfur protein maturation components reveals target specificity and limited functional replacement between the two systems.

Science.gov (United States)

Dos Santos, Patricia C; Johnson, Deborah C; Ragle, Brook E; Unciuleac, Mihaela-Carmen; Dean, Dennis R

2007-04-01

The nitrogen-fixing organism Azotobacter vinelandii contains at least two systems that catalyze formation of [Fe-S] clusters. One of these systems is encoded by nif genes, whose products supply [Fe-S] clusters required for maturation of nitrogenase. The other system is encoded by isc genes, whose products are required for maturation of [Fe-S] proteins that participate in general metabolic processes. The two systems are similar in that they include an enzyme for the mobilization of sulfur (NifS or IscS) and an assembly scaffold (NifU or IscU) upon which [Fe-S] clusters are formed. Normal cellular levels of the Nif system, which supplies [Fe-S] clusters for the maturation of nitrogenase, cannot also supply [Fe-S] clusters for the maturation of other cellular [Fe-S] proteins. Conversely, when produced at the normal physiological levels, the Isc system cannot supply [Fe-S] clusters for the maturation of nitrogenase. In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygen-availability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory iscR gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS.

Utilization of solid coffee waste as a substrate for microbial protein production

Energy Technology Data Exchange (ETDEWEB)

Arue, C; Bahar, S

1986-01-01

The feasibility of using the solid waste from the instant coffee processing industry (NESCAFE) as a substrate for the production of protein from fungi imperfecti in order to be used as an animal feed supplement was studied. Studies on the selected fungi, Paecilomyces elegans, Aspergillus oryzae and Fusarium oxysporum showed that F. oxysporum produces significantly higher protein levels than the other fungi studied. The fungus was grown in batch on the acid hydrolyzed coffee medium. Maximal values for sugar utilization and mycelium production (3-4 mg/ml) were obtained on 0.5% (w/v) acid hydrolyzed substrate (4% w/v) supplemented with 0.05% (w/v) potassium phosphate and 0.2% (w/v) yeast extract. Supplementary nitrogen was not necessary. The fungus was found to require pyridoxine and inositol. Addition of 1.5% (w/v) glucose to the medium increased the biomass production, indicating that the carbon source may be a limiting factor. 37 references.

First principles studies of electron tunneling in proteins

Science.gov (United States)

Hayashi, Tomoyuki; Stuchebrukhov, Alexei A.

2014-01-01

A first principles study of electronic tunneling along the chain of seven Fe/S clusters in respiratory complex I, a key enzyme in the respiratory electron transport chain, is described. The broken-symmetry states of the Fe/S metal clusters calculated at both DFT and semi-empirical ZINDO levels were utilized to examine both the extremely weak electronic couplings between Fe/S clusters and the tunneling pathways, which provide a detailed atomistic-level description of the charge transfer process in the protein. One-electron tunneling approximation was found to hold within a reasonable accuracy, with only a moderate induced polarization of the core electrons. The method is demonstrated to be able to calculate accurately the coupling matrix elements as small as 10−4 cm−1. A distinct signature of the wave properties of electrons is observed as quantum interferences of multiple tunneling pathways. PMID:25383312

Permanent LMN denervation of human skeletal muscle and recovery by h-b FES: management and monitoring

Directory of Open Access Journals (Sweden)

Helmut Kern

2010-09-01

Full Text Available Denervation of a defined skeletal muscle is due to lower motor neuron (LMN or peripheral nerve lesions that have major consequences on the muscle tissue. After early atrophy, the mid- and late-phases presents two very contrasting myofibers populations: beside those severely atrophic with internalized groups of myonuclei, large fast-type muscle fibers continue to be present 4 to 6 years after Spinal Cord Injury (SCI. Recent results of rat experiments provides the rational basis for understanding the residual functional characteristics of the long-term denervated muscle and the molecular explanation of its ability to respond to home-base functional electrical stimulation (h-b FES using custom-designed electrodes and stimulators. Further outcomes of the Vienna-Padova ten-year collaboration are: 1. a world-unique Myo- Bank of muscle biopsies and 2. improved imaging procedures (Color Computer Tomography (CT scan and Functional Echomyography, all demonstrating that h-b FES induces improvements in muscle contractility, tissue composition and mass, despite permanent LMN denervation. The benefits of h-b FES could be extended from patents suffering with complete Conus-Cauda Syndrome to the numerous patients with incomplete LMN denervation of skeletal muscles to determine whether h-b FES reduces secondary complications related to disuse and impaired blood perfusion (reduction in bone density, risk of bone fracture, decubitus ulcers, and pulmonary thromboembolism. We are confident that translation of the results of a clinical experiment, the EU Project RISE, to the larger cohort of incomplete LMN denervated muscles will provide the wanted results.

Biogeochemistry of pyrite and iron sulfide oxidation in marine sediments

DEFF Research Database (Denmark)

Schippers, A.; Jørgensen, BB

2002-01-01

as substrates and NO3- as electron acceptor, in the presence of (FeS2)-Fe-55, to test for co-oxidation of FeS2, but an anaerobic microbial dissolution of (FeS2)-Fe-55, could not been detected. FeS2 and FeS were not oxidized by amorphous Fe(III) oxide in the presence of Fe-complexing organic compounds......Pyrite (FeS2) and iron monosulfide (FeS) play a central role in the sulfur and iron cycles of marine sediments, They may be buried in the sediment or oxidized by O-2 after transport by bioturbation to the sediment surface. FeS2 and FeS may also be oxidized within the anoxic sediment in which NO3...... marine sediments and incubated at different temperatures for > 1 yr. Bacteria could not be enriched with FeS2 as substrate or with FeS and amorphous Fe(III) oxide. With FeS and NO3-, 14 enrichments were obtained. One of these enrichments was further cultivated anaerobically with Fe2+ and S-0...

Characterization of fluorescence quenching in bifluorophoric protease substrates.

Science.gov (United States)

Packard, B Z; Toptygin, D D; Komoriya, A; Brand, L

1997-09-01

NorFES is a relatively rigid, bent undecapeptide which contains an amino acid sequence that is recognized by the serine protease elastase (AspAlaIleProNle downward arrow SerIleProLysGlyTyr ( downward arrow indicates the primary cleavage site)). Covalent attachment of a fluorophore on each side of NorFES's elastase cleavage site enables one to use a change of fluorescence intensity as a measure of enzymatic activity. In this study two bichromophoric NorFES derivatives, D-NorFES-A and D-NorFES-D, were prepared in which D (donor) was tetramethylrhodamine and A (acceptor) was rhodamine-X, two chromophores with characteristics suitable for energy transfer. Absorption and fluorescence spectra were obtained with both the intact and cleaved homodoubly, heterodoubly and singly labeled derivatives. It was found that both the homo and hetero doubly-labeled derivatives form ground-state complexes which exhibit exciton bands. The hetero labeled derivative exhibits little or no resonance energy transfer. Spectral measurements were also done in urea, which partially disrupts ground-state dimers.

Cotranslocational processing of the protein substrate calmodulin by an AAA+ unfoldase occurs via unfolding and refolding intermediates.

Science.gov (United States)

Augustyniak, Rafal; Kay, Lewis E

2018-05-22

Protein remodeling by AAA+ enzymes is central for maintaining proteostasis in a living cell. However, a detailed structural description of how this is accomplished at the level of the substrate molecules that are acted upon is lacking. Here, we combine chemical cross-linking and methyl transverse relaxation-optimized NMR spectroscopy to study, at atomic resolution, the stepwise unfolding and subsequent refolding of the two-domain substrate calmodulin by the VAT AAA+ unfoldase from Thermoplasma acidophilum By engineering intermolecular disulphide bridges between the substrate and VAT we trap the substrate at different stages of translocation, allowing structural studies throughout the translocation process. Our results show that VAT initiates substrate translocation by pulling on intrinsically unstructured N or C termini of substrate molecules without showing specificity for a particular amino acid sequence. Although the B1 domain of protein G is shown to unfold cooperatively, translocation of calmodulin leads to the formation of intermediates, and these differ on an individual domain level in a manner that depends on whether pulling is from the N or C terminus. The approach presented generates an atomic resolution picture of substrate unfolding and subsequent refolding by unfoldases that can be quite different from results obtained via in vitro denaturation experiments.

Coordinating Upper and Lower Body During FES-Assisted Transfers in Persons With Spinal Cord Injury in Order to Reduce Arm Support.

Science.gov (United States)

Jovic, Jovana; Azevedo Coste, Christine; Fraisse, Philippe; Henkous, Sonia; Fattal, Charles

2015-12-01

The goal of this study is to minimize arm forces applied during sit-to-stand (STS) transfers in persons with spinal cord injury (SCI) by using functional electrical stimulation (FES) applied to lower limbs muscles. A new FES system has been used to automatically trigger muscle stimulation of the lower limbs, at the desired moment in regards to trunk motion. The objective was to decrease arm participation during STS motion of a person with complete paraplegia and low-level tetraplegia. Six participants with chronic SCI participated in the study. Participants with SCI were recruited to complete STS movement using a new system for FES-assisted STS transfer. All participants attended one muscle mapping session to test their muscles condition, two training sessions to become familiarized with the experimental setup, and two measurement sessions using the proposed system for FES-assisted STS movement. The applied arm forces during STS movement were recorded and analyzed for different stimulation onset values with respect to the maximal trunk acceleration signal using one-way ANOVA statistical test. Post-hoc analysis was performed using Tukey's method. The results of this study showed that the moment of the stimulation onset has an influence on the arm forces applied during the STS motion. The lowest values of arm forces were obtained for STS movements where the electrical stimulation was triggered before and around the time corresponding to the maximal value of the trunk acceleration signal. Lowest arm forces values were obtained for STS motions that were similar to those of healthy persons in terms of trunk movements and beginning of lower limb movements in regards to maximal trunk acceleration signal. The FES system was able to mimic the rising motion of a healthy individual by triggering the FES at the appropriate moment. This method could prove useful for pivot transfer, therapeutic or functional verticalization. © 2015 International Neuromodulation Society.

Prediction of binding modes between protein L-isoaspartyl (D-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening.

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Oda, Akifumi; Noji, Ikuhiko; Fukuyoshi, Shuichi; Takahashi, Ohgi

2015-12-10

Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, D-α-Asp and D-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of D-β-Asp is the largest and the proportions of l-β-Asp and D-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and D-α-Asp is frequently considered. The protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and D-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and D-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or D-α-Asp residues were investigated using computational protein-ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and D-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and D-α-Asp peptides. In contrast, for peptides containing l-α-Asp or D-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods. Copyright © 2015 Elsevier B.V. All rights reserved.

Abundance in proteins expressed after functional electrical stimulation cycling or arm cycling ergometry training in persons with chronic spinal cord injury.

Science.gov (United States)

Gorgey, Ashraf S; Graham, Zachary A; Bauman, William A; Cardozo, Christopher; Gater, David R

2017-07-01

Longitudinal design. The study determined the effects of two forms of exercise training on the abundance of two proteins, (glucose transporter-4 [GLUT-4], adenosine monophosphate kinase [AMPK]) involved in glucose utilization and the transcriptional coactivator that regulates the genes involved in energy metabolism and mitochondrial biogenesis (peroxisome proliferator-activated receptor (PPAR) coactivator 1 alpha [PGC-1α]), in muscles in men with chronic motor-complete spinal cord injury (SCI). Clinical trial at a Medical Center. Nine men with chronic motor-complete SCI participated in functional electrical stimulation lower extremity cycling (FES-LEC; n = 4) or arm cycling ergometer (arm-cycling ergometer [ACE]; n = 5) 5 days/week for 16 weeks. Whole body composition was measured by dual energy X-ray absorptiometry. An intravenous glucose tolerance test was performed to measure glucose effectiveness (Sg) and insulin sensitivity (Si). Muscle biopsies of the right vastus lateralis (VL) and triceps muscles were collected one week prior to and post the exercise training intervention. Neither training intervention altered body composition or carbohydrate metabolism. GLUT-4 increased by 3.8 fold in the VL after FES training and increased 0.6 fold in the triceps after ACE training. PGC-1α increased by 2.3 fold in the VL after FES training and 3.8 fold in the triceps after ACE training. AMPK increased by 3.4 fold in the VL after FES training and in the triceps after ACE training. FES-LEC and ACE training were associated with greater protein expressions in the trained muscles by effectively influencing the abundance of GLUT-4, AMPK and PGC-1α. Thus, FES-LEC training of paralyzed muscle can modulate protein expression similar to that of trained and innervated muscle.

Monitoring substrate enables real-time regulation of a protein localization pathway.

Science.gov (United States)

Ito, Koreaki; Mori, Hiroyuki; Chiba, Shinobu

2018-06-01

Protein localization machinery supports cell survival and physiology, suggesting the potential importance of its expression regulation. Here, we summarize a remarkable scheme of regulation, which allows real-time feedback regulation of the machinery expression. A class of regulatory nascent polypeptides, called monitoring substrates, undergoes force-sensitive translation arrest. The resulting ribosome stalling on the mRNA then affects mRNA folding to expose the ribosome-binding site of the downstream target gene and upregulate its translation. The target gene encodes a component of the localization machinery, whose physical action against the monitoring substrate leads to arrest cancellation. Thus, this scheme of feedback loop allows the cell to adjust the amount of the machinery to correlate inversely with the effectiveness of the process at a given moment. The system appears to have emerged late in evolution, in which a narrow range of organisms selected a distinct monitoring substrate-machinery combination. Currently, regulatory systems of SecM-SecA, VemP-SecDF2 and MifM-YidC2 are known to occur in different bacterial species.

The interface of the ferromagnetic metal CoS2 and the nonmagnetic semiconductor FeS2

KAUST Repository

Nazir, S.

2010-11-05

The electronic and magnetic properties of the cubic pyriteCoS2/FeS2interface are studied using the all-electron full-potential linearized augmented plane wave method. We find that this contact between a ferromagneticmetal and a nonmagnetic semiconductor shows a metallic character. The CoS2 stays close to half-metallicity at the interface, while the FeS2 becomes metallic. The magnetic moment of the Co atoms at the interface slightly decreases as compared to the bulk value and a small moment is induced on the Fe atoms. Furthermore, at the interfaceferromagnetic ordering is found to be energetically favorable as compared to antiferromagnetic ordering.

Enhanced leaf photosynthesis as a target to increase grain yield: insights from transgenic rice lines with variable Rieske FeS protein content in the cytochrome b6 /f complex.

Science.gov (United States)

Yamori, Wataru; Kondo, Eri; Sugiura, Daisuke; Terashima, Ichiro; Suzuki, Yuji; Makino, Amane

2016-01-01

Although photosynthesis is the most important source for biomass and grain yield, a lack of correlation between photosynthesis and plant yield among different genotypes of various crop species has been frequently observed. Such observations contribute to the ongoing debate whether enhancing leaf photosynthesis can improve yield potential. Here, transgenic rice plants that contain variable amounts of the Rieske FeS protein in the cytochrome (cyt) b6 /f complex between 10 and 100% of wild-type levels have been used to investigate the effect of reductions of these proteins on photosynthesis, plant growth and yield. Reductions of the cyt b6 /f complex did not affect the electron transport rates through photosystem I but decreased electron transport rates through photosystem II, leading to concomitant decreases in CO2 assimilation rates. There was a strong control of plant growth and grain yield by the rate of leaf photosynthesis, leading to the conclusion that enhancing photosynthesis at the single-leaf level would be a useful target for improving crop productivity and yield both via conventional breeding and biotechnology. The data here also suggest that changing photosynthetic electron transport rates via manipulation of the cyt b6 /f complex could be a potential target for enhancing photosynthetic capacity in higher plants. © 2015 John Wiley & Sons Ltd.

Monomeric Yeast Frataxin is an Iron-Binding Protein

International Nuclear Information System (INIS)

Cook, J.; Bencze, K.; Jankovic, A.; Crater, A.; Busch, C.; Bradley, P.; Stemmler, A.; Spaller, M.; Stemmler, T.

2006-01-01

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron

In silico engineering of aggregation-prone recombinant proteins for substrate recognition by the chaperonin GroEL.

Science.gov (United States)

Kumar, Vipul; Punetha, Ankita; Sundar, Durai; Chaudhuri, Tapan K

2012-01-01

Molecular chaperones appear to have been evolved to facilitate protein folding in the cell through entrapment of folding intermediates on the interior of a large cavity formed between GroEL and its co-chaperonin GroES. They bind newly synthesized or non-native polypeptides through hydrophobic interactions and prevent their aggregation. Some proteins do not interact with GroEL, hence even though they are aggregation prone, cannot be assisted by GroEL for their folding. In this study, we have attempted to engineer these non-substrate proteins to convert them as the substrate for GroEL, without compromising on their function. We have used a computational biology approach to generate mutants of the selected proteins by selectively mutating residues in the hydrophobic patch, similar to GroES mobile loop region that are responsible for interaction with GroEL, and compared with the wild counterparts for calculation of their instability and aggregation propensities. The energies of the newly designed mutants were computed through molecular dynamics simulations. We observed increased aggregation propensity of some of the mutants formed after replacing charged amino acid residues with hydrophobic ones in the well defined hydrophobic patch, raising the possibility of their binding ability to GroEL. The newly generated mutants may provide potential substrates for Chaperonin GroEL, which can be experimentally generated and tested for their tendency of aggregation, interactions with GroEL and the possibility of chaperone-assisted folding to produce functional proteins.

Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

Science.gov (United States)

Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

2015-06-01

Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

Science.gov (United States)

Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

2016-01-01

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

Low cost, Lightweight, FeS2-Based Photovoltaic Devices by On Demand Ink Jet Printing

Data.gov (United States)

National Aeronautics and Space Administration — This research projects seeks to develop novel synthesis for iron pyrite, FeS2, nanocrystals and nanorods. The synthesis of the material includes investigating the...

Multi-muscle FES force control of the human arm for arbitrary goals.

Science.gov (United States)

Schearer, Eric M; Liao, Yu-Wei; Perreault, Eric J; Tresch, Matthew C; Memberg, William D; Kirsch, Robert F; Lynch, Kevin M

2014-05-01

We present a method for controlling a neuroprosthesis for a paralyzed human arm using functional electrical stimulation (FES) and characterize the errors of the controller. The subject has surgically implanted electrodes for stimulating muscles in her shoulder and arm. Using input/output data, a model mapping muscle stimulations to isometric endpoint forces measured at the subject's hand was identified. We inverted the model of this redundant and coupled multiple-input multiple-output system by minimizing muscle activations and used this inverse for feedforward control. The magnitude of the total root mean square error over a grid in the volume of achievable isometric endpoint force targets was 11% of the total range of achievable forces. Major sources of error were random error due to trial-to-trial variability and model bias due to nonstationary system properties. Because the muscles working collectively are the actuators of the skeletal system, the quantification of errors in force control guides designs of motion controllers for multi-joint, multi-muscle FES systems that can achieve arbitrary goals.

The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter

NARCIS (Netherlands)

Mulligan, Christopher; Geertsma, Eric R.; Severi, Emmanuele; Kelly, David J.; Poolman, Bert; Thomas, Gavin H.

2009-01-01

Substrate-binding protein-dependent secondary transporters are widespread in prokaryotes and are represented most frequently by members of the tripartite ATP-independent periplasmic (TRAP) transporter family. Here, we report the membrane reconstitution of a TRAP transporter, the sialic acid-specific

Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins

International Nuclear Information System (INIS)

Kirchenbuechler, David; Born, Simone; Kirchgessner, Norbert; Houben, Sebastian; Hoffmann, Bernd; Merkel, Rudolf

2010-01-01

Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile

FES-Rowing versus Zoledronic Acid to Improve BoneHealth in SCI

Science.gov (United States)

2016-12-01

Army position, policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public...no established treatment to prevent bone loss or to induce new bone formation following SCI. The goal of this clinical trial -- FES-Rowing versus...infusion at the VA Boston Healthcare-Jamaica Plain Campus. The nurse practitioner that administered the infusion made follow-up phone calls within 24

Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

Science.gov (United States)

Masuda, Hiro-taka; Ishihara, Seiichiro; Harada, Ichiro; Mizutani, Takeomi; Ishikawa, Masayori; Kawabata, Kazushige; Haga, Hisashi

2014-01-01

We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

Effect of capping ligands on the optical properties and electronic energies of iron pyrite FeS2 nanocrystals and solid thin films

International Nuclear Information System (INIS)

Zhai, Guangmei; Xie, Rongwei; Wang, Heng; Zhang, Jitao; Yang, Yongzhen; Wang, Hua; Li, Xuemin; Liu, Xuguang; Xu, Bingshe

2016-01-01

In this work, the optical and electronic properties of iron pyrite FeS 2 nanocrystals and solid thin films with various capping ligands were systematically investigated by UV–Vis–NIR absorption spectroscopy, cyclic voltammetry and current density–voltage characteristic measurements. The iron pyrite nanocrystals with various ligands have an indirect band gap of around 1.05 eV and broad absorption spanning into the near-infrared region, exhibiting favorable optical properties for their photovoltaic applications. The electron affinities and ionization potentials of FeS 2 nanocrystals determined through cyclic voltammetry measurements show strong ligand dependence. An energy level shift of up to 190 meV was obtained among the pyrite nanocrystals capped with the ligands employed in this work. The iron pyrite nanocrystal films capped with iodide and 1,2-ethanedithiol exhibit the largest band edge energy shift and conductivity, respectively. Our results not only provide several useful optical and electronic parameters of pyrite nanocrystals for their further use in optoelectronic devices as active layers and/or infrared optical absorption materials, but also highlight the relationship between their surface chemistry and electronic energies. - Highlights: • The energy levels of FeS 2 nanocrystals with various ligands were determined via electrochemical measurements. • The energy levels of FeS 2 nanocrystals showed strong ligand-dependence. • An energy level shift of up to 190 meV was obtained for the pyrite nanocrystals studied in the work. • The conductivities of FeS 2 nanocrystals with different ligands were obtained by current density–voltage measurements.

[Structure and evolution of the eukaryotic FANCJ-like proteins].

Science.gov (United States)

Wuhe, Jike; Zefeng, Wu; Sanhong, Fan; Xuguang, Xi

2015-02-01

The FANCJ-like protein family is a class of ATP-dependent helicases that can catalytically unwind duplex DNA along the 5'-3' direction. It is involved in the processes of DNA damage repair, homologous recombination and G-quadruplex DNA unwinding, and plays a critical role in maintaining genome integrity. In this study, we systemically analyzed FNACJ-like proteins from 47 eukaryotic species and discussed their sequences diversity, origin and evolution, motif organization patterns and spatial structure differences. Four members of FNACJ-like proteins, including XPD, CHL1, RTEL1 and FANCJ, were found in eukaryotes, but some of them were seriously deficient in most fungi and some insects. For example, the Zygomycota fungi lost RTEL1, Basidiomycota and Ascomycota fungi lost RTEL1 and FANCJ, and Diptera insect lost FANCJ. FANCJ-like proteins contain canonical motor domains HD1 and HD2, and the HD1 domain further integrates with three unique domains Fe-S, Arch and Extra-D. Fe-S and Arch domains are relatively conservative in all members of the family, but the Extra-D domain is lost in XPD and differs from one another in rest members. There are 7, 10 and 2 specific motifs found from the three unique domains respectively, while 5 and 12 specific motifs are found from HD1 and HD2 domains except the conserved motifs reported previously. By analyzing the arrangement pattern of these specific motifs, we found that RTEL1 and FANCJ are more closer and share two specific motifs Vb2 and Vc in HD2 domain, which are likely related with their G-quadruplex DNA unwinding activity. The evidence of evolution showed that FACNJ-like proteins were originated from a helicase, which has a HD1 domain inserted by extra Fe-S domain and Arch domain. By three continuous gene duplication events and followed specialization, eukaryotes finally possessed the current four members of FANCJ-like proteins.

Dynamic optimization of walker-assisted FES-activated paraplegic walking: simulation and experimental studies.

Science.gov (United States)

Nekoukar, Vahab; Erfanian, Abbas

2013-11-01

In this paper, we propose a musculoskeletal model of walker-assisted FES-activated paraplegic walking for the generation of muscle stimulation patterns and characterization of the causal relationships between muscle excitations, multi-joint movement, and handle reaction force (HRF). The model consists of the lower extremities, trunk, hands, and a walker. The simulation of walking is performed using particle swarm optimization to minimize the tracking errors from the desired trajectories for the lower extremity joints, to reduce the stimulations of the muscle groups acting around the hip, knee, and ankle joints, and to minimize the HRF. The results of the simulation studies using data recorded from healthy subjects performing walker-assisted walking indicate that the model-generated muscle stimulation patterns are in agreement with the EMG patterns that have been reported in the literature. The experimental results on two paraplegic subjects demonstrate that the proposed methodology can improve walking performance, reduce HRF, and increase walking speed when compared to the conventional FES-activated paraplegic walking. Copyright © 2013 IPEM. Published by Elsevier Ltd. All rights reserved.

Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor.

Science.gov (United States)

Lievens, Sam; Gerlo, Sarah; Lemmens, Irma; De Clercq, Dries J H; Risseeuw, Martijn D P; Vanderroost, Nele; De Smet, Anne-Sophie; Ruyssinck, Elien; Chevet, Eric; Van Calenbergh, Serge; Tavernier, Jan

2014-12-01

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Dissection of structural and functional requirements that underlie the interaction of ERdj3 protein with substrates in the endoplasmic reticulum.

Science.gov (United States)

Otero, Joel H; Lizák, Beata; Feige, Matthias J; Hendershot, Linda M

2014-10-03

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes

Energy Technology Data Exchange (ETDEWEB)

Serrano, Pauline N. [Department of Chemistry, University of California, Davis CA 95616 USA; Wang, Hongxin [Department of Chemistry, University of California, Davis CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA; Crack, Jason C. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Prior, Christopher [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Hutchings, Matthew I. [School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ UK; Thomson, Andrew J. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Kamali, Saeed [University of Tennessee Space Institute, Tullahome TN 37388-9700 USA; Yoda, Yoshitaka [Research and Utilization Division, SPring-8/JASRI, 1-1-1 Kouto, Sayo Hyogo 679-5198 Japan; Zhao, Jiyong [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Hu, Michael Y. [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Alp, Ercan E. [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Oganesyan, Vasily S. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Le Brun, Nick E. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Cramer, Stephen P. [Department of Chemistry, University of California, Davis CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA

2016-10-25

The reaction of protein-bound iron–sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe₂(NO)₄(Cys)₂]) and Roussin's Black Salt (RBS, [Fe₄(NO)₇S₃]. In the latter case, the absence of ³²S/³⁴S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

Science.gov (United States)

Loch, Christian M; Strickler, James E

2012-11-01

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution

DEFF Research Database (Denmark)

Wong, W T; Schumacher, C; Salcini, A E

1995-01-01

In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heteroge......In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several...... heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic...... (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins....

FES Science Network Requirements - Report of the Fusion Energy Sciences Network Requirements Workshop Conducted March 13 and 14, 2008

International Nuclear Information System (INIS)

Tierney, Brian; Dart, Eli; Tierney, Brian

2008-01-01

The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the U.S. Department of Energy Office of Science, the single largest supporter of basic research in the physical sciences in the United States of America. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years. In March 2008, ESnet and the Fusion Energy Sciences (FES) Program Office of the DOE Office of Science organized a workshop to characterize the networking requirements of the science programs funded by the FES Program Office. Most sites that conduct data-intensive activities (the Tokamaks at GA and MIT, the supercomputer centers at NERSC and ORNL) show a need for on the order of 10 Gbps of network bandwidth for FES-related work within 5 years. PPPL reported a need for 8 times that (80 Gbps) in that time frame. Estimates for the 5-10 year time period are up to 160 Mbps for large simulations. Bandwidth requirements for ITER range from 10 to 80 Gbps. In terms of science process and collaboration structure, it is clear that the proposed Fusion Simulation Project (FSP) has the potential to significantly impact the data movement patterns and therefore the network requirements for U.S. fusion science. As the FSP is defined over the next two years, these changes will become clearer. Also, there is a clear and present unmet need for better network connectivity between U.S. FES sites and two Asian fusion experiments--the EAST Tokamak in China and the KSTAR Tokamak in South Korea. In addition to achieving its goal of collecting and characterizing the network requirements of the science endeavors funded by the FES Program Office, the workshop emphasized that there is a need for research into better ways of conducting remote

FES Science Network Requirements - Report of the Fusion Energy Sciences Network Requirements Workshop Conducted March 13 and 14, 2008

Energy Technology Data Exchange (ETDEWEB)

Tierney, Brian; Dart, Eli; Tierney, Brian

2008-07-10

The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the U.S. Department of Energy Office of Science, the single largest supporter of basic research in the physical sciences in the United States of America. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years. In March 2008, ESnet and the Fusion Energy Sciences (FES) Program Office of the DOE Office of Science organized a workshop to characterize the networking requirements of the science programs funded by the FES Program Office. Most sites that conduct data-intensive activities (the Tokamaks at GA and MIT, the supercomputer centers at NERSC and ORNL) show a need for on the order of 10 Gbps of network bandwidth for FES-related work within 5 years. PPPL reported a need for 8 times that (80 Gbps) in that time frame. Estimates for the 5-10 year time period are up to 160 Mbps for large simulations. Bandwidth requirements for ITER range from 10 to 80 Gbps. In terms of science process and collaboration structure, it is clear that the proposed Fusion Simulation Project (FSP) has the potential to significantly impact the data movement patterns and therefore the network requirements for U.S. fusion science. As the FSP is defined over the next two years, these changes will become clearer. Also, there is a clear and present unmet need for better network connectivity between U.S. FES sites and two Asian fusion experiments--the EAST Tokamak in China and the KSTAR Tokamak in South Korea. In addition to achieving its goal of collecting and characterizing the network requirements of the science endeavors funded by the FES Program Office, the workshop emphasized that there is a need for research into better ways of conducting remote

Deviation of the typical AAA substrate-threading pore prevents fatal protein degradation in yeast Cdc48.

Science.gov (United States)

Esaki, Masatoshi; Islam, Md Tanvir; Tani, Naoki; Ogura, Teru

2017-07-14

Yeast Cdc48 is a well-conserved, essential chaperone of ATPases associated with diverse cellular activity (AAA) proteins, which recognizes substrate proteins and modulates their conformations to carry out many cellular processes. However, the fundamental mechanisms underlying the diverse pivotal roles of Cdc48 remain unknown. Almost all AAA proteins form a ring-shaped structure with a conserved aromatic amino acid residue that is essential for proper function. The threading mechanism hypothesis suggests that this residue guides the intrusion of substrate proteins into a narrow pore of the AAA ring, thereby becoming unfolded. By contrast, the aromatic residue in one of the two AAA rings of Cdc48 has been eliminated through evolution. Here, we show that artificial retrieval of this aromatic residue in Cdc48 is lethal, and essential features to support the threading mechanism are required to exhibit the lethal phenotype. In particular, genetic and biochemical analyses of the Cdc48 lethal mutant strongly suggested that when in complex with the 20S proteasome, essential proteins are abnormally forced to thread through the Cdc48 pore to become degraded, which was not detected in wild-type Cdc48. Thus, the widely applicable threading model is less effective for wild-type Cdc48; rather, Cdc48 might function predominantly through an as-yet-undetermined mechanism.

Investigation of electronic and magnetic properties of FeS: First principle and Monte Carlo simulations

Science.gov (United States)

Bouachraoui, Rachid; El Hachimi, Abdel Ghafour; Ziat, Younes; Bahmad, Lahoucine; Tahiri, Najim

2018-06-01

Electronic and magnetic properties of hexagonal Iron (II) Sulfide (hexagonal FeS) have been investigated by combining the Density functional theory (DFT) and Monte Carlo simulations (MCS). This compound is constituted by magnetic hexagonal lattice occupied by Fe2+ with spin state (S = 2). Based on ab initio method, we calculated the exchange coupling JFe-Fe between two magnetic atoms Fe-Fe in different directions. Also phase transitions, magnetic stability and magnetizations have been investigated in the framework of Monte Carlo simulations. Within this method, a second phase transition is observed at the Néel temperature TN = 450 K. This finding in good agreement with the reported data in the literature. The effect of the applied different parameters showed how can these parameters affect the critical temperature of this system. Moreover, we studied the density of states and found that the hexagonal FeS will be a promoting material for spintronic applications.

Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking

Directory of Open Access Journals (Sweden)

Tyagi Sadhna

2009-06-01

Full Text Available Abstract Background Previously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions. Results Here we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine. Conclusion The experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand, although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria.

Magnetron-sputter deposition of Fe3S4 thin films and their conversion into pyrite (FeS2) by thermal sulfurization for photovoltaic applications

International Nuclear Information System (INIS)

Liu Hongfei; Chi Dongzhi

2012-01-01

The authors report on the fabrication of FeS 2 (pyrite) thin films by sulfurizing Fe 3 S 4 that were deposited by direct current magnetron sputtering at room temperature. Under the selected sputtering conditions, Fe 3 S 4 nanocrystal films are obtained and the nanocrystals tend to locally cluster and closely pack into ricelike nanoparticles with an increase in film thickness. Meanwhile, the film tends to crack when the film thickness is increased over ∼1.3 μm. The film cracking can be effectively suppressed by an introduction of a 3-nm Cu intermediate layer prior to Fe 3 S 4 deposition. However, an introduction of a 3-nm Al intermediate layer tends to enhance the film cracking. By post-growth thermal sulfurization of the Fe 3 S 4 thin films in a tube-furnace, FeS 2 with high phase purity, as determined by using x ray diffraction, is obtained. Optical absorption spectroscopy was employed to characterize the resultant FeS 2 thin films, which revealed two absorption edges at 0.9 and 1.2 eV, respectively. These two absorption edges are assigned to the direct bandgap (0.9 eV) and the indirect allowed transitions (1.2 eV) of FeS 2 , respectively.

Interplay between grain structure and protein adsorption on functional response of osteoblasts: ultrafine-grained versus coarse-grained substrates.

Science.gov (United States)

Misra, R D K; Nune, C; Pesacreta, T C; Somani, M C; Karjalainen, L P

2013-01-01

The rapid adsorption of proteins is the starting and primary biological response that occurs when a biomedical device is implanted in the physiological system. The biological response, however, depends on the surface characteristics of the device. Considering the significant interest in nano-/ultrafine surfaces and nanostructured coatings, we describe here, the interplay between grain structure and protein adsorption (bovine serum albumin: BSA) on osteoblasts functions by comparing nanograined/ultrafine-grained (NG/UFG) and coarse-grained (CG: grain size in the micrometer range) substrates by investigating cell-substrate interactions. The protein adsorption on NG/UFG surface was beneficial in favorably modulating biological functions including cell attachment, proliferation, and viability, whereas the effect was less pronounced on protein adsorbed CG surface. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on protein adsorbed NG/UFG surface. The functional response followed the sequence: NG/UFG(BSA) > NG/UFG > CG(BSA) > CG. The differences in the cellular response on bare and protein adsorbed NG/UFG and CG surfaces are attributed to cumulative contribution of grain structure and degree of hydrophilicity. The study underscores the potential advantages of protein adsorption on artificial biomedical devices to enhance the bioactivity and regulate biological functions. Copyright © 2012 Wiley Periodicals, Inc.

Utility of {sup 18}F-fluoroestradiol ({sup 18}F-FES) PET/CT imaging as a pharmacodynamic marker in patients with refractory estrogen receptor-positive solid tumors receiving Z-endoxifen therapy

Energy Technology Data Exchange (ETDEWEB)

Lin, Frank I. [National Cancer Institute, NIH, Cancer Imaging Program, Bethesda, MD (United States); National Cancer Institute, Molecular Imaging Program, Bethesda, MD (United States); Gonzalez, E.M.; Kurdziel, K.A.; Ton, A.; Turkbey, B.; Choyke, P.L.; Lindenberg, M.L. [National Cancer Institute, Molecular Imaging Program, Bethesda, MD (United States); Kummar, S.; Do, K.; Collins, J.M.; Doroshow, J.H. [National Cancer Institute, Division of Cancer Treatment and Diagnosis and Center for Cancer Research, Bethesda, MD (United States); Shih, J. [National Cancer Institute, NIH, Biometric Research Program, Bethesda, MD (United States); Adler, S. [Leidos Biomedical Research, Inc., Clinical Research Directorate/Clinical Monitoring Research Program, Frederick, MD (United States); Jacobs, P.M. [National Cancer Institute, NIH, Cancer Imaging Program, Bethesda, MD (United States); Bhattacharyya, S. [Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, MD (United States); Chen, A.P. [National Cancer Institute, Early Clinical Trials Development Program, DCTD, Bethesda, MD (United States)

2017-03-15

Z-endoxifen is the most potent of the metabolites of tamoxifen, and has the potential to be more effective than tamoxifen because it bypasses potential drug resistance mechanisms attributable to patient variability in the expression of the hepatic microsomal enzyme CYP2D6. {sup 18}F-FES is a positron emission tomography (PET) imaging agent which selectively binds to estrogen receptor alpha (ER-α) and has been used for non-invasive in vivo assessment of ER activity in tumors. This study utilizes {sup 18}F-FES PET imaging as a pharmacodynamic biomarker in patients with ER+ tumors treated with Z-endoxifen. Fifteen patients were recruited from a parent therapeutic trial of Z-endoxifen and underwent imaging with {sup 18}F-FES PET at baseline. Eight had positive lesions on the baseline scan and underwent follow-up imaging with {sup 18}F-FES 1-5 days post administration of Z-endoxifen. Statistically significant changes (p = 0.0078) in standard uptake value (SUV)-Max were observed between the baseline and follow-up scans as early as 1 day post drug administration. F-FES PET imaging could serve as a pharmacodynamic biomarker for patients treated with ER-directed therapy. (orig.)

Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

Directory of Open Access Journals (Sweden)

Jeong Y

2015-11-01

Full Text Available Yoon Jeong,1,2 Kwan Hong Lee,1,2 Hansoo Park,3 Jonghoon Choi1,2 1Department of Bionano Technology, Graduate School, Hanyang University, Seoul, 2Department of Bionano Engineering, Hanyang University ERICA, Ansan, 3School of Integrative Engineering, Chung-Ang University, Seoul, South Korea Abstract: We present an evaluation of protein-G-terminated glass slides that may contain a suitable substrate for aligning the orientation of antibodies to obtain better binding moiety to the target antigen. The results of the protein-G-terminated slides were compared with those obtained with epoxy-based slides to evaluate signal enhancement for human immunoglobulin G (IgG targets, and an increase in the average fluorescence intensity was observed for the lowest measurable amount of IgG target in the assay using protein-G-terminated slides. Applying this strategy for signal amplification to single-cell assays improves the limits of detection for human IgG protein and cytokines (interleukin-2 and interferon-γ captured from hybridomas. Our data indicate that protein-G-terminated slides have a higher binding capacity for antigens and have better spot-to-spot consistency than that of traditional epoxy-based slides. These properties would be beneficial in the detection of fine amounts of single-cell-secreted proteins, which may provide key insights into cell–cell communication and immune responses. Keywords: microwell array, antibody’s orientation, single cell analysis, secreted cytokine, protein-G-terminated surface

The interface of the ferromagnetic metal CoS2 and the nonmagnetic semiconductor FeS2

KAUST Repository

Nazir, S.; Schwingenschlö gl, Udo

2010-01-01

semiconductor shows a metallic character. The CoS2 stays close to half-metallicity at the interface, while the FeS2 becomes metallic. The magnetic moment of the Co atoms at the interface slightly decreases as compared to the bulk value and a small moment

The nonstructural proteins of Pneumoviruses are remarkably distinct in substrate diversity and specificity.

Science.gov (United States)

Ribaudo, Michael; Barik, Sailen

2017-11-06

Interferon (IFN) inhibits viruses by inducing several hundred cellular genes, aptly named 'interferon (IFN)-stimulated genes' (ISGs). The only two RNA viruses of the Pneumovirus genus of the Paramyxoviridae family, namely Respiratory Syncytial Virus (RSV) and Pneumonia Virus of Mice (PVM), each encode two nonstructural (NS) proteins that share no sequence similarity but yet suppress IFN. Since suppression of IFN underlies the ability of these viruses to replicate in the host cells, the mechanism of such suppression has become an important area of research. This Short Report is an important extension of our previous efforts in defining this mechanism. We show that, like their PVM counterparts, the RSV NS proteins also target multiple members of the ISG family. While significantly extending the substrate repertoire of the RSV NS proteins, these results, unexpectedly, also reveal that the target preferences of the NS proteins of the two viruses are entirely different. This is surprising since the two Pneumoviruses are phylogenetically close with similar genome organization and gene function, and the NS proteins of both also serve as suppressors of host IFN response. The finding that the NS proteins of the two highly similar viruses suppress entirely different members of the ISG family raises intriguing questions of pneumoviral NS evolution and mechanism of action.

Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology.

Science.gov (United States)

Kottom, Theodore J; Limper, Andrew H

2011-10-01

Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates. Copyright © 2011 John Wiley & Sons, Ltd.

Preparation of Carbon Nanotube/TiO2 Mesoporous Hybrid Photoanode with Iron Pyrite (FeS2) Thin Films Counter Electrodes for Dye-Sensitized Solar Cell

Science.gov (United States)

Kilic, Bayram; Turkdogan, Sunay; Astam, Aykut; Ozer, Oguz Can; Asgin, Mansur; Cebeci, Hulya; Urk, Deniz; Mucur, Selin Pravadili

2016-05-01

Multi-walled carbon nanotube (MWCNT)/TiO2 mesoporous networks can be employed as a new alternative photoanode in dye-sensitized solar cells (DSSCs). By using the MWCNT/TiO2 mesoporous as photoanodes in DSSC, we demonstrate that the MWCNT/TiO2 mesoporous photoanode is promising alternative to standard FTO/TiO2 mesoporous based DSSC due to larger specific surface area and high electrochemical activity. We also show that iron pyrite (FeS2) thin films can be used as an efficient counter electrode (CE), an alternative to the conventional high cost Pt based CE. We are able to synthesis FeS2 nanostructures utilizing a very cheap and easy hydrothermal growth route. MWCNT/TiO2 mesoporous based DSSCs with FeS2 CE achieved a high solar conversion efficiency of 7.27% under 100 mW cm-2 (AM 1.5G 1-Sun) simulated solar irradiance which is considerably (slightly) higher than that of A-CNT/TiO2 mesoporous based DSSCs with Pt CE. Outstanding performance of the FeS2 CE makes it a very promising choice among the various CE materials used in the conventional DSSC and it is expected to be used more often to achieve higher photon-to-electron conversion efficiencies.

Formation mechanism and yield of molecules ejected from ZnS, CdS, and FeS2 during ion bombardment

International Nuclear Information System (INIS)

Nikzad, S.; Calaway, W.F.; Pellin, M.J.; Young, C.E.; Gruen, D.M.; Tombrello, T.A.

1994-01-01

Neutral species ejected from single crystals of ZnS, CdS, and FeS 2 during ion bombardment by 3 keV Ar + were detected by laser post-ionization followed by time-of-flight mass spectrometry. While metal atoms (Fe, Zn, Cd) and S 2 were the dominant species observed, substantial amounts of S, FeS, Zn 2 , ZnS, Cd 2 , and CdS were also detected. The experimental results demonstrate that molecules represent a larger fraction of the sputtered yield than was previously believed from secondary ion mass spectrometry experiments. In addition, the data suggest that the molecules are not necessarily formed from adjacent atoms in the solid and that a modified form of the recombination model could provide a mechanism for their formation

The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4.

Science.gov (United States)

Dlouhy, Adrienne C; Li, Haoran; Albetel, Angela-Nadia; Zhang, Bo; Mapolelo, Daphne T; Randeniya, Sajini; Holland, Ashley A; Johnson, Michael K; Outten, Caryn E

2016-12-13

Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The Escherichia coli genome encodes members of both of these proteins families, namely, the monothiol glutaredoxin Grx4 and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that E. coli Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4-IbaG heterodimer. In vitro interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4-IbaG heterodimer as well as the [2Fe-2S] Grx4-IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe-S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe-S cluster assembly and trafficking in E. coli as demonstrated for other BolA homologues that interact with monothiol Grxs.

Effects of a heat shock protein inducer on the atrial fibrillation substrate caused by acute atrial ischaemia

NARCIS (Netherlands)

Sakabe, Masao; Shiroshita-Takeshita, Akiko; Maguy, Ange; Brundel, Bianca J. J. M.; Fujiki, Akira; Inoue, Hiroshi; Nattel, Stanley

2008-01-01

Aims Heat shock proteins (HSPs) are a set of endogenous cytoprotective factors activated by various pathological conditions. This study addressed the effects of geranylgeranylacetone (GGA), an orally active HSP inducer, on the atrial fibrillation (AF) substrate associated with acute atria( ischaemia

Pengaruh Nisbah Energi-Protein, Nitrogen-Sulfur dan Kalsium-Fosfor Terhadap Produk Metabolisme Rumen dan Kecernaan Substrat

Directory of Open Access Journals (Sweden)

S.N.O. Suwandyastuti

2013-10-01

Full Text Available Influence of the ratio of energy-protein, nitrogen-sulphur, and calsium-phosphor upon rumen metabolism product and digestibility of substrat ABSTRACT. The Rumen microbes are capable to digest the glucosa polymer of plant waste for energy source and can used the Non Protein Nitrogen (NPN for body protein synthesis, if the other precussor (Sulphur, Phospor and Branch Chain of Carbon are available.  To know the effectivity of the utilization of plant waste for energy, an experiment have been conducted by in vitro method, used the Randomized Black Design, four replication, factorial 33.  The factors tested are : (1 three levels of energy : protein (E/P ratio : 4, 5 and 6; (2 three levels of Nitrogen : Sulphur (N/Sratio: 7.5, 10 and 12.5; (3 three levels of Calsium : Phospor (Ca/P ratio : 0.5, 1 and 2. The variables measured are : synthesis of protein microbes (SPM ; production of Volatile Fatty Acid (VFA and Nitrogen Ammonia (N-NH3; the digestibility of substrat. Based on the all variable measured, the experiment can be concluded : (1 the effectivity of the utilization of rice straw will be increased if it used is fortified with 50 percent TDN as energy source (E/P=4, 0.20 percent dry matter of sulphur (N/S=10 and 0.0625 - 0.125 percent dry matter (DM of phospor (Ca/P=1.0 – 2.0; (2 To stimulate the activity of cellulolytic microbes, its need the fortification of sulphur until reach the optimum level (must be investigated.

Progranulin, a glycoprotein deficient in frontotemporal dementia, is a novel substrate of several protein disulfide isomerase family proteins.

Directory of Open Access Journals (Sweden)

Sandra Almeida

Full Text Available The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER and Golgi. Using chemical crosslinking, immunoprecipitation, and mass spectrometry, we found that progranulin is bound to a network of ER Ca(2+-binding chaperones including BiP, calreticulin, GRP94, and four members of the protein disulfide isomerase (PDI family. Loss of ERp57 inhibits progranulin secretion. Thus, progranulin is a novel substrate of several PDI family proteins and modulation of the ER chaperone network may be a therapeutic target for controlling progranulin secretion.

N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

Science.gov (United States)

Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

2015-01-01

The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental disorder.

Science.gov (United States)

Sekiguchi, Mari; Katayama, Syouichi; Hatano, Naoya; Shigeri, Yasushi; Sueyoshi, Noriyuki; Kameshita, Isamu

2013-07-15

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in brain and mutations of its gene are known to be associated with neurodevelopmental disorders such as X-linked West syndrome and Rett syndrome. However, the physiological substrates of CDKL5 that are directly linked to these neurodevelopmental disorders are currently unknown. In this study, we explored endogenous substrates for CDKL5 in mouse brain extracts fractionated by a liquid-phase isoelectric focusing. In conjunction with CDKL5 phosphorylation assay, this approach detected a protein band with an apparent molecular mass of 120kDa that is remarkably phosphorylated by CDKL5. This 120-kDa protein was identified as amphiphysin 1 (Amph1) by LC-MS/MS analysis, and the site of phosphorylation by CDKL5 was determined to be Ser-293. The phosphorylation mimic mutants, Amph1(S293E) and Amph1(S293D), showed significantly reduced affinity for endophilin, a protein involved in synaptic vesicle endocytosis. Introduction of point mutations in the catalytic domain of CDKL5, which are disease-causing missense mutations found in Rett patients, resulted in the impairment of kinase activity toward Amph1. These results suggest that Amph1 is the cytoplasmic substrate for CDKL5 and that its phosphorylation may play crucial roles in the neuronal development. Copyright © 2013 Elsevier Inc. All rights reserved.

Introducing enzyme selectivity as a quantitative parameter to describe the effects of substrate concentration on protein hydrolysis

NARCIS (Netherlands)

Butré, C.I.

2014-01-01

To understand the differences in peptide composition that result from variations in the conditions of enzymatic hydrolysis of proteins (e.g. substrate concentration) the mechanism of hydrolysis needs to be understood in detail. Therefore, methods and tools were developed to characterize and

Effect of administration route on FES uptake into MCF-7 tumors

International Nuclear Information System (INIS)

Downer, Joanna B.; Jones, Lynne A.; Katzenellenbogen, John A.; Welch, Michael J.

2001-01-01

We have observed that intraperitoneal administration of [ 18 F]fluoroestradiol (FES), a radiolabeled estrogen receptor ligand, results in higher abdominal organ uptake and slower blood clearance than intravenous administration in female mice. In SCID mice bearing MCF-7 human tumors SC, IP administration resulted in tumor uptake that was only about one third that obtained with IV administration. Thus, the route of administration of a radiopharmaceutical for imaging or radiotherapy of a tumor in the abdomen, an ovarian tumor, for example, could have a profound effect on the efficiency and selectivity of delivery of the agent to the tumor

Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery

Science.gov (United States)

Maio, Nunziata; Rouault, Tracey. A.

2014-01-01

Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i. e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein- protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. PMID:25245479

Effect of surface Fe-S hybrid structure on the activity of the perfect and reduced α-Fe2O3(001) for chemical looping combustion

Science.gov (United States)

Xiao, Xianbin; Qin, Wu; Wang, Jianye; Li, Junhao; Dong, Changqing

2018-05-01

Sulfurization of the gradually reduced Fe2O3 surfaces is inevitable while Fe2O3 is used as an oxygen carrier (OC) for coal chemical looping combustion (CLC), which will result in formation of Fe-S hybrid structure on the surfaces. The Fe-S hybrid structure will directly alter the reactivity of the surfaces. Therefore, detailed properties of Fe-S hybrid structure over the perfect and reduced Fe2O3(001) surfaces, and its effect on the interfacial interactions, including CO oxidization and decomposition on the surfaces, were investigated by using density functional theory (DFT) calculations. The S atom prefers to chemically bind to Fe site with electron transfer from the surfaces to the S atom, and a deeper reduction of Fe2O3(001) leads to an increasing interaction between S and Fe. The formation of Fe-S hybrid structure alters the electronic properties of the gradually reduced Fe2O3(001) surfaces, promoting CO oxidation on the surfaces ranging from Fe2O3 to FeO, but depressing carbon deposition on the surfaces ranging from FeO to Fe. The sulfurized FeO acts as a watershed to realize relatively high CO oxidation rate and low carbon deposition. Results provided a fundamental understanding for controlling and optimizing the CLC processes.

Substrate channel in nitrogenase revealed by a molecular dynamics approach.

Science.gov (United States)

Smith, Dayle; Danyal, Karamatullah; Raugei, Simone; Seefeldt, Lance C

2014-04-15

Mo-dependent nitrogenase catalyzes the biological reduction of N2 to two NH3 molecules at FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized submicrosecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel. The viability of this observed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, resulting in the discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment and that approaches a face of FeMo-cofactor earlier implicated in substrate binding.

[Comparative characteristics of microbial proteases by the level of hydrolysis of protein substrates].

Science.gov (United States)

Rimareva, L V; Overchenko, M B; Serba, E M; Trifonova, V V

1997-01-01

Screening of enzyme preparations displaying a maximum proteolytic activity at pH 4.0-5.5 and effecting deep proteolysis of plant proteins was performed. Amyloprotooryzin prepared from Aspergillus oryzae 387 containing a complex of proteolytic enzymes was the most effective. The amino acid composition of the hydrolysates obtained was studied. Amyloprotooryzin increased the contents of amino acids by 108-227%, depending on the substrate used. The enzymatic complex of amyloprotooryzin was studied; in addition, proteases, alpha-amylase, exo-beta-glucanase, and xylanase were detected in the complex.

Impact of the environmental conditions and substrate pre-treatment on whey protein hydrolysis: A review.

Science.gov (United States)

Cheison, Seronei Chelulei; Kulozik, Ulrich

2017-01-22

Proteins in solution are subject to myriad forces stemming from interactions with each other as well as with the solvent media. The role of the environmental conditions, namely pH, temperature, ionic strength remains under-estimated yet it impacts protein conformations and consequently its interaction with, and susceptibility to, the enzyme. Enzymes, being proteins are also amenable to the environmental conditions because they are either activated or denatured depending on the choice of the conditions. Furthermore, enzyme specificity is restricted to a narrow regime of optimal conditions while opportunities outside the optimum conditions remain untapped. In addition, the composition of protein substrate (whether mixed or single purified) have been underestimated in previous studies. In addition, protein pre-treatment methods like heat denaturation prior to hydrolysis is a complex phenomenon whose progression is influenced by the environmental conditions including the presence or absence of sugars like lactose, ionic strength, purity of the protein, and the molecular structure of the mixed proteins particularly presence of free thiol groups. In this review, we revisit protein hydrolysis with a focus on the impact of the hydrolysis environment and show that preference of peptide bonds and/or one protein over another during hydrolysis is driven by the environmental conditions. Likewise, heat-denaturing is a process which is dependent on not only the environment but the presence or absence of other proteins.

Determination of the Influence of Substrate Concentration on Enzyme Selectivity Using Whey Protein Isolate and Bacillus licheniformis Protease

NARCIS (Netherlands)

Butré, C.I.; Sforza, S.; Gruppen, H.; Wierenga, P.A.

2014-01-01

Increasing substrate concentration during enzymatic protein hydrolysis results in a decrease in hydrolysis rate. To test if changes in the mechanism of hydrolysis also occur, the enzyme selectivity was determined. The selectivity is defined quantitatively as the relative rate of hydrolysis of each

SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters.

Science.gov (United States)

Lee, Po-Hsien; Kuo, Kuei-Ling; Chu, Pei-Ying; Liu, Eric M; Lin, Jung-Hsin

2009-07-01

Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design.

Na7 [Fe2S6 ] , Na2 [FeS2 ] and Na2 [FeSe2 ] : New 'reduced' sodium chalcogenido ferrates

Science.gov (United States)

Stüble, Pirmin; Peschke, Simon; Johrendt, Dirk; Röhr, Caroline

2018-02-01

Three new 'reduced' FeII containing sodium chalcogenido ferrates were obtained applying a reductive synthetic route. The mixed-valent sulfido ferrate Na7 [Fe2S6 ] , which forms bar-shaped crystals with metallic greenish luster, was synthesized in pure phase from natural pyrite and elemental sodium at a maximum temperature of 800 °C. Its centrosymmetric triclinic structure (SG P 1 bar , a = 764.15(2), b = 1153.70(2), c = 1272.58(3) pm, α = 62.3325 (7) , β = 72.8345 (8) , γ = 84.6394 (8) ° , Z = 3, R1 = 0.0185) exhibits two crystallographically different [Fe2S6 ] 7 - dimers of edge-sharing [FeS4 ] tetrahedra, with somewhat larger Fe-S distances than in the fully oxidized FeIII dimers of e.g. Na6 [Fe2III S6 ] . In contrast to the localized AFM ordered pure di-ferrates(III), the Curie-Weiss behavior of the magnetic susceptibility proves the rarely observed valence-delocalized S = 9/2 state of the mixed-valent FeIII /FeII dimer. The nearly spin-only value of the magnetic moment combined with the chemical bonding not generally differing from that in pure ferrates(II) and (III), provides a striking argument, that the reduction of the local Fe spin moments observed in all condensed sulfido ferrate moieties is connected with the AFM spin ordering. The two isotypic ferrates(II) Na2 [FeS2 ] and Na2 [FeSe2 ] with chain-like structural units (SG Ibam, a = 643.54(8)/ 660.81(1), b = 1140.2(2)/1190.30(2) c = 562.90(6)/585.59(1) pm, Z = 4, R1 = 0.0372/0.0466) crystallize in the K2 [ZnO2 ] -type structure. Although representing merely further members of the common series of chalcogenido metallates(II) Na2 [MIIQ2 ] , these two new phases, together with Na6 [FeS4 ] and Li2 [FeS2 ] , are the only examples of pure FeII alkali chalcogenido ferrates. The new compounds allow for a general comparison of di- and chain ferrates(II) and (III) and mixed-valent analogs concerning the electronic and magnetic properties (including Heisenberg super-exchange and double-exchange interactions

Development of Conformation Independent Computational Models for the Early Recognition of Breast Cancer Resistance Protein Substrates

Directory of Open Access Journals (Sweden)

Melisa Edith Gantner

2013-01-01

Full Text Available ABC efflux transporters are polyspecific members of the ABC superfamily that, acting as drug and metabolite carriers, provide a biochemical barrier against drug penetration and contribute to detoxification. Their overexpression is linked to multidrug resistance issues in a diversity of diseases. Breast cancer resistance protein (BCRP is the most expressed ABC efflux transporter throughout the intestine and the blood-brain barrier, limiting oral absorption and brain bioavailability of its substrates. Early recognition of BCRP substrates is thus essential to optimize oral drug absorption, design of novel therapeutics for central nervous system conditions, and overcome BCRP-mediated cross-resistance issues. We present the development of an ensemble of ligand-based machine learning algorithms for the early recognition of BCRP substrates, from a database of 262 substrates and nonsubstrates compiled from the literature. Such dataset was rationally partitioned into training and test sets by application of a 2-step clustering procedure. The models were developed through application of linear discriminant analysis to random subsamples of Dragon molecular descriptors. Simple data fusion and statistical comparison of partial areas under the curve of ROC curves were applied to obtain the best 2-model combination, which presented 82% and 74.5% of overall accuracy in the training and test set, respectively.

Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate.

Science.gov (United States)

Emanuele, Michael J; Ciccia, Alberto; Elia, Andrew E H; Elledge, Stephen J

2011-06-14

The anaphase-promoting complex/cyclosome (APC/C) is a cell cycle-regulated E3 ubiquitin ligase that controls the degradation of substrate proteins at mitotic exit and throughout the G1 phase. We have identified an APC/C substrate and cell cycle-regulated protein, KIAA0101/PAF15. PAF15 protein levels peak in the G2/M phase of the cell cycle and drop rapidly at mitotic exit in an APC/C- and KEN-box-dependent fashion. PAF15 associates with proliferating cell nuclear antigen (PCNA), and depletion of PAF15 decreases the number of cells in S phase, suggesting a role for it in cell cycle regulation. Following irradiation, PAF15 colocalized with γH2AX foci at sites of DNA damage through its interaction with PCNA. Finally, PAF15 depletion led to an increase in homologous recombination-mediated DNA repair, and overexpression caused sensitivity to UV-induced DNA damage. We conclude that PAF15 is an APC/C-regulated protein involved in both cell cycle progression and the DNA damage response.

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: [Fe-S] cluster-driven protein rearrangement

International Nuclear Information System (INIS)

Martin, A.E.; Burgess, B.K.; Stout, C.D.; Cash, V.L.; Dean, D.R.; Jensen, G.M.; Stephens, P.J.

1990-01-01

Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here the authors report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure

ER-bound protein tyrosine phosphatase PTP1B interacts with Src at the plasma membrane/substrate interface.

Directory of Open Access Journals (Sweden)

Melisa C Monteleone

Full Text Available PTP1B is an endoplasmic reticulum (ER anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC. Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.

The effect of polylysine on casein-kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme

DEFF Research Database (Denmark)

Meggio, F; Boldyreff, B; Marin, O

1992-01-01

, moreover, is variably accounted for by changes in Vmax and/or Km, depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the beta subunit, since the recombinant alpha subunit is much less responsive than CK2 holoenzyme......The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic alpha subunit, either...

Strong 3D and 1D magnetism in hexagonal Fe-chalcogenides FeS and FeSe vs. weak magnetism in hexagonal FeTe.

Science.gov (United States)

Parker, David S

2017-06-13

We present a comparative theoretical study of the hexagonal forms of the Fe-chalcogenides FeS, FeSe and FeTe with their better known tetragonal forms. While the tetragonal forms exhibit only an incipient antiferromagnetism and experimentally show superconductivity when doped, the hexagonal forms of FeS and FeSe display a robust magnetism. We show that this strong magnetism arises from a van Hove singularity associated with the direct Fe-Fe c-axis chains in the generally more three-dimensional NiAs structure. We also find that hexagonal FeTe is much less magnetic than the other two hexagonal materials, so that unconventional magnetically-mediated superconductivity is possible, although a large T c value is unlikely.

Loss of proteostatic control as a substrate for Atrial Fibrillation; a novel target for upstream therapy by Heat Shock Proteins

Directory of Open Access Journals (Sweden)

Roelien Amanda Marjolein Meijering

2012-02-01

Full Text Available Atrial Fibrillation (AF is the most common, sustained clinical tachyarrhythmia associated with significant morbidity and mortality. AF is a persistent condition with progressive structural remodeling of the atrial cardiomyocytes due to the AF itself, resulting in cellular changes commonly observed in ageing and in other heart diseases. While rhythm control by electrocardioversion or drug treatment is the treatment of choice in symptomatic AF patients, its effectiveness is still limited. Current research is directed at preventing new-onset AF by limiting the development of substrates underlying AF promotion and resembles mechanism-based therapy. Upstream therapy refers to the use of non-ion channel anti-arrhythmic drugs that modify the atrial substrate- or target-specific mechanisms of AF, with the ultimate aim to prevent the occurrence (primary prevention or recurrence of the arrhythmia following (spontaneous conversion (secondary prevention.Heat shock proteins (HSPs are molecular chaperones and comprise a large family of proteins involved in the protection against various forms of cellular stress. Their classical function is the conservation of proteostasis via prevention of toxic protein aggregation by binding to (partially unfolded proteins. Our recent data reveal that HSPs prevent electrical, contractile and structural remodeling of cardiomyocytes, thus attenuating the AF substrate in cellular, Drosophila melanogaster and animal experimental models. Furthermore, studies in humans suggest a protective role for HSPs against the progression from paroxysmal AF to persistent AF and in recurrence of AF. In this review, we discuss upregulation of the heat shock response system as a novel target for upstream therapy to prevent derailment of proteostasis and consequently promotion and recurrence of AF.

Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A-Class Dye-Decolorizing Peroxidase from Thermomonospora curvata

Energy Technology Data Exchange (ETDEWEB)

Shrestha, Ruben; Chen, Xuejie; Ramyar, Kasra X.; Hayati, Zahra; Carlson, Eric A.; Bossmann, Stefan H.; Song, Likai; Geisbrecht, Brian V.; Li, Ping (FSU); (KSU)

2016-12-12

Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases in which a catalytic distal aspartate is involved in H₂O₂ activation to catalyze oxidations under acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial TcDyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds. On the basis of the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the TcDyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in TcDyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of the surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the cross-linking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class TcDyP is minimal, possibly due to its extremely small solvent-accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding the DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.

Dynamic substrate enhancement for the identification of specific, second-site-binding fragments targeting a set of protein tyrosine phosphatases

NARCIS (Netherlands)

Schmidt, Marco F; Groves, Matthew R; Rademann, Jörg

2011-01-01

Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets. The development of potent, selective PTP inhibitors has been a difficult challenge mainly due to the high homology of the phosphotyrosine substrate pockets. Here, a strategy of dynamic

FIRST SOUND EVIDENCE OF MUSCLE REGENERATION IN RECOVERY OF FUNCTION OF HUMAN PERMANENT DENERVATED MUSCLES BY A LONG-LASTING FUNCTIONAL ELECTRICAL STIMULATION (FES TRAINING: BIOPSY FINDINGS

Directory of Open Access Journals (Sweden)

Helmut Kern

2004-12-01

Full Text Available Contrary to general believe, in one case of 18month cauda equina lesion four-month electrical stimulation of thigh muscles (impulse energy 1.92 Joule increased stimulation frequency from 2 to 20 Hz, i. e., up to tetanic contractions. After 2 years of treatment, CT-cross sectional area of quadriceps improved 58.3% (right and 44.4% (left with increased muscle density. Mean myofiber size was 37.2 ± 24.8 µm (right and 40.5 ±  24.9 µm (left. Improvement of stimulated knee torque, from zero to 12.0 Nm and 10.5 Nm, respectively, enabled to stand up trials. Surviving myofibers undergo re-growth (they show the chess board appearance of normal muscle, and dying myofibers continuously regenerate (up to 3% are embryonic myosin positive 3-year post-FES. Regeneration events are essential components of the FES rehabilitation protocol due to superior excitability of regenerated myofibers in comparison to long-term denervated, degenerated myofibers, which were almost not excitable before FES training.

Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding.

Science.gov (United States)

Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

2015-01-01

Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.

Thermoluminescence kinetics of pyrite (FeS2)

International Nuclear Information System (INIS)

Silverman, A.N; Levy, P.W.; Kierstead, J.A.

1990-01-01

Thermoluminescence of pyrite (FeS 2 ) has been investigated to study the kinetics of single peak glow curves. The material used normally exhibits one large and four small peaks. However a glow curve can be obtained with only the large single peak that is suitable for testing thermoluminescence kinetics. Glow curves from aliquots of a single natural pyrite crystal studied in detail contain two low intensity thermoluminescence (TL) peaks at ∼90 degree and ∼250 degree C, and two chemiluminescence (CL) peaks at ∼350 degree and ∼430 degree C. The CL peaks are largely removable by initially heating the sample chamber under vacuum, pumping through liquid nitrogen traps, and recording glow curves immediately after helium is introduced, procedures which reduce system contaminants that react with pyrite. The shape, the variation of the temperature of the peak maximum (T max ) with dose, and the retrapping to recombination cross section ratio σ of the large 250 degree C peak are better described by the general one trap (GOT) kinetic equation, the basic equation from which the 1st and 2nd order kinetic equations are obtained as special cases (see text), than by the 1st and 2nd order equations. 12 refs., 7 figs

Strong 3D and 1D magnetism in hexagonal Fe-chalcogenides FeS and FeSe vs. weak magnetism in hexagonal FeTe

Energy Technology Data Exchange (ETDEWEB)

Parker, David S. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

2017-06-13

We present a comparative theoretical study of the hexagonal forms of the Fe-chalcogenides FeS, FeSe and FeTe with their better known tetragonal forms. While the tetragonal forms exhibit only an incipient antiferromagnetism and experimentally show superconductivity when doped, the hexagonal forms of FeS and FeSe display a robust magnetism. We show that this strong magnetism arises from a van Hove singularity associated with the direct Fe-Fe c-axis chains in the generally more three-dimensional NiAs structure. We also find that hexagonal FeTe is much less magnetic than the other two hexagonal materials, so that unconventional magnetically-mediated superconductivity is possible, although a large T_c value is unlikely.

Involvement of the Cra global regulatory protein in the expression of the iscRSUA operon, revealed during studies of tricarballylate catabolism in Salmonella enterica.

Science.gov (United States)

Lewis, Jeffrey A; Boyd, Jeffrey M; Downs, Diana M; Escalante-Semerena, Jorge C

2009-04-01

In Salmonella enterica, tricarballylate (Tcb) catabolism requires function of TcuB, a membrane-bound protein that contains [4Fe-4S] clusters and heme. TcuB transfers electrons from reduced flavin adenine dinucleotide in the Tcb dehydrogenase (TcuA) to electron acceptors in the membrane. We recently showed that functions needed to assemble [Fe-S] clusters (i.e., the iscRSUA-hscBA-fdx operon) compensate for the lack of ApbC during growth of an apbC strain on Tcb. ApbC had been linked to [Fe-S] cluster metabolism, and we showed that an apbC strain had decreased TcuB activity. Here we report findings that expand our understanding of the regulation of expression of the iscRSUA genes in Salmonella enterica. We investigated why low levels of glucose or other saccharides restored growth of an apbC strain on Tcb. Here we report the following findings. (i) A Cra. (iv) Putative Cra binding sites are present in the regulatory region of the iscRSUA operon. (v) Cra protein binds to all three sites in the iscRSUA promoter region in a concentration-dependent fashion. To our knowledge, this is the first report of the involvement of Cra in [Fe-S] cluster assembly.

Embryonal Fyn-associated substrate (EFS) and CASS4: The lesser-known CAS protein family members.

Science.gov (United States)

Deneka, Alexander; Korobeynikov, Vladislav; Golemis, Erica A

2015-10-01

The CAS (Crk-associated substrate) adaptor protein family consists of four members: CASS1/BCAR1/p130Cas, CASS2/NEDD9/HEF1/Cas-L, CASS3/EFS/Sin and CASS4/HEPL. While CAS proteins lack enzymatic activity, they contain specific recognition and binding sites for assembly of larger signaling complexes that are essential for cell proliferation, survival, migration, and other processes. All family members are intermediates in integrin-dependent signaling pathways mediated at focal adhesions, and associate with FAK and SRC family kinases to activate downstream effectors regulating the actin cytoskeleton. Most studies of CAS proteins to date have been focused on the first two members, BCAR1 and NEDD9, with altered expression of these proteins now appreciated as influencing disease development and prognosis for cancer and other serious pathological conditions. For these family members, additional mechanisms of action have been defined in receptor tyrosine kinase (RTK) signaling, estrogen receptor signaling or cell cycle progression, involving discrete partner proteins such as SHC, NSP proteins, or AURKA. By contrast, EFS and CASS4 have been less studied, although structure-function analyses indicate they conserve many elements with the better-known family members. Intriguingly, a number of recent studies have implicated these proteins in immune system function, and the pathogenesis of developmental disorders, autoimmune disorders including Crohn's disease, Alzheimer's disease, cancer and other diseases. In this review, we summarize the current understanding of EFS and CASS4 protein function in the context of the larger CAS family group. Copyright © 2015 Elsevier B.V. All rights reserved.

Protein farnesyltransferase isoprenoid substrate discrimination is dependent on isoprene double bonds and branched methyl groups.

Science.gov (United States)

Micali, E; Chehade, K A; Isaacs, R J; Andres, D A; Spielmann, H P

2001-10-16

Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized the benzyloxyisoprenyl pyrophosphate (BnPP) series of transferable farnesyl pyrophosphate (FPP) analogues (1a-e) to test the length dependence of the isoprenoid substrate on the FTase-catalyzed transfer of lipid to protein substrate. Kinetic analyses show that pyrophosphates 1a-e and geranyl pyrophosphate (GPP) transfer with a lower efficiency than FPP whereas geranylgeranyl pyrophosphate (GGPP) does not transfer at all. While a correlation was found between K(m) and analogue hydrophobicity and length, there was no correlation between k(cat) and these properties. Potential binding geometries of FPP, GPP, GGPP, and analogues 1a-e were examined by modeling the molecules into the active site of the FTase crystal structure. We found that analogue 1d displaces approximately the same volume of the active site as does FPP, whereas GPP and analogues 1a-c occupy lesser volumes and 1e occupies a slightly larger volume. Modeling also indicated that GGPP adopts a different conformation than the farnesyl chain of FPP, partially occluding the space occupied by the Ca(1)a(2)X peptide in the ternary X-ray crystal structure. Within the confines of the FTase pocket, the double bonds and branched methyl groups of the geranylgeranyl chain significantly restrict the number of possible conformations relative to the more flexible lipid chain of analogues 1a-e. The modeling results also provide a molecular explanation for the observation that an aromatic ring is a good isostere for the terminal isoprene of FPP.

Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

Science.gov (United States)

Bezerra, Gustavo Arruda; Ohara-Nemoto, Yuko; Cornaciu, Irina; Fedosyuk, Sofiya; Hoffmann, Guillaume; Round, Adam; Márquez, José A; Nemoto, Takayuki K; Djinović-Carugo, Kristina

2017-06-06

Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

Intracellular protein breakdown. 8

International Nuclear Information System (INIS)

Bohley, P.; Kirschke, H.; Langner, J.; Wiederanders, B.; Ansorge, S.

1976-01-01

Double-labelled proteins from rat liver cytosol ( 14 C in long-lived, 3 H in short-lived proteins after in-vivo-labelling) are used as substrates for unlabelled proteinases in vitro. Differences in the degradation rates of short-lived and long-lived proteins in vitro by different proteinases and after addition of different effectors allow conclusions concerning their importance for the in-vivo-turnover of substrate proteins. The main activity (>90%) of soluble lysosomal proteinases at pH 6.1 and pH 6.9 is caused by thiolproteinases, which degrade preferentially short-lived cytosol proteins. These proteinases are inhibited by leupeptin. Autolysis of double-labelled cell fractions shows a remarkably faster breakdown of short-lived substrate proteins only in the soluble part of lysosomes. Microsomal fractions degrade in vitro preferentially long-lived substrate proteins. (author)

Synthesis and structural insight into ESX-1 Substrate Protein C, an immunodominant Mycobacterium tuberculosis-secreted antigen.

Science.gov (United States)

Son, Soo Jung; Harris, Paul W R; Squire, Chris J; Baker, Edward N; Brimble, Margaret A

2016-05-01

Tuberculosis, the second leading cause of death from a single infectious agent, is recognized as a major threat to human health due to a lack of practicable vaccines against the disease and the widespread occurrence of drug resistance. With a pressing need for a novel protein target as a platform for new vaccine development, ESX-1 Substrate Protein C (EspC) was recently identified as a novel Mycobacterium tuberculosis-secreted antigen that is as immunodominant as the two specific immunodiagnostic T-cell antigens, CFP-10 and ESAT-6. Here, we present the first chemical total synthesis, folding conditions, and circular dichroism data of EspC. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 267-274, 2016. © 2016 Wiley Periodicals, Inc.

Native Liquid Extraction Surface Analysis Mass Spectrometry: Analysis of Noncovalent Protein Complexes Directly from Dried Substrates

Science.gov (United States)

Martin, Nicholas J.; Griffiths, Rian L.; Edwards, Rebecca L.; Cooper, Helen J.

2015-08-01

Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2 4H] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The `contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

Science.gov (United States)

Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

2015-01-01

Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388

The effectiveness of FES-evoked EMG potentials to assess muscle force and fatigue in individuals with spinal cord injury.

Science.gov (United States)

Ibitoye, Morufu Olusola; Estigoni, Eduardo H; Hamzaid, Nur Azah; Wahab, Ahmad Khairi Abdul; Davis, Glen M

2014-07-14

The evoked electromyographic signal (eEMG) potential is the standard index used to monitor both electrical changes within the motor unit during muscular activity and the electrical patterns during evoked contraction. However, technical and physiological limitations often preclude the acquisition and analysis of the signal especially during functional electrical stimulation (FES)-evoked contractions. Hence, an accurate quantification of the relationship between the eEMG potential and FES-evoked muscle response remains elusive and continues to attract the attention of researchers due to its potential application in the fields of biomechanics, muscle physiology, and rehabilitation science. We conducted a systematic review to examine the effectiveness of eEMG potentials to assess muscle force and fatigue, particularly as a biofeedback descriptor of FES-evoked contractions in individuals with spinal cord injury. At the outset, 2867 citations were identified and, finally, fifty-nine trials met the inclusion criteria. Four hypotheses were proposed and evaluated to inform this review. The results showed that eEMG is effective at quantifying muscle force and fatigue during isometric contraction, but may not be effective during dynamic contractions including cycling and stepping. Positive correlation of up to r = 0.90 (p peak-to-peak amplitude of the eEMG and the decline in the force output during fatiguing isometric contractions has been reported. In the available prediction models, the performance index of the eEMG signal to estimate the generated muscle force ranged from 3.8% to 34% for 18 s to 70 s ahead of the actual muscle force generation. The strength and inherent limitations of the eEMG signal to assess muscle force and fatigue were evident from our findings with implications in clinical management of spinal cord injury (SCI) population.

The Effectiveness of FES-Evoked EMG Potentials to Assess Muscle Force and Fatigue in Individuals with Spinal Cord Injury

Directory of Open Access Journals (Sweden)

Morufu Olusola Ibitoye

2014-07-01

Full Text Available The evoked electromyographic signal (eEMG potential is the standard index used to monitor both electrical changes within the motor unit during muscular activity and the electrical patterns during evoked contraction. However, technical and physiological limitations often preclude the acquisition and analysis of the signal especially during functional electrical stimulation (FES-evoked contractions. Hence, an accurate quantification of the relationship between the eEMG potential and FES-evoked muscle response remains elusive and continues to attract the attention of researchers due to its potential application in the fields of biomechanics, muscle physiology, and rehabilitation science. We conducted a systematic review to examine the effectiveness of eEMG potentials to assess muscle force and fatigue, particularly as a biofeedback descriptor of FES-evoked contractions in individuals with spinal cord injury. At the outset, 2867 citations were identified and, finally, fifty-nine trials met the inclusion criteria. Four hypotheses were proposed and evaluated to inform this review. The results showed that eEMG is effective at quantifying muscle force and fatigue during isometric contraction, but may not be effective during dynamic contractions including cycling and stepping. Positive correlation of up to r = 0.90 (p < 0.05 between the decline in the peak-to-peak amplitude of the eEMG and the decline in the force output during fatiguing isometric contractions has been reported. In the available prediction models, the performance index of the eEMG signal to estimate the generated muscle force ranged from 3.8% to 34% for 18 s to 70 s ahead of the actual muscle force generation. The strength and inherent limitations of the eEMG signal to assess muscle force and fatigue were evident from our findings with implications in clinical management of spinal cord injury (SCI population.

Melting phase relations in the Fe-S and Fe-S-O systems at core conditions in small terrestrial bodies

Science.gov (United States)

Pommier, Anne; Laurenz, Vera; Davies, Christopher J.; Frost, Daniel J.

2018-05-01

We report an experimental investigation of phase equilibria in the Fe-S and Fe-S-O systems. Experiments were performed at high temperatures (1400-1850 °C) and high pressures (14 and 20 GPa) using a multi-anvil apparatus. The results of this study are used to understand the effect of sulfur and oxygen on core dynamics in small terrestrial bodies. We observe that the formation of solid FeO grains occurs at the Fe-S liquid - Fe solid interface at high temperature ( > 1400 °C at 20 GPa). Oxygen fugacities calculated for each O-bearing sample show that redox conditions vary from ΔIW = -0.65 to 0. Considering the relative density of each phase and existing evolutionary models of terrestrial cores, we apply our experimental results to the cores of Mars and Ganymede. We suggest that the presence of FeO in small terrestrial bodies tends to contribute to outer-core compositional stratification. Depending on the redox and thermal history of the planet, FeO may also help form a transitional redox zone at the core-mantle boundary.

Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

Science.gov (United States)

Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

2011-01-01

HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

Characterization and evaluation of whey protein-based biofilms as substrates for in vitro cell cultures.

Science.gov (United States)

Gilbert, Vanessa; Rouabhia, Mahmoud; Wang, Hongxum; Arnould, Anne-Lise; Remondetto, Gabriel; Subirade, Muriel

2005-12-01

Whey proteins-based biofilms were prepared using different plasticizers in order to obtain a biomaterial for the human keratinocytes and fibroblasts in vitro culture. The film properties were evaluated by Fourier Transform Infrared Spectroscopy (FTIR) technique and mechanical tests. A relationship was found between the decrease of intermolecular hydrogen bond strength and film mechanical behavior changes, expressed by a breaking stress and Young modulus values diminishing. These results allow stating that the film molecular configuration could induce dissimilarities in its mechanical properties. The films toxicity was assessed by evaluating the cutaneous cells adherence, growth, proliferation and structural stratification. Microscopic observation demonstrated that both keratinocytes and fibroblasts adhered to the biofilms. The trypan blue exclusion test showed that keratinocytes grew at a significantly high rate on all the biofilms. Structural analysis demonstrated that keratinocytes stratified when cultured on the whey protein-based biofilms and gave rise to multi-layered epidermal structures. The most organized epidermis was obtained with whey protein isolate/DEG biofilm. This structure had a well-organized basal layer under supra-basal and corneous layers. This study demonstrated that whey proteins, an inexpensive renewable resource which can be obtained readily, were non-toxic to cutaneous cells and thus they could be useful substrates for a variety of biomedical applications, including tissue engineering.

Virtual half-metallicity at the CoS2/FeS2 interface induced by strain

KAUST Repository

Nazir, Safdar

2013-01-01

Spin polarized ab initio calculations based on density functional theory are performed to investigate the electronic and magnetic properties of the interface between the ferromagnetic metal CoS2 and the nonmagnetic semiconductor FeS2. Relaxation of the interface structure is taken into account by atomic force minimization. We find that both Co and Fe are close to half-metallicity at the interface. Tensile strain is shown to strongly enhance the spin polarization so that a virtually half-metallic interface can be achieved, for comparably moderate strain. © 2012 The Royal Society of Chemistry.

In-situ Density and Thermal Expansion Measurements of Fe and Fe-S Alloying Liquids Under Planetary Core Conditions

Science.gov (United States)

Jing, Z.; Chantel, J.; Yu, T.; Sakamaki, T.; Wang, Y.

2015-12-01

Liquid iron is likely the dominant constituent in the cores of terrestrial planets and icy satellites such as Earth, Mars, Mercury, the Moon, Ganymede, and Io. Suggested by geophysical and geochemical observations, light elements such as S, C, Si, etc., are likely present in planetary cores. These light elements can significantly reduce the density and melting temperature of the Fe cores, and hence their abundances are crucial to our understanding of the structure and thermal history of planetary cores, as well as the generation of intrinsic magnetic fields. Knowledge on the density of Fe-light element alloying liquids at high pressures is critical to place constraints on the composition of planetary cores. However, density data on liquid Fe-light element alloys at core pressures are very limited in pressure and composition and are sometimes controversial. In this study, we extend the density dataset for Fe-rich liquids by measuring the density of Fe, Fe-10wt%S, Fe-20wt%S, Fe-27wt%S, and FeS liquids using the X-ray absorption technique in a DIA-type multianvil apparatus up to 7 GPa and 2173 K. An ion chamber (1D-detector) and a CCD camera (2D-detector) were used to measure intensities of transmitted monochromatic X-rays through molten samples, with the photon energy optimized at 40 keV. The densities were then determined from the Beer-Lambert law using the mass absorption coefficients, calibrated by solid standards using X-ray diffraction. At each pressure, density measurements were conducted at a range of temperatures above the liquidus of the samples, enabling the determination of thermal expansion. Combined with our previous results on the sound velocity of Fe and Fe-S liquids at high pressures (Jing et al., 2014, Earth Planet. Sci. Lett. 396, 78-87), these data provide tight constraints on the equation of state and thermodynamic properties such as the adiabatic temperature gradient for Fe-S liquids. We will discuss these results with implications to planetary

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

Science.gov (United States)

Braun, Clemens; Sakamoto, Atsushi; Fuchs, Holger; Ishiguro, Naoki; Suzuki, Shinobu; Cui, Yunhai; Klinder, Klaus; Watanabe, Michitoshi; Terasaki, Tetsuya; Sauer, Achim

2017-10-02

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. K p,uu,brain and K p,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, K p,uu,brain for the BCRP substrates was low. In contrast, K p,uu,CSF for both BCRP substrates was close to unity, resulting in K p,uu,CSF /K p,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

Science.gov (United States)

Stepchenkova, E I; Tarakhovskaya, E R; Siebler, H M; Pavlov, Y I

2017-01-01

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ. Copyright © 2016

Acidogenic fermentation characteristics of different types of protein-rich substrates in food waste to produce volatile fatty acids.

Science.gov (United States)

Shen, Dongsheng; Yin, Jun; Yu, Xiaoqin; Wang, Meizhen; Long, Yuyang; Shentu, Jiali; Chen, Ting

2017-03-01

In this study, tofu and egg white, representing typical protein-rich substrates in food waste based on vegetable and animal protein, respectively, were investigated for producing volatile fatty acids (VFAs) by acidogenic fermentation. VFA production, composition, conversion pathways and microbial communities in acidogenesis from tofu and egg white with and without hydrothermal (HT) pretreatment were compared. The results showed HT pretreatment could improve the VFA production of tofu but not for egg white. The optimum VFA yields were 0.46g/gVS (tofu with HT) and 0.26g/gVS (egg white without HT), respectively. Tofu could directly produce VFAs through the Stickland reaction, while egg white was converted to lactate and VFAs simultaneously. About 30-40% of total protein remained in all groups after fermentation. Up to 50% of the unconverted soluble protein in the HT groups was protease. More lactate-producing bacteria, mainly Leuconostoc and Lactobacillus, were present during egg white fermentation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Activation and dissociation of CO2 on the (001), (011), and (111) surfaces of mackinawite (FeS): A dispersion-corrected DFT study.

Science.gov (United States)

Dzade, N Y; Roldan, A; de Leeuw, N H

2015-09-07

Iron sulfide minerals, including mackinawite (FeS), are relevant in origin of life theories, due to their potential catalytic activity towards the reduction and conversion of carbon dioxide (CO2) to organic molecules, which may be applicable to the production of liquid fuels and commodity chemicals. However, the fundamental understanding of CO2 adsorption, activation, and dissociation on FeS surfaces remains incomplete. Here, we have used density functional theory calculations, corrected for long-range dispersion interactions (DFT-D2), to explore various adsorption sites and configurations for CO2 on the low-index mackinawite (001), (110), and (111) surfaces. We found that the CO2 molecule physisorbs weakly on the energetically most stable (001) surface but adsorbs relatively strongly on the (011) and (111) FeS surfaces, preferentially at Fe sites. The adsorption of the CO2 on the (011) and (111) surfaces is shown to be characterized by significant charge transfer from surface Fe species to the CO2 molecule, which causes a large structural transformation in the molecule (i.e., forming a negatively charged bent CO2 (-δ) species, with weaker C-O confirmed via vibrational frequency analyses). We have also analyzed the pathways for CO2 reduction to CO and O on the mackinawite (011) and (111) surfaces. CO2 dissociation is calculated to be slightly endothermic relative to the associatively adsorbed states, with relatively large activation energy barriers of 1.25 eV and 0.72 eV on the (011) and (111) surfaces, respectively.

Raman molecular fingerprint of non-structural protein 1 in phosphate buffer saline with gold substrate.

Science.gov (United States)

Radzol, A R M; Lee, Khuan Y; Mansor, W

2013-01-01

SERS is a form of Raman spectroscopy that is enhanced with nano-sensing chip as substrate. It can yield distinct biochemical fingerprint for molecule of solids, liquids and gases. Vice versa, it can be used to identify unknown molecule. It has further advantage of being non-invasive, non-contact and cheap, as compared to other existing laboratory based techniques. NS1 has been clinically accepted as an alternative biomarker to IgM in diagnosing viral diseases carried by virus of flaviviridae. Its presence in the blood serum at febrile stage of the flavivirus infection has been proven. Being an antigen, it allows early detection that can help to reduce the mortality rate. This paper proposes SERS as a technique for detection of NS1 from its scattering spectrum. Contribution from our work so far has never been reported. From our experiments, it is found that NS1 protein is Raman active. Its spectrum exhibits five prominent peaks at Raman shift of 548, 1012, 1180, 1540 and 1650 cm(-1). Of these, peak at 1012 cm(-1) scales the highest intensity. It is singled out as the peak to fingerprint the NS1 protein. This is because its presence is verified by the ring breathing vibration of the benzene ring structure side chain molecule. The characteristic peak is found to vary in proportion to concentration. It is found that for a 99% change in concentration, a 96.7% change in intensity is incurred. This yields a high sensitivity of about one a.u. per ppm. Further investigation from the characterization graph shows a correlation coefficient of 0.9978 and a standard error estimation of 0.02782, which strongly suggests a linear relationship between the concentration and characteristic peak intensity of NS1. Our finding produces favorable evidence to the use of SERS technique for detection of NS1 protein for early detection of flavivirus infected diseases with gold substrate.

Can FES-augmented active cycling training improve locomotion in post-acute elderly stroke patients?

Directory of Open Access Journals (Sweden)

Elisabetta Peri

2016-06-01

Full Text Available Recent studies advocated the use of active cycling coupled with functional electrical stimulation to induce neuroplasticity and enhance functional improvements in stroke adult patients. The aim of this work was to evaluate whether the benefits induced by such a treatment are superior to standard physiotherapy. A single-blinded randomized controlled trial has been performed on post-acute elderly stroke patients. Patients underwent FES-augmented cycling training combined with voluntary pedaling or standard physiotherapy. The intervention consisted of fifteen 30-minutes sessions carried out within 3 weeks. Patients were evaluated before and after training, through functional scales, gait analysis and a voluntary pedaling test. Results were compared with an age-matched healthy group. Sixteen patients completed the training. After treatment, a general improvement of all clinical scales was obtained for both groups. Only the mechanical efficiency highlighted a group effect in favor of the experimental group. Although a group effect was not found for any other cycling or gait parameters, the experimental group showed a higher percentage of change with respect to the control group (e.g. the gait velocity was improved of 35.4% and 25.4% respectively, and its variation over time was higher than minimal clinical difference for the experimental group only. This trend suggests that differences in terms of motor recovery between the two groups may be achieved increasing the training dose. In conclusion, this study, although preliminary, showed that FES-augmented active cycling training seems to be effective in improving cycling and walking ability in post-acute elderly stroke patients. A higher sample size is required to confirm results.

Substrate-driven mapping of the degradome by comparison of sequence logos.

Directory of Open Access Journals (Sweden)

Julian E Fuchs

Full Text Available Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available.

Activation and dissociation of CO2 on the (001), (011), and (111) surfaces of mackinawite (FeS): A dispersion-corrected DFT study

NARCIS (Netherlands)

Dzade, N.Y.; Roldan, Alberto; de Leeuw, N.H.

2015-01-01

Iron sulfide minerals, including mackinawite (FeS), are relevant in origin of life theories, due to their potential catalytic activity towards the reduction and conversion of carbon dioxide (CO2) to organic molecules, which may be applicable to the production of liquid fuels and commodity chemicals.

Utilization of palm oil processing effluents as substrates for microbial protein production by the fungus Aspergillus oryzae

Energy Technology Data Exchange (ETDEWEB)

Barker, T.; Worgan, J.T.

1981-01-01

The filamentous fungus Aspergillus oryzae grew well on the effluents produced during the extraction of palm oil. Biomass yields of approximately 50 g/100 g organic matter were obtained which contained 40% crude protein and had BOD reductions of 85% and COD reductions of 75-80% in batch culture following optimization of growth conditions. Supplementation with an inorganic N source was necessary. The more resistant substrate constituents to biodegradation were water-soluble carbohydrate and nitrogenous material, possibly Maillard reaction products, and polyphenols.

Proteomic strategy for the analysis of the polychlorobiphenyl-degrading cyanobacterium Anabaena PD-1 exposed to Aroclor 1254.

Directory of Open Access Journals (Sweden)

Hangjun Zhang

Full Text Available The cyanobacterium Anabaena PD-1, which was originally isolated from polychlorobiphenyl (PCB-contaminated paddy soils, has capabilities for dechlorinatin and for degrading the commercial PCB mixture Aroclor 1254. In this study, 25 upregulated proteins were identified using 2D electrophoresis (2-DE coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS. These proteins were involved in (i PCB degradation (i.e., 3-chlorobenzoate-3,4-dioxygenase; (ii transport processes [e.g., ATP-binding cassette (ABC transporter substrate-binding protein, amino acid ABC transporter substrate-binding protein, peptide ABC transporter substrate-binding protein, putrescine-binding protein, periplasmic solute-binding protein, branched-chain amino acid uptake periplasmic solute-binding protein, periplasmic phosphate-binding protein, phosphonate ABC transporter substrate-binding protein, and xylose ABC transporter substrate-binding protein]; (iii energetic metabolism (e.g., methanol/ethanol family pyrroloquinoline quinone (PQQ-dependent dehydrogenase, malate-CoA ligase subunit beta, enolase, ATP synthase β subunit, FOF1 ATP synthase subunit beta, ATP synthase α subunit, and IMP cyclohydrolase; (iv electron transport (cytochrome b6f complex Fe-S protein; (v general stress response (e.g., molecular chaperone DnaK, elongation factor G, and translation elongation factor thermostable; (vi carbon metabolism (methanol dehydrogenase and malate-CoA ligase subunit beta; and (vii nitrogen reductase (nitrous oxide reductase. The results of real-time polymerase chain reaction showed that the genes encoding for dioxygenase, ABC transporters, transmembrane proteins, electron transporter, and energetic metabolism proteins were significantly upregulated during PCB degradation. These genes upregulated by 1.26- to 8.98-fold. These findings reveal the resistance and adaptation of cyanobacterium to the presence of PCBs, shedding light on the

Which fault destroyed Fes city (Morocco) in 1755? A new insight from the Holocene deformations observed along the southern border of Gibraltar arc

Science.gov (United States)

Poujol, Antoine; Ritz, Jean-François; Vernant, Philippe; Huot, Sebastien; Maate, Soufian; Tahayt, Abdelilah

2017-08-01

In this paper, we present the first estimate of the Holocene deformation along the southern front of Gibraltar arc (Morocco) and the first field constraints on the local 1755 CE Fes-Meknes surface rupturing earthquake which could be associated to the "Great Lisbon Earthquake" (M > 8.5) in November 1st, 1755. Using satellite imagery, aerial photographs and field investigations, we carried out a morphotectonic study along the 150 km-long Southern Rif Front (SRF) to identify the most recent evidences of tectonic activity. Analyzed offset alluvial deposits confirm that (i) the last 5 ka cumulative deformation leading to a slip rate of 3.5 ± 1 mm/yr for this segment of the SRF is consistent with the GPS derived horizontal shortening rate of 2-4 mm/yr and (ii) a recent major earthquake ruptured a 30 km-long segment along the SRF. Based on deposits dating and historical seismicity we propose that this seismic event occurred in 1755 as a local earthquake. Even though this 1755 local event cannot be considered as a strong aftershock of the main Lisbon seismic event (M > 8.5), their temporal closeness, their occurrence under the same convergent stress regime ( NNW-SSE-oriented compression) and the fact that Fes-Meknes area was strongly shaken during the Lisbon earthquake, raises the question of the possible triggering of the Fes earthquake. Anyway, our new results suggest that most of the Nubia-Rif belt convergence is accommodated by the SRF, making it potentially the most destructive structure of the Rif.

PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

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Jiangning Song

Full Text Available The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s. Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

KAUST Repository

Rashid, Fahad

2017-02-23

Human flap endonuclease 1 (FEN1) and related structure-specific 5\\'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5\\'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually \\'locks\\' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men

DEFF Research Database (Denmark)

Vigelsø Hansen, Andreas; Gram, Martin; Wiuff, Caroline

2016-01-01

by aerobic training in young and older men. METHODS: 17 young (23 ± 1 years, 24 ± 1 kg m(-2), and 20 ± 2% body fat) and 15 older men (68 ± 1 years; 27 ± 1 kg m(-2), and 29 ± 2% body fat) underwent 2 weeks' one leg immobilization followed by 6 weeks' cycle training. Biopsies were obtained from m. vastus...... lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl...... GS (74%) protein compared to the older men. Immobilization decreased and training restored HAD activity, GS and SNAP23 protein content in young and older men. CONCLUSION: Evidence of age-related metabolic inflexibility is presented, seen as body fat and IMTG accumulation. The question arises...

Three extracellular dipeptidyl peptidases found in Aspergillus oryzae show varying substrate specificities.

Science.gov (United States)

Maeda, Hiroshi; Sakai, Daisuke; Kobayashi, Takuji; Morita, Hiroto; Okamoto, Ayako; Takeuchi, Michio; Kusumoto, Ken-Ichi; Amano, Hitoshi; Ishida, Hiroki; Yamagata, Youhei

2016-06-01

Three extracellular dipeptidyl peptidase genes, dppB, dppE, and dppF, were unveiled by sequence analysis of the Aspergillus oryzae genome. We investigated their differential enzymatic profiles, in order to gain an understanding of the diversity of these genes. The three dipeptidyl peptidases were expressed using Aspergillus nidulans as the host. Each recombinant enzyme was purified and subsequently characterized. The enzymes displayed similar optimum pH values, but optimum temperatures, pH stabilities, and substrate specificities varied. DppB was identified as a Xaa-Prolyl dipeptidyl peptidase, while DppE scissile substrates were similar to the substrates for Aspergillus fumigatus DPPV (AfDPPV). DppF was found to be a novel enzyme that could digest both substrates for A. fumigatus DPPIV and AfDPPV. Semi-quantitative PCR revealed that the transcription of dppB in A. oryzae was induced by protein substrates and repressed by the addition of an inorganic nitrogen source, despite the presence of protein substrates. The transcription of dppE depended on its growth time, while the transcription of dppF was not affected by the type of the nitrogen source in the medium, and it started during the early stage of the fungal growth. Based on these results, we conclude that these enzymes may represent the nutrition acquisition enzymes. Additionally, DppF may be one of the sensor peptidases responsible for the detection of the protein substrates in A. oryzae environment. DppB may be involved in nitrogen assimilation control, since the transcription of dppB was repressed by NaNO3, despite the presence of protein substrates.

FeS2-doped MoS2 nanoflower with the dominant 1T-MoS2 phase as an excellent electrocatalyst for high-performance hydrogen evolution

International Nuclear Information System (INIS)

Zhao, Xue; Ma, Xiao; Lu, Qingqing; Li, Qun; Han, Ce; Xing, Zhicai; Yang, Xiurong

2017-01-01

Well-established methods to improve the hydrogen evolution reaction (HER) performances include, but are not limited to, tailoring the morphology and electronic structure of transition metal dichalcogenides (TMDs), and doping of earth abundant chemicals such as iron pyrite FeS 2 into existing TMDs. In this work, MoS 2 nanoflowers with the majority being octahedral MoS 2 (1T-MoS 2 ) and doped with FeS 2 were prepared and applied to HER. The as-prepared catalysts were characterized by X-ray absorption fine structure at the K-edge of Mo, S, and Fe to probe the local electronic structures. The resulting nanomaterial was identified to be FeS 2 doped MoS 2 nanoflower (denoted as Fe-MoS 2 NF) with 66% 1T-MoS 2 which was the metallic phase and could drastically boost the HER properties. The Fe-MoS 2 NF exhibited high HER performance with a Tafel slope of 82 mV dec −1 and it needs 136 mV to achieve a current density of 10 mA cm −2 . The synthesis of Fe-MoS 2 NF with refined morphology and active electronic structure is expected to open a new era for improving the catalytic activity and stability of MoS 2 .

Evaluation of the Weevil-damaged Sweet Potato as Substrate for Microbial Protein Obtaining

Directory of Open Access Journals (Sweden)

Lic. Antonio Montes-de-Oca-Olivares

2015-11-01

Full Text Available The production of microbial protein from agricultural and agroindustrial wastes is an important way to supply the demand of this essential nutritional principle. Sweet potato (Ipomea batata tubercles damaged by weevil (Cylas formicarius are considered a waste due to their unpleasant flavor. This research deal in the characterization of sweet potato damaged by weevil, as an alternative substratefor the culture of the fodder yeast Candida utilis. It was found that the damaged tubercle had a similar composition that the healthy one, concerning dry matter, total reducing sugars, nitrogen and minerals; the high content of reducing sugars (30-40 % dry weight recommends the use of this waste as a substrate for single cell protein production. Several fungal strains were assayed to enzymatic degradation of sweet potato polysaccharides; from these ones, Aspergillus oryzae H/28-1 and Neurospora sp. were the more actives to release reducing sugars to the culture medium, being the last one the more prominent. Theyeast Candida utilis showed a satisfactory growth in media formulated in basis to weevil-damaged sweet potato, reaching reducing sugar consumptions over 80 % and biomass yields of 37-58 %; addition of urea as nitrogen source improved both parameters of the growth. The fermentation’s end-product acquired a pleasant flavor, which suggests a better palatability.

Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

Science.gov (United States)

Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

1994-11-04

Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

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Trey K Sato

2016-10-01

Full Text Available The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3, a component of MAP Kinase (MAPK signaling (HOG1, a regulator of Protein Kinase A (PKA signaling (IRA2, and a scaffolding protein for mitochondrial iron-sulfur (Fe-S cluster biogenesis (ISU1. Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

Science.gov (United States)

Sato, Trey K; Tremaine, Mary; Parreiras, Lucas S; Hebert, Alexander S; Myers, Kevin S; Higbee, Alan J; Sardi, Maria; McIlwain, Sean J; Ong, Irene M; Breuer, Rebecca J; Avanasi Narasimhan, Ragothaman; McGee, Mick A; Dickinson, Quinn; La Reau, Alex; Xie, Dan; Tian, Mingyuan; Reed, Jennifer L; Zhang, Yaoping; Coon, Joshua J; Hittinger, Chris Todd; Gasch, Audrey P; Landick, Robert

2016-10-01

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

PROTEIN ENRICHMENT OF SPENT SORGHUM RESIDUE USING ...

African Journals Online (AJOL)

BSN

The optimum concentration of spent sorghum for protein enrichment with S. cerevisiae was 7.Sg/100 ml. Th.: protein ... production of single sell protein using Candida utilis and cassava starch effluem as substrate. ... wastes as substrates, Kluyveromyces fragilis and milk whey coconut water as substrate (Rahmat et al.,. 1995 ...

Structural, optical, and hydrogenation properties of ZnO nanowall networks grown on a Si (1 1 1) substrate by plasma-assisted molecular beam epitaxy

International Nuclear Information System (INIS)

Su, S.C.; Lu, Y.M.; Zhang, Z.Z.; Li, B.H.; Shen, D.Z.; Yao, B.; Zhang, J.Y.; Zhao, D.X.; Fan, X.W.

2008-01-01

ZnO nanowall networks were grown on a Si (1 1 1) substrate by plasma-assisted molecular beam epitaxy (P-MBE) without using catalysts. Scanning electronic microscopy (FE-SEM) confirmed the formation of nanowalls with a thickness of about 10-20 nm. X-ray diffraction (XRD) showed that the ZnO nanowall networks were crystallized in a wurtzite structure with their height parallel to the direction. Photoluminescence (PL) of the ZnO nanowall networks exhibited free excitons (FEs), donor-bound exciton (D 0 X), donor-acceptor pair (DAP), and free exciton to acceptor (FA) emissions. The growth mechanism of the ZnO nanowall networks was discussed, and their hydrogenation was also studied

Protein-based stable isotope probing.

Science.gov (United States)

Jehmlich, Nico; Schmidt, Frank; Taubert, Martin; Seifert, Jana; Bastida, Felipe; von Bergen, Martin; Richnow, Hans-Hermann; Vogt, Carsten

2010-12-01

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.

Recovery of supraspinal control of leg movement in a chronic complete flaccid paraplegic man after continuous low-frequency pelvic nerve stimulation and FES-assisted training

DEFF Research Database (Denmark)

Possover, Marc; Forman, Axel

2017-01-01

INTRODUCTION: More than 30 years ago, functional electrical stimulation (FES) was developed as an orthotic system to be used for rehabilitation for SCI patients. In the present case report, FES-assisted training was combined with continuous low-frequency stimulation of the pelvic somatic nerves...... in a SCI patient. CASE PRESENTATION: We report on unexpected findings in a 41-year-old man with chronic complete flaccid paraplegia, since he was 18 years old, who underwent spinal stem cell therapy and a laparoscopic implantation of neuroprosthesis (LION procedure) in the pelvic lumbosacral nerves....... The patient had complete flaccid sensomotoric paraplegia T12 as a result of a motor vehicle accident in 1998. In June 2011, he underwent a laparoscopic implantation of stimulation electrodes to the sciatic and femoral nerves for continuous low-frequency electrical stimulation and functional electrical...

Chaperone activity of human small heat shock protein-GST fusion proteins.

Science.gov (United States)

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

A novel motion sensor-driven control system for FES-assisted walking after spinal cord injury: A pilot study.

Science.gov (United States)

Braz, Gustavo P; Russold, Michael F; Fornusek, Che; Hamzaid, Nur Azah; Smith, Richard M; Davis, Glen M

2016-11-01

This pilot study reports the development of a novel closed-loop (CL) FES-gait control system, which employed a finite-state controller that processed kinematic feedback from four miniaturized motion sensors. This strategy automated the control of knee extension via quadriceps and gluteus stimulation during the stance phase of gait on the supporting leg, and managed the stimulation delivered to the common peroneal nerve (CPN) during swing-phase on the contra-lateral limb. The control system was assessed against a traditional open-loop (OL) system on two sensorimotor 'complete' paraplegic subjects. A biomechanical analysis revealed that the closed-loop control of leg swing was efficient, but without major advantages compared to OL. CL automated the control of knee extension during the stance phase of gait and for this reason was the method of preference by the subjects. For the first time, a feedback control system with a simplified configuration of four miniaturized sensors allowed the addition of instruments to collect the data of multiple physiological and biomechanical variables during FES-evoked gait. In this pilot study of two sensorimotor complete paraplegic individuals, CL ameliorated certain drawbacks of current OL systems - it required less user intervention and accounted for the inter-subject differences in their stimulation requirements. Copyright © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.

Regulation of energy substrate utilization and hepatic insulin sensitivity by phosphatidylcholine transfer protein/StarD2.

Science.gov (United States)

Scapa, Erez F; Pocai, Alessandro; Wu, Michele K; Gutierrez-Juarez, Roger; Glenz, Lauren; Kanno, Keishi; Li, Hua; Biddinger, Sudha; Jelicks, Linda A; Rossetti, Luciano; Cohen, David E

2008-07-01

Phosphatidylcholine transfer protein (PC-TP, also known as StarD2) is a highly specific intracellular lipid binding protein with accentuated expression in oxidative tissues. Here we show that decreased plasma concentrations of glucose and free fatty acids in fasting PC-TP-deficient (Pctp(-/-)) mice are attributable to increased hepatic insulin sensitivity. In hyperinsulinemic-euglycemic clamp studies, Pctp(-/-) mice exhibited profound reductions in hepatic glucose production, gluconeogenesis, glycogenolysis, and glucose cycling. These changes were explained in part by the lack of PC-TP expression in liver per se and in part by marked alterations in body fat composition. Reduced respiratory quotients in Pctp(-/-) mice were indicative of preferential fatty acid utilization for energy production in oxidative tissues. In the setting of decreased hepatic fatty acid synthesis, increased clearance rates of dietary triglycerides and increased hepatic triglyceride production rates reflected higher turnover in Pctp(-/-) mice. Collectively, these data support a key biological role for PC-TP in the regulation of energy substrate utilization.

Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates

Directory of Open Access Journals (Sweden)

Saddler Jack N

2011-02-01

Full Text Available Abstract A range of lignocellulosic feedstocks (including agricultural, softwood and hardwood substrates were pretreated with either sulfur dioxide-catalyzed steam or an ethanol organosolv procedure to try to establish a reliable assessment of the factors governing the minimum protein loading that could be used to achieve efficient hydrolysis. A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass (poplar, douglas fir and lodgepole pine. Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic

Irradiation of FeS: Implications for the Lifecycle of Sulfur in the Interstellar Medium and Presolar FeS Grains

Science.gov (United States)

Keller, Lindsay P.; Loeffler, M. J.; Christoffersen, R.; Dukes, C.; Rahman, Z.; Baragiola, R.

2010-01-01

Fe(Ni) sulfides are ubiquitous in chondritic meteorites and cometary samples where they are the dominant host of sulfur. Despite their abundance in these early solar system materials, their presence in interstellar and circumstellar environments is poorly understood. Fe-sulfides have been reported from astronomical observations of pre- and post-main sequence stars [1, 2] and occur as inclusions in bonafide circumstellar silicate grains [3, 4]. In cold, dense molecular cloud (MC) environments, sulfur is highly depleted from the gas phase [e.g. 5], yet observations of sulfur-bearing molecules in dense cores find a total abundance that is only a small fraction of the sulfur seen in diffuse regions [6], therefore the bulk of the depletion must reside in an abundant unobserved phase. In stark contrast, sulfur is essentially undepleted from the gas phase in the diffuse interstellar medium (ISM) [7-9], indicating that little sulfur is incorporated into solid grains in this environment. This is a rather puzzling observation unless Fe-sulfides are not produced in significant quantities in stellar outflows, or their lifetime in the ISM is very short due to rapid destruction. The main destruction mechanism is sputtering due to supernova shocks in the warm, diffuse ISM [10]. This process involves the reduction of Fe-sulfide with the production of Fe metal as a by-product and returning S to the gas phase. In order to test this hypothesis, we irradiated FeS and analyzed the resulting material using X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM).

Electrophoretic transfer protein zymography.

Science.gov (United States)

Pan, Daniel; Hill, Adam P; Kashou, Anthony; Wilson, Karl A; Tan-Wilson, Anna

2011-04-15

Zymography detects and characterizes proteolytic enzymes by electrophoresis of protease-containing samples into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing a copolymerized protein substrate. The usefulness of zymography for molecular weight determination and proteomic analysis is hampered by the fact that some proteases exhibit slower migration through a gel that contains substrate protein. This article introduces electrophoretic transfer protein zymography as one solution to this problem. In this technique, samples containing proteolytic enzymes are first resolved in nonreducing SDS-PAGE on a gel without protein substrate. The proteins in the resolving gel are then electrophoretically transferred to a receiving gel previously prepared with a copolymerized protein substrate. The receiving gel is then developed as a zymogram to visualize clear or lightly stained bands in a dark background. Band intensities are linearly related to the amount of protease, extending the usefulness of the technique so long as conditions for transfer and development of the zymogram are kept constant. Conditions of transfer, such as the pore sizes of resolving and receiving gels and the transfer time relative to the molecular weight of the protease, are explored. Copyright © 2011 Elsevier Inc. All rights reserved.

Substrate-Trapped Interactors of PHD3 and FIH Cluster in Distinct Signaling Pathways

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Javier Rodriguez

2016-03-01

Full Text Available Amino acid hydroxylation is a post-translational modification that regulates intra- and inter-molecular protein-protein interactions. The modifications are regulated by a family of 2-oxoglutarate- (2OG dependent enzymes and, although the biochemistry is well understood, until now only a few substrates have been described for these enzymes. Using quantitative interaction proteomics, we screened for substrates of the proline hydroxylase PHD3 and the asparagine hydroxylase FIH, which regulate the HIF-mediated hypoxic response. We were able to identify hundreds of potential substrates. Enrichment analysis revealed that the potential substrates of both hydroxylases cluster in the same pathways but frequently modify different nodes of signaling networks. We confirm that two proteins identified in our screen, MAPK6 (Erk3 and RIPK4, are indeed hydroxylated in a FIH- or PHD3-dependent mechanism. We further determined that FIH-dependent hydroxylation regulates RIPK4-dependent Wnt signaling, and that PHD3-dependent hydroxylation of MAPK6 protects the protein from proteasomal degradation.

Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: Application to proenkephalin processing enzymes

Energy Technology Data Exchange (ETDEWEB)

Irvine, J.W.; Roberts, S.F.; Lindberg, I. (Louisiana State Univ. Medical Center, New Orleans (USA))

1990-10-01

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as ({sup 35}S)methionine-labeled proenkephalin or {sup 125}I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of ({sup 35}S)proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved ({sup 35}S)methionine-labeled proenkephalin but not {sup 125}I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.

Trapping and proteomic identification of cellular substrates of the ClpP protease in Staphylococcus aureus

DEFF Research Database (Denmark)

Feng, Jingyuan; Michalik, Stephan; Varming, Anders Nissen

2013-01-01

In the important human pathogen Staphylococcus aureus the cytoplasmic ClpP protease is essential for mounting cellular stress responses and for virulence. To directly identify substrates of the ClpP protease, we expressed in vivo a proteolytic inactive form of ClpP (ClpP(trap)) that will retain...... but not degrade substrates translocated into its proteolytic chamber. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry. In total, approximately 70 proteins were trapped in both of the two S. aureus strains NCTC8325......A, and the cell division protein FtsZ. Newly identified ClpP substrates include the global transcriptional regulators PerR and HrcA, proteins involved in DNA damage repair (RecA, UvrA, UvrB), and proteins essential for protein synthesis (RpoB and Tuf). Our study hence underscores the central role of Clp...

5´AMP activated protein kinase α2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase 4

DEFF Research Database (Denmark)

Fritzen, Andreas Mæchel; Lundsgaard, Annemarie; Jeppesen, Jacob

2015-01-01

after prolonged exercise and during the following six hours post exercise in 5´AMP activated protein kinase (AMPK)α2 and α1 knock-out (KO) and wild type (WT) mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post...

Proteins interacting with the 26S proteasome

DEFF Research Database (Denmark)

Hartmann-Petersen, R; Gordon, C

2004-01-01

The 26S proteasome is the multi-protein protease that recognizes and degrades ubiquitinylated substrates targeted for destruction by the ubiquitin pathway. In addition to the well-documented subunit organization of the 26S holoenzyme, it is clear that a number of other proteins transiently...... associate with the 26S complex. These transiently associated proteins confer a number of different roles such as substrate presentation, cleavage of the multi-ubiquitin chain from the protein substrate and turnover of misfolded proteins. Such activities are essential for the 26S proteasome to efficiently...... fulfill its intracellular function in protein degradation....

Myristylation of gag-onc fusion proteins in mammalian transforming retroviruses

International Nuclear Information System (INIS)

Schultz, A.; Oroszlan, S.

1984-01-01

Four cell lines producing transforming proteins encoded by three mammalian oncogenes (fes, abl, and ras) were investigated for incorporation of [ 3 H]myristate into gag-onc fusion proteins. Using 5-min pulse-labelings, fusion proteins of Abelson murine leukemia virus, Gardner-Arnstein strain of feline sarcoma virus (FeSV), and Snyder-Theilen strain of FeSV were shown to be myristylated. In a 4-hr pulse, p29gag-ras of rat sarcoma virus (RaSV) was also shown to incorporate radiolabel. The fatty acid was recovered from this labeled protein by acid hydrolysis, and identified by reverse-phase thin-layer chromatography to be [ 3 H]myristic acid. The results indicate that substitution of viral gag sequences by cellular oncogene sequences does not abolish their ability to become post-translationally modified by this long chain fatty acid. It is assumed that in the fusion proteins the myristyl moiety is linked through an amide linkage to the amino-terminal glycine as previously found for several retroviral gag precursor polyproteins. The possible role of myristylation of transforming proteins is discussed

Myristylation of gag-onc fusion proteins in mammalian transforming retroviruses

Energy Technology Data Exchange (ETDEWEB)

Schultz, A.; Oroszlan, S.

1984-03-01

Four cell lines producing transforming proteins encoded by three mammalian oncogenes (fes, abl, and ras) were investigated for incorporation of (/sup 3/H)myristate into gag-onc fusion proteins. Using 5-min pulse-labelings, fusion proteins of Abelson murine leukemia virus, Gardner-Arnstein strain of feline sarcoma virus (FeSV), and Snyder-Theilen strain of FeSV were shown to be myristylated. In a 4-hr pulse, p29gag-ras of rat sarcoma virus (RaSV) was also shown to incorporate radiolabel. The fatty acid was recovered from this labeled protein by acid hydrolysis, and identified by reverse-phase thin-layer chromatography to be (/sup 3/H)myristic acid. The results indicate that substitution of viral gag sequences by cellular oncogene sequences does not abolish their ability to become post-translationally modified by this long chain fatty acid. It is assumed that in the fusion proteins the myristyl moiety is linked through an amide linkage to the amino-terminal glycine as previously found for several retroviral gag precursor polyproteins. The possible role of myristylation of transforming proteins is discussed.

Integration of statistical modeling and high-content microscopy to systematically investigate cell-substrate interactions.

Science.gov (United States)

Chen, Wen Li Kelly; Likhitpanichkul, Morakot; Ho, Anthony; Simmons, Craig A

2010-03-01

Cell-substrate interactions are multifaceted, involving the integration of various physical and biochemical signals. The interactions among these microenvironmental factors cannot be facilely elucidated and quantified by conventional experimentation, and necessitate multifactorial strategies. Here we describe an approach that integrates statistical design and analysis of experiments with automated microscopy to systematically investigate the combinatorial effects of substrate-derived stimuli (substrate stiffness and matrix protein concentration) on mesenchymal stem cell (MSC) spreading, proliferation and osteogenic differentiation. C3H10T1/2 cells were grown on type I collagen- or fibronectin-coated polyacrylamide hydrogels with tunable mechanical properties. Experimental conditions, which were defined according to central composite design, consisted of specific permutations of substrate stiffness (3-144 kPa) and adhesion protein concentration (7-520 microg/mL). Spreading area, BrdU incorporation and Runx2 nuclear translocation were quantified using high-content microscopy and modeled as mathematical functions of substrate stiffness and protein concentration. The resulting response surfaces revealed distinct patterns of protein-specific, substrate stiffness-dependent modulation of MSC proliferation and differentiation, demonstrating the advantage of statistical modeling in the detection and description of higher-order cellular responses. In a broader context, this approach can be adapted to study other types of cell-material interactions and can facilitate the efficient screening and optimization of substrate properties for applications involving cell-material interfaces. Copyright 2009 Elsevier Ltd. All rights reserved.

A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-Crk signaling

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Hanafusa Hidesaburo

2002-07-01

Full Text Available Abstract Background The adaptor protein p130Cas (Cas has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals. Results We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk. Conclusion Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

Cytosolic iron-sulphur protein assembly is functionally conserved and essential in procyclic and bloodstream Trypanosoma brucei

Czech Academy of Sciences Publication Activity Database

Basu, Somsuvro; Netz, D. J.; Haindrich, A. C.; Herlerth, N.; Lagny, T. J.; Pierik, A. J.; Lill, R.; Lukeš, Julius

2014-01-01

Roč. 93, č. 5 (2014), s. 897-910 ISSN 0950-382X R&D Projects: GA ČR(CZ) GAP305/11/2179; GA MŠk LH12104; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : inducible expression system * Cfd1- Nbp35 complex * DNA metabolism * Fe/S proteins * transfer-RNA * cluster * mitochondrial * maturation * biogenesis * yeast Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.419, year: 2014

Proton NMR Studies of a Large Protein. pH, Substrate Titrations, and NOESY Experiments with Perdeuterated Yeast Phosphoglycerate Kinase Containing [ 1H]Histidine Residues

Science.gov (United States)

Pappu, K. M.; Serpersu, E. H.

Fully deuterated yeast phosphoglycerate kinase ([ 2H]PGK) was prepared biosynthetically with only histidine side chains of normal ( 1H) isotopic composition. The 1H NMR spectrum of this enzyme([ 1H]His[ 2H]PGK) showed that the histidine side chains are clearly visible as sharp signals. Thus detailed structural studies by 1H NMR became feasible with isotope-hybrid phosphoglycerate kinase which is otherwise too large ( Mr ˜ 46,000) for conventional 1H NMR studies. Proton signals of bound substrates were visible in the 1H NMR spectrum even with a substrate-to-enzyme ratio of less than 1/2 (mol/mol). The 2D NOESY spectrum of enzyme-MgdATP-glycerol 3-phosphate complex showed that, although protein concentration was very high (1.5 m M), no intraprotein cross peaks were observed other than those of intraresidue histidine NOE cross peaks. In addition, intrasubstrate NOEs and intermolecular NOEs between histidine and substrate protons were visible at a 1.5/1 substrate/enzyme (mol/mol) ratio. Paramagnetic effects of a substrate analog, Cr(III)ATP, on some of the histidine side chains indicated that the formation of the ternary enzyme-substrate complex causes large conformational changes in the enzyme.

Activation of lower back muscles via FES for pressure sores prevention in paraplegia: a case study.

Science.gov (United States)

Vanoncini, M; Holderbaum, W; Andrews, B J

2010-04-01

The aim of this paper is to show the feasibility of the use of functional electrical stimulation (FES) applied to the lower back muscles for pressure sores prevention in paraplegia. The hypothesis under study is that FES induces a change in the pressure distribution on the contact area during sitting. Tests were conducted on a paraplegic subject (T5), sitting on a standard wheelchair and cushion. Trunk extensors (mainly the erector spinae) were stimulated using surface electrodes placed on the skin. A pressure mapping system was used to measure the pressure on the sitting surface in four situations: (a) no stimulation; (b) stimulation on one side of the spine only; (c) stimulation on both sides, at different levels; and (d) stimulation at the same level on both sides, during pressure-relief manoeuvres. A session of prolonged stimulation was also conducted. The experimental results show that the stimulation of the erector spinae on one side of the spine can induce a trunk rotation on the sagittal plane, which causes a change in the pressure distribution. A decrease of pressure on the side opposite to the stimulation was recorded. The phenomenon is intensified when different levels of stimulation are applied to the two sides, and such change can be sustained for a considerable time (around 5 minutes). The stimulation did not induce changes during pressure-relief manoeuvres. Finally, from this research we can conclude that the stimulation of the trunk extensors can be a useful tool for pressure sores prevention, and can potentially be used in a routine for pressure sores prevention based on periodical weight shifts.

Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein

Directory of Open Access Journals (Sweden)

Yang Ting

2008-11-01

Full Text Available Abstract Background Deposition of amyloid-β protein (Aβ is a major pathological hallmark of Alzheimer's disease (AD. Aβ is generated from γ-secretase cleavage of amyloid precursor protein (APP. In addition to APP, γ-secretase also cleaves other type I integral membrane proteins, including the Notch receptor, a key molecule involved in embryonic development. Results To explore selective γ-secretase inhibitors, a combination of five methods was used to systematically determine these inhibitors' profiles on the γ-secretase cleavage of APP and Notch. When two potent γ-secretase inhibitors, compound E (cpd E and DAPT, were used in a conventional in vitro γ-secretase activity assay, cpd E completely blocked Aβ generation from the cleavage of substrate APP C100, but only had a minor effect on Notch cleavage and NICD generation. Next, cpd E and DAPT were applied to HEK293 cells expressing a truncated Notch substrate NotchΔE. Both cpd E and DAPT were more potent in blocking Aβ generation than NICD generation. Third, a reporter construct was created that carried the NICD targeting promoter with three Su(H binding sequences followed by the luciferase gene. We found that the inhibition of NICD generation by cpd E and DAPT was consistent with the reduced expression of luciferase gene driven by this Notch targeting promoter. Fourth, levels of "Notch-Aβ-like" (Nβ* peptide derived from two previously reported chimeric APP with its transmembrane domain or the juxtamembrane portion replaced by the Notch sequence were quantified. Measurement of Nβ* peptides by ELISA confirmed that EC50's of cpd E were much higher for Nβ* than Aβ. Finally, the expression levels of Notch target gene her6 in cpd E or DAPT-treated zebrafish were correlated with the degree of tail curvature due to defective somitogenesis, a well characterized Notch phenotype in zebrafish. Conclusion Our ELISA-based quantification of Aβ and Nβ* in combination with the test in

Iron Isotopic Compositions of Troilite (FeS) Inclusions from Iron Meteorites

Energy Technology Data Exchange (ETDEWEB)

Cook, David L.; Schönbächler, Maria, E-mail: david.cook@erdw.ethz.ch [Institut für Geochemie und Petrologie, ETH Zürich, Clausiusstrasse 25, 8092 Zürich (Switzerland)

2017-10-01

We report non-mass-dependent Fe isotopic data for troilite (FeS) inclusions from 10 iron meteorites, representing both non-magmatic (IAB) and magmatic groups (IIAB, IIIAB, IVA). No resolvable variations are present in the most neutron-rich isotope ({sup 58}Fe), but small deficits (≈−0.1 ε ) in {sup 56}Fe were observed in several inclusions. With the exception of several Ca–Al-rich inclusions in primitive meteorites, these are the first reported non-mass-dependent variations in Fe isotopes for material formed in the early solar system. Nucleosynthetic variations in Ni isotopes were previously reported in these same samples. The effects in Fe isotopes are not correlated with those in Ni, which suggests that the origins of the isotopic variations are decoupled from one another. The {sup 56}Fe deficits may represent incomplete mixing of the precursor dust in the protoplanetary disk. Alternatively, a parent body process (e.g., irradiation by galactic cosmic rays) may have modified the Fe isotopic compositions of some inclusions, which initially had homogeneous Fe isotopic compositions.

AAA-ATPases in Protein Degradation

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Ravikiran S. Yedidi

2017-06-01

Full Text Available Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

AAA-ATPases in Protein Degradation.

Science.gov (United States)

Yedidi, Ravikiran S; Wendler, Petra; Enenkel, Cordula

2017-01-01

Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

Enzyme functional evolution through improved catalysis of ancestrally nonpreferred substrates

Science.gov (United States)

Huang, Ruiqi; Hippauf, Frank; Rohrbeck, Diana; Haustein, Maria; Wenke, Katrin; Feike, Janie; Sorrelle, Noah; Piechulla, Birgit; Barkman, Todd J.

2012-01-01

In this study, we investigated the role for ancestral functional variation that may be selected upon to generate protein functional shifts using ancestral protein resurrection, statistical tests for positive selection, forward and reverse evolutionary genetics, and enzyme functional assays. Data are presented for three instances of protein functional change in the salicylic acid/benzoic acid/theobromine (SABATH) lineage of plant secondary metabolite-producing enzymes. In each case, we demonstrate that ancestral nonpreferred activities were improved upon in a daughter enzyme after gene duplication, and that these functional shifts were likely coincident with positive selection. Both forward and reverse mutagenesis studies validate the impact of one or a few sites toward increasing activity with ancestrally nonpreferred substrates. In one case, we document the occurrence of an evolutionary reversal of an active site residue that reversed enzyme properties. Furthermore, these studies show that functionally important amino acid replacements result in substrate discrimination as reflected in evolutionary changes in the specificity constant (kcat/KM) for competing substrates, even though adaptive substitutions may affect KM and kcat separately. In total, these results indicate that nonpreferred, or even latent, ancestral protein activities may be coopted at later times to become the primary or preferred protein activities. PMID:22315396

Structural basis for substrate placement by an archaeal box C/D ribonucleoprotein particle.

Science.gov (United States)

Xue, Song; Wang, Ruiying; Yang, Fangping; Terns, Rebecca M; Terns, Michael P; Zhang, Xinxin; Maxwell, E Stuart; Li, Hong

2010-09-24

Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2'-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a half-mer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modeling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs. Copyright © 2010 Elsevier Inc. All rights reserved.

Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization

DEFF Research Database (Denmark)

Coda, L; Salcini, A E; Confalonieri, S

1998-01-01

in NIH-3T3 cells overexpressing the receptor, even at low levels of receptor occupancy, thus behaving as physiological substrates. A role for eps15R in clathrin-mediated endocytosis is suggested by its localization in plasma membrane-coated pits and in vivo association to the coated pits' adapter protein...... AP-2. Finally, we demonstrate that a sizable fraction of eps15R exists in the cell as a complex with eps15 and that its EH domains exhibit binding specificities that are partially distinct from those of eps15. We propose that eps15 and eps15R are multifunctional binding proteins that serve...

Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells.

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Masato Nagaoka

Full Text Available Maintenance and differentiation of human pluripotent stem cells (hPSCs usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth-Holm-Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs.

EXTRACELLULAR PROTEINS PRODUCED BY DIFFERENT SPECIES OF THE FUNGUS TRICHODERMA ON SECONDARY PAPER MILL SLUDGE SUBSTRATE

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Ida Vaskova,

2012-01-01

Full Text Available Kraft pulping is the most commonly used pulping process in the pulp and paper industry. In this process wood chips are chemically delignified using sodium sulfide and sodium hydroxide. Delignification is usually followed by mechanical fiberization and a bleaching process of the resulting wood pulp. In addition to lignin-free wood pulp, this process also produces waste that contains residues of used chemicals, lignin, cellulose, hemicelluloses, and small amounts of other wood components. Because of the worldwide large-scale production of paper, the sludge from paper mills contributes significantly to environmental pollution. Although there have been great efforts being made to utilize this lignin-rich material, sludge is mostly disposed in landfills or incinerated in a boiler. This research project used secondary sludge as a substrate for 7 wood-decay fungi taxonomically belonging to the genus Trichoderma. The examined fungi expressed the capability of consuming sludge components as a carbon source to produce extracellular proteins. The proteins were separated by gel electrophoresis. Before and after fungi cultivation, the sludge was analyzed by Fourier transform infrared spectroscopy (FTIR.

Production of fungal protein from cellulosic plant materials

Energy Technology Data Exchange (ETDEWEB)

Sitaram, N; Kunhi, A A.M.; Geethadevi, B R; Rao, T N.R.

1979-01-01

The ability of 5 Aspergillus niger strains, a Penicillium chrysogenum strain, a Pestalotia strain, and a basidiomycete to produce microbial protein on 3 alkali-treated cellulosic substrates (rice straw, bagasse, and peanut shells) was evaluated. Most strains grew better on rice straw than on the other 2 substrates. Penicillium chrysogenum St-F3B produced more protein on all 3 substrates than did any of the other strains with a maximum production on rice straw of 85 mg/g substrate after 72 h incubation on a rotary shaker at pH 3.5 to 6.0. An inverse relation between substrate concentration and protein production per g substrate was observed with this organism.

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

KAUST Repository

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.; Sobhy, Mohamed Abdelmaboud; Joudeh, Luay I; Yan, Chunli; Piwonski, Hubert Marek; Tsutakawa, Susan E; Ivanov, Ivaylo; Tainer, John A; Habuchi, Satoshi; Hamdan, Samir

2017-01-01

that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme

Complementary methods for the identification of substrates of proteolysis.

Science.gov (United States)

Pham, Victoria C; Anania, Veronica G; Phung, Qui T; Lill, Jennie R

2014-01-01

Proteolysis describes the cleavage of proteins into smaller components, which in vivo occurs typically to either activate or impair the functionality of cellular proteins. Proteolysis can occur during cellular homeostasis or can be induced due to external stress stimuli such as heat, biological or chemical insult, and is mediated by the activity of cellular enzymes, namely, proteases. Proteolytic cleavage of proteins can influence protein activation by exposing an active site or disrupting inhibitor binding. Conversely, proteolytic cleavage of many proteins has also been shown to lead to protein degradation resulting in inactivation of the substrate. Thousands of proteolytic events are known to take place in regulated cellular processes such as apoptosis and pyroptosis, however, their individual contribution to these processes remains poorly understood. Additionally, many cellular homeostatic processes are regulated by proteolytic events, however, in some cases, few proteolytic substrates have been identified. To gain further insight into the mechanism of action of these cellular processes, and to characterize biomarkers of cell death and other pathological indications, it is imperative to utilize a complete arsenal of tools for studying proteolysis events in vivo and in vitro. In this chapter, we focus on alternative methodologies to N-terminomics for profiling substrates of proteolysis and describe an additional suite of tools including orthogonal biophysical separation techniques such as COFRADIC or GASSP, and affinity capture tools that can enrich for newly formed C-termini (C-terminomics) generated as a result of caspase-mediated proteolysis. © 2014 Elsevier Inc. All rights reserved.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

Science.gov (United States)

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-01

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Ultrasound-Enhanced Biogas Production from Different Substrates

DEFF Research Database (Denmark)

González-Fernández, Cristina; Timmers, Rudolphus Antonius; Ruiz, Begona

2015-01-01

Among the biofuel production processes using different substrates, the biogas generation process is one of the simplest. Compared with bioethanol or biodiesel production processes, anaerobic digestion is a process where all the organic matter (carbohydrates, lipids and proteins) can be biologically...... production. The present chapter is dedicated to providing a review of ultrasound pretreatment applied to different substrates (lignocelullosic materials, manures, sludge and microalgae). The advantages and constraints, that ultrasound pretreatment exhibit towards biogas production, are discussed and compared...

Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

Science.gov (United States)

Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

2014-01-01

Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous depletion of Atm and Mdl rebalances cytosolic Fe-S cluster assembly but not heme import into the mitochondrion of Trypanosoma brucei

Czech Academy of Sciences Publication Activity Database

Horáková, Eva; Changmai, Piya; Paris, Zdeněk; Salmon, D.; Lukeš, Julius

2015-01-01

Roč. 282, č. 21 (2015), s. 4157-4175 ISSN 1742-464X R&D Projects: GA ČR(CZ) GAP305/11/2179; GA ČR GJ15-21450Y; GA MŠk LH12104 EU Projects: European Commission(XE) 316304 Institutional support: RVO:60077344 Keywords : Atm * Fe-S cluster * heme * Mdl * Trypanosoma Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.237, year: 2015

Enhancing trichloroethylene degradation using non-aromatic compounds as growth substrates.

Science.gov (United States)

Kim, Seungjin; Hwang, Jeongmin; Chung, Jinwook; Bae, Wookeun

2014-06-30

The effect of non-aromatic compounds on the trichloroethylene (TCE) degradation of toluene-oxidizing bacteria were evaluated using Burkholderia cepacia G4 that expresses toluene 2-monooxygenase and Pseudomonas putida that expresses toluene dioxygenase. TCE degradation rates for B. cepacia G4 and P. putida with toluene alone as growth substrate were 0.144 and 0.123 μg-TCE/mg-protein h, respectively. When glucose, acetate and ethanol were fed as additional growth substrates, those values increased up to 0.196, 0.418 and 0.530 μg-TCE/mg-protein h, respectively for B. cepacia G4 and 0.319, 0.219 and 0.373 μg-TCE/mg-protein h, respectively for P. putida. In particular, the addition of ethanol resulted in a high TCE degradation rate regardless of the initial concentration. The use of a non-aromatic compound as an additional substrate probably enhanced the TCE degradation because of the additional supply of NADH that is consumed in co-metabolic degradation of TCE. Also, it is expected that the addition of a non-aromatic substrate can reduce the necessary dose of toluene and, subsequently, minimize the potential competitive inhibition upon TCE co-metabolism by toluene. Copyright © 2014 Elsevier B.V. All rights reserved.

Mass spectrometry-assisted protease substrate screening

DEFF Research Database (Denmark)

Schlüter, Hartmut; Rykl, Jana; Thiemann, Joachim

2007-01-01

-phase chromatography they are analyzed by tandem mass spectrometry and the substrates identified by database searching. The proof of principle in this study is demonstrated by incubating immobilized human plasma proteins with thrombin and by identifying by tandem mass spectrometry the fibrinopeptides, released...

Turning Escherichia coli into a Frataxin-Dependent Organism.

Directory of Open Access Journals (Sweden)

Béatrice Roche

2015-05-01

Full Text Available Fe-S bound proteins are ubiquitous and contribute to most basic cellular processes. A defect in the ISC components catalyzing Fe-S cluster biogenesis leads to drastic phenotypes in both eukaryotes and prokaryotes. In this context, the Frataxin protein (FXN stands out as an exception. In eukaryotes, a defect in FXN results in severe defects in Fe-S cluster biogenesis, and in humans, this is associated with Friedreich's ataxia, a neurodegenerative disease. In contrast, prokaryotes deficient in the FXN homolog CyaY are fully viable, despite the clear involvement of CyaY in ISC-catalyzed Fe-S cluster formation. The molecular basis of the differing importance in the contribution of FXN remains enigmatic. Here, we have demonstrated that a single mutation in the scaffold protein IscU rendered E. coli viability strictly dependent upon a functional CyaY. Remarkably, this mutation changed an Ile residue, conserved in prokaryotes at position 108, into a Met residue, conserved in eukaryotes. We found that in the double mutant IscUIM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes. Biochemical analyses of the "eukaryotic-like" IscUIM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters. Finally, bioinformatic studies of prokaryotic IscU proteins allowed us to trace back the source of FXN-dependency as it occurs in present-day eukaryotes. We propose an evolutionary scenario in which the current mitochondrial Isu proteins originated from the IscUIM version present in the ancestor of the Rickettsiae. Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108th position of the scaffold.

Uncovering a Role for the Tail of the Dictyostelium discoideum SadA Protein in Cell-Substrate Adhesion ▿ †

OpenAIRE

Kowal, Anthony S.; Chisholm, Rex L.

2011-01-01

Previous work from our laboratory showed that the Dictyostelium discoideum SadA protein plays a central role in cell-substrate adhesion. SadA null cells exhibit a loss of adhesion, a disrupted actin cytoskeleton, and a cytokinesis defect. How SadA mediates these phenotypes is unknown. This work addresses the mechanism of SadA function, demonstrating an important role for the C-terminal cytoplasmic tail in SadA function. We found that a SadA tailless mutant was unable to rescue the sadA adhesi...

Substrate Discrimination by ClpB and Hsp104

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Danielle M. Johnston

2017-05-01

Full Text Available ClpB of E. coli and yeast Hsp104 are homologous molecular chaperones and members of the AAA+ (ATPases Associated with various cellular Activities superfamily of ATPases. They are required for thermotolerance and function in disaggregation and reactivation of aggregated proteins that form during severe stress conditions. ClpB and Hsp104 collaborate with the DnaK or Hsp70 chaperone system, respectively, to dissolve protein aggregates both in vivo and in vitro. In yeast, the propagation of prions depends upon Hsp104. Since protein aggregation and amyloid formation are associated with many diseases, including neurodegenerative diseases and cancer, understanding how disaggregases function is important. In this study, we have explored the innate substrate preferences of ClpB and Hsp104 in the absence of the DnaK and Hsp70 chaperone system. The results suggest that substrate specificity is determined by nucleotide binding domain-1.

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

Science.gov (United States)

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

From UBE3A to Angelman syndrome: a substrate perspective

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Gabrielle L Sell

2015-09-01

Full Text Available Angelman syndrome (AS is a debilitating neurodevelopmental disorder that is characterized by motor dysfunction, intellectual disability, speech impairment, seizures and common features of autism spectrum disorders (ASDs. Some of these AS related phenotypes can be seen in other neurodevelopmental disorders (Williams, 2011;Tan et al., 2014. AS patients commonly carry mutations that render the maternally inherited UBE3A gene nonfunctional. Duplication of the chromosomal region containing the UBE3A gene is associated with ASDs. Although the causative role for UBE3A gene mutations in AS is well established, a long-standing challenge in AS research has been to identify neural substrates of UBE3A, an E3 ubiquitin ligase. A prevailing hypothesis is that changes in UBE3A protein levels would alter the levels of a collection of protein substrates, giving rise to the unique phenotypic aspects of AS and possibly UBE3A associated ASDs. Interestingly, proteins altered in AS are linked to additional ASDs that are not previously associated with changes in UBE3A, indicating a possible molecular overlap underlying the broad-spectrum phenotypes of these neurogenetic disorders. This idea raises the possibility that there may exist a one-size-fits-all approach to the treatment of neurogenetic disorders with phenotypes overlapping AS. Furthermore, while a comprehensive list of UBE3A substrates and downstream affected pathways should be developed, this is only part of the story. The timing of when UBE3A protein functions, through either changes in UBE3A or possibly substrate expression patterns, appears to be critical for AS phenotype development. These data call for further investigation of UBE3A substrates and their timing of action relevant to AS phenotypes.

Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2.

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Jeong-Hun Kang

Full Text Available Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of (32P [counts per minute (CPM] for each peptide substrate was determined by the radiolabel assay using [γ-(32P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135 were phosphorylated by other enzymes (PKA, PKCα, and ERK1, R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2.

Disparate subcellular location of putative sortase substrates in Clostridium difficile.

Science.gov (United States)

Peltier, Johann; Shaw, Helen A; Wren, Brendan W; Fairweather, Neil F

2017-08-23

Clostridium difficile is a gastrointestinal pathogen but how the bacterium colonises this niche is still little understood. Sortase enzymes covalently attach specific bacterial proteins to the peptidoglycan cell wall and are often involved in colonisation by pathogens. Here we show C. difficile proteins CD2537 and CD3392 are functional substrates of sortase SrtB. Through manipulation of the C-terminal regions of these proteins we show the SPKTG motif is essential for covalent attachment to the cell wall. Two additional putative substrates, CD0183 which contains an SPSTG motif, and CD2768 which contains an SPQTG motif, are not cleaved or anchored to the cell wall by sortase. Finally, using an in vivo asymmetric cleavage assay, we show that despite containing a conserved SPKTG motif, in the absence of SrtB these proteins are localised to disparate cellular compartments.

Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes.

Science.gov (United States)

Nillegoda, Nadinath B; Stank, Antonia; Malinverni, Duccio; Alberts, Niels; Szlachcic, Anna; Barducci, Alessandro; De Los Rios, Paolo; Wade, Rebecca C; Bukau, Bernd

2017-05-15

Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes.

Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways.

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Francesca eSacco

2014-05-01

Full Text Available Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI#K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26 respectively.

NMR of proteins (4Fe-4S): structural properties and intramolecular electron transfer

International Nuclear Information System (INIS)

Huber, J.G.

1996-01-01

NMR started to be applied to Fe-S proteins in the seventies. Its use has recently been enlarged as the problems arising from the paramagnetic polymetallic clusters ware overcome. Applications to [4Fe-4S] are presented herein. The information derived thereof deepens the understanding of the redox properties of these proteins which play a central role in the metabolism of bacterial cells. The secondary structure elements and the overall folding of Chromatium vinosum ferredoxin (Cv Fd) in solution have been established by NMR. The unique features of this sequence have been shown to fold as an α helix at the C-terminus and as a loop between two cysteines ligand of one cluster: these two parts localize in close proximity from one another. The interaction between nuclear and electronic spins is a source of additional structural information for (4Fe-AS] proteins. The conformation of the cysteine-ligands, as revealed by the Fe-(S γ -C β -H β )Cys dihedral angles, is related to the chemical shifts of the signals associated with the protons of these residues. The longitudinal relaxation times of the protons depend on their distance to the cluster. A quantitative relationship has been established and used to show that the solution structure of the high-potential ferredoxin from Cv differs significantly from the crystal structure around Phe-48. Both parameters (chemical shifts and longitudinal relaxation times) give also insight into the electronic and magnetic properties of the [4Fe-4S] clusters. The rate of intramolecular electron transfer between the two [4FE-4S] clusters of ferredoxins has been measured by NMR. It is far slower in the case of Cv Fd than for shorter ferredoxins. The difference may be associated with changes in the magnetic and/or electronic properties of one cluster. The strong paramagnetism of the [4Fe-4S] clusters, which originally limited the applicability of NMR to proteins containing these cofactors, has been proven instrumental in affording new

Functional Echomyography: thickness, ecogenicity, contraction and perfusion of the LMN denervated human muscle before and during h-bFES

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Riccardo Zanato

2010-03-01

Full Text Available Permanent denervated muscles were evaluated by ultrasound to monitor changes in morphology, thickness, contraction-relaxation kinetics and perfusion due to the electrical stimulation program of the Rise2-Italy project. In a case of monolateral lesion, morphology and ultrasonographic structure of the denervated muscles changed during the period of stimulation from a pattern typical of complete denervation-induced muscle atrophy to a pattern which might be considered “normal” when detected in an old patient. Thickness improved significantly more in the middle third of the denervated muscle, reaching the same value as the contralateral innervated muscle. Contraction-relaxation kinetics, measured by recording the muscle movements during electrical stimulation, showed an abnormal behavior of the chronically denervated muscle during the relaxation phase, which resulted to be significantly longer than in normal muscle. The long-term denervated muscles analyzed with Echo Doppler showed at rest a low resistance arterial flow that became pulsed during and after electrical stimulation. As expected, the ultra sound measured electrical stimulation-induced hyperemia lasted longer than the stimulation period. The higher than normal energy of the delivered electrical stimuli of the Vienna home-based Functional Electrical Stimulation strategy (h-b FES demonstrate that the explored muscles were still almost completely denervated during the one-year of training. In conclusion, this pilot study confirms the usefulness of Functional Echomyography in the follow-up and the positive effects of h-b FES of denervated muscles.

High protein complementation with high fiber substrates for oyster ...

African Journals Online (AJOL)

Agricultural residues have been world widely accepted for oyster mushroom culture. In this study, we used wheat straw, barley straw, maize stem residue, and lawn residue as substrates coupled with wheat bran, rice bran and soybean powder as complements for the growth of Pleurotus florida and Pleurotus ostreatus as ...

GSHSite: exploiting an iteratively statistical method to identify s-glutathionylation sites with substrate specificity.

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Yi-Ju Chen

Full Text Available S-glutathionylation, the covalent attachment of a glutathione (GSH to the sulfur atom of cysteine, is a selective and reversible protein post-translational modification (PTM that regulates protein activity, localization, and stability. Despite its implication in the regulation of protein functions and cell signaling, the substrate specificity of cysteine S-glutathionylation remains unknown. Based on a total of 1783 experimentally identified S-glutathionylation sites from mouse macrophages, this work presents an informatics investigation on S-glutathionylation sites including structural factors such as the flanking amino acids composition and the accessible surface area (ASA. TwoSampleLogo presents that positively charged amino acids flanking the S-glutathionylated cysteine may influence the formation of S-glutathionylation in closed three-dimensional environment. A statistical method is further applied to iteratively detect the conserved substrate motifs with statistical significance. Support vector machine (SVM is then applied to generate predictive model considering the substrate motifs. According to five-fold cross-validation, the SVMs trained with substrate motifs could achieve an enhanced sensitivity, specificity, and accuracy, and provides a promising performance in an independent test set. The effectiveness of the proposed method is demonstrated by the correct identification of previously reported S-glutathionylation sites of mouse thioredoxin (TXN and human protein tyrosine phosphatase 1b (PTP1B. Finally, the constructed models are adopted to implement an effective web-based tool, named GSHSite (http://csb.cse.yzu.edu.tw/GSHSite/, for identifying uncharacterized GSH substrate sites on the protein sequences.

Secretomic survey of Trichoderma harzianum grown on plant biomass substrates.

Science.gov (United States)

Gómez-Mendoza, Diana Paola; Junqueira, Magno; do Vale, Luis Henrique Ferreira; Domont, Gilberto Barbosa; Ferreira Filho, Edivaldo Ximenes; Sousa, Marcelo Valle de; Ricart, Carlos André Ornelas

2014-04-04

The present work aims at characterizing T. harzianum secretome when the fungus is grown in synthetic medium supplemented with one of the four substrates: glucose, cellulose, xylan, and sugarcane bagasse (SB). The characterization was done by enzymatic assays and proteomic analysis using 2-DE/MALDI-TOF and gel-free shotgun LC-MS/MS. The results showed that SB induced the highest cellulolytic and xylanolytic activities when compared with the other substrates, while remarkable differences in terms of number and distribution of protein spots in 2-DE gels were also observed among the samples. Additionally, treatment of the secretomes with PNGase F revealed that most spot trails in 2-DE gels corresponded to N-glycosylated proteoforms. The LC-MS/MS analysis of the samples identified 626 different protein groups, including carbohydrate-active enzymes and accessory, noncatalytic, and cell-wall-associated proteins. Although the SB-induced secretome displayed the highest cellulolytic and xylanolytic activities, it did not correspond to a higher proteome complexity because CM-cellulose-induced secretome was significantly more diverse. Among the identified proteins, 73% were exclusive to one condition, while only 5% were present in all samples. Therefore, this study disclosed the variation of T. harzianum secretome in response to different substrates and revealed the diversity of the fungus enzymatic toolbox.

Protein kinase CK2 mutants defective in substrate recognition. Purification and kinetic analysis

DEFF Research Database (Denmark)

Sarno, S; Vaglio, P; Meggio, F

1996-01-01

Five mutants of protein kinase CK2 alpha subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition...... of the recombinant beta subunit. By this latter procedure five mutated tetrameric holoenzymes were obtained as judged from their subunit composition, sedimentation coefficient on sucrose gradient ultracentrifugation, and increased activity toward a specific peptide substrate as compared with the isolated alpha......191A,R195A, K198A; K79A,R80A,K83A; and K74A,K75A, K76A,K77A are assayed with the peptides RRRADDSADDDD, RRRADDSDDADD, and RRRADDSDDDAA, respectively. In contrast, the phosphorylation efficiencies of the other substituted peptides decrease more markedly with these mutants than with CK2 wild type...

Binding of mouse immunoglobulin G to polylysine-coated glass substrate for immunodiagnosis

Science.gov (United States)

Vashist, Sandeep Kumar; Tewari, Rupinder; Bajpai, Ram Prakash; Bharadwaj, Lalit Mohan; Raiteri, Roberto

2006-12-01

We report a method for immobilizing mouse immunoglobulin G (IgG) on polylysine-coated glass substrate for immunodiagnostic applications. Mouse IgG molecules were immobilized on polylysine-coated glass substrate employing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and protein A. The amino groups of the polylysine-coated glass slide were cross linked to the carboxyl groups of protein A employing EDC crosslinker. Protein A was employed as it binds to the constant Fc region of antibodies keeping their antigen binding sites on the variable F ab region free to bind to antigens. The qualitative analysis of surface immobilized mouse IgG was done by fluorescent microscopy employing fluorescein isothiocyanate (FITC) labeled mouse IgG molecules. The immobilization densities of protein A and mouse IgG were determined by 3, 3', 4, 4'-tetramethyl benzidine (TMB) substrate assay employing horse radish peroxidise labelled molecules and were found to be 130 +/- 17 ng/cm2 and 596 +/- 31 ng/cm2 respectively. The biomolecular coatings analyzed by atomic force microscopy (AFM) were found to be uniform.

Surface enhanced Raman spectroscopy detection of biomolecules using EBL fabricated nanostructured substrates.

Science.gov (United States)

Peters, Robert F; Gutierrez-Rivera, Luis; Dew, Steven K; Stepanova, Maria

2015-03-20

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

Cellular reprogramming through mitogen-activated protein kinases

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Justin eLee

2015-10-01

Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Chemical composition of Pleurotus pulmonarius (Fr. Quél., substrates and residue after cultivation

Directory of Open Access Journals (Sweden)

Silva Sueli Oliveira

2002-01-01

Full Text Available The cultivation of Pleurotus pulmonarius was carried out on different substrate: cotton waste (A leaves of Cymbopogon citratus (B and leaves of Panicum maximum Jacq. (C. The mushroom had a varied chemical composition; nevertheless they contained a good composition for to be used as a good source of protein for human kind. The results showed the higher protein content (29.19% and fibre (9.0% for the mushroom that were cultivated on cotton peel. The substrate composition and the residue composition after the mushroom harvest were: Protein for the substrate "A" (10.63% and 9.35%, "B" (7.87% and 4.24% and "C" (7.55% and 5.90 %; Lipids "A" (4.17% and 2.03%, "B"(2.77% and 3.20% and "C" (0.91% and 2.05%; Fibres "A" (49.02% and 37.02%, "B" (28.40% and 23.26% and "C" (37.50% and 26.66% respectively. The substrate "A" showed 0.048% of iron and "C" 0.14% of magnesium, 0.31% of potassium and 0.26% of calcium. In the substrate "B" the quantity of the minerals were very low in comparison to the other substrates. The residues after the harvest of mushroom could be used as fertiliser or as complement in the composition for animal feed.

[Acute myocardial infarction in Morocco: FES-AMI registry data].

Science.gov (United States)

Akoudad, H; El Khorb, N; Sekkali, N; Mechrafi, A; Zakari, N; Ouaha, L; Lahlou, I

2015-12-01

Acute myocardial infarction is the most dangerous complication of coronary atherothrombosis. There are several disparities in regard to its management around the world. The aim of this study is to analyze the specificities of management of acute myocardial infarction in Morocco. FES-AMI (Fès Acute Myocardial Infarction) is a prospective monocentric registry conducted in cardiology department of Hassan II university hospital in Fès. In this registry, we enrolled patients with acute myocardial infarction who presented within 5 days after symptom onset. From January 2005 to August 2015, we enrolled 1835 patients. Seventy-five percent of patients were males and mean age was 60 years old. Fifty-one percent of patients were smokers, 27% were hypertensives and 14% were diabetics. Sixty-six percent of patients had more than 2 risk factors. Time from symptom onset to hospital admission was less than six hours for 40% of the patients. Thirty-six percent of patients were admitted more than twelve hours after the onset of chest pain. Only 37% of patients received reperfusion therapy, 31% with in-hospital thrombolysis and 6% with primary angioplasty. In-hospital mortality was 7.6%. The patients enrolled in our registry have late presentation of acute myocardial infarction and less rate of reperfusion therapy. Furthermore, the majority of our patients have multiple risk factors and this result underlines the failure of preventive interventions. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Functional relevance of AcrB Trimerization in pump assembly and substrate binding.

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Wei Lu

Full Text Available AcrB is a multidrug transporter in the inner membrane of Escherichia coli. It is an obligate homotrimer and forms a tripartite efflux complex with AcrA and TolC. AcrB is the engine of the efflux machinery and determines substrate specificity. Active efflux depends on several functional features including proton translocation across the inner membrane through a proton relay pathway in the transmembrane domain of AcrB; substrate binding and migration through the substrate translocation pathway; the interaction of AcrB with AcrA and TolC; and the formation of AcrB homotrimer. Here we investigated two aspects of the inter-correlation between these functional features, the dependence of AcrA-AcrB interaction on AcrB trimerization, and the reliance of substrate binding and penetration on protein-protein interaction. Interaction between AcrA and AcrB was investigated through chemical crosslinking, and a previously established in vivo fluorescent labeling method was used to probe substrate binding. Our data suggested that dissociation of the AcrB trimer drastically decreased its interaction with AcrA. In addition, while substrate binding with AcrB seemed to be irrelevant to the presence or absence of AcrA and TolC, the capability of trimerization and conduction of proton influx did affect substrate binding at selected sites along the substrate translocation pathway in AcrB.

Substrate Handbook for Biogas Production; Substrathandbok foer biogasproduktion

Energy Technology Data Exchange (ETDEWEB)

Carlsson, My; Uldal, Martina (AnoxKaldnes AB, Lund (Sweden))

2009-02-15

Today, co-digestion plants in Sweden treat a broad range of different substrates, of which some have not previously been used for anaerobic digestion. The major part of this organic waste derives from households, restaurants, food industries and farms. When evaluating a new substrate as feed for anaerobic digestion, several different aspects need to be taken into consideration, such as anaerobic degradability, TS/VS content, nutrient composition and risk for mechanical problems. Consequently, there is a need for practical guidelines on how to evaluate new substrates as raw materials for biogas production, including not only gas yield but also what practical and microbiological problems that may arise when the specific substrate is treated together with other substrates in the plant. The aim with this handbook is to provide a basis on how to evaluate new substrates as feed for anaerobic digestion. The intention is that this material will save time and effort for the personnel at the plant when they come in contact with new types of waste. Also, the aim is to facilitate the process of identifying new substrates within the ABP-regulation (1774/2002) and what requirements are then demanded on handling. The work with the handbook has been divided in three different parts; (1) an extensive literature study and a compilation of the achieved results, (2) interviews with personnel at most of the Swedish co-digestion plants to identify substrates and problems of interest, and (3) lab tests of selected substrates. The lab tests included Bio Methane Potential (BMP) tests as well as a simple characterization of each substrate based on fat/protein/carbohydrate content. All data origins from anaerobic digestion within the mesophilic temperature range, but the results and discussion are applicable also for thermophilic anaerobic digestion. The result of this work is a written report together with an Excel file which are to be directly used by the biogas plants as a basis in the

2008 GRC Iron Sulfur Enzymes-Conference to be held June 8-13, 2008

Energy Technology Data Exchange (ETDEWEB)

Cramer, Stephen [Univ. of California, Davis, CA (United States); Gray, Nancy Ryan [Gordon Research Conferences, West Kingston, RI (United States)

2009-01-01

Iron-sulfur proteins are among the most common and ancient enzymes and electron-transfer agents in nature. They play key roles in photosynthesis, respiration, and the metabolism of small molecules such as H2, CO, and N2. The Iron Sulfur Enzyme Gordon Research Conference evolved from an earlier GRC on Nitrogen Fixation that began in 1994. The scope of the current meeting has broadened to include all enzymes or metalloproteins in which Fe-S bonds play a key role. This year's meeting will focus on the biosynthesis of Fe-S clusters, as well as the structure and mechanism of key Fe-S enzymes such as hydrogenase, nitrogenase and its homologues, radical SAM enzymes, and aconitase-related enzymes. Recent progress on the role of Fe-S enzymes in health, disease, DNA/RNA-processing, and alternative bio-energy systems will also be highlighted. This conference will assemble a broad, diverse, and international group of biologists and chemists who are investigating fundamental issues related to Fe-S enzymes, on atomic, molecular, organism, and environmental scales. The topics to be addressed will include: Biosynthesis & Genomics of Fe-S Enzymes; Fundamental Fe-S Chemistry; Hydrogen and Fe-S Enzymes; Nitrogenase & Homologous Fe-S Enzymes; Fe-S Enzymes in Health & Disease; Radical SAM and Aconitase-Related Fe-S Enzymes; Fe-S Enzymes and Synthetic Analogues in BioEnergy; and Fe-S Enzymes in Geochemistry and the Origin of Life.

Aerobic lineage of the oxidative stress response protein rubrerythrin emerged in an ancient microaerobic, (hyperthermophilic environment

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Juan Pablo Cardenas

2016-11-01

Full Text Available Rubrerythrins (RBRs are non-heme di-iron proteins belonging to the ferritin-like superfamily (FLSF. They are involved in oxidative stress defense as peroxide scavengers in a wide range of organisms. The vast majority of RBRs, including classical forms of this protein, contain a C-terminal rubredoxin-like domain involved in electron transport that is used during catalysis in anaerobic conditions. Rubredoxin is an ancient and large protein family of short length (<100 residues that contains a Fe-S center involved in electron transfer. However, functional forms of the enzyme lacking the rubredoxin-like domain have been reported (e.g., sulerythrin and ferriperoxin. In this study, phylogenomic evidence is presented that suggests that a complete lineage of rubrerythrins, lacking the rubredoxin-like domain, arose in an ancient microaerobic and (hyperthermophilic environments in the ancestors of the Archaea Thermoproteales and Sulfolobales. This lineage (termed the aerobic-type lineage subsequently evolved to become adapted to environments with progressively lower temperatures and higher oxygen concentrations via the acquisition of two co-localized genes, termed DUF3501 and RFO, encoding a conserved protein of unknown function and a predicted Fe-S oxidoreductase respectively. Proposed Horizontal Gene Transfer (HGT events from these archaeal ancestors to Bacteria expanded the opportunities for further evolution of this RBR including adaption to lower temperatures. The second lineage (termed the cyanobacterial lineage is proposed to have evolved in cyanobacterial ancestors, maybe in direct response to the production of oxygen via oxygenic photosynthesis during the Great Oxygen Event (GOE. It is hypothesized that both lineages of RBR emerged in a largely anaerobic world with whiffs of oxygen and that their subsequent independent evolutionary trajectories allowed microorganisms to transition from this anaerobic world to an aerobic one.

Relationship between the climbing up and climbing down stairs domain scores on the FES-DMD, the score on the Vignos Scale, age and timed performance of functional activities in boys with Duchenne muscular dystrophy

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Lilian A. Y. Fernandes

2014-12-01

Full Text Available BACKGROUND: Knowing the potential for and limitations of information generated using different evaluation instruments favors the development of more accurate functional diagnoses and therapeutic decision-making. OBJECTIVE: To investigate the relationship between the number of compensatory movements when climbing up and going down stairs, age, functional classification and time taken to perform a tested activity (TA of going up and down stairs in boys with Duchenne muscular dystrophy (DMD. METHOD: A bank of movies featuring 30 boys with DMD performing functional activities was evaluated. Compensatory movements were assessed using the climbing up and going down stairs domain of the Functional Evaluation Scale for Duchenne Muscular Dystrophy (FES-DMD; age in years; functional classification using the Vignos Scale (VS, and TA using a timer. Statistical analyses were performed using the Spearman correlation test. RESULTS: There is a moderate relationship between the climbing up stairs domain of the FES-DMD and age (r=0.53, p=0.004 and strong relationships with VS (r=0.72, p=0.001 and TA for this task (r=0.83, p<0.001. There were weak relationships between the going down stairs domain of the FES-DMD-going down stairs with age (r=0.40, p=0.032, VS (r=0.65, p=0.002 and TA for this task (r=0.40, p=0.034. CONCLUSION: These findings indicate that the evaluation of compensatory movements used when climbing up stairs can provide more relevant information about the evolution of the disease, although the activity of going down stairs should be investigated, with the aim of enriching guidance and strengthening accident prevention. Data from the FES-DMD, age, VS and TA can be used in a complementary way to formulate functional diagnoses. Longitudinal studies and with broader age groups may supplement this information.

Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase.

Science.gov (United States)

Deville, Célia; Carroni, Marta; Franke, Kamila B; Topf, Maya; Bukau, Bernd; Mogk, Axel; Saibil, Helen R

2017-08-01

Refolding aggregated proteins is essential in combating cellular proteotoxic stress. Together with Hsp70, Hsp100 chaperones, including Escherichia coli ClpB, form a powerful disaggregation machine that threads aggregated polypeptides through the central pore of tandem adenosine triphosphatase (ATPase) rings. To visualize protein disaggregation, we determined cryo-electron microscopy structures of inactive and substrate-bound ClpB in the presence of adenosine 5'- O -(3-thiotriphosphate), revealing closed AAA+ rings with a pronounced seam. In the substrate-free state, a marked gradient of resolution, likely corresponding to mobility, spans across the AAA+ rings with a dynamic hotspot at the seam. On the seam side, the coiled-coil regulatory domains are locked in a horizontal, inactive orientation. On the opposite side, the regulatory domains are accessible for Hsp70 binding, substrate targeting, and activation. In the presence of the model substrate casein, the polypeptide threads through the entire pore channel and increased nucleotide occupancy correlates with higher ATPase activity. Substrate-induced domain displacements indicate a pathway of regulated substrate transfer from Hsp70 to the ClpB pore, inside which a spiral of loops contacts the substrate. The seam pore loops undergo marked displacements, along with ordering of the regulatory domains. These asymmetric movements suggest a mechanism for ATPase activation and substrate threading during disaggregation.

Unique structural modulation of a non-native substrate by cochaperone DnaJ.

Science.gov (United States)

Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik; Mapa, Koyeli

2013-02-12

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK-DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.

Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2.

Science.gov (United States)

Keresztessy, Zsolt; Csosz, Eva; Hársfalvi, Jolán; Csomós, Krisztián; Gray, Joe; Lightowlers, Robert N; Lakey, Jeremy H; Balajthy, Zoltán; Fésüs, László

2006-11-01

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q(6), Q(8), and Q(22) are modified by TG2. Kinetic parameters of SnQ1 transamidation (K(M)(app) = 250 microM, k(cat) = 18.3 sec(-1), and k(cat)/K(M)(app) = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.

Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

International Nuclear Information System (INIS)

Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

1988-01-01

The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate [Gpp(NH)p] on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed [ 32 P]ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-α/sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-α/sub i/ or anti-α 0 antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed

Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.

Directory of Open Access Journals (Sweden)

Hirotaka Takahashi

Full Text Available Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3. Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1 targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein.

Radical SAM, A Novel Protein Superfamily Linking Unresolved Steps in Familiar Biosynthetic Pathways with Radical Mechanisms: Functional Characterization Using New Analysis and Information Visualization Methods

Energy Technology Data Exchange (ETDEWEB)

Sofia, Heidi J.; Chen, Guang; Hetzler, Elizabeth G.; Reyes Spindola, Jorge F.; Miller, Nancy E.

2001-03-01

A large protein superfamily with over 500 members has been discovered and analyzed using powerful new bioinformatics and information visualization methods. Evidence exists that these proteins generate a 5?-deoxyadenosyl radical by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. Radical SAM superfamily proteins function in DNA precursor, vitamin, cofactor, antibiotic, and herbicide biosynthesis in a collection of basic and familiar pathways. One of the members is interferon-inducible and is considered a candidate drug target for osteoporosis. The identification of this superfamily suggests that radical-based catalysis is important in a number of previously well-studied but unresolved biochemical pathways.

De Novo Construction of Redox Active Proteins.

Science.gov (United States)

Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

2016-01-01

Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. © 2016 Elsevier Inc. All rights reserved.

Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease.

Science.gov (United States)

Rampello, Anthony J; Glynn, Steven E

2017-03-24

The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Information encoded in non-native states drives substrate-chaperone pairing.

Science.gov (United States)

Mapa, Koyeli; Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik

2012-09-05

Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identifications of Putative PKA Substrates with Quantitative Phosphoproteomics and Primary-Sequence-Based Scoring.

Science.gov (United States)

Imamura, Haruna; Wagih, Omar; Niinae, Tomoya; Sugiyama, Naoyuki; Beltrao, Pedro; Ishihama, Yasushi

2017-04-07

Protein kinase A (PKA or cAMP-dependent protein kinase) is a serine/threonine kinase that plays essential roles in the regulation of proliferation, differentiation, and apoptosis. To better understand the functions of PKA, it is necessary to elucidate the direct interplay between PKA and their substrates in living human cells. To identify kinase target substrates in a high-throughput manner, we first quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drug stimulations. LC-MS/MS analyses identified 2755 and 3191 phosphopeptides from experiments with activator or inhibitor of PKA. To exclude potential indirect targets of PKA, we built a computational model to characterize the kinase sequence specificity toward the substrate target site based on known kinase-substrate relationships. Finally, by combining the sequence recognition model with the quantitative changes in phosphorylation measured in the two drug perturbation experiments, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 previously known substrates. Moreover, 18 of these sites were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed a confident list of PKA substrate candidates, expanding our knowledge of PKA signaling network.

Composition of Overlapping Protein-Protein and Protein-Ligand Interfaces.

Directory of Open Access Journals (Sweden)

Ruzianisra Mohamed

Full Text Available Protein-protein interactions (PPIs play a major role in many biological processes and they represent an important class of targets for therapeutic intervention. However, targeting PPIs is challenging because often no convenient natural substrates are available as starting point for small-molecule design. Here, we explored the characteristics of protein interfaces in five non-redundant datasets of 174 protein-protein (PP complexes, and 161 protein-ligand (PL complexes from the ABC database, 436 PP complexes, and 196 PL complexes from the PIBASE database and a dataset of 89 PL complexes from the Timbal database. In all cases, the small molecule ligands must bind at the respective PP interface. We observed similar amino acid frequencies in all three datasets. Remarkably, also the characteristics of PP contacts and overlapping PL contacts are highly similar.

Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to resurrected ancestor protein ancMALS of yeast maltases and isomaltases

DEFF Research Database (Denmark)

Viigand, Katrin; Visnapuu, Triinu; Mardo, Karin

2016-01-01

and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino......, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α-glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars....

Caffeine degradation by Rhizopus delemar in packed bed column bioreactor using coffee husk as substrate Degradação de cafeína por Rhizopus delemar em biorreator de colunas usando casca de café como substrato

Directory of Open Access Journals (Sweden)

Cristiane Vanessa Tagliari

2003-11-01

Full Text Available Various microorganisms including bacteria, yeast and fungi can degrade caffeine. There are few publications about caffeine degradation pathway in filamentous fungi, mainly by solid-state fermentation (SSF. Studies were carried out on degradation of caffeine and their metabolites by filamentous fungi in SSF using coffee husk as substrate. The purpose of this work was to investigate the caffeine degradation pathway by Rhizopus delemar in packed bed column fermenter and to compare this degradation metabolism with glass flasks fermentation. The methylxanthines were quantified by HPLC analysis. The experiments were realized with the optimized conditions in previous experiments: pH 6.5, 28ºC, inoculation rate 10(6 spores/g substrate, aeration rate 60 mL/min and initial moisture 73%. Under these conditions, after 72 hous of fermentation was achieved only 0.19% of caffeine and 0.014% of theophylline in the coffee husk. The strain proved to be able for caffeine and theophylline degradation by SSF in packed bed column bioreactor.Diversos microrganismos incluindo bactérias, fungos e leveduras são capazes de assimilar a cafeína de meios sintéticos ou de resíduos de café. Existem poucos trabalhos sobre a via de degradação da cafeína em fungos filamentosos, principalmente por fermentação no estado sólido (FES. Estudos de degradação da cafeína por fungos filamentosos em FES usando casca de café como substrato vêm sendo realizados. O objetivo deste trabalho foi investigar a via de degradação da cafeína por Rhizopus delemar em biorreator de colunas aeradas e comparar este metabolismo de degradação com o da fermentação em frascos de vidro. As metilxantinas foram quantificadas por análises em HPLC. Os experimentos foram realizados com as condições otimizadas previamente: pH 6,5, 28ºC, 10(6 espores/g substrato, vazão de ar 60 mL/min e 73% de umidade inicial. Após 90 horas de fermentação, 65% da cafeína foi reduzida, resultando 0

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

Directory of Open Access Journals (Sweden)

Jose L S Lopes

Full Text Available Diacylglycerol acyltransferase 1 (DGAT1 is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Biofunctionalization on alkylated silicon substrate surfaces via "click" chemistry.

Science.gov (United States)

Qin, Guoting; Santos, Catherine; Zhang, Wen; Li, Yan; Kumar, Amit; Erasquin, Uriel J; Liu, Kai; Muradov, Pavel; Trautner, Barbara Wells; Cai, Chengzhi

2010-11-24

Biofunctionalization of silicon substrates is important to the development of silicon-based biosensors and devices. Compared to conventional organosiloxane films on silicon oxide intermediate layers, organic monolayers directly bound to the nonoxidized silicon substrates via Si-C bonds enhance the sensitivity of detection and the stability against hydrolytic cleavage. Such monolayers presenting a high density of terminal alkynyl groups for bioconjugation via copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC, a "click" reaction) were reported. However, yields of the CuAAC reactions on these monolayer platforms were low. Also, the nonspecific adsorption of proteins on the resultant surfaces remained a major obstacle for many potential biological applications. Herein, we report a new type of "clickable" monolayers grown by selective, photoactivated surface hydrosilylation of α,ω-alkenynes, where the alkynyl terminal is protected with a trimethylgermanyl (TMG) group, on hydrogen-terminated silicon substrates. The TMG groups on the film are readily removed in aqueous solutions in the presence of Cu(I). Significantly, the degermanylation and the subsequent CuAAC reaction with various azides could be combined into a single step in good yields. Thus, oligo(ethylene glycol) (OEG) with an azido tag was attached to the TMG-alkyne surfaces, leading to OEG-terminated surfaces that reduced the nonspecific adsorption of protein (fibrinogen) by >98%. The CuAAC reaction could be performed in microarray format to generate arrays of mannose and biotin with varied densities on the protein-resistant OEG background. We also demonstrated that the monolayer platform could be functionalized with mannose for highly specific capturing of living targets (Escherichia coli expressing fimbriae) onto the silicon substrates.

Influence of substrate temperature on certain physical properties ...

Indian Academy of Sciences (India)

2016-11-12

Nov 12, 2016 ... with increasing substrate temperature was explained on the basis of the Zener pinning effect. ... the inactivation of proteins as investigated by Feng et al [9] and in that .... ing 30 ml of nutrient agar medium for bacterial growth.

Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

Science.gov (United States)

Yunus, Ali A.; Lima, Christopher D.

2009-01-01

SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation. PMID:19107417

Decoding P4-ATPase substrate interactions.

Science.gov (United States)

Roland, Bartholomew P; Graham, Todd R

Cellular membranes display a diversity of functions that are conferred by the unique composition and organization of their proteins and lipids. One important aspect of lipid organization is the asymmetric distribution of phospholipids (PLs) across the plasma membrane. The unequal distribution of key PLs between the cytofacial and exofacial leaflets of the bilayer creates physical surface tension that can be used to bend the membrane; and like Ca 2+ , a chemical gradient that can be used to transduce biochemical signals. PL flippases in the type IV P-type ATPase (P4-ATPase) family are the principle transporters used to set and repair this PL gradient and the asymmetric organization of these membranes are encoded by the substrate specificity of these enzymes. Thus, understanding the mechanisms of P4-ATPase substrate specificity will help reveal their role in membrane organization and cell biology. Further, decoding the structural determinants of substrate specificity provides investigators the opportunity to mutationally tune this specificity to explore the role of particular PL substrates in P4-ATPase cellular functions. This work reviews the role of P4-ATPases in membrane biology, presents our current understanding of P4-ATPase substrate specificity, and discusses how these fundamental aspects of P4-ATPase enzymology may be used to enhance our knowledge of cellular membrane biology.

Unfolded Protein Response-regulated Drosophila Fic (dFic) Protein Reversibly AMPylates BiP Chaperone during Endoplasmic Reticulum Homeostasis*

Science.gov (United States)

Ham, Hyeilin; Woolery, Andrew R.; Tracy, Charles; Stenesen, Drew; Krämer, Helmut; Orth, Kim

2014-01-01

Drosophila Fic (dFic) mediates AMPylation, a covalent attachment of adenosine monophosphate (AMP) from ATP to hydroxyl side chains of protein substrates. Here, we identified the endoplasmic reticulum (ER) chaperone BiP as a substrate for dFic and mapped the modification site to Thr-366 within the ATPase domain. The level of AMPylated BiP in Drosophila S2 cells is high during homeostasis, whereas the level of AMPylated BiP decreases upon the accumulation of misfolded proteins in the ER. Both dFic and BiP are transcriptionally activated upon ER stress, supporting the role of dFic in the unfolded protein response pathway. The inactive conformation of BiP is the preferred substrate for dFic, thus endorsing a model whereby AMPylation regulates the function of BiP as a chaperone, allowing acute activation of BiP by deAMPylation during an ER stress response. These findings not only present the first substrate of eukaryotic AMPylator but also provide a target for regulating the unfolded protein response, an emerging avenue for cancer therapy. PMID:25395623

Chitin and stress induced protein kinase activation

DEFF Research Database (Denmark)

Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

2017-01-01

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein

International Nuclear Information System (INIS)

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-01-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[α- 32 P]triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an α subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera

Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men.

Science.gov (United States)

Vigelsø, Andreas; Gram, Martin; Wiuff, Caroline; Hansen, Christina Neigaard; Prats, Clara; Dela, Flemming; Helge, Jørn Wulff

2016-03-01

Aging and inactivity lead to skeletal muscle metabolic inflexibility, but the underlying molecular mechanisms are not entirely elucidated. Therefore, we investigated how muscle lipid and glycogen stores and major regulatory proteins were affected by short-term immobilization followed by aerobic training in young and older men. 17 young (23 ± 1 years, 24 ± 1 kg m(-2), and 20 ± 2% body fat) and 15 older men (68 ± 1 years; 27 ± 1 kg m(-2), and 29 ± 2% body fat) underwent 2 weeks' one leg immobilization followed by 6 weeks' cycle training. Biopsies were obtained from m. vastus lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl-CoA dehydrogenase (HAD) activity The older men had higher intramuscular triglyceride (IMTG) (73 %) and Glycogen (16%) levels compared to the young men, and IMTG tended to increase with immobilization. PLIN2 and 3 protein content increased with immobilization in the older men only. The young men had higher GS (74%) protein compared to the older men. Immobilization decreased and training restored HAD activity, GS and SNAP23 protein content in young and older men. Evidence of age-related metabolic inflexibility is presented, seen as body fat and IMTG accumulation. The question arises as to whether IMTG accumulation in the older men is caused by or leading to the increase in PLIN2 and 3 protein content. Training decreased body fat and IMTG levels in both young and older men; hence, training should be prioritized to reduce the detrimental effect of aging on metabolism.

Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity.

Science.gov (United States)

Dickey, Seth W; Baker, Rosanna P; Cho, Sangwoo; Urban, Siniša

2013-12-05

Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in more than a dozen diseases. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. Remarkably, rhomboid proteases displayed no physiological affinity for substrates (K(d) ~190 μM/0.1 mol%). Instead, ~10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in k(cat) values alone. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Ultimately, a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but are strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications. Copyright © 2013 Elsevier Inc. All rights reserved.

Low affinity uniporter carrier proteins can increase net substrate uptake rate by reducing efflux

NARCIS (Netherlands)

Bosdriesz, Evert; Wortel, Meike T.; Haanstra, Jurgen R.; Wagner, Marijke J.; De La Torre Cortés, Pilar; Teusink, Bas

2018-01-01

Many organisms have several similar transporters with different affinities for the same substrate. Typically, high-affinity transporters are expressed when substrate is scarce and low-affinity ones when it is abundant. The benefit of using low instead of high-affinity transporters remains unclear,

Low affinity uniporter carrier proteins can increase net substrate uptake rate by reducing efflux

NARCIS (Netherlands)

Bosdriesz, Evert; Wortel, M.T.; Haanstra, Jurgen R.; Wagner, Marijke J.; De La Torre, P.; Teusink, Bas

2018-01-01

Many organisms have several similar transporters with different affinities for the same substrate. Typically, high-affinity transporters are expressed when substrate is scarce and low-affinity ones when it is abundant. The benefit of using low instead of high-affinity transporters remains

Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris

Directory of Open Access Journals (Sweden)

Bader Oliver

2008-07-01

Full Text Available Abstract Background Kexin-like proteinases are a subfamily of the subtilisin-like serine proteinases with multiple regulatory functions in eukaryotes. In the yeast Saccharomyces cerevisiae the Kex2 protein is biochemically well investigated, however, with the exception of a few well known proteins such as the α-pheromone precursors, killer toxin precursors and aspartic proteinase propeptides, very few substrates are known. Fungal kex2 deletion mutants display pleiotropic phenotypes that are thought to result from the failure to proteolytically activate such substrates. Results In this study we have aimed at providing an improved assembly of Kex2 target proteins to explain the phenotypes observed in fungal kex2 deletion mutants by in vitro digestion of recombinant substrates from Candida albicans and C. glabrata. We identified CaEce1, CA0365, one member of the Pry protein family and CaOps4-homolog proteins as novel Kex2 substrates. Conclusion Statistical analysis of the cleavage sites revealed extended subsite recognition of negatively charged residues in the P1', P2' and P4' positions, which is also reflected in construction of the respective binding pockets in the ScKex2 enzyme. Additionally, we provide evidence for the existence of structural constrains in potential substrates prohibiting proteolysis. Furthermore, by using purified Kex2 proteinases from S. cerevisiae, P. pastoris, C. albicans and C. glabrata, we show that while the substrate specificity is generally conserved between organisms, the proteinases are still distinct from each other and are likely to have additional unique substrate recognition.

Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum

International Nuclear Information System (INIS)

Kino, Kuniki; Kuratsu, Shoko; Noguchi, Atsushi; Kokubo, Masahiro; Nakazawa, Yuji; Arai, Toshinobu; Yagasaki, Makoto; Kirimura, Kohtaro

2007-01-01

Glutathione (GSH) is synthesized by γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed γ-GCS-GS catalyzing both γ-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the γ-GCS activity, S. agalactiae γ-GCS-GS had different substrate specificities from those of Escherichia coli γ-GCS. Furthermore, S. agalactiae γ-GCS-GS synthesized several kinds of γ-glutamyltripeptide, γ-Glu-X aa -Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding γ-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae γ-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed γ-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of γ-glutamyltripeptide, γ-Glu-Cys-X aa . Whereas the substrate specificities of γ-GCS domain protein and GS domain protein of S. agalactiae γ-GCS-GS were the same as those of S. agalactiae γ-GCS-GS

Herp enhances ER-associated protein degradation by recruiting ubiquilins

International Nuclear Information System (INIS)

Kim, Tae-Yeon; Kim, Eunmin; Yoon, Sungjoo Kim; Yoon, Jong-Bok

2008-01-01

ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates

Protein N-myristoylation in Escherichia coli: Reconstitution of a eukaryotic protein modification in bacteria

International Nuclear Information System (INIS)

Duronio, R.J.; Jackson-Machelski, E.; Heuckeroth, R.O.; Gordon, J.I.; Olins, P.O.; Devine, C.S.; Yonemoto, W.; Slice, L.W.; Taylor, S.S.

1990-01-01

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH 2 -terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. The authors have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH 2 -terminal 39 amino acids. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed

Anaerobic digestion of cattle offal: protein and lipid-rich substrate degradation and population dynamics of acidogens and methanogens.

Science.gov (United States)

Lee, Joonyeob; Koo, Taewoan; Han, Gyuseong; Shin, Seung Gu; Hwang, Seokhwan

2015-12-01

Anaerobic digestion of cattle offal was investigated in batch reactors at 35 °C to determine the feasibility of using cattle offal as a feedstock. The organic content [i.e., volatile solids (VS)] of the cattle offal was mainly composed of protein (33.9%) and lipids (46.1%). Hydrolysis along with acidogenesis was monitored to investigate the substrate degradation and generation of intermediate products (e.g., volatile fatty acids, ammonia). Acetate (2.03 g/L), propionate (0.60 g/L), n-butyrate (0.39 g/L), and iso-valerate (0.37 g/L) were major acidogenesis products (91% of total volatile fatty acid concentration). Overall protein and lipid degradation were 82.9 and 81.8%, respectively. Protein degraded first, and four times faster (0.28 day(-1)) than lipid (0.07 day(-1)). Methane yields were 0.52 L CH4/g VSadded and 0.65 L CH4/g VSremoved, indicating that anaerobic digestion of the offal was feasible. A quantitative QPCR assay was conducted to understand the microbial dynamics. The variation patt erns in the gene concentrations successfully indicated the population dynamics of proteolytic and lipolytic acidogens. A fourth-order Runge-Kutta approximation was used to determine the kinetics of the acidogens. The molecular biotechnology approach was appropriate for the evaluation of the acidogenic biokinetics. The maximum growth rate, μ m, halfsaturation coefficients, K s, microbial yield coefficient, Y, cell mass decay rate coefficient, k d, of the proteolytic acidogens were 9.9 day(-1), 37.8 g protein/L, 1.1 × 10(10) copies/g protein, and 3.8 × 10(-1), respectively. Those for the lipolytic acidogens were 1.2 × 10(-1) day(-1), 8.3 g lipid/L, 1.5 × 10(9) copies/g lipid, and 9.9 × 10(-3) day(-1), respectively.

Enhanced protein adsorption and patterning on nanostructured latex-coated paper.

Science.gov (United States)

Juvonen, Helka; Määttänen, Anni; Ihalainen, Petri; Viitala, Tapani; Sarfraz, Jawad; Peltonen, Jouko

2014-06-01

Specific interactions of extracellular matrix proteins with cells and their adhesion to the substrate are important for cell growth. A nanopatterned latex-coated paper substrate previously shown to be an excellent substrate for cell adhesion and 2D growth was studied for directed immobilization of proteins. The nanostructured latex surface was formed by short-wavelength IR irradiation of a two-component latex coating consisting of a hydrophilic film-forming styrene butadiene acrylonitrile copolymer and hydrophobic polystyrene particles. The hydrophobic regions of the IR-treated latex coating showed strong adhesion of bovine serum albumin (cell repelling protein), fibronectin (cell adhesive protein) and streptavidin. Opposite to the IR-treated surface, fibronectin and streptavidin had a poor affinity toward the untreated pristine latex coating. Detailed characterization of the physicochemical surface properties of the latex-coated substrates revealed that the observed differences in protein affinity were mainly due to the presence or absence of the protein repelling polar and charged surface groups. The protein adsorption was assisted by hydrophobic (dehydration) interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Na+/substrate Coupling in the Multidrug Antiporter NorM Probed with a Spin-labeled Substrate

Science.gov (United States)

Steed, P. Ryan; Stein, Richard A.; Mishra, Smriti; Goodman, Michael C.; Mchaourab, Hassane S.

2013-01-01

NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na+ gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal to characterize mechanistically relevant binding interactions with NorM and to directly address the coupling of ion and drug binding. Fluorescence and EPR experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na+. Using double electron-electron resonance (DEER) spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na+ did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na+ does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na+ gradient across the native membrane, govern Na+/drug coupling during antiport. PMID:23902581

Evaluation of various substrates and supplements for biological ...

African Journals Online (AJOL)

An experiment was conducted to determine the effects of different substrates namely wheat straw (Triticum aestivum), maize stover (Zea mays L), thatch grass (Hyparrhenia filipendula) and oil/protein rich supplements (maize bran, cottonseed hull [Gossypium hirsutum]) on biological efficiency of two oyster mushroom ...

Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays.

NARCIS (Netherlands)

Krumpochova, P; Sapthu, S.; Brouwers, J.F.H.M.; de Haas, M.; de Vos, R.; Borst, P.; van de Wetering, K.

2013-01-01

ABSTRACT The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

Energy Technology Data Exchange (ETDEWEB)

Khadempour, Lily [Department of Bacteriology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Zoology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison WI 53706 USA; Burnum-Johnson, Kristin E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Baker, Erin S. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Nicora, Carrie D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Webb-Robertson, Bobbie-Jo M. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; White, Richard A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Monroe, Matthew E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Huang, Eric L. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Smith, Richard D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Currie, Cameron R. [Department of Bacteriology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison WI 53706 USA

2016-10-26

Herbivores use symbiotic microbes to help gain access to energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, having tremendous impact on their ecosystems as dominant generalist herbivores through cultivation of a fungus, Leucoagaricus gongylophorous. Here we examine how this mutualism could facilitate the flexible substrate incorporation of the ants by providing leaf-cutter ant subcolonies four substrate types: leaves, flowers, oats, and a mixture of all three. Through metaproteomic analysis of the fungus gardens, we were able to identify and quantify 1766 different fungal proteins, including 161 biomass-degrading enzymes. This analysis revealed that fungal protein profiles were significantly different between subcolonies fed different substrates with the highest abundance of cellulolytic enzymes observed in the leaf and flower treatments. When the fungus garden is provided with leaves and flowers, which contain the majority of their energy in recalcitrant material, it increases its production of proteins that break down cellulose: endoglucanases, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, the mixed treatment closely resembled the treatment with oats alone. This suggests that when provided a mixture of substrates, the fungus garden preferentially produces enzymes necessary for breakdown of simpler, more digestible substrates. This flexible, substrate-specific response of the fungal cultivar allows the leaf-cutter ants to derive energy from a wide range of substrates, which may contribute to their ability to be dominant generalist herbivores.

Quantitative Photochemical Immobilization of Biomolecules on Planar and Corrugated Substrates: A Versatile Strategy for Creating Functional Biointerfaces

Science.gov (United States)

Martin, Teresa A.; Herman, Christine T.; Limpoco, Francis T.; Michael, Madeline C.; Potts, Gregory K.; Bailey, Ryan C.

2014-01-01

Methods for the generation of substrates presenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retention of function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor-ligand interactions. This molecularly general photochemical patterning method is an enabling tool that will allow the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces. PMID:21793535

Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate.

OpenAIRE

Tomasiewicz, H G; Cook-Deegan, R; Chikaraishi, D M

1984-01-01

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase.

Science.gov (United States)

Lee, Yong-Jik; Lee, Sang-Jae; Kim, Seong-Bo; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

2014-03-18

Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilus L-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of L-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Comparison of metabolic substrates in alligators and several birds of prey.

Science.gov (United States)

Sweazea, Karen L; McMurtry, John P; Elsey, Ruth M; Redig, Patrick; Braun, Eldon J

2014-08-01

On average, avian blood glucose concentrations are 1.5-2 times those of mammals of similar mass and high concentrations of insulin are required to lower blood glucose. Whereas considerable data exist for granivorous species, few data are available for plasma metabolic substrate and glucoregulatory hormone concentrations for carnivorous birds and alligators. Birds and mammals with carnivorous diets have higher metabolic rates than animals consuming diets with less protein whereas alligators have low metabolic rates. Therefore, the present study was designed to compare substrate and glucoregulatory hormone concentrations in several birds of prey and a phylogenetically close relative of birds, the alligator. The hypothesis was that the combination of carnivorous diets and high metabolic rates favored the evolution of greater protein and fatty acid utilization leading to insulin resistance and high plasma glucose concentrations in carnivorous birds. In contrast, it was hypothesized that alligators would have low substrate utilization attributable to a low metabolic rate. Fasting plasma substrate and glucoregulatory hormone concentrations were compared for bald eagles (Haliaeetus leucocephalus), great horned owls (Bubo virginianus), red-tailed hawks (Buteo jamaicensis), and American alligators (Alligator mississippiensis). Avian species had high circulating β-hydroxybutyrate (10-21 mg/dl) compared to alligators (2.81 ± 0.16 mg/dl). In mammals high concentrations of this byproduct of fatty acid utilization are correlated with insulin resistance. Fasting glucose and insulin concentrations were positively correlated in eagles whereas no relationship was found between these variables for owls, hawks or alligators. Additionally, β-hydroxybutyrate concentrations were low in alligators. Similar to carnivorous mammals, ingestion of a high protein diet may have favored the utilization of fatty acids and protein for energy thereby promoting the development of insulin

Uncovering a role for the tail of the Dictyostelium discoideum SadA protein in cell-substrate adhesion.

Science.gov (United States)

Kowal, Anthony S; Chisholm, Rex L

2011-05-01

Previous work from our laboratory showed that the Dictyostelium discoideum SadA protein plays a central role in cell-substrate adhesion. SadA null cells exhibit a loss of adhesion, a disrupted actin cytoskeleton, and a cytokinesis defect. How SadA mediates these phenotypes is unknown. This work addresses the mechanism of SadA function, demonstrating an important role for the C-terminal cytoplasmic tail in SadA function. We found that a SadA tailless mutant was unable to rescue the sadA adhesion deficiency, and overexpression of the SadA tail domain reduced adhesion in wild-type cells. We also show that SadA is closely associated with the actin cytoskeleton. Mutagenesis studies suggested that four serine residues in the tail, S924/S925 and S940/S941, may regulate association of SadA with the actin cytoskeleton. Glutathione S-transferase pull-down assays identified at least one likely interaction partner of the SadA tail, cortexillin I, a known actin bundling protein. Thus, our data demonstrate an important role for the carboxy-terminal cytoplasmic tail in SadA function and strongly suggest that a phosphorylation event in this tail regulates an interaction with cortexillin I. Based on our data, we propose a model for the function of SadA.

Entropy-driven crystal formation on highly strained substrates

KAUST Repository

Savage, John R.

2013-05-20

In heteroepitaxy, lattice mismatch between the deposited material and the underlying surface strongly affects nucleation and growth processes. The effect of mismatch is well studied in atoms with growth kinetics typically dominated by bond formation with interaction lengths on the order of one lattice spacing. In contrast, less is understood about how mismatch affects crystallization of larger particles, such as globular proteins and nanoparticles, where interparticle interaction energies are often comparable to thermal fluctuations and are short ranged, extending only a fraction of the particle size. Here, using colloidal experiments and simulations, we find particles with short-range attractive interactions form crystals on isotropically strained lattices with spacings significantly larger than the interaction length scale. By measuring the free-energy cost of dimer formation on monolayers of increasing uniaxial strain, we show the underlying mismatched substrate mediates an entropy-driven attractive interaction extending well beyond the interaction length scale. Remarkably, because this interaction arises from thermal fluctuations, lowering temperature causes such substrate-mediated attractive crystals to dissolve. Such counterintuitive results underscore the crucial role of entropy in heteroepitaxy in this technologically important regime. Ultimately, this entropic component of lattice mismatched crystal growth could be used to develop unique methods for heterogeneous nucleation and growth of single crystals for applications ranging from protein crystallization to controlling the assembly of nanoparticles into ordered, functional superstructures. In particular, the construction of substrates with spatially modulated strain profiles would exploit this effect to direct self-assembly, whereby nucleation sites and resulting crystal morphology can be controlled directly through modifications of the substrate.

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

Science.gov (United States)

Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

2018-01-01

Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono

Molecular docking simulations provide insights in the substrate binding sites and possible substrates of the ABCC6 transporter.

Directory of Open Access Journals (Sweden)

Mohammad Jakir Hosen

Full Text Available The human ATP-binding cassette family C member 6 (ABCC6 gene encodes an ABC transporter protein (ABCC6, primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE, an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS and leukotriene C4 (LTC4 were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3 was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE.

Sumo-dependent substrate targeting of the SUMO protease Ulp1

Directory of Open Access Journals (Sweden)

Westerbeck Jason W

2011-10-01

Full Text Available Abstract Background In the yeast Saccharomyces cerevisiae, the essential small ubiquitin-like modifier (SUMO protease Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes but has also been shown to deconjugate sumoylated septins at the bud-neck of dividing cells. How Ulp1 is directed to bud-neck localized septins and other cytoplasmic deconjugation targets is not well understood. Results Using a structure/function approach, we set out to elucidate features of Ulp1 that are required for substrate targeting. To aid our studies, we took advantage of a catalytically inactive mutant of Ulp1 that is greatly enriched at the septin ring of dividing yeast cells. We found that the localization of Ulp1 to the septins requires both SUMO and specific structural features of Ulp1's catalytic domain. Our analysis identified a 218-amino acid, substrate-trapping mutant of the catalytic domain of Ulp1, Ulp1(3(C580S, that is necessary and sufficient for septin localization. We also used the targeting and SUMO-binding properties of Ulp1(3(C580S to purify Smt3-modified proteins from cell extracts. Conclusions Our study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates in vivo and in vitro. Furthermore, we found that a substrate-trapping Ulp1(3(C580S interacts robustly with human SUMO1, SUMO2 and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins.

Activation of the DnaK-ClpB Complex is Regulated by the Properties of the Bound Substrate.

Science.gov (United States)

Fernández-Higuero, Jose Angel; Aguado, Alejandra; Perales-Calvo, Judit; Moro, Fernando; Muga, Arturo

2018-04-11

The chaperone ClpB in bacteria is responsible for the reactivation of aggregated proteins in collaboration with the DnaK system. Association of these chaperones at the aggregate surface stimulates ATP hydrolysis, which mediates substrate remodeling. However, a question that remains unanswered is whether the bichaperone complex can be selectively activated by substrates that require remodeling. We find that large aggregates or bulky, native-like substrates activates the complex, whereas a smaller, permanently unfolded protein or extended, short peptides fail to stimulate it. Our data also indicate that ClpB interacts differently with DnaK in the presence of aggregates or small peptides, displaying a higher affinity for aggregate-bound DnaK, and that DnaK-ClpB collaboration requires the coupled ATPase-dependent remodeling activities of both chaperones. Complex stimulation is mediated by residues at the β subdomain of DnaK substrate binding domain, which become accessible to the disaggregase when the lid is allosterically detached from the β subdomain. Complex activation also requires an active NBD2 and the integrity of the M domain-ring of ClpB. Disruption of the M-domain ring allows the unproductive stimulation of the DnaK-ClpB complex in solution. The ability of the DnaK-ClpB complex to discrimínate different substrate proteins might allow its activation when client proteins require remodeling.

Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

Energy Technology Data Exchange (ETDEWEB)

Wang, Chengliang [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Zhang, Qiongdi [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Hang, Tianrong [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Tao, Yue [Shanghai Children’s Medical Center, 1678 Dongfang Road, Pudong, Shanghai 200120, People’s Republic of (China); Ma, Xukai [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Wu, Minhao; Zhang, Xuan, E-mail: xuanzbin@ustc.edu.cn; Zang, Jianye, E-mail: xuanzbin@ustc.edu.cn [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China)

2015-08-28

The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66{sup 176–C} complexed with Rpl8{sup 204–224} in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

Interactions between Casein kinase Iepsilon (CKIepsilon and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.

Directory of Open Access Journals (Sweden)

Caroline Lund Dahlberg

Full Text Available Members of the Casein Kinase I (CKI family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways.CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding.The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis

Directory of Open Access Journals (Sweden)

Nicola V. Huck

2017-12-01

Full Text Available Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

PREFACE: Cell-substrate interactions Cell-substrate interactions

Science.gov (United States)

Gardel, Margaret; Schwarz, Ulrich

2010-05-01

One of the most striking achievements of evolution is the ability to build cellular systems that are both robust and dynamic. Taken by themselves, both properties are obvious requirements: robustness reflects the fact that cells are there to survive, and dynamics is required to adapt to changing environments. However, it is by no means trivial to understand how these two requirements can be implemented simultaneously in a physical system. The long and difficult quest to build adaptive materials is testimony to the inherent difficulty of this goal. Here materials science can learn a lot from nature, because cellular systems show that robustness and dynamics can be achieved in a synergetic fashion. For example, the capabilities of tissues to repair and regenerate are still unsurpassed in the world of synthetic materials. One of the most important aspects of the way biological cells adapt to their environment is their adhesive interaction with the substrate. Numerous aspects of the physiology of metazoan cells, including survival, proliferation, differentiation and migration, require the formation of adhesions to the cell substrate, typically an extracellular matrix protein. Adhesions guide these diverse processes both by mediating force transmission from the cell to the substrate and by controlling biochemical signaling pathways. While the study of cell-substrate adhesions is a mature field in cell biology, a quantitative biophysical understanding of how the interactions of the individual molecular components give rise to the rich dynamics and mechanical behaviors observed for cell-substrate adhesions has started to emerge only over the last decade or so. The recent growth of research activities on cell-substrate interactions was strongly driven by the introduction of new physical techniques for surface engineering into traditional cell biological work with cell culture. For example, microcontact printing of adhesive patterns was used to show that cell fate depends

WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates in pathogenic mycobacteria.

KAUST Repository

Abdallah, Abdallah

2018-04-09

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages, suggesting an important role in ESX-1-mediated virulence during the early phase of infection. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.

WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates in pathogenic mycobacteria.

KAUST Repository

Abdallah, Abdallah; Weerdenburg, Eveline; Guan, Qingtian; Ummels, Roy; Borggreve, S; Adroub, Sabir; Malas, Tareq; Naeem, Raeece; Zhang, Huoming; Otto, Thomas; Bitter, Wilbert; Pain, Arnab

2018-01-01

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages, suggesting an important role in ESX-1-mediated virulence during the early phase of infection. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.

In Situ Demonstration and Characteristic Analysis of the Protease Using Substrate Immersing Zymography.

Science.gov (United States)

He, HaiLun; Li, Hao; Liu, Dan

2017-01-01

Zymography, the detection of proteolytic activities on the basis of protein substrate degradation, has been a technique described in the literature for at least in the past 50 years. In this study, we used substrate immersing zymography to analyze proteolysis of proteases. Instead of being directly added into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, the substrates were added into the immersing solution after electrophoresis. With substrate immersing zymography, some characters of proteases, such as enzyme forms, potential proteolytic activity, molecular weights, presence of complexes, and potentially active enzyme fragments in complex biological samples, can be determined.

Analysis of Protein Phosphorylation and Its Functional Impact on Protein-Protein Interactions via Text Mining of the Scientific Literature.

Science.gov (United States)

Wang, Qinghua; Ross, Karen E; Huang, Hongzhan; Ren, Jia; Li, Gang; Vijay-Shanker, K; Wu, Cathy H; Arighi, Cecilia N

2017-01-01

Post-translational modifications (PTMs) are one of the main contributors to the diversity of proteoforms in the proteomic landscape. In particular, protein phosphorylation represents an essential regulatory mechanism that plays a role in many biological processes. Protein kinases, the enzymes catalyzing this reaction, are key participants in metabolic and signaling pathways. Their activation or inactivation dictate downstream events: what substrates are modified and their subsequent impact (e.g., activation state, localization, protein-protein interactions (PPIs)). The biomedical literature continues to be the main source of evidence for experimental information about protein phosphorylation. Automatic methods to bring together phosphorylation events and phosphorylation-dependent PPIs can help to summarize the current knowledge and to expose hidden connections. In this chapter, we demonstrate two text mining tools, RLIMS-P and eFIP, for the retrieval and extraction of kinase-substrate-site data and phosphorylation-dependent PPIs from the literature. These tools offer several advantages over a literature search in PubMed as their results are specific for phosphorylation. RLIMS-P and eFIP results can be sorted, organized, and viewed in multiple ways to answer relevant biological questions, and the protein mentions are linked to UniProt identifiers.

Can microbes compete with cows for sustainable protein production - A feasibility study on high quality protein

DEFF Research Database (Denmark)

Vestergaard, Mike; Chan, Siu Hung Joshua; Jensen, Peter Ruhdal

2016-01-01

An increasing population and their increased demand for high-protein diets will require dramatic changes in the food industry, as limited resources and environmental issues will make animal derived foods and proteins, gradually more unsustainable to produce. To explore alternatives to animal...... derived proteins, an economic model was built around the genome-scale metabolic network of E. coli to study the feasibility of recombinant protein production as a food source. Using a novel model, we predicted which microbial production strategies are optimal for economic return, by capturing the tradeoff...... between the market prices of substrates, product output and the efficiency of microbial production. A case study with the food protein, Bovine Alpha Lactalbumin was made to evaluate the upstream economic feasibilities. Simulations with different substrate profiles at maximum productivity were used...

Crius: A Novel Fragment-Based Algorithm of De Novo Substrate Prediction for Enzymes.

Science.gov (United States)

Yao, Zhiqiang; Jiang, Shuiqin; Zhang, Lujia; Gao, Bei; He, Xiao; Zhang, John Z H; Wei, Dongzhi

2018-05-03

The study of enzyme substrate specificity is vital for developing potential applications of enzymes. However, the routine experimental procedures require lot of resources in the discovery of novel substrates. This article reports an in silico structure-based algorithm called Crius, which predicts substrates for enzyme. The results of this fragment-based algorithm show good agreements between the simulated and experimental substrate specificities, using a lipase from Candida antarctica (CALB), a nitrilase from Cyanobacterium syechocystis sp. PCC6803 (Nit6803), and an aldo-keto reductase from Gluconobacter oxydans (Gox0644). This opens new prospects of developing computer algorithms that can effectively predict substrates for an enzyme. This article is protected by copyright. All rights reserved. © 2018 The Protein Society.

Nutritional composition of black soldier fly (Hermetia illucens) prepupae reared on different organic waste substrates.

Science.gov (United States)

Spranghers, Thomas; Ottoboni, Matteo; Klootwijk, Cindy; Ovyn, Anneke; Deboosere, Stefaan; De Meulenaer, Bruno; Michiels, Joris; Eeckhout, Mia; De Clercq, Patrick; De Smet, Stefaan

2017-06-01

Black soldier fly larvae are converters of organic waste into edible biomass, of which the composition may depend on the substrate. In this study, larvae were grown on four substrates: chicken feed, vegetable waste, biogas digestate, and restaurant waste. Samples of prepupae and substrates were freeze-dried and proximate, amino acid, fatty acid and mineral analyses were performed. Protein content of prepupae varied between 399 and 431 g kg -1 dry matter (DM) among treatments. Differences in amino acid profile of prepupae were small. On the other hand, the ether extract (EE) and ash contents differed substantially. Prepupae reared on digestate were low in EE and high in ash (218 and 197 g kg -1 DM, respectively) compared to those reared on vegetable waste (371 and 96 g kg -1 DM, respectively), chicken feed (336 and 100 g kg -1 DM, respectively) and restaurant waste (386 and 27 g kg -1 DM, respectively). Prepupal fatty acid profiles were characterised by high levels of C12:0 in all treatments. Since protein content and quality were high and comparable for prepupae reared on different substrates, black soldier fly could be an interesting protein source for animal feeds. However, differences in EE and ash content as a function of substrate should be considered. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Substrate-Na{sup +} complex formation: Coupling mechanism for {gamma}-aminobutyrate symporters

Energy Technology Data Exchange (ETDEWEB)

Pallo, Anna; Simon, Agnes [Department of Neurochemistry, Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences (Hungary); Bencsura, Akos [Department of Theoretical Chemistry, Institute of Structural Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest (Hungary); Heja, Laszlo [Department of Neurochemistry, Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences (Hungary); Kardos, Julianna, E-mail: jkardos@chemres.hu [Department of Neurochemistry, Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences (Hungary)

2009-07-24

Crystal structures of transmembrane transport proteins belonging to the important families of neurotransmitter-sodium symporters reveal how they transport neurotransmitters across membranes. Substrate-induced structural conformations of gated neurotransmitter-sodium symporters have been in the focus of research, however, a key question concerning the mechanism of Na{sup +} ion coupling remained unanswered. Homology models of human glial transporter subtypes of the major inhibitory neurotransmitter {gamma}-aminobutyric acid were built. In accordance with selectivity data for subtype 2 vs. 3, docking and molecular dynamics calculations suggest similar orthosteric substrate (inhibitor) conformations and binding crevices but distinguishable allosteric Zn{sup 2+} ion binding motifs. Considering the occluded conformational states of glial human {gamma}-aminobutyric acid transporter subtypes, we found major semi-extended and minor ring-like conformations of zwitterionic {gamma}-aminobutyric acid in complex with Na{sup +} ion. The existence of the minor ring-like conformation of {gamma}-aminobutyric acid in complex with Na{sup +} ion may be attributed to the strengthening of the intramolecular H-bond by the electrostatic effect of Na{sup +} ion. Coupling substrate uptake into cells with the thermodynamically favorable Na{sup +} ion movement through substrate-Na{sup +} ion complex formation may be a mechanistic principle featuring transmembrane neurotransmitter-sodium symporter proteins.

Structure based protein engineering of Bacillus stearothermophilus {alpha}-amylase: toward a new substrate specificity

Energy Technology Data Exchange (ETDEWEB)

Rasera, Ana Claudia [Sao Paulo Univ., SP (Brazil). Inst. de Ciencias Biomedicas; Iulek, Jorge [Universidade Estadual de Ponta Grossa, PR (Brazil). Inst. de Quimica; Delboni, Luis Fernando; Barbosa, Valma Martins Barbosa [Parana Univ., Curitiba, PR (Brazil). Dept. de Bioquimica

1997-12-31

Full text. Structural similarity is observed in all members of {alpha}-amylase family but different products are generated during hydrolysis of starch due to different affinities for intermediate dextrins. In order to understand the structural determinants for this property and to introduce different specificity to {alpha}-amylase of Bacillus stearothermophilus we intend to solve the three dimensional structure by X-ray crystallography of the native protein by using synchrotron radiation at Brazilian National Synchrotron Light Laboratory (LNLS). Protein was over expressed in E. coli, purified and crystallization experiments were carried out by using sparse matrix Crystal Screen and Crystal Screen II from Hampton Research (Laguna Hills, CA, USA). Several condition have produced crystals with some defined characteristic: MDP seems to be important to the crystallization: the preferential pH is around 7.5 with organic buffer (HEPES); organic solvent as 2-propanol seems to be also important for the crystallization. On those condition crystals appeared as cluster of needles or small crystals with high number of nucleation. New conditions are being prepared to improve the site and quality of crystals. Data collection of native of Bacillus stearothermophilus {alpha}-amylase will e done at Protein Crystallography Station at LNLS. Crystal structure of mutated {alpha}-amylase bu site direct mutagenesis of residues suggested by the native crystal structure will be obtained. Co-crystallization of Bacillus stearothermophilus {alpha}-amylase and oligosaccharide will be carried out to identify residues involved in the binding site to plan new mutation. In another series of mutation the putative binding site residues, once identified, will be mutated to residues observed in TAKA amylase to confer different specificity to {alpha}-amylase. Based on the available TAKA amylase structure, in the primary sequence homology and in the three dimensional model of Bacillus stearothermophilus

Structure based protein engineering of Bacillus stearothermophilus α-amylase: toward a new substrate specificity

International Nuclear Information System (INIS)

Rasera, Ana Claudia; Iulek, Jorge; Delboni, Luis Fernando; Barbosa, Valma Martins Barbosa

1997-01-01

Full text. Structural similarity is observed in all members of α-amylase family but different products are generated during hydrolysis of starch due to different affinities for intermediate dextrins. In order to understand the structural determinants for this property and to introduce different specificity to α-amylase of Bacillus stearothermophilus we intend to solve the three dimensional structure by X-ray crystallography of the native protein by using synchrotron radiation at Brazilian National Synchrotron Light Laboratory (LNLS). Protein was over expressed in E. coli, purified and crystallization experiments were carried out by using sparse matrix Crystal Screen and Crystal Screen II from Hampton Research (Laguna Hills, CA, USA). Several condition have produced crystals with some defined characteristic: MDP seems to be important to the crystallization: the preferential pH is around 7.5 with organic buffer (HEPES); organic solvent as 2-propanol seems to be also important for the crystallization. On those condition crystals appeared as cluster of needles or small crystals with high number of nucleation. New conditions are being prepared to improve the site and quality of crystals. Data collection of native of Bacillus stearothermophilus α-amylase will e done at Protein Crystallography Station at LNLS. Crystal structure of mutated α-amylase bu site direct mutagenesis of residues suggested by the native crystal structure will be obtained. Co-crystallization of Bacillus stearothermophilus α-amylase and oligosaccharide will be carried out to identify residues involved in the binding site to plan new mutation. In another series of mutation the putative binding site residues, once identified, will be mutated to residues observed in TAKA amylase to confer different specificity to α-amylase. Based on the available TAKA amylase structure, in the primary sequence homology and in the three dimensional model of Bacillus stearothermophilus α-amylase (using Bacillus

Towards complete sets of farnesylated and geranylgeranylated proteins.

Directory of Open Access Journals (Sweden)

Sebastian Maurer-Stroh

2007-04-01

Full Text Available Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein-protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase and can be queried for verification status, type of modifying enzymes (anchor type, and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins-for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain-containing FLJ32421 (termed BROFTI, and Rab28 (short isoform as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for

Structural Basis for Target Protein Regcognition by Thiredoxin

DEFF Research Database (Denmark)

Maeda, Kenji

2007-01-01

Ser) and a mutant of an in vitro substrate alpha-amylase/subtilisin inhibitor (BASI) (Cys144Ser), as a reaction intermediate-mimic of Trx-catalyzed disulfide reduction. The resultant structure showed a sequence of BASI residues along a conserved hydrophobic groove constituted of three loop segments...... of Trx-fold proteins glutaredoxin and glutathione transferase. This study suggests that the features of main chain conformation as well as charge property around disulfide bonds in protein substrates are important factors for interaction with Trx. Moreover, this study describes a detailed structural......Thioredoxin (Trx) is an ubiquitous protein disulfide reductase that possesses two redox active cysteines in the conserved active site sequence motif, Trp-CysN-Gly/Pro-Pro-CysC situated in the so called Trx-fold. The lack of insight into the protein substrate recognition mechanism of Trx has to date...

Evaluación de la dinámica de conservación del producto final de un alimento obtenido por fermentación en estado sólido de la papa (Fes-papa

Directory of Open Access Journals (Sweden)

Luis Miguel Borras-Sandoval

2015-01-01

Full Text Available Se expone el efecto de la fermentación en estado sólido sobre un alimento preparado a base del tubérculo de la papa (Solanum tuberosum, y su posible utilización en la alimentación animal. La papa fresca y picada se mezcló con un material fibroso secante (harina de alfalfa, melaza, urea, un preparado microbiano, premezcla mineral, carbonato de calcio y sulfato de sodio, y se dejó fermentar, acorde con el tiempo y la temperatura previamente establecidos (48 h y 20°C, en bolsas plásticas de 50 kg. El producto Fes-papa se muestreó el día uno de elaboración y a los treinta y noventa días. El producto Fes-papa presentó modificaciones significativas en los indicadores fermentativos evaluados. El pH descendió constantemente desde el inicio (6.3 hasta terminar la evaluación (pH 4.86; algo similar ocurrió con la materia seca (MSI y el componente fibroso (FDN-FDA, los cuales descendieron con el tiempo de fermentación, mejorando sensiblemente la digestibilidad del producto. La Fes-papa es un proceso biotecnológico sencillo para aprovechar los tubérculos de los residuos de cosecha y generar un alimento energético-proteico que, acorde con los indicadores fermentativos y contenido en MS, pudiera ser empleado en la alimentación animal; además, contrarrestaría la contaminación ambiental.

Retuning Rieske-type Oxygenases to Expand Substrate Range

Energy Technology Data Exchange (ETDEWEB)

Mohammadi, Mahmood; Viger, Jean-François; Kumar, Pravindra; Barriault, Diane; Bolin, Jeffrey T.; Sylvestre, Michel (INRS); (Purdue)

2012-09-17

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE{sub p4}, a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE{sub LB400} by the double substitution T335A/F336M, and BphAE{sub RR41}, obtained by changing Asn{sup 338}, Ile{sup 341}, and Leu{sup 409} of BphAE{sub p4} to Gln{sup 338}, Val{sup 341}, and Phe{sup 409}, metabolize dibenzofuran two and three times faster than BphAE{sub LB400}, respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE{sub LB400} showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.

Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

Science.gov (United States)

Prudova, Anna; auf dem Keller, Ulrich; Butler, Georgina S; Overall, Christopher M

2010-05-01

Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses

Specialized activities and expression differences for Clostridium thermocellum biofilm and planktonic cells.

Science.gov (United States)

Dumitrache, Alexandru; Klingeman, Dawn M; Natzke, Jace; Rodriguez, Miguel; Giannone, Richard J; Hettich, Robert L; Davison, Brian H; Brown, Steven D

2017-02-27

Clostridium (Ruminiclostridium) thermocellum is a model organism for its ability to deconstruct plant biomass and convert the cellulose into ethanol. The bacterium forms biofilms adherent to lignocellulosic feedstocks in a continuous cell-monolayer in order to efficiently break down and uptake cellulose hydrolysates. We developed a novel bioreactor design to generate separate sessile and planktonic cell populations for omics studies. Sessile cells had significantly greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division consistent with substrate replete conditions. Planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. This study demonstrates that microbial attachment to cellulose substrate produces widespread gene expression changes for critical functions of this organism and provides physiological insights for two cells populations relevant for engineering of industrially-ready phenotypes.

Substrate specificity determinants of class III nucleotidyl cyclases.

Science.gov (United States)

Bharambe, Nikhil G; Barathy, Deivanayaga V; Syed, Wajeed; Visweswariah, Sandhya S; Colaςo, Melwin; Misquith, Sandra; Suguna, Kaza

2016-10-01

The two second messengers in signalling, cyclic AMP and cyclic GMP, are produced by adenylyl and guanylyl cyclases respectively. Recognition and discrimination of the substrates ATP and GTP by the nucleotidyl cyclases are vital in these reactions. Various apo-, substrate- or inhibitor-bound forms of adenylyl cyclase (AC) structures from transmembrane and soluble ACs have revealed the catalytic mechanism of ATP cyclization reaction. Previously reported structures of guanylyl cyclases represent ligand-free forms and inactive open states of the enzymes and thus do not provide information regarding the exact mode of substrate binding. The structures we present here of the cyclase homology domain of a class III AC from Mycobacterium avium (Ma1120) and its mutant in complex with ATP and GTP in the presence of calcium ion, provide the structural basis for substrate selection by the nucleotidyl cyclases at the atomic level. Precise nature of the enzyme-substrate interactions, novel modes of substrate binding and the ability of the binding pocket to accommodate diverse conformations of the substrates have been revealed by the present crystallographic analysis. This is the first report to provide structures of both the nucleotide substrates bound to a nucleotidyl cyclase. Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 5D15 (Ma1120 CHD +ATP.Ca 2+ ), 5D0E (Ma1120 CHD +GTP.Ca 2+ ), 5D0H (Ma1120 CHD (KDA→EGY)+ATP.Ca 2+ ), 5D0G (Ma1120 CHD (KDA→EGY)+GTP.Ca 2+ ). Adenylyl cyclase (EC number: 4.6.1.1). © 2016 Federation of European Biochemical Societies.

Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

DEFF Research Database (Denmark)

Sol, E-ri Maria; Wagner, Sebastian A; Weinert, Brian T

2012-01-01

Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates...... of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site...... by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases....

Molecular dynamics simulations of protein-tyrosine phosphatase 1B. I. Ligand-induced changes in the protein motions

DEFF Research Database (Denmark)

Peters, Günther H. J.; Frimurer, T.M.; Andersen, J.N.

1999-01-01

Activity of enzymes, such as protein tyrosine phosphatases (PTPs), is often associated with structural changes in the enzyme, resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate on the motions occurring in PTPs, we have performed...... molecular dynamics simulations of PTP1B and PTP1B complexed with a high-affinity peptide DADEpYL, where pY stands for phosphorylated tyrosine. The peptide sequence is derived from the epidermal growth factor receptor (EGFR(988-993)). Simulations were performed in water for 1 ns, and the concerted motions...... in the protein were analyzed using the essential dynamics technique. Our results indicate that the predominately internal motions in PTP1B occur in a subspace of only a few degrees of freedom. Upon substrate binding, the flexibility of the protein is reduced by similar to 10%. The largest effect is found...

Purification of a large molecular weight transglutaminase substrate from liver plasma membranes

International Nuclear Information System (INIS)

Slife, C.W.; Morris, G.S.; Tyrrell, D.J.

1986-01-01

Transglutaminases are enzymes which catalyze the covalent crosslinking of proteins by forming epsilon(γ-glutamyl)lysine isopeptide linkages. In earlier studies, the authors reported that a large molecular weight protein aggregate in rat liver plasma membranes served as a substrate for a plasma membrane-associated transglutaminase. The enzyme specifically incorporated a lysine analog, [ 3 H]putrescine, into a protein complex which remained at the top of an acrylamide gel upon electrophoresis in SDS and reducing agents. The complex has now been isolated by extracting the plasma membranes with detergent (octylglucoside) resuspending the detergent insoluble residues in 6 M guanidine HCl and chromatographing the residue on a 4% agarose column in 6 M guanidine HCl. Most of the radioactivity is found in the void volume fractions from the column. SDS polyacrylamide gel electrophoresis shows that these fractions contain mostly proteins that do not enter the acrylamide gel. Since this purification procedure is essentially the same as that used to isolate a rat hepatocyte adhesion factor from rat liver plasma membranes it is possible that the large molecular weight transglutaminase substrate and the adhesion factor are contained in the same protein aggregate

Effect of External Electric Field on Substrate Transport of a Secondary Active Transporter.

Science.gov (United States)

Zhang, Ji-Long; Zheng, Qing-Chuan; Yu, Li-Ying; Li, Zheng-Qiang; Zhang, Hong-Xing

2016-08-22

Substrate transport across a membrane accomplished by a secondary active transporter (SAT) is essential to the normal physiological function of living cells. In the present research, a series of all-atom molecular dynamics (MD) simulations under different electric field (EF) strengths was performed to investigate the effect of an external EF on the substrate transport of an SAT. The results show that EF both affects the interaction between substrate and related protein's residues by changing their conformations and tunes the timeline of the transport event, which collectively reduces the height of energy barrier for substrate transport and results in the appearance of two intermediate conformations under the existence of an external EF. Our work spotlights the crucial influence of external EFs on the substrate transport of SATs and could provide a more penetrating understanding of the substrate transport mechanism of SATs.

Structural Basis for Substrate Recognition by the Ankyrin Repeat Domain of Human DHHC17 Palmitoyltransferase

Energy Technology Data Exchange (ETDEWEB)

Verardi, Raffaello; Kim, Jin-Sik; Ghirlando, Rodolfo; Banerjee, Anirban

2017-09-01

DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment of Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most physiologically relevant substrates.

Experimental studies on the electronic structure of pyrite FeS2 films prepared by thermally sulfurizing iron films

International Nuclear Information System (INIS)

Zhang Hui; Wang Baoyi; Zhang Rengang; Zhang Zhe; Wei Long; Qian Haijie; Su Run; Kui Rexi

2006-01-01

Pyrite FeS 2 films have been prepared by thermally sulfurizing iron films deposited by magnetron sputtering. The electronic structures were studies by X-ray absorption near edge structure and X-ray photoemission spectrum. The results show that an S 3p valence band with relatively higher intensity compared to the calculation exists in 2-10 eV range and a high density below the Fermi level of Fe 3d states were detected. A second gap of 2.8 eV in the unoccupied density of states was found above the conduction band which was 2.4 eV by experimentally calculation. The difference between t 2g and e g which were formed in an octahedral crystal field was computed to be 2.1 eV. (authors)

The role of Aspergillus flavus veA in the production of extracellular proteins during growth on starch substrates.

Science.gov (United States)

Duran, Rocio M; Gregersen, Scott; Smith, Timothy D; Bhetariya, Preetida J; Cary, Jeffrey W; Harris-Coward, Pamela Y; Mattison, Christopher P; Grimm, Casey; Calvo, Ana M

2014-06-01

The aflatoxin-producer and opportunistic plant pathogenic, filamentous fungus Aspergillus flavus is responsible for the contamination of corn and other important agricultural commodities. In order to obtain nutrients from the host A. flavus produces a variety of extracellular hydrolytic enzymes. Interestingly, A. flavus amylase and protease activity are dependent on the global regulator veA, a gene known to regulate morphogenesis and secondary metabolism in numerous fungi. Analysis of starch degradation by fungal enzymes secreted into broths of starch- or corn kernel-based media showed a notable accumulation of glucose in samples of the A. flavus control strain while the deletion veA sample accumulated high levels of maltose and maltotriose and only a small amount of glucose. Furthermore, SDS-PAGE and proteomics analysis of culture broths from starch- or corn kernel-based media demonstrated differential production of a number of proteins that included a reduction in the amount of a glucoamylase protein in the veA mutant compared to the control strain, while an alpha-amylase was produced in greater quantities in the veA mutant. Quantitative real-time PCR and western blot analyses using anti-glucoamylase or alpha-amylase antisera supported the proteomics results. Additionally, an overall reduction in protease activity was observed in the veA mutant including production of the alkaline protease, oryzin, compared to the control strain. These findings contribute to our knowledge of mechanisms controlling production of hydrolases and other extracellular proteins during growth of A. flavus on natural starch-based substrates.

Pathways of Unconventional Protein Secretion

NARCIS (Netherlands)

Rabouille, Catherine

2017-01-01

Secretory proteins are conventionally transported through the endoplasmic reticulum to the Golgi and then to the plasma membrane where they are released into the extracellular space. However, numerous substrates also reach these destinations using unconventional pathways. Unconventional protein

Pathways of Unconventional Protein Secretion

NARCIS (Netherlands)

Rabouille, Catherine

2016-01-01

Secretory proteins are conventionally transported through the endoplasmic reticulum to the Golgi and then to the plasma membrane where they are released into the extracellular space. However, numerous substrates also reach these destinations using unconventional pathways. Unconventional protein

Can microbes compete with cows for sustainable protein production - A feasibility study on high quality protein.

Science.gov (United States)

Vestergaard, Mike; Chan, Siu Hung Joshua; Jensen, Peter Ruhdal

2016-11-08

An increasing population and their increased demand for high-protein diets will require dramatic changes in the food industry, as limited resources and environmental issues will make animal derived foods and proteins, gradually more unsustainable to produce. To explore alternatives to animal derived proteins, an economic model was built around the genome-scale metabolic network of E. coli to study the feasibility of recombinant protein production as a food source. Using a novel model, we predicted which microbial production strategies are optimal for economic return, by capturing the tradeoff between the market prices of substrates, product output and the efficiency of microbial production. A case study with the food protein, Bovine Alpha Lactalbumin was made to evaluate the upstream economic feasibilities. Simulations with different substrate profiles at maximum productivity were used to explore the feasibility of recombinant Bovine Alpha Lactalbumin production coupled with market prices of utilized materials. We found that recombinant protein production could be a feasible food source and an alternative to traditional sources.

Can microbes compete with cows for sustainable protein production - A feasibility study on high quality protein

Science.gov (United States)

Vestergaard, Mike; Chan, Siu Hung Joshua; Jensen, Peter Ruhdal

2016-11-01

An increasing population and their increased demand for high-protein diets will require dramatic changes in the food industry, as limited resources and environmental issues will make animal derived foods and proteins, gradually more unsustainable to produce. To explore alternatives to animal derived proteins, an economic model was built around the genome-scale metabolic network of E. coli to study the feasibility of recombinant protein production as a food source. Using a novel model, we predicted which microbial production strategies are optimal for economic return, by capturing the tradeoff between the market prices of substrates, product output and the efficiency of microbial production. A case study with the food protein, Bovine Alpha Lactalbumin was made to evaluate the upstream economic feasibilities. Simulations with different substrate profiles at maximum productivity were used to explore the feasibility of recombinant Bovine Alpha Lactalbumin production coupled with market prices of utilized materials. We found that recombinant protein production could be a feasible food source and an alternative to traditional sources.

Profiling Y561-dependent and -independent substrates of CSF-1R in epithelial cells.

Directory of Open Access Journals (Sweden)

Melodie L Knowlton

2010-10-01

Full Text Available Receptor tyrosine kinases (RTKs activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs, which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK.

Comprehensive structural and substrate specificity classification of the Saccharomyces cerevisiae methyltransferome.

Science.gov (United States)

Wlodarski, Tomasz; Kutner, Jan; Towpik, Joanna; Knizewski, Lukasz; Rychlewski, Leszek; Kudlicki, Andrzej; Rowicka, Maga; Dziembowski, Andrzej; Ginalski, Krzysztof

2011-01-01

Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.

Comprehensive structural and substrate specificity classification of the Saccharomyces cerevisiae methyltransferome.

Directory of Open Access Journals (Sweden)

Tomasz Wlodarski

Full Text Available Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity. Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.

Power electronics substrate for direct substrate cooling

Science.gov (United States)

Le, Khiet [Mission Viejo, CA; Ward, Terence G [Redondo Beach, CA; Mann, Brooks S [Redondo Beach, CA; Yankoski, Edward P [Corona, CA; Smith, Gregory S [Woodland Hills, CA

2012-05-01

Systems and apparatus are provided for power electronics substrates adapted for direct substrate cooling. A power electronics substrate comprises a first surface configured to have electrical circuitry disposed thereon, a second surface, and a plurality of physical features on the second surface. The physical features are configured to promote a turbulent boundary layer in a coolant impinged upon the second surface.

Protein quality of insects as potential ingredients for dog and cat foods.

Science.gov (United States)

Bosch, Guido; Zhang, Sheng; Oonincx, Dennis G A B; Hendriks, Wouter H

2014-01-01

Insects have been proposed as a high-quality, efficient and sustainable dietary protein source. The present study evaluated the protein quality of a selection of insect species. Insect substrates were housefly pupae, adult house cricket, yellow mealworm larvae, lesser mealworm larvae, Morio worm larvae, black soldier fly larvae and pupae, six spot roach, death's head cockroach and Argentinean cockroach. Reference substrates were poultry meat meal, fish meal and soyabean meal. Substrates were analysed for DM, N, crude fat, ash and amino acid (AA) contents and for in vitro digestibility of organic matter (OM) and N. The nutrient composition, AA scores as well as in vitro OM and N digestibility varied considerably between insect substrates. For the AA score, the first limiting AA for most substrates was the combined requirement for Met and Cys. The pupae of the housefly and black soldier fly were high in protein and had high AA scores but were less digestible than other insect substrates. The protein content and AA score of house crickets were high and similar to that of fish meal; however, in vitro N digestibility was higher. The cockroaches were relatively high in protein but the indispensable AA contents, AA scores and the in vitro digestibility values were relatively low. In addition to the indices of protein quality, other aspects such as efficiency of conversion of organic side streams, feasibility of mass-production, product safety and pet owner perception are important for future dog and cat food application of insects as alternative protein source.

Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate.

Science.gov (United States)

Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

2013-08-01

Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed.