Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability.

PubMed

Hu, Xiao-Pan; Yang, Yi; Ma, Bin-Guang

2015-06-09

Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

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

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which oftenmore » takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.« less

NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.

PubMed

D'Onofrio, Mariapina; Ragona, Laura; Fessas, Dimitrios; Signorelli, Marco; Ugolini, Raffaella; Pedò, Massimo; Assfalg, Michael; Molinari, Henriette

2009-01-01

The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.

Protein vivisection reveals elusive intermediates in folding

PubMed Central

Zheng, Zhongzhou; Sosnick, Tobin R.

2010-01-01

Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu→Glu−) to destabilize and unfold a specific region of the protein. We apply this strategy to Ubiquitin, reversibly trapping a folding intermediate in which the β5 strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high energy states. PMID:20144618

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses

DOE PAGES

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; ...

2015-09-04

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. Furthemore, these analyses, which were confirmed usingmore » bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. Our analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases.« less

Fatty acid transfer between multilamellar liposomes and fatty acid-binding proteins.

PubMed

Brecher, P; Saouaf, R; Sugarman, J M; Eisenberg, D; LaRosa, K

1984-11-10

A simple experimental system was developed for studying the movement of long-chain fatty acids between multilamellar liposomes and soluble proteins capable of binding fatty acids. Oleic acid was incorporated into multilamellar liposomes containing cholesterol and egg yolk lecithin and incubated with albumin or hepatic fatty acid-binding protein. It was found that the fatty acid transferred from the liposomes to either protein rapidly and selectively under conditions where phospholipid and cholesterol transfer did not occur. More than 50% of the fatty acid contained within liposomes could become protein bound, suggesting that the fatty acid moved readily between and across phospholipid bilayers. Transfer was reduced at low pH, and this reduction appeared to result from decreased dissociation of the protonated fatty acid from the bilayer. Liposomes made with dimyristoyl or dipalmitoyl lecithin and containing 1 mol per cent palmitic acid were used to show the effect of temperature on fatty acid transfer. Transfer to either protein did not occur at temperatures where the liposomes were in a gel state but occurred rapidly at temperatures at or above the transition temperatures of the phospholipid used.

Protein and amino acid nutrition

USDA-ARS?s Scientific Manuscript database

Dairy cow protein and amino acid nutrition have a significant role in sustainable dairying. Protein, amino acids, and nitrogen are inextricably linked through effects in the rumen, metabolism of the cow, and environmental nutrient management. Feeding systems have been making progress toward emphasiz...

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

PubMed Central

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

2015-01-01

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

PubMed

Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

2014-05-20

Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.

Urinary Loss of Tricarboxylic Acid Cycle Intermediates As Revealed by Metabolomics Studies: An Underlying Mechanism to Reduce Lipid Accretion by Whey Protein Ingestion?

PubMed Central

2015-01-01

Whey protein intake is associated with the modulation of energy metabolism and altered body composition both in human subjects and in animals, but the underlying mechanisms are not yet elucidated. We fed obesity-prone C57BL/6J mice high-fat diets with either casein (HF casein) or whey (HF whey) for 6 weeks. At equal energy intake and apparent fat and nitrogen digestibility, mice fed HF whey stored less energy as lipids, evident both as lower white adipose tissue mass and as reduced liver lipids, compared with HF-casein-fed mice. Explorative analyses of 48 h urine, both by 1H NMR and LC–MS metabolomic platforms, demonstrated higher urinary excretion of tricarboxylic acid (TCA) cycle intermediates citric acid and succinic acid (identified by both platforms), and cis-aconitic acid and isocitric acid (identified by LC–MS platform) in the HF whey, relative to in the HF-casein-fed mice. Targeted LC–MS analyses revealed higher citric acid and cis-aconitic acid concentrations in fed state plasma, but not in liver of HF-whey-fed mice. We propose that enhanced urinary loss of TCA cycle metabolites drain available substrates for anabolic processes, such as lipogenesis, thereby leading to reduced lipid accretion in HF-whey-fed compared to HF-casein-fed mice. PMID:24702026

Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

PubMed

Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

2016-06-03

Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria.

Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

PubMed Central

Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

2012-01-01

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an “open-cap” conformation or a “swivel-back” mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics. PMID:22713574

The interaction of albumin and fatty-acid-binding protein with membranes: oleic acid dissociation.

PubMed

Catalá, A

1984-10-01

Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

PubMed Central

Grange, T; de Sa, C M; Oddos, J; Pictet, R

1987-01-01

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis. Images PMID:2885805

Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein

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

Arpino, James A. J.; Rizkallah, Pierre J., E-mail: rizkallahp@cardiff.ac.uk; Jones, D. Dafydd, E-mail: rizkallahp@cardiff.ac.uk

2014-08-01

The beneficial engineered single-amino-acid deletion variants EGFP{sup D190Δ} and EGFP{sup A227Δ} have been studied. Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFP{sup D190Δ} containing amore » deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP{sup A227Δ} revealed that a ‘flipping’ mechanism was used to adjust for residue deletion at the end of a β-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.« less

Protein Design Using Unnatural Amino Acids

NASA Astrophysics Data System (ADS)

Bilgiçer, Basar; Kumar, Krishna

2003-11-01

With the increasing availability of whole organism genome sequences, understanding protein structure and function is of capital importance. Recent developments in the methodology of incorporation of unnatural amino acids into proteins allow the exploration of proteins at a very detailed level. Furthermore, de novo design of novel protein structures and function is feasible with unprecedented sophistication. Using examples from the literature, this article describes the available methods for unnatural amino acid incorporation and highlights some recent applications including the design of hyperstable protein folds.

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB.

PubMed

Malki, Abderrahim; Le, Hai-Tuong; Milles, Sigrid; Kern, Renée; Caldas, Teresa; Abdallah, Jad; Richarme, Gilbert

2008-05-16

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

Molecular characterization of amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87.

PubMed

Esmaelizad, Majid; Jelokhani-Niaraki, Saber; Hashemnejad, Khadije; Kamalzadeh, Morteza; Lotfi, Mohsen

2011-12-01

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D(pol)) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D(pol) coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp(26)→Glu substitution in a beta sheet located within a small groove of the 3D(pol) protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.

Oleic acid transfer from microsomes to egg lecithin liposomes: participation of fatty acid binding protein.

PubMed

Catalá, A; Avanzati, B

1983-11-01

Oleic acid transfer from microsomes or mitochondria to egg lecithin liposomes was stimulated by fatty acid binding protein. By gel filtration, it could be demonstrated that this protein incorporates oleic acid into liposomes. Fatty acid binding protein transfer activity was higher using microsomes rather than mitochondria, which suggests a selective interaction with different kinds of membranes. Transfer of oleic acid by this soluble protein is greater than that of stearic acid. The results indicate that fatty acid binding protein may participate in the intracellular transport of fatty acids.

Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle

PubMed Central

Zhang, Youjun; Beard, Katherine F. M.; Swart, Corné; Bergmann, Susan; Krahnert, Ina; Nikoloski, Zoran; Graf, Alexander; Ratcliffe, R. George; Sweetlove, Lee J.; Fernie, Alisdair R.; Obata, Toshihiro

2017-01-01

Protein complexes of sequential metabolic enzymes, often termed metabolons, may permit direct channelling of metabolites between the enzymes, providing increased control over metabolic pathway fluxes. Experimental evidence supporting their existence in vivo remains fragmentary. In the present study, we test binary interactions of the proteins constituting the plant tricarboxylic acid (TCA) cycle. We integrate (semi-)quantitative results from affinity purification-mass spectrometry, split-luciferase and yeast-two-hybrid assays to generate a single reliability score for assessing protein–protein interactions. By this approach, we identify 158 interactions including those between catalytic subunits of sequential enzymes and between subunits of enzymes mediating non-adjacent reactions. We reveal channelling of citrate and fumarate in isolated potato mitochondria by isotope dilution experiments. These results provide evidence for a functional TCA cycle metabolon in plants, which we discuss in the context of contemporary understanding of this pathway in other kingdoms. PMID:28508886

Support Vector Machine-based classification of protein folds using the structural properties of amino acid residues and amino acid residue pairs.

PubMed

Shamim, Mohammad Tabrez Anwar; Anwaruddin, Mohammad; Nagarajaram, H A

2007-12-15

Fold recognition is a key step in the protein structure discovery process, especially when traditional sequence comparison methods fail to yield convincing structural homologies. Although many methods have been developed for protein fold recognition, their accuracies remain low. This can be attributed to insufficient exploitation of fold discriminatory features. We have developed a new method for protein fold recognition using structural information of amino acid residues and amino acid residue pairs. Since protein fold recognition can be treated as a protein fold classification problem, we have developed a Support Vector Machine (SVM) based classifier approach that uses secondary structural state and solvent accessibility state frequencies of amino acids and amino acid pairs as feature vectors. Among the individual properties examined secondary structural state frequencies of amino acids gave an overall accuracy of 65.2% for fold discrimination, which is better than the accuracy by any method reported so far in the literature. Combination of secondary structural state frequencies with solvent accessibility state frequencies of amino acids and amino acid pairs further improved the fold discrimination accuracy to more than 70%, which is approximately 8% higher than the best available method. In this study we have also tested, for the first time, an all-together multi-class method known as Crammer and Singer method for protein fold classification. Our studies reveal that the three multi-class classification methods, namely one versus all, one versus one and Crammer and Singer method, yield similar predictions. Dataset and stand-alone program are available upon request.

Alterations in the lenticular protein profile in experimental selenite-induced cataractogenesis and prevention by ellagic acid.

PubMed

Sakthivel, Muniyan; Geraldine, Pitchairaj; Thomas, Philip A

2011-08-01

Accumulating evidence suggests that oxidative stress underlies age-related formation of cataract, and that antioxidants retard cataractogenesis. This study aimed to evaluate whether ellagic acid, a natural polyphenol with antioxidant properties, prevents alterations in the lenticular protein profile in an experimental model of selenite cataract. Alterations in lenticular protein were determined by two-dimensional electrophoresis (2DE) and image analysis. Eluted αA-crystallin spots were analyzed by mass spectrometry. Western blot analysis was also performed to confirm the differential expression of certain crystallins and cytoskeletal proteins. In cataractous lenses, 2DE and image analysis revealed approximately 45 and 60 prominent spots in soluble and insoluble protein fractions respectively. Analysis of the pI and molecular weight of protein spots revealed differences in the expression of crystallin proteins in soluble and insoluble fractions. Western blot analysis confirmed changes in the expression of αA- and βB1- crystallins in both soluble and insoluble protein fractions, while mass spectrometry confirmed the degradation of αA-crystallin in selenite cataractous lenses. Western blot analysis also confirmed the occurrence of altered expression of certain cytoskeletal proteins in insoluble fractions. However, the lenticular protein profile in lenses from selenite-challenged, ellagic acid-treated rats was essentially similar to that noted in lenses from normal rats. The present study confirms the importance of structural and cytoskeletal proteins in the maintenance of lenticular transparency; the results also suggest that ellagic acid prevents lenticular protein alterations induced by selenite in an experimental setting.

A dominant conformational role for amino acid diversity in minimalist protein–protein interfaces

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

Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobodymore » bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.« less

Regulation of intestinal protein metabolism by amino acids.

PubMed

Bertrand, Julien; Goichon, Alexis; Déchelotte, Pierre; Coëffier, Moïse

2013-09-01

Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50% per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.

Hypochlorous and peracetic acid induced oxidation of dairy proteins.

PubMed

Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

2011-02-09

Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

Identification of Bombyx mori bidensovirus VD1-ORF4 reveals a novel protein associated with viral structural component.

PubMed

Li, Guohui; Hu, Zhaoyang; Guo, Xuli; Li, Guangtian; Tang, Qi; Wang, Peng; Chen, Keping; Yao, Qin

2013-06-01

Bombyx mori bidensovirus (BmBDV) VD1-ORF4 (open reading frame 4, ORF4) consists of 3,318 nucleotides, which codes for a predicted 1,105-amino acid protein containing a conserved DNA polymerase motif. However, its functions in viral propagation remain unknown. In the current study, the transcription of VD1-ORF4 was examined from 6 to 96 h postinfection (p.i.) by RT-PCR, 5'-RACE revealed the transcription initiation site of BmBDV ORF4 to be -16 nucleotides upstream from the start codon, and 3'-RACE revealed the transcription termination site of VD1-ORF4 to be +7 nucleotides downstream from termination codon. Three different proteins were examined in the extracts of BmBDV-infected silkworms midguts by Western blot using raised antibodies against VD1-ORF4 deduced amino acid, and a specific protein band about 53 kDa was further detected in purified virions using the same antibodies. Taken together, BmBDV VD1-ORF4 codes for three or more proteins during the viral life cycle, one of which is a 53 kDa protein and confirmed to be a component of BmBDV virion.

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui. Comparison of the ribosomal protein S11 family.

PubMed

Kimura, M; Kimura, J; Hatakeyama, T

1988-11-21

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45-49%) than to the eubacterial counterparts (35%).

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

PubMed Central

Zook, James D.; Molugu, Trivikram R.; Jacobsen, Neil E.; Lin, Guangxin; Soll, Jürgen; Cherry, Brian R.; Brown, Michael F.; Fromme, Petra

2013-01-01

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the 13C, 15N, 2H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, HN, CO, Cα, and Cβ chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein. PMID:24205117

Structures and functions of proteins and nucleic acids in protein biosynthesis

NASA Astrophysics Data System (ADS)

Miyazawa, Tatsuo; Yokoyama, Shigeyuki

Infrared and Raman spectroscopy is useful for studying helical conformations of polypeptides, which are determined by molecular structure parameters. Nuclear magnetic resonance spectroscopy, as well as X-ray analysis, is now established to be important for conformation studies of proteins and nucleic acids in solution. This article is mainly concerned with the conformational aspect and function regulation in protein biosynthesis. The strict recognition of transfer ribonucleic acid (tRNA) by aminoacyl-tRNA synthetase (ARS) is achieved by multi-step mutual adaptation. The conformations of ARS-bound amino acids have been elucidated by transferred nuclear Overhauser effect analysis. Aminoacyl-tRNA takes the 3‧-isomeric form in the polypeptide chain elongation cycle. The regulation of codon recognition by post-transcriptional modification is achieved by conversion of the conformational characteristic of the anticodon of tRNA. The cytidine → lysidine modification of the anticodon of minor isoleucine tRNA concurrently converts the amino acid specificity and the codon specificity. As novel protein engineering, a basic strategy has been established for in vivo biosynthesis of proteins that are substituted with unnatural amino acids (alloproteins).

Identification of novel phosphatidic acid-binding proteins in the rat brain.

PubMed

Park, ChiHu; Kang, Du-Seock; Shin, Geon-Hoon; Seo, Jeongkon; Kim, Hyein; Suh, Pann-Ghill; Bae, Chang-Dae; Shin, Joo-Ho

2015-05-19

Phosphatidic acid (PA) is an abundant negatively-charged phospholipid and has long been considered to be an important signaling molecule in diverse cellular events. Thus, the identification of proteins that specifically interact with PA is of considerable interest to understand the regulatory roles of PA. Herein, lipid-affinity purification and mass spectrometric analysis reveals 43 proteins, 19 known and 24 novel, as PA-binding proteins. A lipid-protein overlay assay confirmed that GDI1, PACSIN1, and DPYSL2 interact with not only with PA but also with other phospholipids. These results might be helpful for deciphering the functional effect of PA in the brain. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Emerging branches of the N-end rule pathways are revealing the sequence complexities of N-termini dependent protein degradation.

PubMed

Eldeeb, Mohamed A; Leitao, Luana C A; Fahlman, Richard P

2018-06-01

The N-end rule links the identity of the N-terminal amino acid of a protein to its in vivo half-life, as some N-terminal residues confer metabolic instability to a protein via their recognition by the cellular machinery that targets them for degradation. Since its discovery, the N-end rule has generally been defined as set of rules of whether an N-terminal residue is stabilizing or not. However, recent studies are revealing that the N-terminal code of amino acids conferring protein instability is more complex than previously appreciated, as recent investigations are revealing that the identity of adjoining downstream residues can also influence the metabolic stability of N-end rule substrate. This is exemplified by the recent discovery of a new branch of N-end rule pathways that target proteins bearing N-terminal proline. In addition, recent investigations are demonstrating that the molecular machinery in N-termini dependent protein degradation may also target proteins for lysosomal degradation, in addition to proteasome-dependent degradation. Herein, we describe some of the recent advances in N-end rule pathways and discuss some of the implications regarding the emerging additional sequence requirements.

Structure of Zebrafish IRBP Reveals Fatty Acid Binding

PubMed Central

Ghosh, Debashis; Haswell, Karen M.; Sprada, Molly; Gonzalez-Fernandez, Federico

2015-01-01

Interphotoreceptor retinoid-binding protein (IRBP) has a remarkable role in targeting and protecting all-trans and 11-cis retinol, and 11-cis retinal during the rod and cone visual cycles. Little is known about how the correct retinoid is efficiently delivered and removed from the correct cell at the required time. It has been proposed that different fatty composition at that the outer-segments and retinal-pigmented epithelium could have an important role is regulating the delivery and uptake of the visual cycle retinoids at the cell-interphotoreceptor-matrix interface. Although this suggests intriguing mechanisms for the role of local fatty acids in visual-cycle retinoid trafficking, nothing is known about the structural basis of IRBP-fatty acid interactions. Such regulation may be mediated through IRBP’s unusual repeating homologous modules, each containing about 300 amino acids. We have been investigating structure-function relationships of Zebrafish IRBP (zIRBP), which has only two tandem modules (z1 and z2), as a model for the more complex four-module mammalian IRBP’s. Here we report the first X-ray crystal structure of a teleost IRBP, and the only structure with a bound ligand. The X-ray structure of z1, determined at 1.90Å resolution, reveals a two-domain organization of the module (domains A and B). A deep hydrophobic pocket was identified within the N-terminal domain A. In fluorescence titrations assays, oleic acid displaced all-trans retinol from zIRBP. Our study, which provides the first structure of an IRBP with bound ligand, supports a potential role for fatty acids in regulating retinoid binding. PMID:26344741

Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

PubMed Central

Niskanen, Einari A; Hytönen, Vesa P; Grapputo, Alessandro; Nordlund, Henri R; Kulomaa, Markku S; Laitinen, Olli H

2005-01-01

Background A chicken egg contains several biotin-binding proteins (BBPs), whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins. PMID:15777476

Interactions between Therapeutic Proteins and Acrylic Acid Leachable.

PubMed

Liu, Dengfeng; Nashed-Samuel, Yasser; Bondarenko, Pavel V; Brems, David N; Ren, Da

2012-01-01

Leachables are chemical compounds that migrate from manufacturing equipment, primary containers and closure systems, and packaging components into biopharmaceutical and pharmaceutical products. Acrylic acid (at concentration around 5 μg/mL) was detected as leachable in syringes from one of the potential vendors (X syringes). In order to evaluate the potential impact of acrylic acid on therapeutic proteins, an IgG 2 molecule was filled into a sterilized X syringe and then incubated at 45 °C for 45 days in a pH 5 acetate buffer. We discovered that acrylic acid can interact with proteins at three different sites: (1) the lysine side chain, (2) the N-terminus, and (3) the histidine side chain, by the Michael reaction. In this report, the direct interactions between acrylic acid leachable and a biopharmaceutical product were demonstrated and the reaction mechanism was proposed. Even thought a small amount (from 0.02% to 0.3%) of protein was found to be modified by acrylic acid, the modified protein can potentially be harmful due to the toxicity of acrylic acid. After being modified by acrylic acid, the properties of the therapeutic protein may change due to charge and hydrophobicity variations. Acrylic acid was detected to migrate from syringes (Vendor X) into a therapeutic protein solution (at a concentration around 5 μg/mL). In this study, we discovered that acrylic acid can modify proteins at three different sites: (1) the lysine side chain, 2) the N-terminus, and 3) the histidine side chain, by the Michael reaction. In this report, the direct interactions between acrylic acid leachable and a biopharmaceutical product were demonstrated and the reaction mechanism was proposed.

FAX1, a Novel Membrane Protein Mediating Plastid Fatty Acid Export

PubMed Central

Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin

2015-01-01

Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734

Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis

PubMed Central

Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

2011-01-01

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

Effect of a keto acid-amino acid supplement on the metabolism and renal elimination of branched-chain amino acids in patients with chronic renal insufficiency on a low protein diet.

PubMed

Teplan, V; Schück, O; Horácková, M; Skibová, J; Holecek, M

2000-10-27

The aim of our study was to evaluate the effect of a low-protein diet supplemented with keto acids-amino acids on renal function and urinary excretion of branched-chain amino acids (BCAA) in patients with chronic renal insufficiency (CRI). In a prospective investigation 28 patients with CRI (16 male, 12 female, aged 28-66 yrs, CCr 18.6 +/- 10.2 ml/min) on a low-protein diet (0.6 g of protein /kg BW/day and energy intake 140 kJ/kg BW/day) for a period of one month were included. Subsequently, this low protein diet was supplemented with keto acids-amino acids at a dose of 0.1 g/kg BW/day orally for a period of 3 months. Examinations performed at baseline and at the end of the follow-up period revealed significant increase in the serum levels of BCAA leucine (p < 0.02), isoleucine (p < 0.03), and valine (p < 0.02) while their renal fractional excretion declined (p < 0.02, p < 0.01 resp.). Keto acid-amino acid administration had no effect on renal function and on the clearance of inulin, para-aminohippuric acid. Endogenous creatinine and urea clearance remained unaltered. A significant correlation between fractional excretion of sodium and leucine (p < 0.05) and a hyperbolic relationship between inulin clearance and fractional excretion of BCAA (p < 0.01) were seen. Moreover, a significant decrease in proteinuria (p < 0.02), plasma urea concentration and renal urea excretion and a rise in albumin level (p < 0.03) were noted. We conclude that in patients with CRI on a low protein diet the supplementation of keto acids-amino acids does not affect renal hemodynamics, but is associated--despite increases in plasma concentrations--with a reduction of renal amino acid and protein excretion suggesting induction of alterations in the tubular transport mechanisms.

Analysis of Protein–Protein Interactions in MCF-7 and MDA-MB-231 Cell Lines Using Phthalic Acid Chemical Probes

PubMed Central

Liang, Shih-Shin; Wang, Tsu-Nai; Tsai, Eing-Mei

2014-01-01

Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein–protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively. PMID:25402641

Statistical potential-based amino acid similarity matrices for aligning distantly related protein sequences.

PubMed

Tan, Yen Hock; Huang, He; Kihara, Daisuke

2006-08-15

Aligning distantly related protein sequences is a long-standing problem in bioinformatics, and a key for successful protein structure prediction. Its importance is increasing recently in the context of structural genomics projects because more and more experimentally solved structures are available as templates for protein structure modeling. Toward this end, recent structure prediction methods employ profile-profile alignments, and various ways of aligning two profiles have been developed. More fundamentally, a better amino acid similarity matrix can improve a profile itself; thereby resulting in more accurate profile-profile alignments. Here we have developed novel amino acid similarity matrices from knowledge-based amino acid contact potentials. Contact potentials are used because the contact propensity to the other amino acids would be one of the most conserved features of each position of a protein structure. The derived amino acid similarity matrices are tested on benchmark alignments at three different levels, namely, the family, the superfamily, and the fold level. Compared to BLOSUM45 and the other existing matrices, the contact potential-based matrices perform comparably in the family level alignments, but clearly outperform in the fold level alignments. The contact potential-based matrices perform even better when suboptimal alignments are considered. Comparing the matrices themselves with each other revealed that the contact potential-based matrices are very different from BLOSUM45 and the other matrices, indicating that they are located in a different basin in the amino acid similarity matrix space.

Cartilage acidic protein 1, a new member of the beta-propeller protein family with amyloid propensity.

PubMed

Anjos, Liliana; Morgado, Isabel; Guerreiro, Marta; Cardoso, João C R; Melo, Eduardo P; Power, Deborah M

2017-02-01

Cartilage acidic protein1 (CRTAC1) is an extracellular matrix protein of chondrogenic tissue in humans and its presence in bacteria indicate it is of ancient origin. Structural modeling of piscine CRTAC1 reveals it belongs to the large family of beta-propeller proteins that in mammals have been associated with diseases, including amyloid diseases such as Alzheimer's. In order to characterize the structure/function evolution of this new member of the beta-propeller family we exploited the unique characteristics of piscine duplicate genes Crtac1a and Crtac1b and compared their structural and biochemical modifications with human recombinant CRTAC1. We demonstrate that CRTAC1 has a beta-propeller structure that has been conserved during evolution and easily forms high molecular weight thermo-stable aggregates. We reveal for the first time the propensity of CRTAC1 to form amyloid-like structures, and hypothesize that the aggregating property of CRTAC1 may be related to its disease-association. We further contribute to the general understating of CRTAC1's and beta-propeller family evolution and function. Proteins 2017; 85:242-255. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

PubMed

Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

2016-01-29

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

NPIDB: Nucleic acid-Protein Interaction DataBase.

PubMed

Kirsanov, Dmitry D; Zanegina, Olga N; Aksianov, Evgeniy A; Spirin, Sergei A; Karyagina, Anna S; Alexeevski, Andrei V

2013-01-01

The Nucleic acid-Protein Interaction DataBase (http://npidb.belozersky.msu.ru/) contains information derived from structures of DNA-protein and RNA-protein complexes extracted from the Protein Data Bank (3846 complexes in October 2012). It provides a web interface and a set of tools for extracting biologically meaningful characteristics of nucleoprotein complexes. The content of the database is updated weekly. The current version of the Nucleic acid-Protein Interaction DataBase is an upgrade of the version published in 2007. The improvements include a new web interface, new tools for calculation of intermolecular interactions, a classification of SCOP families that contains DNA-binding protein domains and data on conserved water molecules on the DNA-protein interface.

Glutamic Acid as a Precursor to N-Terminal Pyroglutamic Acid in Mouse Plasmacytoma Protein

PubMed Central

Twardzik, Daniel R.; Peterkofsky, Alan

1972-01-01

Cell suspensions derived from a mouse plasmacytoma (RPC-20) that secretes an immunoglobulin light chain containing N-terminal pyroglutamic acid can synthesize protein in vitro. Chromatographic examination of an enzymatic digest of protein labeled with glutamic acid shows only labeled glutamic acid and pyroglutamic acid; hydrolysis of protein from cells labeled with glutamine, however, yields substantial amounts of glutamic acid in addition to glutamine and pyroglutamic acid. The absence of glutamine synthetase and presence of glutaminase in plasmacytoma homogenates is consistent with these findings. These data indicate that N-terminal pyroglutamic acid can be derived from glutamic acid without prior conversion of glutamic acid to glutamine. Since free or bound forms of glutamine cyclize nonezymatically to pyroglutamate with ease, while glutamic acid does not, the data suggest that N-terminal pyroglutamic acid formation from glutamic acid is enzymatic rather than spontaneous. Images PMID:4400295

Automated protein hydrolysis delivering sample to a solid acid catalyst for amino acid analysis.

PubMed

Masuda, Akiko; Dohmae, Naoshi

2010-11-01

In this study, we developed an automatic protein hydrolysis system using strong cation-exchange resins as solid acid catalysts. Examining several kinds of inorganic solid acids and cation-exchange resins, we found that a few cation-exchange resins worked as acid catalysts for protein hydrolysis when heated in the presence of water. The most efficient resin yielded amounts of amino acids that were over 70% of those recovered after conventional hydrolysis with hydrochloric acid and resulted in amino acid compositions matching the theoretical values. The solid-acid hydrolysis was automated by packing the resin into columns, combining the columns with a high-performance liquid chromatography system, and heating them. The amino acids that constitute a protein can thereby be determined, minimizing contamination from the environment.

NMR studies of protein-nucleic acid interactions.

PubMed

Varani, Gabriele; Chen, Yu; Leeper, Thomas C

2004-01-01

Protein-DNA and protein-RNA complexes play key functional roles in every living organism. Therefore, the elucidation of their structure and dynamics is an important goal of structural and molecular biology. Nuclear magnetic resonance (NMR) studies of protein and nucleic acid complexes have common features with studies of protein-protein complexes: the interaction surfaces between the molecules must be carefully delineated, the relative orientation of the two species needs to be accurately and precisely determined, and close intermolecular contacts defined by nuclear Overhauser effects (NOEs) must be obtained. However, differences in NMR properties (e.g., chemical shifts) and biosynthetic pathways for sample productions generate important differences. Chemical shift differences between the protein and nucleic acid resonances can aid the NMR structure determination process; however, the relatively limited dispersion of the RNA ribose resonances makes the process of assigning intermolecular NOEs more difficult. The analysis of the resulting structures requires computational tools unique to nucleic acid interactions. This chapter summarizes the most important elements of the structure determination by NMR of protein-nucleic acid complexes and their analysis. The main emphasis is on recent developments (e.g., residual dipolar couplings and new Web-based analysis tools) that have facilitated NMR studies of these complexes and expanded the type of biological problems to which NMR techniques of structural elucidation can now be applied.

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

PubMed

Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

2014-08-03

Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

Vanillic acid from Actinidia deliciosa impedes virulence in Serratia marcescens by affecting S-layer, flagellin and fatty acid biosynthesis proteins.

PubMed

Sethupathy, Sivasamy; Ananthi, Sivagnanam; Selvaraj, Anthonymuthu; Shanmuganathan, Balakrishnan; Vigneshwari, Loganathan; Balamurugan, Krishnaswamy; Mahalingam, Sundarasamy; Pandian, Shunmugiah Karutha

2017-11-27

Serratia marcescens is one of the important nosocomial pathogens which rely on quorum sensing (QS) to regulate the production of biofilm and several virulence factors. Hence, blocking of QS has become a promising approach to quench the virulence of S. marcescens. For the first time, QS inhibitory (QSI) and antibiofilm potential of Actinidia deliciosa have been explored against S. marcescens clinical isolate (CI). A. deliciosa pulp extract significantly inhibited the virulence and biofilm production without any deleterious effect on the growth. Vanillic acid was identified as an active lead responsible for the QSI activity. Addition of vanillic acid to the growth medium significantly affected the QS regulated production of biofilm and virulence factors in a concentration dependent mode in S. marcescens CI, ATCC 14756 and MG1. Furthermore vanillic acid increased the survival of Caenorhabditis elegans upon S. marcescens infection. Proteomic analysis and mass spectrometric identification of differentially expressed proteins revealed the ability of vanillic acid to modulate the expression of proteins involved in S-layers, histidine, flagellin and fatty acid production. QSI potential of the vanillic acid observed in the current study paves the way for exploring it as a potential therapeutic candidate to treat S. marcescens infections.

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

PubMed

Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

2006-05-19

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions

PubMed Central

Shashikanth, Nitesh; Kisting, Meridith A.; Leckband, Deborah E.

2016-01-01

The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions. PMID:27009566

Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue.

PubMed Central

Wilkinson, T C; Wilton, D C

1986-01-01

Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method. Images Fig. 1. PMID:3800946

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

NASA Technical Reports Server (NTRS)

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

1992-01-01

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

Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

PubMed Central

Roy, Sushmita; Martinez, Diego; Platero, Harriett; Lane, Terran; Werner-Washburne, Margaret

2009-01-01

Background Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information. Results AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins. Conclusion AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains. PMID:19936254

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides

PubMed Central

Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

2011-01-01

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrPC) into the aggregated misfolded scrapie isoform, named PrPSc. Recent studies on the physiological role of PrPC revealed that this protein has probably multiple functions, notably in cell–cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5′-GACACAAGCCGA-3′ was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities. PMID:21737432

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides.

PubMed

Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

2011-10-01

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.

Co-localisation of advanced glycation end products and D-β-aspartic acid-containing proteins in gelatinous drop-like corneal dystrophy.

PubMed

Kaji, Yuichi; Oshika, Tetsuro; Takazawa, Yutaka; Fukayama, Masashi; Fujii, Noriko

2012-08-01

Gelatinous drop-like corneal dystrophy (GDLD), also known as familial subepithelial corneal amyloidosis, is an autosomal recessive disorder that causes progressive corneal opacity due to accumulation of amyloid fibrils in the corneal stroma. Genetic analyses have revealed that a mutation in membrane component chromosome 1 surface marker 1 gene is responsible for GDLD. However, the mechanism of amyloid formation in the corneal stroma remains unclear. The present study attempted to reveal the role of advanced glycation end products (AGE) and d-amino acids in amyloid formation in GDLD. Informed consent was obtained from five patients with GDLD, three patients with bullous keratopathy and three patients with interstitial keratitis and all the specimens were analysed. Localisation of amyloid fibrils was analysed using Congo-red and thioflavin T staining. In addition, the localisation of AGE (N(ε)-carboxy(methyl)-L-lysine, pyrraline and pentosidine) and D-β-aspartic acid-containing proteins, a major form of d-amino acid-containing proteins, was analysed immunohistochemically. In all GDLD specimens, strong immunoreactivity to AGE and D-β-aspartic acid-containing proteins was detected in the subepithelial amyloid-rich region. In contrast, amyloid fibrils, AGE, or D-amino acid-containing proteins were slightly detected in the corneal stroma of patients with bullous keratopathy and interstitial keratitis. Abnormally accumulated proteins rich in AGE and D-β-aspartic acid co-localise in the amyloid lesions in GDLD. These results indicate that non-enzymatic post-translational modifications of proteins, including AGE formation and isomerisation of aspartyl residues, will be the cause as well as the result of amyloid fibril formations in GDLD.

Proteomic profiling of maize opaque endosperm mutants reveals selective accumulation of lysine-enriched proteins

PubMed Central

Morton, Kyla J.; Jia, Shangang; Zhang, Chi; Holding, David R.

2016-01-01

Reduced prolamin (zein) accumulation and defective endoplasmic reticulum (ER) body formation occurs in maize opaque endosperm mutants opaque2 (o2), floury2 (fl2), defective endosperm*B30 (DeB30), and Mucronate (Mc), whereas other opaque mutants such as opaque1 (o1) and floury1 (fl1) are normal in these regards. This suggests that other factors contribute to kernel texture. A liquid chromatography approach coupled with tandem mass spectrometry (LC-MS/MS) proteomics was used to compare non-zein proteins of nearly isogenic opaque endosperm mutants. In total, 2762 proteins were identified that were enriched for biological processes such as protein transport and folding, amino acid biosynthesis, and proteolysis. Principal component analysis and pathway enrichment suggested that the mutants partitioned into three groups: (i) Mc, DeB30, fl2 and o2; (ii) o1; and (iii) fl1. Indicator species analysis revealed mutant-specific proteins, and highlighted ER secretory pathway components that were enriched in selected groups of mutants. The most significantly changed proteins were related to stress or defense and zein partitioning into the soluble fraction for Mc, DeB30, o1, and fl1 specifically. In silico dissection of the most significantly changed proteins revealed novel qualitative changes in lysine abundance contributing to the overall lysine increase and the nutritional rebalancing of the o2 and fl2 endosperm. PMID:26712829

Proteomic Investigation of Aphid Honeydew Reveals an Unexpected Diversity of Proteins

PubMed Central

Haubruge, Eric; Hance, Thierry; Thonart, Philippe; De Pauw, Edwin; Francis, Frédéric

2013-01-01

Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige) approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora). Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu), and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective. PMID:24086359

Conditional knock-out of lipoic acid protein ligase 1 reveals redundancy pathway for lipoic acid metabolism in Plasmodium berghei malaria parasite.

PubMed

Wang, Min; Wang, Qiong; Gao, Xiang; Su, Zhong

2017-06-27

Lipoic acid is a cofactor for α-keto acid dehydrogenase system that is involved in the central energy metabolism. In the apicomplexan parasite, Plasmodium, lipoic acid protein ligase 1 (LplA1) and LplA2 catalyse the ligation of acquired lipoic acid to the dehydrogenase complexes in the mitochondrion. The enzymes LipB and LipA mediate lipoic acid synthesis and ligation to the enzymes in the apicoplast. These enzymes in the lipoic acid metabolism machinery have been shown to play important roles in the biology of Plasmodium parasites, but the relationship between the enzymes is not fully elucidated. We used an anhydrotetracycline (ATc)-inducible transcription system to generate transgenic P. berghei parasites in which the lplA1 gene was conditionally knocked out (LplA1-cKO). Phenotypic changes and the lplA1 and lplA2 gene expression profiles of cloned LplA1-cKO parasites were analysed. LplA1-cKO parasites showed severely impaired growth in vivo in the first 8 days of infection, and retarded blood-stage development in vitro, in the absence of ATc. However, these parasites resumed viability in the late stage of infection and mounted high levels of parasitemia leading to the death of the hosts. Although lplA1 mRNA expression was regulated tightly by ATc during the whole course of infection, lplA2 mRNA expression was significantly increased in the late stage of infection only in the LplA1-cKO parasites that were not exposed to ATc. The lplA2 gene can be activated as an alternative pathway to compensate for the loss of LplA1 activity and to maintain lipoic acid metabolism.

Effects of Acids, Bases, and Heteroatoms on Proximal Radial Distribution Functions for Proteins

PubMed Central

Nguyen, Bao Linh; Pettitt, B. Montgomery

2015-01-01

The proximal distribution of water around proteins is a convenient method of quantifying solvation. We consider the effect of charged and sulfur-containing amino acid side-chain atoms on the proximal radial distribution function (pRDF) of water molecules around proteins using side-chain analogs. The pRDF represents the relative probability of finding any solvent molecule at a distance from the closest or surface perpendicular protein atom. We consider the near-neighbor distribution. Previously, pRDFs were shown to be universal descriptors of the water molecules around C, N, and O atom types across hundreds of globular proteins. Using averaged pRDFs, a solvent density around any globular protein can be reconstructed with controllable relative error. Solvent reconstruction using the additional information from charged amino acid side-chain atom types from both small models and protein averages reveals the effects of surface charge distribution on solvent density and improves the reconstruction errors relative to simulation. Solvent density reconstructions from the small-molecule models are as effective and less computationally demanding than reconstructions from full macromolecular models in reproducing preferred hydration sites and solvent density fluctuations. PMID:26388706

Effects of Acids, Bases, and Heteroatoms on Proximal Radial Distribution Functions for Proteins.

PubMed

Nguyen, Bao Linh; Pettitt, B Montgomery

2015-04-14

The proximal distribution of water around proteins is a convenient method of quantifying solvation. We consider the effect of charged and sulfur-containing amino acid side-chain atoms on the proximal radial distribution function (pRDF) of water molecules around proteins using side-chain analogs. The pRDF represents the relative probability of finding any solvent molecule at a distance from the closest or surface perpendicular protein atom. We consider the near-neighbor distribution. Previously, pRDFs were shown to be universal descriptors of the water molecules around C, N, and O atom types across hundreds of globular proteins. Using averaged pRDFs, a solvent density around any globular protein can be reconstructed with controllable relative error. Solvent reconstruction using the additional information from charged amino acid side-chain atom types from both small models and protein averages reveals the effects of surface charge distribution on solvent density and improves the reconstruction errors relative to simulation. Solvent density reconstructions from the small-molecule models are as effective and less computationally demanding than reconstructions from full macromolecular models in reproducing preferred hydration sites and solvent density fluctuations.

The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

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

Peisach, D.; Gee, P.; Kent, K.

2010-03-08

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 {angstrom} crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline bindingmore » site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.« less

Phospholipase D and phosphatidic acid in plant defence response: from protein-protein and lipid-protein interactions to hormone signalling.

PubMed

Zhao, Jian

2015-04-01

Phospholipase Ds (PLDs) and PLD-derived phosphatidic acids (PAs) play vital roles in plant hormonal and environmental responses and various cellular dynamics. Recent studies have further expanded the functions of PLDs and PAs into plant-microbe interaction. The molecular diversities and redundant functions make PLD-PA an important signalling complex regulating lipid metabolism, cytoskeleton dynamics, vesicle trafficking, and hormonal signalling in plant defence through protein-protein and protein-lipid interactions or hormone signalling. Different PLD-PA signalling complexes and their targets have emerged as fast-growing research topics for understanding their numerous but not yet established roles in modifying pathogen perception, signal transduction, and downstream defence responses. Meanwhile, advanced lipidomics tools have allowed researchers to reveal further the mechanisms of PLD-PA signalling complexes in regulating lipid metabolism and signalling, and their impacts on jasmonic acid/oxylipins, salicylic acid, and other hormone signalling pathways that essentially mediate plant defence responses. This review attempts to summarize the progress made in spatial and temporal PLD/PA signalling as well as PLD/PA-mediated modification of plant defence. It presents an in-depth discussion on the functions and potential mechanisms of PLD-PA complexes in regulating actin filament/microtubule cytoskeleton, vesicle trafficking, and hormonal signalling, and in influencing lipid metabolism-derived metabolites as critical signalling components in plant defence responses. The discussion puts PLD-PA in a broader context in order to guide future research. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Amino acid selective unlabeling for sequence specific resonance assignments in proteins

PubMed Central

Krishnarjuna, B.; Jaipuria, Garima; Thakur, Anushikha

2010-01-01

Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective ‘unlabeling’ or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly 13C/15N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {12COi–15Ni+1}-filtered HSQC, which aids in linking the 1HN/15N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i − 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to 2H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of 14N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies. Electronic supplementary material The online version of this article (doi:10.1007/s10858-010-9459-z) contains supplementary material, which is available to authorized users. PMID:21153044

Echinococcus granulosus fatty acid binding proteins subcellular localization.

PubMed

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Changes of synovial fluid protein concentrations in supra-patellar bursitis patients after the injection of different molecular weights of hyaluronic acid.

PubMed

Chen, Carl P C; Hsu, Chih Chin; Pei, Yu-Cheng; Chen, Ruo Li; Zhou, Shaobo; Shen, Hsuan-Chen; Lin, Shih-Cherng; Tsai, Wen Chung

2014-04-01

Knee pain is commonly seen in orthopedic and rehabilitation outpatient clinical settings, and in the aging population. Bursitis of the knee joint, especially when the volume of the synovial fluid is large enough, can compress and distend the nearby soft tissues, causing pain in the knee joint. Out of all the bursae surrounding the knee joint, supra-patellar bursitis is most often associated with knee pain. Treatment strategies in managing supra-patellar bursitis include the aspiration of joint synovial fluid and then followed by steroid injection into the bursa. When supra-patellar bursitis is caused by degenerative disorders, the concept of viscosupplementation treatment may be effective by injecting hyaluronic acid into the bursa. However, the rheology or the changes in the concentrations of proteins (biomarkers) that are related to the development of bursitis in the synovial fluid is virtually unexplored. Therefore, this study aimed to identify the concentration changes in the synovial fluid total protein amount and individual proteins associated with supra-patellar bursitis using the Bradford protein assay and western immunoglobulin methods. A total of 20 patients were divided into two groups with 10 patients in each group. One group received the high molecular weight hyaluronic acid product of Synvisc Hylan G-F 20 and the other group received the low molecular weight hyaluronic acid product of Hya-Joint Synovial Fluid Supplement once per week injection into the bursa for a total of 3 weeks. Significant decreases in the synovial fluid total protein concentrations were observed after the second dosage of high molecular weight hyaluronic acid injections. Apolipoprotein A-I, interleukin 1 beta, alpha 1 antitrypsin, and matrix metalloproteinase 1 proteins revealed a trend of decreasing western immunoblotting band densities after hyaluronic acid injections. The decreases in apolipoprotein A-I and interleukin 1 beta protein band densities were significant in the high

Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

PubMed

Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E; Levesque, Brié; Pedersen, Stine B; Bartels, Lina; Wapenaar, Hannah; Ye, Fei; Zhang, Mingjie; Bowen, Mark E; Strømgaard, Kristian

2017-09-15

The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

Comparative proteomic analyses reveal that the regulators of G-protein signaling proteins regulate amino acid metabolism of the rice blast fungus Magnaporthe oryzae.

PubMed

Zhang, Haifeng; Ma, Hongyu; Xie, Xin; Ji, Jun; Dong, Yanhan; Du, Yan; Tang, Wei; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

2014-11-01

The rice blast fungus Magnaporthe oryzae encodes eight regulators of G-protein (GTP-binding protein) signaling (RGS) proteins MoRgs1-MoRgs8 that orchestrate the growth, asexual/sexual production, appressorium differentiation, and pathogenicity. To address the mechanisms by which MoRgs proteins function, we conducted a 2DE proteome study and identified 82 differentially expressed proteins by comparing five ∆Morgs mutants with wild-type Guy11 strain. We found that the abundances of eight amino acid (AA) biosynthesis or degradation associated proteins were markedly altered in five ∆Morgs mutants, indicating one of the main collective roles for the MoRgs proteins is to influence AA metabolism. We showed that MoRgs proteins have distinct roles in AA metabolism and nutrient responses from growth assays. In addition, we characterized MoLys20 (Lys is lysine), a homocitrate synthase, whose abundance was significantly decreased in the ∆Morgs mutants. The ∆Molys20 mutant is auxotrophic for lys and exogenous lys could partially rescue its auxotrophic defects. Deletion of MoLYS20 resulted in defects in conidiation and infection, as well as pathogenicity on rice. Overall, our results indicate that one of the critical roles for MoRgs proteins is to regulate AA metabolism, and that MoLys20 may be directly or indirectly regulated by MoRgs and participated in lys biosynthesis, thereby affecting fungal development and pathogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fatty acids and small organic compounds bind to mineralo-organic nanoparticles derived from human body fluids as revealed by metabolomic analysis

NASA Astrophysics Data System (ADS)

Martel, Jan; Wu, Cheng-Yeu; Hung, Cheng-Yu; Wong, Tsui-Yin; Cheng, Ann-Joy; Cheng, Mei-Ling; Shiao, Ming-Shi; Young, John D.

2016-03-01

Nanoparticles entering the human body instantly become coated with a ``protein corona'' that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an ``organic corona'' containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body.Nanoparticles entering the human body instantly become coated with a ``protein corona'' that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an ``organic corona'' containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body. Electronic supplementary information (ESI) available. See

Co-localisation of advanced glycation end products and d-β-aspartic acid-containing proteins in gelatinous drop-like corneal dystrophy

PubMed Central

Oshika, Tetsuro; Takazawa, Yutaka; Fukayama, Masashi; Fujii, Noriko

2012-01-01

Purpose Gelatinous drop-like corneal dystrophy (GDLD), also known as familial subepithelial corneal amyloidosis, is an autosomal recessive disorder that causes progressive corneal opacity due to accumulation of amyloid fibrils in the corneal stroma. Genetic analyses have revealed that a mutation in membrane component chromosome 1 surface marker 1 gene is responsible for GDLD. However, the mechanism of amyloid formation in the corneal stroma remains unclear. The present study attempted to reveal the role of advanced glycation end products (AGE) and d-amino acids in amyloid formation in GDLD. Methods Informed consent was obtained from five patients with GDLD, three patients with bullous keratopathy and three patients with interstitial keratitis and all the specimens were analysed. Localisation of amyloid fibrils was analysed using Congo-red and thioflavin T staining. In addition, the localisation of AGE (Nɛ-carboxy(methyl)-l-lysine, pyrraline and pentosidine) and d-β-aspartic acid-containing proteins, a major form of d-amino acid-containing proteins, was analysed immunohistochemically. Results In all GDLD specimens, strong immunoreactivity to AGE and d-β-aspartic acid-containing proteins was detected in the subepithelial amyloid-rich region. In contrast, amyloid fibrils, AGE, or d-amino acid-containing proteins were slightly detected in the corneal stroma of patients with bullous keratopathy and interstitial keratitis. Conclusions Abnormally accumulated proteins rich in AGE and d-β-aspartic acid co-localise in the amyloid lesions in GDLD. These results indicate that non-enzymatic post-translational modifications of proteins, including AGE formation and isomerisation of aspartyl residues, will be the cause as well as the result of amyloid fibril formations in GDLD. PMID:22694960

High Dietary Protein Intake and Protein-Related Acid Load on Bone Health.

PubMed

Cao, Jay J

2017-12-01

Consumption of high-protein diets is increasingly popular due to the benefits of protein on preserving lean mass and controlling appetite and satiety. The paper is to review recent clinical research assessing dietary protein on calcium metabolism and bone health. Epidemiological studies show that long-term, high-protein intake is positively associated with bone mineral density and reduced risk of bone fracture incidence. Short-term interventional studies demonstrate that a high-protein diet does not negatively affect calcium homeostasis. Existing evidence supports that the negative effects of the acid load of protein on urinary calcium excretion are offset by the beneficial skeletal effects of high-protein intake. Future research should focus on the role and the degree of contribution of other dietary and physiological factors, such as intake of fruits and vegetables, in reducing the acid load and further enhancing the anabolic effects of protein on the musculoskeletal system.

The role of beta-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower.

PubMed

González-Mellado, Damián; von Wettstein-Knowles, Penny; Garcés, Rafael; Martínez-Force, Enrique

2010-05-01

The beta-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.

Hydrogen-deuterium exchange mass spectrometry reveals folding and allostery in protein-protein interactions.

PubMed

Ramirez-Sarmiento, Cesar A; Komives, Elizabeth A

2018-04-06

Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra performance liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS. In this review, we focus on studies that have used a combination of approaches to reveal (re)folding of proteins as well as on long-distance allosteric changes upon interaction. Copyright © 2018 Elsevier Inc. All rights reserved.

Fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C.

PubMed

Lin, Zhaoyu; Liu, Fei; Shi, Peiliang; Song, Anying; Huang, Zan; Zou, Dayuan; Chen, Qin; Li, Jianxin; Gao, Xiang

2018-02-26

Changes in metabolic pathway preferences are key events in the reprogramming process of somatic cells to induced pluripotent stem cells (iPSCs). The optimization of metabolic conditions can enhance reprogramming; however, the detailed underlying mechanisms are largely unclear. By comparing the gene expression profiles of somatic cells, intermediate-phase cells, and iPSCs, we found that carnitine palmitoyltransferase (Cpt)1b, a rate-limiting enzyme in fatty acid oxidation, was significantly upregulated in the early stage of the reprogramming process. Mouse embryonic fibroblasts isolated from transgenic mice carrying doxycycline (Dox)-inducible Yamanaka factor constructs were used for reprogramming. Various fatty acid oxidation-related metabolites were added during the reprogramming process. Colony counting and fluorescence-activated cell sorting (FACS) were used to calculate reprogramming efficiency. Fatty acid oxidation-related metabolites were measured by liquid chromatography-mass spectrometry. Seahorse was used to measure the level of oxidative phosphorylation. We found that overexpression of cpt1b enhanced reprogramming efficiency. Furthermore, palmitoylcarnitine or acetyl-CoA, the primary and final products of Cpt1-mediated fatty acid oxidation, also promoted reprogramming. In the early reprogramming process, fatty acid oxidation upregulated oxidative phosphorylation and downregulated protein kinase C activity. Inhibition of protein kinase C also promoted reprogramming. We demonstrated that fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C activity in the early stage of the reprogramming process. This study reveals that fatty acid oxidation is crucial for the reprogramming efficiency.

Proteomic Analysis Reveals Differentially Regulated Protein Acetylation in Human Amyotrophic Lateral Sclerosis Spinal Cord

PubMed Central

Azadzoi, Kazem; Yang, Yun; Fei, Zhou; Dou, Kefeng; Kowall, Neil W.; Choi, Han-Pil; Vieira, Fernando; Yang, Jing-Hua

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord. Histone deacetylase (HDAC) inhibitors have neuroprotective effects potentially useful for the treatment of neurodegenerative diseases including ALS; however, the molecular mechanisms underlying their potential efficacy is not well understood. Here we report that protein acetylation in urea-soluble proteins is differently regulated in post-mortem ALS spinal cord. Two-dimensional electrophoresis (2-DE) analysis reveals several protein clusters with similar molecular weight but different charge status. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identifies glial fibrillary acidic protein (GFAP) as the dominant component in the protein clusters. Further analysis indicates six heavily acetylated lysine residues at positions 89, 153, 189, 218, 259 and 331 of GFAP. Immunoprecipitation followed by Western blotting confirms that the larger form of GFAP fragments are acetylated and upregulated in ALS spinal cord. Further studies demonstrate that acetylation of the proteins additional to GFAP is differently regulated, suggesting that acetylation and/or deacetylation play an important role in pathogenesis of ALS. PMID:24312501

Optical tweezers reveal how proteins alter replication

NASA Astrophysics Data System (ADS)

Chaurasiya, Kathy

Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

PubMed Central

Terwilliger, Austen; Swick, Michelle C.; Pflughoeft, Kathryn J.; Pomerantsev, Andrei; Lyons, C. Rick; Koehler, Theresa M.

2015-01-01

ABSTRACT Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed “nutritional immunity.” Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis, the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCE The mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood—hemoglobin and serum albumin—can satiate the amino acid requirement for Bacillus anthracis, the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for

Folic acid modulates eNOS activity via effects on posttranslational modifications and protein–protein interactions☆

PubMed Central

Taylor, Sarah Y.; Dixon, Hannah M.; Yoganayagam, Shobana; Price, Natalie; Lang, Derek

2013-01-01

Folic acid enhances endothelial function and improves outcome in primary prevention of cardiovascular disease. The exact intracellular signalling mechanisms involved remain elusive and were therefore the subject of this study. Particular focus was placed on folic acid-induced changes in posttranslational modifications of endothelial nitric oxide synthase (eNOS). Cultured endothelial cells were exposed to folic acid in the absence or presence of phosphatidylinositol-3' kinase/Akt (PI3K/Akt) inhibitors. The phosphorylation status of eNOS was determined via western blotting. The activities of eNOS and PI3K/Akt were evaluated. The interaction of eNOS with caveolin-1, Heat-Shock Protein 90 and calmodulin was studied using co-immunoprecipitation. Intracellular localisation of eNOS was investigated using sucrose gradient centrifugation and confocal microscopy. Folic acid promoted eNOS dephosphorylation at negative regulatory sites, and increased phosphorylation at positive regulatory sites. Modulation of phosphorylation status was concomitant with increased cGMP concentrations, and PI3K/Akt activity. Inhibition of PI3K/Akt revealed specific roles for this kinase pathway in folic acid-mediated eNOS phosphorylation. Regulatory protein and eNOS protein associations were altered in favour of a positive regulatory effect in the absence of bulk changes in intracellular eNOS localisation. Folic acid-mediated eNOS activation involves the modulation of eNOS phosphorylation status at multiple residues and positive changes in important protein–protein interactions. Such intracellular mechanisms may in part explain improvements in clinical vascular outcome following folic acid treatment. PMID:23796957

Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium.

PubMed

Shishkov, Alexander; Bogacheva, Elena; Fedorova, Natalia; Ksenofontov, Alexander; Badun, Gennadii; Radyukhin, Victor; Lukashina, Elena; Serebryakova, Marina; Dolgov, Alexey; Chulichkov, Alexey; Dobrov, Evgeny; Baratova, Lyudmila

2011-12-01

The structure of the C-terminal domain of the influenza virus A matrix M1 protein, for which X-ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally-activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C-terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C-terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C-terminal domain is an almost flat layer with a three-α-helical structure. To explain the high level of tritium label incorporation into the C-terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI-TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C-terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell. © 2011 The Authors Journal compilation © 2011 FEBS.

Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score of three Mediterranean cephalopods.

PubMed

Zlatanos, Spiros; Laskaridis, Kostas; Feist, Christian; Sagredos, Angelos

2006-10-01

Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score (PDCAAS) in three commercially important cephalopods of the Mediterranean sea (cuttlefish, octopus and squid) were determined. The results of the proximate analysis showed that these species had very high protein:fat ratios similar to lean beef. Docosahexaenoic, palmitic and eicosipentaenoic acid were the most abundant fatty acids among analyzed species. The amount of n-3 fatty acids was higher than that of saturated, monounsaturated and n-6 fatty acids. Despite the fact that cephalopods contain small amounts of fat they were found quite rich in n-3 fatty acids. Finally, PDCAAS indicated that these organisms had a very good protein quality.

Interaction of perfluoroalkyl acids with human liver fatty acid-binding protein.

PubMed

Sheng, Nan; Li, Juan; Liu, Hui; Zhang, Aiqian; Dai, Jiayin

2016-01-01

Perfluoroalkyl acids (PFAAs) are highly persistent and bioaccumulative, resulting in their broad distribution in humans and the environment. The liver is an important target for PFAAs, but the mechanisms behind PFAAs interaction with hepatocyte proteins remain poorly understood. We characterized the binding of PFAAs to human liver fatty acid-binding protein (hL-FABP) and identified critical structural features in their interaction. The binding interaction of PFAAs with hL-FABP was determined by fluorescence displacement and isothermal titration calorimetry (ITC) assay. Molecular simulation was conducted to define interactions at the binding sites. ITC measurement revealed that PFOA/PFNA displayed a moderate affinity for hL-FABP at a 1:1 molar ratio, a weak binding affinity for PFHxS and no binding for PFHxA. Moreover, the interaction was mainly mediated by electrostatic attraction and hydrogen bonding. Substitution of Asn111 with Asp caused loss of binding affinity to PFAA, indicating its crucial role for the initial PFAA binding to the outer binding site. Substitution of Arg122 with Gly caused only one molecule of PFAA to bind to hL-FABP. Molecular simulation showed that substitution of Arg122 increased the volume of the outer binding pocket, making it impossible to form intensive hydrophobic stacking and hydrogen bonds with PFOA, and highlighting its crucial role in the binding process. The binding affinity of PFAAs increased significantly with their carbon number. Arg122 and Asn111 played a pivotal role in these interactions. Our findings may help understand the distribution pattern, bioaccumulation, elimination, and toxicity of PFAAs in humans.

Overexpression of Human Fatty Acid Transport Protein 2/Very Long Chain Acyl-CoA Synthetase 1 (FATP2/Acsvl1) Reveals Distinct Patterns of Trafficking of Exogenous Fatty Acids

PubMed Central

Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

2014-01-01

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

PubMed

Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

2013-11-01

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

Interaction of milk whey protein with common phenolic acids

NASA Astrophysics Data System (ADS)

Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

2014-01-01

Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

Site specific incorporation of keto amino acids into proteins

DOEpatents

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

2011-03-22

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

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

2008-10-07

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

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

2011-12-06

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

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

2012-02-14

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Amino acid repletion does not decrease muscle protein catabolism during hemodialysis.

PubMed

Raj, Dominic S C; Adeniyi, Oladipo; Dominic, Elizabeth A; Boivin, Michel A; McClelland, Sandra; Tzamaloukas, Antonios H; Morgan, Nancy; Gonzales, Lawrence; Wolfe, Robert; Ferrando, Arny

2007-06-01

Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol.min(-1).100 ml (-1)) was more negative during HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.

LC-MS display of the total modified amino acids in cataract lens proteins and in lens proteins glycated by ascorbic acid in vitro.

PubMed

Cheng, Rongzhu; Feng, Qi; Ortwerth, Beryl J

2006-05-01

We previously reported chromatographic evidence supporting the similarity of yellow chromophores isolated from aged human lens proteins, early brunescent cataract lens proteins and calf lens proteins ascorbylated in vitro [Cheng, R. et al. Biochimica et Biophysica Acta 1537, 14-26, 2001]. In this paper, new evidence supporting the chemical identity of the modified amino acids in these protein populations were collected by using a newly developed two-dimensional LC-MS mapping technique supported by tandem mass analysis of the major species. The pooled water-insoluble proteins from aged normal human lenses, early stage brunescent cataract lenses and calf lens proteins reacted with or without 20 mM ascorbic acid in air for 4 weeks were digested with a battery of proteolytic enzymes under argon to release the modified amino acids. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column and four major A330nm-absorbing peaks were collected. Peaks 1, 2 and 3, which contained most of the modified amino acids were concentrated and subjected to RP-HPLC/ESI-MS, and the mass elution maps were determined. The samples were again analyzed and those peaks with a 10(4) - 10(6) response factor were subjected to MS/MS analysis to identify the daughter ions of each modification. Mass spectrometric maps of peaks 1, 2 and 3 from cataract lenses showed 58, 40 and 55 mass values, respectively, ranging from 150 to 600 Da. Similar analyses of the peaks from digests of the ascorbylated calf lens proteins gave 81, 70 and 67 mass values, respectively, of which 100 were identical to the peaks in the cataract lens proteins. A total of 40 of the major species from each digest were analyzed by LC-MS/MS and 36 were shown to be identical. Calf lens proteins incubated without ascorbic acid showed several similar mass values, but the response factors were 100 to 1000-fold less for every modification. Based upon these data, we conclude

Proximate composition and fatty acid analysis of Lablab purpureus (L.) legume seed: implicates to both protein and essential fatty acid supplementation.

PubMed

Hossain, Shahdat; Ahmed, Rashed; Bhowmick, Sujan; Mamun, Abdullah Al; Hashimoto, Michio

2016-01-01

The high mortality rate in Bangladesh is related to poverty, which results in protein malnutrition, essential fatty acid deficiency and lacks in adequate vitamins, minerals and calorie. Exploring new food items with improved dietary nutrition factors may, therefore, help to decrease the mortality rate in the poor countries like Bangladesh. Accordingly, the present study was a proximate composition and fatty acid analysis of L. purpureus seed-a legume seed which is given no careful attention locally, though it might be a good source of valuable nutrition factors for both animals and humans. The purpose of the study was, therefore, to generate awareness that L. purpureus could also act as a good source of food components essential for good health. Proximate analysis revealed that the seed powder contained 8.47 ± 0.52% moisture; 3.50 ± 0.0.07% ash; 1.02 ± 0.06% total fat; 23.95 ± 0.15% total protein; 1.21 ± 0.16% total dietary fiber; 61.86 ± 0.70% total carbohydrate and 352.4 ± 2.66 kcal/100 g energy. Phytic acid content (%) was 1.014 ± 0.048. Major fatty acid composition (%): the essential fatty acid linoleic acid (C18:2, ω-6) was 9.50 ± 0.68, while the linolenic acid (C18:3, ω-3) was 1.95 ± 0.18. Palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1) were, respectively, 2.96 ± 0.19, 0.77 ± 0.04 and 1.10 ± 0.06. Lignoceric acid (C24:0) was 0.11 ± 0.007%. Monounsaturated palmitoleic acid (0.006 ± 0.0), docosapentaenoic acid (DPA, C22:5, ω-3) and nervonic acid (0.002 ± 0.0) were present in trace amounts. Arachidonic acid (AA, C20:4, ω-6), eicosapentaenoic acid (C20:5, ω-3), and docosahexaenoic acid (C22:6, ω-3) were not detected. The fatty acid profile, thus, suggests that essential omega-6 fatty acid linoleic acid (C18:3, ω-6) and omega-3 linolenic acid (C18:3, ω-3) were the major polyunsaturated fatty acids (PUFA) present in L. purpureus seed. In addition, the seed contained high amount of proteins

Fatty Acid Binding Protein 5 Modulates Docosahexaenoic Acid-Induced Recovery in Rats Undergoing Spinal Cord Injury

PubMed Central

Figueroa, Johnny D.; Serrano-Illan, Miguel; Licero, Jenniffer; Cordero, Kathia; Miranda, Jorge D.

2016-01-01

Abstract Omega-3 polyunsaturated fatty acids (n-3 PUFAs) promote functional recovery in rats undergoing spinal cord injury (SCI). However, the precise molecular mechanism coupling n-3 PUFAs to neurorestorative responses is not well understood. The aim of the present study was to determine the spatiotemporal expression of fatty acid binding protein 5 (FABP5) after contusive SCI and to investigate whether this protein plays a role in n-3 PUFA–mediated functional recovery post-SCI. We found that SCI resulted in a robust spinal cord up-regulation in FABP5 mRNA levels (556 ± 187%) and protein expression (518 ± 195%), when compared to sham-operated rats, at 7 days post-injury (dpi). This upregulation coincided with significant alterations in the metabolism of fatty acids in the injured spinal cord, as revealed by metabolomics-based lipid analyses. In particular, we found increased levels of the n-3 series PUFAs, particularly docosahexaenoic acid (DHA; 22:6 n-3) and eicosapentaenoic acid (EPA; 20:5 n-3) at 7 dpi. Animals consuming a diet rich in DHA and EPA exhibited a significant upregulation in FABP5 mRNA levels at 7 dpi. Immunofluorescence showed low basal FABP5 immunoreactivity in spinal cord ventral gray matter NeuN+ neurons of sham-operated rats. SCI resulted in a robust induction of FABP5 in glial (GFAP+, APC+, and NG2+) and precursor cells (DCX+, nestin+). We found that continuous intrathecal administration of FABP5 silencing with small interfering RNA (2 μg) impaired spontaneous open-field locomotion post-SCI. Further, FABP5 siRNA administration hindered the beneficial effects of DHA to ameliorate functional recovery at 7 dpi. Altogether, our findings suggest that FABP5 may be an important player in the promotion of cellular uptake, transport, and/or metabolism of DHA post-SCI. Given the beneficial roles of n-3 PUFAs in ameliorating functional recovery, we propose that FABP5 is an important contributor to basic repair mechanisms in the

SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type Protein Kinase, Is Important for Abscisic Acid Responses in Arabidopsis through Phosphorylation of ABSCISIC ACID-INSENSITIVE51[OPEN

PubMed Central

Zhou, Xiaona; Hao, Hongmei; Zhang, Yuguo; Bai, Yili; Zhu, Wenbo; Qin, Yunxia; Yuan, Feifei; Zhao, Feiyi; Wang, Mengyao; Hu, Jingjiang; Xu, Hong; Guo, Aiguang; Zhao, Huixian; Zhao, Yang; Cao, Cuiling; Yang, Yongqing; Schumaker, Karen S.; Guo, Yan; Xie, Chang Gen

2015-01-01

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-RELATED PROTEIN KINASE3-type protein kinase, SOS2-LIKE PROTEIN KINASE5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, ABSCISIC ACID-INSENSITIVE5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-LIKE CALCIUM BINDING PROTEINs) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana). PMID:25858916

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein

PubMed Central

Darwiche, Rabih; Mène-Saffrané, Laurent; Gfeller, David; Asojo, Oluwatoyin A.; Schneiter, Roger

2017-01-01

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro. Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites. PMID:28365570

[Metabolism of cholesterol and fatty acids in nephrotic syndrome and its regulation by sterol regulatory element binding proteins (SREBP's). Effect of soy protein consumption].

PubMed

Tovar, Armando; Manzano, Natalia; Torres, Nimbe

2005-01-01

Hyperlipidemia occurs during nephrotic syndrome (NS). It is known that cholesterol and fatty acid biosynthesis is controlled by the transcription factors sterol regulatory element binding proteins (SREBPs). Soy protein consumption reduces the concentration of these lipids, although its mechanism of action is not well known. The aim of the present study was to establish whether soy protein consumption reduces cholesterol and triglycerides levels by regulating of SREBPs. Male Wistar rats with experimental NS were studied for 64 days. The results showed that rats fed with soy protein had significantly lower plasma cholesterol and triglyceride concentrations as well as proteinuria than rats fed with casein diet. These decrements were associated with a decrease in the expression of SREBP-1 and fatty acid biosynthetic enzymes. In addition, Western blot analysis revealed that in nuclear extracts from hepatocytes of rats fed with soy protein, there was a lower concentration of SREBP-1 than in rats fed with casein. The results of this study indicate that consumption of a soy protein diet has beneficial effects on nephrotic syndrome.

Uric acid contributes greatly to hepatic antioxidant capacity besides protein.

PubMed

Mikami, T; Sorimachi, M

2017-12-20

Uric acid is the end-product of purine nucleotide metabolism and an increase in uric acid concentration in the body results in hyperuricemia, ultimately leading to gout. However, uric acid is a potent antioxidant and interacts with reactive oxygen species (ROS) to be non-enzymatically converted to allantoin. Uric acid accounts for approximately 60 % of antioxidant capacity in the plasma; however, its contribution to tissue antioxidant capacity is unknown. In this study, the contribution of uric acid to tissue antioxidant capacity and its conversion to allantoin by scavenging ROS in tissue were examined. The results showed that a decrease in hepatic uric acid content via allopurinol administration significantly reduced hepatic total-radical trapping antioxidant parameter (TRAP) content in protein-free cytosol. Additionally, treating protein-free cytosol with uricase led to a further reduction of hepatic TRAP content. Allantoin was also detected in the solution containing protein-free cytosol that reacted with ROS. These findings suggest that in the absence of protein, uric acid contributes greatly to antioxidant capacity in the liver, where uric acid is converted to allantoin by scavenging ROS.

Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action.

PubMed

Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J; Zhang, Huiming; Tao, W Andy; Zhu, Jian-Kang

2013-07-02

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

Conformational Entropy of Intrinsically Disordered Proteins from Amino Acid Triads

PubMed Central

Baruah, Anupaul; Rani, Pooja; Biswas, Parbati

2015-01-01

This work quantitatively characterizes intrinsic disorder in proteins in terms of sequence composition and backbone conformational entropy. Analysis of the normalized relative composition of the amino acid triads highlights a distinct boundary between globular and disordered proteins. The conformational entropy is calculated from the dihedral angles of the middle amino acid in the amino acid triad for the conformational ensemble of the globular, partially and completely disordered proteins relative to the non-redundant database. Both Monte Carlo (MC) and Molecular Dynamics (MD) simulations are used to characterize the conformational ensemble of the representative proteins of each group. The results show that the globular proteins span approximately half of the allowed conformational states in the Ramachandran space, while the amino acid triads in disordered proteins sample the entire range of the allowed dihedral angle space following Flory’s isolated-pair hypothesis. Therefore, only the sequence information in terms of the relative amino acid triad composition may be sufficient to predict protein disorder and the backbone conformational entropy, even in the absence of well-defined structure. The predicted entropies are found to agree with those calculated using mutual information expansion and the histogram method. PMID:26138206

Rewiring protein synthesis: From natural to synthetic amino acids.

PubMed

Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

2017-11-01

The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of protein synthesis by amino acids in muscle of neonates

PubMed Central

Suryawan, Agus; Davis, Teresa A.

2011-01-01

The marked increase in skeletal muscle mass during the neonatal period is largely due to a high rate of postprandial protein synthesis that is modulated by an enhanced sensitivity to insulin and amino acids. The amino acid signaling pathway leading to the stimulation of protein synthesis has not been fully elucidated. Among the amino acids, leucine is considered to be a principal anabolic agent that regulates protein synthesis. mTORC1, which controls protein synthesis, has been implicated as a target for leucine. Until recently, there have been few studies exploring the role of amino acids in enhancing muscle protein synthesis in vivo. In this review, we discuss amino acid-induced protein synthesis in muscle in the neonate, focusing on current knowledge of the role of amino acids in the activation of mTORC1 leading to mRNA translation. The role of the amino acid transporters, SNAT2, LAT1, and PAT, in the modulation of mTORC1 activation and the role of amino acids in the activation of putative regulators of mTORC1, i.e., raptor, Rheb, MAP4K3, Vps34, and Rag GTPases, are discussed. PMID:21196241

Comparative proteomic analysis of differentially expressed proteins in β-aminobutyric acid enhanced Arabidopsis thaliana tolerance to simulated acid rain.

PubMed

Liu, Tingwu; Jiang, Xinwu; Shi, Wuliang; Chen, Juan; Pei, Zhenming; Zheng, Hailei

2011-05-01

Acid rain is a worldwide environmental issue that has seriously destroyed forest ecosystems. As a highly effective and broad-spectrum plant resistance-inducing agent, β-aminobutyric acid could elevate the tolerance of Arabidopsis when subjected to simulated acid rain. Using comparative proteomic strategies, we analyzed 203 significantly varied proteins of which 175 proteins were identified responding to β-aminobutyric acid in the absence and presence of simulated acid rain. They could be divided into ten groups according to their biological functions. Among them, the majority was cell rescue, development and defense-related proteins, followed by transcription, protein synthesis, folding, modification and destination-associated proteins. Our conclusion is β-aminobutyric acid can lead to a large-scale primary metabolism change and simultaneously activate antioxidant system and salicylic acid, jasmonic acid, abscisic acid signaling pathways. In addition, β-aminobutyric acid can reinforce physical barriers to defend simulated acid rain stress. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dietary fatty acids and membrane protein function.

PubMed

Murphy, M G

1990-02-01

In recent years, there has been growing public awareness of the potential health benefits of dietary fatty acids, and of the distinction between the effects of the omega6 and omega3 polyunsaturated fatty acids that are concentrated in vegetable and fish oils, respectively. A part of the biologic effectiveness of the two families of polyunsaturated fatty acids resides in their relative roles as precursors of the eicosanoids. However, we are also beginning to appreciate that as the major components of the hydrophobic core of the membrane bilayer, they can interact with and directly influence the functioning of select integral membrane proteins. Among the most important of these are the enzymes, receptors, and ion channels that are situated in the plasma membrane of the cell, since they carry out the communication and homeostatic processes that are necessary for normal cell function. This review examines current information regarding the effects of diet-induced changes in plasma membrane fatty acid composition on several specific enzymes (adenylate cyclase, 5'-nucleotidase, Na(+)/K(+)-ATPase) and cell-surface receptors (opiate, adrenergic, insulin). Dietary manipulation studies have demonstrated a sensitivity of each to a fatty acid environment that is variably dependent on the nature of the fatty acid(s) and/or source of the membrane. The molecular mechanisms appear to involve fatty acid-dependent effects on protein conformation, on the "fluidity" and/or thickness of the membrane, or on protein synthesis. Together, the results of these studies reinforce the concept that dietary fats have the potential to regulate physiologic function and to further our understanding of how this occurs at a membrane level.

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

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

Melton, Elaina M.; Center for Cardiovascular Sciences, Albany Medical College, Albany, NY; Cerny, Ronald L.

Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4,more » for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

Modulating protein adsorption onto hydroxyapatite particles using different amino acid treatments

PubMed Central

Lee, Wing-Hin; Loo, Ching-Yee; Van, Kim Linh; Zavgorodniy, Alexander V.; Rohanizadeh, Ramin

2012-01-01

Hydroxyapatite (HA) is a material of choice for bone grafts owing to its chemical and structural similarities to the mineral phase of hard tissues. The combination of osteogenic proteins with HA materials that carry and deliver the proteins to the bone-defective areas will accelerate bone regeneration. The study investigated the treatment of HA particles with different amino acids such as serine (Ser), asparagine (Asn), aspartic acid (Asp) and arginine (Arg) to enhance the adsorption ability of HA carrier for delivering therapeutic proteins to the body. The crystallinity of HA reduced when amino acids were added during HA preparation. Depending on the types of amino acid, the specific surface area of the amino acid-functionalized HA particles varied from 105 to 149 m2 g–1. Bovine serum albumin (BSA) and lysozyme were used as model proteins for adsorption study. The protein adsorption onto the surface of amino acid-functionalized HA depended on the polarities of HA particles, whereby, compared with lysozyme, BSA demonstrated higher affinity towards positively charged Arg-HA. Alternatively, the binding affinity of lysozyme onto the negatively charged Asp-HA was higher when compared with BSA. The BSA and lysozyme adsorptions onto the amino acid-functionalized HA fitted better into the Freundlich than Langmuir model. The amino acid-functionalized HA particles that had higher protein adsorption demonstrated a lower protein-release rate. PMID:21957116

Impact of antinutritional factors in food proteins on the digestibility of protein and the bioavailability of amino acids and on protein quality.

PubMed

Sarwar Gilani, G; Wu Xiao, Chao; Cockell, Kevin A

2012-08-01

Dietary antinutritional factors have been reported to adversely affect the digestibility of protein, bioavailability of amino acids and protein quality of foods. Published data on these negative effects of major dietary antinutritional factors are summarized in this manuscript. Digestibility and the quality of mixed diets in developing countries are considerably lower than of those in developed regions. For example, the digestibility of protein in traditional diets from developing countries such as India, Guatemala and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94 %). Poor digestibility of protein in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, is due to the presence of less digestible protein fractions, high levels of insoluble fibre, and/or high concentrations of antinutritional factors present endogenously or formed during processing. Examples of naturally occurring antinutritional factors include glucosinolates in mustard and canola protein products, trypsin inhibitors and haemagglutinins in legumes, tannins in legumes and cereals, gossypol in cottonseed protein products, and uricogenic nucleobases in yeast protein products. Heat/alkaline treatments of protein products may yield Maillard reaction compounds, oxidized forms of sulphur amino acids, D-amino acids and lysinoalanine (LAL, an unnatural nephrotoxic amino acid derivative). Among common food and feed protein products, soyabeans are the most concentrated source of trypsin inhibitors. The presence of high levels of dietary trypsin inhibitors from soyabeans, kidney beans or other grain legumes have been reported to cause substantial reductions in protein and amino acid digestibility (up to 50 %) and protein quality (up to 100 %) in rats and/or pigs. Similarly, the presence of high levels of tannins in sorghum and other cereals, fababean and other grain legumes can cause

Fatty acid-binding protein 5 (FABP5) promotes lipolysis of lipid droplets, de novo fatty acid (FA) synthesis and activation of nuclear factor-kappa B (NF-κB) signaling in cancer cells.

PubMed

Senga, Shogo; Kobayashi, Narumi; Kawaguchi, Koichiro; Ando, Akira; Fujii, Hiroshi

2018-06-12

Fatty acid-binding proteins (FABPs) are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting them to the appropriate compartments in the cell. Epidermal fatty acid-binding protein (FABP5) is an intracellular lipid-binding protein that is abundantly expressed in adipocytes and macrophages. Previous studies have revealed that the FABP5 expression level is closely related to malignancy in various types of cancer. However, its precise functions in the metabolisms of cancer cells remain unclear. Here, we revealed that FABP5 knockdown significantly induced downregulation of the genes expression, such as hormone-sensitive lipase (HSL), monoacylglycerol lipase (MAGL), elongation of long-chain fatty acid member 6 (Elovl6), and acyl-CoA synthetase long-chain family member 1 (ACSL1), which are involved in altered lipid metabolism, lipolysis, and de novo FA synthesis in highly aggressive prostate and breast cancer cells. Moreover, we demonstrated that FABP5 induced inflammation and cytokine production through the nuclear factor-kappa B signaling pathway activated by reactive oxygen species and protein kinase C in PC-3 and MDA-MB-231 cells. Thus, FABP5 might regulate lipid quality and/or quantity to promote aggressiveness such as cell growth, invasiveness, survival, and inflammation in prostate and breast cancer cells. In the present study, we have revealed for the first time that high expression of FABP5 plays a critical role in alterations of lipid metabolism, leading to cancer development and metastasis in highly aggressive prostate and breast cancer cells. Copyright © 2018. Published by Elsevier B.V.

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

PubMed Central

Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

2005-01-01

CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

Decreased eicosapentaenoic acid levels in acne vulgaris reveals the presence of a proinflammatory state.

PubMed

Aslan, İbrahim; Özcan, Filiz; Karaarslan, Taner; Kıraç, Ebru; Aslan, Mutay

2017-01-01

This study aimed to determine circulating levels of polyunsaturated fatty acids (PUFAs), secretory phospholipase A2 (sPLA2), lipoprotein lipase (LPL) and measure circulating protein levels of angiopoietin-like protein 3 (ANGPTL3), ANGPTL4, cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in patients with acne vulgaris. Serum from 21 control subjects and 31 acne vulgaris patients were evaluated for levels of arachidonic acid (AA, C20:4n- 6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). PUFA levels were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods Serum EPA levels were significantly decreased while AA/EPA and DGLA/EPA ratio were significantly increased in acne vulgaris patients compared to controls. Serum levels of AA, DGLA and DHA showed no significant difference while activity of sPLA2 and LPL were significantly increased in acne vulgaris compared to controls. Results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly decreased serum EPA levels and increased activity of sPLA2, AA/EPA and DGLA/EPA ratio. Increased LPL activity in the serum of acne vulgaris patients can be protective through its anti-dyslipidemic actions. This is the first study reporting altered EPA levels and increased sPLA2 activity in acne vulgaris and supports the use of omega-3 fatty acids as adjuvant treatment for acne patients. Copyright © 2016 Elsevier Inc. All rights reserved.

Protein and metabolic engineering for the production of organic acids.

PubMed

Liu, Jingjing; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng; Chen, Jian

2017-09-01

Organic acids are natural metabolites of living organisms. They have been widely applied in the food, pharmaceutical, and bio-based materials industries. In recent years, biotechnological routes to organic acids production from renewable raw materials have been regarded as very promising approaches. In this review, we provide an overview of current developments in the production of organic acids using protein and metabolic engineering strategies. The organic acids include propionic acid, pyruvate, itaconic acid, succinic acid, fumaric acid, malic acid and citric acid. We also expect that rapid developments in the fields of systems biology and synthetic biology will accelerate protein and metabolic engineering for microbial organic acid production in the future. Copyright © 2017. Published by Elsevier Ltd.

Evolutionary biochemistry: revealing the historical and physical causes of protein properties

PubMed Central

Harms, Michael J.; Thornton, Joseph W.

2014-01-01

The repertoire of proteins and nucleic acids in the living world is determined by evolution; their properties are determined by the laws of physics and chemistry. Explanations of these two kinds of causality — the purviews of evolutionary biology and biochemistry, respectively — are typically pursued in isolation, but many fundamental questions fall squarely at the interface of fields. Here we articulate the paradigm of evolutionary biochemistry, which aims to dissect the physical mechanisms and evolutionary processes by which biological molecules diversified and to reveal how their physical architecture facilitates and constrains their evolution. We show how an integration of evolution with biochemistry moves us towards a more complete understanding of why biological molecules have the properties that they do. PMID:23864121

Induced oxidative stress management in wounds through phenolic acids engineered fibrous protein: An in vitro assessment using polymorphonuclear (PMN) cells.

PubMed

Thiruselvi, T; Thirupathi Kumara Raja, S; Shanuja, S K; Iswarya, S; Gnanamani, A

2017-03-01

The present study explores the preparation, characterization and the role of phenolic acid tethered fibrous protein in the management of induced oxidative stress studied under in vitro conditions. In brief, the biomaterial is prepared by engineering the fibrous protein with dihydroxy and trihydroxy phenolic acid moieties and subjected to characterization to ensure the tethering. The resultant biomaterial studied for its efficacy as a free radical scavenger using polymorphonuclear (PMN) cells with induced oxidative stress and also as an agent for cell migration using fibroblasts cells. Results revealed that induced oxidative stress in PMN cells after exposure to UVB radiation managed well with the prepared biomaterial by reducing the levels of superoxide anion, oxygen and hydroxyl radicals. Further, the protein and the phenolic acid interaction supports the cell migration as evidenced from the scratch assay. In conclusion, though phenolic acids are well known for their antimicrobial and antioxidant potential, indenting these acids directly to the wounds is not sensible, but tethering to protein explored the scavenging activity as expected. The present study infers that phenolic acid engineered protein has a significant role in managing the imbalance in the redox state prevailing in wounds and supports the healing at appreciable level. Copyright © 2016 Elsevier B.V. All rights reserved.

Utilizing knowledge base of amino acids structural neighborhoods to predict protein-protein interaction sites.

PubMed

Jelínek, Jan; Škoda, Petr; Hoksza, David

2017-12-06

Protein-protein interactions (PPI) play a key role in an investigation of various biochemical processes, and their identification is thus of great importance. Although computational prediction of which amino acids take part in a PPI has been an active field of research for some time, the quality of in-silico methods is still far from perfect. We have developed a novel prediction method called INSPiRE which benefits from a knowledge base built from data available in Protein Data Bank. All proteins involved in PPIs were converted into labeled graphs with nodes corresponding to amino acids and edges to pairs of neighboring amino acids. A structural neighborhood of each node was then encoded into a bit string and stored in the knowledge base. When predicting PPIs, INSPiRE labels amino acids of unknown proteins as interface or non-interface based on how often their structural neighborhood appears as interface or non-interface in the knowledge base. We evaluated INSPiRE's behavior with respect to different types and sizes of the structural neighborhood. Furthermore, we examined the suitability of several different features for labeling the nodes. Our evaluations showed that INSPiRE clearly outperforms existing methods with respect to Matthews correlation coefficient. In this paper we introduce a new knowledge-based method for identification of protein-protein interaction sites called INSPiRE. Its knowledge base utilizes structural patterns of known interaction sites in the Protein Data Bank which are then used for PPI prediction. Extensive experiments on several well-established datasets show that INSPiRE significantly surpasses existing PPI approaches.

Carbonyl-based blue autofluorescence of proteins and amino acids

PubMed Central

Niyangoda, Chamani; Miti, Tatiana; Breydo, Leonid; Uversky, Vladimir

2017-01-01

Intrinsic protein fluorescence is inextricably linked to the near-UV autofluorescence of aromatic amino acids. Here we show that a novel deep-blue autofluorescence (dbAF), previously thought to emerge as a result of protein aggregation, is present at the level of monomeric proteins and even poly- and single amino acids. Just as its aggregation-related counterpart, this autofluorescence does not depend on aromatic residues, can be excited at the long wavelength edge of the UV and emits in the deep blue. Differences in dbAF excitation and emission peaks and intensities from proteins and single amino acids upon changes in solution conditions suggest dbAF’s sensitivity to both the chemical identity and solution environment of amino acids. Autofluorescence comparable to dbAF is emitted by carbonyl-containing organic solvents, but not those lacking the carbonyl group. This implicates the carbonyl double bonds as the likely source for the autofluorescence in all these compounds. Using beta-lactoglobulin and proline, we have measured the molar extinction coefficients and quantum yields for dbAF in the monomeric state. To establish its potential utility in monitoring protein biophysics, we show that dbAF emission undergoes a red-shift comparable in magnitude to tryptophan upon thermal denaturation of lysozyme, and that it is sensitive to quenching by acrylamide. Carbonyl dbAF therefore provides a previously neglected intrinsic optical probe for investigating the structure and dynamics of amino acids, proteins and, by extension, DNA and RNA. PMID:28542206

Sunflower (Helianthus annuus) fatty acid synthase complex: β-hydroxyacyl-[acyl carrier protein] dehydratase genes.

PubMed

González-Thuillier, Irene; Venegas-Calerón, Mónica; Sánchez, Rosario; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

2016-02-01

Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

Cinnamic acid and its derivatives inhibit fructose-mediated protein glycation.

PubMed

Adisakwattana, Sirichai; Sompong, Weerachat; Meeprom, Aramsri; Ngamukote, Sathaporn; Yibchok-Anun, Sirintorn

2012-01-01

Cinnamic acid and its derivatives have shown a variety of pharmacologic properties. However, little is known about the antiglycation properties of cinnamic acid and its derivatives. The present study sought to characterize the protein glycation inhibitory activity of cinnamic acid and its derivatives in a bovine serum albumin (BSA)/fructose system. The results demonstrated that cinnamic acid and its derivatives significantly inhibited the formation of advanced glycation end products (AGEs) by approximately 11.96-63.36% at a concentration of 1 mM. The strongest inhibitory activity against the formation of AGEs was shown by cinnamic acid. Furthermore, cinnamic acid and its derivatives reduced the level of fructosamine, the formation of N(ɛ)-(carboxymethyl) lysine (CML), and the level of amyloid cross β-structure. Cinnamic acid and its derivatives also prevented oxidative protein damages, including effects on protein carbonyl formation and thiol oxidation of BSA. Our findings may lead to the possibility of using cinnamic acid and its derivatives for preventing AGE-mediated diabetic complications.

Cinnamic Acid and Its Derivatives Inhibit Fructose-Mediated Protein Glycation

PubMed Central

Adisakwattana, Sirichai; Sompong, Weerachat; Meeprom, Aramsri; Ngamukote, Sathaporn; Yibchok-anun, Sirintorn

2012-01-01

Cinnamic acid and its derivatives have shown a variety of pharmacologic properties. However, little is known about the antiglycation properties of cinnamic acid and its derivatives. The present study sought to characterize the protein glycation inhibitory activity of cinnamic acid and its derivatives in a bovine serum albumin (BSA)/fructose system. The results demonstrated that cinnamic acid and its derivatives significantly inhibited the formation of advanced glycation end products (AGEs) by approximately 11.96–63.36% at a concentration of 1 mM. The strongest inhibitory activity against the formation of AGEs was shown by cinnamic acid. Furthermore, cinnamic acid and its derivatives reduced the level of fructosamine, the formation of Nɛ-(carboxymethyl) lysine (CML), and the level of amyloid cross β-structure. Cinnamic acid and its derivatives also prevented oxidative protein damages, including effects on protein carbonyl formation and thiol oxidation of BSA. Our findings may lead to the possibility of using cinnamic acid and its derivatives for preventing AGE-mediated diabetic complications. PMID:22408423

Enterocyte fatty acid-binding proteins (FABPs): different functions of liver and intestinal FABPs in the intestine.

PubMed

Gajda, Angela M; Storch, Judith

2015-02-01

Fatty acid-binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both liver- (LFABP; FABP1) and intestinal FABPs (IFABP; FABP2) are expressed. These proteins display high-affinity binding for long-chain fatty acids (FA) and other hydrophobic ligands; thus, they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences in ligand-binding stoichiometry and specificity, and in mechanisms of FA transfer to membranes, and it has been hypothesized that LFABP and IFABP have different functions in the enterocyte. Studies directly comparing LFABP- and IFABP-null mice have revealed markedly different phenotypes, indicating that these proteins indeed have different functions in intestinal lipid metabolism and whole body energy homeostasis. In this review, we discuss the evolving knowledge of the functions of LFABP and IFABP in the intestinal enterocyte. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Nonribosomal Landscape in the Nucleolus Revealed by the Stem Cell Protein Nucleostemin

PubMed Central

Politz, Joan C. Ritland; Polena, Ilvin; Trask, Ian; Bazett-Jones, David P.; Pederson, Thoru

2005-01-01

Nucleostemin is a p53-interactive cell cycle progression factor that shuttles between the nucleolus and nucleoplasm, but it has no known involvement in ribosome synthesis. We found the dynamic properties of nucleostemin differed strikingly from fibrillarin (a protein directly involved in rRNA processing) both in response to rRNA transcription inhibition and in the schedule of reentry into daughter nuclei and the nucleolus during late telophase/early G1. Furthermore, nucleostemin was excluded from the nucleolar domains in which ribosomes are born—the fibrillar centers and dense fibrillar component. Instead it was concentrated in rRNA-deficient sites within the nucleolar granular component. This finding suggests that the nucleolus may be more subcompartmentalized than previously thought. In support of this concept, electron spectroscopic imaging studies of the nitrogen and phosphorus distribution in the nucleolar granular component revealed regions that are very rich in protein and yet devoid of nucleic acid. Together, these results suggest that the ultrastructural texture of the nucleolar granular component represents not only ribosomal particles but also RNA-free zones populated by proteins or protein complexes that likely serve other functions. PMID:15857956

Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation.

PubMed Central

Konrad, Zvia; Eichler, Jerry

2002-01-01

Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg(2+)-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [(3)H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane. PMID:12069685

Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

PubMed Central

Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

2014-01-01

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control. PMID:24495932

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type.

PubMed

Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

2014-02-05

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

NASA Astrophysics Data System (ADS)

Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

2014-02-01

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

Electrostatic interactions during acidic phospholipid reactivation of DnaA protein, the Escherichia coli initiator of chromosomal replication.

PubMed

Kitchen, J L; Li, Z; Crooke, E

1999-05-11

The initiation of Escherichia coli chromosomal replication by DnaA protein is strongly influenced by the tight binding of the nucleotides ATP and ADP. Anionic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA protein to replicatively active ATP-DnaA protein in vitro, and thus likely play a key role in regulating DnaA activity. Previous studies have revealed that, during this reactivation, a specific region of DnaA protein inserts into the hydrophobic portion of the lipid bilayer in an acidic phospholipid-dependent manner. To elucidate the requirement for acidic phospholipids in the reactivation process, the contribution of electrostatic forces in the interaction of DnaA and lipid was examined. DnaA-lipid binding required anionic phospholipids, and DnaA-lipid binding as well as lipid-mediated release of DnaA-bound nucleotide were inhibited by increased ionic strength, suggesting the involvement of electrostatic interactions in these processes. As the vesicular content of acidic phospholipids was increased, both nucleotide release and DnaA-lipid binding increased in a linear, parallel manner. Given that DnaA-membrane binding, the insertion of DnaA into the membrane, and the consequent nucleotide release all require anionic phospholipids, the acidic headgroup may be necessary to recruit DnaA protein to the membrane for insertion and subsequent reactivation for replication.

Protein and Amino Acid Requirements during Pregnancy.

PubMed

Elango, Rajavel; Ball, Ronald O

2016-07-01

Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg(-1) · d(-1), respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg(-1) · d(-1) during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14-18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. © 2016 American Society for Nutrition.

Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-linking*

PubMed Central

Wong, Shenq-Huey; King, Chih-Yen

2015-01-01

Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-l-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1–61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine substitutions and two alanine substitutions were introduced at fixed positions in Sup(1–61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala-substituted Sup(1–61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and intermolecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amounts of 14N- and 15N-labeled protein. Fibers propagating the VL strain type exhibited intra- and intermolecular cross-linking between amino acid residues 3 and 28, as well as intra- and intermolecular linking between 32 and 55. Inter- and intramolecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain folds of different [PSI] strains. PMID:26265470

Parsing the life-shortening effects of dietary protein: effects of individual amino acids

PubMed Central

Bouchebti, Sofia; Bazazi, Sepideh; Le Hesran, Sophie; Puga, Camille; Latil, Gérard; Simpson, Stephen J.

2017-01-01

High-protein diets shorten lifespan in many organisms. Is it because protein digestion is energetically costly or because the final products (the amino acids) are harmful? To answer this question while circumventing the life-history trade-off between reproduction and longevity, we fed sterile ant workers on diets based on whole proteins or free amino acids. We found that (i) free amino acids shortened lifespan even more than proteins; (ii) the higher the amino acid-to-carbohydrate ratio, the shorter ants lived and the lower their lipid reserves; (iii) for the same amino acid-to-carbohydrate ratio, ants eating free amino acids had more lipid reserves than those eating whole proteins; and (iv) on whole protein diets, ants seem to regulate food intake by prioritizing sugar, while on free amino acid diets, they seem to prioritize amino acids. To test the effect of the amino acid profile, we tested diets containing proportions of each amino acid that matched the ant's exome; surprisingly, longevity was unaffected by this change. We further tested diets with all amino acids under-represented except one, finding that methionine, serine, threonine and phenylalanine are especially harmful. All together, our results show certain amino acids are key elements behind the high-protein diet reduction in lifespan. PMID:28053059

Parsing the life-shortening effects of dietary protein: effects of individual amino acids.

PubMed

Arganda, Sara; Bouchebti, Sofia; Bazazi, Sepideh; Le Hesran, Sophie; Puga, Camille; Latil, Gérard; Simpson, Stephen J; Dussutour, Audrey

2017-01-11

High-protein diets shorten lifespan in many organisms. Is it because protein digestion is energetically costly or because the final products (the amino acids) are harmful? To answer this question while circumventing the life-history trade-off between reproduction and longevity, we fed sterile ant workers on diets based on whole proteins or free amino acids. We found that (i) free amino acids shortened lifespan even more than proteins; (ii) the higher the amino acid-to-carbohydrate ratio, the shorter ants lived and the lower their lipid reserves; (iii) for the same amino acid-to-carbohydrate ratio, ants eating free amino acids had more lipid reserves than those eating whole proteins; and (iv) on whole protein diets, ants seem to regulate food intake by prioritizing sugar, while on free amino acid diets, they seem to prioritize amino acids. To test the effect of the amino acid profile, we tested diets containing proportions of each amino acid that matched the ant's exome; surprisingly, longevity was unaffected by this change. We further tested diets with all amino acids under-represented except one, finding that methionine, serine, threonine and phenylalanine are especially harmful. All together, our results show certain amino acids are key elements behind the high-protein diet reduction in lifespan. © 2017 The Author(s).

Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

PubMed

Sultana, Azmiri; Lee, Jeffrey E

2015-02-02

Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. Copyright © 2015 John Wiley & Sons, Inc.

Persulfidation proteome reveals the regulation of protein function by hydrogen sulfide in diverse biological processes in Arabidopsis.

PubMed

Aroca, Angeles; Benito, Juan M; Gotor, Cecilia; Romero, Luis C

2017-10-13

Hydrogen sulfide-mediated signaling pathways regulate many physiological and pathophysiological processes in mammalian and plant systems. The molecular mechanism by which hydrogen sulfide exerts its action involves the post-translational modification of cysteine residues to form a persulfidated thiol motif, a process called protein persulfidation. We have developed a comparative and quantitative proteomic analysis approach for the detection of endogenous persulfidated proteins in wild-type Arabidopsis and L-CYSTEINE DESULFHYDRASE 1 mutant leaves using the tag-switch method. The 2015 identified persulfidated proteins were isolated from plants grown under controlled conditions, and therefore, at least 5% of the entire Arabidopsis proteome may undergo persulfidation under baseline conditions. Bioinformatic analysis revealed that persulfidated cysteines participate in a wide range of biological functions, regulating important processes such as carbon metabolism, plant responses to abiotic and biotic stresses, plant growth and development, and RNA translation. Quantitative analysis in both genetic backgrounds reveals that protein persulfidation is mainly involved in primary metabolic pathways such as the tricarboxylic acid cycle, glycolysis, and the Calvin cycle, suggesting that this protein modification is a new regulatory component in these pathways. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Isolation and characterization of a novel acidic matrix protein hic22 from the nacreous layer of the freshwater mussel, Hyriopsis cumingii.

PubMed

Liu, X J; Jin, C; Wu, L M; Dong, S J; Zeng, S M; Li, J L

2016-07-29

Matrix proteins that either weakly acidic or unusually highly acidic have important roles in shell biomineralization. In this study, we have identified and characterized hic22, a weakly acidic matrix protein, from the nacreous layer of Hyriopsis cumingii. Total protein was extracted from the nacre using 5 M EDTA and hic22 was purified using a DEAE-sepharose column. The N-terminal amino acid sequence of hic22 was determined and the complete cDNA encoding hic22 was cloned and sequenced by rapid amplification of cDNA ends-polymerase chain reaction. Finally, the localization and distribution of hic22 was determined by in situ hybridization. Our results revealed that hic22 encodes a 22-kDa protein composed of 185 amino acids. Tissue expression analysis and in situ hybridization indicated that hic22 is expressed in the dorsal epithelial cells of the mantle pallial; moreover, significant expression levels of hic22 were observed after the early formation of the pearl sac (days 19-77), implying that hic22 may play an important role in biomineralization of the nacreous layer.

Fatty acids and small organic compounds bind to mineralo-organic nanoparticles derived from human body fluids as revealed by metabolomic analysis.

PubMed

Martel, Jan; Wu, Cheng-Yeu; Hung, Cheng-Yu; Wong, Tsui-Yin; Cheng, Ann-Joy; Cheng, Mei-Ling; Shiao, Ming-Shi; Young, John D

2016-03-14

Nanoparticles entering the human body instantly become coated with a "protein corona" that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an "organic corona" containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body.

Alkali-catalyzed low temperature wet crosslinking of plant proteins using carboxylic acids.

PubMed

Reddy, Narendra; Li, Ying; Yang, Yiqi

2009-01-01

We report the development of a new method of alkali-catalyzed low temperature wet crosslinking of plant proteins to improve their breaking tenacity without using high temperatures or phosphorus-containing catalysts used in conventional poly(carboxylic acid) crosslinking of cellulose and proteins. Carboxylic acids are preferred over aldehyde-containing crosslinkers for crosslinking proteins and cellulose because of their low toxicity and cost and ability to improve the desired properties of the materials. However, current knowledge in carboxylic acid crosslinking of proteins and cellulose requires the use of carboxylic acids with at least three carboxylic groups, toxic phosphorous-containing catalysts and curing at high temperatures (150-185 degrees C). The use of high temperatures and low pH in conventional carboxylic acid crosslinking has been reported to cause substantial strength loss and/or undesired changes in the properties of the crosslinked materials. In this research, gliadin, soy protein, and zein fibers have been crosslinked with malic acid, citric acid, and butanetetracarboxylic acid to improve the tenacity of the fibers without using high temperatures and phosphorus-containing catalysts. The new method of wet crosslinking using carboxylic acids containing two or more carboxylic groups will be useful to crosslink proteins for various industrial applications.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [Austin, TX

2011-08-30

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

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

Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

2012-02-14

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta; Lital , Schultz; Peter G. , Zhang; Zhiwen

2010-10-12

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

2009-02-24

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid.

PubMed

Capaldi, Stefano; Guariento, Mara; Perduca, Massimiliano; Di Pietro, Santiago M; Santomé, José A; Monaco, Hugo L

2006-07-01

The family of the liver bile acid-binding proteins (L-BABPs), formerly called liver basic fatty acid-binding proteins (Lb-FABPs) shares fold and sequence similarity with the paralogous liver fatty acid-binding proteins (L-FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X-ray structure of a member of the L-BABP family, axolotl (Ambystoma mexicanum) L-BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L-BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L-BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent. (c) 2006 Wiley-Liss, Inc.

Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

PubMed

Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

2017-05-01

Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

PLASMA PROTEIN PRODUCTION INFLUENCED BY AMINO ACID MIXTURES AND LACK OF ESSENTIAL AMINO ACIDS

PubMed Central

Madden, S. C.; Anderson, F. W.; Donovan, J. C.; Whipple, G. H.

1945-01-01

When blood plasma proteins are depleted by bleeding with return of red cells suspended in saline (plasmapheresis) it is possible to bring dogs to a steady state of hypoproteinemia and a constant level of plasma protein production if the diet nitrogen intake is controlled and limited. Such dogs are outwardly normal but have a lowered resistance to infection and intoxication and probably to vitamin deficiency. When the diet nitrogen is provided by certain mixtures of the ten growth essential amino acids plus glycine, given intravenously at a rapid rate, plasma protein production is good. The same mixture absorbed subcutaneously at a slower rate may be slightly better utilized. Fed orally the same mixture is better utilized and associated with a lower urinary nitrogen excretion. An ample amino acid mixture for the daily intake of a 10 kilo dog may contain in grams dl-threonine 1.4, dl-valine 3, dl-leucine 3, dl-isoleucine 2, l(+)-lysine·HCl·H2O 2.2, dl-tryptophane 0.3, dl-phenylalanine 2, dl-methionine 1.2, l(+)-histidine·HCl·H2O 1, l(+)-arginine·HCl 1, and glycine 2. Half this quantity is inadequate and not improved by addition of a mixture of alanine, serine, norleucine, proline, hydroxyproline, and tyrosine totalling 1.4 gm. Aspartic acid appears to induce vomiting when added to a mixture of amino acids. The same response has been reported for glutamic acid (8). Omission from the intake of leucine or of leucine and isoleucine results in negative nitrogen balance and rapid weight loss but plasma protein production may be temporarily maintained. It is possible that leucine may be captured from red blood cell destruction. Tryptophane deficiency causes an abrupt decline in plasma protein production. No decline occurred during 2 weeks of histidine deficiency but the urinary nitrogen increased to negative balance. Plasma protein production may be impaired during conditions of dietary deficiency not related to the protein or amino acid intake. Skin lesions and liver

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies

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

Peters, J.; Peters, M.; Lottspeich, F.

1987-11-01

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate (HPI))-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and M/sub r/ estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%)more » of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.« less

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

PubMed Central

Pawlowski, David R.; Karalus, Richard J.

2012-01-01

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4. By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while

Electricity-free, sequential nucleic acid and protein isolation.

PubMed

Pawlowski, David R; Karalus, Richard J

2012-05-15

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification

The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

PubMed

Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

1992-02-01

The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs

Fatty acid-amino acid conjugates are essential for systemic activation of salicylic acid-induced protein kinase and accumulation of jasmonic acid in Nicotiana attenuata.

PubMed

Hettenhausen, Christian; Heinrich, Maria; Baldwin, Ian T; Wu, Jianqiang

2014-11-28

Herbivory induces the activation of mitogen-activated protein kinases (MAPKs), the accumulation of jasmonates and defensive metabolites in damaged leaves and in distal undamaged leaves. Previous studies mainly focused on individual responses and a limited number of systemic leaves, and more research is needed for a better understanding of how different plant parts respond to herbivory. In the wild tobacco Nicotiana attenuata, FACs (fatty acid-amino acid conjugates) in Manduca sexta oral secretions (OS) are the major elicitors that induce herbivory-specific signaling but their role in systemic signaling is largely unknown. Here, we show that simulated herbivory (adding M. sexta OS to fresh wounds) dramatically increased SIPK (salicylic acid-induced protein kinase) activity and jasmonic acid (JA) levels in damaged leaves and in certain (but not all) undamaged systemic leaves, whereas wounding alone had no detectable systemic effects; importantly, FACs and wounding are both required for activating these systemic responses. In contrast to the activation of SIPK and elevation of JA in specific systemic leaves, increases in the activity of an important anti-herbivore defense, trypsin proteinase inhibitor (TPI), were observed in all systemic leaves after simulated herbivory, suggesting that systemic TPI induction does not require SIPK activation and JA increases. Leaf ablation experiments demonstrated that within 10 minutes after simulated herbivory, a signal (or signals) was produced and transported out of the treated leaves, and subsequently activated systemic responses. Our results reveal that N. attenuata specifically recognizes herbivore-derived FACs in damaged leaves and rapidly send out a long-distance signal to phylotactically connected leaves to activate MAPK and JA signaling, and we propose that FACs that penetrated into wounds rapidly induce the production of another long-distance signal(s) which travels to all systemic leaves and activates TPI defense.

Conversion of amino-acid sequence in proteins to classical music: search for auditory patterns

PubMed Central

2007-01-01

We have converted genome-encoded protein sequences into musical notes to reveal auditory patterns without compromising musicality. We derived a reduced range of 13 base notes by pairing similar amino acids and distinguishing them using variations of three-note chords and codon distribution to dictate rhythm. The conversion will help make genomic coding sequences more approachable for the general public, young children, and vision-impaired scientists. PMID:17477882

The Urine Preservative Acetic Acid Degrades Urine Protein: Implications for Urine Biorepositories and the AASK Cohort Study.

PubMed

Almaani, Salem; Hebert, Lee A; Rovin, Brad H; Birmingham, Daniel J

2017-05-01

Patients enrolled in the African American Study of Kidney Disease and Hypertension (AASK) Cohort Study who exhibited overt proteinuria have been reported to show high nonalbumin proteinuria (NAP), which is characteristic of a tubulopathy. To determine whether African American Study of Kidney Disease and Hypertension nephropathy (AASK-N) is a tubulopathy, we obtained urine samples of 37 patients with AASK-N, with 24-hour protein-to-creatinine ratios (milligrams per milligram) ranging from 0.2 to 1.0, from the National Institute of Diabetes and Digestive Kidney Diseases repository and tested for seven markers of tubular proteinuria. By protocol, each sample had been collected in acetic acid (0.5%; mean final concentration). Compared with samples from patients with lupus nephritis or healthy black controls, AASK-N samples had lower amounts of six markers. Four markers (albumin, β -2-microglobulin, cystatin C, and osteopontin) were undetectable in most AASK-N samples. Examination by SDS-PAGE followed by protein staining revealed protein profiles indicative of severe protein degradation in 34 of 37 AASK-N urine samples. Treatment of lupus nephritis urine samples with 0.5% acetic acid produced the same protein degradation profile as that of AASK-N urine. We conclude that the increased NAP in AASK-N is an artifact of acetic acid-mediated degradation of albumin. The AASK-N repository urine samples have been compromised by the acetic acid preservative. Copyright © 2017 by the American Society of Nephrology.

Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

PubMed

Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

2006-05-01

Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase.

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

PubMed Central

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

2016-01-01

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

Differential Epitope Mapping by STD NMR Spectroscopy To Reveal the Nature of Protein-Ligand Contacts.

PubMed

Monaco, Serena; Tailford, Louise E; Juge, Nathalie; Angulo, Jesus

2017-11-27

Saturation transfer difference (STD) NMR spectroscopy is extensively used to obtain epitope maps of ligands binding to protein receptors, thereby revealing structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. Herein, we report the development of the novel method differential epitope mapping by STD NMR (DEEP-STD NMR) for identifying the type of protein residues contacting the ligand. The method produces differential epitope maps through 1) differential frequency STD NMR and/or 2) differential solvent (D 2 O/H 2 O) STD NMR experiments. The two approaches provide different complementary information on the binding pocket. We demonstrate that DEEP-STD NMR can be used to readily obtain pharmacophore information on the protein. Furthermore, if the 3D structure of the protein is known, this information also helps in orienting the ligand in the binding pocket. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Crude protein and essential amino acid requirements in chicks during the first week posthatch.

PubMed

Sklan, D; Noy, Y

2003-05-01

1. This study examined optimal lysine and sulphur amino acid supply in the first week posthatch in broilers and the relationship between essential amino acids and dietary crude protein during the first week posthatch on performance at 7 d and through marketing. 2. The optimal supply during the 7 d posthatch using a 230 g/kg crude protein diet for sulphur amino acids was 9.1 and for lysine was 10.3-10.8 g/kg with maximal body weight (BW) or feed efficiency as the criteria. 3. Feeding diets with crude protein content ranging from 200 to 260 g/kg with either constant amounts of essential amino acids at different crude protein levels or constant ratios of essential amino acids to crude protein resulted in enhanced performance at 7 but not at 4 d with high protein intake and proportionally increased essential amino acids. 4. Performance on diets with crude protein ranging from 160 to 280 g/kg, with constant ratios of essential amino acid to crude protein, was much enhanced with the high crude protein diets at 7 d. All chicks were transferred to standard diets after 7 d and the BW advantage due to the balanced amino acid-high crude protein diet remained through marketing. 5. Thus increasing essential amino acids in a constant ratio to crude protein enhanced performance during the 7 d posthatch.

NIAS-Server: Neighbors Influence of Amino acids and Secondary Structures in Proteins.

PubMed

Borguesan, Bruno; Inostroza-Ponta, Mario; Dorn, Márcio

2017-03-01

The exponential growth in the number of experimentally determined three-dimensional protein structures provide a new and relevant knowledge about the conformation of amino acids in proteins. Only a few of probability densities of amino acids are publicly available for use in structure validation and prediction methods. NIAS (Neighbors Influence of Amino acids and Secondary structures) is a web-based tool used to extract information about conformational preferences of amino acid residues and secondary structures in experimental-determined protein templates. This information is useful, for example, to characterize folds and local motifs in proteins, molecular folding, and can help the solution of complex problems such as protein structure prediction, protein design, among others. The NIAS-Server and supplementary data are available at http://sbcb.inf.ufrgs.br/nias .

Interaction of melanin with proteins--the importance of an acidic intramelanosomal pH.

PubMed

Mani, I; Sharma, V; Tamboli, I; Raman, G

2001-06-01

Melanin is a highly irregular heteropolymer consisting of monomeric units derived from the enzymatic oxidation of the amino acid tyrosine. The process of melanin formation takes place in specialized acidic organelles (melanosomes) in melanocytes. The process of melanin polymerization requires an alkaline pH in vitro, and therefore, the purpose of an acidic environment in vivo remains a mystery. It is known that melanin is always bound to protein in vivo. It is also seen that polymerization in vitro at an acidic pH necessarily requires the presence of proteins. The effect of various model proteins on melanin synthesis and their interaction with melanin was studied. It was seen that many proteins could increase melanin synthesis at an acidic pH, and that different proteins resulted in the formation of different states of melanin, i.e., a precipitate or a soluble, protein-bound form. We also present evidence to show that soluble protein-bound melanin is present in vivo (in B16 cells as well as in B16 melanoma tissue). An acidic pH appeared to be necessary to ensure the formation of a uniform, very high molecular weight melano-protein complex. The interaction between melanin and proteins appears to be largely charge-dependent as evidenced by zeta potential measurements, and this interaction is also increased in an acidic pH. Thus, it appears that an acidic intramelanosomal pH is essential to ensure maximum interaction between protein and melanin, and also to ensure that all the melanin formed is protein-bound.

Membrane protein properties revealed through data-rich electrostatics calculations

PubMed Central

Guerriero, Christopher J.; Brodsky, Jeffrey L.; Grabe, Michael

2015-01-01

SUMMARY The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem including: full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane induced pKa shifts, calculation of non-polar energies, and command-line scripting for large scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane potentially revealing interesting functional information. PMID:26118532

Predicting nucleic acid binding interfaces from structural models of proteins.

PubMed

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2012-02-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

Predicting nucleic acid binding interfaces from structural models of proteins

PubMed Central

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2011-01-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767

Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling*

PubMed Central

Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

2015-01-01

The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the “lipolysome.” Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome. PMID:25953897

Database of amino acid-nucleotide contacts in contacts in DNA-homeodomain protein

NASA Astrophysics Data System (ADS)

Grokhlina, T. I.; Zrelov, P. V.; Ivanov, V. V.; Polozov, R. V.; Chirgadze, Yu. N.; Sivozhelezov, V. S.

2013-09-01

The analysis of amino acid-nucleotide contacts in interfaces of the protein-DNA complexes, intended to find consistencies in the protein-DNA recognition, is a complex problem that requires an analysis of the physicochemical characteristics of these contacts and the positions of the participating amino acids and nucleotides in the chains of the protein and the DNA, respectively, as well as conservatism of these contacts. Thus, those heterogeneous data should be systematized. For this purpose we have developed a database of amino acid-nucleotide contacts ANTPC (Amino acid Nucleotide Type Position Conservation) following the archetypal example of the proteins in the homeodomain family. We show that it can be used to compare and classify the interfaces of the protein-DNA complexes.

Matrix-assisted laser desorption/ionization-mass spectrometry of hydrophobic proteins in mixtures using formic acid, perfluorooctanoic acid, and sorbitol.

PubMed

Loo, Rachel R Ogorzalek; Loo, Joseph A

2007-02-01

Three MALDI-MS sample/matrix preparation approaches were evaluated for their ability to enhance hydrophobic protein detection from complex mixtures: (1) formic acid-based formulations, (2) perfluorooctanoic acid (PFOA) surfactant addition, and (3) sorbitol addition. While MALDI-MS of Escherichia coli cells desorbed from a standard sinapinic acid matrix displayed 94 (M + H)+ ions, 119 were observed from a formic acid-based matrix with no more than 10 common to both. Formic acid matrix revealed many lipoproteins and an 8282 m/z ion proposed to be the abundant, water-insoluble ATPase proteolipid. Among the formic acid-based cocktails examined, the slowest rate of serine/threonine formylation was found for 50% H2O/33% 2-propanol/17% formic acid. Faster formylation was observed from cocktails containing more formic acid and from mixtures including CH3CN. Sinapinic, ferulic, DHB, 4-hydroxybenzylidene malononitrile, and 2-mercaptobenzothiazole matrixes performed well in formic acid formulations. Dramatic differences in mixture spectra were also observed from PFOA/sinapinic acid, at detergent concentrations exceeding the critical micelle concentration, although these matrix cocktails proved difficult to crystallize. E. coli ions observed from these matrix conditions are listed in Tables S-1 and S-3 (Supporting Information). Similar complementarity was observed for M. acetivorans whole-cell mixtures. Including sorbitol in the sinapinic acid matrix was found to promote homogeneous crystallization and to enhance medium and higher m/z ion detection from dilute E. coli cellular mixtures.

Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

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

Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.

2015-08-07

Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extractionmore » improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.« less

Imbalance of plasma amino acids, metabolites and lipids in patients with lysinuric protein intolerance (LPI).

PubMed

Kurko, Johanna; Tringham, Maaria; Tanner, Laura; Näntö-Salonen, Kirsti; Vähä-Mäkilä, Mari; Nygren, Heli; Pöhö, Päivi; Lietzen, Niina; Mattila, Ismo; Olkku, Anu; Hyötyläinen, Tuulia; Orešič, Matej; Simell, Olli; Niinikoski, Harri; Mykkänen, Juha

2016-09-01

Lysinuric protein intolerance (LPI [MIM 222700]) is an aminoaciduria with defective transport of cationic amino acids in epithelial cells in the small intestine and proximal kidney tubules due to mutations in the SLC7A7 gene. LPI is characterized by protein malnutrition, failure to thrive and hyperammonemia. Many patients also suffer from combined hyperlipidemia and chronic kidney disease (CKD) with an unknown etiology. Here, we studied the plasma metabolomes of the Finnish LPI patients (n=26) and healthy control individuals (n=19) using a targeted platform for analysis of amino acids as well as two analytical platforms with comprehensive coverage of molecular lipids and polar metabolites. Our results demonstrated that LPI patients have a dichotomy of amino acid profiles, with both decreased essential and increased non-essential amino acids. Altered levels of metabolites participating in pathways such as sugar, energy, amino acid and lipid metabolism were observed. Furthermore, of these metabolites, myo-inositol, threonic acid, 2,5-furandicarboxylic acid, galactaric acid, 4-hydroxyphenylacetic acid, indole-3-acetic acid and beta-aminoisobutyric acid associated significantly (P<0.001) with the CKD status. Lipid analysis showed reduced levels of phosphatidylcholines and elevated levels of triacylglycerols, of which long-chain triacylglycerols associated (P<0.01) with CKD. This study revealed an amino acid imbalance affecting the basic cellular metabolism, disturbances in plasma lipid composition suggesting hepatic steatosis and fibrosis and novel metabolites correlating with CKD in LPI. In addition, the CKD-associated metabolite profile along with increased nitrite plasma levels suggests that LPI may be characterized by increased oxidative stress and apoptosis, altered microbial metabolism in the intestine and uremic toxicity. Copyright © 2016 Elsevier Inc. All rights reserved.

Apparent growth phase-dependent phosphorylation of malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a major fatty acid synthase II component in Mycobacterium bovis BCG.

PubMed

Sinha, Indrajit; Boon, Calvin; Dick, Thomas

2003-10-10

Probing protein extracts from exponentially growing and stationary phase cultures of Mycobacterium bovis BCG with anti-phospho amino acid antibodies revealed a 31-kDa anti-phospho threonine antibody-reactive protein specific to growing culture. The corresponding protein was purified via two-dimensional gel electrophoresis and identified via mass spectrometry to be malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a component of the fatty acid biosynthetic pathway. MCAT tagged with histidine reacted with anti-phospho threonine antibody and was positive in an in-gel chemical assay for phospho proteins. Analysis of the growth phase dependence of MCAT-His phosphorylation and protein levels showed that phosphorylated MCAT-His can be detected only in growing culture. In contrast, MCAT-His protein level was growth phase-independent. These results suggest that MCAT may be a substrate of a protein kinase and phosphatase, and that aspects of fatty acid synthesis in tubercle bacilli are regulated by protein phosphorylation.

Cloning and characteristics of fish glial fibrillary acidic protein: implications for optic nerve regeneration.

PubMed

Cohen, I; Shani, Y; Schwartz, M

1993-08-15

Mammalian central nervous system neurons do not regenerate after axonal injury, unlike their counterparts in fish and amphibians. After axonal injury, glial cells in mammals do not support regrowth of axons, while in fish they support the regeneration process. Controversy exists as to whether or not the intact fish optic nerve expresses glial fibrillary acidic protein, a well-known marker for mature astrocytes, and thus whether its astrocytes differ in this respect from those of the brain and spinal cord, as well as from optic nerve astrocytes of other species. In an attempt to resolve this question we cloned fish glial fibrillary acidic protein. Two different complementary DNA clones were isolated from a carp brain complementary DNA library, each encoding a different form of glial fibrillary acidic protein apparently originating from different genes. Monospecific polyclonal antibodies were raised against a peptide synthesized according to the predicted amino acid sequence, and used to identify and localize the fish glial fibrillary acidic protein. Two glial fibrillary acidic proteins (of 49 kDa and 51 kDa) were identified by the antibodies in all tested fish central nervous system tissues. The antibodies were then used to examine glial fibrillary acidic protein immunoreactivity in sections taken from uninjured and injured optic nerves of goldfish. Injury was followed by an elevation in glial fibrillary acidic protein immunoreactivity along the whole length of the nerve, except at the site of the injury, where--as in the case of vimentin--no immunoreactivity was detectable. However, in contrast to vimentin-positive glial cells, which repopulate the site of the injury soon after the optic nerve is injured, glial fibrillary acidic protein-positive glial cells remained outside the injury site for as long as 6 weeks after the injury. Despite the injury-induced changes in glial fibrillary acidic protein immunoreactivity, no change was observed in the level of transcript

Improving protein complex classification accuracy using amino acid composition profile.

PubMed

Huang, Chien-Hung; Chou, Szu-Yu; Ng, Ka-Lok

2013-09-01

Protein complex prediction approaches are based on the assumptions that complexes have dense protein-protein interactions and high functional similarity between their subunits. We investigated those assumptions by studying the subunits' interaction topology, sequence similarity and molecular function for human and yeast protein complexes. Inclusion of amino acids' physicochemical properties can provide better understanding of protein complex properties. Principal component analysis is carried out to determine the major features. Adopting amino acid composition profile information with the SVM classifier serves as an effective post-processing step for complexes classification. Improvement is based on primary sequence information only, which is easy to obtain. Copyright © 2013 Elsevier Ltd. All rights reserved.

Acid extraction and purification of recombinant spider silk proteins.

PubMed

Mello, Charlene M; Soares, Jason W; Arcidiacono, Steven; Butler, Michelle M

2004-01-01

A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers.

Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle.

PubMed

Reidy, P T; Walker, D K; Dickinson, J M; Gundermann, D M; Drummond, M J; Timmerman, K L; Cope, M B; Mukherjea, R; Jennings, K; Volpi, E; Rasmussen, B B

2014-06-01

Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. Copyright © 2014 the American Physiological Society.

Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

PubMed Central

Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.

2014-01-01

Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. PMID:24699854

Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa.

PubMed

Trevino, Saul R; Scholtz, J Martin; Pace, C Nick

2007-02-16

Poor protein solubility is a common problem in high-resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all 20 amino acids to protein solubility has not been done. Here, 20 variants at the completely solvent-exposed position 76 of ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II beta-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine.

Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa

PubMed Central

Trevino, Saul R.; Scholtz, J. Martin; Pace, C. Nick

2009-01-01

SUMMARY Poor protein solubility is a common problem in high resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all twenty amino acids to protein solubility has not been done. Here, twenty variants at the completely solvent-exposed position 76 of Ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II β-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine. PMID:17174328

Organization, chromosomal localization and promoter analysis of the gene encoding human acidic fibroblast growth factor intracellular binding protein.

PubMed Central

Kolpakova, E; Frengen, E; Stokke, T; Olsnes, S

2000-01-01

Acidic fibroblast growth factor (aFGF) intracellular binding protein (FIBP) is a protein found mainly in the nucleus that might be involved in the intracellular function of aFGF. Here we present a comparative analysis of the deduced amino acid sequences of human, murine and Drosophila FIBP analogues and demonstrate that FIBP is an evolutionarily conserved protein. The human gene spans more than 5 kb, comprising ten exons and nine introns, and maps to chromosome 11q13.1. Two slightly different splice variants found in different tissues were isolated and characterized. Sequence analysis of the region surrounding the translation start revealed a CpG island, a classical feature of widely expressed genes. Functional studies of the promoter region with a luciferase reporter system suggested a strong transcriptional activity residing within 600 bp of the 5' flanking region. PMID:11104667

Structural characterization and physicochemical properties of protein extracted from soybean meal assisted by steam flash-explosion with dilute acid soaking.

PubMed

Zhang, Yanpeng; Yang, Ruijin; Zhang, Weinong; Hu, Zhixiong; Zhao, Wei

2017-03-15

The aim of this work was to analyze the influence of steam flash-explosion (SFE) with dilute acid soaking pretreatment on the structural characteristics and physiochemical properties of protein from soybean meal (SBM). The pretreatment led to depolymerisation of soy protein isolate (SPI) and formation of new protein aggregation through non-disulfide covalent bonds, which resulted in broader MW distribution of SPI. The analysis of CD spectroscopy showed that the SFE treatment induced minor changes in secondary structure, however, the intrinsic tryptophan fluorescence revealed that acid soaking and SFE treatment pronouncedly altered the tertiary structure of SPI. The protein zeta potential was shown to be increased after SFE treatment attributed to the changes in protein structure and the covalent coupling between carbohydrate and protein. These results contribute to clarifying the mechanisms of the effect of pretreatment on SPI structure, thus moving further toward implementing SFE in the processing chain of SPI. Copyright © 2016 Elsevier Ltd. All rights reserved.

Endogenous fatty acids in olfactory hairs influence pheromone binding protein structure and function in Lymantria dispar.

PubMed

Nardella, Jason; Terrado, Mailyn; Honson, Nicolette S; Plettner, Erika

2015-08-01

The gypsy moth utilizes a pheromone, (7R,8S)-2-methyl-7,8-epoxyoctadecane, for mate location. The pheromone is detected by sensory hairs (sensilla) on the antennae of adult males. Sensilla contain the dendrites of olfactory neurons bathed in lymph, which contains pheromone binding proteins (PBPs). We have extracted and identified free fatty acids from lymph of sensory hairs, and we demonstrate that these function as endogenous ligands for gypsy moth PBP1 and PBP2. Homology modeling of both PBPs, and docking of fatty acids reveal multiple binding sites: one internal, the others external. Pheromone binding assays suggest that these fatty acids increase PBP-pheromone binding affinity. We show that fatty acid binding causes an increase in α-helix content in the N-terminal domain, but not in the C-terminal peptide of both proteins. The C-terminal peptide was shown to form a α-helix in a hydrophobic, homogeneous environment, but not in the presence of fatty acid micelles. Through partition assays we show that the fatty acids prevent adsorption of the pheromone on hydrophobic surfaces and facilitate pheromone partition into an aqueous phase. We propose that lymph is an emulsion of fatty acids and PBP that influence each other and thereby control the partition equilibria of hydrophobic odorants. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein and Amino Acid Requirements during Pregnancy123

PubMed Central

Elango, Rajavel; Ball, Ronald O

2016-01-01

Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg−1 · d−1, respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg−1 · d−1 during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14–18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. PMID:27422521

Neuromuscular electrical stimulation prior to presleep protein feeding stimulates the use of protein-derived amino acids for overnight muscle protein synthesis.

PubMed

Dirks, Marlou L; Groen, Bart B L; Franssen, Rinske; van Kranenburg, Janneau; van Loon, Luc J C

2017-01-01

Short periods of muscle disuse result in substantial skeletal muscle atrophy. Recently, we showed that both neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. In this study, we test our hypothesis that NMES can augment the use of presleep protein-derived amino acids for overnight muscle protein synthesis in older men. Twenty healthy, older [69 ± 1 (SE) yr] men were subjected to 24 h of bed rest, starting at 8:00 AM. In the evening, volunteers were subjected to 70-min 1-legged NMES, while the other leg served as nonstimulated control (CON). Immediately following NMES, 40 g of intrinsically l-[1- 13 C]-phenylalanine labeled protein was ingested prior to sleep. Blood samples were taken throughout the night, and muscle biopsies were obtained from both legs in the evening and the following morning (8 h after protein ingestion) to assess dietary protein-derived l-[1- 13 C]-phenylalanine enrichments in myofibrillar protein. Plasma phenylalanine concentrations and plasma l-[1- 13 C]-phenylalanine enrichments increased significantly following protein ingestion and remained elevated for up to 6 h after protein ingestion (P < 0.05). During overnight sleep, myofibrillar protein-bound l-[1- 13 C]-phenylalanine enrichments (MPE) increased to a greater extent in the stimulated compared with the control leg (0.0344 ± 0.0019 vs. 0.0297 ± 0.0016 MPE, respectively; P < 0.01), representing 18 ± 6% greater incorporation of presleep protein-derived amino acids in the NMES compared with CON leg. In conclusion, application of NMES prior to presleep protein feeding stimulates the use of dietary protein-derived amino acids for overnight muscle protein synthesis in older men. Neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. Here we demonstrate that in older

Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling.

PubMed

Trusov, Yuri; Sewelam, Nasser; Rookes, James Edward; Kunkel, Matt; Nowak, Ekaterina; Schenk, Peer Martin; Botella, José Ramón

2009-04-01

Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

The histone-like protein HU has a role in gene expression during the acid adaptation response in Helicobacter pylori.

PubMed

Álvarez, Alhejandra; Toledo, Héctor

2017-08-01

Gastritis, ulcers, and gastric malignancy have been linked to human gastric epithelial colonization by Helicobacter pylori. Characterization of the mechanisms by which H. pylori adapts to the human stomach environment is of crucial importance to understand H. pylori pathogenesis. In an effort to extend our knowledge of these mechanisms, we used proteomic analysis and qRT-PCR to characterize the role of the histone-like protein HU in the response of H. pylori to low pH. Proteomic analysis revealed that genes involved in chemotaxis, oxidative stress, or metabolism are under control of the HU protein. Also, expression of the virulence factors Ggt and NapA is affected by the null mutation of hup gene both at neutral and acid pH, as evidenced by qRT-PCR analysis. Those results showed that H. pylori gene expression is altered by shift to low pH, thus confirming that acid exposure leads to profound changes in genomic expression, and suggest that the HU protein is a regulator that may help the bacterium adapt to the acid stress. In accordance with previous reports, we found that the HU protein participates in gene expression regulation when the microorganism is exposed to acid stress. Such transcriptional regulation underlies protein accumulation in the H. pylori cell. © 2017 John Wiley & Sons Ltd.

Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

PubMed Central

Hellman, Lance M.; Fried, Michael G.

2009-01-01

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep

PubMed Central

Rozance, Paul J.; Thorn, Stephanie R.; Friedman, Jacob E.; Hay, William W.

2012-01-01

Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion. PMID:22649066

Relationship between Acute Phase Proteins and Serum Fatty Acid Composition in Morbidly Obese Patients

PubMed Central

Fernandes, Ricardo; Beserra, Bruna Teles Soares; Cunha, Raphael Salles Granato; Hillesheim, Elaine; Camargo, Carolina de Quadros; Pequito, Danielle Cristina Tonello; de Castro, Isabela Coelho; Fernandes, Luiz Cláudio; Nunes, Everson Araújo; Trindade, Erasmo Benício Santos de Moraes

2013-01-01

Background. Obesity is considered a low-grade inflammatory state and has been associated with increased acute phase proteins as well as changes in serum fatty acids. Few studies have assessed associations between acute phase proteins and serum fatty acids in morbidly obese patients. Objective. To investigate the relationship between acute phase proteins (C-Reactive Protein, Orosomucoid, and Albumin) and serum fatty acids in morbidly obese patients. Methods. Twenty-two morbidly obese patients were enrolled in this study. Biochemical and clinical data were obtained before bariatric surgery, and fatty acids measured in preoperative serum. Results. Orosomucoid was negatively correlated with lauric acid (P = 0.027) and eicosapentaenoic acid (EPA) (P = 0.037) and positively with arachidonic acid (AA) (P = 0.035), AA/EPA ratio (P = 0.005), and n-6/n-3 polyunsaturated fatty acids ratio (P = 0.035). C-Reactive Protein (CRP) was negatively correlated with lauric acid (P = 0.048), and both CRP and CRP/Albumin ratio were negatively correlated with margaric acid (P = 0.010, P = 0.008, resp.). Albumin was positively correlated with EPA (P = 0.027) and margaric acid (P = 0.008). Other correlations were not statistically significant. Conclusion. Our findings suggest that serum fatty acids are linked to acute phase proteins in morbidly obese patients. PMID:24167354

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui.

PubMed

Hatakeyama, T; Kimura, M

1988-03-15

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.

Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

PubMed

Marcoline, Frank V; Bethel, Neville; Guerriero, Christopher J; Brodsky, Jeffrey L; Grabe, Michael

2015-08-04

The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of tannic acid on the synthesis of protein and nucleic acid by rat liver

PubMed Central

Badawy, A. A.-B.; White, Audrey E.; Lathe, G. H.

1969-01-01

1. As early as 1hr. after the intraperitoneal administration of tannic acid to rats, it could be demonstrated in the liver. At 3hr. the nuclear fraction contained the largest amount of tannic acid. 2. Nuclear RNA synthesis was inhibited in vivo 2hr. after the administration of tannic acid. Induction by cortisol of tryptophan pyrrolase was 90% inhibited at 24hr. 3. Incorporation of [1-14C]leucine into protein by liver slices from treated rats was decreased by 50% after 24hr. Its incorporation into postmitochondrial supernatant from treated animals was not inhibited. Incorporation into slices and postmitochondrial supernatants were inhibited in vitro by tannic acid. 4. The sequence of events: concentration of tannic acid in nuclei, inhibition of nuclear RNA synthesis, inhibition of protein synthesis and production of necrosis, is discussed. PMID:5808319

Tauroursodeoxycholic acid binds to the G-protein site on light activated rhodopsin.

PubMed

Lobysheva, E; Taylor, C M; Marshall, G R; Kisselev, O G

2018-05-01

The heterotrimeric G-protein binding site on G-protein coupled receptors remains relatively unexplored regarding its potential as a new target of therapeutic intervention or as a secondary site of action by the existing drugs. Tauroursodeoxycholic acid bears structural resemblance to several compounds that were previously identified to specifically bind to the light-activated form of the visual receptor rhodopsin and to inhibit its activation of transducin. We show that TUDCA stabilizes the active form of rhodopsin, metarhodopsin II, and does not display the detergent-like effects of common amphiphilic compounds that share the cholesterol scaffold structure, such as deoxycholic acid. Computer docking of TUDCA to the model of light-activated rhodopsin revealed that it interacts using similar mode of binding to the C-terminal domain of transducin alpha subunit. The ring regions of TUDCA made hydrophobic contacts with loop 3 region of rhodopsin, while the tail of TUDCA is exposed to solvent. The results show that TUDCA interacts specifically with rhodopsin, which may contribute to its wide-ranging effects on retina physiology and as a potential therapeutic compound for retina degenerative diseases. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Nucleic acid chaperone activity of retroviral Gag proteins.

PubMed

Rein, Alan

2010-01-01

Retrovirus particles in which the Gag protein has not yet been cleaved by the viral protease are termed immature particles. The viral RNA within these particles shows clear evidence of the action of a nucleic acid chaperone (NAC): the genomic RNA is dimeric, and a cellular tRNA molecule is annealed, by its 3' 18 nucleotides, to a complementary stretch in the viral RNA, in preparation for priming reverse transcription in the next round of infection. It seems very likely that the NAC that has catalyzed dimerization and tRNA annealing is the NC domain of the Gag protein itself. However, neither the dimeric linkage nor the tRNA:viral RNA complex has the same structure as those in mature virus particles: thus the conformational effects of Gag within the particles are not equivalent to those of the free NC protein present in mature particles. It is not known whether these dissimilarities reflect intrinsic differences in the NAC activities of Gag and NC, or limitations on Gag imposed by the structure of the immature particle. Analysis of the interactions of recombinant Gag proteins with nucleic acids is complicated by the fact that they result in assembly of virus-like particles. Nevertheless, the available data indicates that the affinity of Gag for nucleic acids can be considerably higher than that of free NC. This enhanced affinity may be due to contributions of the matrix domain, a positively charged region at the N-terminus of Gag; interactions of neighboring Gag molecules with each other may also increase the affinity due to cooperativity of the binding. Recombinant HIV-1 Gag protein clearly exhibits NAC activity. In two well-studied experimental systems, Gag was more efficient than NC, as its NAC effects could be detected at a significantly lower molar ratio of protein to nucleotide than with NC. In one system, binding of nucleic acid by the matrix domain of Gag retarded the Gag-induced annealing of two RNAs; this effect could be ameliorated by the competitive

Folic acid-conjugated soybean protein-based nanoparticles mediate efficient antitumor ability in vitro.

PubMed

Yao, Weijing; Zha, Qian; Cheng, Xu; Wang, Xin; Wang, Jun; Tang, Rupei

2016-11-23

In this study, soy protein isolate was hydrolyzed by compound enzymes to give aqueous soy protein with low molecular weights. Folic acid modified and free soy protein nanoparticles were successfully prepared by a desolvation method as target-specific drug delivery, respectively. Ultraviolet spectrophotometry demonstrated that folic acid was successfully grafted onto soy protein. The shape and size of folic acid modified soy protein nanoparticles were detected by transmission electron microscopy, scanning electron microscope, and dynamic light scattering. In addition, a series of characteristics including kinetic stability, pH stability, and time stability were also performed. Doxorubicin was successfully loaded into folic acid modified soy protein nanoparticles, and the encapsulation and loading efficiencies were 96.7% and 23%, respectively. Doxorubicin-loaded folic acid modified soy protein nanoparticles exhibited faster drug release rate than soy protein nanoparticles in PBS solution (pH = 5). The tumor penetration and antitumor experiments were done using three-dimensional multicellular tumor spheroids as the in vitro model. The results proved that folic acid modified soy protein nanoparticles display higher penetration and accumulation than soy protein nanoparticles, therefore possessing efficient growth inhibitory ability against multicellular tumor spheroids. © The Author(s) 2016.

Role of Protein and Amino Acids in Infant and Young Child Nutrition: Protein and Amino Acid Needs and Relationship with Child Growth.

PubMed

Uauy, Ricardo; Kurpad, Anura; Tano-Debrah, Kwaku; Otoo, Gloria E; Aaron, Grant A; Toride, Yasuhiko; Ghosh, Shibani

2015-01-01

Over a third of all deaths of children under the age of five are linked to undernutrition. At a 90% coverage level, a core group of ten interventions inclusive of infant and young child nutrition could save one million lives of children under 5 y of age (15% of all deaths) (Lancet 2013). The infant and young child nutrition package alone could save over 220,000 lives in children under 5 y of age. High quality proteins (e.g. milk) in complementary, supplementary and rehabilitation food products have been found to be effective for good growth. Individual amino acids such as lysine and arginine have been found to be factors linked to growth hormone release in young children via the somatotropic axis and high intakes are inversely associated with fat mass index in pre-pubertal lean girls. Protein intake in early life is positively associated with height and weight at 10 y of age. This paper will focus on examining the role of protein and amino acids in infant and young child nutrition by examining protein and amino acid needs in early life and the subsequent relationship with stunting.

Proteolysis of chloroplast proteins is responsible for accumulation of free amino acids in dark-treated tea (Camellia sinensis) leaves.

PubMed

Chen, Yiyong; Fu, Xiumin; Mei, Xin; Zhou, Ying; Cheng, Sihua; Zeng, Lanting; Dong, Fang; Yang, Ziyin

2017-03-22

Shade management (dark treatment) on tea (Camellia sinensis) plants is a common approach to improve free amino acids in raw materials of tea leaves. However, the reason for amino acid accumulation in dark-treated tea leaves is still unknown. In the present study, dark treatment significantly increased content of free amino acids and reduced content of soluble proteins in tea leaves. Quantitative proteomics analysis showed that most enzymes involved in biosyntheses of amino acids were down-accumulated by dark treatment. Chloroplast numbers reduced in dark-treated leaves and the content of soluble proteins reduced in the chloroplasts isolated from dark-treated leaves compared to control. These suggest that proteolysis of chloroplast proteins contributed to amino acid accumulation in dark-treated leaves. Two chloroplasts proteases, ATP-dependent Clp protease proteolytic subunit 3 and protease Do-like 2, were up-accumulated in dark-treated leaves. This study firstly elucidated the mechanism of accumulation of amino acids in dark-treated tea leaves. Effect of dark on crop growth has been widely studied, while less attention has been paid to effect of dark on quality-related metabolites in crops. Shade management (dark treatment) on tea plants is a common approach to improve free amino acids in tea leaves. However, the reason for accumulation of free amino acids in dark-treated tea leaves is still unknown. In the present study, an iTRAQ-based quantitative proteomic analysis was performed and the results revealed the accumulation of free amino acids in dark-treated tea leaves was not due to activation of biosyntheses of amino acids, but resulted from proteolysis of chloroplast proteins. The information will advance our understanding of formation of quality or function-related metabolites in agricultural crops exposed to dark stress/shade management. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of antinutritional factors on protein digestibility and amino acid availability in foods.

PubMed

Gilani, G Sarwar; Cockell, Kevin A; Sepehr, Estatira

2005-01-01

Digestibility of protein in traditional diets from developing countries such as India, Guatemala, and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94%). The presence of less digestible protein fractions, high levels of insoluble fiber, and high concentrations of antinutritional factors in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, are responsible for poor digestibility of protein. The effects of the presence of some of the important antinutritional factors on protein and amino digestibilities of food and feed products are reviewed in this chapter. Food and feed products may contain a number of antinutritional factors that may adversely affect protein digestibility and amino acid availability. Antinutritional factors may occur naturally, such as glucosinolates in mustard and rapeseed protein products, trypsin inhibitors and hemagglutinins in legumes, tannins in legumes and cereals, phytates in cereals and oilseeds, and gossypol in cottonseed protein products. Antinutritional factors may also be formed during heat/alkaline processing of protein products, yielding Maillard compounds, oxidized forms of sulfur amino acids, D-amino acids, and lysinoalanine (LAL, an unnatural amino acid derivative). The presence of high levels of dietary trypsin inhibitors from soybeans, kidney beans, or other grain legumes can cause substantial reductions in protein and amino acid digestibilities (up to 50%) in rats and pigs. Similarly, the presence of high levels of tannins in cereals, such as sorghum, and grain legumes, such as fababean (Vicia faba L.), can result in significantly reduced protein and amino acid digestibilities (up to 23%) in rats, poultry, and pigs. Studies involving phytase supplementation of production rations for swine or poultry have provided indirect evidence that normally encountered levels of phytates in cereals and legumes

Omics approaches on fresh-cut lettuce reveal global molecular responses to sodium hypochlorite and peracetic acid treatment.

PubMed

Daddiego, Loretta; Bianco, Linda; Capodicasa, Cristina; Carbone, Fabrizio; Dalmastri, Claudia; Daroda, Lorenza; Del Fiore, Antonella; De Rossi, Patrizia; Di Carli, Mariasole; Donini, Marcello; Lopez, Loredana; Mengoni, Alessio; Paganin, Patrizia; Perrotta, Gaetano; Bevivino, Annamaria

2018-01-01

Lettuce is a leafy vegetable that is extensively commercialized as a ready-to-eat product because of its widespread use in human nutrition as salad. It is well known that washing treatments can severely affect the quality and shelf-life of ready-to-eat vegetables. The study presented here evaluated the effect of two washing procedures on fresh-cut lettuce during storage. An omics approach was applied to reveal global changes at molecular level induced by peracetic acid washing in comparison with sodium hypochlorite treatment. Microbiological analyses were also performed to quantify total bacterial abundance and composition. The study revealed wide metabolic alterations induced by the two sanitizers. In particular, transcriptomic and proteomic analyses pointed out a number of transcripts and proteins differentially accumulated in response to peracetic acid washing, mainly occurring on the first day of storage. In parallel, different microbiota composition and significant reduction in total bacterial load following washing were also observed. The results provide useful information for the fresh-cut industry to select an appropriate washing procedure preserving fresh-like attributes as much as possible during storage of the end product. Molecular evidence indicated peracetic acid to be a valid alternative to sodium hypochlorite as sanitizer solution. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

The peritumoural adipose tissue microenvironment and cancer. The roles of fatty acid binding protein 4 and fatty acid binding protein 5.

PubMed

Guaita-Esteruelas, S; Gumà, J; Masana, L; Borràs, J

2018-02-15

The adipose tissue microenvironment plays a key role in tumour initiation and progression because it provides fatty acids and adipokines to tumour cells. The fatty acid-binding protein (FABP) family is a group of small proteins that act as intracellular fatty acid transporters. Adipose-derived FABPs include FABP4 and FABP5. Both have an important role in lipid-related metabolic processes and overexpressed in many cancers, such as breast, prostate, colorectal and ovarian. Moreover, their expression in peritumoural adipose tissue is deregulated, and their circulating levels are upregulated in some tumours. In this review, we discuss the role of the peritumoural adipose tissue and the related adipokines FABP4 and FABP5 in cancer initiation and progression and the possible pathways implicated in these processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of Long-Term Protein Restriction on Meat Quality, Muscle Amino Acids, and Amino Acid Transporters in Pigs.

PubMed

Yin, Jie; Li, Yuying; Zhu, Xiaotong; Han, Hui; Ren, Wenkai; Chen, Shuai; Bin, Peng; Liu, Gang; Huang, Xingguo; Fang, Rejun; Wang, Bin; Wang, Kai; Sun, Liping; Li, Tiejun; Yin, Yulong

2017-10-25

This study aimed to investigate the long-term effects of protein restriction from piglets to finishing pigs for 16 weeks on meat quality, muscle amino acids, and amino acid transporters. Thirty-nine piglets were randomly divided into three groups: a control (20-18-16% crude protein, CP) and two protein restricted groups (17-15-13% CP and 14-12-10% CP). The results showed that severe protein restriction (14-12-10% CP) inhibited feed intake and body weight, while moderate protein restriction (17-15-13% CP) had little effect on growth performance in pigs. Meat quality (i.e., pH, color traits, marbling, water-holding capacity, and shearing force) were tested, and the results exhibited that 14-12-10% CP treatment markedly improved muscle marbling score and increased yellowness (b*). pH value (45 min) was significantly higher in 17-15-13% CP group than that in other groups. In addition, protein restriction reduced muscle histone, arginine, valine, and isoleucine abundances and enhanced glycine and lysine concentrations compared with the control group, while the RT-PCR results showed that protein restriction downregulated amino acids transporters. Mechanistic target of rapamycin (mTOR) signaling pathway was inactivated in the moderate protein restricted group (17-15-13% CP), while severe protein restriction with dietary 14-12-10% CP markedly enhanced mTOR phosphorylation. In conclusion, long-term protein restriction affected meat quality and muscle amino acid metabolism in pigs, which might be associated with mTOR signaling pathway.

Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins

PubMed Central

Fromont-Racine, Micheline; Mayes, Andrew E.; Brunet-Simon, Adeline; Rain, Jean-Christophe; Colley, Alan; Dix, Ian; Decourty, Laurence; Joly, Nicolas; Ricard, Florence; Beggs, Jean D.

2000-01-01

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale. PMID:10900456

Proteomic analysis of the intestinal adaptation response reveals altered expression of fatty acid binding proteins following massive small bowel resection.

PubMed

Stephens, Andrew N; Pereira-Fantini, Prue M; Wilson, Guineva; Taylor, Russell G; Rainczuk, Adam; Meehan, Katie L; Sourial, Magdy; Fuller, Peter J; Stanton, Peter G; Robertson, David M; Bines, Julie E

2010-03-05

Intestinal adaptation in response to the loss of the small intestine is essential to restore enteral autonomy in patients who have undergone massive small bowel resection (MSBR). In a proportion of patients, intestinal function is not restored, resulting in chronic intestinal failure (IF). Early referral of such patients for transplant provides the best prognosis; however, the molecular mechanisms underlying intestinal adaptation remain elusive and there is currently no convenient marker to predict whether patients will develop IF. We have investigated the adaptation response in a well-characterized porcine model of intestinal adaptation. 2D DIGE analysis of ileal epithelium from piglets recovering from massive small bowel resection (MSBR) identified over 60 proteins that changed specifically in MSBR animals relative to nonoperational or sham-operated controls. Three fatty acid binding proteins (L-FABP, FABP-6, and I-FABP) showed changes in MSBR animals. The expression changes and localization of each FABP were validated by immunoblotting and immunohistochemical analysis. FABP expression changes in MSBR animals occurred concurrently with altered triglyceride and bile acid metabolism as well as weight gain. The observed FABP expression changes in the ileal epithelium occur as part of the intestinal adaptation response and could provide a clinically useful marker to evaluate adaptation following MSBR.

Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

PubMed

D'Ovidio, Renato; Raiola, Alessandro; Capodicasa, Cristina; Devoto, Alessandra; Pontiggia, Daniela; Roberti, Serena; Galletti, Roberta; Conti, Eric; O'Sullivan, Donal; De Lorenzo, Giulia

2004-08-01

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2010-09-07

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2012-07-10

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Total amino acid stabilization during cell-free protein synthesis reactions.

PubMed

Calhoun, Kara A; Swartz, James R

2006-05-17

Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75-250% relative to cell-free expression using the control extract.

AFAL: a web service for profiling amino acids surrounding ligands in proteins

NASA Astrophysics Data System (ADS)

Arenas-Salinas, Mauricio; Ortega-Salazar, Samuel; Gonzales-Nilo, Fernando; Pohl, Ehmke; Holmes, David S.; Quatrini, Raquel

2014-11-01

With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate.

AFAL: a web service for profiling amino acids surrounding ligands in proteins.

PubMed

Arenas-Salinas, Mauricio; Ortega-Salazar, Samuel; Gonzales-Nilo, Fernando; Pohl, Ehmke; Holmes, David S; Quatrini, Raquel

2014-11-01

With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html ). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate.

Reasons for the occurrence of the twenty coded protein amino acids

NASA Technical Reports Server (NTRS)

Weber, A. L.; Miller, S. L.

1981-01-01

Factors involved in the selection of the 20 protein L-alpha-amino acids during chemical evolution and the early stages of Darwinian evolution are discussed. The selection is considered on the basis of the availability in the primitive ocean, function in proteins, the stability of the amino acid and its peptides, stability to racemization, and stability on the transfer RNA. It is concluded that aspartic acid, glutamic acid, arginine, lysine, serine and possibly threonine are the best choices for acidic, basic and hydroxy amino acids. The hydrophobic amino acids are reasonable choices, except for the puzzling absences of alpha-amino-n-butyric acid, norvaline and norleucine. The choices of the sulfur and aromatic amino acids seem reasonable, but are not compelling. Asparagine and glutamine are apparently not primitive. If life were to arise on another planet, it would be expected that the catalysts would be poly-alpha-amino acids and that about 75% of the amino acids would be the same as on the earth.

A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

PubMed Central

Martín-Acebes, Miguel A.; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

2013-01-01

West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses. PMID:23874963

Protein painting reveals solvent-excluded drug targets hidden within native protein–protein interfaces

PubMed Central

Luchini, Alessandra; Espina, Virginia; Liotta, Lance A.

2014-01-01

Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein–protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets. PMID:25048602

The Urine Preservative Acetic Acid Degrades Urine Protein: Implications for Urine Biorepositories and the AASK Cohort Study

PubMed Central

Almaani, Salem; Hebert, Lee A.; Rovin, Brad H.

2017-01-01

Patients enrolled in the African American Study of Kidney Disease and Hypertension (AASK) Cohort Study who exhibited overt proteinuria have been reported to show high nonalbumin proteinuria (NAP), which is characteristic of a tubulopathy. To determine whether African American Study of Kidney Disease and Hypertension nephropathy (AASK-N) is a tubulopathy, we obtained urine samples of 37 patients with AASK-N, with 24-hour protein-to-creatinine ratios (milligrams per milligram) ranging from 0.2 to 1.0, from the National Institute of Diabetes and Digestive Kidney Diseases repository and tested for seven markers of tubular proteinuria. By protocol, each sample had been collected in acetic acid (0.5%; mean final concentration). Compared with samples from patients with lupus nephritis or healthy black controls, AASK-N samples had lower amounts of six markers. Four markers (albumin, β-2-microglobulin, cystatin C, and osteopontin) were undetectable in most AASK-N samples. Examination by SDS-PAGE followed by protein staining revealed protein profiles indicative of severe protein degradation in 34 of 37 AASK-N urine samples. Treatment of lupus nephritis urine samples with 0.5% acetic acid produced the same protein degradation profile as that of AASK-N urine. We conclude that the increased NAP in AASK-N is an artifact of acetic acid–mediated degradation of albumin. The AASK-N repository urine samples have been compromised by the acetic acid preservative. PMID:28104821

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

PubMed

Reiz, Bela; Li, Liang

2010-09-01

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

PubMed Central

Shankaran, Mahalakshmi; King, Chelsea L.; Angel, Thomas E.; Holmes, William E.; Li, Kelvin W.; Colangelo, Marc; Price, John C.; Turner, Scott M.; Bell, Christopher; Hamilton, Karyn L.; Miller, Benjamin F.; Hellerstein, Marc K.

2015-01-01

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

Designing Light-Activated Charge-Separating Proteins with a Naphthoquinone Amino Acid

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

Lichtenstein, Bruce R.; Bialas, Chris; Cerda, José F.

2015-09-14

The first principles design of manmade redox-protein maquettes is used to clarify the physical/chemical engineering supporting the mechanisms of natural enzymes with a view to recapitulate and surpass natural performance. Herein, we use intein-based protein semisynthesis to pair a synthetic naphthoquinone amino acid (Naq) with histidine-ligated photoactive metal–tetrapyrrole cofactors, creating a 100 μs photochemical charge separation unit akin to photosynthetic reaction centers. By using propargyl groups to protect the redox-active para-quinone during synthesis and assembly while permitting selective activation, we gain the ability to employ the quinone amino acid redox cofactor with the full set of natural amino acids inmore » protein design. Direct anchoring of quinone to the protein backbone permits secure and adaptable control of intraprotein electron-tunneling distances and rates.« less

Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives.

PubMed

Zhang, Hua; Lu, Haiyan; Chingin, Konstantin; Chen, Huanwen

2015-07-21

Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

Engineering acyl carrier protein to enhance production of shortened fatty acids.

PubMed

Liu, Xueliang; Hicks, Wade M; Silver, Pamela A; Way, Jeffrey C

2016-01-01

The acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids. We constructed a homology model of the Synechococcus elongatus ACP, showing a hydrophobic pocket harboring the growing acyl chain. Amino acids within the pocket were mutated to increase steric hindrance to the acyl chain. Certain mutant ACPs, when over-expressed in Escherichia coli, increased the proportion of shorter chain lipids; I75 W and I75Y showed the strongest effects. Expression of I75 W and I75Y mutant ACPs also increased production of lauric acid in E. coli that expressed the C12-specific acyl-ACP thioesterase from Cuphea palustris. We engineered the specificity of the ACP, an essential protein of fatty acid metabolism, to alter the E. coli lipid pool and enhance production of medium-chain fatty acids as biofuel precursors. These results indicate that modification of ACP itself could be combined with enzymes affecting length specificity in fatty acid synthesis to enhance production of commodity chemicals based on fatty acids.

Overexpression of heart-type fatty acid binding protein enhances fatty acid-induced podocyte injury.

PubMed

Gao, Qing; Sarkar, Alhossain; Chen, Yizhi; Xu, Bo; Zhu, Xiaojuan; Yuan, Yang; Guan, Tianjun

2018-02-01

Deregulated lipid metabolism is a characteristic of metabolic diseases including type 2 diabetes and obesity, and likely contributes to podocyte injury and end-stage kidney disease. Heart-type fatty acid binding protein (H-FABP) was reported to be associated with lipid metabolism. The present study investigated whether H-FABP contributes to podocyte homeostasis. Podocytes were transfected by lentiviral vector to construct a cell line which stably overexpressed H-FABP. Small interfering RNA capable of effectively silencing H-FABP was introduced into podocytes to construct a cell line with H-FABP knockdown. Certain groups were treated with palmitic acid (PA) and the fat metabolism, as well as inflammatory and oxidative stress markers were measured. PA accelerated lipid metabolism derangement, inflammatory reaction and oxidative stress in podocytes. Overexpression of H-FABP enhanced the PA-induced disequilibrium in podocytes. The mRNA and protein expression levels of acyl-coenzyme A oxidase 3 and monocyte chemotactic protein 1, and the protein expression levels of 8-hydroxy-2'-deoxyguanosine and 4-hydroxynonenal were upregulated in the H-FABP overexpression group, while the mRNA and protein expression of peroxisome proliferator activated receptor α was downregulated. Knockdown of H-FABP inhibited the PA-induced injury and lipid metabolism derangement, as well as the inflammatory reaction and oxidative stress in podocytes. These results indicated that overexpression of H-FABP enhances fatty acid-induced podocyte injury, while H-FABP inhibition may represent a potential therapeutic strategy for the prevention of lipid metabolism-associated podocyte injury.

A simple atomic-level hydrophobicity scale reveals protein interfacial structure.

PubMed

Kapcha, Lauren H; Rossky, Peter J

2014-01-23

Many amino acid residue hydrophobicity scales have been created in an effort to better understand and rapidly characterize water-protein interactions based only on protein structure and sequence. There is surprisingly low consistency in the ranking of residue hydrophobicity between scales, and their ability to provide insightful characterization varies substantially across subject proteins. All current scales characterize hydrophobicity based on entire amino acid residue units. We introduce a simple binary but atomic-level hydrophobicity scale that allows for the classification of polar and non-polar moieties within single residues, including backbone atoms. This simple scale is first shown to capture the anticipated hydrophobic character for those whole residues that align in classification among most scales. Examination of a set of protein binding interfaces establishes good agreement between residue-based and atomic-level descriptions of hydrophobicity for five residues, while the remaining residues produce discrepancies. We then show that the atomistic scale properly classifies the hydrophobicity of functionally important regions where residue-based scales fail. To illustrate the utility of the new approach, we show that the atomic-level scale rationalizes the hydration of two hydrophobic pockets and the presence of a void in a third pocket within a single protein and that it appropriately classifies all of the functionally important hydrophilic sites within two otherwise hydrophobic pores. We suggest that an atomic level of detail is, in general, necessary for the reliable depiction of hydrophobicity for all protein surfaces. The present formulation can be implemented simply in a manner no more complex than current residue-based approaches. © 2013.

Effect of acid- and alkaline-aided extractions on functional and rheological properties of proteins recovered from mechanically separated turkey meat (MSTM).

PubMed

Hrynets, Yuliya; Omana, Dileep A; Xu, Yan; Betti, Mirko

2010-09-01

Functional and rheological characteristics of acid- and alkali-extracted proteins from mechanically separated turkey meat (MSTM) have been investigated. Extractions were carried out at 4 pH values (2.5, 3.5, 10.5, and 11.5). The study demonstrated that alkali and acid extractions resulted in significant (P < 0.0001) decreases of cooking and water loss compared to raw MSTM; however, the cooking loss was found to be similar (P = 0.5699) among the different protein isolates. Proteins extracted at pH 10.5 showed the lowest (P = 0.0249) water loss. Emulsion and foaming properties were found to be slightly higher in alkali-extracted proteins compared to those for acid extractions. The myofibrillar protein fraction showed better ability to form and stabilize emulsions compared to sarcoplasmic proteins. Myofibrillar proteins also showed better foam expansion; however, foam volume stability was similar for both myofibrillar and sarcoplasmic protein fractions. Textural characteristics (hardness, chewiness, springiness, and cohesiveness) of recovered proteins were found to be unaffected (P > 0.05) by different extraction pH. The protein extracted at pH 3.5 formed a highly viscoelastic gel network as evidenced by storage modulus (G') values, whereas the gel formed from proteins extracted at pH 10.5 was found to be the weakest. The work also revealed that acid treatments were more effective for removal of total heme pigments from MSTM. Color characteristics of protein isolates were markedly improved compared to the initial material and tended to be better when subjected to acid extractions. Mechanically separated meat is one of the cheapest sources of protein obtained by grinding meat and bones together and forcing the mixture through a perforated drum. The use of mechanically separated turkey meat (MSTM) for the production of further processed poultry products is limited due to its undesirable color and textural properties. Recovery of proteins from MSTM using pH shifting

Structural and functional interaction of fatty acids with human liver fatty acid-binding protein (L-FABP) T94A variant.

PubMed

Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Landrock, Kerstin K; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

2014-05-01

The human liver fatty acid-binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride levels. How this amino acid substitution elicits these effects is not known. This issue was addressed using human recombinant wild-type (WT) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC and CC). The T94A substitution did not alter or only slightly altered L-FABP binding affinities for saturated, monounsaturated or polyunsaturated long chain fatty acids, nor did it change the affinity for intermediates of triglyceride synthesis. Nevertheless, the T94A substitution markedly altered the secondary structural response of L-FABP induced by binding long chain fatty acids or intermediates of triglyceride synthesis. Finally, the T94A substitution markedly decreased the levels of induction of peroxisome proliferator-activated receptor α-regulated proteins such as L-FABP, fatty acid transport protein 5 and peroxisome proliferator-activated receptor α itself meditated by the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in cultured primary human hepatocytes. Thus, although the T94A substitution did not alter the affinity of human L-FABP for long chain fatty acids, it significantly altered human L-FABP structure and stability, as well as the conformational and functional response to these ligands. © 2014 FEBS.

Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

PubMed

González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

2015-01-01

Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

PubMed

Gai, Yingping; Yuan, Shuo-Shuo; Liu, Zhao-Yang; Zhao, Huai-Ning; Liu, Qi; Qin, Yong-Li; Fang, Li-Jing; Ji, Xian-Ling

2018-05-30

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signalling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 ( MuMLPL329 ) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 ( Pst. DC3000 ) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Towards the elucidation of molecular determinants of cooperativity in the liver bile acid binding protein.

PubMed

Pedò, Massimo; D'Onofrio, Mariapina; Ferranti, Pasquale; Molinari, Henriette; Assfalg, Michael

2009-11-15

Bile acid binding proteins (BABPs) are cytosolic lipid chaperones contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Liver BABPs act in parallel with ileal transporters to ensure vectorial transport of bile salts in hepatocytes and enterocytes, respectively. We describe the investigation of ligand binding to liver BABP, an essential step in the understanding of intracellular bile salt transport. Binding site occupancies were monitored in NMR titration experiments using (15)N-labelled ligand, while the relative populations of differently bound BABP forms were assessed by mass spectrometry. This site-specific information allowed the determination of intrinsic thermodynamic parameters and the identification of an extremely high cooperativity between two binding sites. Protein-observed NMR experiments revealed a global structural rearrangement which suggests an allosteric mechanism at the basis of the observed cooperativity. The view of a molecular tool capable of buffering against significant concentrations of free bile salts in a large range of solution conditions emerges from the observed pH-dependence of binding. We set to determine the molecular determinants of cooperativity by analysing the binding properties of a protein containing a mutated internal histidine. Both mass spectrometry and NMR experiments are consistent with an overall decreased binding affinity of the mutant, while the measured diffusion coefficients of ligand species reveal that the affinity loss concerns essentially one of the two binding sites. We therefore identified a mutation able to disrupt energetic communication functional to efficient binding and conclude that the buried histidine establishes contacts that stabilize the ternary complex. 2009 Wiley-Liss, Inc.

Sequence characterization of S100A8 gene reveals structural differences of protein and transcriptional factor binding sites in water buffalo and yak.

PubMed

Kathiravan, P; Goyal, S; Kataria, R S; Mishra, B P; Jayakumar, S; Joshi, B K

2011-01-01

The present study was undertaken to characterize the structure of S100A8 gene and its promoter in water buffalo and yak. Sequence data of 2.067 kb, 2.071 kb, and 2.052 kb with respect to complete S100A8 gene including 5' flanking region was generated in river buffalo, swamp buffalo, and yak, respectively. BLAST analysis of coding DNA sequences (CDS) of S100A8 gene revealed 95% homology of buffalo sequence with cattle, 85% with pig and horse, 83% with dog, 72-73% with murines, and around 79% with primates and humans. Phylogenetic analysis of predicted CDS revealed distinct clustering of murines, primates, and domestic animals with bovines and bubalines forming a subcluster among farm animals. In silico translation of predicted CDS revealed a sequence of 89 amino acids with 7 amino acid changes between cattle and buffalo and 2 changes between cattle and yak. The search for Pfam family revealed the N-terminal calcium binding domain and the noncanonical EF hand domain in the carboxy terminus, with more variations being observed in the N-terminal domain among different species. Two amino acid changes observed in carboxy terminal EF hand domain resulted in altered secondary structure of yak S100A8 protein. Analysis of S100A8 gene promoter revealed 14 putative motifs for transcriptional factor binding sites. Two putative motifs viz. C/EBP and v-Myb were found to be absent in swamp buffalo as compared to river buffalo and cattle. Differences in the structure of S100A8 protein and the transcriptional factor binding sites identified in the present study need to be analyzed further for their functional significance in yak and swamp buffalo respectively. Copyright © Taylor & Francis Group, LLC

Chlorogenic Acid Decreases Intestinal Permeability and Increases Expression of Intestinal Tight Junction Proteins in Weaned Rats Challenged with LPS

PubMed Central

Ruan, Zheng; Liu, Shiqiang; Zhou, Yan; Mi, Shumei; Liu, Gang; Wu, Xin; Yao, Kang; Assaad, Houssein; Deng, Zeyuan; Hou, Yongqing; Wu, Guoyao; Yin, Yulong

2014-01-01

Chlorogenic acid, a natural phenolic acid present in fruits and plants, provides beneficial effects for human health. The objectives of this study were to investigate whether chlorogenic acid (CHA) could improve the intestinal barrier integrity for weaned rats with lipopolysaccharide (LPS) challenge. Thirty-two weaned male Sprague Dawley rats (21±1 d of age; 62.26±2.73 g) were selected and randomly allotted to four treatments, including weaned rat control, LPS-challenged and chlorogenic acid (CHA) supplemented group (orally 20 mg/kg and 50 mg/kg body). Dietary supplementation with CHA decreased (P<0.05) the concentrations of urea and albumin in the serum, compared to the LPS-challenged group. The levels of IFN-γ and TNF-α were lower (P<0.05) in the jejunal and colon of weaned rats receiving CHA supplementation, in comparison with the control group. CHA supplementation increased (P<0.05) villus height and the ratio of villus height to crypt depth in the jejunal and ileal mucosae under condictions of LPS challenge. CHA supplementation decreased (P<0.05) intestinal permeability, which was indicated by the ratio of lactulose to mannitol and serum DAO activity, when compared to weaned rats with LPS challenge. Immunohistochemical analysis of tight junction proteins revealed that ZO-1 and occludin protein abundances in the jejunum and colon were increased (P<0.05) by CHA supplementation. Additionally, results of immunoblot analysis revealed that the amount of occludin in the colon was also increased (P<0.05) in CHA-supplemented rats. In conclusion, CHA decreases intestinal permeability and increases intestinal expression of tight junction proteins in weaned rats challenged with LPS. PMID:24887396

Thermal stability and gel quality of myofibrillar protein as affected by soy protein isolates subjected to an acidic pH and mild heating.

PubMed

Niu, Haili; Xia, Xiufang; Wang, Chao; Kong, Baohua; Liu, Qian

2018-03-01

Thermal stability and gel quality of myofibrillar protein were evaluated with regard to the addition of native soy protein isolates (SPI) and SPI subjected to acidic pH and mild heating (modified SPI). Compared with the control, the addition of modified SPI increased the compression force of the protein gel and decreased water loss (P<0.05). Differential scanning calorimetry results showed that an addition of 0.75% native SPI decreased the first transition temperature (P<0.05), and addition of 0.5% and 0.75% modified SPI exhibited no appreciable changes on it (P>0.05), indicating that a higher concentration of modified SPI would not damage the protein thermal stability. Moreover, the addition of modified SPI enhanced hydrogen bonding and disulphide linkages. Atomic force microscopy analysis revealed that the addition of modified SPI decreased the roughness of the mixed myofibrillar protein gels. Overall, modified SPI has the potential to improve myofibrillar protein gel texture and water holding capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tolerance to acetic acid is improved by mutations of the TATA-binding protein gene.

PubMed

An, Jieun; Kwon, Hyeji; Kim, Eunjung; Lee, Young Mi; Ko, Hyeok Jin; Park, Hongjae; Choi, In-Geol; Kim, Sooah; Kim, Kyoung Heon; Kim, Wankee; Choi, Wonja

2015-03-01

Screening a library of overexpressing mutant alleles of the TATA-binding gene SPT15 yielded two Saccharomyces cerevisiae strains (MRRC 3252 and 3253) with enhanced tolerance to acetic acid. They were also tolerant to propionic acid and hydrogen peroxide. Transcriptome profile analysis identified 58 upregulated genes and 106 downregulated genes in MRRC 3252. Stress- and protein synthesis-related transcription factors were predominantly enriched in the upregulated and downregulated genes respectively. Eight deletion mutants for some of the highly downregulated genes were acetic acid-tolerant. The level of intracellular reactive oxygen species was considerably lessened in MRRC 3252 and 3253 upon exposure to acetic acid. Metabolome profile analysis revealed that intracellular concentrations of 5 and 102 metabolites were increased and decreased, respectively, in MRRC 3252, featuring a large increase of urea and a significant decrease of amino acids. The dur1/2Δmutant, in which the urea degradation gene DUR1/2 is deleted, displayed enhanced tolerance to acetic acid. Enhanced tolerance to acetic acid was also observed on the medium containing a low concentration of amino acids. Taken together, this study identified two SPT15 alleles, nine gene deletions and low concentration of amino acids in the medium that confer enhanced tolerance to acetic acid. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids.

PubMed

Villa, Jordan K; Tran, Hong-Anh; Vipani, Megha; Gianturco, Stephanie; Bhasin, Konark; Russell, Brent L; Harbron, Elizabeth J; Young, Douglas D

2017-07-16

The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2013-03-12

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel sythetases molecules, methods for identifying and making the novel synthetases, methods for producing containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lapidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

PubMed

Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

2008-02-15

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. Copyright 2007 Wiley-Liss, Inc.

Influence of the Amino Acid Sequence on Protein-Mineral Interactions in Soil

NASA Astrophysics Data System (ADS)

Chacon, S. S.; Reardon, P. N.; Purvine, S.; Lipton, M. S.; Washton, N.; Kleber, M.

2017-12-01

The intimate associations between protein and mineral surfaces have profound impacts on nutrient cycling in soil. Proteins are an important source of organic C and N, and a subset of proteins, extracellular enzymes (EE), can catalyze the depolymerization of soil organic matter (SOM). Our goal was to determine how variation in the amino acid sequence could influence a protein's susceptibility to become chemically altered by mineral surfaces to infer the fate of adsorbed EE function in soil. We hypothesized that (1) addition of charged amino acids would enhance the adsorption onto oppositely charged mineral surfaces (2) addition of aromatic amino acids would increase adsorption onto zero charged surfaces (3) Increase adsorption of modified proteins would enhance their susceptibility to alterations by redox active minerals. To test these hypotheses, we generated three engineered proxies of a model protein Gb1 (IEP 4.0, 6.2 kDA) by inserting either negatively charged, positively charged or aromatic amino acids in the second loop. These modified proteins were allowed to interact with functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnessite) at pH 5 and 7. We used LC-MS/MS and solution-state Heteronuclear Single Quantum Coherence Spectroscopy NMR to observe modifications on engineered proteins as a consequence to mineral interactions. Preliminary results indicate that addition of any amino acids to a protein increase its susceptibility to fragmentation and oxidation by redox active mineral surfaces, and alter adsorption to the other mineral surfaces. This suggest that not all mineral surfaces in soil may act as sorbents for EEs and chemical modification of their structure should also be considered as an explanation for decrease in EE activity. Fragmentation of proteins by minerals can bypass the need to produce proteases, but microbial acquisition of other nutrients that require enzymes such as cellulases, ligninases or phosphatases

A sensitive method for measuring protein turnover based on the measurement of 2-3H-labelled amino acids in protein.

PubMed Central

Humphrey, T J; Davies, D D

1976-01-01

A method for measuring the rate of protein degradation is described. The method measures the change in 2-3H content of protein with time by racemization of the protein hydrolysate with acetic anhydride. The 3H on C-2 of amino acids is stable in proteins but becomes labile, owing to the action of transaminases, once the amino acids are released by proteolysis. The specific measurement of 2-3H in amino acids largely overcomes problems due to compartmentation and isotope recycling and evidence to support this claim is presented. Values for the half-life of Lemna minor (duckweed) protein determined by the new method are compared with values obtained by other methods. PMID:949338

Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

PubMed

Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

2017-08-01

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

pH shift protein recovery with organic acids on texture and color of cooked gels.

PubMed

Paker, Ilgin; Beamer, Sarah; Jaczynski, Jacek; Matak, Kristen E

2015-01-01

Isoelectric solubilization and precipitation (ISP) processing uses pH shifts to separate protein from fish frames, which may increase commercial interest for silver carp. Texture and color properties of gels made from silver carp protein recovered at different pH strategies and organic acid types were compared. ISP was applied to headed gutted silver carp using 10 mol L(-1) sodium hydroxide (NaOH) and either glacial acetic acid (AA) or a (1:1) formic and lactic acid combination (F&L). Protein gels were made with recovered protein and standard functional additives. Texture profile analysis and the Kramer shear test showed that protein gels made from protein solubilized at basic pH values were firmer, harder, more cohesive, gummier and chewier (P < 0.05) than proteins solubilized under acidic conditions. Acidic solubilization led to whiter (P < 0.05) gels, and using F&L during ISP yielded whiter gels under all treatments (P < 0.05). Gels made from ISP-recovered silver carp protein using organic acids show potential for use as a functional ingredient in restructured foods. © 2014 Society of Chemical Industry.

Characterization of the Complex Locus of Bean Encoding Polygalacturonase-Inhibiting Proteins Reveals Subfunctionalization for Defense against Fungi and Insects1

PubMed Central

D'Ovidio, Renato; Raiola, Alessandro; Capodicasa, Cristina; Devoto, Alessandra; Pontiggia, Daniela; Roberti, Serena; Galletti, Roberta; Conti, Eric; O'Sullivan, Donal; De Lorenzo, Giulia

2004-01-01

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects. PMID:15299124

Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

PubMed Central

Hesse, Almut

2016-01-01

Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

An OmpA family protein, a target of the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius, controls acetic acid fermentation.

PubMed

Iida, Aya; Ohnishi, Yasuo; Horinouchi, Sueharu

2008-07-01

Via N-acylhomoserine lactones, the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius NCI1051, a gram-negative acetic acid bacterium, represses acetic acid and gluconic acid fermentation. Two-dimensional polyacrylamide gel electrophoretic analysis of protein profiles of strain NCI1051 and ginI and ginR mutants identified a protein that was produced in response to the GinI/GinR regulatory system. Cloning and nucleotide sequencing of the gene encoding this protein revealed that it encoded an OmpA family protein, named GmpA. gmpA was a member of the gene cluster containing three adjacent homologous genes, gmpA to gmpC, the organization of which appeared to be unique to vinegar producers, including "Gluconacetobacter polyoxogenes." In addition, GmpA was unique among the OmpA family proteins in that its N-terminal membrane domain forming eight antiparallel transmembrane beta-strands contained an extra sequence in one of the surface-exposed loops. Transcriptional analysis showed that only gmpA of the three adjacent gmp genes was activated by the GinI/GinR quorum-sensing system. However, gmpA was not controlled directly by GinR but was controlled by an 89-amino-acid protein, GinA, a target of this quorum-sensing system. A gmpA mutant grew more rapidly in the presence of 2% (vol/vol) ethanol and accumulated acetic acid and gluconic acid in greater final yields than strain NCI1051. Thus, GmpA plays a role in repressing oxidative fermentation, including acetic acid fermentation, which is unique to acetic acid bacteria and allows ATP synthesis via ethanol oxidation. Consistent with the involvement of gmpA in oxidative fermentation, its transcription was also enhanced by ethanol and acetic acid.

STRUCTURAL AND FUNCTIONAL INTERACTION OF FATTY ACIDS WITH HUMAN LIVER FATTY ACID BINDING PROTEIN (L-FABP) T94A VARIANT

PubMed Central

Huang, Huan; McIntosh, Avery L.; Martin, Gregory G.; Landrock, Kerstin K.; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P.; Kier, Ann B.; Schroeder, Friedhelm

2014-01-01

The human liver fatty acid binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride (TG) levels. How this amino acid substitution elicits these effects is not known. This issue was addressed with human recombinant wild-type (WT, T94T) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC, and CC). T94A substitution did not or only slightly alter L-FABP binding affinities for saturated, monounsaturated, or polyunsaturated long chain fatty acids (LCFA), nor did it change the affinity for intermediates in TG synthesis. Nevertheless, T94A substitution markedly altered the secondary structural response of L-FABP induced by binding LCFA or intermediates of TG synthesis. Finally, T94A substitution markedly diminished polyunsaturated fatty acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), induction of peroxisome proliferator-activated receptor alpha (PPARα) - regulated proteins such as L-FABP, fatty acid transport protein 5 (FATP5), and PPARα itself in cultured primary human hepatocytes. Thus, while T94A substitution did not alter the affinity of human L-FABP for LCFAs, it significantly altered human L-FABP structure and stability as well as conformational and functional response to these ligands. PMID:24628888

Atomic force microscopy reveals the mechanical design of a modular protein

PubMed Central

Li, Hongbin; Oberhauser, Andres F.; Fowler, Susan B.; Clarke, Jane; Fernandez, Julio M.

2000-01-01

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering. PMID:10823913

Atomic force microscopy reveals the mechanical design of a modular protein.

PubMed

Li, H; Oberhauser, A F; Fowler, S B; Clarke, J; Fernandez, J M

2000-06-06

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering.

Overexpression of heart-type fatty acid binding protein enhances fatty acid-induced podocyte injury

PubMed Central

Gao, Qing; Sarkar, Alhossain; Chen, Yizhi; Xu, Bo; Zhu, Xiaojuan; Yuan, Yang; Guan, Tianjun

2018-01-01

Deregulated lipid metabolism is a characteristic of metabolic diseases including type 2 diabetes and obesity, and likely contributes to podocyte injury and end-stage kidney disease. Heart-type fatty acid binding protein (H-FABP) was reported to be associated with lipid metabolism. The present study investigated whether H-FABP contributes to podocyte homeostasis. Podocytes were transfected by lentiviral vector to construct a cell line which stably overexpressed H-FABP. Small interfering RNA capable of effectively silencing H-FABP was introduced into podocytes to construct a cell line with H-FABP knockdown. Certain groups were treated with palmitic acid (PA) and the fat metabolism, as well as inflammatory and oxidative stress markers were measured. PA accelerated lipid metabolism derangement, inflammatory reaction and oxidative stress in podocytes. Overexpression of H-FABP enhanced the PA-induced disequilibrium in podocytes. The mRNA and protein expression levels of acyl-coenzyme A oxidase 3 and monocyte chemotactic protein 1, and the protein expression levels of 8-hydroxy-2′-deoxyguanosine and 4-hydroxynonenal were upregulated in the H-FABP overexpression group, while the mRNA and protein expression of peroxisome proliferator activated receptor α was downregulated. Knockdown of H-FABP inhibited the PA-induced injury and lipid metabolism derangement, as well as the inflammatory reaction and oxidative stress in podocytes. These results indicated that overexpression of H-FABP enhances fatty acid-induced podocyte injury, while H-FABP inhibition may represent a potential therapeutic strategy for the prevention of lipid metabolism-associated podocyte injury. PMID:29434805

Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

DOE PAGES

Smith, Steven D.; Bridou, Romain; Johs, Alexander; ...

2015-02-27

Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation

PubMed Central

Epperson, L. Elaine; Rose, James C.; Carey, Hannah V.

2010-01-01

Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast. PMID:19923364

[Analysis of proteins, amino acids and inorganic elements in Holotrichia diomphalia from different areas].

PubMed

Cao, Wei; Liu, Dan; Zhang, Yi-Kai; Wang, Xiao-Yu; Chang, Yan-Rong; Yang, Qian; Wang, Si-Wang

2010-10-01

To analyze the content of proteins,amino acids and inorganic elements of Holotrichia diomphalia in different growing areas as the references for quality evaluation and reasonable application of them. The contents of proteins were determined using semi-micro Kjeldahl method. The contents of seventeen amino acids and inorganic elements were determined with amino acid analyzer and atomic absorption spectrometer and elemental analyzer, respectively. The contents of protein were 33.4%-44.4%, and that in Jiangxi were the highest in five different areas. There were seventeen kinds of amino acids in Holotrichia diomphalia. Among them, seven amino acids were essential to human life. The content of glutamic acid was the highest in seventeen amino acids. In inorganic elements, the content of Mg, Ca was higher in macroelements and Fe, Zn was higher in microelements. There are many kinds of necessary amino acids and inorganic elements for man kind in Holotrichia diomphalia. The contents of proteins, amino acids and inorganic elements have some difference in Holotrichia diomphalia of different growing areas.

Comparative characterization of random-sequence proteins consisting of 5, 12, and 20 kinds of amino acids

PubMed Central

Tanaka, Junko; Doi, Nobuhide; Takashima, Hideaki; Yanagawa, Hiroshi

2010-01-01

Screening of functional proteins from a random-sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random-sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random-sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random-sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279–284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120-amino acid, random-sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random-sequence proteins arbitrarily chosen from these libraries. We found that random-sequence proteins constructed with the 12-member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20-member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids. PMID:20162614

Multi‐omic profiling ­of EPO‐producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

PubMed Central

Ley, Daniel; Seresht, Ali Kazemi; Engmark, Mikael; Magdenoska, Olivera; Nielsen, Kristian Fog; Kildegaard, Helene Faustrup

2015-01-01

ABSTRACT Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi‐omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO‐K1 cells under growth‐limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO‐producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)+, adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT‐PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)+ and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post‐translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time‐course analysis of high‐ and low‐producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID

Cellular Fatty Acid Composition, Soluble-Protein Profile, and Antimicrobial Resistance Pattern of Eubacterium lentum

PubMed Central

Mosca, Adriana; Summanen, Paula; Finegold, Sydney M.; De Michele, Giampiero; Miragliotta, Giuseppe

1998-01-01

Phenotypic heterogeneity among isolates of Eubacterium lentum has been recognized for many years. To better delineate their taxonomic relatedness, 29 clinical isolates of E. lentum were examined for soluble-protein content, cellular fatty acid profile, and antimicrobial resistance pattern in order to ascertain whether differences in these characteristics could be correlated with differences in biochemical activities. Among 29 isolates we could identify 6 that were different from all the others. These strains were coccobacilli with translucent colonies; they were catalase and H2S negative, not fluorescent under UV light, and susceptible to beta-lactam drugs; growth was not stimulated by arginine; and fatty acid analysis revealed the presence of straight-chain fatty acids. The remainder of the strains, including the type species, were pleomorphic bacilli with speckled colonies and were catalase and H2S positive; all but two were fluorescent under UV light; they were resistant to beta-lactam antibiotics; growth was greatly stimulated by arginine; and they demonstrated saturated branched-chain fatty acids. Our data suggest that E. lentum can be further differentiated into different types. PMID:9508307

Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5

PubMed Central

Ladurner, Angela; Zehl, Martin; Grienke, Ulrike; Hofstadler, Christoph; Faur, Nadina; Pereira, Fátima C.; Berry, David; Dirsch, Verena M.; Rollinger, Judith M.

2017-01-01

Worldwide, metabolic diseases such as obesity and type 2 diabetes have reached epidemic proportions. A major regulator of metabolic processes that gained interest in recent years is the bile acid receptor TGR5 (Takeda G protein-coupled receptor 5). This G protein-coupled membrane receptor can be found predominantly in the intestine, where it is mainly responsible for the secretion of the incretins glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). The aim of this study was (i) to identify plant extracts with TGR5-activating potential, (ii) to narrow down their activity to the responsible constituents, and (iii) to assess whether the intestinal microbiota produces transformed metabolites with a different activity profile. Chenodeoxycholic acid (CDCA) served as positive control for both, the applied cell-based luciferase reporter gene assay for TGR5 activity and the biotransformation assay using mouse fecal slurry. The suitability of the workflow was demonstrated by the biotransformation of CDCA to lithocholic acid resulting in a distinct increase in TGR5 activity. Based on a traditional Tibetan formula, 19 plant extracts were selected and investigated for TGR5 activation. Extracts from the commonly used spices Syzygium aromaticum (SaroE, clove), Pimenta dioica (PdioE, allspice), and Kaempferia galanga (KgalE, aromatic ginger) significantly increased TGR5 activity. After biotransformation, only KgalE showed significant differences in its metabolite profile, which, however, did not alter its TGR5 activity compared to non-transformed KgalE. UHPLC-HRMS (high-resolution mass spectrometry) analysis revealed triterpene acids (TTAs) as the main constituents of the extracts SaroE and PdioE. Identification and quantification of TTAs in these two extracts as well as comparison of their TGR5 activity with reconstituted TTA mixtures allowed the attribution of the TGR5 activity to TTAs. EC50s were determined for the main TTAs, i.e., oleanolic acid (2.2 ± 1.6 μM), ursolic

Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

PubMed Central

Mendoza, Vanessa Leah; Vachet, Richard W.

2009-01-01

For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS).

PubMed

Mathai, John K; Liu, Yanhong; Stein, Hans H

2017-02-01

An experiment was conducted to compare values for digestible indispensable amino acid scores (DIAAS) for four animal proteins and four plant proteins with values calculated as recommended for protein digestibility-corrected amino acid scores (PDCAAS), but determined in pigs instead of in rats. Values for standardised total tract digestibility (STTD) of crude protein (CP) and standardised ileal digestibility (SID) of amino acids (AA) were calculated for whey protein isolate (WPI), whey protein concentrate (WPC), milk protein concentrate (MPC), skimmed milk powder (SMP), pea protein concentrate (PPC), soya protein isolate (SPI), soya flour and whole-grain wheat. The PDCAAS-like values were calculated using the STTD of CP to estimate AA digestibility and values for DIAAS were calculated from values for SID of AA. Results indicated that values for SID of most indispensable AA in WPI, WPC and MPC were greater (P<0·05) than for SMP, PPC, SPI, soya flour and wheat. With the exception of arginine and tryptophan, the SID of all indispensable AA in SPI was greater (P<0·05) than in soya flour, and with the exception of threonine, the SID of all indispensable AA in wheat was less (P<0·05) than in all other ingredients. If the same scoring pattern for children between 6 and 36 months was used to calculate PDCAAS-like values and DIAAS, PDCAAS-like values were greater (P<0·05) than DIAAS values for SMP, PPC, SPI, soya flour and wheat indicating that PDCAAS-like values estimated in pigs may overestimate the quality of these proteins.

Distribution of stable free radicals among amino acids of isolated soy proteins.

PubMed

Lei, Qingxin; Liebold, Christopher M; Boatright, William L; Shah Jahan, M

2010-09-01

Application of deuterium sulfide to powdered isolated soy proteins (ISP) was used to quench stable free radicals and produce a single deuterium label on amino acids where free radicals reside. The deuterium labels rendered increases of isotope ratio for the specific ions of radical-bearing amino acids. Isotope ratio measurements were achieved by gas chromatography/mass spectrometry (GC/MS) analyses after the amino acids were released by acidic hydrolysis and converted to volatile derivatives with propyl chloroformate. The isotope enrichment data showed the stable free radicals were located on Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp but not on Val, Pro, Met, Phe, Lys, and His. Due to the low abundance of Ser, Thr, and Cys derivatives and the impossibility to accurately measure their isotope ratios, the radical bearing status for these amino acids remained undetermined even though their derivatives were positively identified from ISP hydrolysates. The relative isotope enrichment for radical-bearing amino acids Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp were 8.67%, 2.96%, 2.90%, 3.94%, 6.03%, 3.91%, and 21.48%, respectively. Isotope ratio increase for Tyr was also observed but further investigation revealed such increase was mainly from nonspecific deuterium-hydrogen exchange not free radical quenching. The results obtained from the present study provide important information for a better understanding of the mechanisms of free radical formation and stabilization in "dry" ISP.

Fatty acid binding proteins and the nervous system: Their impact on mental conditions.

PubMed

Matsumata, Miho; Inada, Hitoshi; Osumi, Noriko

2016-01-01

The brain is rich in lipid and fatty molecules. In this review article, we focus on fatty acid binding proteins (Fabps) that bind to fatty acids such as arachidonic acid and docosahexianoic acid and transfer these lipid ligands within the cytoplasm. Among Fabp family molecules, Fabp3, Fabp5, and Fabp7 are specifically localized in neural stem/progenitor cells, neurons and glia in a cell-type specific manner. Quantitative trait locus analysis has revealed that Fabp7 is related with performance of prepulse inhibition (PPI) that is used as an endophenotype of psychiatric diseases such as schizophrenia. Fabp5 and Fabp7 play important roles on neurogenesis and differentially regulate acoustic startle response and PPI. However, other behavior performances including spatial memory, anxiety-like behavior, and diurnal changes in general activity were not different in mice deficient for Fabp7 or Fabp5. Considering the importance of fatty acids in neurogenesis, we would like to emphasize that lipid nutrition and its dynamism via Fabps play significant roles in mental conditions. This might provide a good example of how nutritional environment can affect psychiatric conditions at the molecular level. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

An Arabidopsis mitochondria-localized RRL protein mediates abscisic acid signal transduction through mitochondrial retrograde regulation involving ABI4.

PubMed

Yao, Xuan; Li, Juanjuan; Liu, Jianping; Liu, Kede

2015-10-01

The molecular mechanisms of abscisic acid (ABA) signalling have been studied for many years; however, how mitochondria-localized proteins play roles in ABA signalling remains unclear. Here an Arabidopsis mitochondria-localized protein RRL (RETARDED ROOT GROWTH-LIKE) was shown to function in ABA signalling. A previous study had revealed that the Arabidopsis mitochondria-localized protein RRG (RETARDED ROOT GROWTH) is required for cell division in the root meristem. RRL shares 54% and 57% identity at the nucleotide and amino acid sequences, respectively, with RRG; nevertheless, RRL shows a different function in Arabidopsis. In this study, disruption of RRL decreased ABA sensitivity whereas overexpression of RRL increased ABA sensitivity during seed germination and seedling growth. High expression levels of RRL were found in germinating seeds and developing seedlings, as revealed by β-glucuronidase (GUS) staining of ProRRL-GUS transgenic lines. The analyses of the structure and function of mitochondria in the knockout rrl mutant showed that the disruption of RRL causes extensively internally vacuolated mitochondria and reduced ABA-stimulated reactive oxygen species (ROS) production. Previous studies have revealed that the expression of alternative oxidase (AOX) in the alternative respiratory pathway is increased by mitochondrial retrograde regulation to regain ROS levels when the mitochondrial electron transport chain is impaired. The APETALA2 (AP2)-type transcription factor ABI4 is a regulator of ALTERNATIVE OXIDASE1a (AOX1a) in mitochondrial retrograde signalling. This study showed that ABA-induced AOX1a and ABI4 expression was inhibited in the rrl mutant, suggesting that RRL is probably involved in ABI4-mediated mitochondrial retrograde signalling. Furthermore, the results revealed that ABI4 is a downstream regulatory factor in RRL-mediated ABA signalling in seed germination and seedling growth. © The Author 2015. Published by Oxford University Press on behalf of

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

USGS Publications Warehouse

Nicholas, K.R.; Fisher, J.A.; Muths, E.; Trott, J.; Janssens, P.A.; Reich, C.; Shaw, D.C.

2001-01-01

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of α-lactalbumin, β-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

USGS Publications Warehouse

Nicholas, K.R.; Fisher, J.A.; Muths, E.; Trott, J.; Janssens, P.A.; Reich, C.; Shaw, D.C.

2001-01-01

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of ??-lactalbumin, ??-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young. ?? 2001 Elsevier Science Inc.

Discovering amino acid patterns on binding sites in protein complexes

PubMed Central

Kuo, Huang-Cheng; Ong, Ping-Lin; Lin, Jung-Chang; Huang, Jen-Peng

2011-01-01

Discovering amino acid (AA) patterns on protein binding sites has recently become popular. We propose a method to discover the association relationship among AAs on binding sites. Such knowledge of binding sites is very helpful in predicting protein-protein interactions. In this paper, we focus on protein complexes which have protein-protein recognition. The association rule mining technique is used to discover geographically adjacent amino acids on a binding site of a protein complex. When mining, instead of treating all AAs of binding sites as a transaction, we geographically partition AAs of binding sites in a protein complex. AAs in a partition are treated as a transaction. For the partition process, AAs on a binding site are projected from three-dimensional to two-dimensional. And then, assisted with a circular grid, AAs on the binding site are placed into grid cells. A circular grid has ten rings: a central ring, the second ring with 6 sectors, the third ring with 12 sectors, and later rings are added to four sectors in order. As for the radius of each ring, we examined the complexes and found that 10Å is a suitable range, which can be set by the user. After placing these recognition complexes on the circular grid, we obtain mining records (i.e. transactions) from each sector. A sector is regarded as a record. Finally, we use the association rule to mine these records for frequent AA patterns. If the support of an AA pattern is larger than the predetermined minimum support (i.e. threshold), it is called a frequent pattern. With these discovered patterns, we offer the biologists a novel point of view, which will improve the prediction accuracy of protein-protein recognition. In our experiments, we produced the AA patterns by data mining. As a result, we found that arginine (arg) most frequently appears on the binding sites of two proteins in the recognition protein complexes, while cysteine (cys) appears the fewest. In addition, if we discriminate the shape

A Complex Prime Numerical Representation of Amino Acids for Protein Function Comparison.

PubMed

Chen, Duo; Wang, Jiasong; Yan, Ming; Bao, Forrest Sheng

2016-08-01

Computationally assessing the functional similarity between proteins is an important task of bioinformatics research. It can help molecular biologists transfer knowledge on certain proteins to others and hence reduce the amount of tedious and costly benchwork. Representation of amino acids, the building blocks of proteins, plays an important role in achieving this goal. Compared with symbolic representation, representing amino acids numerically can expand our ability to analyze proteins, including comparing the functional similarity of them. Among the state-of-the-art methods, electro-ion interaction pseudopotential (EIIP) is widely adopted for the numerical representation of amino acids. However, it could suffer from degeneracy that two different amino acid sequences have the same numerical representation, due to the design of EIIP. In light of this challenge, we propose a complex prime numerical representation (CPNR) of amino acids, inspired by the similarity between a pattern among prime numbers and the number of codons of amino acids. To empirically assess the effectiveness of the proposed method, we compare CPNR against EIIP. Experimental results demonstrate that the proposed method CPNR always achieves better performance than EIIP. We also develop a framework to combine the advantages of CPNR and EIIP, which enables us to improve the performance and study the unique characteristics of different representations.

Plasma membrane proteins Yro2 and Mrh1 are required for acetic acid tolerance in Saccharomyces cerevisiae.

PubMed

Takabatake, Akiko; Kawazoe, Nozomi; Izawa, Shingo

2015-03-01

Yro2 and its paralogous protein Mrh1 of Saccharomyces cerevisiae have seven predicted transmembrane domains and predominantly localize to the plasma membrane. Their physiological functions and regulation of gene expression have not yet been elucidated in detail. We herein demonstrated that MRH1 was constitutively expressed, whereas the expression of YRO2 was induced by acetic acid stress and entering the stationary phase. Fluorescence microscopic analysis revealed that Mrh1 and Yro2 were distributed as small foci in the plasma membrane under acetic acid stress conditions. The null mutants of these genes (mrh1∆, yro2∆, and mrh1∆yro2∆) showed delayed growth and a decrease in the productivity of ethanol in the presence of acetic acid, indicating that Yro2 and Mrh1 are involved in tolerance to acetic acid stress.

Interplay of protein and DNA structure revealed in simulations of the lac operon.

PubMed

Czapla, Luke; Grosner, Michael A; Swigon, David; Olson, Wilma K

2013-01-01

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

A nine-country study of the protein content and amino acid composition of mature human milk

PubMed Central

Feng, Ping; Gao, Ming; Burgher, Anita; Zhou, Tian Hui; Pramuk, Kathryn

2016-01-01

Background Numerous studies have evaluated protein and amino acid levels in human milk. However, research in this area has been limited by small sample sizes and study populations with little ethnic or racial diversity. Objective Evaluate the protein and amino acid composition of mature (≥30 days) human milk samples collected from a large, multinational study using highly standardized methods for sample collection, storage, and analysis. Design Using a single, centralized laboratory, human milk samples from 220 women (30–188 days postpartum) from nine countries were analyzed for amino acid composition using Waters AccQ-Tag high-performance liquid chromatography and total nitrogen content using the LECO FP-528 nitrogen analyzer. Total protein was calculated as total nitrogen×6.25. True protein, which includes protein, free amino acids, and peptides, was calculated from the total amino acids. Results Mean total protein from individual countries (standard deviation [SD]) ranged from 1,133 (125.5) to 1,366 (341.4) mg/dL; the mean across all countries (SD) was 1,192 (200.9) mg/dL. Total protein, true protein, and amino acid composition were not significantly different across countries except Chile, which had higher total and true protein. Amino acid profiles (percent of total amino acids) did not differ across countries. Total and true protein concentrations and 16 of 18 amino acid concentrations declined with the stage of lactation. Conclusions Total protein, true protein, and individual amino acid concentrations in human milk steadily decline from 30 to 151 days of lactation, and are significantly higher in the second month of lactation compared with the following 4 months. There is a high level of consistency in the protein content and amino acid composition of human milk across geographic locations. The size and diversity of the study population and highly standardized procedures for the collection, storage, and analysis of human milk support the validity and

Systematic Analysis of Compositional Order of Proteins Reveals New Characteristics of Biological Functions and a Universal Correlate of Macroevolution

PubMed Central

Persi, Erez; Horn, David

2013-01-01

We present a novel analysis of compositional order (CO) based on the occurrence of Frequent amino-acid Triplets (FTs) that appear much more than random in protein sequences. The method captures all types of proteomic compositional order including single amino-acid runs, tandem repeats, periodic structure of motifs and otherwise low complexity amino-acid regions. We introduce new order measures, distinguishing between ‘regularity’, ‘periodicity’ and ‘vocabulary’, to quantify these phenomena and to facilitate the identification of evolutionary effects. Detailed analysis of representative species across the tree-of-life demonstrates that CO proteins exhibit numerous functional enrichments, including a wide repertoire of particular patterns of dependencies on regularity and periodicity. Comparison between human and mouse proteomes further reveals the interplay of CO with evolutionary trends, such as faster substitution rate in mouse leading to decrease of periodicity, while innovation along the human lineage leads to larger regularity. Large-scale analysis of 94 proteomes leads to systematic ordering of all major taxonomic groups according to FT-vocabulary size. This is measured by the count of Different Frequent Triplets (DFT) in proteomes. The latter provides a clear hierarchical delineation of vertebrates, invertebrates, plants, fungi and prokaryotes, with thermophiles showing the lowest level of FT-vocabulary. Among eukaryotes, this ordering correlates with phylogenetic proximity. Interestingly, in all kingdoms CO accumulation in the proteome has universal characteristics. We suggest that CO is a genomic-information correlate of both macroevolution and various protein functions. The results indicate a mechanism of genomic ‘innovation’ at the peptide level, involved in protein elongation, shaped in a universal manner by mutational and selective forces. PMID:24278003

Gross and true ileal digestible amino acid contents of several animal body proteins and their hydrolysates.

PubMed

Cui, J; Chong, B; Rutherfurd, S M; Wilkinson, B; Singh, H; Moughan, P J

2013-07-01

Amino acid compositions of ovine muscle, ovine myofibrillar protein, ovine spleen, ovine liver, bovine blood plasma, bovine blood globulins and bovine serum albumin and the amino acid compositions and in vivo (laboratory rat) true ileal amino acid digestibilities of hydrolysates (sequential hydrolysis with Neutrase, Alcalase and Flavourzyme) of these protein sources were determined. True ileal amino acid digestibility differed (P<0.05) among the seven protein hydrolysates. The ovine myofibrillar protein and liver hydrolysates were the most digestible, with a mean true ileal digestibility across all amino acids of 99%. The least digestible protein hydrolysate was bovine serum albumin with a comparable mean true ileal digestibility of 93%. When the digestible amino acid contents were expressed as proportions relative to lysine, considerable differences, across the diverse protein sources, were found in the pattern of predicted absorbed amino acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enamel proteins mitigate mechanical and structural degradations in mature human enamel during acid attack

NASA Astrophysics Data System (ADS)

Lubarsky, Gennady V.; Lemoine, Patrick; Meenan, Brian J.; Deb, Sanjukta; Mutreja, Isha; Carolan, Patrick; Petkov, Nikolay

2014-04-01

A hydrazine deproteination process was used to investigate the role of enamel proteins in the acid erosion of mature human dental enamel. Bright field high resolution transmission electron micrographs and x-ray diffraction analysis show no crystallographic changes after the hydrazine treatment with similar nanoscale hydroxyapatite crystallite size and orientation for sound and de-proteinated enamel. However, the presence of enamel proteins reduces the erosion depth, the loss of hardness and the loss of structural order in enamel, following exposure to citric acid. Nanoindentation creep is larger for sound enamel than for deproteinated enamel but it reduces in sound enamel after acid attack. These novel results are consistent with calcium ion-mediated visco-elasticty in enamel matrix proteins as described previously for nacre, bone and dental proteins. They are also in good agreement with a previous double layer force spectroscopy study by the authors which found that the proteins electrochemically buffer enamel against acid attack. Finally, this suggests that acid attack, and more specifically dental erosion, is influenced by ionic permeation through the enamel layer and that it is mitigated by the enamel protein matrix.

Amino acids of Diclidophora merlangi (Monogenea).

PubMed

Arme, C; Whyte, A

1975-02-01

The level of free amino acids in Diclidophora merlangi is high, comprising over 500 mu moles/g ethanol extracted dry weight. A single amino acid, proline, constitutes some 70% of the total pool. Analysis of parasite protein and host blood and mucus revealed low proline levels, suggesting that the high free pool content was not related to a requirement for protein systhesis or to its abundance in the diet of the worm. Experiments revealed that proline was not involved specifically in osmoregulation, and the reasons for the large amounts present in Diclidophora remain unknown.

The Abundant Class III Chitinase Homolog in Young Developing Banana Fruits Behaves as a Transient Vegetative Storage Protein and Most Probably Serves as an Important Supply of Amino Acids for the Synthesis of Ripening-Associated Proteins1

PubMed Central

Peumans, Willy J.; Proost, Paul; Swennen, Rony L.; Van Damme, Els J.M.

2002-01-01

Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening-associated proteins. Analyses of individual proteins revealed that a thaumatin-like protein, a β-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas. PMID:12376669

Factor VII and protein C are phosphatidic acid-binding proteins.

PubMed

Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H

2013-08-20

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

An OmpA Family Protein, a Target of the GinI/GinR Quorum-Sensing System in Gluconacetobacter intermedius, Controls Acetic Acid Fermentation▿ †

PubMed Central

Iida, Aya; Ohnishi, Yasuo; Horinouchi, Sueharu

2008-01-01

Via N-acylhomoserine lactones, the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius NCI1051, a gram-negative acetic acid bacterium, represses acetic acid and gluconic acid fermentation. Two-dimensional polyacrylamide gel electrophoretic analysis of protein profiles of strain NCI1051 and ginI and ginR mutants identified a protein that was produced in response to the GinI/GinR regulatory system. Cloning and nucleotide sequencing of the gene encoding this protein revealed that it encoded an OmpA family protein, named GmpA. gmpA was a member of the gene cluster containing three adjacent homologous genes, gmpA to gmpC, the organization of which appeared to be unique to vinegar producers, including “Gluconacetobacter polyoxogenes.” In addition, GmpA was unique among the OmpA family proteins in that its N-terminal membrane domain forming eight antiparallel transmembrane β-strands contained an extra sequence in one of the surface-exposed loops. Transcriptional analysis showed that only gmpA of the three adjacent gmp genes was activated by the GinI/GinR quorum-sensing system. However, gmpA was not controlled directly by GinR but was controlled by an 89-amino-acid protein, GinA, a target of this quorum-sensing system. A gmpA mutant grew more rapidly in the presence of 2% (vol/vol) ethanol and accumulated acetic acid and gluconic acid in greater final yields than strain NCI1051. Thus, GmpA plays a role in repressing oxidative fermentation, including acetic acid fermentation, which is unique to acetic acid bacteria and allows ATP synthesis via ethanol oxidation. Consistent with the involvement of gmpA in oxidative fermentation, its transcription was also enhanced by ethanol and acetic acid. PMID:18487322

Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

PubMed

Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

2015-09-15

Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

An Integrated Phosphoproteomics Work Flow Reveals Extensive Network Regulation in Early Lysophosphatidic Acid Signaling*

PubMed Central

Schreiber, Thiemo B.; Mäusbacher, Nina; Kéri, György; Cox, Jürgen; Daub, Henrik

2010-01-01

Lysophosphatidic acid (LPA) induces a variety of cellular signaling pathways through the activation of its cognate G protein-coupled receptors. To investigate early LPA responses and assess the contribution of epidermal growth factor (EGF) receptor transactivation in LPA signaling, we performed phosphoproteomics analyses of both total cell lysate and protein kinase-enriched fractions as complementary strategies to monitor phosphorylation changes in A498 kidney carcinoma cells. Our integrated work flow enabled the identification and quantification of more than 5,300 phosphorylation sites of which 224 were consistently regulated by LPA. In addition to induced phosphorylation events, we also obtained evidence for early dephosphorylation reactions due to rapid phosphatase regulation upon LPA treatment. Phosphorylation changes induced by direct heparin-binding EGF-like growth factor-mediated EGF receptor activation were typically weaker and only detected on a subset of LPA-regulated sites, indicating signal integration among EGF receptor transactivation and other LPA-triggered pathways. Our results reveal rapid phosphoregulation of many proteins not yet implicated in G protein-coupled receptor signaling and point to various additional mechanisms by which LPA might regulate cell survival and migration as well as gene transcription on the molecular level. Moreover, our phosphoproteomics analysis of both total lysate and kinase-enriched fractions provided highly complementary parts of the LPA-regulated signaling network and thus represents a useful and generic strategy toward comprehensive signaling studies on a system-wide level. PMID:20071362

Interactions between Human Liver Fatty Acid Binding Protein and Peroxisome Proliferator Activated Receptor Selective Drugs

PubMed Central

Velkov, Tony

2013-01-01

Fatty acid binding proteins (FABPs) act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs). PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L-) FABP displays a high binding affinity for PPAR subtype selective drugs. NMR chemical shift perturbation mapping and proteolytic protection experiments show that the binding of the PPAR subtype selective drugs produces conformational changes that stabilize the portal region of L-FABP. NMR chemical shift perturbation studies also revealed that L-FABP can form a complex with the PPAR ligand binding domain (LBD) of PPARα. This protein-protein interaction may represent a mechanism for facilitating the activation of PPAR transcriptional activity via the direct channeling of ligands between the binding pocket of L-FABP and the PPARαLBD. The role of L-FABP in the delivery of ligands directly to PPARα via this channeling mechanism has important implications for regulatory pathways that mediate xenobiotic responses and host protection in tissues such as the small intestine and the liver where L-FABP is highly expressed. PMID:23476633

Inferring Selection on Amino Acid Preference in Protein Domains

PubMed Central

Durbin, Richard

2009-01-01

Models that explicitly account for the effect of selection on new mutations have been proposed to account for “codon bias” or the excess of “preferred” codons that results from selection for translational efficiency and/or accuracy. In principle, such models can be applied to any mutation that results in a preferred allele, but in most cases, the fitness effect of a specific mutation cannot be predicted. Here we show that it is possible to assign preferred and unpreferred states to amino acid changing mutations that occur in protein domains. We propose that mutations that lead to more common amino acids (at a given position in a domain) can be considered “preferred alleles” just as are synonymous mutations leading to codons for more abundant tRNAs. We use genome-scale polymorphism data to show that alleles for preferred amino acids in protein domains occur at higher frequencies in the population, as has been shown for preferred codons. We show that this effect is quantitative, such that there is a correlation between the shift in frequency of preferred alleles and the predicted fitness effect. As expected, we also observe a reduction in the numbers of polymorphisms and substitutions at more important positions in domains, consistent with stronger selection at those positions. We examine the derived allele frequency distribution and polymorphism to divergence ratios of preferred and unpreferred differences and find evidence for both negative and positive selections acting to maintain protein domains in the human population. Finally, we analyze a model for selection on amino acid preferences in protein domains and find that it is consistent with the quantitative effects that we observe. PMID:19095755

Refinement of Generalized Born Implicit Solvation Parameters for Nucleic Acids and their Complexes with Proteins

PubMed Central

Nguyen, Hai; Pérez, Alberto; Bermeo, Sherry; Simmerling, Carlos

2016-01-01

The Generalized Born (GB) implicit solvent model has undergone significant improvements in accuracy for modeling of proteins and small molecules. However, GB still remains a less widely explored option for nucleic acid simulations, in part because fast GB models are often unable to maintain stable nucleic acid structures, or they introduce structural bias in proteins, leading to difficulty in application of GB models in simulations of protein-nucleic acid complexes. Recently, GB-neck2 was developed to improve the behavior of protein simulations. In an effort to create a more accurate model for nucleic acids, a similar procedure to the development of GB-neck2 is described here for nucleic acids. The resulting parameter set significantly reduces absolute and relative energy error relative to Poisson Boltzmann for both nucleic acids and nucleic acid-protein complexes, when compared to its predecessor GB-neck model. This improvement in solvation energy calculation translates to increased structural stability for simulations of DNA and RNA duplexes, quadruplexes, and protein-nucleic acid complexes. The GB-neck2 model also enables successful folding of small DNA and RNA hairpins to near native structures as determined from comparison with experiment. The functional form and all required parameters are provided here and also implemented in the AMBER software. PMID:26574454

Nucleic acid encoding DS-CAM proteins and products related thereto

DOEpatents

Korenberg, Julie R.

2005-11-01

In accordance with the present invention, there are provided Down Syndrome-Cell Adhesion Molecule (DS-CAM) proteins. Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention DS-CAM proteins can be employed in a variety of ways, for example, for the production of anti-DS-CAM antibodies thereto, in therapeutic compositions and methods employing such proteins and/or antibodies. DS-CAM proteins are also useful in bioassays to identify agonists and antagonists thereto.

Identification of Rosmarinic Acid-Adducted Sites in Meat Proteins in a Gel Model under Oxidative Stress by Triple TOF MS/MS.

PubMed

Tang, Chang-Bo; Zhang, Wan-Gang; Wang, Yao-Song; Xing, Lu-Juan; Xu, Xing-Lian; Zhou, Guang-Hong

2016-08-24

Triple TOF MS/MS was used to identify adducts between rosmarinic acid (RosA)-derived quinones and meat proteins in a gel model under oxidative stress. Seventy-five RosA-modified peptides responded to 67 proteins with adduction of RosA. RosA conjugated with different amino acids in proteins, and His, Arg, and Lys adducts with RosA were identified for the first time in meat. A total of 8 peptides containing Cys, 14 peptides containing His, 48 peptides containing Arg, 64 peptides containing Lys, and 5 peptides containing N-termini that which participated in adduction reaction with RosA were identified, respectively. Seventy-seven adduction sites were subdivided into all adducted proteins including 2 N-terminal adduction sites, 3 Cys adduction sites, 4 His adduction sites, 29 Arg adduction sites, and 39 Lys adduction sites. Site occupancy analyses showed that approximately 80.597% of the proteins carried a single RosA-modified site, 14.925% retained two sites, 1.492% contained three sites, and the rest 2.985% had four or more sites. Large-scale triple TOF MS/MS mapping of RosA-adducted sites reveals the adduction regulations of quinone and different amino acids as well as the adduction ratios, which clarify phenol-protein adductions and pave the way for industrial meat processing and preservation.

One-Pot Procedure for Recovery of Gallic Acid from Wastewater and Encapsulation within Protein Particles.

PubMed

Nourbakhsh, Himan; Madadlou, Ashkan; Emam-Djomeh, Zahra; Wang, Yi-Cheng; Gunasekaran, Sundaram; Mousavi, Mohammad E

2016-02-24

A whey protein isolate solution was heat-denatured and treated with the enzyme transglutaminase, which cross-linked ≈26% of the amino groups and increased the magnitude of the ζ-potential value. The protein solution was microemulsified, and then the resulting water-in-oil microemulsion was dispersed within a gallic acid-rich model wastewater. Gallic acid extraction by the outlined microemulsion liquid membrane (MLM) from the exterior aqueous phase (wastewater) and accumulation within the internal aqueous nanodroplets induced protein cold-set gelation and resulted in the formation of gallic acid-enveloping nanoparticles. Measurements with a strain-controlled rheometer indicated a progressive increase in the MLM viscosity during gallic acid recovery corresponding to particle formation. The mean hydrodynamic size of the nanoparticles made from the heat-denatured and preheated enzymatically cross-linked proteins was 137 and 122 nm, respectively. The enzymatic cross-linking of whey proteins led to a higher gallic acid recovery yield and increased the glass transition enthalpy and temperature. A similar impact on glass transition indices was observed by the gallic acid-induced nanoparticulation of proteins. Scanning electron microscopy showed the existence of numerous jammed/fused nanoparticles. It was suggested on the basis of the results of Fourier transform infrared spectroscopy that the in situ nanoparticulation of proteins shifted the C-N stretching and C-H bending peaks to higher wavenumbers. X-ray diffraction results proposed a decreased β-sheet content for proteins because of the acid-induced particulation. The nanoparticles made from the enzymatically cross-linked protein were more stable against the in vitro gastrointestinal digestion and retained almost 19% of the entrapped gallic acid after 300 min sequential gastric and intestinal digestions.

Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

PubMed

Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

2013-12-01

Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient ( i.e ., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs . 252 ± 43 m, p < 0.0001) in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides) subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA) of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly ( p < 0.05) more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH) was 1

Proteomic-based stable isotope probing reveals taxonomically Distinct Patterns in Amino Acid Assimilation by Coastal Marine Bacterioplankton

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

Bryson, Samuel; Li, Zhou; Pett-Ridge, Jennifer

Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual global primary production, yet defining how substrate utilization preferences and resource partitioning structure these microbial communities remains a challenge. In this study, we utilized proteomics-based stable isotope probing (proteomic SIP) to characterize the assimilation of amino acids by coastal marine bacterioplankton populations. We incubated microcosms of seawater collected from Newport, OR and Monterey Bay, CA with 1 M 13C-amino acids for 15 and 32 hours. Subsequent analysis of 13C incorporation into protein biomass quantified the frequency and extent of isotope enrichment for identifiedmore » proteins. Using these metrics we tested whether amino acid assimilation patterns were different for specific bacterioplankton populations. Proteins associated with Rhodobacterales and Alteromonadales tended to have a significantly high number of tandem mass spectra from 13C-enriched peptides, while Flavobacteriales and SAR11 proteins generally had significantly low numbers of 13C-enriched spectra. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of 13C-enriched spectra at time-point 2, while Alteromonadales ribosomal proteins were 13C- enriched across time-points. Overall, proteomic SIP facilitated quantitative comparisons of dissolved free amino acids assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population-level metabolic responses to resource acquisition in complex microbial communities.« less

Proteomic-based stable isotope probing reveals taxonomically Distinct Patterns in Amino Acid Assimilation by Coastal Marine Bacterioplankton

DOE PAGES

Bryson, Samuel; Li, Zhou; Pett-Ridge, Jennifer; ...

2016-04-26

Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual global primary production, yet defining how substrate utilization preferences and resource partitioning structure these microbial communities remains a challenge. In this study, we utilized proteomics-based stable isotope probing (proteomic SIP) to characterize the assimilation of amino acids by coastal marine bacterioplankton populations. We incubated microcosms of seawater collected from Newport, OR and Monterey Bay, CA with 1 M 13C-amino acids for 15 and 32 hours. Subsequent analysis of 13C incorporation into protein biomass quantified the frequency and extent of isotope enrichment for identifiedmore » proteins. Using these metrics we tested whether amino acid assimilation patterns were different for specific bacterioplankton populations. Proteins associated with Rhodobacterales and Alteromonadales tended to have a significantly high number of tandem mass spectra from 13C-enriched peptides, while Flavobacteriales and SAR11 proteins generally had significantly low numbers of 13C-enriched spectra. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of 13C-enriched spectra at time-point 2, while Alteromonadales ribosomal proteins were 13C- enriched across time-points. Overall, proteomic SIP facilitated quantitative comparisons of dissolved free amino acids assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population-level metabolic responses to resource acquisition in complex microbial communities.« less

HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO

EPA Science Inventory

HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO. MR Blanton and ES Hunter. Reproductive Toxicology Division, NHEERL, ORD, US EPA, RTP, NC, USA.
Sponsor: JM Rogers.
Haloacetic Acids (HAAs) formed during the disinfection process are present in drin...

Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

PubMed

James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

2015-01-01

Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury.

Amino acid sequence of the smaller basic protein from rat brain myelin

PubMed Central

Dunkley, Peter R.; Carnegie, Patrick R.

1974-01-01

1. The complete amino acid sequence of the smaller basic protein from rat brain myelin was determined. This protein differs from myelin basic proteins of other species in having a deletion of a polypeptide of 40 amino acid residues from the centre of the molecule. 2. A detailed comparison is made of the constant and variable regions in a group of myelin basic proteins from six species. 3. An arginine residue in the rat protein was found to be partially methylated. The ratio of methylated to unmethylated arginine at this position differed from that found for the human basic protein. 4. Three tryptic peptides were isolated in more than one form. The differences between the two forms of each peptide are discussed in relation to the electrophoretic heterogeneity of myelin basic proteins, which is known to occur at alkaline pH values. 5. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50029 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4141893

The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics.

PubMed

Liu, Xu; Shepherd, Tyson R; Murray, Ann M; Xu, Zhen; Fuentes, Ernesto J

2013-03-05

PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms. The crystal structure of the PDZ/SDC1 complex indicates that syndecan affinity is derived from amino acids beyond the four C-terminal residues. Remarkably, the crystal structure of the PDZ/pSDC1 complex reveals a binding pocket that accommodates the phosphoryl group. Methyl relaxation experiments of PDZ/SCD1 and PDZ/pSDC1 complexes reveal that PDZ-phosphoryl interactions dampen dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data are consistent with a selection model by which specificity and phosphorylation regulate PDZ/syndecan interactions and signaling events. Importantly, our relaxation data demonstrate that PDZ/phospho-ligand interactions regulate protein dynamics and their coupling to distal sites. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nitro-fatty acid metabolome: saturation, desaturation, beta-oxidation, and protein adduction.

PubMed

Rudolph, Volker; Schopfer, Francisco J; Khoo, Nicholas K H; Rudolph, Tanja K; Cole, Marsha P; Woodcock, Steven R; Bonacci, Gustavo; Groeger, Alison L; Golin-Bisello, Franca; Chen, Chen-Shan; Baker, Paul R S; Freeman, Bruce A

2009-01-16

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via beta-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO2 hydration or nitric oxide (*NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added beta-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet

Quasi-elastic neutron scattering reveals ligand-induced protein dynamics of a G-protein-coupled receptor

DOE PAGES

Shrestha, Utsab R.; Perera, Suchithranga M. D. C.; Bhowmik, Debsindhu; ...

2016-09-15

Light activation of the visual G-protein-coupled receptor (GPCR) rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane yielding the activation of cognate G-protein (transducin), which initiates biological signaling. Here, we report a quasi-elastic neutron scattering study of the activation of rhodopsin as a GPCR prototype. Our results reveal a broadly distributed relaxation of hydrogen atom dynamics of rhodopsin on a picosecond–nanosecond time scale, crucial for protein function, as only observed for globular proteins previously. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differencesmore » can be attributed to the influence of the covalently bound retinal ligand. Moreover, an idea of the generic free-energy landscape is used to explain the GPCR dynamics of ligand-binding and ligand-free protein conformations, which can be further applied to other GPCR systems.« less

Quasi-elastic neutron scattering reveals ligand-induced protein dynamics of a G-protein-coupled receptor

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

Shrestha, Utsab R.; Perera, Suchithranga M. D. C.; Bhowmik, Debsindhu

Light activation of the visual G-protein-coupled receptor (GPCR) rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane yielding the activation of cognate G-protein (transducin), which initiates biological signaling. Here, we report a quasi-elastic neutron scattering study of the activation of rhodopsin as a GPCR prototype. Our results reveal a broadly distributed relaxation of hydrogen atom dynamics of rhodopsin on a picosecond–nanosecond time scale, crucial for protein function, as only observed for globular proteins previously. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differencesmore » can be attributed to the influence of the covalently bound retinal ligand. Moreover, an idea of the generic free-energy landscape is used to explain the GPCR dynamics of ligand-binding and ligand-free protein conformations, which can be further applied to other GPCR systems.« less

Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment.

PubMed

Gruber, David F; Gaffney, Jean P; Mehr, Shaadi; DeSalle, Rob; Sparks, John S; Platisa, Jelena; Pieribone, Vincent A

2015-01-01

We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein's fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment.

Omega-3 fatty acid deficiency disrupts endocytosis, neuritogenesis, and mitochondrial protein pathways in the mouse hippocampus

PubMed Central

English, Jane A.; Harauma, Akiko; Föcking, Melanie; Wynne, Kieran; Scaife, Caitriona; Cagney, Gerard; Moriguchi, Toru; Cotter, David R.

2013-01-01

Omega-3 fatty acid (n-3 FA) deficiency is an environmental risk factor for schizophrenia, yet characterization of the consequences of deficiency at the protein level in the brain is limited. We aimed to identify the protein pathways disrupted as a consequence of chronic n-3 deficiency in the hippocampus of mice. Fatty acid analysis of the hippocampus following chronic dietary deficiency revealed a 3-fold decrease (p < 0.001) in n-3 FA levels. Label free LC-MS/MS analysis identified and profiled 1008 proteins, of which 114 were observed to be differentially expressed between n-3 deficient and control groups (n = 8 per group). The cellular processes that were most implicated were neuritogenesis, endocytosis, and exocytosis, while specific protein pathways that were most significantly dysregulated were mitochondrial dysfunction and clathrin mediated endocytosis (CME). In order to characterize whether these processes and pathways are ones influenced by antipsychotic medication, we used LC-MS/MS to test the differential expression of these 114 proteins in the hippocampus of mice chronically treated with the antipsychotic agent haloperidol. We observed 23 of the 114 proteins to be differentially expressed, 17 of which were altered in the opposite direction to that observed following n-3 deficiency. Overall, our findings point to disturbed synaptic function, neuritogenesis, and mitochondrial function as a consequence of dietary deficiency in n-3 FA. This study greatly aids our understanding of the molecular mechanism by which n-3 deficiency impairs normal brain function, and provides clues as to how n-3 FA exert their therapeutic effect in early psychosis. PMID:24194745

The effect of feeding high corn oil on fatty-acid-binding-protein isolated from rat liver.

PubMed

Catalá, A

1987-12-01

Fatty-acid-binding-protein isolated from liver of rats receiving normal or high fat diet was studied by three different methods. The effect of high fat diet on the thermal stability of the protein was determined employing differential scanning calorimetry. Fatty acids have a stabilizing effect on the thermal stability of the protein. In order to determine the relative binding affinity of native and delipidated protein a Sephadex G-50 assay was employed using [1-14C] oleate as ligand. The delipidated protein exhibited greater binding of oleate than did the native material. Increases in the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes in vitro were also observed when protein obtained from both sources were delipidated. The results suggest that high corn oil diet would modify the properties of fatty-acid-binding-protein in the uptake and cytosolic transport of long-chain fatty acids.

Evaluation of hyaluronic acid-protein conjugates for polymer masked-unmasked protein therapy.

PubMed

Ferguson, Elaine L; Alshame, Alshame M J; Thomas, David W

2010-12-15

Bioresponsive polymers may effectively be utilized to enhance the circulation time and stability of biologically active proteins and peptides, while reducing their immunogenicity and toxicity. Recently, dextrin-epidermal growth factor (EGF) conjugates, which make use of the Polymer-masked UnMasked Protein Therapy (PUMPT) concept, have been developed and shown potential as modulators of impaired wound healing. This study investigated the potential of PUMPT using hyaluronic acid (HA) conjugates to mask activity and enhance protein stability, while allowing restoration of biological activity following triggered degradation. HA fragments (Mw ∼90,000g/mol), obtained by acid hydrolysis of Rooster comb HA, were conjugated to trypsin as a model enzyme or to EGF as a model growth factor. Conjugates contained 2.45 and 0.98% (w/w) trypsin or EGF, respectively, and contained <5% free protein. HA conjugation did not significantly alter trypsin's activity. However, incubation of the conjugate with physiological concentrations of HAase increased its activity to ∼145% (p<0.001) that of the free enzyme. In contrast, when HA-EGF conjugates were tested in vitro, no effect on cell proliferation was seen, even in the presence of HAase. HA conjugates did not display typical masking/unmasking behavior, HA-trypsin conjugates exhibited ∼52% greater stability in the presence of elastase, compared to free trypsin, demonstrating the potential of HA conjugates for further development as modulators of tissue repair. Copyright © 2010 Elsevier B.V. All rights reserved.

Mitochondrial genetic codes evolve to match amino acid requirements of proteins.

PubMed

Swire, Jonathan; Judson, Olivia P; Burt, Austin

2005-01-01

Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular--and contrary to the predictions of the mutation-drift hypothesis--the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins.

Enantiomeric separation of non-protein amino acids by electrokinetic chromatography.

PubMed

Pérez-Míguez, Raquel; Marina, María Luisa; Castro-Puyana, María

2016-10-07

New analytical methodologies enabling the enantiomeric separation of a group of non-protein amino acids of interest in the pharmaceutical and food analysis fields were developed in this work using Electrokinetic Chromatography. The use of FMOC as derivatization reagent and the subsequent separation using acidic conditions (formate buffer at pH 2.0) and anionic cyclodextrins as chiral selectors allowed the chiral separation of eight from the ten non-protein amino acids studied. Pyroglutamic acid, norvaline, norleucine, 3,4-dihydroxyphenilalanine, 2-aminoadipic acid, and selenomethionine were enantiomericaly separated using sulfated-α-CD while sulfated-γ-CD enabled the enantiomeric separation of norvaline, 3,4-dihydroxyphenilalanine, 2-aminoadipic acid, selenomethionie, citrulline, and pipecolic acid. Moreover, the potential of the developed methodologies was demonstrated in the analysis of citrulline and its enantiomeric impurity in food supplements. For that purpose, experimental and instrumental variables were optimized and the analytical characteristics of the proposed method were evaluated. LODs of 2.1×10 -7 and 1.8×10 -7 M for d- and l-citrulline, respectively, were obtained. d-Cit was not detectable in any of the six food supplement samples analyzed showing that the effect of storage time on the racemization of citrulline was negligible. Copyright © 2016 Elsevier B.V. All rights reserved.

Acidic Ribosomal Proteins from the Extreme ’Halobacterium cutirubrum’,

DTIC Science & Technology

the extreme halophilic bacterium, Halobacterium cutirubrum. The identification of the protein moieties involved in these and other interactions in...the halophile ribosome requires a rapid and reproducible screening method for the separation, enumeration and identification of these acidic...polypeptides in the complex ribosomal protein mixtures. In this paper the authors present the results of analyses of the halophile ribosomal proteins using a

The fragile X mental retardation protein has nucleic acid chaperone properties

PubMed Central

Gabus, Caroline; Mazroui, Rachid; Tremblay, Sandra; Khandjian, Edouard W.; Darlix, Jean-Luc

2004-01-01

The fragile X syndrome is the most common cause of inherited mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP contains two K-homology (KH) domains and one RGG box that are landmarks characteristic of RNA-binding proteins. In agreement with this, FMRP associates with messenger ribonucleoparticles (mRNPs) within actively translating ribosomes, and is thought to regulate translation of target mRNAs, including its own transcript. To investigate whether FMRP might chaperone nucleic acid folding and hybridization, we analysed the annealing and strand exchange activities of DNA oligonucleotides and the enhancement of ribozyme-directed RNA substrate cleavage by FMRP and deleted variants relative to canonical nucleic acid chaperones, such as the cellular YB-1/p50 protein and the retroviral nucleocapsid protein HIV-1 NCp7. FMRP was found to possess all the properties of a potent nucleic acid chaperone, requiring the KH motifs and RGG box for optimal activity. These findings suggest that FMRP may regulate translation by acting on RNA–RNA interactions and thus on the structural status of mRNAs. PMID:15096575

The fragile X mental retardation protein has nucleic acid chaperone properties.

PubMed

Gabus, Caroline; Mazroui, Rachid; Tremblay, Sandra; Khandjian, Edouard W; Darlix, Jean-Luc

2004-01-01

The fragile X syndrome is the most common cause of inherited mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP contains two K-homology (KH) domains and one RGG box that are landmarks characteristic of RNA-binding proteins. In agreement with this, FMRP associates with messenger ribonucleoparticles (mRNPs) within actively translating ribosomes, and is thought to regulate translation of target mRNAs, including its own transcript. To investigate whether FMRP might chaperone nucleic acid folding and hybridization, we analysed the annealing and strand exchange activities of DNA oligonucleotides and the enhancement of ribozyme-directed RNA substrate cleavage by FMRP and deleted variants relative to canonical nucleic acid chaperones, such as the cellular YB-1/p50 protein and the retroviral nucleocapsid protein HIV-1 NCp7. FMRP was found to possess all the properties of a potent nucleic acid chaperone, requiring the KH motifs and RGG box for optimal activity. These findings suggest that FMRP may regulate translation by acting on RNA-RNA interactions and thus on the structural status of mRNAs.

Miniaturized technology for protein and nucleic acid point-of-care testing.

PubMed

Olasagasti, Felix; Ruiz de Gordoa, Juan Carlos

2012-11-01

The field of point-of-care (POC) testing technology is developing quickly and producing instruments that are increasingly reliable, while their size is being gradually reduced. Proteins are a common target for POC analyses and the detection of protein markers typically involves immunoassays aimed at detecting different groups of proteins such as tumor markers, inflammation proteins, and cardiac markers; but other techniques can also be used to analyze plasma proteins. In the case of nucleic acids, hybridization and amplification strategies can be used to record electromagnetic or electric signals. These techniques allow for the identification of specific viral or bacterial infections as well as specific cancers. In this review, we consider some of the latest advances in the analysis of specific nucleic acid and protein biomarkers, taking into account their trend toward miniaturization and paying special attention to the technology that can be implemented in future applications, such as lab-on-a-chip instruments. Copyright © 2012 Mosby, Inc. All rights reserved.

Amino Acid Distribution Rules Predict Protein Fold: Protein Grammar for Beta-Strand Sandwich-Like Structures

PubMed Central

Kister, Alexander

2015-01-01

We present an alternative approach to protein 3D folding prediction based on determination of rules that specify distribution of “favorable” residues, that are mainly responsible for a given fold formation, and “unfavorable” residues, that are incompatible with that fold, in polypeptide sequences. The process of determining favorable and unfavorable residues is iterative. The starting assumptions are based on the general principles of protein structure formation as well as structural features peculiar to a protein fold under investigation. The initial assumptions are tested one-by-one for a set of all known proteins with a given structure. The assumption is accepted as a “rule of amino acid distribution” for the protein fold if it holds true for all, or near all, structures. If the assumption is not accepted as a rule, it can be modified to better fit the data and then tested again in the next step of the iterative search algorithm, or rejected. We determined the set of amino acid distribution rules for a large group of beta sandwich-like proteins characterized by a specific arrangement of strands in two beta sheets. It was shown that this set of rules is highly sensitive (~90%) and very specific (~99%) for identifying sequences of proteins with specified beta sandwich fold structure. The advantage of the proposed approach is that it does not require that query proteins have a high degree of homology to proteins with known structure. So long as the query protein satisfies residue distribution rules, it can be confidently assigned to its respective protein fold. Another advantage of our approach is that it allows for a better understanding of which residues play an essential role in protein fold formation. It may, therefore, facilitate rational protein engineering design. PMID:25625198

Role of the Acidic Tail of High Mobility Group Protein B1 (HMGB1) in Protein Stability and DNA Bending

PubMed Central

Belgrano, Fabricio S.; de Abreu da Silva, Isabel C.; Bastos de Oliveira, Francisco M.; Fantappié, Marcelo R.; Mohana-Borges, Ronaldo

2013-01-01

High mobility group box (HMGB) proteins are abundant nonhistone proteins found in all eukaryotic nuclei and are capable of binding/bending DNA. The human HMGB1 is composed of two binding motifs, known as Boxes A and B, are L-shaped alpha-helix structures, followed by a random-coil acidic tail that consists of 30 Asp and Glu residues. This work aimed at evaluating the role of the acidic tail of human HMGB1 in protein stability and DNA interactions. For this purpose, we cloned, expressed and purified HMGB1 and its tailless form, HMGB1ΔC, in E. coli strain. Tryptophan fluorescence spectroscopy and circular dichroism (CD) experiments clearly showed an increase in protein stability promoted by the acidic tail under different conditions, such as the presence of the chemical denaturant guanidine hydrochloride (Gdn.HCl), high temperature and low pH. Folding intermediates found at low pH for both proteins were denatured only in the presence of chemical denaturant, thus showing a relatively high stability. The acidic tail did not alter the DNA-binding properties of the protein, although it enhanced the DNA bending capability from 76° (HMGB1ΔC) to 91° (HMGB1), as measured using the fluorescence resonance energy transfer technique. A model of DNA bending in vivo was proposed, which might help to explain the interaction of HMGB1 with DNA and other proteins, i.e., histones, and the role of that protein in chromatin remodeling. PMID:24255708

Δ98Δ, a minimalist model of antiparallel β-sheet proteins based on intestinal fatty acid binding protein

PubMed Central

Curto, Lucrecia María; Caramelo, Julio Javier; Franchini, Gisela Raquel; Delfino, José María

2009-01-01

The design of β-barrels has always been a formidable challenge for de novo protein design. For instance, a persistent problem is posed by the intrinsic tendency to associate given by free edges. From the opposite standpoint provided by the redesign of natural motifs, we believe that the intestinal fatty acid binding protein (IFABP) framework allows room for intervention, giving rise to abridged forms from which lessons on β-barrel architecture and stability could be learned. In this context, Δ98Δ (encompassing residues 29–126 of IFABP) emerges as a monomeric variant that folds properly, retaining functional activity, despite lacking extensive stretches involved in the closure of the β-barrel. Spectroscopic probes (fluorescence and circular dichroism) support the existence of a form preserving the essential determinants of the parent structure, albeit endowed with enhanced flexibility. Chemical and physical perturbants reveal cooperative unfolding transitions, with evidence of significant population of intermediate species in equilibrium, structurally akin to those transiently observed in IFABP. The recognition by the natural ligand oleic acid exerts a mild stabilizing effect, being of a greater magnitude than that found for IFABP. In summary, Δ98Δ adopts a monomeric state with a compact core and a loose periphery, thus pointing to the nonintuitive notion that the integrity of the β-barrel can indeed be compromised with no consequence on the ability to attain a native-like and functional fold. PMID:19309727

PLASMA PROTEIN AND HEMOGLOBIN PRODUCTION : DELETION OF INDIVIDUAL AMINO ACIDS FROM GROWTH MIXTURE OF TEN ESSENTIAL AMINO ACIDS. SIGNIFICANT CHANGES IN URINARY NITROGEN.

PubMed

Robscheit-Robbins, F S; Miller, L L; Whipple, G H

1947-02-28

Given healthy dogs fed abundant iron and protein-free or low protein diets with sustained anemia and hypoproteinemia, we can study the capacity of these animals to produce simultaneously new hemoglobin and plasma protein. Reserve stores of blood protein-building materials are measurably depleted and levels of 6 to 8 gm. per cent for hemoglobin and 4 to 5 gm. per cent for plasma protein can be maintained for weeks or months depending upon the intake of food proteins or amino acid mixtures. These dogs are very susceptible to infection and various poisons. Dogs tire of these diets and loss of appetite terminates many experiments. Under these conditions (double depletion) standard growth mixtures of essential amino acids are tested to show the response in blood protein output and urinary nitrogen balance. As a part of each tabulated experiment one of the essential amino acids is deleted from the complete growth mixture to compare such response with that of the whole mixture. Methionine, threonine, phenylalanine, and tryptophane when singly eliminated from the complete amino acid mixture do effect a sharp rise in urinary nitrogen. This loss of urinary nitrogen is corrected when the individual amino acid is replaced in the mixture. Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when these individual amino acids are deleted from the complete growth mixture of amino acids during 3 to 4 week periods. Tryptophane and to a less extent phenylalanine and threonine when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output (Table 4). Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output. Various amino acid mixtures under these conditions may give a positive

Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber.

PubMed Central

Pieper-Fürst, U.; Madkour, M. H.; Mayer, F.; Steinbüchel, A.

1994-01-01

The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated M(r)-15,500 protein of Rhodococcus ruber (the GA14 protein) was analyzed. The sequence revealed that the corresponding structural gene is represented by open reading frame 3, encoding a protein with a calculated M(r) of 14,175 which was recently localized downstream of the PHA synthase gene (U. Pieper and A. Steinbüchel, FEMS Microbiol. Lett. 96:73-80, 1992). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10*) with open reading frame 3 overexpressed the GA14 protein. The GA14 protein was subsequently purified in a three-step procedure including chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopic studies indicates that a tetrameric structure of the recombinant, native GA14 protein is most likely. Immunoelectron microscopy demonstrated a localization of the GA14 protein at the periphery of PHA granules as well as close to the cell membrane in R. ruber. Investigations of PHA-leaky and PHA-negative mutants of R. ruber indicated that expression of the GA14 protein depended strongly on PHA synthesis. Images PMID:8021220

Effects of heat, cold, acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1.

PubMed

Chen, Ming-Ju; Tang, Hsin-Yu; Chiang, Ming-Lun

2017-09-01

Lactobacillus kefiranofaciens M1 is a probiotic strain isolated from Taiwanese kefir grains. The present study evaluated the effects of heat, cold, acid and bile salt adaptations on the stress tolerance of L. kefiranofaciens M1. The regulation of protein expression of L. kefiranofaciens M1 under these adaptation conditions was also investigated. The results showed that adaptation of L. kefiranofaciens M1 to heat, cold, acid and bile salts induced homologous tolerance and cross-protection against heterologous challenge. The extent of induced tolerance varied depending on the type and condition of stress. Proteomic analysis revealed that 27 proteins exhibited differences in expression between non-adapted and stress-adapted L. kefiranofaciens M1 cells. Among these proteins, three proteins involved in carbohydrate metabolism (triosephosphate isomerase, enolase and NAD-dependent glycerol-3-phosphate dehydrogenase), two proteins involved in pH homeostasis (ATP synthase subunits AtpA and AtpB), two stress response proteins (chaperones DnaK and GroEL) and one translation-related protein (30S ribosomal protein S2) were up-regulated by three of the four adaptation treatments examined. The increased synthesis of these stress proteins might play a critical protective role in the cellular defense against heat, cold, acid and bile salt stresses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chemical Functionalization of Germanium with Dextran Brushes for Immobilization of Proteins Revealed by Attenuated Total Reflection Fourier Transform Infrared Difference Spectroscopy.

PubMed

Schartner, Jonas; Hoeck, Nina; Güldenhaupt, Jörn; Mavarani, Laven; Nabers, Andreas; Gerwert, Klaus; Kötting, Carsten

2015-07-21

Protein immobilization studied by attenuated total reflection Fourier transform infrared (ATR-FT-IR) difference spectroscopy is an emerging field enabling the study of proteins at atomic detail. Gold or glass surfaces are frequently used for protein immobilization. Here, we present an alternative method for protein immobilization on germanium. Because of its high refractive index and broad spectral window germanium is the best material for ATR-FT-IR spectroscopy of thin layers. So far, this technique was mainly used for protein monolayers, which lead to a limited signal-to-noise ratio. Further, undesired protein-protein interactions can occur in a dense layer. Here, the germanium surface was functionalized with thiols and stepwise a dextran brush was generated. Each step was monitored by ATR-FT-IR spectroscopy. We compared a 70 kDa dextran with a 500 kDa dextran regarding the binding properties. All surfaces were characterized by atomic force microscopy, revealing thicknesses between 40 and 110 nm. To analyze the capability of our system we utilized N-Ras on mono-NTA (nitrilotriacetic acid) functionalized dextran, and the amount of immobilized Ras corresponded to several monolayers. The protein stability and loading capacity was further improved by means of tris-NTA for immobilization. Small-molecule-induced changes were revealed with an over 3 times higher signal-to-noise ratio compared to monolayers. This improvement may allow the observation of very small and so far hidden changes in proteins upon stimulus. Furthermore, we immobilized green fluorescent protein (GFP) and mCherry simultaneously enabling an analysis of the surface by fluorescence microscopy. The absence of a Förster resonance energy transfer (FRET) signal demonstrated a large protein-protein distance, indicating an even distribution of the protein within the dextran.

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition.

PubMed

Bourgeois, Michael; Jacquin, Françoise; Savois, Vincent; Sommerer, Nicolas; Labas, Valérie; Henry, Céline; Burstin, Judith

2009-01-01

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post-translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.

Protein Solubility, Digestibility and Fractionation after Germination of Sorghum Varieties

PubMed Central

Afify, Abd El-Moneim M. R.; El-Beltagi, Hossam S.; Abd El-Salam, Samiha M.; Omran, Azza A.

2012-01-01

The changes in crude protein, free amino acids, amino acid composition, protein solubility, protein fractionation and protein digestibility after germination of sorghum were investigated. Sorghum varieties (Dorado, Shandaweel-6, Giza-15) were soaked for 20 h followed by germination for 72 h; the results revealed that crude protein and free amino acids in raw sorghum varieties ranged from 10.62 to 12.46% and 0.66 to 1.03 mg/g, respectively. Shandaweel-6 was the highest variety in crude protein and free amino acids content. After germination, crude protein was decreased and free amino acids were increased. There was an increase in content of valine and phenylalanine amino acids after germination. On the other hand, there was a decrease in most of amino acids after germination. After germination protein solubility was significantly increased. Regarding protein fractions, there was an increase in albumin, globulin and kafirin proteins and a decrease in cross linked kafirin and cross linked glutelin after germination. PMID:22319611

Effects of non-protein amino acids on survival and locomotion of Osmia bicornis.

PubMed

Felicioli, Antonio; Sagona, Simona; Galloni, Marta; Bortolotti, Laura; Bogo, Gherardo; Guarnieri, Massimo; Nepi, Massimo

2018-04-17

To investigate the effects of two non-protein amino acids, β-alanine and γ-amino butyric acid (GABA), on Osmia bicornis survival and locomotion, two groups of caged bees were fed with sugar syrup enriched with β-alanine and GABA, respectively. A further control group was fed with sugar syrup. Five behavioural categories were chosen according to the principle of parsimony and intrinsic unitary consistency from start to end, and recorded by scan sampling: two states (remaining under paper or in tubes) and three events (walking on net, feeding from flower and flying). We also analysed the amino acid content of haemolymph sampled from an additional 45 bees fed the same diets (15 per diet type). Bees fed with ß-alanine had a significantly shorter survival than those fed with control and GABA diets. The GABA diet induced higher locomotion than β-alanine. The former non-protein amino acid was only detected in the haemolymph of bees fed GABA. The results suggest that insects consuming non-protein amino-acid-rich diets absorb and transfer these substances to the haemolymph and that non-protein amino acids affect survival and locomotion. Ecological consequences are discussed in the framework of plant reproductive biology. This article is protected by copyright. All rights reserved. © 2018 The Royal Entomological Society.

[Evaluation of the adjusted amino acid score by digestibility for estimating the protein quality and protein available in food and diets].

PubMed

Pak, N; Vera, G; Araya, H

1985-03-01

The purpose of the present study was to evaluate the amino acid score adjusted by digestibility to estimate protein quality and utilizable protein in foods and diets, considering net protein utilization (NPU) as a biological reference method. Ten foods of vegetable origin and ten of animal origin, as well as eight mixtures of foods of vegetable and animal origin were studied. When all the foods were considered, a positive (r = 0.83) and highly significant correlation (p less than 0.001) between NPU and the amino acid score adjusted by digestibility was found. When the foods were separated according to their origin, this correlation was positive only for the foods of vegetable origin (r = 0.93) and statistically significant (p less than 0.001). Also, only in those foods were similar values found between NPU and amino acid score adjusted by digestibility, as well as in utilizable protein estimated considering both methods. Caution is required to interpret protein quality and utilizable protein values of foods of animal origin and mixtures of foods of vegetable and animal origin when the amino acid score method adjusted by digestibility, or NPU, are utilized.

Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

PubMed

Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

2015-10-08

Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

Whole-body protein turnover reveals the cost of detoxification of secondary metabolites in a vertebrate browser.

PubMed

Au, Jessie; Marsh, Karen J; Wallis, Ian R; Foley, William J

2013-10-01

The detoxification limitation hypothesis predicts that the metabolism and biotransformation of plant secondary metabolites (PSMs) elicit a cost to herbivores. There have been many attempts to estimate these costs to mammalian herbivores in terms of energy, but this ignores what may be a more important cost-increases in protein turnover and concomitant losses of amino acids. We measured the effect of varying dietary protein concentrations on the ingestion of two PSMs (1,8 cineole-a monoterpene, and benzoic acid-an aromatic carboxylic acid) by common brushtail possums (Trichosurus vulpecula). The dietary protein concentration had a small effect on how much cineole possums ingested. In contrast, protein had a large effect on how much benzoate they ingested, especially at high dietary concentrations of benzoate. This prompted us to measure the effects of dietary protein and benzoate on whole-body protein turnover using the end-product method following an oral dose of [(15)N] glycine. Increasing the concentration of dietary protein in diets without PSMs improved N balance but did not influence whole-body protein turnover. In contrast, feeding benzoate in a low-protein diet pushed animals into negative N balance. The concomitant increases in the rates of whole-body protein turnover in possums eating diets with more benzoate were indicative of a protein cost of detoxification. This was about 30 % of the dietary N intake and highlights the significant effects that PSMs can have on nutrient metabolism and retention.

Cinnamic Acid Derivatives as Inhibitors of Oncogenic Protein Kinases--Structure, Mechanisms and Biomedical Effects.

PubMed

Mielecki, Marcin; Lesyng, Bogdan

2016-01-01

Cinnamic acid belongs to phenolic-acid class of polyphenols, one of the most abundant plant secondary metabolites. These substances are widely studied because of plethora of their biological activities. In particular, their inhibition of protein kinases contributes to the pleiotropic effects in the cell. Protein kinases are essential in controlling cell signaling networks. Selective targeting of oncogenic protein kinases increases clinical anticancer efficacy. Cinnamic acid and related compounds have inspired researchers in the design of numerous synthetic and semisynthetic inhibitors of oncogenic protein kinases for the past three decades. Interest in cinnamoyl-scaffold-containing compounds revived in recent years, which was stimulated by modern drug design and discovery methodologies such as in vitro and in silico HTS. This review presents cinnamic acid derivatives and analogs for which direct inhibition of protein kinases was identified. We also summarize significance of the above protein kinase families - validated or promising targets for anticancer therapies. The inhibition mode may vary from ATP-competitive, through bisubstrate-competitive and mixedcompetitive, to non-competitive one. Kinase selectivity is often correlated with subtle chemical modifications, and may also be steered by an additional non-cinnamoyl fragment of the inhibitor. Specific cinnamic acid congeners may synergize their effects in the cell by a wider range of activities, like suppression of additional enzymes, e.g. deubiquitinases, influencing the same signaling pathways (e.g. JAK2/STAT). Cinnamic acid, due to its biological and physicochemical properties, provides nature-inspired ideas leading to novel inhibitors of oncogenic protein kinases and related enzymes, capable to target a variety of cancer cells.

Structure of the WipA protein reveals a novel tyrosine protein phosphatase effector from Legionella pneumophila.

PubMed

Pinotsis, Nikos; Waksman, Gabriel

2017-06-02

Legionnaires' disease is a severe form of pneumonia caused by the bacterium Legionella pneumophila. L. pneumophila pathogenicity relies on secretion of more than 300 effector proteins by a type IVb secretion system. Among these Legionella effectors, WipA has been primarily studied because of its dependence on a chaperone complex, IcmSW, for translocation through the secretion system, but its role in pathogenicity has remained unknown. In this study, we present the crystal structure of a large fragment of WipA, WipA435. Surprisingly, this structure revealed a serine/threonine phosphatase fold that unexpectedly targets tyrosine-phosphorylated peptides. The structure also revealed a sequence insertion that folds into an α-helical hairpin, the tip of which adopts a canonical coiled-coil structure. The purified protein was a dimer whose dimer interface involves interactions between the coiled coil of one WipA molecule and the phosphatase domain of another. Given the ubiquity of protein-protein interaction mediated by interactions between coiled-coils, we hypothesize that WipA can thereby transition from a homodimeric state to a heterodimeric state in which the coiled-coil region of WipA is engaged in a protein-protein interaction with a tyrosine-phosphorylated host target. In conclusion, these findings help advance our understanding of the molecular mechanisms of an effector involved in Legionella virulence and may inform approaches to elucidate the function of other effectors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

PubMed

Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

2014-09-01

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Silk protein aggregation kinetics revealed by Rheo-IR.

PubMed

Boulet-Audet, Maxime; Terry, Ann E; Vollrath, Fritz; Holland, Chris

2014-02-01

The remarkable mechanical properties of silk fibres stem from a multi-scale hierarchical structure created when an aqueous protein "melt" is converted to an insoluble solid via flow. To directly relate a silk protein's structure and function in response to flow, we present the first application of a Rheo-IR platform, which couples cone and plate rheology with attenuated total reflectance infrared spectroscopy. This technique provides a new window into silk processing by linking shear thinning to an increase in molecular alignment, with shear thickening affecting changes in the silk protein's secondary structure. Additionally, compared to other static characterization methods for silk, Rheo-IR proved particularly useful at revealing the intrinsic difference between natural (native) and reconstituted silk feedstocks. Hence Rheo-IR offers important novel insights into natural silk processing. This has intrinsic academic merit, but it might also be useful when designing reconstituted silk analogues alongside other polymeric systems, whether natural or synthetic. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

PubMed

Huang, Tengfang; Jander, Georg

2017-10-01

Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of

Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

DOEpatents

Xie, Jianming [San Diego, CA; Wang, Lei [San Diego, CA; Wu, Ning [Boston, MA; Schultz, Peter G [La Jolla, CA

2008-07-15

Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

PubMed

Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

2014-03-01

Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

Quantitative Protein Localization Signatures Reveal an Association between Spatial and Functional Divergences of Proteins

PubMed Central

Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

2014-01-01

Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

Serum protein adsorption and platelet adhesion on aspartic-acid-immobilized polysulfone membranes.

PubMed

Higuchi, Akon; Hashiba, Hirokazu; Hayashi, Rika; Yoon, Boo Ok; Sakurai, Masaru; Hara, Mariko

2004-01-01

Polysulfone (PSf) membranes that covalently conjugated with aspartic acid (ASP-PSf) were prepared and analyzed for hemocompatability. Compared to PSf or other types of surface-modified PSf membranes, the ASP-PSf membranes had a reduced ability to adsorb protein from either a plasma solution or a mixed solution of albumin, globulin and fibrinogen. This appears to be due to the creation of a hydrophilic surface by the aspartic acid zwitterion immobilized on the ASP-PSf membranes. Furthermore, the analyses of membrane protein adsorption showed that a mixed protein solution recapitulates the cooperative adsorption of proteins that occurs in plasma. We also found that the number of adhering platelets was the lowest on the ASP-PSf membranes and, in general, that platelet adhesion decreased in parallel with fibrinogen adsorption. In summary, aspartic acid immobilized on the ASP-PSf membranes, which have zwitterions with a net zero charge, effectively contributes to the hydrophilic and hemocompatible sites on the surface of the hydrophobic PSf membranes.

Genomic and transcriptomic analyses reveal adaptation mechanisms of an Acidithiobacillus ferrivorans strain YL15 to alpine acid mine drainage.

PubMed

Peng, Tangjian; Ma, Liyuan; Feng, Xue; Tao, Jiemeng; Nan, Meihua; Liu, Yuandong; Li, Jiaokun; Shen, Li; Wu, Xueling; Yu, Runlan; Liu, Xueduan; Qiu, Guanzhou; Zeng, Weimin

2017-01-01

Acidithiobacillus ferrivorans is an acidophile that often occurs in low temperature acid mine drainage, e.g., that located at high altitude. Being able to inhabit the extreme environment, the bacterium must possess strategies to copy with the survival stress. Nonetheless, information on the strategies is in demand. Here, genomic and transcriptomic assays were performed to illuminate the adaptation mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine drainage environment in Yulong copper mine in southwest China. Genomic analysis revealed that strain has a gene repertoire for metal-resistance, e.g., genes coding for the mer operon and a variety of transporters/efflux proteins, and for low pH adaptation, such as genes for hopanoid-synthesis and the sodium:proton antiporter. Genes for various DNA repair enzymes and synthesis of UV-absorbing mycosporine-like amino acids precursor indicated hypothetical UV radiation-resistance mechanisms in strain YL15. In addition, it has two types of the acquired immune system-type III-B and type I-F CRISPR/Cas modules against invasion of foreign genetic elements. RNA-seq based analysis uncovered that strain YL15 uses a set of mechanisms to adapt to low temperature. Genes involved in protein synthesis, transmembrane transport, energy metabolism and chemotaxis showed increased levels of RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher RNA transcript counts at 6°C, possibly implicated in protecting DNA against oxidative stress at low temperature. The study represents the first to comprehensively unveil the adaptation mechanisms of an acidophilic bacterium to the acid mine drainage in alpine regions.

Genomic and transcriptomic analyses reveal adaptation mechanisms of an Acidithiobacillus ferrivorans strain YL15 to alpine acid mine drainage

PubMed Central

Ma, Liyuan; Feng, Xue; Tao, Jiemeng; Nan, Meihua; Liu, Yuandong; Li, Jiaokun; Shen, Li; Wu, Xueling; Yu, Runlan; Liu, Xueduan; Qiu, Guanzhou; Zeng, Weimin

2017-01-01

Acidithiobacillus ferrivorans is an acidophile that often occurs in low temperature acid mine drainage, e.g., that located at high altitude. Being able to inhabit the extreme environment, the bacterium must possess strategies to copy with the survival stress. Nonetheless, information on the strategies is in demand. Here, genomic and transcriptomic assays were performed to illuminate the adaptation mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine drainage environment in Yulong copper mine in southwest China. Genomic analysis revealed that strain has a gene repertoire for metal-resistance, e.g., genes coding for the mer operon and a variety of transporters/efflux proteins, and for low pH adaptation, such as genes for hopanoid-synthesis and the sodium:proton antiporter. Genes for various DNA repair enzymes and synthesis of UV-absorbing mycosporine-like amino acids precursor indicated hypothetical UV radiation—resistance mechanisms in strain YL15. In addition, it has two types of the acquired immune system–type III-B and type I-F CRISPR/Cas modules against invasion of foreign genetic elements. RNA-seq based analysis uncovered that strain YL15 uses a set of mechanisms to adapt to low temperature. Genes involved in protein synthesis, transmembrane transport, energy metabolism and chemotaxis showed increased levels of RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher RNA transcript counts at 6°C, possibly implicated in protecting DNA against oxidative stress at low temperature. The study represents the first to comprehensively unveil the adaptation mechanisms of an acidophilic bacterium to the acid mine drainage in alpine regions. PMID:28542527

Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals.

PubMed

Liu, Fobang; Lai, Senchao; Tong, Haijie; Lakey, Pascale S J; Shiraiwa, Manabu; Weller, Michael G; Pöschl, Ulrich; Kampf, Christopher J

2017-03-01