Protein Function Research Papers - Academia.edu (original) (raw)

Mena, an actin regulatory protein, functions at the convergence of motility pathways that drive breast cancer cell invasion and migration in vivo. The tumor microenvironment spontaneously induces both increased expression of the Mena INV and decreased expression of Mena11a isoforms in invasive and migratory tumor cells. Tumor cells with this Mena expression pattern participate with macrophages in migration and intravasation in mouse mammary tumors in vivo. Consistent with these findings, anatomical sites containing tumor cells with high levels of Mena expression associated with perivascular macrophages were identified in human invasive ductal breast carcinomas and called TMEM. The number of TMEM sites positively correlated with the development of distant metastasis in humans. Here we demonstrate that mouse mammary tumors generated from EGFP-Mena INV expressing tumor cells are significantly less cohesive and have discontinuous cell-cell contacts compared to Mena11a xenografts. Using the mouse PyMT model we show that metastatic mammary tumors express 8.7 fold more total Mena and 7.5 fold more Mena INV mRNA than early non-metastatic ones. Furthermore, Mena INV expression in fine needle aspiration biopsy (FNA) samples of human invasive ductal carcinomas correlate with TMEM score while Mena11a does not. These results suggest that Mena INV is the isoform associated with breast cancer cell discohesion, invasion and intravasation in mice and in humans. They also imply that Mena INV expression and TMEM score measure related aspects of a common tumor cell dissemination mechanism and provide new insight into metastatic risk.

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- Cell Adhesion, Tumor microenvironment, Mice, Protein Function

Fibrillins are nuclear-encoded, plastid proteins associated with chromoplast fibrils and chloroplast plastoglobules, thylakoids, photosynthetic antenna complexes, and stroma. There are 12 sub-families of fibrillins. However, only three of these sub-families have been characterized genetically or functionally. We review evidence indicating that fibrillins are involved in plastoglobule structural development, chromoplast pigment accumulation, hormonal responses, protection of the photosynthetic apparatus from photodamage, and plant resistance to a range of biotic and abiotic stresses. The area of fibrillin research has substantial growth potential and will contribute to better understanding of mechanisms of plant stress tolerance and plastid structure and function.

- by Dharmendra Singh
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- Photosynthesis, Plant Biology, Gene expression, Phylogeny

† E.P. and R.A. contributed equally to this work.

- by Christina Othon
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- Thermodynamics, Fluorescence Spectroscopy, Water, Protein Folding
- by Dale Oxidase-edmondson
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- Science, Multidisciplinary, Plastics, Disinfectants
- by Hesham Ali
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- Genetics, Computer Science, Genetic Algorithms, Molecular Biophysics

Sequence determination and analysis began on proteins in the 1950s, with RNA starting about a decade later and DNA a similar period later still. Hence many of the concepts for function prediction were first developed by looking at amino acid sequences. Over time these methods have become much more sophisticated, allowing better discrimination of only weak similarities. The most recent developments concern an examination of contextual information, such as operon structure, metabolic reconstruction or co-expression profiles.

- by Charlie Hodgman
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- Bioinformatics, Research, Sequence Analysis, Biological Sciences

P-glycoprotein (P-gp) belongs to superfamily of ABC (ATP-binding cassette) drug transporters. It is expressed in intestines, brain, kidneys, testes, liver, adrenal gland, lungs heart and eyes. This protein functions as a biological barrier by extruding toxic substances and xenobiotics out of cells. P-gp could modulate pharmacokinetics of antibacterial drugs through limitation of their oral absorption and penetration into target organs. Drug-drug interactions with antibacterials could be mediated by inhibition or induction of P-gp. Among the antibiotics recognized as substrates and modulators of P-gp are structurally unrelated compounds as fluoroquinolones, macrolides, ansamycines, tetracyclines and antracyclines. These findings provide a basis for the understanding of the pharmacokinetics, pharmacodynamics and toxicodynamics of the antibiotics in healthy and diseased individuals.

- by Aneliya Milanova
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- P-glycoprotein, Protein Function, Adrenal Gland, Drug Interaction

The eect of rigor temperature (RT, 35 versus 10 C), increased ionic concentration (MS: 0 versus 0 . 2% mixed salts), oxidized lipids (OL, 0 versus 1% added oxidized fat), increased free amino acids (FAA, 0 versus 0 . 3% added mixture of amino acids), fat content (FC, 0 versus 15% added fat) and excluding atmospheric gases (V, 0 versus 99 . 9% vacuum) on protein functionality in mince stored 1 month at À20 C was studied using a complete 2 6 factorial design. All factors studied aected the functional properties of beef. Fresh 24 hr samples that entered rigor at 35 C had lower total (TPS), myo®brillar (MPS) and sarcoplasmic (SPS) protein solubilities than samples entering rigor at 10 C (p<0 . 01). During frozen storage, RT, MS and OL alone did not aect protein solubilities, FAA increased TPS but did not aect MPS and SPS, increasing the meat's fat content raised TPS and SPS but did not aect MPS, applying a vacuum increased TPS and MPS but lowered SPS (p<0 . 05). Cook yield of frozen stored mince increased with higher FAA but decreased with higher fat content, and the peak force of patties made from frozen stored mince was lowered by increased MS and low FC during storage. Various signi®cant interactions are tabulated and discussed. #

- by Red Hunter
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- Chemical Engineering, Meat Science, Animal Production, Factorial Design

A filter based on a set of unsupervised neural networks trained with a winner-take-all strategy discloses signals along the coding sequences of G-protein coupled receptors. By comparing with the existing experimental data it appears that these signals correlate with putative functional domains of the proteins. After protein alignment within subfamilies, signals cluster in protein regions which, according to the presently available experimental results, are described as possible functional domains of the folded proteins. The mapping procedure reveals characteristic regions in the coding sequences common and/or characteristic of the receptor subtype. This is particularly noticeable for the third cytoplasmic loop, which is likely to be involved in the molecular coupling of all the subfamilies with G-proteins. The results indicate that our mapping can highlight intrinsic representative features of the coding sequences which, in the case of G-protein coupled receptors, are characteristic of protein functional regions and suggest a possible application of the filter for predicting functional determinants in proteins starting from the coding sequence.

- by Piero Fariselli
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- Genetics, Neural Networks, Neural Network, Sequence Analysis

We re-sequenced HPV16 genome (~6 kb) implicated in cervical carcinogenesis (LCR, E2, E5, E6, E7, L1, L2) to prioritize sequence variants for functional validation as biomarkers, using CaCx cases (n = 74) and asymptomatic controls (n = 24). Of the nucleotide variations recorded (n = 271), non-synonymous changes in L2 region were significantly higher (p = 0.005) among cases (2.67%) compared to controls (1.27%). Using SIFT database, 29 non-synonymous changes (frequency = 0.01-0.03) predicted as deleterious to protein functions were identified. Haplotype analysis considering 110 polymorphic variations (frequency ≥ 0.05) within intact viral isolates (53 CaCx cases and 21 controls) using NETWORK software, confirmed Asian-American (AA, 14.86%) and European (E, 85.14%) variants, differing at 78 positions. The E-variants portrayed thirty-six haplotypes, of which, E-12 was most prevalent within cases (38.1%; 16 / 42) and controls (28.57%; 6 / 21) harboring polymorphic variations at 10 positions, in contrast to HPV16R. Cases of the E-12 haplotype harbored 7 deleterious mutations distributed within L1 (n = 1), E2 (n = 1), E5 (n = 1), and L2 (n = 4), while none within similar controls. Thus rare deleterious variations within genes implicated in productive infection over the E-12 haplotype background of intact HPV16 isolates might be of causal relevance for CaCx development.

- by Sharmila Sengupta
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- Polymorphism, Adolescent, India, Biological Sciences

This paper reports a study of the influence of a Konjac glucomannan aqueous dispersion (KAD) as ingredient, at different alkalinity levels, on the thermal stability of low-quality squid surimi (Dosidicus gigas) and the viscoelastic properties of its gel. An increase in elastic (G 0 ) and viscous (G 00 ) moduli at T < 50°C, reflecting protein aggregation, and a strong decrease at T > 50°C, reflecting structural damage, were observed in thermal gelation profiles of low-quality squid surimi. The contribution of 1% KAD (10%) to enhancement of gelation ability was assessed by evaluating the viscoelastic properties of the gels, with and without KAD, at increasing alkalinity levels. Gels with KAD at high-pH had the best rheological properties. Small amplitude oscillatory shear (SAOS) tests showed a significant decrease in rigidity, an increase in strain amplitude and a decrease in the frequency-dependence of G 0 and G 00 . These results were in agreement with instrumental texture analyses, meaning that KAD may be used to overcome the negative effect of the poor protein functionality of low-quality squid surimi and achieve better gels from them.* Ó

- by Javier Borderías
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- Food Engineering, Protein Aggregation, Protein Function, Rheological properties

Potato fruit juice, i.e. the stream resulting after the extraction of the starch from the potato, contains up to 2.5% [w/w] of proteins that are potentially valuable for the food market. However, today the recovery of protein from the potato fruit juice with reverse osmosis membranes results in a protein concentrate that is not suitable for human consumption. The described research shows that the use of ultrafiltration with additional diafiltration is able to produce a higher quality protein. Tests with the produced protein show that the quality depends on the rate of diafiltration used and that the product has functional properties that are equal or better than the compared commercial food product that are currently used.

- by Antoine Kemperman and +1
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- Engineering, Potato, Desalination, Protein Function
- by eric chevet
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- Bioinformatics, Genomics, Computational Biology, Mass Spectrometry

Experimental and computational approaches to estimate solubility and permeability in discovery and development settings are described. In the discovery setting 'the rule of 5' predicts that poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight (MWT) is greater than 500 and the calculated Log P (CLogP) is greater than 5 (or MlogP . 4.15). Computational methodology for the rule-based Moriguchi Log P (MLogP) calculation is described. Turbidimetric solubility measurement is described and applied to known drugs. High throughput screening (HTS) leads tend to have higher MWT and Log P and lower turbidimetric solubility than leads in the pre-HTS era. In the development setting, solubility calculations focus on exact value prediction and are difficult because of polymorphism. Recent work on linear free energy relationships and Log P approaches are critically reviewed. Useful predictions are possible in closely related analog series when coupled with experimental thermodynamic solubility measurements.

- by Christopher Lipinski
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- Pharmacology, Thermodynamics, Physical Chemistry, Polymorphism

A practical course was given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering" at the Federal University of Sã o Carlos (UFSCar), Sã o Paulo, Brazil. The goal of the course was to teach current molecular biology tools applied to a real research situation that could be reported by the students themselves. The purpose was to produce a plant recombinant protein and demonstrate a heretofore unreported biological activity. Cystatins, natural inhibitors of cysteine proteases, were proposed for these studies. Initially, the students searched for plant cystatin cDNA sequences in the NCBI databases and selected the Oryzacystatin I gene (ocI) from rice, Oriza sativa, as the target gene for this study. Total RNA was extracted from rice-germinating seeds and primers containing restriction sites for NdeI and EcoRI were designed based on the ocI cDNA sequence and then used to amplify the open reading frame (ORF). RT-PCR amplification provided a band of the expected size for ocI ORF (309 bp). The PCR product was cut with NdeI and EcoRI restriction enzymes and cloned directly in the pET28a expression vector digested with the same enzymes. A pET28-ocI recombinant clone was selected, checked by sequencing, and used to transform Escherichia coli BL21 (DE3) expression strain. After induction of the bacteria with isopropylthiogalactoside and cellular disruption, the His-tagged OCI protein, present mainly in the soluble fraction, was purified by affinity chromatography in a nickel column. The purified protein was successfully used to inhibit fungal growth (Trichoderma reesei). The results were discussed extensively and the students contributed to the writing of this article, of which they are co-authors.

Background One of the strategies for protein function annotation is to search particular structural motifs that are known to be shared by proteins with a given function. Results Here, we present a systematic extraction of structural motifs of seven residues from protein loops and we explore their correspondence with functional sites. Our approach is based on the structural alphabet HMM-SA (Hidden Markov Model - Structural Alphabet), which allows simplification of protein structures into uni-dimensional sequences, and advanced pattern statistics adapted to short sequences. Structural motifs of interest are selected by looking for structural motifs significantly over-represented in SCOP superfamilies in protein loops. We discovered two types of structural motifs significantly over-represented in SCOP superfamilies: (i) ubiquitous motifs, shared by several superfamilies and (ii) superfamily-specific motifs, over-represented in few superfamilies. A comparison of ubiquitous words with known small structural motifs shows that they contain well-described motifs as turn, niche or nest motifs. A comparison between superfamily-specific motifs and biological annotations of Swiss-Prot reveals that some of them actually correspond to functional sites involved in the binding sites of small ligands, such as ATP/GTP, NAD(P) and SAH/SAM. Conclusions Our findings show that statistical over-representation in SCOP superfamilies is linked to functional features. The detection of over-represented motifs within structures simplified by HMM-SA is therefore a promising approach for prediction of functional sites and annotation of uncharacterized proteins.

- by Leslie REGAD
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- Biological Sciences, Markov chains, Mathematical Sciences, hidden Markov model

Modifications of neuronal circuits allow the brain to adapt and change with experience. This plasticity manifests during development and throughout life, and can be remarkably long lasting. Evidence has linked activity-regulated gene expression to the long-term structural and electrophysiological adaptations that take place during developmental critical periods, learning and memory, and alterations to sensory map representations in the adult. In all these cases, the cellular response to neuronal activity integrates multiple tightly coordinated mechanisms to precisely orchestrate long-lasting, functional and structural changes in brain circuits. Experience-dependent plasticity is triggered when neuronal excitation activates cellular signaling pathways from the synapse to the nucleus that initiate new programs of gene expression. The protein products of activity-regulated genes then work via a diverse array of cellular mechanisms to modify neuronal functional properties. Synaptic stre...

- by Elly Nedivi
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- Cognitive Science, Gene expression, Synaptic Plasticity, Signal Transduction

Each intracellular organelle critically depends on maintaining its specific lipid composition that in turn contributes to the biophysical properties of the membrane. With our knowledge increasing about the organization of membranes with defined microdomains of different lipid compositions, questions arise regarding the molecular mechanisms that underlie the targeting to/segregation from microdomains of a given protein. In addition to specific lipid-transmembrane segment interactions as a basis for partitioning, the presence in a given microdomain may alter the conformation of proteins and, thus, the activity and availability for regulatory modifications. However, for most proteins, the specific lipid environment of transmembrane segments as well as its relevance to protein function and overall membrane organization are largely unknown. To help fill this gap, we have synthesized a novel photoactive sphingolipid precursor that, together with a precursor for phosphoglycerolipids and with photo-cholesterol, was investigated in vivo with regard to specific protein transmembrane spanlipid interactions. As a proof of principle, we show specific labeling of the ceramide transporter with the sphingolipid probe and describe specific in vivo interactions of lipids with caveolin-1, phosphatidylinositol transfer protein b, and the mature form of nicastrin. This novel photolabile sphingolipid probe allows the detection of proteinsphingolipid interactions within the membrane bilayer of living cells.-Haberkant,

- by Constanze Reinhard
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- Molecular Mechanics, Cell line, Proteins, Sphingolipids

Pulses (pea, chickpea, lentil, bean) are an important source of food proteins. They contain high amounts of lysine, leucine, aspartic acid, glutamic acid and arginine and provide well balanced essential amino acid profiles when consumed with cereals and other foods rich in sulphur-containing amino acids and tryptophan. The protein content of most pulse legumes fall within the range of 17-30% (d.w.b.). Apart from their nutritional properties, pulse proteins also possess functional properties that play an important role in food formulation and processing. Examples of such functional properties include solubility, water and fat binding capacity and foaming. Various research studies indicate that some functional properties of pulse proteins may be comparable to those of other frequently used proteins such as soy and whey. The functional properties of pulse proteins have been exploited in the preparation and development of products such as bakery products, soups, extruded products and ready to eat snacks. The growing body of research on the health benefits associated with the consumption of pulses has increased interest in developing innovative technologies to expand the use of pulses in food products. At the same time, there are growing global food security challenges and protein malnutrition continues to be a problem in many countries around the world. Pulses, especially when blended with cereal proteins, may offer a promising alternative source for nutritional and functional proteins. This review provides an overview of the characteristics of pulse proteins, current and emerging techniques for their fractionation, their major functional properties and opportunities for their use in various applications.

- by Fatemeh Zare
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- Chemical Engineering, Food, Food Security, Properties

The 18th international papillomavirus conference took place in Barcelona, Spain in July 2000. The HPV clinical workshop was jointly organised with the annual meeting of the Spanish Association of Cervical Pathology and Colposcopy. The conference included 615 abstracts describing ongoing research in epidemiology, diagnosis/screening, treatment/prognosis, immunology/human immunodeficiency virus, vaccine development/

- by Ian Frazer
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- Clinical Pathology, Spain, Clinical, Protein Function

Scorpion toxins are common experimental tools for studies of biochemical and pharmacological properties of ion channels. The number of functionally annotated scorpion toxins is steadily growing, but the number of identified toxin sequences is increasing at much faster pace. With an estimated 100,000 different variants, bioinformatic analysis of scorpion toxins is becoming a necessary tool for their systematic functional analysis. Here, we report a bioinformatics-driven system involving scorpion toxin structural classification, functional annotation, database technology, sequence comparison, nearest neighbour analysis, and decision rules which produces highly accurate predictions of scorpion toxin functional properties. #

- by Seng Hong Too
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- Functional Analysis, Algorithms, Molecular, Computer Software

844ins68 is a frequent polymorphism of the cystathionine ␤-synthase gene (CBS) that consists of a 68-bp insertion duplicating the 3 splice site of intron 7 and the 5-end of exon 8. The presence of two identical 3 splice sites spaced by 68 bp should lead to either a selection of the proximal site or to at least two alternatively spliced CBS mRNA variants. Instead, an accurate selection of the distal 3 splice site is observed in the 844ins68 carriers. The duplication has generated a gene re-arrangement at the 3 splice site where two GGGG runs have been brought close to each other. Using a minigene system, we have investigated the effect this peculiar configuration might have on the selection of the 3 splice site of intron 7 in the CBS gene. Minimal disruption of the G runs resulted in a dramatic shift toward the proximal 3 splice site selection with inclusion of the 68-bp insertion and a consequent change of the reading frame. The insertional event created this peculiar configuration of two G repeats close to each other that subsequently acquired the ability to strongly bind heterogeneous nuclear ribonucleoprotein (hnRNP) H1, a specific trans-acting factor. The interaction of hnRNP H1 with G runs within the 844ins68 context might interfere with the recruitment of splicing factors to the proximal 3 splice site thus favoring the selection of the distal 3 splice site. Our results therefore suggest the possibility that the insertion was an evolutionary event that allowed the rescue of the wild-type sequence, so preserving protein function.

- by Maurizio Romano
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- RNA, Polymorphism, Biological Chemistry, Biological Sciences

Proteins are increasingly being utilized to perform functional roles in food formulations. Common food proteins possess good functional properties including solubility, gelation, emulsification and foaming. The functional properties of proteins are impaired near their pl (isoelectric point), as is the case in most acidic foods. Enzymatic modification of food proteins by controlled proteolysis can enhance their functional properties over a wide pH range, and other processing conditions. Choosing the right proteolytic enzyme, environmental conditions for hydrolysis and degree of hydrolysis is crucial for enhancing the functional properties of proteins. Understanding the molecular properties required for protein functionality and the development of strategies to achieve them are critical for developing and utilizing modified protein ingredients. Numerous reports on enhancing the functionality of food proteins by limited proteolysis have been published, some of which are reviewed in this article.

- by D. Panyam and +1
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- Optimization, Proteins, Protein Function, Environmental Conditions

This work shows that the recently described Escherichia coli BtuE peroxidase protects the bacterium against oxidative stress that is generated by tellurite and by other reactive oxygen species elicitors (ROS). Cells lacking btuE (DbtuE) displayed higher sensitivity to K 2 TeO 3 and other oxidative stress-generating agents than did the isogenic, parental, wild-type strain. They also exhibited increased levels of cytoplasmic reactive oxygen species, oxidized proteins, thiobarbituric acid reactive substances, and lipoperoxides. E. coli DbtuE that was exposed to tellurite or H 2 O 2 did not show growth changes relative to wild type cells either in aerobic or anaerobic conditions. Nevertheless, the elimination of btuE from cells deficient in catalases/ peroxidases (Hpx 2 ) resulted in impaired growth and resistance to these toxicants only in aerobic conditions, suggesting that BtuE is involved in the defense against oxidative damage. Genetic complementation of E. coli DbtuE restored toxicant resistance to levels exhibited by the wild type strain. As expected, btuE overexpression resulted in decreased amounts of oxidative damage products as well as in lower transcriptional levels of the oxidative stress-induced genes ibpA, soxS and katG.

- by Juan Sandoval
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- Genetics, Oxidative Stress, Multidisciplinary, Peroxidase

Protein crosslinking can have a profound eect on the structure and function of proteins in food. Dehydroascorbic acid (DHA) has been shown to be involved in Maillard type chemistry that leads to protein crosslinking. In this study, the eect of temperature on the rate of this reaction was studied. The reaction was shown to proceed rapidly at temperatures that may be encountered during food processing. In order to assess the relative reactivity of DHA and its breakdown products, ®ve known degradation products were reacted with protein and their crosslinking ability, via Maillard chemistry, was assessed. Oxalic acid did not eect protein crosslinking. Threose, glyoxal, diacetyl and methyl glyoxal all reacted faster than DHA. The main crosslinking reaction observed was shown to involve a lysine residue. Our results suggest that these molecules may be important in determining the modi®cation of protein functionality during food processing. #

- by Lyall Simmons
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- Food Chemistry, Multidisciplinary, Food Processing, Protein Structure and Function

BRCTs are protein-docking modules involved in eukaryotic DNA repair. They are characterized by low sequence homology with generally well-conserved structure organization. In a considerable number of proteins, a pair of BRCT structural repeats occurs, connected with inter-BRCT linkers, variable in length, sequence and structure. Linkers may separate and control the relative position of BRCT domains as well as protect and stabilize the hydrophobic inter-BRCT interface region. Their vital role in protein function has been demonstrated by recent findings associating missense mutations in the inter-repeat linker region of the BRCT domain of BRCA1 (BRCA1-BRCT) to hereditary breast/ovarian cancer. The interaction of 53BP1 with the core domain of the p53 tumor suppressor involves the C-terminal BRCT repeat as well as the inert-BRCT linker of the tandem BRCT domain of 53BP1 (53BP1-BRCT). High-accuracy differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to characterize the heatinduced unfolding of 53BP1-BRCT domain. The calorimetric results provide evidence for unfolding to an intermediate, only partly unfolded state, which, based on the CD results, retains the secondary structural characteristics of the native protein. A direct comparison with the corresponding thermal processes for BRAC1-BRCT and BARD1-BRCT provides evidence that the observed behavior is analogous to BRCA1-BRCT even though the two domains differ substantially in the linker structure. Moreover, chemical denaturation experiments of the untagged 53BP1-BRCT and comparison with BRCA1 and BARD1 BRCTs show that no clear association can be drawn between the structural organization of the inter-BRCT linkers and the overall stability of the BRCT domains.

- by Angelos Thanassoulas
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- Thermodynamics, Fluorescence Spectroscopy, DNA repair, Ovarian Cancer

Ultra high pressure (UHP) processing is an attractive non-thermal technique for food treatment and preservation at room temperature, with the potential to achieve interesting functional effects. The majority of UHP process applications in food systems have focused on shelf-life extension associated with non-thermal sterilization and a reduction or increase in enzymatic activity. Only a few studies have investigated modifications of structural characteristics and/or protein functionalities. Despite the rapid expansion of UHP applications in food systems, limited information is available on the effects of UHP on the structural and physicochemical properties of starch and/or its chemical derivatives included in most processed foods as major ingredients or minor additives. Starch and its chemical derivatives are responsible for textural and physical properties of food systems, impacting their end-use quality and/or shelf-life. This article reviews UHP processes for native (unmodified) starch granules and their effects on the physicochemical properties of UHP-treated starch. Furthermore, functional roles of UHP in acid-hydrolysis, hydroxypropylation, acetylation, and cross-linking reactions of starch granules, as well as the physicochemical properties of UHP-assisted starch chemical derivatives, are discussed.

- by Geera Store
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- Nutrition and Dietetics, Collagen Cross-linking, Starch, Animal Production

The most critical step in maize domestication (Zea mays ssp. mays) was the liberation of the kernel from the hardened, protective casing that envelops the kernel in the maize progenitor, teosinte 1 . This evolutionary step exposed the kernel on the surface of the ear such that it could be readily utilized as a food source by humans. Here, we show that this key event in maize domestication is controlled by a single gene (teosinte glume architecture; tga1) belonging to the SBP-domain family 2 of transcriptional regulators. The factor controlling the phenotypic difference between maize and teosinte maps to a 1 kilobase region within which maize and teosinte show only six fixed differences in their DNA sequences. One of these differences encodes a non-conservative amino acid substitution and may affect protein function, while the other five differences potentially affect gene regulation. Molecular evolution analyses show that this region was the target of selection during maize domestication. Our results demonstrate that modest genetic changes in single genes can induce dramatic changes in phenotype during domestication and evolution.

- by Yves Vigouroux
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- Genetics, Gene regulation, Molecular Evolution, Agriculture

Identification of antigens that induce an immune response against tick infestations is required for the development of vaccines against these economically important ectoparasites. In order to identify protective antigens, we constructed a cDNA expression library from a continuous Ixodes scapularis cell line (IDE8) that was initially derived from tick embryos. cDNA clones were subjected to several rounds of screening in which mice were immunized with individual pools and then challenge-exposed by allowing I. scapularis larvae to feed on the immunized and control mice. Immunity against tick infestation was determined by the reduction in the ability of the larvae to feed to repletion and molt to the nymphal stage. Individual clones in pools that induced immunity to larval infestations were partially sequenced and grouped according to their putative protein function by comparison with sequence databases. The screening identified several individual antigens that induced a protective immune response against I. scapularis infestations. Our studies demonstrated for the first time that cDNA expression library immunization (ELI) combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in vaccines against ticks.

- by Katherine Kocan
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- Immune response, Sequence Analysis, Cell Culture, Biological Sciences
- by Sudandiradoss C
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- Genetics, Cystic Fibrosis, Protein Structure and Function, Protein structure

distinct pathophysiologic entities, but rather represent Eva Nelis, 10 Christine Van Broeckhoven, 10 a spectrum of related "myelinopathies" due to an unand James R. Lupski 1, 11, 12 derlying defect in myelination. Furthermore, we hy-1 Department of Molecular and Human Genetics pothesize the differences in clinical severity seen with 3 Department of Neurology mutations in MPZ are related to the type of mutation 11 Department of Pediatrics and its subsequent effect on protein function (i.e., loss

- by D. Witt
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- Cognitive Science, Protein Function, Peripheral Neuropathy, Spectrum

- by Poul Poulsen
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- Engineering, Potato, Desalination, Protein Function

Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.

- by Mathias Uhlén
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- Fluorescence Microscopy, Proteomics, Mitochondria, Confocal Microscopy

Ten years of experience with molecular classspecific information systems (MCSIS) such as with the hand-curated G protein-coupled receptor database (GPCRDB) or the semiautomatically generated nuclear receptor database has made clear that a wide variety of questions can be answered when protein-related data from many different origins can be flexibly combined. MCSISes revolve around a multiple sequence alignment (MSA) that includes ''all'' available sequences from the entire superfamily, and it has been shown at many occasions that the quality of these alignments is the most crucial aspect of the MCSIS approach. We describe here a system called 3DM that can automatically build an entire MCSIS. 3DM bases the MSA on a multiple structure alignment, which implies that the availability of a large number of superfamily members with a known three-dimensional structure is a requirement for 3DM to succeed well. Thirteen MCSISes were constructed and placed on the Internet for examination. These systems have been instrumental in a large series of research projects related to enzyme activity or the understanding and engineering of specificity, protein stability engineering, DNA-diagnostics, drug design, and so forth. Proteins 2010; 78:2101-2113. V V C 2010 Wiley-Liss, Inc.

- by Henk-Jan Joosten and +5
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- Microbiology, Computational Biology, Protein Engineering, Protein Stability

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- by Vajira Dissanayake
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- Genetics, Data Mining, Molecular Dynamics Simulation, Polymorphism

This study aimed to determine the effect of pretreating defatted soy flakes with ultrasound on soy protein isolate (SPI) yield and functionality. Defatted soy flakes dispersed into water (16%, w/w) were sonicated for 30, 60 and 120 s at ultrasonic amplitudes of 21 and 84 µmpp (peak to peak amplitude in µm), representing low and high power, respectively. The power densities were 0.30 and 2.56 W mL−1, respectively. The SPI yield increased by 13 and 34%, after sonication for 120 s at low and high power, respectively. The sonication of defatted soy flakes for 120 s at the higher power level improved the SPI solubility by 34% at pH 7.0, while decreasing emulsification and foaming capacities by 12 and 26%, respectively, when compared to control SPI. Rheological behavior of the SPI was also modified with significant loss in consistency coefficient due to sonication. Some of these results could be explained by the loss of the protein native state with increased sonication time and power.

- by Bishnu Karki
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- Chemical Engineering, High Pressure, Heat Treatment, Ultrasound

Hepatocyte invasion by malaria parasites is mediated by specific molecular interactions. Several lines of evidence suggest the importance of the surface plasmodial circumsporozoite (CS) protein in the sporozoite invasion of hepatocytes. Identification of the sequences involved in binding to hepatocytes is an important step towards understanding the structural basis for the sporozoitehepatocyte interaction. In this study, binding assays between Plasmodium falciparum CS peptides and HepG2 cells were performed. Fifteen overlapping residue 20 mer long peptides, spanning the entire CS sequence, were tested in HepG2 cell binding assays. Five High Binding Activity Peptides (HBAPs) to HepG2 cells were identified: 4593, (NANPNANPNANP); 4383, (N 6 SRSLGEND 6 DGN 6 NEDNEKLR); 4388, (GNGQGHNM 6 PNDPNRNVD 6 ENA 6 ); 4389, (HNM 6 PNDPNRNVD 6 ENA 6 NAN 6 SA 6 ) and 4390, (DPNRNVD 6 ENA 6 NAN 6 SA 6 VKNN 6 N). The HBAP HepG2 interaction is independent of charge and amino-acid composition, but sequence dependent. Four HBAPs (4383, 4388, 4389 and 4390) are bound with similar affinity to a 50 kDa molecule. These HBAPs define three Hepatocyte Binding Sequences (HBSs): HBS-1, located between residues 68 and 87 (HBAP 4383); HBS-11, the repeat NANP region (HBAP 4593), for which anti repeat antibodies are able to specifically inhibit sporozoite invasion of hepatocytes have been reported; and HBS-111, between residues 286 and 315 (HBAPs 4388, 4388 and 4390), respectively. Interestingly, HBS 111 carries two earlier-reported B-epitopes (underlined) in peptides 4388, 4389 and 4390 (GNGQGHNMPNDPNRNVD ENANANSAVKNN) in its sequence. The HBSs reported here show lesser interspecie-variability than the entire protein in species invading the same kind of hepatic cells. This data supports these HBSs' important role in CS-protein function; they could be used as ligand by the sporozoite to invade hepatic cells.

- by Marisol Ocampo
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- Biological Sciences, Hepatocellular Carcinoma, Vaccine, Peptides

The florigen paradigm implies a universal flowering-inducing hormone that is common to all flowering plants. Recent work identified FT orthologues as originators of florigen and their polypeptides as the likely systemic agent. However, the developmental processes targeted by florigen remained unknown. Here we identify local balances between SINGLE FLOWER TRUSS (SFT), the tomato precursor of florigen, and SELF-PRUNING (SP), a potent SFT-dependent SFT inhibitor as prime targets of mobile florigen. The graft-transmissible impacts of florigen on organ-specific traits in perennial tomato show that in addition to import by shoot apical meristems, florigen is imported by organs in which SFT is already expressed. By modulating local SFT/SP balances, florigen confers differential flowering responses of primary and secondary apical meristems, regulates the reiterative growth and termination cycles typical of perennial plants, accelerates leaf maturation, and influences the complexity of compound leaves, the growth of stems and the formation of abscission zones. Florigen is thus established as a plant protein functioning as a general growth hormone. Developmental interactions and a phylogenetic analysis suggest that the SFT/SP regulatory hierarchy is a recent evolutionary innovation unique to flowering plants.

- by A. Goldshmidt
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- Multidisciplinary, Growth Hormone, Phylogeny, Plant Growth Regulators
- by sandeep kumar
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- Thermodynamics, Protein Folding, Kinetics, Protein Science
- by Roy Weber
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- Data Analysis, Kinetics, Link analysis, Oligochaeta

Identifying relationships between function, amino acid sequence and protein structure represents a major challenge. In this study we propose a bioinformatics approach that identifies functional keywords in the Swiss-Prot database that correlate with intrinsic disorder. A statistical evaluation is employed to rank the significance of these correlations. Protein sequence data redundancy and the relationship between protein length and protein structure were taken into consideration to ensure the quality of the statistical inferences. Over 200,000 proteins from Swiss-Prot database were analyzed using this approach. The predictions of intrinsic disorder were carried out using PONDR VL3E predictor of long disordered regions that achieves an accuracy of above 86%. Overall, out of the 710 Swiss-Prot functional keywords that were each associated with at least 20 proteins, 238 were found to be strongly positively correlated with predicted long intrinsically disordered regions, whereas 302 were strongly negatively correlated with such regions. The remaining 170 keywords were ambiguous without strong positive or negative correlation with the disorder predictions. These functions cover a large variety of biological activities and imply that disordered regions are characterized by a wide functional repertoire. Our results agree well with literature findings, as we were able to find at least one illustrative example of functional disorder or order shown experimentally for the vast majority of keywords showing the strongest positive or negative correlation with intrinsic disorder. This work opens a series of three papers, which enriches the current view of protein structure-function relationships, especially with regards to functionalities of intrinsically disordered proteins and provides researchers with a novel tool that could be used to improve the understanding of the relationships between protein structure and function. The first paper of the series describes our statistical approach, outlines the major findings and provides illustrative examples of biological processes and functions positively and negatively correlated with intrinsic disorder.

- by Lilia Iakoucheva
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- Algorithms, Computational Biology, Biological Sciences, Statistical Inference

The gene cluster containing the nitrile hydratase (NHase) and amidase genes of a moderate thermophile, B. pallidus RAPc8 has been cloned and sequenced. The (5.9 kb) section of cloned DNA contained eight complete open reading frames, encoding (in order), amidase (belonging to the nitrilase related aliphatic amidase family), nitrile hydratase h and a subunits (of the cobalt containing class), a 122amino acid accessory protein, designated P14K, a homologue of the 2Fe-2S class of ferredoxins and three putative proteins with distinct homology to the cobalt uptake proteins cbiM, cbiN and cbiQ of the S. typhimurium LT2 cobalamin biosynthesis pathway. The amidase and nitrile hydratase genes were subcloned and inducibly expressed in Escherichia coli, to levels of approximately 37 U/mg and 49 U/ mg, respectively, without the co-expression of additional flanking genes. However, co-expression of P14K with the NHase structural genes significantly enhanced the specific activity of the recombinant NHase. This is the first description of an accessory protein involved in thermostable NHase expression. Modelling of the P14K protein structure has suggested that this protein functions as a subunit-specific chaperone, aiding in the folding of the NHase a subunit prior to a-h subunit association and the formation of a 2 h 2 NHase holoenzyme. D

- by Don A Cowan
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- Biological Sciences, Protein Design, Escherichia coli, Nitriles

Epitope tagging is a powerful method for the rapid analysis of protein function. In Saccharomyces cerevisiae epitope tags are introduced easily into chromosomal loci by homologous recombination using a simple PCR-based strategy. Although quite a number of tools exist for C-terminal tagging as well as N-terminal tagging of proteins expressed by heterologous promoters, there are only very limited possibilities to tag proteins at the N-terminus and retain the endogenous expression level. Furthermore, no PCR-templates for internal tagging have been reported. Here we describe new modules that are suitable for both the repeated N-terminal and internal tagging of proteins, leaving their endogenous promoters intact. The tags include 6xHA, 9xMyc, yEGFP, TEV-GST-6xHIS, ProtA, TEV-ProtA and TEV-ProtA-7xHIS in conjunction with different heterologous selection markers.

- by Thomas Sommer
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- Microbiology, Industrial Biotechnology, Saccharomyces cerevisiae, Epitope mapping
- by Giovanni Chiappetta
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- Proteomics, Proteins, Protein Function, Redox Regulation

Most methods annotating protein function utilise sequence homology to proteins of experimentally known function. Such a homology-based annotation transfer is problematic and limited in scope. Therefore, computational biologists have begun to develop ab initio methods that predict aspects of function, including subcellular localization, post-translational modifications, functional type and protein-protein interactions. For the first two cases, the most accurate approaches rely on

- by Burkhard Rost
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- Physiology, Computational Biology, Neural Network, Sequence Analysis