Cupin Research Articles

The molecular architecture of QdtA, a sugar 3,4-ketoisomerase fromThermoanaerobacterium thermosaccharolyticum

Unusual di- and trideoxysugars are often found on the O-antigens of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on various natural products. One such sugar is 3-acetamido-3,6-dideoxy-D-glucose. A key step in its biosynthesis, catalyzed by a 3,4-ketoisomerase, is the conversion of thymidine diphosphate (dTDP)-4-keto-6-deoxyglucose to dTDP-3-keto-6-deoxyglucose. Here we report an X-ray analysis of a 3,4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum. For this investigation, the wild-type enzyme, referred to as QdtA, was crystallized in the presence of dTDP and its structure solved to 2.0-Å resolution. The dimeric enzyme adopts a three-dimensional architecture that is characteristic for proteins belonging to the cupin superfamily. In order to trap the dTDP-4-keto-6-deoxyglucose substrate into the active site, a mutant protein, H51N, was subsequently constructed, and the structure of this protein in complex with the dTDP-sugar ligand was solved to 1.9-Å resolution. Taken together, the structures suggest that His 51 serves as a catalytic base, that Tyr 37 likely functions as a catalytic acid, and that His 53 provides a proton shuttle between the C-3' hydroxyl and the C-4' keto group of the hexose. This study reports the first three-dimensional structure of a 3,4-ketoisomerase in complex with its dTDP-sugar substrate and thus sheds new molecular insight into this fascinating class of enzymes.

Peanut allergens.

The earliest known evidence of peanut farming dates back 7,600 years. With a prevalence of roughly 1%, peanut allergy is a diagnostic and treatment challenge, but is also a very good model for studying all aspects of food allergy, including its molecular basis and pathomechanisms. Therefore, the very starting point for elucidating all these aspects is the identification of peanut allergens with subsequent clearing of their structure and their preparation as pure recombinant and/or natural allergens. This is the basis for in vitro diagnostic tests as well as the development of immunotherapeutic drugs. With regard to class I food allergy, peanut allergy affects by far the largest group of patients. In peanuts, 12 allergens have been identified and their molecular characteristics are described herein. Ara h 1, Ara h 3.01 and Ara h 3.02 (the former Ara h 4) belong to the cupin superfamily. The conglutins Ara h 2, Ara h 6 and Ara h 7, and the non-specific lipid transfer protein Ara h 9 belong to the prolamin superfamily. Ara h 5 (profilin) and Ara h 8 (Bet v 1-homologous protein) cause class II food allergies and are associated with inhalation allergy to pollen via the sequential and/or conformational similarity of molecules. Two peanut oleosins are listed as Ara h 10 and Ara h 11 and two defensins as Ara h 12 and Ara h 13 by the WHO/IUIS Allergen Nomenclature Subcommittee. The effect of the above-specified allergens has to be considered in the context of their matrix, which is influenced by processing factors and the individual's immune system.

Protein Similarity Networks Reveal Relationships among Sequence, Structure, and Function within the Cupin Superfamily

The cupin superfamily is extremely diverse and includes catalytically inactive seed storage proteins, sugar-binding metal-independent epimerases, and metal-dependent enzymes possessing dioxygenase, decarboxylase, and other activities. Although numerous proteins of this superfamily have been structurally characterized, the functions of many of them have not been experimentally determined. We report the first use of protein similarity networks (PSNs) to visualize trends of sequence and structure in order to make functional inferences in this remarkably diverse superfamily. PSNs provide a way to visualize relatedness of structure and sequence among a given set of proteins. Structure- and sequence-based clustering of cupin members reflects functional clustering. Networks based only on cupin domains and networks based on the whole proteins provide complementary information. Domain-clustering supports phylogenetic conclusions that the N- and C-terminal domains of bicupin proteins evolved independently. Interestingly, although many functionally similar enzymatic cupin members bind the same active site metal ion, the structure and sequence clustering does not correlate with the identity of the bound metal. It is anticipated that the application of PSNs to this superfamily will inform experimental work and influence the functional annotation of databases.

The analysis of specific allergenicity of food allergens families

Classification of food allergens based on protein's structure and function contributes to the study of the relationship between bioinformatics and potential allergenicity of allergens, as well as the evaluation of novel proteins' allergenicity. Some researches were focused on classification within plant or animal food allergens respectively, but there is not any classification of the wholefood allergens. In this article, we classified all the food allergens included in the SDAP into different food allergen families and analysed their specific allergenicity. According to allergen families in AllFam Database, food allergens taken from SDAP (Structural Database of Allergenic Proteins) are classified into different allergen families. Moreover, Protean of DNAStar was applied to analyzing the allergenicity of food allergens. 60% of food allergens are included in the five allergen families: Prolamin superfamily, EF hand domain, Cupin superfamily Profilin and Bet v 1-related protein. Besides, three other cross-food allergen families are found: EF hand domain, Serpin serine protease inhibitor and Triose phosphate isomerase, which include food allergens from both plants and animals. Common characters of food allergens from the same family were easy to find. For instance, they share the same specific peptides and potential T lymphocyte antigen epitope. This classification and characterization of allergen allergenicity in different food allergen families provide foundation for studying the structure and function of novel food allergens as well as the data for the assessment of potential allergenicity.

Temporal expression of pecan allergens during nut development

SummaryPecan nuts are rich in healthy oils, proteins, carbohydrates, and essential nutrients, but can cause food allergies that are rarely outgrown in susceptible individuals. Three conserved seed-storage proteins from the prolamin and cupin superfamilies, including 2S albumin, 7S vicillin, and 11S legumin are commonly identified as allergens in nuts. We have characterised the temporal expression of the genes encoding these proteins during nut development in pecan. Transcript levels from the Car i 1, Car i 4, and the pecan 7S vicillin homologue (Car i 7S) genes were investigated in the cultivars ‘Desirable’ and ‘Sumner’ using quantitative real-time PCR (RT-qPCR).Values for Car i 1 and Car i 4 gene expression generally peaked in September, during the dough stage, with lower values at other times. There was a less distinct pattern of expression for Car i 7S, with lower values in August than in mid-September in ‘Sumner’, and lower values in August than in September and October in ‘Desirable’. Both cultivars generally had similar levels of transcripts, except for higher levels of Car i 1 and Car i 4 in ‘Desirable’ in August. Understanding the timing of allergen gene transcription could assist plant breeders to develop cultivars with lower levels of allergens.

Evaluation of In Silico Prediction Possibility of Epitope Sequences Using Experimental Data Concerning Allergenic Food Proteins Summarized in BIOPEP Database.

The aim of the study was to evaluate the possibility of predicting potential epitope sequences and location in allergenic proteins from food using EVALLER program by comparison with experimental epitopes summarised in the BIOPEP database of allergenic proteins. Sequences of experimental epitopes from food allergens, present in the BIOPEP database of allergenic proteins were used in the study. Sequences of potential epitopes were found using EVALLER program. The Positive Predictive Value (PPV) has been used as a measure of prediction quality. The potential epitopes fully or partially overlapping with the experimental ones were considered as true positive results whereas these unrelated to the experimental ones as false positive results. The PPV for entire dataset containing 310 potential epitopes was 80.6%. The PPV varied signifi cantly among particular allergen families defi ned according to the AllFam database. Caseins revealed PPV=100% (with one exception), proteins from tropomyosin family and proteins from papain-like cystein protease family – exceeding 50%. The last two families possess also relatively low frequency of epitope occurrence. The predictive potential was poor (less than 50%) for plant allergens from cupin superfamily. Families such as lipocalins from milk and EF-hand family (parvalbumins) revealed high variability within family. The EVALLER program may be used as a tool for the prediction of epitope location although its potential varies considerably among allergen families. High PPV is associated with a high number of known experimental epitopes (such as in caseins) and/or a high degree of sequence conservation within family (caseins, tropomyosins).

Ring-Cleaving Dioxygenases with a Cupin Fold

Ring-cleaving dioxygenases catalyze key reactions in the aerobic microbial degradation of aromatic compounds. Many pathways converge to catecholic intermediates, which are subject to ortho or meta cleavage by intradiol or extradiol dioxygenases, respectively. However, a number of degradation pathways proceed via noncatecholic hydroxy-substituted aromatic carboxylic acids like gentisate, salicylate, 1-hydroxy-2-naphthoate, or aminohydroxybenzoates. The ring-cleaving dioxygenases active toward these compounds belong to the cupin superfamily, which is characterized by a six-stranded β-barrel fold and conserved amino acid motifs that provide the 3His or 2- or 3His-1Glu ligand environment of a divalent metal ion. Most cupin-type ring cleavage dioxygenases use an Fe(II) center for catalysis, and the proposed mechanism is very similar to that of the canonical (type I) extradiol dioxygenases. The metal ion is presumed to act as an electron conduit for single electron transfer from the metal-bound substrate anion to O(2), resulting in activation of both substrates to radical species. The family of cupin-type dioxygenases also involves quercetinase (flavonol 2,4-dioxygenase), which opens up two C-C bonds of the heterocyclic ring of quercetin, a wide-spread plant flavonol. Remarkably, bacterial quercetinases are capable of using different divalent metal ions for catalysis, suggesting that the redox properties of the metal are relatively unimportant for the catalytic reaction. The major role of the active-site metal ion could be to correctly position the substrate and to stabilize transition states and intermediates rather than to mediate electron transfer. The tentative hypothesis that quercetinase catalysis involves direct electron transfer from metal-bound flavonolate to O(2) is supported by model chemistry.

Characterization of Two Bacterial Hydroxynitrile Lyases with High Similarity to Cupin Superfamily Proteins

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity.

Crystallization and X-ray data collection of HP0902 from Helicobacter pylori 26695.

HP0902 from Helicobacter pylori 26695 belongs to the cupin superfamily of proteins, which encompasses proteins with a great diversity in function. In this work, two types of recombinant HP0902 protein were crystallized: one with an N-terminal His(6) tag ((H6)HP0902) and the other with a C-terminal His(6) tag (HP0902(H6)). The (H6)HP0902 crystal diffracted to 1.40 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.5, b = 78.6, c = 41.4 Å. The HP0902(H6) crystal belonged to space group P4(3)2(1)2 or P4(1)2(1)2 and diffracted to 2.5 Å resolution, with unit-cell parameters a = b = 50.4, c = 142.0 Å.

Food allergen protein families and their structural characteristics: new data from Europrevail

In the past decade the number of identified food allergens has tremendously increased. Nevertheless, these proteins can be assigned to only a limited number of protein families with certain biological functions such as hydrolysis of proteins/polysaccharides, binding, transport and storage of ligands and cytoskeleton association. In depth knowledge about the 3D structures of individual food allergens, their biological activity and stability will help to fine-tune in vitro diagnosis of food allergy and assess the risk of cross reactive allergies to other food sources. Therefore, the Europrevall project set out for a p collection of highly purified, characterised and authenticated food allergens from animal and plant food sources. The allergen library included both, recombinant and natural proteins. A catalogue of harmonised quality criteria of the purified allergens was agreed and up to date methodologies such as mass spectroscopy, circular dichroism spectroscopy, fourier-transform infrared spectroscopy and 1D-NMR analysis were applied to define them. Plant food allergens were purified from fruits (apple, peach, and kiwi), vegetables (carrot, celeriac, tomato and soybean) and grains, nuts and spices (wheat, hazelnut, peanut, walnut, sesame seeds, sunflower seeds and mustard). These allergens derived from the most important protein families such as the seed storage proteins from the prolamin and cupin superfamily, profilins, Bet v 1 related proteins, and oleosins. From animal foods allergenic proteins from cow’s and goat’s milk, hen’s egg, fish and shrimp were purified and included members of the following protein families: lipocalins, caseins, C-type lysozymes, various protease inhibitors, tropomyosins, and parvalbumins. Subsequently selected sets of allergens e.g .from kiwi or celeriac were used to compare conventional in vitro diagnosis with the allergen specific approach. Additionally, these well defined proteins were enabled further in depth studies investigating the impact of food processing or digestion on the structural features of the individual allergen.

Evolution of seed storage globulins and cupin superfamily

An extensive superfamily of cupins (clan cl09118) currently combines thousands of functionally and structurally diverse prokaryote and eukaryote proteins, which contain a beta-barrel of antiparallel beta-strands (cupin module). Possible ways of the formation of the cupin superfamily were suggested based on the comparison of primary and tertiary structures of proteins from several conserved families of cupins including seed storage globulins and plant oxalate oxydases (germins), and bacterial oxalate decarboxylases, gentisate dioxygenases and epimerases. The origin of two-domain structure of seed storage globulins from cyanobacterial two-domain oxalate decarboxylases has been deduced. The evolutionary pathway of single-domain germins previously suggested to be immediate progenitors of storage globulins was traced back. Common evolutionary roots of germins and oxalate decarboxylases descend from recent bacterial and archaebacterial proteins whose primitive structure is restricted to the cupin module. These root proteins reflect the hypothetical structure of a pro-cupin that probably gave rise to at least a part of the total diversity of members of the cupin superfamily (for instance, to the cupin module of gentisate dioxygenases). The major dilemma for the description of the cupin superfamily is distinguishing evolutionary divergence from convergence. The structural convergence can be exemplified by formation of a beta-barrel inside extremely conserved structures of the otherwise unrelated epimerases from Archaea and bacteria.

Potential allergens of green gram (Vigna radiata L. Millsp) identified as members of cupin superfamily and seed albumin

No systematic study on allergenicity of green gram seed proteins have been performed so far, although incidences of IgE-mediated reaction to green gram seedlings have been reported. We sought to investigate the allergenic potential of green gram, followed by identification and characterization of its relevant allergens using proteomic approaches. BALB/c mice were sensitized intraperitoneally with green gram proteins, and levels of specific Igs, Th2 cytokines, histamine, anaphylactic symptoms and histopathological responses were studied. Twelve naso-bronchial allergic patients with a history of sensitization to green gram were selected on the basis of positive skin prick test and elevated specific IgE levels. Green gram allergens were identified and characterized by their ability to endure pepsin, by IgE immunoblot of two-dimensional (2D) gels in combination with mass spectrometry and by bioinformatics approaches. Increased specific IgE, IgG1, Th2 cytokine and histamine levels, high anaphylactic scores and histological changes in lungs and spleen of green gram crude protein extract-treated mice are indicative of its sensitization ability. Four proteins (molecular weights: 52, 50, 30 and 18 kDa) showed pepsin resistance and IgE-binding capability with sensitized human and mice sera. The four proteins tentatively named as Vig r2 (52 kDa, pI 5.7), Vig r3 (50 kDa, pI 5.8), Vig r4 (30 kDa, pI 6.6) and Vig r5 (18 kDa, pI 5.5) showed significant sequence similarity with known allergens of soybean, lentil, pea, lupin, etc. Mass spectrometric analysis identified Vig r2 as 8S globulin β-isoform precursor, Vig r3 as 8S globulin α-isoform precursor and Vig r4 as seed albumin. Green gram seeds contain at least four clinically relevant allergenic proteins, namely Vig r2, Vig r3, Vig r4 and Vig r5 that were capable of inducing strong IgE-mediated reactions. One of the most important steps towards diagnostic and therapeutic approaches to deal effectively with food allergy is continued identification of newer food allergens and their characterization. The significance of this study can be enormous as the data generated may work as basic biology data in developing a green gram species modified genetically that may have reduced allergenicity.

Proteomic analysis of known and candidate rice allergens between non-transgenic and transgenic plants

Salt-soluble proteins extracted from non-transgenic and transgenic rice were evaluated for the presence of known and potential allergens by proteomic techniques. The salt-soluble proteins were extracted, separated by 1D and 2D electrophoresis, and analyzed by Western blotting. 1D immunoblot analysis with patients’ sera revealed few qualitative differences between the IgE-binding proteins of the non-transgenic and transgenic rice. 1D immunoblot with antigen-specific-animal sera revealed no qualitative or quantitative differences in two known allergens, RAG2 and glyoxalase I, between non-transgenic and transgenic rice. Multiple spots containing known and novel IgE-binding proteins were detected among the salt-soluble proteins of non-transgenic rice by 2D immunoblotting. Two globulin-like proteins, a 52kDa protein and a 63kDa protein, were identified as novel IgE-binding proteins that are candidates for rice allergens. These globulin-like proteins were homologous to Cupin superfamily allergens. Quantitative analysis of 19, 52, and 63kDa globulins with protein-specific-animal sera showed no significant differences in the expression of these proteins between the transgenic rice and non-transgenic rice. These results indicate that none of the known or novel endogeneous IgE-binding proteins detected in this study appear to be altered by genetic modification.

Structure-Based Annotation of a Novel Sugar Isomerase from the Pathogenic E. coli O157:H7

Prokaryotes can use a variety of sugars as carbon sources in order to provide a selective survival advantage. The gene z5688 found in the pathogenic Escherichia coli O157:H7 encodes a “hypothetical” protein of unknown function. Sequence analysis identified the gene product as a putative member of the cupin superfamily of proteins, but no other functional information was known. We have determined the crystal structure of the Z5688 protein at 1.6 Å resolution and identified the protein as a novel E. coli sugar isomerase (EcSI) through overall fold analysis and secondary-structure matching. Extensive substrate screening revealed that EcSI is capable of acting on d-lyxose and d-mannose. The complex structure of EcSI with fructose allowed the identification of key active-site residues, and mutagenesis confirmed their importance. The structure of EcSI also suggested a novel mechanism for substrate binding and product release in a cupin sugar isomerase. Supplementation of a nonpathogenic E. coli strain with EcSI enabled cell growth on the rare pentose d-lyxose.

Crystal structures of the apo and GDP‐bound forms of a cupin‐like protein BbDUF985 from Branchiostoma belcheri tsingtauense

Cupins are ubiquitous proteins existing in all three kingdoms of life and share a conserved b-barrel fold with a characteristic pocket located at the center of the b-barrel.1,2 Despite sharing the highly conserved topology, cupins show remarkable variations in their sequences, architecture of domains (comprising either one or two cupin domains), quaternary assembly, and the nature of bound metal ion as well.3 In the Pfam database (http://pfam.sanger.ac.uk/), cupins are classified into 35 protein families, with greatly diversified functions such as isomerases, epimerase, dioxygenase, nonenzymatic storage proteins, and it is suggested that cupin is one of the most functionally diverse protein superfamily.2,4 In recent years, large-scale sequencing of genomes and cDNAs has elicited a great number of hypothetical cupins whose biological functions need to be established. The cephalochordate amphioxus is a modern survivor of an ancient chordate lineage and acts as a living fossil between invertebrates and vertebrates. With the completion of the draft genome of Florida amphioxus Branchiostoma floridae in 2008, it was revealed that they are not only the most primitive chordates but also the genomic features appear to have a great deal in common with vertebrates as well.5 BbDUF985 is a hypothetical protein of 172 residues from Branchiostoma belcheri tsingtauense belonging to the cupin_5 family (Pfam entry: PF06172) of the cupin superfamily.4 Interestingly, although BbDUF985 is a protein from eukaryote, sequence homology analysis indicates that all its closest homologs are prokaryotic and more distantly kindred proteins from higher species. Previously, we solved the structure of YML079w (sharing 33% sequence identity with BbDUF985) from Saccharomyces cerevisiae in the guanosine triphosphate (GTP)-bound form,6 whereas the apo form structure is absent and further biochemical study was not undertook then. Here, we present the crystal structures of BbDUF985 in both the apo and guanosine diphosphate (GDP)-bound forms, which are the first pair of structures (with and without the ligand) from the cupin_5 family. Moreover, using fluorescence spectrometry, we determined the dissociation constant (KD) of GDP and investigated the spectra of BbDUF985 with several proposed ligands binding to the proteins in cupin superfamily. Combined with sequence analysis, we speculate that BbDUF985 and its homologs in the cupin_5 family might be involved in the nucleotide transport or metabolism.

English

Germin and germin-like proteins (GLPs) the members of cupin superfamily of proteins, which are functionally most diverse proteins. Germin and GLPs have some unique features as they are highly resistant to proteases and to degradation by heat, high pH and detergents like Sodium dodecyl Sulphate (SDS). They are water soluble extracellular enzymatic protein that may also have Oxalate Oxidase (OxO), Superoxide dismutase (SOD) or ADP-glucose pyrophosphate or phosphodiestrase (AGPPase) activities. At the moment seven GLP gene promoter from different organisms have been studied and published. These all promoter sequences have been analyzed in this study. It was observed that these promoters have important regulatory elements, which are involved in various important functions. These elements have been compared on the basis of location, copy number, and distributed on positive and negative strands. It was also observed that some of these elements are common and remained conserved among all GLP promoters during evolution. Such regulatory elements are commonly observed in seed storage proteins, dehydration in response to light, senescence observed on exposure to dark and in elements specific for expression in pollen. Moreover, these common elements are reported to be expressed under environmental stresses (salt and pathogen attack) and to growth regulators.

Characterization of the expression profile of a wheat aci-reductone-dioxygenase-like gene in response to stripe rust pathogen infection and abiotic stresses

The methionine salvage pathway is conserved from prokaryotes to high eukaryotes. The reaction catalyzed by aci-reductone-dioxygenase (ARD) represents a branch point in the methionine salvage pathway. A novel aci-reductone-dioxygenase gene, designed as TaARD, was identified in a subtraction library constructed with RNA isolated from wheat leaves infected with the stripe rust pathogen. TaARD was predicted to encode a 197 amino acid protein that belongs to the cupin superfamily. In transient expression assays with onion epidermal cells, the TaARD-GFP fusion protein localized to the nucleus and cytoplasm. Southern blot analysis showed that the wheat genome had multiple copies of TaARD. Quantitative real-time RT-PCR (qRT-PCR) analyses revealed that the TaARD transcript was induced in wheat leaves infected with a compatible stripe rust strain. However, its expression was reduced or suppressed in incompatible interactions and by ABA, ethephon (ET), or salicylic acid (SA) treatments. With methyl jasmonate (MeJA) treatment, TaARD transcript level was suppressed in the first 6h but increased afterwards. The expression of TaARD also was inhibited by wounding and environmental stimuli, including high salinity and low temperature. Because of the role of ARD in the methionine salvage pathway, these results suggest that TaARD may be involved in ethylene synthesis and ethylene signaling in response to biotic and abiotic stresses.

Thiol dioxygenases: unique families of cupin proteins

Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a six-stranded β-barrel core, and this canonical cupin or "jelly roll" β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active-site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative fingerprint motif for ADOs, or DUF1637 family members, is proposed. In ADOs, the conserved glutamate residue in cupin motif 1 is replaced by either glycine or valine. Both ADOs and CDOs appear to represent unique clades within the cupin superfamily.

Pirin delocalization in melanoma progression identified by high content immuno-detection based approaches

BackgroundPirin (PIR) is a highly conserved nuclear protein originally isolated as an interactor of NFI/CTF1 transcription/replication factor. It is a member of the functionally diverse cupin superfamily and its activity has been linked to different biological and molecular processes, such as regulation of transcription, apoptosis, stress response and enzymatic processes. Although its precise role in these functions has not yet been defined, PIR expression is known to be deregulated in several human malignancies.ResultsWe performed immunohistochemical analysis of PIR expression in primary samples from normal human tissues and tumors and identified a dislocation of PIR to the cytoplasm in a subset of melanomas, and a positive correlation between cytoplasmic PIR levels and melanoma progression. PIR localization was subsequently analyzed in vitro in melanoma cell lines through a high content immunofluorescence based approach (ImmunoCell-Array).ConclusionsThe high consistency between in vivo and in vitro results obtained by immunohistochemistry and ImmunoCell-Array provides a validation of the potential of ImmunoCell-Array technology for the rapid screening of putative biological markers, and suggests that cytoplasmic localization of PIR may represent a characteristic of melanoma progression.

293 cells over‐expressing human ADI1 and CD81 are permissive for serum‐derived hepatitis C virus infection

Human aci-reductone dioxygenase 1 (ADI1) is a member of the Cupin superfamily. It binds to and inhibits the activities of membrane-type 1 matrix metalloproteinase, a protein known to interact with the tight junction protein, claudin-1. Previously, a variant protein, named submergence-induced protein-like factor (Sip-L), consisting of ADI1 amino acids 64-179, was found to support hepatitis C virus (HCV) infection and replication in 293 cells. In the present study, it was discovered that over-expression of human ADI1 in 293 cells (293-ADI1 cells) also supported HCV infection and replication. Using serum-derived HCV as an infectious source, enhanced cell uptake of HCV to a Northern blot detectable level was found in 293 cells over-expressing both CD81 and ADI1 (293-ADI1-CD81 cells). The enhanced cell entry was confirmed by the use of the vesicular stomatitis virus-based HCV pseudotype particles. However, transfection of HCV replicon RNA by electroporation into naïve 293 and 293-ADI1 cells revealed no difference in replication efficiency. Using the infectious J6/JFH chimera as an infectious source, the infectivity was compared between 293-ADI1-CD81 and Huh-7.5 cells. More infection foci were formed in the 293-ADI1-CD81 cells in the first round of infection. In conclusion, human ADI1 over-expression in 293 cells enhances cell entry but not replication of HCV. 293-ADI1-CD81 cells are permissive for serum-derived HCV infection.