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Human Proteins (human + protein)
Selected AbstractsMUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay,HUMAN MUTATION, Issue 2 2010Sara Molatore Abstract MUTYH -associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh,/, mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1,8, 2009. © 2009 Wiley-Liss, Inc. [source] Paralog of the formylglycine-generating enzyme , retention in the endoplasmic reticulum by canonical and noncanonical signalsFEBS JOURNAL, Issue 6 2008Santosh Lakshmi Gande Formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the oxidation of a specific cysteine residue to formylglycine, which is the catalytic residue required for sulfate ester hydrolysis. This post-translational modification occurs in the endoplasmic reticulum (ER), and is an essential step in the biogenesis of this enzyme family. A paralog of FGE (pFGE) also localizes to the ER. It shares many properties with FGE, but lacks formylglycine-generating activity. There is evidence that FGE and pFGE act in concert, possibly by forming complexes with sulfatases and one another. Here we show that human pFGE, but not FGE, is retained in the ER through its C - terminal tetrapeptide PGEL, a noncanonical variant of the classic KDEL ER-retention signal. Surprisingly, PGEL, although having two nonconsensus residues (PG), confers efficient ER retention when fused to a secretory protein. Inducible coexpression of pFGE at different levels in FGE-expressing cells did not significantly influence the kinetics of FGE secretion, suggesting that pFGE is not a retention factor for FGE in vivo. PGEL is accessible at the surface of the pFGE structure. It is found in 21 mammalian species with available pFGE sequences. Other species carry either canonical signals (eight mammals and 26 nonmammals) or different noncanonical variants (six mammals and six nonmammals). Among the latter, SGEL was tested and found to also confer ER retention. Although evolutionarily conserved for mammalian pFGE, the PGEL signal is found only in one further human protein entering the ER. Its consequences for KDEL receptor-mediated ER retrieval and benefit for pFGE functionality remain to be fully resolved. [source] Folding and turnover of human iron regulatory protein 1 depend on its subcellular localizationFEBS JOURNAL, Issue 4 2007Alain Martelli Aconitases are iron,sulfur hydrolyases catalysing the interconversion of citrate and isocitrate in a wide variety of organisms. Eukaryotic aconitases have been assigned additional roles, as in the case of the metazoan dual activity cytosolic aconitase,iron regulatory protein 1 (IRP1). This human protein was produced in yeast mitochondria to probe IRP1 folding in this organelle where iron,sulfur synthesis originates. The behaviour of human IRP1 was compared with that of genuine mitochondrial (yeast or human) aconitases. All enzymes were functional in yeast mitochondria, but IRP1 was found to form dense particles as detected by electron microscopy. MS analysis of purified inclusion bodies evidenced the presence of human IRP1 and ,-ketoglutarate dehydrogenase complex component 1 (KGD1), one of the subunits of ,-ketoglutarate dehydrogenase. KGD1 triggered formation of the mitochondrial aggregates, because the latter were absent in a KGD1, mutant, but it did not efficiently do so in the cytosol. Despite the iron-binding capacity of IRP1 and the readily synthesis of iron,sulfur clusters in mitochondria, the dense particles were not iron-rich, as indicated by elemental analysis of purified mitochondria. The data show that proper folding of dual activity IRP1-cytosolic aconitase is deficient in mitochondria, in contrast to genuine mitochondrial aconitases. Furthermore, efficient clearance of the aggregated IRP1,KGD1 complex does not occur in the organelle, which emphasizes the role of molecular interactions in determining the fate of IRP1. Thus, proper folding of human IRP1 strongly depends on its cellular environment, in contrast to other members of the aconitase family. [source] Novel brain 14-3-3 interacting proteins involved in neurodegenerative diseaseFEBS JOURNAL, Issue 16 2005Shaun Mackie We isolated two novel 14-3-3 binding proteins using 14-3-3 , as bait in a yeast two-hybrid screen of a human brain cDNA library. One of these encoded the C-terminus of a neural specific armadillo-repeat protein, ,-catenin (neural plakophilin-related arm-repeat protein or neurojungin). ,-Catenin from brain lysates was retained on a 14-3-3 affinity column. Mutation of serine 1072 in the human protein and serine 1094 in the equivalent site in the mouse homologue (in a consensus binding motif for 14-3-3) abolished 14-3-3 binding to ,-catenin in vitro and in transfected cells. ,-catenin binds to presenilin-1, encoded by the gene most commonly mutated in familial Alzheimer's disease. The other clone was identified as the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). Human IRSp53 interacts with the gene product implicated in dentatorubral-pallidoluysian atrophy, an autosomal recessive disorder associated with glutamine repeat expansion of atrophin-1. [source] Ebp2p, yeast homologue of a human protein that interacts with Epstein,Barr virus Nuclear Antigen 1, is required for pre-rRNA processing and ribosomal subunit assemblyGENES TO CELLS, Issue 7 2000Rota Tsujii Background A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response. Results EBP2, encoding the yeast homologue of a human protein that interacts with Epstein,Barr virus Nuclear Antigen 1, was cloned in a two-hybrid screen using RRS1 as a bait. The rrs1-1 mutation, which produces Rrs1p without the C-terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p. Ebp2p is essential for growth and is mainly localized in the nucleolus. The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p-depleted cells, the rate of pre-rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild-type cells. The polysome pattern indicates that Ebp2p-depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half-mer polysomes. Conclusions Ebp2p is required for the maturation of 25S rRNA and 60S subunit assembly. Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis. [source] Isolation and functional identification of a novel human hepatic growth factor: Hepatopoietin Cn,HEPATOLOGY, Issue 3 2008Chun-Ping Cui Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion-exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [3H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30-kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine-rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. Conclusion: HPPCn is a novel hepatic growth factor that plays a role in liver regeneration. (HEPATOLOGY 2008;47:986,995.) [source] Studies on the association between immunoglobulin E autoreactivity and immunoglobulin E-dependent histamine-releasing factorsIMMUNOLOGY, Issue 2 2002Ilona Kleine Budde Summary It has been reported that serum immunoglobulin E (IgE) from certain atopic patients can sensitize basophils to release histamine in response to IgE-dependent histamine-releasing factors (HRFs). It has also been shown that patients suffering from severe forms of atopy may contain IgE autoantibodies. It was investigated whether HRF-responsive sera contained IgE autoantibodies and if there was an association between IgE autoreactivity and IgE-dependent responsiveness to HRF. The presence of HRF-responsive IgE (IgE+) in serum of patients with respiratory atopy was determined by stimulating stripped human basophils sensitized by serum with peripheral blood mononuclear cell (PBMC)-derived HRF, and measuring the release of histamine. In parallel, these sera were screened for the presence of IgE autoantibodies to nitrocellulose-blotted human cellular extracts. The capacity of IgE autoantigen-containing preparations to induce histamine release was tested in the stripped basophil assay. Eleven out of 52 sera contained IgE autoantibodies to blotted cellular extracts of human PBMCs or of the human epithelial cell line A431. No significant association was found between IgE autoreactivity and IgE-dependent responsiveness to HRF: 7/26 IgE+ sera contained IgE to human cellular extracts, and 4/26 of the sera without IgE+ did also. IgE autoantigen-containing extracts did not induce histamine release of appropriately sensitized basophils. By size-exclusion chromatography it was shown that a 32,000 MW autoantigen eluted in the >55,000 MW fraction, which indicates that this protein forms polymers or complexes with other macromolecules. This might explain the discrepancy between binding and histamine-releasing activity. A 20,000 MW IgE-defined autoantigen cross-reacted with a shrimp allergen. Our results indicate that IgE-reactivity to immunoblotted human protein and IgE-dependent HRF activity are distinct entities that may co-occur in atopic patients. [source] Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved proteinPROTEIN SCIENCE, Issue 6 2002Abel Baerga-Ortiz Abstract The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen. [source] First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical valueACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2006L. Banci The EC `Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, ,170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples. [source] Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Ting-Kuo Huang Abstract Plant cell culture production of recombinant products offers a number of advantages over traditional eukaryotic expression systems, particularly if the product can be targeted to and purified from the cell culture broth. However, one of the main obstacles is product degradation by proteases that are produced during cell culture, and/or the loss of biological activity of secreted (extracellular) products as a result of alteration in the protein conformation. Because proteolysis activity and target protein stability can be significantly influenced by culture conditions, it is important to evaluate bioprocess conditions that minimize these effects. In this study, a bioreactor strategy using a protocol involving pH adjustment and medium exchange during plant cell culture is proposed for improving the production of functional recombinant ,1 -antitrypsin (rAAT), a human blood protein, produced using several alternative expression systems, including a Cauliflower mosaic virus (CaMV) 35S constitutive promoter expression system, a chemically inducible, estrogen receptor-based promoter (XVE) expression system, and a novel Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva) expression system developed by our group. We have demonstrated that higher medium pH help reduce protease activity derived from cell cultures and improve the inherent stability of human AAT protein as well. This strategy resulted in a fourfold increase in the productivity of extracellular functional rAAT (100 µg/L) and a twofold increase in the ratio of functional rAAT to total rAAT (48%) in transgenic N. benthamiana cell cultures using a chemically inducible viral amplicon expression system. Biotechnol. Bioeng. 2009;102: 508,520. © 2008 Wiley Periodicals, Inc. [source] Structure of cyclophilin from Leishmania donovani at 1.97,Ĺ resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2007V. Venugopal The crystal structure of cyclophilin from Leishmania donovani (LdCyp) has been determined and refined at 1.97,Ĺ resolution to a crystallographic R factor of 0.178 (Rfree = 0.197). The structure was solved by molecular replacement using cyclophilin from Trypanosoma cruzi as the search model. LdCyp exhibits complete structural conservation of the cyclosporin-binding site with respect to the homologous human protein, as anticipated from LdCyp,cyclosporin binding studies. Comparisons with other cyclophilins show deviations primarily in the loop regions. The solvent structure encompassing the molecule has also been analyzed in some detail. [source] Focused proteomics: Monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stainELECTROPHORESIS, Issue 15 2004James Murray Abstract We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue. [source] Ability of human CDC25B phosphatase splice variants to replace the function of the fission yeast Cdc25 cell cycle regulatorFEMS YEAST RESEARCH, Issue 3 2004Matthieu Lemaire Abstract CDC25 phosphatases are essential and evolutionary-conserved actors of the eukaryotic cell cycle control. To examine and compare the properties of three splicing variants of human CDC25B, recombinant fission yeast strains expressing the human proteins in place of the endogenous Cdc25 were generated and characterized. We report, that the three CDC25B variants: (i) efficiently replace the yeast counterpart in vegetative growth, (ii) partly restore the , and UV radiation DNA damage-activated checkpoint, (iii) fail to restore the DNA replication checkpoint activated by hydroxyurea. Although these yeast strains do not reveal the specific functions of the human CDC25B variants, they should provide useful screening tools for the identification of new cell cycle regulators and pharmacological inhibitors of CDC25 phosphatase. [source] MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay,HUMAN MUTATION, Issue 2 2010Sara Molatore Abstract MUTYH -associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh,/, mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1,8, 2009. © 2009 Wiley-Liss, Inc. [source] Molecular cloning of several rat ABC transporters including a new ABC transporter, Abcb8, and their expression in rat testisINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2006Nathalie Melaine Summary Several members of the ABC transporter superfamily play an important role in testicular physiology and defence against anticancer drugs. Using a reverse transcription-polymerase chain reaction strategy with degenerate primers and rat testis RNA as template, we have looked for the presence of other members of this superfamily. Of the six partial cDNA found, five corresponded to ABC transporters already known ,Mdr1b, Mrp1, Tapl/Abcb9, Umat/Abcb6 and Sur2/Abcc9, and one presented a strong homology with mouse and human ABCB8. Using a 5, and 3, RACE approach, we cloned the full-length cDNA and found that the predicted protein presented 92% and 80% homology with the mouse and human proteins respectively. Strong expression of rat abcb8 was found in heart, brain and testis when compared with liver, lung and spleen. In the testis, rat abcb8 was expressed both in the somatic Sertoli cells and peritubular cells and in the germline (spermatogonia and pachytene spermatocytes). Furthermore, Umat/Abcb6 was very highly expressed in the testis (high amounts in meiotic pachytene spermatocytes and low amount in post-meiotic early spermatids). In conclusion, we confirm the presence of several ABC transporters in the testis and also provide evidence of the presence of Abcb8 in the organ. [source] QSAR model for alignment-free prediction of human breast cancer biomarkers based on electrostatic potentials of protein pseudofolding HP-lattice networksJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 16 2008Santiago Vilar Abstract Network theory allows relationships to be established between numerical parameters that describe the molecular structure of genes and proteins and their biological properties. These models can be considered as quantitative structure,activity relationships (QSAR) for biopolymers. The work described here concerns the first QSAR model for 122 proteins that are associated with human breast cancer (HBC), as identified experimentally by Sjöblom et al. (Science 2006, 314, 268) from over 10,000 human proteins. In this study, the 122 proteins related to HBC (HBCp) and a control group of 200 proteins that are not related to HBC (non-HBCp) were forced to fold in an HP lattice network. From these networks a series of electrostatic potential parameters (,k) was calculated to describe each protein numerically. The use of ,k as an entry point to linear discriminant analysis led to a QSAR model to discriminate between HBCp and non-HBCp, and this model could help to predict the involvement of a certain gene and/or protein in HBC. In addition, validation procedures were carried out on the model and these included an external prediction series and evaluation of an additional series of 1000 non-HBCp. In all cases good levels of classification were obtained with values above 80%. This study represents the first example of a QSAR model for the computational chemistry inspired search of potential HBC protein biomarkers. © 2008 Wiley Periodicals, Inc. J Comput Chem 2008 [source] Deciphering the human nucleolar proteome,MASS SPECTROMETRY REVIEWS, Issue 2 2006Yohann Couté Abstract Nucleoli are plurifunctional nuclear domains involved in the regulation of several major cellular processes such as ribosome biogenesis, the biogenesis of non-ribosomal ribonucleoprotein complexes, cell cycle, and cellular aging. Until recently, the protein content of nucleoli was poorly described. Several proteomic analyses have been undertaken to discover the molecular bases of the biological roles fulfilled by nucleoli. These studies have led to the identification of more than 700 proteins. Extensive bibliographic and bioinformatic analyses allowed the classification of the identified proteins into functional groups and suggested potential functions of 150 human proteins previously uncharacterized. The combination of improvements in mass spectrometry technologies, the characterization of protein complexes, and data mining will assist in furthering our understanding of the role of nucleoli in different physiological and pathological cell states. © 2005 Wiley Periodicals, Inc. Mass Spec Rev 25:215,234, 2006 [source] Production of Melanocyte-Specific Antibodies to Human Melanosomal Proteins: Expression Patterns in Normal Human Skin and in Cutaneous Pigmented LesionsPIGMENT CELL & MELANOMA RESEARCH, Issue 4 2001Victoria Virador Multiple factors affect skin pigmentation, including those that regulate melanocyte and/or keratinocyte function. Such factors, particularly those that operate at the level of the melanosome, are relatively well characterized in mice, but the expression and function of structural and enzymatic proteins in melanocytes in human skin are not as well known. Some years ago, we generated peptide-specific antibodies to murine melanosomal proteins that proved to be instrumental in elucidating melanocyte development and differentiation in mice, but cross-reactivity of those antibodies with the corresponding human proteins often was weak or absent. In an effort to characterize the roles of melanosomal proteins in human skin pigmentation, and to understand the underlying mechanism(s) of abnormal skin pigmentation, we have now generated polyclonal antibodies against the human melanocyte-specific markers, tyrosinase, tyrosinase-related protein 1 (TYRP1), Dopachrome tautomerase (DCT) and Pmel17 (SILV, also known as GP100). We used these antibodies to determine the distribution and function of melanosomal proteins in normal human skin (adult and newborn) and in various cutaneous pigmented lesions, such as intradermal nevi, lentigo simplex, solar lentigines and malignant melanomas. We also examined cytokeratin expression in these same samples to assess keratinocyte distribution and function. Immunohistochemical staining reveals distinct patterns of melanocyte distribution and function in normal skin and in various types of cutaneous pigmented lesions. Those differences in the expression patterns of melanocyte markers provide important clues to the roles of melanocytes in normal and in disrupted skin pigmentation. [source] Grp94, the endoplasmic reticulum Hsp90, has a similar solution conformation to cytosolic Hsp90 in the absence of nucleotidePROTEIN SCIENCE, Issue 9 2009Kristin A. Krukenberg Abstract The molecular chaperone, Hsp90, is an essential eukaryotic protein that assists in the maturation and activation of client proteins. Hsp90 function depends upon the binding and hydrolysis of ATP, which causes large conformational rearrangements in the chaperone. Hsp90 is highly conserved from bacteria to eukaryotes, and similar nucleotide-dependent conformations have been demonstrated for the bacterial, yeast, and human proteins. There are, however, important species-specific differences in the ability of nucleotide to shift the conformation from one state to another. Although the role of nucleotide in conformation has been well studied for the cytosolic yeast and human proteins, the conformations found in the absence of nucleotide are less well understood. In contrast to cytosolic Hsp90, crystal structures of the endoplasmic reticulum homolog, Grp94, show the same conformation in the presence of both ADP and AMPPNP. This conformation differs from the yeast AMPPNP-bound crystal state, suggesting that Grp94 may have a different conformational cycle. In this study, we use small angle X-ray scattering and rigid body modeling to study the nucleotide free states of cytosolic yeast and human Hsp90s, as well as mouse Grp94. We show that all three proteins adopt an extended, chair-like conformation distinct from the extended conformation observed for the bacterial Hsp90. For Grp94, we also show that nucleotide causes a small shift toward the crystal state, although the extended state persists as the major population. These results provide the first evidence that Grp94 shares a conformational state with other Hsp90 homologs. [source] Novel affinity tag system using structurally defined antibody-tag interaction: Application to single-step protein purificationPROTEIN SCIENCE, Issue 12 2008Terukazu Nogi Abstract Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody,peptide interaction, opening opportunities for further improvements/modifications. [source] The epitope space of the human proteomePROTEIN SCIENCE, Issue 4 2008Lisa Berglund Abstract In the post-genome era, there is a great need for protein-specific affinity reagents to explore the human proteome. Antibodies are suitable as reagents, but generation of antibodies with low cross-reactivity to other human proteins requires careful selection of antigens. Here we show the results from a proteome-wide effort to map linear epitopes based on uniqueness relative to the entire human proteome. The analysis was based on a sliding window sequence similarity search using short windows (8, 10, and 12 amino acid residues). A comparison of exact string matching (Hamming distance) and a heuristic method (BLAST) was performed, showing that the heuristic method combined with a grid strategy allows for whole proteome analysis with high accuracy and feasible run times. The analysis shows that it is possible to find unique antigens for a majority of the human proteins, with relatively strict rules involving low sequence identity of the possible linear epitopes. The implications for human antibody-based proteomics efforts are discussed. [source] Host,pathogen protein interactions predicted by comparative modelingPROTEIN SCIENCE, Issue 12 2007Fred P. Davis Abstract Pathogens have evolved numerous strategies to infect their hosts, while hosts have evolved immune responses and other defenses to these foreign challenges. The vast majority of host,pathogen interactions involve protein,protein recognition, yet our current understanding of these interactions is limited. Here, we present and apply a computational whole-genome protocol that generates testable predictions of host,pathogen protein interactions. The protocol first scans the host and pathogen genomes for proteins with similarity to known protein complexes, then assesses these putative interactions, using structure if available, and, finally, filters the remaining interactions using biological context, such as the stage-specific expression of pathogen proteins and tissue expression of host proteins. The technique was applied to 10 pathogens, including species of Mycobacterium, apicomplexa, and kinetoplastida, responsible for "neglected" human diseases. The method was assessed by (1) comparison to a set of known host,pathogen interactions, (2) comparison to gene expression and essentiality data describing host and pathogen genes involved in infection, and (3) analysis of the functional properties of the human proteins predicted to interact with pathogen proteins, demonstrating an enrichment for functionally relevant host,pathogen interactions. We present several specific predictions that warrant experimental follow-up, including interactions from previously characterized mechanisms, such as cytoadhesion and protease inhibition, as well as suspected interactions in hypothesized networks, such as apoptotic pathways. Our computational method provides a means to mine whole-genome data and is complementary to experimental efforts in elucidating networks of host,pathogen protein interactions. [source] Determination of the human type I interferon receptor binding site on human interferon-,2 by cross saturation and an NMR-based model of the complexPROTEIN SCIENCE, Issue 11 2006Sabine R. Quadt-Akabayov Abstract Type I interferons (IFNs) are a family of homologous helical cytokines that exhibit pleiotropic effects on a wide variety of cell types, including antiviral activity and antibacterial, antiprozoal, immunomodulatory, and cell growth regulatory functions. Consequently, IFNs are the human proteins most widely used in the treatment of several kinds of cancer, hepatitis C, and multiple sclerosis. All type I IFNs bind to a cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. The structure of the extracellular domain of IFNAR2 (R2-EC) was solved recently. Here we study the complex and the binding interface of IFN,2 with R2-EC using multidimensional NMR techniques. NMR shows that IFN,2 does not undergo significant structural changes upon binding to its receptor, suggesting a lock-and-key mechanism for binding. Cross saturation experiments were used to determine the receptor binding site upon IFN,2. The NMR data and previously published mutagenesis data were used to derive a docking model of the complex with an RMSD of 1 Ĺ, and its well-defined orientation between IFN,2 and R2-EC and the structural quality greatly improve upon previously suggested models. The relative ligand,receptor orientation is believed to be important for interferon signaling and possibly one of the parameters that distinguish the different IFN I subtypes. This structural information provides important insight into interferon signaling processes and may allow improvement in the development of therapeutically used IFNs and IFN-like molecules. [source] Affinity reagent resources for human proteome detection: Initiatives and perspectivesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2007Oda Stoevesandt Abstract Essential to the ambition of characterising fully the human proteome are systematic and comprehensive collections of specific affinity reagents directed against all human proteins, including splice variants and modifications. Although a large number of affinity reagents are available commercially, their quality is often questionable and only a fraction of the proteome is covered. In order for more targets to be examined, there is a need for broad availability of panels of affinity reagents, including binders against proteins of unknown functions. The most familiar affinity reagents are antibodies and their fragments, but engineered forms of protein scaffolds and nucleic acid aptamers with similar diversity and binding properties are becoming viable alternatives. Recent initiatives in Europe and the USA have been established to improve both the availability and quality of reagents for affinity proteomics, with the ultimate aim of creating standardised collections of well-validated binding molecules for proteome analysis. As well as coordinating affinity reagent production through existing resources and technology providers, these projects aim to benchmark key molecular entities, tools, and applications, and establish the bioinformatics framework and databases needed. The benefits of such reagent resources will be seen in basic research, medicine and the biotechnology and pharmaceutical industries. [source] Shotgun proteomic analysis of human-induced sputumPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2006Ben Nicholas Dr. Abstract Induced sputum is a readily accessible biological fluid whose composition may alter as a consequence of disease. To date, however, the proteins that routinely populate this biofluid are largely unknown, in part due to the technical difficulties in processing such mucin-rich samples. To provide a catalogue of sputum proteins, we have surveyed the proteome of human-induced sputum (sputome). A combination of 2-D gel analysis and GeLC-MS/MS allowed a total of 191 human proteins to be confidently assigned. In addition to the expected components, several hitherto unreported proteins were found to be present, including three members of the annexin family, kallikreins 1 and 11, and peroxiredoxins 1, 2 and 5. Other sets of proteins identified included four proteins previously annotated as hypothetical or conserved hypothetical. Taken together, these data represent the first extensive survey of the proteome of induced sputum and provide a platform for future identification of biomarkers of lung disease. [source] Immobilization of diverse foreign proteins in viral polyhedra and potential application for protein microarraysPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2006Keiko Ikeda Abstract Cypoviruses are insect viruses that produce a cytoplasmic crystalline particle called the polyhedron in which progeny virions are occluded. The virion structural protein, VP3, is implicated in the occlusion of viral particles into polyhedra. In this study, we determined the amino acid sequence of VP3 required for occlusion of viral particles into polyhedra and proposed that this sequence could be used as an immobilization signal to direct the stable incorporation of foreign proteins into polyhedra. A large-scale survey revealed that the immobilization signal could, in fact, direct the incorporation of a variety of human proteins into polyhedra. Immune reactivity and protein,protein interactions were detected on the surface of polyhedra containing immobilized foreign proteins, and these particles were shown to be highly stabilized against dehydration. We showed that these particles could be arrayed onto a glass slide by standard spotting and laser manipulation methods. Thus, this approach is well suited for protein expression, purification, and the development of protein microarrays. [source] Immunoaffinity separation of plasma proteins by IgY microbeads: Meeting the needs of proteomic sample preparation and analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2005Lei Huang Abstract Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95,99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein,protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics. [source] The Human Protein Atlas,a tool for pathology,THE JOURNAL OF PATHOLOGY, Issue 4 2008F Pontén Abstract Tissue-based diagnostics and research is incessantly evolving with the development of new molecular tools. It has long been realized that immunohistochemistry can add an important new level of information on top of morphology and that protein expression patterns in a cancer may yield crucial diagnostic and prognostic information. We have generated an immunohistochemistry-based map of protein expression profiles in normal tissues, cancer and cell lines. For each antibody, altogether 708 spots of tissues and cells are analysed and the resulting images and data are presented as freely available in the Human Protein Atlas (www.proteinatlas.org). The new version 4 of the atlas, including more than 5 million images of immunohistochemically stained tissues and cells, is based on 6122 antibodies, representing 5011 human proteins encoded by approximately 25% of the human genome. The gene-centric database includes a putative classification of proteins in various protein classes, both functional classes, such as kinases or transcription factors and project-related classes, such as candidate genes for cancer or cardiovascular diseases. For each of the internally generated antibodies, the exact antigen sequence is presented, together with a visualization of application-specific validation data, including a protein array assay, western blot analysis, immunohistochemistry and, in most cases, immunofluorescent-based confocal microscopy. The updated version also includes new search algorithms to allow complex queries regarding expression profiles, protein classes and chromosome location. Thus, the presented Human Protein Atlas provides a resource for pathology-based biomedical research, including protein science and biomarker discovery. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Bracovirus gene products are highly divergent from insect proteinsARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2008Annie Bézier Abstract Recently, several polydnavirus (PDV) genomes have been completely sequenced. The dsDNA circles enclosed in virus particles and injected by wasps into caterpillars appear to mainly encode virulence factors potentially involved in altering host immunity and/or development, thereby allowing the survival of the parasitoid larvae within the host tissues. Parasitoid wasps generally inject virulence factors produced in the venom gland. As PDV genomes are inherited vertically by wasps through a proviral form, wasp virulence genes may have been transferred to this chromosomal form, leading to their incorporation into virus particles. Indeed, many gene products from Cotesia congregata bracovirus (CcBV), such as PTPs, I,B-like, and cystatins, contain protein domains conserved in metazoans. Surprisingly however, CcBV virulence gene products are not more closely related to insect proteins than to human proteins. To determine whether the distance between CcBV and insect proteins is a specific feature of BV proteins or simply reflects a general high divergence of parasitoid wasp products, which might be due to parasitic lifestyle, we have analyzed the sequences of wasp genes obtained from a cDNA library. Wasp sequences having a high similarity with Apis mellifera genes involved in a variety of biological functions could be identified indicating that the high level of divergence observed for BV products is a hallmark of these viral proteins. We discuss how this divergence might be explained in the context of the current hypotheses on the origin and evolution of wasp-bracovirus associations. Arch. Insect Biochem. Physiol. 67:172,187, 2008. © 2008 Wiley-Liss, Inc. [source] Inhibition of Amyloid Fibrillization of Hen Egg-White Lysozymes by Rifampicin and p -BenzoquinoneBIOTECHNOLOGY PROGRESS, Issue 3 2007Valerie H. Lieu It has been reported that more than 20 different human proteins can fold abnormally, resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The current research, utilizing hen egg-white lysozymes as a model system, is aimed at exploring inhibitory activities of two potential molecules against lysozyme fibril formation. We first demonstrated that the formation of lysozyme amyloid fibrils at pH 2.0 was markedly enhanced by the presence of agitation in comparison with its quiescent counterpart. Next, via numerous spectroscopic techniques and transmission electron microscopy, our results revealed that the inhibition of lysozyme amyloid formation by either rifampicin or its analogue p -benzoquinone followed a concentration-dependent fashion. Furthermore, while both inhibitors were shown to acquire an anti-aggregating and a disaggregating activity, rifampicin, in comparison with p -benzoquinone, served as a more effective inhibitor against in vitro amyloid fibrillogenesis of lysozyme. It is our belief that the data reported in this work will not only reinforce the findings validated by others that rifampicin and p -benzoquinone serve as two promising preventive molecules against amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases. [source] | ||||||||||||||||||||||||||||