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Sample records for human hedgehog-interacting protein

  1. Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

    PubMed Central

    Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

    2013-01-01

    Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

  2. Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples

    PubMed Central

    Shahi, Mehdi H.; Zazpe, Idoya; Afzal, Mohammad; Sinha, Subrata; Rebhun, Robert B.; Meléndez, Bárbara; Rey, Juan A.

    2016-01-01

    Glioma constitutes one of the most common groups of brain tumors, and its prognosis is influenced by different genetic and epigenetic modulations. In this study, we demonstrated low or no expression of hedgehog interacting protein (HHIP) in most of the cell lines and primary glioma tumor samples. We further proceeded to promoter methylation study of this gene in the same cell lines and primary tumor samples and found 87 % (7/8) HHIP methylation in glioblastoma cell lines and 75 % (33/44) in primary tumor samples. These methylation pattern correlates with low or unexpressed HHIP in both cell lines and primary tumor samples. Our results suggest the possibility of epigenetic regulation of this gene in glioma, similarly to medulloblastoma, gastric, hepatic, and pancreatic cancers. Also, HHIP might be a diagnostic or prognostic marker in glioma and help to the detection of these tumors in early stages of disease. PMID:25416442

  3. Cysteine-rich domains related to Frizzled receptors and Hedgehog-interacting proteins

    PubMed Central

    Pei, Jimin; Grishin, Nick V

    2012-01-01

    Frizzled and Smoothened are homologous seven-transmembrane proteins functioning in the Wnt and Hedgehog signaling pathways, respectively. They harbor an extracellular cysteine-rich domain (FZ-CRD), a mobile evolutionary unit that has been found in a number of other metazoan proteins and Frizzled-like proteins in Dictyostelium. Domains distantly related to FZ-CRDs, in Hedgehog-interacting proteins (HHIPs), folate receptors and riboflavin-binding proteins (FRBPs), and Niemann-Pick Type C1 proteins (NPC1s), referred to as HFN-CRDs, exhibit similar structures and disulfide connectivity patterns compared with FZ-CRDs. We used computational analyses to expand the homologous set of FZ-CRDs and HFN-CRDs, providing a better understanding of their evolution and classification. First, FZ-CRD-containing proteins with various domain compositions were identified in several major eukaryotic lineages including plants and Chromalveolata, revealing a wider phylogenetic distribution of FZ-CRDs than previously recognized. Second, two new and distinct groups of highly divergent FZ-CRDs were found by sensitive similarity searches. One of them is present in the calcium channel component Mid1 in fungi and the uncharacterized FAM155 proteins in metazoans. Members of the other new FZ-CRD group occur in the metazoan-specific RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) proteins that are putative tumor suppressors acting as inhibitors of matrix metalloproteases. Finally, sequence and three-dimensional structural comparisons helped us uncover a divergent HFN-CRD in glypicans, which are important morphogen-binding heparan sulfate proteoglycans. Such a finding reinforces the evolutionary ties between the Wnt and Hedgehog signaling pathways and underscores the importance of gene duplications in creating essential signaling components in metazoan evolution. PMID:22693159

  4. Stromal cells expressing hedgehog-interacting protein regulate the proliferation of myeloid neoplasms

    PubMed Central

    Kobune, M; Iyama, S; Kikuchi, S; Horiguchi, H; Sato, T; Murase, K; Kawano, Y; Takada, K; Ono, K; Kamihara, Y; Hayashi, T; Miyanishi, K; Sato, Y; Takimoto, R; Kato, J

    2012-01-01

    Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh-signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasms is unknown. In this study, we assessed the expression of Hh-related genes in primary human CD34+ cells, CD34+ blastic cells and BM stromal cells. Both Indian Hh (Ihh) and its signal transducer, smoothened (SMO), were expressed in CD34+ acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)-derived cells. However, Ihh expression was relatively low in BM stromal cells. Remarkably, expression of the intrinsic Hh-signaling inhibitor, human Hh-interacting protein (HHIP) in AML/MDS-derived stromal cells was markedly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression levels in BM stromal cells highly correlated with their supporting activity for SMO+ leukemic cells. Knockdown of HHIP gene in stromal cells increased their supporting activity although control cells marginally supported SMO+ leukemic cell proliferation. The demethylating agent, 5-aza-2′-deoxycytidine rescued HHIP expression via demethylation of HHIP gene and reduced the leukemic cell-supporting activity of AML/MDS-derived stromal cells. This indicates that suppression of stromal HHIP could be associated with the proliferation of AML/MDS cells. PMID:22961059

  5. Endothelial microparticles carrying hedgehog-interacting protein induce continuous endothelial damage in the pathogenesis of acute graft-versus-host disease.

    PubMed

    Nie, Di-Min; Wu, Qiu-Ling; Zheng, Peng; Chen, Ping; Zhang, Ran; Li, Bei-Bei; Fang, Jun; Xia, Ling-Hui; Hong, Mei

    2016-05-15

    Accumulating evidence suggests that endothelial microparticles (EMPs), a marker of endothelial damage, are elevated in acute graft-versus-host disease (aGVHD), and that endothelial damage is implicated in the pathogenesis of aGVHD, but the mechanisms remain elusive. In this study, we detected the plasma EMP levels and endothelial damage in patients and mice with aGVHD in vivo and then examined the effects of EMPs derived from injured endothelial cells (ECs) on endothelial damage and the role of hedgehog-interacting protein (HHIP) carried by EMPs in these effects in vitro. Our results showed that EMPs were persistently increased in the early posttransplantation phase in patients and mice with aGVHD. Meanwhile, endothelial damage was continuous in aGVHD mice, but was temporary in non-aGVHD mice after transplantation. In vitro, EMPs induced endothelial damage, including increased EC apoptosis, enhanced reactive oxygen species, decreased nitric oxide production and impaired angiogenic activity. Enhanced expression of HHIP, an antagonist for the Sonic hedgehog (SHH) signaling pathway, was observed in patients and mice with aGVHD and EMPs from injured ECs. The endothelial damage induced by EMPs was reversed when the HHIP incorporated into EMPs was silenced with an HHIP small interfering RNA or inhibited with the SHH pathway agonist, Smoothened agonist. This work supports a feasible vicious cycle in which EMPs generated during endothelial injury, in turn, aggravate endothelial damage by carrying HHIP into target ECs, contributing to the continuously deteriorating endothelial damage in the development of aGVHD. EMPs harboring HHIP would represent a potential therapeutic target for aGVHD. PMID:27009877

  6. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  7. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

    Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images PMID:468991

  8. Human MSH2 protein

    DOEpatents

    Chapelle, A. de la; Vogelstein, B.; Kinzler, K.W.

    1997-01-07

    The human MSH2 gene, responsible for hereditary non-polyposis colorectal cancer, was identified by virtue of its homology to the MutS class of genes, which are involved in DNA mismatch repair. The sequence of cDNA clones of the human gene are provided, and the sequence of the gene can be used to demonstrate the existence of germ line mutations in hereditary non-polyposis colorectal cancer (HNPCC) kindreds, as well as in replication error{sup +} (RER{sup +}) tumor cells. 19 figs.

  9. Human MSH2 protein

    DOEpatents

    de la Chapelle, Albert; Vogelstein, Bert; Kinzler, Kenneth W.

    1997-01-01

    The human MSH2 gene, responsible for hereditary non-polyposis colorectal cancer, was identified by virtue of its homology to the MutS class of genes, which are involved in DNA mismatch repair. The sequence of cDNA clones of the human gene are provided, and the sequence of the gene can be used to demonstrate the existence of germ line mutations in hereditary non-polyposis colorectal cancer (HNPCC) kindreds, as well as in replication error.sup.+ (RER.sup.+) tumor cells.

  10. Proteins aggregation and human diseases

    NASA Astrophysics Data System (ADS)

    Hu, Chin-Kun

    2015-04-01

    Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease.

  11. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  12. Protein Evolution of Human Milk.

    PubMed

    Thakkar, Sagar K; Giuffrida, Francesca; Bertschy, Emmanuelle; De Castro, Antonio; Destaillats, Frédéric; Lee, Le Ye

    2016-01-01

    Given the documented short- and long-term advantages of breastfeeding, human milk (HM) as a sole source of nutrition for the first few months of newborn life is considered a normative standard. Each macroconstituent of HM plays a crucial role in the growth and development of the baby. Lipids are largely responsible for providing more than 50% of the energy as well as providing essential fatty acids and minor lipids that are integral to all cell membranes. Carbohydrates can be broadly divided into lactose and oligosaccharides, which are a readily digestible source of glucose and indigestible nonnutritive components, respectively. Proteins in HM provide essential amino acids indispensable for the growth of infants. What is more interesting is that protein concentration profoundly changes from colostrum to mature milk. In this report, we share data from an observatory, single-center, longitudinal trial assessing the constituents of HM collected 30, 60 and 120 days postpartum from 50 mothers (singleton deliveries: 25 male and 25 female infants). The protein content decreased with evolving stages of lactation from an average of 1.45 to 1.38 g/100 ml. The data did not show any gender differences as it was reported for lipid content at 120 days postpartum by our group. Additionally, we also share consolidated literature data on protein evolution of HM during the first year of lactation. PMID:27336906

  13. Human antimicrobial peptides and proteins.

    PubMed

    Wang, Guangshun

    2014-05-13

    As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between -3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized medicine to combat

  14. Human Antimicrobial Peptides and Proteins

    PubMed Central

    Wang, Guangshun

    2014-01-01

    As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized medicine to

  15. Protein phosphorylation systems in postmortem human brain

    SciTech Connect

    Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P. )

    1989-01-01

    Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.

  16. Increased Adipose Protein Carbonylation in Human Obesity

    PubMed Central

    Frohnert, Brigitte I.; Sinaiko, Alan R.; Serrot, Federico J.; Foncea, Rocio E.; Moran, Antoinette; Ikramuddin, Sayeed; Choudry, Umar; Bernlohr, David A.

    2015-01-01

    Insulin resistance is associated with obesity but mechanisms controlling this relationship in humans are not fully understood. Studies in animal models suggest a linkage between adipose reactive oxygen species (ROS) and insulin resistance. ROS oxidize cellular lipids to produce a variety of lipid hydroperoxides that in turn generate reactive lipid aldehydes that covalently modify cellular proteins in a process termed carbonylation. Mammalian cells defend against reactive lipid aldehydes and protein carbonylation by glutathionylation using glutathione-S-transferase A4 (GSTA4) or carbonyl reduction/oxidation via reductases and/or dehydrogenases. Insulin resistance in mice is linked to ROS production and increased level of protein carbonylation, mitochondrial dysfunction, decreased insulin-stimulated glucose transport, and altered adipokine secretion. To assess protein carbonylation and insulin resistance in humans, eight healthy participants underwent subcutaneous fat biopsy from the periumbilical region for protein analysis and frequently sampled intravenous glucose tolerance testing to measure insulin sensitivity. Soluble proteins from adipose tissue were analyzed using two-dimensional gel electrophoresis and the major carbonylated proteins identified as the adipocyte and epithelial fatty acid–binding proteins. The level of protein carbonylation was directly correlated with adiposity and serum free fatty acids (FFAs). These results suggest that in human obesity oxidative stress is linked to protein carbonylation and such events may contribute to the development of insulin resistance. PMID:21593812

  17. Structure of mutant human oncogene protein determined

    SciTech Connect

    Baum, R.

    1989-01-16

    The protein encoded by a mutant human oncogene differs only slightly in structure from the native protein that initiates normal cell division, a finding that may complicate efforts to develop inhibitors of the mutant protein. Previously, the x-ray structure of the protein encoded by the normal c-Ha-ras gene, a protein believed to signal cells to start or stop dividing through its interaction with guanosine triphosphate (GTP), was reported. The structure of the protein encoded by a transforming c-Ha-ras oncogene, in which a valine codon replaces the normal glycine codon at position 12 in the gene, has now been determined. The differences in the structures of the mutant and normal proteins are located primarily in a loop that interacts with the /beta/-phosphate of a bound guanosine diphosphate (GDP) molecule.

  18. Expression of human milk proteins in plants.

    PubMed

    Lönnerdal, Bo

    2002-06-01

    Human milk proteins are believed to have a multitude of biological activities benefiting the newborn infant. Such functions include antibacterial and antiviral activities, enhancement of the immune system and increased nutrient absorption. To date, only breast-fed infants have been exposed to these proteins. However, by using genetic engineering it is now possible to express these proteins in plants, such as rice, at very high levels. Recombinant human milk proteins can subsequently be added to infant formula and baby foods. Prior to such addition, safety tests and efficacy trials need to be conducted. The safety tests will initially be done in rats and then in humans. The efficacy trials should also evaluate stability against heat treatment (processing), pH (stomach conditions) and proteolytic enzymes (digestion). To date, we have expressed recombinant human lactoferrin, lysozyme and alpha1-antitrypsin in rice at very high expression levels. These recombinant proteins showed a stability and activities similar to those of the native milk proteins, suggesting that they may be able to exert biological activities in infants when added to formula or baby foods.

  19. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Human Serum Albumin. Contributes to many transport and regulatory processes and has multifunctional binding properties which range from various metals, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The most abundant protein of the circulatory system. It binds and transports an incredible variety of biological and pharmaceutical ligands throughout the blood stream. Principal Investigator on STS-26 was Larry DeLucas.

  20. New Anthocyanin-Human Salivary Protein Complexes.

    PubMed

    Ferrer-Gallego, Raúl; Soares, Susana; Mateus, Nuno; Rivas-Gonzalo, Julián; Escribano-Bailón, M Teresa; de Freitas, Victor

    2015-08-01

    The interaction between phenolic compounds and salivary proteins is considered the basis of the poorly understood phenomenon of astringency. Furthermore, this interaction is an important factor in relation to their bioavailability. In this work, interactions between anthocyanin and human salivary protein fraction were studied by mass spectrometry (MALDI-TOF-MS and FIA-ESI-MS) and saturation-transfer difference (STD) NMR spectroscopy. Anthocyanins were able to interact with saliva proteins. The dissociation constant (KD) between malvidin 3-glucoside and salivary proline-rich proteins was 1.92 mM for the hemiketal form (pH 3.4) and 1.83 mM for the flavylium cation (pH 1.0). New soluble complexes between these salivary proteins and malvidin 3-glucoside were identified for the first time.

  1. [Cow's milk protein allergy through human milk].

    PubMed

    Denis, M; Loras-Duclaux, I; Lachaux, A

    2012-03-01

    Cow's milk protein allergy (CMPA) is the first allergy that affects infants. In this population, the incidence rate reaches 7.5%. The multiplicity and aspecificity of the symptoms makes its diagnosis sometimes complicated, especially in the delayed type (gastrointestinal, dermatological, and cutaneous). CMPA symptoms can develop in exclusively breastfed infants with an incidence rate of 0.5%. It, therefore, raises questions about sensitization to cow's milk proteins through breast milk. Transfer of native bovine proteins such as β-lactoglobulin into the breast milk is controversial: some authors have found bovine proteins in human milk but others point to cross-reactivity between human milk proteins and cow's milk proteins. However, it seems that a small percentage of dietary proteins can resist digestion and become potentially allergenic. Moreover, some authors suspect the transfer of some of these dietary proteins from the maternal bloodstream to breast milk, but the mechanisms governing sensitization are still being studied. Theoretically, CMPA diagnosis is based on clinical observations, prick-test or patch-test results, and cow's milk-specific IgE antibody concentration. A positive food challenge test usually confirms the diagnosis. No laboratory test is available to make a certain diagnosis, but the detection of eosinophil cationic protein (ECP) in the mother's milk, for example, seems to be advantageous since it is linked to CMA. Excluding cow's milk from the mother's diet is the only cure when she still wants to breastfeed. Usually, cow's milk proteins are reintroduced after 6 months of exclusion. Indeed, the prognosis for infants is very good: 80% acquire a tolerance before the age of 3 or 4 years. Mothers should not avoid dairy products during pregnancy and breastfeeding as preventive measures against allergy.

  2. Dietary protein intake and human health.

    PubMed

    Wu, Guoyao

    2016-03-01

    A protein consists of amino acids (AA) linked by peptide bonds. Dietary protein is hydrolyzed by proteases and peptidases to generate AA, dipeptides, and tripeptides in the lumen of the gastrointestinal tract. These digestion products are utilized by bacteria in the small intestine or absorbed into enterocytes. AA that are not degraded by the small intestine enter the portal vein for protein synthesis in skeletal muscle and other tissues. AA are also used for cell-specific production of low-molecular-weight metabolites with enormous physiological importance. Thus, protein undernutrition results in stunting, anemia, physical weakness, edema, vascular dysfunction, and impaired immunity. Based on short-term nitrogen balance studies, the Recommended Dietary Allowance of protein for a healthy adult with minimal physical activity is currently 0.8 g protein per kg body weight (BW) per day. To meet the functional needs such as promoting skeletal-muscle protein accretion and physical strength, dietary intake of 1.0, 1.3, and 1.6 g protein per kg BW per day is recommended for individuals with minimal, moderate, and intense physical activity, respectively. Long-term consumption of protein at 2 g per kg BW per day is safe for healthy adults, and the tolerable upper limit is 3.5 g per kg BW per day for well-adapted subjects. Chronic high protein intake (>2 g per kg BW per day for adults) may result in digestive, renal, and vascular abnormalities and should be avoided. The quantity and quality of protein are the determinants of its nutritional values. Therefore, adequate consumption of high-quality proteins from animal products (e.g., lean meat and milk) is essential for optimal growth, development, and health of humans. PMID:26797090

  3. Protein supplementation of human IVF culture media.

    PubMed

    Blake, Deborah; Svalander, Peter; Jin, Meishan; Silversand, Christer; Hamberger, Lars

    2002-03-01

    This review travels the road of protein supplementation in embryo culture development-from whole crude plasma in the mid Twentieth century moving through to the completely genetically engineered human albumin with successful births at the beginning of the Twenty-first. PMID:12005309

  4. Protein Phosphatase 1α Interacting Proteins in the Human Brain

    PubMed Central

    Esteves, Sara L.C.; Domingues, Sara C.; da Cruz e Silva, Odete A.B.; da Cruz e Silva, Edgar F.

    2012-01-01

    Abstract Protein Phosphatase 1 (PP1) is a major serine/threonine-phosphatase whose activity is dependent on its binding to regulatory subunits known as PP1 interacting proteins (PIPs), responsible for targeting PP1 to a specific cellular location, specifying its substrate or regulating its action. Today, more than 200 PIPs have been described involving PP1 in panoply of cellular mechanisms. Moreover, several PIPs have been identified that are tissue and event specific. In addition, the diversity of PP1/PIP complexes can further be achieved by the existence of several PP1 isoforms that can bind preferentially to a certain PIP. Thus, PP1/PIP complexes are highly specific for a particular function in the cell, and as such, they are excellent pharmacological targets. Hence, an in-depth survey was taken to identify specific PP1α PIPs in human brain by a high-throughput Yeast Two-Hybrid approach. Sixty-six proteins were recognized to bind PP1α, 39 being novel PIPs. A large protein interaction databases search was also performed to integrate with the results of the PP1α Human Brain Yeast Two-Hybrid and a total of 246 interactions were retrieved. PMID:22321011

  5. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  6. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Plasma Protein Fraction (Human). 640.90 Section 640...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  7. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  8. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  9. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  10. Human complement protein C99 is a calcium binding protein

    SciTech Connect

    Thielens, N.M.; Lohner, K.; Esser, A.F.

    1988-05-15

    Human complement protein C9 is shown to be a metalloprotein that binds 1 mol of Ca/sup 2 +//mol of C9 with a dissociation constant of 3 ..mu..m as measured by equilibrium dialysis. Incubation with EDTA removes the bound calcium, resulting in a apoprotein with decreased thermal stability. This loss in stability leads to aggregation and, therefore, to loss of hemolytic activity upon heating to a few degrees above the physiological temperature. Heat-induced aggregation of apoC9 can be prevented by salts that stabilize proteins according to the Hofmeister series of lyotropic ions, suggesting that the ion in native C9 may ligand with more than one structural element of domain of the protein. Ligand blotting indicates that the calcium binding site is located in the amino-terminal half of the protein. Removal of calcium by inclusion of EDTA in assay mixtures has no effect on the hemolytic activity of C9, and its capacity to bind to C8 in solution, or to small unilamellar lipid vesicles at temperatures at or below the physiological range. Although the precise structural and functional role of the bound calcium is not know, it is clear that it provides thermal stability to C9 and it may have a function in regulation of membrane insertion.

  11. Cow's milk proteins in human milk.

    PubMed

    Coscia, A; Orrù, S; Di Nicola, P; Giuliani, F; Rovelli, I; Peila, C; Martano, C; Chiale, F; Bertino, E

    2012-01-01

    Cow's milk proteins (CMPs) are among the best characterized food allergens. Cow's milk contains more than twenty five different proteins, but only whey proteins alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin (BSA), and lactoferrin, as well as the four caseins, have been identified as allergens. Aim of this study was to investigate by proteomics techniques cow's milk allergens in human colostrum of term and preterm newborns' mothers, not previously detected, in order to understand if such allergens could be cause of sensitization during lactation. Term colostrum samples from 62 healthy mothers and preterm colostrum samples from 11 healthy mothers were collected for this purpose. The most relevant finding was the detection of the intact bovine alpha-S1-casein in both term and preterm colostrum. Using this method, which allows direct proteins identification, beta-lactoglobulin was not detected in any of colostrum samples. According to our results bovine alpha 1 casein that is considered a major cow's milk allergen is readily secreted in human milk: further investigations are needed in order to clarify if alpha-1-casein has a major role in sensitization or tolerance to cow's milk of exclusively breastfed predisposed infants.

  12. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  13. Defining the Protein-Protein Interaction Network of the Human Protein Tyrosine Phosphatase Family.

    PubMed

    Li, Xu; Tran, Kim My; Aziz, Kathryn E; Sorokin, Alexey V; Chen, Junjie; Wang, Wenqi

    2016-09-01

    Protein tyrosine phosphorylation, which plays a vital role in a variety of human cellular processes, is coordinated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Genomic studies provide compelling evidence that PTPs are frequently mutated in various human cancers, suggesting that they have important roles in tumor suppression. However, the cellular functions and regulatory machineries of most PTPs are still largely unknown. To gain a comprehensive understanding of the protein-protein interaction network of the human PTP family, we performed a global proteomic study. Using a Minkowski distance-based unified scoring environment (MUSE) for the data analysis, we identified 940 high confidence candidate-interacting proteins that comprise the interaction landscape of the human PTP family. Through a gene ontology analysis and functional validations, we connected the PTP family with several key signaling pathways or cellular functions whose associations were previously unclear, such as the RAS-RAF-MEK pathway, the Hippo-YAP pathway, and cytokinesis. Our study provides the first glimpse of a protein interaction network for the human PTP family, linking it to a number of crucial signaling events, and generating a useful resource for future studies of PTPs.

  14. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  15. Protein oxidation, UVA and human DNA repair.

    PubMed

    Karran, Peter; Brem, Reto

    2016-08-01

    Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency - with particular reference to NER and skin cancer risk.

  16. Human skeletal muscle protein breakdown during spaceflight

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Schluter, M. D.

    1997-01-01

    Human spaceflight is associated with a loss of body protein. Excretion of 3-methylhistidine (3-MH) in the urine is a useful measurement of myofibrillar protein breakdown. Bed rest, particularly with 6 degrees head-down tilt, is an accepted ground-based model for human spaceflight. The objectives of this report were to compare 3-MH excretion from two Life Sciences shuttle missions (duration 9.5 and 15 days, n = 9) and from 17 days of bed rest (n = 7) with 6 degrees head-down tilt. The bed rest study was designed to mimic an actual Life Sciences spaceflight and so incorporated an extensive battery of physiological tests focused on the musculoskeletal system. Results showed that nitrogen retention, based on excretion of nitrogen in the urine, was reduced during both bed rest [from 22 +/- 1 to 1 +/- 5 mg N x kg(-1) x day(-1) (n = 7; P < 0.05)] and spaceflight [from 57 +/- 9 to 19 +/- 3 mg N x kg(-1) x day(-1) (n = 9; P < 0.05)]. 3-MH excretion was unchanged with either bed rest [pre-bed rest 5.30 +/- 0.29 vs. bed rest 5.71 +/- 0.30 micromol 3-MH x kg(-1) x day(-1), n = 7; P = not significant (NS)] or spaceflight [preflight 4.98 +/- 0.37 vs. 4.59 +/- 0.39 micromol 3-MH x kg(-1) x day(-1) in-flight, n = 9; P = NS]. We conclude that 1) 3-MH excretion was unaffected by spaceflight on the shuttle or with bed rest plus exercise, and 2) because protein breakdown (elevated 3-MH) was increased on Skylab but not on the shuttle, it follows that muscle protein breakdown is not an inevitable consequence of spaceflight.

  17. Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

    PubMed

    Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

    2016-06-01

    The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms. PMID:27543868

  18. Proteins of human milk. I. Identification of major components

    SciTech Connect

    Anderson, N.G.; Powers, M.T.; Tollaksen, S.L.

    1982-04-01

    Traditionally, human milk proteins are identified largely by reference to bovine milk. Hence, to identify the major proteins in human milk, we subjected human and bovine milk, in parallel, to high-resolution two-dimensional electrophoresis. Isoelectric precipitation at pH 4.6 was our criterion for distinguishing whey proteins from those of the casein complex. The ..cap alpha..- and..beta..-caseins were identified on the basis of relative abundance, relative molecular mass, and relative isoelectric points. No protein disappeared from ISO-DALT patterns of human milk after rennin treatment, and no new protein comparable to bovine para K-casein appeared in the BASO-DALT patterns; this suggests that K-casein is absent from human milk. The proteins identified in human milk patterns include the ..cap alpha.. and ..beta.. casein families, lactalbumin, albumin, transferrin, IgA, and lactoferrin. Numerous additional proteins seen in patterns for human milk remain to be identified.

  19. Functional prediction of hypothetical proteins in human adenoviruses.

    PubMed

    Dorden, Shane; Mahadevan, Padmanabhan

    2015-01-01

    Assigning functional information to hypothetical proteins in virus genomes is crucial for gaining insight into their proteomes. Human adenoviruses are medium sized viruses that cause a range of diseases. Their genomes possess proteins with uncharacterized function known as hypothetical proteins. Using a wide range of protein function prediction servers, functional information was obtained about these hypothetical proteins. A comparison of functional information obtained from these servers revealed that some of them produced functional information, while others provided little functional information about these human adenovirus hypothetical proteins. The PFP, ESG, PSIPRED, 3d2GO, and ProtFun servers produced the most functional information regarding these hypothetical proteins. PMID:26664031

  20. A physical interaction network of dengue virus and human proteins.

    PubMed

    Khadka, Sudip; Vangeloff, Abbey D; Zhang, Chaoying; Siddavatam, Prasad; Heaton, Nicholas S; Wang, Ling; Sengupta, Ranjan; Sahasrabudhe, Sudhir; Randall, Glenn; Gribskov, Michael; Kuhn, Richard J; Perera, Rushika; LaCount, Douglas J

    2011-12-01

    Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.

  1. The human RBPome: from genes and proteins to human disease.

    PubMed

    Neelamraju, Yaseswini; Hashemikhabir, Seyedsasan; Janga, Sarath Chandra

    2015-09-01

    RNA binding proteins (RBPs) play a central role in mediating post transcriptional regulation of genes. However less is understood about them and their regulatory mechanisms. In this study, we construct a catalogue of 1344 experimentally confirmed RBPs. The domain architecture of RBPs enabled us to classify them into three groups - Classical (29%), Non-classical (19%) and unclassified (52%). A higher percentage of proteins with unclassified domains reveals the presence of various uncharacterised motifs that can potentially bind RNA. RBPs were found to be highly disordered compared to Non-RBPs (p<2.2e-16, Fisher's exact test), suggestive of a dynamic regulatory role of RBPs in cellular signalling and homeostasis. Evolutionary analysis in 62 different species showed that RBPs are highly conserved compared to Non-RBPs (p<2.2e-16, Wilcox-test), reflecting the conservation of various biological processes like mRNA splicing and ribosome biogenesis. The expression patterns of RBPs from human proteome map revealed that ~40% of them are ubiquitously expressed and ~60% are tissue-specific. RBPs were also seen to be highly associated with several neurological disorders, cancer and inflammatory diseases. Anatomical contexts like B cells, T-cells, foetal liver and foetal brain were found to be strongly enriched for RBPs, implying a prominent role of RBPs in immune responses and different developmental stages. The catalogue and meta-analysis presented here should form a foundation for furthering our understanding of RBPs and the cellular networks they control, in years to come. This article is part of a Special Issue entitled: Proteomics in India. PMID:25982388

  2. Bioactive properties of milk proteins in humans: A review.

    PubMed

    Nongonierma, Alice B; FitzGerald, Richard J

    2015-11-01

    Many studies have demonstrated that milk protein consumption has benefits in terms of promoting human health. This review assesses the intervention studies which have evaluated potential health enhancing effects in humans following the ingestion of milk proteins. The impact of milk protein ingestion has been studied to asses their satiating, hypotensive, antimicrobial, anti-inflammatory, anticancer, antioxidant and insulinotropic properties as well as their impact on morphological modifications (e.g., muscle and fat mass) in humans. Consistent health promoting effects appear to have been observed in certain instances (i.e., muscle protein synthesis, insulinotropic and hypotensive activity). However, controversial outcomes have also been reported (i.e., antimicrobial, anti-inflammatory, anticancer and antioxidant properties). Several factors including interindividual differences, the timing of protein ingestion as well as the potency of the active components may explain these differences. In addition, processing conditions have been reported, in certain instances, to affect milk protein structure and therefore modify their bioactive potential. It is thought that the health promoting properties of milk proteins are linked to the release of bioactive peptides (BAPs) during gastrointestinal digestion. There is a need for further research to develop a more in-depth understanding on the possible mechanisms involved in the observed physiological effects. In addition, more carefully controlled and appropriately powered human intervention studies are required to demonstrate the health enhancing properties of milk proteins in humans.

  3. The evolution of human cells in terms of protein innovation.

    PubMed

    Sardar, Adam J; Oates, Matt E; Fang, Hai; Forrest, Alistair R R; Kawaji, Hideya; Gough, Julian; Rackham, Owen J L

    2014-06-01

    Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type-specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type-specific domain architectures.

  4. Cloning and expression of special F protein from human liver

    PubMed Central

    Liu, Shu-Ye; Yu, Xin-Da; Song, Chun-Juan; Lu, Wei; Zhang, Jian-Dong; Shi, Xin-Rong; Duan, Ying; Zhang, Ju

    2007-01-01

    AIM: To clone human liver special F protein and to express it in a prokaryotic system. METHODS: Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this, cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein’s cDNA was subcloned into the expression vector pET-15b and transformed into E. coli BL21 (DE3) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein. RESULTS: The cDNA clone of human liver special F protein (1134bp) was successfully produced, with the cDNA sequence being published in Gene-bank: DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted. CONCLUSION: F protein expresses cDNA clone in a prokaryotic system, which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein. PMID:17465469

  5. Prioritizing protein complexes implicated in human diseases by network optimization

    PubMed Central

    2014-01-01

    Background The detection of associations between protein complexes and human inherited diseases is of great importance in understanding mechanisms of diseases. Dysfunctions of a protein complex are usually defined by its member disturbance and consequently result in certain diseases. Although individual disease proteins have been widely predicted, computational methods are still absent for systematically investigating disease-related protein complexes. Results We propose a method, MAXCOM, for the prioritization of candidate protein complexes. MAXCOM performs a maximum information flow algorithm to optimize relationships between a query disease and candidate protein complexes through a heterogeneous network that is constructed by combining protein-protein interactions and disease phenotypic similarities. Cross-validation experiments on 539 protein complexes show that MAXCOM can rank 382 (70.87%) protein complexes at the top against protein complexes constructed at random. Permutation experiments further confirm that MAXCOM is robust to the network structure and parameters involved. We further analyze protein complexes ranked among top ten for breast cancer and demonstrate that the SWI/SNF complex is potentially associated with breast cancer. Conclusions MAXCOM is an effective method for the discovery of disease-related protein complexes based on network optimization. The high performance and robustness of this approach can facilitate not only pathologic studies of diseases, but also the design of drugs targeting on multiple proteins. PMID:24565064

  6. Mineral-bound noncollagenous proteins in archaeological human skeletons.

    PubMed

    Freundorfer, S; Grupe, G; Weickmann, D

    1995-05-01

    Archaeometric approaches to archaelogical human bone also include the extraction, identification and molecular analysis of surviving bone proteins. Due to its abundance as a matrix protein, most studies focus on collagen (e.g. radiocarbon dating). Also, a variety of serum proteins are detectable in excavated skeletons. Very limited knowledge still exists on mineral-bound noncollagenous bone proteins from ancient bones because, in the mature tissue, they occur in trace amounts only. Moreover, post-mortem decomposition is likely to change characteristic features of the molecules. Due to their suggested role as growth and developmental factors, identification and quantification of such proteins should be valuable for both physical anthropology and epidemiology. We present a valid method for the detection of small amounts of surviving mineral-bound noncollagenous proteins in excavated human bones up to 7500 years of age.

  7. Mitochondrial Protein Import and Human Health and Disease

    PubMed Central

    MacKenzie, James A.; Mark Payne, R.

    2009-01-01

    The targeting and assembly of nuclear-encoded mitochondrial proteins are essential processes because the energy supply of humans is dependent upon the proper functioning of mitochondria. Defective import of mitochondrial proteins can arise from mutations in the targeting signals within precursor proteins, from mutations that disrupt the proper functioning of the import machinery, or from deficiencies in the chaperones involved in the proper folding and assembly of proteins once they are imported. Defects in these steps of import have been shown to lead to oxidative stress, neurodegenerative diseases, and metabolic disorders. In addition, protein import into mitochondria has been found to be a dynamically regulated process that varies in response to conditions such as oxidative stress, aging, drug treatment, and exercise. This review focuses on how mitochondrial protein import affects human health and disease. PMID:17300922

  8. Development of Blood Analog Fluids Using Human Hair Protein Particles

    NASA Astrophysics Data System (ADS)

    Kobayashi, Shunichi; Morikawa, Hirohisa; Ishii, Shinji; Fujii, Toshihiro

    Model experiments of blood flow are very important in the study of mechanical aspects in cardiovascular research and the development of artificial organs. Several blood analog fluids, such as non-Newtonian fluids have been developed and used in model experiments. However, little is known about blood substitutes with biocompatible properties. We have developed novel procedures for preparing human hair protein films, and have fabricated protein particle suspensions from the films, by mechanical stimulation, for use as blood analog fluid. The average diameter of the protein particles was controlled and microscopic observations were done using a confocal microscope. The Casson’s plot patterns of the suspension containing the protein particles were similar to those of human blood. The protein particles also worked well as ultrasound contrast agents in the ultrasound Doppler flow velocity measurements in the model experiments. Therefore, the protein particle system is a promising alternative for blood cells in artificial blood.

  9. Human protein reference database as a discovery resource for proteomics

    PubMed Central

    Peri, Suraj; Navarro, J. Daniel; Kristiansen, Troels Z.; Amanchy, Ramars; Surendranath, Vineeth; Muthusamy, Babylakshmi; Gandhi, T. K. B.; Chandrika, K. N.; Deshpande, Nandan; Suresh, Shubha; Rashmi, B. P.; Shanker, K.; Padma, N.; Niranjan, Vidya; Harsha, H. C.; Talreja, Naveen; Vrushabendra, B. M.; Ramya, M. A.; Yatish, A. J.; Joy, Mary; Shivashankar, H. N.; Kavitha, M. P.; Menezes, Minal; Choudhury, Dipanwita Roy; Ghosh, Neelanjana; Saravana, R.; Chandran, Sreenath; Mohan, Sujatha; Jonnalagadda, Chandra Kiran; Prasad, C. K.; Kumar-Sinha, Chandan; Deshpande, Krishna S.; Pandey, Akhilesh

    2004-01-01

    The rapid pace at which genomic and proteomic data is being generated necessitates the development of tools and resources for managing data that allow integration of information from disparate sources. The Human Protein Reference Database (http://www.hprd.org) is a web-based resource based on open source technologies for protein information about several aspects of human proteins including protein–protein interactions, post-translational modifications, enzyme–substrate relationships and disease associations. This information was derived manually by a critical reading of the published literature by expert biologists and through bioinformatics analyses of the protein sequence. This database will assist in biomedical discoveries by serving as a resource of genomic and proteomic information and providing an integrated view of sequence, structure, function and protein networks in health and disease. PMID:14681466

  10. Immunoprecipitation of membrane proteins of cultured human sarcoma cells.

    PubMed

    Grófová, M; Forchhammer, J; Lizonová, A; Popovic, M

    1981-01-01

    Human sarcoma associated antigens (HSAA) have previously been identified by indirect immune fluorescence in human sarcoma cells in culture using sera from patients bearing different types of sarcoma. To further characterize these HSAA, surface proteins of cultured cells were labeled with 125Iodine, [3H]-glucosamine and [35S]-methionine and solubilized. After immunoprecipitation labeled proteins were detected in immune complexes by SDS polyacrylamide gel electrophoresis and autoradiography, which allowed comparison with antigens described by other groups. A surface protein (Mr 96 000) was precipitated with sera from sarcoma bearing patients, and two glycoproteins (Mr 115 000 and 85 000) were preferentially precipitated with antisera from rabbits immunized with membranes from two human sarcoma cell lines. At least two of these proteins were found in each of five human sarcoma cell lines studied (U-4SS, U-3930S, U-20S, B-5GT and B-6FS). None of the proteins were precipitated with three human control sera, and only occasionally a faint band was observed in immunoprecipitates from control cells (B-25F, B-41B, B-42FC, U-2S, and U-393S with the immune sera. These proteins are probably some of the antigens responsible for the immune fluorescence observed in determination of HSAA. However, purification of the proteins and competition experiments are needed before this can be finally established.

  11. Spaceflight and protein metabolism, with special reference to humans

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Gaprindashvili, T.

    1994-01-01

    Human space missions have shown that human spaceflight is associated with a loss of body protein. Specific changes include a loss of lean body mass, decreased muscle mass in the calves, decreased muscle strength, and changes in plasma proteins and amino acids. The major muscle loss is believed to be associated with the antigravity (postural) muscle. The most significant loss of protein appears to occur during the first month of flight. The etiology is believed to be multifactorial with contributions from disuse atrophy, undernutrition, and a stress type of response. This article reviews the results of American and Russian space missions to investigate this problem in humans, monkeys, and rats. The relationship of the flight results with ground-based models including bedrest for humans and hindlimb unweighting for rats is also discussed. The results suggest that humans adapt to spaceflight much better than either monkeys or rats.

  12. Heat Shock Proteins in the Human Eye

    PubMed Central

    Urbak, Lærke; Vorum, Henrik

    2010-01-01

    Heat shock proteins (Hsps) are believed to primarily protect and maintain cell viability under stressful conditions such as those occurring during thermal and oxidative challenges chiefly by refolding and stabilizing proteins. Hsps are found throughout the various tissues of the eye where they are thought to confer protection from disease states such as cataract, glaucoma, and cancer. This minireview summarizes the placement, properties, and roles of Hsps in the eye and aims to provide a better comprehension of their function and involvement in ocular disease pathogenesis. PMID:22084677

  13. Statistical Approaches for the Construction and Interpretation of Human Protein-Protein Interaction Network

    PubMed Central

    Hu, Yang; Zhang, Ying; Ren, Jun

    2016-01-01

    The overall goal is to establish a reliable human protein-protein interaction network and develop computational tools to characterize a protein-protein interaction (PPI) network and the role of individual proteins in the context of the network topology and their expression status. A novel and unique feature of our approach is that we assigned confidence measure to each derived interacting pair and account for the confidence in our network analysis. We integrated experimental data to infer human PPI network. Our model treated the true interacting status (yes versus no) for any given pair of human proteins as a latent variable whose value was not observed. The experimental data were the manifestation of interacting status, which provided evidence as to the likelihood of the interaction. The confidence of interactions would depend on the strength and consistency of the evidence.

  14. Statistical Approaches for the Construction and Interpretation of Human Protein-Protein Interaction Network.

    PubMed

    Hu, Yang; Zhang, Ying; Ren, Jun; Wang, Yadong; Wang, Zhenzhen; Zhang, Jun

    2016-01-01

    The overall goal is to establish a reliable human protein-protein interaction network and develop computational tools to characterize a protein-protein interaction (PPI) network and the role of individual proteins in the context of the network topology and their expression status. A novel and unique feature of our approach is that we assigned confidence measure to each derived interacting pair and account for the confidence in our network analysis. We integrated experimental data to infer human PPI network. Our model treated the true interacting status (yes versus no) for any given pair of human proteins as a latent variable whose value was not observed. The experimental data were the manifestation of interacting status, which provided evidence as to the likelihood of the interaction. The confidence of interactions would depend on the strength and consistency of the evidence. PMID:27648447

  15. Statistical Approaches for the Construction and Interpretation of Human Protein-Protein Interaction Network

    PubMed Central

    Hu, Yang; Zhang, Ying; Ren, Jun

    2016-01-01

    The overall goal is to establish a reliable human protein-protein interaction network and develop computational tools to characterize a protein-protein interaction (PPI) network and the role of individual proteins in the context of the network topology and their expression status. A novel and unique feature of our approach is that we assigned confidence measure to each derived interacting pair and account for the confidence in our network analysis. We integrated experimental data to infer human PPI network. Our model treated the true interacting status (yes versus no) for any given pair of human proteins as a latent variable whose value was not observed. The experimental data were the manifestation of interacting status, which provided evidence as to the likelihood of the interaction. The confidence of interactions would depend on the strength and consistency of the evidence. PMID:27648447

  16. Protein dynamics in individual human cells: experiment and theory.

    PubMed

    Cohen, Ariel Aharon; Kalisky, Tomer; Mayo, Avi; Geva-Zatorsky, Naama; Danon, Tamar; Issaeva, Irina; Kopito, Ronen Benjamine; Perzov, Natalie; Milo, Ron; Sigal, Alex; Alon, Uri

    2009-01-01

    A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

  17. Human plasma protein N-glycosylation.

    PubMed

    Clerc, Florent; Reiding, Karli R; Jansen, Bas C; Kammeijer, Guinevere S M; Bondt, Albert; Wuhrer, Manfred

    2016-06-01

    Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level. PMID:26555091

  18. Intracellular localization of VAMP-1 protein in human neutrophils.

    PubMed

    Nabokina, S M

    2001-02-01

    We studied the intracellular localization of vesicle-associated membrane protein VAMP-1 in human neutrophils. VAMP-1 was associated with membranes of gelatinase and specific secretory granules rapidly mobilized during exocytosis. VAMP-1 probably acts as a component of the SNARE complex during exocytosis of gelatinase and specific granules in human neutrophils.

  19. Genetic alterations of protein tyrosine phosphatases in human cancers

    PubMed Central

    Zhao, Shuliang; Sedwick, David; Wang, Zhenghe

    2014-01-01

    Protein tyrosine phosphatases (PTPs) are enzymes that remove phosphate from tyrosine residues in proteins. Recent whole-exome sequencing of human cancer genomes reveals that many PTPs are frequently mutated in a variety of cancers. Among these mutated PTPs, protein tyrosine phosphatase T (PTPRT) appears to be the most frequently mutated PTP in human cancers. Beside PTPN11 which functions as an oncogene in leukemia, genetic and functional studies indicate that most of mutant PTPs are tumor suppressor genes. Identification of the substrates and corresponding kinases of the mutant PTPs may provide novel therapeutic targets for cancers harboring these mutant PTPs. PMID:25263441

  20. Human tissue profiling with multidimensional protein identification technology.

    PubMed

    Cagney, Gerard; Park, Stephen; Chung, Clement; Tong, Bianca; O'Dushlaine, Colm; Shields, Denis C; Emili, Andrew

    2005-01-01

    Profiling of tissues and cell types through systematic characterization of expressed genes or proteins shows promise as a basic research tool, and has potential applications in disease diagnosis and classification. We used multidimensional protein identification protein identification technology (MudPIT) to analyze proteomes for enriched nuclear extracts of eight human tissues: brain, heart, liver, lung, muscle, pancreas, spleen, and testis. We show that the method is approximately 80% reproducible. We address issues of relative abundance, tissue-specificity, and selectivity, and the significance of proteins whose expression does not correlate with that of the corresponding mRNA. Surprisingly, most proteins are detected in a single tissue. These proteins tend to fulfill specialist (and potentially tissue-specific) functions compared to proteins expressed in two or more tissues.

  1. Cytoskeletal proteins inside human immunodeficiency virus type 1 virions.

    PubMed Central

    Ott, D E; Coren, L V; Kane, B P; Busch, L K; Johnson, D G; Sowder, R C; Chertova, E N; Arthur, L O; Henderson, L E

    1996-01-01

    We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of alpha-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and viral proteins. PMID:8892894

  2. Identification of binary interactions between human cytomegalovirus virion proteins.

    PubMed

    Phillips, Stacia L; Bresnahan, Wade A

    2011-01-01

    Human cytomegalovirus (HCMV) virions are composed of a DNA-containing nucleocapsid surrounded by a tegument layer and host-derived lipid envelope studded with virally encoded glycoproteins. These complex virions are estimated to be composed of more than 50 viral proteins. Assembly of HCMV virions is poorly understood, especially with respect to acquisition of the tegument; however, it is thought to involve the stepwise addition of virion components through protein-protein interactions. We sought to identify interactions among HCMV virion proteins using yeast two-hybrid analysis. Using 33 known capsid and tegument proteins, we tested 1,089 pairwise combinations for binary interaction in the two-hybrid assay. We identified 24 interactions among HCMV virion proteins, including 13 novel interactions among tegument proteins and one novel interaction between capsid proteins. Several of these novel interactions were confirmed by coimmunoprecipitation of protein complexes from transfected cells. In addition, we demonstrate three of these interactions in the context of HCMV infection. This study reveals several new protein-protein interactions among HCMV tegument proteins, some of which are likely important for HCMV replication and pathogenesis. PMID:20962080

  3. Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

    PubMed Central

    Malik, A; Egan, J E; Houghten, R A; Sadoff, J C; Hoffman, S L

    1991-01-01

    Cytotoxic T lymphocytes (CTL) against the circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodium falciparum CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falciparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P. falciparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368-390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8+ T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B10.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria. PMID:1707538

  4. LGI Proteins and Epilepsy in Human and Animals.

    PubMed

    Pakozdy, A; Patzl, M; Zimmermann, L; Jokinen, T S; Glantschnigg, U; Kelemen, A; Hasegawa, D

    2015-01-01

    Leucine-rich glioma-inactivated (LGI) protein was first thought to have a suppressor effect in the formation of some cancers. Developments in physiology and medicine made it possible to characterize the function of the LGI protein family and its crucial role in different conditions more precisely. These proteins play an important role in synaptic transmission, and dysfunction may cause hyperexcitability. Genetic mutation of LGI1 was confirmed to be the cause of autosomal dominant lateral temporal lobe epilepsy in humans. The LGI2 mutation was identified in benign familial juvenile epilepsy in Lagotto Romagnolo (LR) dogs. Cats with familial spontaneous temporal lobe epilepsy have been reported, and the etiology might be associated with LGI protein family dysfunction. In addition, an autoimmune reaction against LGI1 was detected in humans and cats with limbic encephalitis. These advances prompted a review of LGI protein function and its role in different seizure disorders.

  5. Induction of Mycobacterium avium proteins upon infection of human macrophages.

    PubMed

    Brunori, Lara; Giannoni, Federico; Bini, Luca; Liberatori, Sabrina; Frota, Cristiane; Jenner, Peter; Thoresen, Ove Fredrik; Orefici, Graziella; Fattorini, Lanfranco

    2004-10-01

    Induction of Mycobacterium avium proteins labelled with [35S]methionine and mRNAs upon infection of the human macrophage cell line THP-1 was investigated by two-dimensional gel electrophoresis-mass spectrometry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. M. avium overexpressed proteins within the macrophages that are involved in fatty acids metabolism (FadE2, FixA), cell wall synthesis (KasA), and protein synthesis (EF-tu). The correlation of differential protein and mRNA expression varied between good and no correlation. Overall, these four proteins may be involved in the adaptation and survival of M. avium within human macrophages. PMID:15378697

  6. Deep conservation of human protein tandem repeats within the eukaryotes.

    PubMed

    Schaper, Elke; Gascuel, Olivier; Anisimova, Maria

    2014-05-01

    Tandem repeats (TRs) are a major element of protein sequences in all domains of life. They are particularly abundant in mammals, where by conservative estimates one in three proteins contain a TR. High generation-scale duplication and deletion rates were reported for nucleic TR units. However, it is not known whether protein TR units can also be frequently lost or gained providing a source of variation for rapid adaptation of protein function, or alternatively, tend to have conserved TR unit configurations over long evolutionary times. To obtain a systematic picture, we performed a proteome-wide analysis of the mode of evolution for human protein TRs. For this purpose, we propose a novel method for the detection of orthologous TRs based on circular profile hidden Markov models. For all detected TRs, we reconstructed bispecies TR unit phylogenies across 61 eukaryotes ranging from human to yeast. Moreover, we performed additional analyses to correlate functional and structural annotations of human TRs with their mode of evolution. Surprisingly, we find that the vast majority of human TRs are ancient, with TR unit number and order preserved intact since distant speciation events. For example, ≥ 61% of all human TRs have been strongly conserved at least since the root of all mammals, approximately 300 Ma. Further, we find no human protein TR that shows evidence for strong recent duplications and deletions. The results are in contrast to the high generation-scale mutability of nucleic TRs. Presumably, most protein TRs fold into stable and conserved structures that are indispensable for the function of the TR-containing protein. All of our data and results are available for download from http://www.atgc-montpellier.fr/TRE.

  7. Deep Conservation of Human Protein Tandem Repeats within the Eukaryotes

    PubMed Central

    Schaper, Elke; Gascuel, Olivier; Anisimova, Maria

    2014-01-01

    Tandem repeats (TRs) are a major element of protein sequences in all domains of life. They are particularly abundant in mammals, where by conservative estimates one in three proteins contain a TR. High generation-scale duplication and deletion rates were reported for nucleic TR units. However, it is not known whether protein TR units can also be frequently lost or gained providing a source of variation for rapid adaptation of protein function, or alternatively, tend to have conserved TR unit configurations over long evolutionary times. To obtain a systematic picture, we performed a proteome-wide analysis of the mode of evolution for human protein TRs. For this purpose, we propose a novel method for the detection of orthologous TRs based on circular profile hidden Markov models. For all detected TRs, we reconstructed bispecies TR unit phylogenies across 61 eukaryotes ranging from human to yeast. Moreover, we performed additional analyses to correlate functional and structural annotations of human TRs with their mode of evolution. Surprisingly, we find that the vast majority of human TRs are ancient, with TR unit number and order preserved intact since distant speciation events. For example, ≥61% of all human TRs have been strongly conserved at least since the root of all mammals, approximately 300 Ma. Further, we find no human protein TR that shows evidence for strong recent duplications and deletions. The results are in contrast to the high generation-scale mutability of nucleic TRs. Presumably, most protein TRs fold into stable and conserved structures that are indispensable for the function of the TR-containing protein. All of our data and results are available for download from http://www.atgc-montpellier.fr/TRE. PMID:24497029

  8. Human Proteinpedia enables sharing of human protein data

    SciTech Connect

    Mathivanan, Suresh; Ahmed, Mukhtar; Ahn, Natalie G.; Alexandre, Hainard; Amanchy, Ramars; Andrews, Philip C.; Bader, Joel S.; Balgley, Brian M.; Bantscheff, Marcus; Bennett, Keiryn; Bjorling, Erik; Blagoev, Blagoy; Bose , Ron; Brahmachari, Samir K.; Burlingame, Alma S.; Bustelo, Xos R.; Cagney, Gerard; Cantin, Greg T; Cardasis, Helene L; Celis, Julio E; Chaerkady, Raghothama; Chu, Feixia; Cole, Phillip A.; Costello, Catherine E; Cotter , Robert J.; Crockett, David; DeLany , James P.; De Marzo, Angelo M; DeSouza, Leroi V; Deutsch, Eric W.; Dransfield , Eric; Drewes , Gerard; Droit , Arnaud; Dunn, Michael; Elenitoba-Johnson, Kojo; Ewing, Rob M.; Van Eyk , Jennifer; Faca , Vitor; Falkner , Jayson; Fang, Xiangming; Fenselau , Catherine; Figeys , Daniel; Gagne , Pierre; Gelfi , Cecilia; Gevaert , Kris; Gimble , Jeffrey; Gnad , Florian; Goel, Renu; Gromov , Pavel; Hanash, Samir M.; Hancock, William S.; Harsha , HC; Hart , Gerald; Faith , Hays; He , Fuchu; Hebbar , Prashantha; Helsens , Kenny; Hermeking , Heiko; Hide , Winston; Hjerno, Karin; Hochstrasser, Denis F.; Hofmann, Oliver; Horn , David M.; Hruban , Ralph H.; Ibarrola , Nieves; James , Peter; Jensen , Ole N.; Jensen, Pia H.; Jung , Peter; Kandasamy, Kumaran; Kheterpal , Indu; Kikuno , Reiko; Korf, Ulrike; Korner, Roman; Kuster, Bernhard; Kwon , Min-Seok; Lee , Hyoung-Joo; Lee , Young - Jin; Lefevre , Michael; Lehvaslaiho, Minna; Lescuyer, Pierre; Levander, Fredrik; Lim, Megan S.; Lobke, Christian; Loo, Joseph; Mann, Matthias; Martens , Lennart; Martinez-Heredia, Juan; McComb, Mark E.; McRedmond , James; Mehrle, Alexander; Menon, Rajasree; Miller, Christine A.; Mischak, Harald; Mohan, S Sujatha; Mohmood , Riaz; Molina , Henrik; Moran , Michael F.; Morgan, James D.; Moritz , Robert; Morzel, Martine; Muddiman, David C.; Nalli , Anuradha; Navarro, J. D.; Neubert , Thomas A.; Ohara , Osamu; Oliva, Rafael; Omenn, Gilbert; Oyama , Masaaki; Paik, Young-Ki; Pennington , Kyla; Pepperkok, Rainer; Periaswamy, Balamurugan; Petricoin, Emanuel F.; Poirier, Guy G.; Prasad, T S Keshava; Purvine, Samuel O.; Rahiman , B Abdul; Ramachandran, Prasanna; Ramachandra , Y L; Rice, Robert H.; Rick , Jens; Ronnholm , Ragna H.; Salonen , Johanna; Sanchez , Jean - Charles; Sayd , Thierry; Seshi, Beerelli; Shankari, Kripa; Sheng , Shi Jun; Shetty , Vivekananda; Shivakumar, K.; Simpson, Richard J.; Sirdeshmukh, Ravi; Siu , K W Michael; Smith, Jeffrey C.; Smith, Richard D.; States, David J.; Sugano, Sumio; Sullivan , Matthew; Superti - Furga, Giulio; Takatalo , Maarit; Thongboonkerd , Visith; Trinidad , Jonathan C.; Uhlen , Mathias; Vandekerckhove, Joel; Vasilescu , Julian; Veenstra, Timothy D.; Vidal - Taboada, Jose - Manuel; Vihinen, Mauno; Wait , Robin; Wang, Xiaoyue; Wiemann, Stefan; Wu , Billy; Xu, Tao; Yates, John R.; Zhong, Jun; Zhou, Ming; Zhu, Yunping; Zurbig, Petra; Pandey, Akhilesh

    2008-02-01

    Proteomic technologies, such as yeast twohybrid, mass spectrometry (MS), protein/ peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories. Even more difficult is the annotation and display of pertinent information in the context of the corresponding proteins. Wikipedia, an online encyclopedia that anyone can edit, has already proven quite successful1 and can be used as a model for sharing biological data. However, the need for experimental evidence, data standardization and ownership of data creates scientific obstacles.

  9. Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

    PubMed

    Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

    2016-02-01

    Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. PMID:26673105

  10. Canola proteins for human consumption: extraction, profile, and functional properties.

    PubMed

    Tan, Siong H; Mailer, Rodney J; Blanchard, Christopher L; Agboola, Samson O

    2011-01-01

    Canola protein isolate has been suggested as an alternative to other proteins for human food use due to a balanced amino acid profile and potential functional properties such as emulsifying, foaming, and gelling abilities. This is, therefore, a review of the studies on the utilization of canola protein in human food, comprising the extraction processes for protein isolates and fractions, the molecular character of the extracted proteins, as well as their food functional properties. A majority of studies were based on proteins extracted from the meal using alkaline solution, presumably due to its high nitrogen yield, followed by those utilizing salt extraction combined with ultrafiltration. Characteristics of canola and its predecessor rapeseed protein fractions such as nitrogen yield, molecular weight profile, isoelectric point, solubility, and thermal properties have been reported and were found to be largely related to the extraction methods. However, very little research has been carried out on the hydrophobicity and structure profiles of the protein extracts that are highly relevant to a proper understanding of food functional properties. Alkaline extracts were generally not very suitable as functional ingredients and contradictory results about many of the measured properties of canola proteins, especially their emulsification tendencies, have also been documented. Further research into improved extraction methods is recommended, as is a more systematic approach to the measurement of desired food functional properties for valid comparison between studies.

  11. The Nucleocapsid Protein of Human Coronavirus NL63

    PubMed Central

    Zuwała, Kaja; Golda, Anna; Kabala, Wojciech; Burmistrz, Michał; Zdzalik, Michal; Nowak, Paulina; Kedracka-Krok, Sylwia; Zarebski, Mirosław; Dobrucki, Jerzy; Florek, Dominik; Zeglen, Sławomir; Wojarski, Jacek; Potempa, Jan; Dubin, Grzegorz; Pyrc, Krzysztof

    2015-01-01

    Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV. PMID:25700263

  12. The nucleocapsid protein of human coronavirus NL63.

    PubMed

    Zuwała, Kaja; Golda, Anna; Kabala, Wojciech; Burmistrz, Michał; Zdzalik, Michal; Nowak, Paulina; Kedracka-Krok, Sylwia; Zarebski, Mirosław; Dobrucki, Jerzy; Florek, Dominik; Zeglen, Sławomir; Wojarski, Jacek; Potempa, Jan; Dubin, Grzegorz; Pyrc, Krzysztof

    2015-01-01

    Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.

  13. Canola Proteins for Human Consumption: Extraction, Profile, and Functional Properties

    PubMed Central

    Tan, Siong H; Mailer, Rodney J; Blanchard, Christopher L; Agboola, Samson O

    2011-01-01

    Canola protein isolate has been suggested as an alternative to other proteins for human food use due to a balanced amino acid profile and potential functional properties such as emulsifying, foaming, and gelling abilities. This is, therefore, a review of the studies on the utilization of canola protein in human food, comprising the extraction processes for protein isolates and fractions, the molecular character of the extracted proteins, as well as their food functional properties. A majority of studies were based on proteins extracted from the meal using alkaline solution, presumably due to its high nitrogen yield, followed by those utilizing salt extraction combined with ultrafiltration. Characteristics of canola and its predecessor rapeseed protein fractions such as nitrogen yield, molecular weight profile, isoelectric point, solubility, and thermal properties have been reported and were found to be largely related to the extraction methods. However, very little research has been carried out on the hydrophobicity and structure profiles of the protein extracts that are highly relevant to a proper understanding of food functional properties. Alkaline extracts were generally not very suitable as functional ingredients and contradictory results about many of the measured properties of canola proteins, especially their emulsification tendencies, have also been documented. Further research into improved extraction methods is recommended, as is a more systematic approach to the measurement of desired food functional properties for valid comparison between studies. PMID:21535703

  14. Molecular interactions of graphene oxide with human blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Kenry, Affa Affb Affc; Loh, Kian Ping; Lim, Chwee Teck

    2016-04-01

    We investigate the molecular interactions between graphene oxide (GO) and human blood plasma proteins. To gain an insight into the bio-physico-chemical activity of GO in biological and biomedical applications, we performed a series of biophysical assays to quantify the molecular interactions between GO with different lateral size distributions and the three essential human blood plasma proteins. We elucidate the various aspects of the GO-protein interactions, particularly, the adsorption, binding kinetics and equilibrium, and conformational stability, through determination of quantitative parameters, such as GO-protein association constants, binding cooperativity, and the binding-driven protein structural changes. We demonstrate that the molecular interactions between GO and plasma proteins are significantly dependent on the lateral size distribution and mean lateral sizes of the GO nanosheets and their subtle variations may markedly influence the GO-protein interactions. Consequently, we propose the existence of size-dependent molecular interactions between GO nanosheets and plasma proteins, and importantly, the presence of specific critical mean lateral sizes of GO nanosheets in achieving very high association and fluorescence quenching efficiency of the plasma proteins. We anticipate that this work will provide a basis for the design of graphene-based and other related nanomaterials for a plethora of biological and biomedical applications.

  15. Antibody Response to Cryptococcus neoformans Proteins in Rodents and Humans

    PubMed Central

    Chen, Lin-Chi; Goldman, David L.; Doering, Tamara L.; Pirofski, Liise-anne; Casadevall, Arturo

    1999-01-01

    The prevalence and specificity of serum antibodies to Cryptococcus neoformans proteins was studied in mice and rats with experimental infection, in individuals with or without a history of potential laboratory exposure to C. neoformans, human immunodeficiency virus (HIV)-positive individuals who developed cryptococcosis, in matched samples from HIV-positive individuals who did not develop cryptococcosis, and in HIV-negative individuals. Rodents had little or no serum antibody reactive with C. neoformans proteins prior to infection. The intensity and specificity of the rodent antibody response were dependent on the species, the mouse strain, and the viability of the inoculum. All humans had serum antibodies reactive with C. neoformans proteins regardless of the potential exposure, the HIV infection status, or the subsequent development of cryptococcosis. Our results indicate (i) a high prevalence of antibodies reactive with C. neoformans proteins in the sera of rodents after cryptococcal infection and in humans with or without HIV infection; (ii) qualitative and quantitative differences in the antibody profiles of HIV-positive individuals; and (iii) similarities and differences between humans, mice, and rats with respect to the specificity of the antibodies reactive with C. neoformans proteins. The results are consistent with the view that C. neoformans infections are common in human populations, and the results have implications for the development of vaccination strategies against cryptococcosis. PMID:10225877

  16. ProNormz--an integrated approach for human proteins and protein kinases normalization.

    PubMed

    Subramani, Suresh; Raja, Kalpana; Natarajan, Jeyakumar

    2014-02-01

    The task of recognizing and normalizing protein name mentions in biomedical literature is a challenging task and important for text mining applications such as protein-protein interactions, pathway reconstruction and many more. In this paper, we present ProNormz, an integrated approach for human proteins (HPs) tagging and normalization. In Homo sapiens, a greater number of biological processes are regulated by a large human gene family called protein kinases by post translational phosphorylation. Recognition and normalization of human protein kinases (HPKs) is considered to be important for the extraction of the underlying information on its regulatory mechanism from biomedical literature. ProNormz distinguishes HPKs from other HPs besides tagging and normalization. To our knowledge, ProNormz is the first normalization system available to distinguish HPKs from other HPs in addition to gene normalization task. ProNormz incorporates a specialized synonyms dictionary for human proteins and protein kinases, a set of 15 string matching rules and a disambiguation module to achieve the normalization. Experimental results on benchmark BioCreative II training and test datasets show that our integrated approach achieve a fairly good performance and outperforms more sophisticated semantic similarity and disambiguation systems presented in BioCreative II GN task. As a freely available web tool, ProNormz is useful to developers as extensible gene normalization implementation, to researchers as a standard for comparing their innovative techniques, and to biologists for normalization and categorization of HPs and HPKs mentions in biomedical literature. URL: http://www.biominingbu.org/pronormz.

  17. [Proteins of human milk involved in immunological processes].

    PubMed

    Lis, Jolanta; Orczyk-Pawiłowicz, Magdalena; Kątnik-Prastowska, Iwona

    2013-05-31

    Human milk contains a lot of components (i.e. proteins, carbohydrates, lipids, inorganic elements) which provide basic nutrients for infants during the first period of their lives. Qualitative composition of milk components of healthy mothers is similar, but their levels change during lactation stages. Colostrum is the fluid secreted during the first days postpartum by mammary epithelial cells. Colostrum is replaced by transitional milk during 5-15 days postpartum and from 15 days postpartum mature milk is produced. Human milk, apart from nutritional components, is a source of biologically active molecules, i.e. immunoglobulins, growth factors, cytokines, acute phase proteins, antiviral and antibacterial proteins. Such components of human milk are responsible for specific biological activities of human milk. This secretion plays an important role in growth and development of newborns. Bioactive molecules present in the milk support the immature immune system of the newborn and also protect against the development of infection. In this article we describe the pathways involved in the production and secretion of human milk, the state of knowledge on the proteome of human milk, and the contents of components of milk during lactation. Moreover, some growth factors and proteins involved in innate and specific immunity, intercellular communication, immunomodulation, and inflammatory processes have been characterized.

  18. Crystal Structure of Human Retinoblastoma Binding Protein 9

    SciTech Connect

    Vorobiev, S.; Su, M; Seetharaman, J; Huang, Y; Chen, C; Maglaqui, M; Janjua, H; Montelione, G; Tong, L; et. al.

    2009-01-01

    As a step towards better integrating protein three-dimensional (3D) structural information in cancer systems biology, the Northeast Structural Genomics Consortium (NESG) (www.nesg.org) has constructed a Human Cancer Pathway Protein Interaction Network (HCPIN) by analysis of several classical cancer-associated signaling pathways and their physical protein-protein interactions. Many well-known cancer-associated proteins play central roles as hubs or bottlenecks in the HCPIN (http://nmr.cabm.rutgers.edu/hcpin). NESG has selected more than 1000 human proteins and protein domains from the HCPIN for sample production and 3D structure determination. The long-range goal of this effort is to provide a comprehensive 3D structure-function database for human cancer-associated proteins and protein complexes, in the context of their interaction networks. Human retinoblastoma binding protein 9 (RBBP9) is one of the HCPIN proteins targeted by NESG. RBBP9 was initially identified as the product of a new gene, Bog (for B5T over-expressed gene), in several transformed rat liver epithelial cell lines resistant to the growth-inhibitory effect of TGF-1 as well as in primary human liver tumors. RBBP9 contains the retinoblastoma (Rb) binding motif LxCxE in its sequence, and was shown to interact with Rb by yeast two-hybrid and coimmunoprecipitation experiments. Mutation of the Leu residue in this motif to Gln blocked the binding to Rb. RBBP9 can displace E2F1 from E2F1-Rb complexes, and over expression of RBBP9 overcomes TGF-1 induced growth arrest and results in transformation of rat liver epithelial cells leading to hepatoblastoma-like tumors in nude mice. RBBP9 may also play a role in cellular responses to chronic low dose radiation. A close homolog of RBBP9, sharing 93% amino acid sequence identity and also known as RBBP10, interacts with a protein with sua5-yciO-yrdC domains.

  19. Bacterial protein toxins in human cancers.

    PubMed

    Rosadi, Francesca; Fiorentini, Carla; Fabbri, Alessia

    2016-02-01

    Many bacteria causing persistent infections produce toxins whose mechanisms of action indicate that they could have a role in carcinogenesis. Some toxins, like CDT and colibactin, directly attack the genome by damaging DNA whereas others, as for example CNF1, CagA and BFT, impinge on key eukaryotic processes, such as cellular signalling and cell death. These bacterial toxins, together with other less known toxins, mimic carcinogens and tumour promoters. The aim of this review is to fulfil an up-to-date analysis of toxins with carcinogenic potential that have been already correlated to human cancers. Bacterial toxins-induced carcinogenesis represents an emerging aspect in bacteriology, and its significance is increasingly recognized.

  20. Ribosomal protein gene mapping and human chromosomal disorders

    SciTech Connect

    Kenmochi, N.; Goodman, N.; Page, D.C.

    1994-09-01

    In Drosophila, the Minute phenotype (reduced body size, diminished viability and fertility, and short, thin bristles) results from heterozygous deficiencies (deletions) at any one of 50 loci scattered about the genome. A handful of these Minute loci have been molecularly characterized, and all have been found to encode ribosomal proteins. Thus, the Minute phenotype appears to result from reduced protein synthetic capacity in flies with one rather than two copies of a given ribosomal protein (rp) gene. We are pursuing the possibility that similar reductions in protein synthetic capacity--again resulting from rp gene deficiencies--might underlie phenotypes associated with certain chromosomal disorders in humans. We and our colleagues have reported findings consistent with a role for RPS4 deficiency in the etiology of certain features of Turner syndrome, a complex human disorder classically associated with an XO karyotype. We are intrigued by the possibility that deficiencies of other human rp genes might cause phenotypic abnormalities similar to those seen in Turner syndrome--just as deficiencies of any of a number of Drosophila rp genes cause the Minute phenotype. We must first learn the chromosomal map position of each of the estimated 83 human rp genes. The task of mapping the functional (intron-containing) rp genes is complicated by the existence of processed pseudogenes elsewhere in the genome. To date, we have assigned (or confirmed the previous assignment of) 38 rp genes to individual human chromosomes by PCR analysis of human-rodent somatic cell hybrids containing subsets of human chromosomes, with all but four chromosomes carrying at least one rp gene. We have also identified more than 100 large-insert human YAC (yeast artificial chromosome) clones that contain individual rp genes. Such screening of YAC libraries will result in precise positioning of the rp genes on the emerging physical map of the human genome.

  1. Exploiting Bacterial Operons To Illuminate Human Iron-Sulfur Proteins.

    PubMed

    Andreini, Claudia; Banci, Lucia; Rosato, Antonio

    2016-04-01

    Organisms from all kingdoms of life use iron-sulfur proteins (FeS-Ps) in a multitude of functional processes. We applied a bioinformatics approach to investigate the human portfolio of FeS-Ps. Sixty-one percent of human FeS-Ps bind Fe4S4 clusters, whereas 39% bind Fe2S2 clusters. However, this relative ratio varies significantly depending on the specific cellular compartment. We compared the portfolio of human FeS-Ps to 12 other eukaryotes and to about 700 prokaryotes. The comparative analysis of the organization of the prokaryotic homologues of human FeS-Ps within operons allowed us to reconstruct the human functional networks involving the conserved FeS-Ps common to prokaryotes and eukaryotes. These functional networks have been maintained during evolution and thus presumably represent fundamental cellular processes. The respiratory chain and the ISC machinery for FeS-P biogenesis are the two conserved processes that involve the majority of human FeS-Ps. Purine metabolism is another process including several FeS-Ps, in which BOLA proteins possibly have a regulatory role. The analysis of the co-occurrence of human FeS-Ps with other proteins highlighted numerous links between the iron-sulfur cluster machinery and the response mechanisms to cell damage, from repair to apoptosis. This relationship probably relates to the production of reactive oxygen species within the biogenesis and degradation of FeS-Ps.

  2. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

    PubMed Central

    Yu, Xiaobo; LaBaer, Joshua

    2015-01-01

    Summary AMPylation (adenylylation) has been recognized as an important post translational modification employed by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes and is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method to identify new substrates using protein microarrays, which can significantly expand the list of potential substrates. Here, we describe procedures to detect AMPylated and auto-AMPylated proteins in a sensitive, high throughput, and non-radioactive manner. The approach employs high-density protein microarrays fabricated using NAPPA (Nucleic Acid Programmable Protein Arrays) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide–alkyne cycloaddition. The assay can be accomplished within 11 hours. PMID:25881200

  3. Structural studies of human glioma pathogenesis-related protein 1

    SciTech Connect

    Asojo, Oluwatoyin A.; Koski, Raymond A.; Bonafé, Nathalie

    2011-10-01

    Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

  4. Human muscle proteins: analysis by two-dimensional electrophoresis

    SciTech Connect

    Giometti, C.S.; Danon, M.J.; Anderson, N.G.

    1983-09-01

    Proteins from single frozen sections of human muscle were separated by two-dimensional gel electrophoresis and detected by fluorography or Coomassie Blue staining. The major proteins were identical in different normal muscles obtained from either sex at different ages, and in Duchenne and myotonic dystrophy samples. Congenital myopathy denervation atrophy, polymyositis, and Becker's muscular dystrophy samples, however, showed abnormal myosin light chain compositions, some with a decrease of fast-fiber myosin light chains and others with a decrease of slow-fiber light chains. These protein alterations did not correlate with any specific disease, and may be cause by generalized muscle-fiber damage.

  5. ABC proteins protect the human body and maintain optimal health.

    PubMed

    Ueda, Kazumitsu

    2011-01-01

    Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

  6. A rapid method of reconstituting human erythrocyte sugar transport proteins.

    PubMed

    Carruthers, A; Melchior, D L

    1984-06-01

    A rapid reconstitution procedure for human erythrocyte hexose transfer activity is described. The procedure (reverse-phase evaporation) avoids exposure of the isolated proteins to detergent, organic solvent, sonication, or freeze-thaw steps during insertion into synthetic membranes and may be effected within 15 min. The so-formed vesicles are unilamellar structures with a large encapsulated volume, narrow size range, and low passive permeabilities. Contamination by carry-through of endogenous (red cell) lipids is less than 1%. Reconstituted hexose transfer activity was examined by using unfractionated proteins (bands 3, 4.5, and 6) and purified proteins (bands 4.5 and 3). With unfractionated proteins, hexose transport activity is low [0.34 mumol X (mg of protein)-1 X min-1], is inhibited by cytochalasin B, and increases monotonically with protein concentration. Kinetic analysis indicates that Vmax values for both influx and efflux of D-glucose are identical. Reconstitution of the cytochalasin B binding protein (band 4.5) results in hexose transport with high specific activity [5 mumol X (mg of protein)-1 X min-1] and symmetry in transfer kinetics. Band 3 proteins also appear to mediate cytochalasin B sensitive D-glucose transport activity.

  7. [Chemistry and clinical significance of human plasma proteins].

    PubMed

    Haupt, H

    1990-10-01

    Enormous progress has been made in the course of the past few years in the various fields of plasma protein research. The primary and disulfide bridge structures are now known for almost all of the 120 proteins thus far isolated from human plasma, including trace and ultratrace proteins as well as a number of genetic variants. Genetic cloning and the derivation of the amino-acid sequence from the nucleotide sequence have played a decisive role here. However, we are only in possession of the exact three-dimensional structure for a small number of plasma proteins. The major problem in this respect is, at present, the lack of suitable protein crystals for X-ray structure analysis. We still do not know the physiological function of a large number of plasma proteins, despite the fact that they, in part, have been well characterised both physically and chemically and could be assigned to their respective protein families on the basis of their amino-acid sequence. The development of techniques for protein structure determination is relatively well advanced today, yet we lack methods of illuminating the structure-function relationship. There are more than 20 different highly purified protein preparations in virus-safe form available today for substitution therapy. To this effect new purification procedures have been developed which pay particular attention to virus elimination and inactivation. Should present indications be confirmed, one may assume that further plasma proteins (e. g. proteinase inhibitors, apolipoproteins, fibronectin) could be of significance in therapy and prophylaxis. Unlimited amounts of human blood are not available. Gene technology offers a promising alternative, at least for the production of plasma protein administered to patients in small amounts. Work is being done intensively on various blood coagulation factors and proteinase inhibitors at the moment, and factor VIII: C is already being successfully used for the treatment of patients with

  8. Evaluation of silica nanoparticle binding to major human blood proteins

    NASA Astrophysics Data System (ADS)

    Hata, Katsutomo; Higashisaka, Kazuma; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Yoshioka, Yasuo; Tsutsumi, Yasuo

    2014-12-01

    Nanomaterials are used for various biomedical applications because they are often more effective than conventional materials. Recently, however, it has become clear that the protein corona that forms on the surface of nanomaterials when they make contact with biological fluids, such as blood, influences the pharmacokinetics and biological responses induced by the nanomaterials. Therefore, when evaluating nanomaterial safety and efficacy, it is important to analyze the interaction between nanomaterials and proteins in biological fluids and to evaluate the effects of the protein corona. Here, we evaluated the interaction of silica nanoparticles, a commonly used nanomaterial, with the human blood proteins albumin, transferrin, fibrinogen, and IgG. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the amount of albumin, transferrin, and IgG binding to the silica particles increased as the particle size decreased under conditions where the silica particle mass remained the same. However, under conditions in which the specific surface area remained constant, there were no differences in the binding of human plasma proteins to the silica particles tested, suggesting that the binding of silica particles with human plasma proteins is dependent on the specific surface area of the silica particles. Furthermore, the amount of albumin, transferrin, and IgG binding to silica nanoparticles with a diameter of 70 nm (nSP70) and a functional amino group was lower than that with unmodified nSP70, although there was no difference in the binding between nSP70 with the surface modification of a carboxyl functional group and nSP70. These results suggest that the characteristics of nanomaterials are important for binding with human blood proteins; this information may contribute to the development of safe and effective nanomaterials.

  9. Improving the Understanding of Pathogenesis of Human Papillomavirus 16 via Mapping Protein-Protein Interaction Network

    PubMed Central

    Dong, Yongcheng; Kuang, Qifan; Dai, Xu; Li, Rong; Wu, Yiming; Leng, Weijia; Li, Yizhou; Li, Menglong

    2015-01-01

    The human papillomavirus 16 (HPV16) has high risk to lead various cancers and afflictions, especially, the cervical cancer. Therefore, investigating the pathogenesis of HPV16 is very important for public health. Protein-protein interaction (PPI) network between HPV16 and human was used as a measure to improve our understanding of its pathogenesis. By adopting sequence and topological features, a support vector machine (SVM) model was built to predict new interactions between HPV16 and human proteins. All interactions were comprehensively investigated and analyzed. The analysis indicated that HPV16 enlarged its scope of influence by interacting with human proteins as much as possible. These interactions alter a broad array of cell cycle progression. Furthermore, not only was HPV16 highly prone to interact with hub proteins and bottleneck proteins, but also it could effectively affect a breadth of signaling pathways. In addition, we found that the HPV16 evolved into high carcinogenicity on the condition that its own reproduction had been ensured. Meanwhile, this work will contribute to providing potential new targets for antiviral therapeutics and help experimental research in the future. PMID:25961044

  10. Human TAFII31 protein is a transcriptional coactivator of the p53 protein.

    PubMed Central

    Lu, H; Levine, A J

    1995-01-01

    The p53 protein activates transcription of a target gene by binding to a specific DNA response element and interacting with the transcriptional apparatus of RNA polymerase II. The amino-terminal domain of p53 interacts with a component of the TFIID basal transcription complex. The human TATA-binding-protein-associated factor TAFII31, a component of TFIID, has been identified as a critical protein required for p53-mediated transcriptional activation. TAFII31 and p53 proteins bind to each other via amino acid residues in the amino-terminal domain of p53 that are essential for transcription. Antibodies directed against TAFII31 protein inhibit p53-activated but not basal transcription in vitro. These results demonstrate that TAFII31 is a coactivator for the p53 protein. Images Fig. 3 Fig. 4 Fig. 6 PMID:7761466

  11. A visual intracellular classification strategy for uncharacterized human proteins.

    PubMed

    Hoja, M R; Wahlestedt, C; Höög, C

    2000-08-25

    The human cDNA and genomic sequencing projects will result in the identification and isolation of some 140,000 genes, the majority of which lack predicted functions and for which the cellular localizations are not known. The identification and characterization of protein components of specific cell structures and machineries are essential steps not only toward defining functions of genes but also toward understanding cell function and regulation. We describe here a new approach, termed PROLOC, which uses full-length cDNAs for systematic classification of novel proteins as a functional pointer. We have PCR-amplified 25 uncharacterized human genes and expressed the encoded proteins as GFP fusions in a human cell line. This pilot project has identified novel proteins associated with the nucleolus, mitochondria, the ER, the ER-Golgi-intermediate compartment (ERGIC), the GC, the plasma membrane, and cytoplasmic foci. This visual classification approach may be scaled up to handle a large number of novel genes and permit the generation of a global cellular protein localization map. Such information should be valuable for many aspects of functional genomics and cell biology. PMID:10942595

  12. Expression cloning of genes encoding human peroxisomal proteins

    SciTech Connect

    Spathaky, J.M.; Tate, A.W.; Cox, T.M.

    1994-09-01

    Numerous metabolic disorders associated with diverse peroxisomal defects have been identified but their molecular characterization has been hampered by difficulties associated with the purification of proteins from this fragile organelle. We have utilized antibodies directed against the C-terminal tripeptide peroxisomal targeting signal to detect hitherto unknown peroxisomal proteins in tissue fractions and to isolate genes encoding peroxisonal proteins from human expression libraries. We immunized rabbits with a peptide conjugate encompassing the C-terminal nine amino acids of rat peroxisomal acyl CoA oxidase. Immunoprecipitation assays using radio-labelled peptide showed that the antibody specifically recognizes the terminal SKL motif as well as C-terminal SHL and SRL but not SHL at an internal position. Affinity-purified antibody was used to probe Western blots of crude and peroxisome-enriched monkey liver preparations and detected 8-10 proteins specifically in the peroxisome fractions. 100 positive clones were identified on screening a human liver cDNA expression library in {lambda}-gt11. Sequence analysis has confirmed the identity of cDNA clones for human acyl CoA oxidase and epoxide hydrolase. Four clones show no sequence identity and their putative role in the human peroxisome is being explored.

  13. Efficient expression and purification of biologically active human cystatin proteins.

    PubMed

    Chauhan, Sakshi; Tomar, Raghuvir S

    2016-02-01

    Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.

  14. An extensive library of surrogate peptides for all human proteins.

    PubMed

    Mohammed, Yassene; Borchers, Christoph H

    2015-11-01

    Selecting the most appropriate surrogate peptides to represent a target protein is a major component of experimental design in Multiple Reaction Monitoring (MRM). Our software PeptidePicker with its v-score remains distinctive in its approach of integrating information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM that is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our "best knowledge" for selecting candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it has previously been observed. Here we present an updated approach where we have already compiled a list of all possible surrogate peptides of the human proteome. Using our stringent selection criteria, the list includes 165k suitable MRM peptides covering 17k proteins of the human reviewed proteins in UniProtKB. Compared to average of 2-4min per protein for retrieving and integrating the information, the precompiled list includes all peptides available instantly. This allows a more cohesive and faster design of a multiplexed MRM experiment and provides insights into evidence for a protein's existence. We will keep this list up-to-date as proteomics data repositories continue to grow. This article is part of a Special Issue entitled: Computational Proteomics. PMID:26232110

  15. Nuclear and nucleolar targeting of human ribosomal protein S6.

    PubMed Central

    Schmidt, C; Lipsius, E; Kruppa, J

    1995-01-01

    Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6. Images PMID:8590812

  16. Annexin A6 protein is downregulated in human hepatocellular carcinoma.

    PubMed

    Meier, Elisabeth M; Rein-Fischboeck, Lisa; Pohl, Rebekka; Wanninger, Josef; Hoy, Andrew J; Grewal, Thomas; Eisinger, Kristina; Krautbauer, Sabrina; Liebisch, Gerhard; Weiss, Thomas S; Buechler, Christa

    2016-07-01

    Annexin A6 (AnxA6) is a lipid-binding protein highly expressed in the liver, regulating cholesterol homeostasis and signaling pathways with a role in liver physiology. Here, we analyzed whether hepatic AnxA6 levels are affected by pathological conditions that are associated with liver dysfunction and liver injury. AnxA6 levels in the fatty liver of mice fed a high-fat diet, in ob/ob and db/db animals and in human fatty liver are comparable to controls. Similarly, AnxA6 levels appear unaffected in murine nonalcoholic steatohepatitis and human liver fibrosis. Accordingly, adiponectin, lysophosphatidylcholine, palmitate, and TGFbeta, all of which have a role in liver injury, do not affect AnxA6 expression in human hepatocytes. Likewise, adiponectin and IL8 do not alter AnxA6 levels in primary human hepatic stellate cells. However, in hepatic tumors of 18 patients, AnxA6 protein levels are substantially reduced compared to nontumorous tissues. AnxA6 mRNA is even increased in the tumors suggesting that posttranscriptional mechanisms are involved herein. Lipidomic analysis shows trends toward elevated cholesteryl ester and sphingomyelin in the tumor samples, yet the ratio of tumor to nontumorous AnxA6 does not correlate with these lipids. The current study shows that AnxA6 is specifically reduced in human hepatocellular carcinoma suggesting a role of this protein in hepatocarcinogenesis. PMID:27334756

  17. Expression and Localization of Lung Surfactant Proteins in Human Testis

    PubMed Central

    Wagner, Walter; Matthies, Cord; Ruf, Christian; Hartmann, Arndt; Garreis, Fabian; Paulsen, Friedrich

    2015-01-01

    Background Surfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions. Objective The aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated. Results Surfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig. Conclusion Surfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP's was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor. PMID:26599233

  18. Antibody humanization by structure-based computational protein design

    PubMed Central

    Choi, Yoonjoo; Hua, Casey; Sentman, Charles L; Ackerman, Margaret E; Bailey-Kellogg, Chris

    2015-01-01

    Antibodies derived from non-human sources must be modified for therapeutic use so as to mitigate undesirable immune responses. While complementarity-determining region (CDR) grafting-based humanization techniques have been successfully applied in many cases, it remains challenging to maintain the desired stability and antigen binding affinity upon grafting. We developed an alternative humanization approach called CoDAH (“Computationally-Driven Antibody Humanization”) in which computational protein design methods directly select sets of amino acids to incorporate from human germline sequences to increase humanness while maintaining structural stability. Retrospective studies show that CoDAH is able to identify variants deemed beneficial according to both humanness and structural stability criteria, even for targets lacking crystal structures. Prospective application to TZ47, a murine anti-human B7H6 antibody, demonstrates the approach. Four diverse humanized variants were designed, and all possible unique VH/VL combinations were produced as full-length IgG1 antibodies. Soluble and cell surface expressed antigen binding assays showed that 75% (6 of 8) of the computationally designed VH/VL variants were successfully expressed and competed with the murine TZ47 for binding to B7H6 antigen. Furthermore, 4 of the 6 bound with an estimated KD within an order of magnitude of the original TZ47 antibody. In contrast, a traditional CDR-grafted variant could not be expressed. These results suggest that the computational protein design approach described here can be used to efficiently generate functional humanized antibodies and provide humanized templates for further affinity maturation. PMID:26252731

  19. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    SciTech Connect

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  20. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  1. Protein Stability, Folding and Misfolding in Human PGK1 Deficiency.

    PubMed

    Valentini, Giovanna; Maggi, Maristella; Pey, Angel L

    2013-01-01

    Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein folding and misfolding. Expression analyses and in vitro biophysical studies indicate that disease-causing mutations enhance protein aggregation propensity. We found a strong correlation among protein aggregation propensity, thermodynamic stability, cooperativity and dynamics. Comparison of folding and unfolding properties with previous reports in PGKs from other species suggests that hPGK1 is very sensitive to mutations leading to enhance protein aggregation through changes in protein folding cooperativity and the structure of the relevant denaturation transition state for aggregation. Overall, we provide a mechanistic framework for protein misfolding of hPGK1, which is insightful to develop new therapeutic strategies aimed to target native state stability and foldability in hPGK1 deficient patients. PMID:24970202

  2. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.

    PubMed

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-04-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  3. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

    PubMed Central

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-01-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  4. Identification of lectin binding proteins in human tears.

    PubMed

    Kuizenga, A; van Haeringen, N J; Kijlstra, A

    1991-12-01

    The identity of glycoproteins in stimulated normal human tears was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of tears onto minigels, blotting, and subsequent incubation with different biotinylated lectins (concanavalin A [Con A], peanut agglutinin [PNA], glycine max agglutinin [SBA], Phaseolus vulgaris agglutinin, wheat germ agglutinin [WGA, native form], Artocarpus integrifolia agglutinin [Jacalin], and Pisum sativum agglutinin). Control proteins included purified secretory immunoglobulin A (sIgA) from human colostrum, human milk lactoferrin, and chicken-egg lysozyme. All samples were prepared in a denaturing (SDS) buffer under nonreducing and reducing conditions. The sIgA in tears and IgA (alpha) heavy chain fragments (reduced sample) were identified with most of the lectins tested. A particular high molecular weight (greater than 200 kD) protein fraction in tears that just entered the separation gel on SDS-PAGE was detected with WGA and Jacalin. This fraction stain poorly with silver. Tear lactoferrin was identified with all lectins used, although binding was low with SBA. Purified milk lactoferrin showed a poor reaction with Jacalin, but a protein in tears of similar mobility bound this lectin (nonreduced samples). Under both nonreducing and reducing conditions, tear-specific prealbumin in tears did not bind any of the lectins tested. Tear lysozyme only reacted with lectin after reduction. The techniques described may provide additional valuable information in addition to commonly used methods for tear protein analysis and further knowledge concerning the role of glycoproteins on the ocular surface.

  5. Identification of the major capsid protein gene of human cytomegalovirus.

    PubMed Central

    Chee, M; Rudolph, S A; Plachter, B; Barrell, B; Jahn, G

    1989-01-01

    The coding region for the major capsid protein (MCP) of human cytomegalovirus (HCMV) was identified by comparing the protein sequence with the respective sequences of herpes simplex virus (HSV), Epstein-Barr virus, and varicella-zoster virus. The predicted length of the HCMV MCP was 1,370 amino acids. Comparison of the MCP sequences of the different human herpesviruses showed a homology of 25% to the MCP of HSV type 1, a homology of 29% to the MCP of Epstein-Barr virus, and a homology of 23% to the MCP of varicella-zoster virus. A subfragment of the HSV type 1 KpnI i fragment encoding the MCP VP5 cross-hybridized with the HCMV HindIII U fragment containing part of the MCP gene. Northern (RNA) blot analyses with subclones out of the coding region for the HCMV MCP detected one large transcript of about 8 kilobases. A portion of the open reading frame was expressed in Escherichia coli plasmid pBD2 IC2OH as a beta-galactosidase fusion protein and was used to generate polyclonal antibodies in New Zealand White rabbits. The obtained antisera reacted in Western immunoblots with the MCP of purified HCMV virions. A monoclonal antibody against the human MCP and a monospecific rabbit antiserum against strain Colburn of simian cytomegalovirus detected the fusion protein as well as the MCP of purified virions in immunoblots. Images PMID:2536837

  6. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    NASA Astrophysics Data System (ADS)

    Laulumaa, Saara; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  7. The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase.

    PubMed

    Francis, Kevin; Sapienza, Paul J; Lee, Andrew L; Kohen, Amnon

    2016-02-23

    Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H → C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs), which are sensitive to the physical nature of the chemical step, and protein mass modulation, which slows down fast dynamics (femto- to picosecond time scale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5 to 45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction's transition state (or tunneling ready state, TRS). Mass modulation of these enzymes through isotopic labeling with (13)C, (15)N, and (2)H at nonexchangeable hydrogens yields an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass modulation of the human DHFR affects neither DAD distribution nor the DAD's conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H → C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically modulated heavy enzymes in general.

  8. The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase.

    PubMed

    Francis, Kevin; Sapienza, Paul J; Lee, Andrew L; Kohen, Amnon

    2016-02-23

    Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H → C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs), which are sensitive to the physical nature of the chemical step, and protein mass modulation, which slows down fast dynamics (femto- to picosecond time scale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5 to 45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction's transition state (or tunneling ready state, TRS). Mass modulation of these enzymes through isotopic labeling with (13)C, (15)N, and (2)H at nonexchangeable hydrogens yields an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass modulation of the human DHFR affects neither DAD distribution nor the DAD's conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H → C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically modulated heavy enzymes in general. PMID:26813442

  9. INCAP studies of vegetable proteins for human consumption.

    PubMed

    Bressani, Ricardo

    2010-03-01

    This article describes the efforts of the Institute of Nutrition of Central America and Panama (INCAP) to develop a relatively low-cost vegetable protein mixture suitable as a complementary food for infants and young children. As it turned out, the resulting product became popular with older children and adults, and its superior nutritional benefits were widely recognized by the population. This effort led to broader studies by INCAP of the nutritional quality of vegetable protein mixtures, including raw materials, processing to convert them into human-grade products, product formulation, and commercialization. PMID:20461907

  10. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    SciTech Connect

    Blackburn, W.D.; Tallant, E.A.; Wallace, R.W.

    1986-05-01

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca/sup 2 +/. One mechanism by which Ca/sup 2 +/ may trigger neutrophil activation is through Ca/sup 2 +//calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca/sup 2 +//CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and /sup 125/I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound /sup 125/I-CaM in a Ca/sup 2 +/-dependent manner. One predominant region of /sup 125/I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca/sup 2 +/-dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 ..mu..g/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca/sup 2 +/-dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca/sup 2 +//calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase.

  11. Solution structure of the human signaling protein RACK1

    PubMed Central

    2010-01-01

    Background The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 106 M-1 and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10-6 M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions. PMID:20529362

  12. Specificity of botulinum protease for human VAMP family proteins.

    PubMed

    Yamamoto, Hideyuki; Ida, Tomoaki; Tsutsuki, Hiroyasu; Mori, Masatoshi; Matsumoto, Tomoko; Kohda, Tomoko; Mukamoto, Masafumi; Goshima, Naoki; Kozaki, Shunji; Ihara, Hideshi

    2012-04-01

    The botulinum neurotoxin light chain (BoNT-LC) is a zinc-dependent metalloprotease that cleaves neuronal SNARE proteins such as SNAP-25, VAMP2, and Syntaxin1. This cleavage interferes with the neurotransmitter release of peripheral neurons and results in flaccid paralysis. SNAP, VAMP, and Syntaxin are representative of large families of proteins that mediate most membrane fusion reactions, as well as both neuronal and non-neuronal exocytotic events in eukaryotic cells. Neuron-specific SNARE proteins, which are target substrates of BoNT, have been well studied; however, it is unclear whether other SNARE proteins are also proteolyzed by BoNT. Herein, we define the substrate specificity of BoNT-LC/B, /D, and /F towards recombinant human VAMP family proteins. We demonstrate that LC/B, /D, and /F are able to cleave VAMP1, 2, and 3, but no other VAMP family proteins. Kinetic analysis revealed that all LC have higher affinity and catalytic activity for the non-neuronal SNARE isoform VAMP3 than for the neuronal VAMP1 and 2 isoforms. LC/D in particular exhibited extremely low catalytic activity towards VAMP1 relative to other interactions, which we determined through point mutation analysis to be a result of the Ile present at residue 48 of VAMP1. We also identified the VAMP3 cleavage sites to be at the Gln 59-Phe 60 (LC/B), Lys 42-Leu 43 (LC/D), and Gln 41-Lys 42 (LC/F) peptide bonds, which correspond to those of VAMP1 or 2. Understanding the substrate specificity and kinetic characteristics of BoNT towards human SNARE proteins may aid in the development of novel therapeutic uses for BoNT.

  13. Characterization of Inhibitor of differentiation (Id) proteins in human cornea.

    PubMed

    Mohan, Rajiv R; Morgan, Brandie R; Anumanthan, Govindaraj; Sharma, Ajay; Chaurasia, Shyam S; Rieger, Frank G

    2016-05-01

    Inhibitor of differentiation (Id) proteins are DNA-binding transcription factors involved in cellular proliferation, migration, inflammation, angiogenesis and fibrosis. However, their expression and role in the cornea is unknown. The present study was undertaken to characterize the expression of Id proteins and their interactions with the pro-fibrotic cytokine Transforming Growth Factor β1 (TGFβ1) and anti-fibrotic cytokine, bone morphogenic protein 7 (BMP7) in human cornea. Human donor corneas procured from Eye Bank were used. Id proteins were localized in human corneal sections using immunofluorescence. Primary cultures of human corneal fibroblasts (HCF) were established and treated with either TGFβ1 (5 ng/ml) or BMP7 (10 ng/ml) for 24 h in serum free medium. Expression of Id's in response to TGFβ1, BMP7 and TGFβ1 + BMP7 was analyzed by quantitative real time PCR (qRT-PCR) and western blot analysis. Id1 and Id2 proteins were ubiquitously expressed in the epithelial cells and stromal keratocytes in human cornea. The Id1 was localized to the basal epithelial cells as seen by immunohistochemistry. HCF expressed all known mammalian Id genes (Id1-Id4). In addition, Id1 and Id2 are selectively expressed in HCF. Treatment of human recombinant TGFβ1 (5 ng/ml) to serum-starved HCF showed a significant increase in Id genes (Id1, Id2 and Id4) at 2 h time point compared to BMP7 treatment, which showed time dependent increase in the expression of Id1-Id3 at 24-48 h. Combined treatment with TGFβ1 + BMP7 to HCF showed a significant increase in Id1 transcript and an increasing trend in Id3 and Id4 expression. The results of this study suggest that Id family of genes (Id1-Id4) are localized in the human cornea and expressed in the corneal fibroblasts. Also, Id's were differentially regulated with TGFβ1 and/or BMP7 in a time dependent manner and might serve as a therapeutic target in corneal fibrosis.

  14. Transposable elements have contributed to thousands of human proteins.

    PubMed

    Britten, Roy

    2006-02-01

    This is a report of many distant but significant protein sequence relationships between human proteins and transposable elements (TEs). The libraries of human repeated sequences contain the DNA sequences of many TEs. These were translated in all reading frames, ignoring stop codons, and were used as amino acid sequence probes to search with BLASTP for similar sequences in a library of 25,193 human proteins. The probes show regions of significant amino acid sequence similarity to 1,950 different human genes, with an expectation of <10(-3). In comparison with previous REPEATMASKER (Institute for Systems Biology, Seattle) studies, these probes detect many more TE sequences in more human coding sequences with greater length than previous work using DNA sequences. If the criterion is opened, very many matches are found occurring on 4,653 different genes after correction for the number seen with random amino acid sequence probes. The processes that led to these extensive sets of sequence relationships between TEs and coding sequences of human genes have been a major source of variation and novel genes during evolution. This paper lists the number of sequence similarities seen by amino acid sequence comparison, which is surely an underestimate of the actual number of significant relationships. It appears that many of these are the result of past events of duplication of genes or gene regions, rather than a direct result of TE insertion. This report of observable relationships leaves to the future the functional implications as well as the detection of the events of TE insertion. PMID:16443682

  15. HPMV: human protein mutation viewer - relating sequence mutations to protein sequence architecture and function changes.

    PubMed

    Sherman, Westley Arthur; Kuchibhatla, Durga Bhavani; Limviphuvadh, Vachiranee; Maurer-Stroh, Sebastian; Eisenhaber, Birgit; Eisenhaber, Frank

    2015-10-01

    Next-generation sequencing advances are rapidly expanding the number of human mutations to be analyzed for causative roles in genetic disorders. Our Human Protein Mutation Viewer (HPMV) is intended to explore the biomolecular mechanistic significance of non-synonymous human mutations in protein-coding genomic regions. The tool helps to assess whether protein mutations affect the occurrence of sequence-architectural features (globular domains, targeting signals, post-translational modification sites, etc.). As input, HPMV accepts protein mutations - as UniProt accessions with mutations (e.g. HGVS nomenclature), genome coordinates, or FASTA sequences. As output, HPMV provides an interactive cartoon showing the mutations in relation to elements of the sequence architecture. A large variety of protein sequence architectural features were selected for their particular relevance to mutation interpretation. Clicking a sequence feature in the cartoon expands a tree view of additional information including multiple sequence alignments of conserved domains and a simple 3D viewer mapping the mutation to known PDB structures, if available. The cartoon is also correlated with a multiple sequence alignment of similar sequences from other organisms. In cases where a mutation is likely to have a straightforward interpretation (e.g. a point mutation disrupting a well-understood targeting signal), this interpretation is suggested. The interactive cartoon can be downloaded as standalone viewer in Java jar format to be saved and viewed later with only a standard Java runtime environment. The HPMV website is: http://hpmv.bii.a-star.edu.sg/ .

  16. Cellular Localization of Engineered Human LINE-1 RNA and Proteins.

    PubMed

    Doucet, Aurélien J; Basyuk, Eugénia; Gilbert, Nicolas

    2016-01-01

    The human LINE-1 retrotransposon has the ability to mobilize into a new genomic location through an intracellular replication cycle. Immunofluorescence and in situ hybridization experiments have been developed to detect subcellular localization of retrotransposition intermediates (i.e., ORF1p, ORF2p, and L1 mRNA). Currently, these protocols are also used to validate the interaction between retrotransposition complex components and potential cellular partners involved in L1 replication. Here, we describe in details methods for the identification of LINE-1 proteins and/or RNA in cells transfected with vectors expressing engineered human LINE-1 elements. PMID:26895060

  17. Protein and amino acid metabolism in the human newborn.

    PubMed

    Kalhan, Satish C; Bier, Dennis M

    2008-01-01

    Birth and adaptation to extrauterine life involve major shifts in the protein and energy metabolism of the human newborn. These include a shift from a state of continuous supply of nutrients including amino acids from the mother to cyclic periodic oral intake, a change in the redox state of organs, thermogenesis, and a significant change in the mobilization and use of oxidative substrates. The development of safe, stable isotopic tracer methods has allowed the study of protein and amino acid metabolism not only in the healthy newborn but also in those born prematurely and of low birth weight. These studies have identified the unique and quantitative aspects of amino acid/protein metabolism in the neonate, thus contributing to rational nutritional care of these babies. The present review summarizes the contemporary data on some of the significant developments in essential and dispensable amino acids and their relationship to overall protein metabolism. Specifically, the recent data of kinetics of leucine, phenylalanine, glutamine, sulfur amino acid, and threonine and their relation to whole-body protein turnover are presented. Finally, the physiological rationale and the impact of nutrient (amino acids) interventions on the dynamics of protein metabolism are discussed.

  18. Effect of protein level and protein source on zinc absorption in humans

    SciTech Connect

    Sandstroem, B.A.; Almgren, A.; Kivistoe, B.C.; Cederblad, A.

    1989-01-01

    The effect of increasing levels of various protein sources on zinc absorption from a legume-based meal was studied in humans with the use of a radionuclide technique. The meals were extrinsically labelled with 65Zn and absorption was determined from measurements of the whole-body retention of the isotope. The mean fractional zinc absorption for the 13 meals was 24.7 +/- 6.9% and was only influenced by the protein content of the meal to a limited extent (r = 0.45). However, the amount of zinc absorbed from the meals was strongly correlated with both the protein (r = 0.85) and zinc content (r = 0.86): 5.9 +/- 1.7 mumol of zinc was absorbed from the basal bean meal which had the lowest protein content; the addition of low zinc chicken doubled the protein content and increased zinc absorption to 10.3 +/- 2.0 mumol; the addition of zinc-rich beef also doubled the protein content, however, zinc absorption was increased to 15.9 +/- 4.7 mumol. It is concluded that the zinc content of the main protein source of the diet determines the amount of zinc absorbed to a large extent. However, relatively small amounts of animal protein can significantly improve the value of a legume-based meal as a source of zinc.

  19. Protein carbonylation and heat shock proteins in human skeletal muscle: relationships to age and sarcopenia.

    PubMed

    Beltran Valls, Maria R; Wilkinson, Daniel J; Narici, Marco V; Smith, Kenneth; Phillips, Bethan E; Caporossi, Daniela; Atherton, Philip J

    2015-02-01

    Aging is associated with a gradual loss of muscle mass termed sarcopenia, which has significant impact on quality-of-life. Because oxidative stress is proposed to negatively impact upon musculoskeletal aging, we investigated links between human aging and markers of oxidative stress, and relationships to muscle mass and strength in young and old nonsarcopenic and sarcopenic adults. Sixteen young and 16 old males (further subdivided into "old" and "old sarcopenic") were studied. The abundance of protein carbonyl adducts within skeletal muscle sarcoplasmic, myofibrillar, and mitochondrial protein subfractions from musculus vastus lateralis biopsies were determined using Oxyblot immunoblotting techniques. In addition, concentrations of recognized cytoprotective proteins (eg, heat shock proteins [HSP], αβ-crystallin) were also assayed. Aging was associated with increased mitochondrial (but not myofibrillar or sarcoplasmic) protein carbonyl adducts, independently of (stage-I) sarcopenia. Correlation analyses of all subjects revealed that mitochondrial protein carbonyl abundance negatively correlated with muscle strength ([1-repetition maximum], p = .02, r (2) = -.16), but not muscle mass (p = .13, r (2) = -.08). Abundance of cytoprotective proteins, including various HSPs (HSP 27 and 70), were unaffected by aging/sarcopenia. To conclude, these data reveal that mitochondrial protein carbonylation increases moderately with age, and that this increase may impact upon skeletal muscle function, but is not a hallmark of (stage-I) sarcopenia, per se.

  20. The human mannose-binding protein functions as an opsonin

    PubMed Central

    1989-01-01

    The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains: a cysteine-rich NH2-terminal domain that stabilizes the alpha-helix of the second collagen-like domain, and a third COOH-terminal carbohydrate binding region. The function of MBP is unknown, although a role in host defense is suggested by its ability to bind yeast mannans. In this report we show that native and recombinant human MBP can serve in an opsonic role in serum and thereby enhance clearance of mannose rich pathogens by phagocytes. MBP binds to wild-type virulent Salmonella montevideo that express a mannose-rich O- polysaccharide. Interaction of MBP with these organisms results in attachment, uptake, and killing of the opsonized bacteria by phagocytes. These results demonstrate that MBP plays a role in first line host defense against certain pathogenic organisms. PMID:2469767

  1. Preparation and application of vegetable proteins, especially proteins from sunflower seed, for human consumption. An approach.

    PubMed

    Gassmann, B

    1983-01-01

    About 80% of the world protein production are of vegetable origin. More than half the vegetable protein is fed to animals, whereas merely 10 kg protein per capita are obtained from meat, milk and eggs per year. Therefore, and because of rising prices for raw materials and energy the production and the firsthand utilisation of proteinacous plants for foodstuffs are a worldwide problem. As future source of protein for human nutrition sunflower seed and oil extraction residues from sunflower seed, respectively, are of great significance. Sunflower seed does not contain anti-nutritive and toxical compounds. After crossing of species having a high oil content, the today cultivated sunflower hybrids bring seeds containing 17-22% crude protein and 30-52% oil. The cultivation also has led to a considerable reduction of the hull content. In processing of sunflower proteins colour problems occur resulting from finely ground particles of dark hulls and from polyphenolic acids which are easily oxidized and converted into brown polymerics. Essential components of the sunflower protein production are, therefore, the at least 98% dehulling before processing as well as the separation of polyphenolic acids and/or the prevention of their oxidation. In principle, the variation and combination of technological steps in pre-treating and defatting of sunflower seed, in extracting, precipitating, washing and drying of proteins, the chemical modification of proteins obtained, the interaction with neutral salts or complexing agents, and the admixture of lysine or proteins of high lysine content allow to adapt sunflower proteins to each type of application.

  2. Determination of total protein in human seminal plasma.

    PubMed

    Hernvann, A; Gonzales, J; Diemert, M C; Galli, J

    1987-06-01

    Methodological problems are encountered in determinating human seminal protein content since results are influenced especially by techniques which are used, and by modifications due to liquefaction process. In order to test these two points, three methods (Folin, Biuret and Meulemans's reactions) were applied to the same seminal samples and results were compared. The differences seem principally due to the high level of glycoproteins. To determine the modifications of total protein content with time after semen collection, each sample was separated in aliquot fractions and proteolysis was blocked at different times for each fraction. There is an increase of the protein concentration during the first fifteen minutes that follows semen collection. The cause of this phenomenon is discussed.

  3. The p53 gene and protein in human brain tumors

    SciTech Connect

    Louis, D.N. )

    1994-01-01

    Because p53 gene alterations are commonplace in human tumors and because p53 protein is involved in a number of important cellular pathways, p53 has become a topic of intensive investigation, both by basic scientists and clinicians. p53 was initially identified by two independent laboratories in 1979 as a 53 kilodalton (kD) protein that complexes with the large T antigen of SV40 virus. Shortly thereafter, it was shown that the E1B oncoprotein of adenovirus also binds p53. The binding of two different oncogenic viral tumor proteins to the same cellular protein suggested that p53 might be integral to tumorigenesis. The human p53 cDNA and gene were subsequently cloned in the mid-1980s, and analysis of p53 gene alterations in human tumors followed a few year later. During these 10 years, researchers grappling with the vagaries of p53 first characterized the gene as an oncogene, then as a tumor suppressor gene, and most recently as both a tumor suppressor gene and a so-called [open quotes]dominant negative[close quotes] oncogene. The last few years have seen an explosion in work on this single gene and its protein product. A review of a computerized medical database revealed approximately 650 articles on p53 in 1992 alone. p53 has assumed importance in neuro-oncology because p53 mutations and protein alterations are frequent in the common diffuse, fibrillary astrocytic tumors of adults. p53 mutations in astrocytomas were first described in 1989 and were followed by more extensive analyses of gene mutations and protein alterations in adult astrocytomas. The gene has also been studied in less common brain tumors. Elucidating the role of p53 in brain tumorigenesis will not only enhance understanding of brain tumor biology but may also contribute to improved diagnosis and therapy. This discussion reviews key aspects of the p53 gene and protein, and describe their emerging roles in central nervous system neoplasia. 102 refs., 6 figs., 1 tab.

  4. Nitrogen and food production: proteins for human diets.

    PubMed

    Smil, Vaclav

    2002-03-01

    Nitrogen was the most commonly yield-limiting nutrient in all pre-industrial agricultures. Only the Haber-Bosch synthesis of ammonia broke this barrier. The rising dependence on nitrogenous fertilizers, which represents the largest human interference in the biospheric N cycle, has two different roles. In affluent nations it helps to produce excess of food in general, and of animal foods in particular, and it boosts agricultural exports. But for at least a third of humanity in the world's most populous countries the use of N fertilizers makes the difference between malnutrition and adequate diet. Our understanding of human N (protein) needs has undergone many revisions and although some uncertainties still remain it is clear that average protein intakes are excessive in rich countries and inadequate for hundreds of millions of people in Asia, Africa, and Latin America. More dietary protein will be needed to eliminate these disparities but the future global use of N fertilizers can be moderated not just by better agronomic practices but also by higher feeding efficiencies and by gradual changes of prevailing diets. As a result, it could be possible to supply adequate nutrition to the world's growing population without any massive increases of N inputs. PMID:12078001

  5. Tunable protein synthesis by transcript isoforms in human cells

    PubMed Central

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-01

    Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. DOI: http://dx.doi.org/10.7554/eLife.10921.001 PMID:26735365

  6. Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

    PubMed Central

    Molnár, János; Szakács, Gergely; Tusnády, Gábor E.

    2016-01-01

    Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations. PMID:26986070

  7. Human Glycolipid Transfer Protein (GLTP) Expression Modulates Cell Shape

    PubMed Central

    Gao, Yongguang; Chung, Taeowan; Zou, Xianqiong; Pike, Helen M.; Brown, Rhoderick E.

    2011-01-01

    Glycolipid transfer protein (GLTP) accelerates glycosphingolipid (GSL) intermembrane transfer via a unique lipid transfer/binding fold (GLTP-fold) that defines the GLTP superfamily and is the prototype for GLTP-like domains in larger proteins, i.e. phosphoinositol 4-phosphate adaptor protein-2 (FAPP2). Although GLTP-folds are known to play roles in the nonvesicular intracellular trafficking of glycolipids, their ability to alter cell phenotype remains unexplored. In the present study, overexpression of human glycolipid transfer protein (GLTP) was found to dramatically alter cell phenotype, with cells becoming round between 24 and 48 h after transfection. By 48 h post transfection, ∼70% conversion to the markedly round shape was evident in HeLa and HEK-293 cells, but not in A549 cells. In contrast, overexpression of W96A-GLTP, a liganding-site point mutant with abrogated ability to transfer glycolipid, did not alter cell shape. The round adherent cells exhibited diminished motility in wound healing assays and an inability to endocytose cholera toxin but remained viable and showed little increase in apoptosis as assessed by poly(ADP-ribose) polymerase cleavage. A round cell phenotype also was induced by overexpression of FAPP2, which binds/transfers glycolipid via its C-terminal GLTP-like fold, but not by a plant GLTP ortholog (ACD11), which is incapable of glycolipid binding/transfer. Screening for human protein partners of GLTP by yeast two hybrid screening and by immuno-pulldown analyses revealed regulation of the GLTP-induced cell rounding response by interaction with δ-catenin. Remarkably, while δ-catenin overexpression alone induced dendritic outgrowths, coexpression of GLTP along with δ-catenin accelerated transition to the rounded phenotype. The findings represent the first known phenotypic changes triggered by GLTP overexpression and regulated by direct interaction with a p120-catenin protein family member. PMID:21625605

  8. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins

    PubMed Central

    Adachi, Jun; Kumar, Chanchal; Zhang, Yanling; Olsen, Jesper V; Mann, Matthias

    2006-01-01

    Background Urine is a desirable material for the diagnosis and classification of diseases because of the convenience of its collection in large amounts; however, all of the urinary proteome catalogs currently being generated have limitations in their depth and confidence of identification. Our laboratory has developed methods for the in-depth characterization of body fluids; these involve a linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer. Here we applied these methods to the analysis of the human urinary proteome. Results We employed one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography for protein separation and fractionation. Fractionated proteins were digested in-gel or in-solution, and digests were analyzed with the LTQ-FT and LTQ-Orbitrap at parts per million accuracy and with two consecutive stages of mass spectrometric fragmentation. We identified 1543 proteins in urine obtained from ten healthy donors, while essentially eliminating false-positive identifications. Surprisingly, nearly half of the annotated proteins were membrane proteins according to Gene Ontology (GO) analysis. Furthermore, extracellular, lysosomal, and plasma membrane proteins were enriched in the urine compared with all GO entries. Plasma membrane proteins are probably present in urine by secretion in exosomes. Conclusion Our analysis provides a high-confidence set of proteins present in human urinary proteome and provides a useful reference for comparing datasets obtained using different methodologies. The urinary proteome is unexpectedly complex and may prove useful in biomarker discovery in the future. PMID:16948836

  9. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h. PMID:25881200

  10. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

  11. Prefusion structure of a human coronavirus spike protein

    PubMed Central

    Kirchdoerfer, Robert N.; Cottrell, Christopher A.; Wang, Nianshuang; Pallesen, Jesper; Yassine, Hadi M.; Turner, Hannah L.; Corbett, Kizzmekia S.; Graham, Barney S.; McLellan, Jason S.; Ward, Andrew B.

    2016-01-01

    HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease1 and is related to the zoonotic SARS2 and MERS3 betacoronaviruses that have high fatality rates and pandemic potential. Cell tropism and host range is determined in part by the coronavirus spike (S) protein4, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a molecular understanding of its function and limiting development of effective interventions. Here we present the 4.0 Å resolution structure of the trimeric HKU1 S protein determined using single-particle cryo-electron microscopy. In the prefusion conformation, the receptor-binding subunits, S1, rest atop the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known to bind protein receptors in other coronaviruses. These features, along with the location of the two protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. Additionally, these studies should serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens. PMID:26935699

  12. Leucine incorporation into mixed skeletal muscle protein in humans

    SciTech Connect

    Nair, K.S.; Halliday, D.; Griggs, R.C. Clinical Research Centre, Harrow )

    1988-02-01

    Fractional mixed skeletal muscle protein synthesis (FMPS) was estimated in 10 postabsorptive healthy men by determining the increment in the abundance of ({sup 13}C)-leucine in quadriceps muscle protein during an intravenous infusion of L-(1-{sup 13}C)leucine. Whole-body muscle protein synthesis (MPS) was calculated based on the estimation of muscle mass from creatinine excretion and compared with whole-body protein synthesis (WBPS) calculated from the nonoxidative portion of leucine flux. A significant correlation was found between MPS. The contribution of MPS to WBPS was 27 {plus minus} 1%, which is comparable to the reports in other species. Morphometric analyses of adjacent muscle samples in eight subjects demonstrated that the biopsy specimens consisted of 86.5 {plus minus} 2% muscular as opposed to other tissues. Because fiber type composition varies between biopsies, the authors examined the relationship between proportions of each fiber type and FMPS. Variation in the composition of biopsies and in fiber-type proportion did not affect the estimation of muscle protein synthesis rate. They conclude that stable isotope techniques using serial needle biopsies permit the direct measurement of FMPS in humans and that this estimation is correlated with an indirect estimation of WBPS.

  13. Bioinformatics Annotation of Human Y Chromosome-Encoded Protein Pathways and Interactions.

    PubMed

    Rengaraj, Deivendran; Kwon, Woo-Sung; Pang, Myung-Geol

    2015-09-01

    We performed a comprehensive analysis of human Y chromosome-encoded proteins, their pathways, and their interactions using bioinformatics tools. From the NCBI annotation release 107 of human genome, we retrieved a total of 66 proteins encoded on Y chromosome. Most of the retrieved proteins were also matched with the proteins listed in the core databases of the Human Proteome Project including neXtProt, PeptideAtlas, and the Human Protein Atlas. When we examined the pathways of human Y-encoded proteins through KEGG database and Pathway Studio software, many of proteins fall into the categories related to cell signaling pathways. Using the STRING program, we found a total of 49 human Y-encoded proteins showing strong/medium interaction with each other. While using the Pathway studio software, we found that a total of 16 proteins interact with other chromosome-encoded proteins. In particular, the SRY protein interacted with 17 proteins encoded on other chromosomes. Additionally, we aligned the sequences of human Y-encoded proteins with the sequences of chimpanzee and mouse Y-encoded proteins using the NCBI BLAST program. This analysis resulted in a significant number of orthologous proteins between human, chimpanzee, and mouse. Collectively, our findings provide the scientific community with additional information on the human Y chromosome-encoded proteins.

  14. Species specificity of the NS1 protein of influenza B virus: NS1 binds only human and non-human primate ubiquitin-like ISG15 proteins.

    PubMed

    Sridharan, Haripriya; Zhao, Chen; Krug, Robert M

    2010-03-12

    Influenza B viruses, which cause a highly contagious respiratory disease every year, are restricted to humans, but the basis for this restriction had not been determined. Here we provide one explanation for this restriction: the species specificity exhibited by the NS1 protein of influenza B virus (NS1B protein). This viral protein combats a major host antiviral response by binding the interferon-alpha/beta-induced, ubiquitin-like ISG15 protein and inhibiting its conjugation to an array of proteins. We demonstrate that the NS1B protein exhibits species-specific binding; it binds human and non-human primate ISG15 but not mouse or canine ISG15. In both transfection assays and virus-infected cells, the NS1B protein binds and relocalizes only human and non-human primate ISG15 from the cytoplasm to nuclear speckles. Human and non-human primate ISG15 proteins consist of two ubiquitin-like domains separated by a short hinge linker of five amino acids. Remarkably, this short hinge plays a large role in the species-specific binding by the NS1B protein. The hinge of human and non-human primate ISG15, which has a sequence that differs from that of other mammalian ISG15 proteins, including mouse and canine ISG15, is absolutely required for binding the NS1B protein. Consequently, the ISG15 proteins of humans and non-human primates are the only mammalian ISG15 proteins that would bind NS1B.

  15. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

  16. Stimulus-dependent secretion of plasma proteins from human neutrophils.

    PubMed Central

    Borregaard, N; Kjeldsen, L; Rygaard, K; Bastholm, L; Nielsen, M H; Sengeløv, H; Bjerrum, O W; Johnsen, A H

    1992-01-01

    In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli. Images PMID:1378856

  17. Engineered cell-laden human protein-based elastomer

    PubMed Central

    Annabi, Nasim; Mithieux, Suzanne M.; Zorlutuna, Pinar; Camci-Unal, Gulden; Weiss, Anthony S.; Khademhosseini, Ali

    2013-01-01

    Elastic tissue equivalence is a vital requirement of synthetic materials proposed for many resilient, soft tissue engineering applications. Here we present a bioelastomer made from tropoelastin, the human protein that naturally facilitates elasticity and cell interactions in all elastic tissues. We combined this protein’s innate versatility with fast non-toxic fabrication techniques to make highly extensible, cell compatible hydrogels. These hydrogels can be produced in less than a minute through photocrosslinking of methacrylated tropoelastin (MeTro) in an aqueous solution. The fabricated MeTro gels exhibit high extensibility (up to 400%) and superior mechanical properties that outperform other photocrosslinkable hydrogels. MeTro gels were used to encapsulate cells within a flexible 3D environment and to manufacture highly elastic 2D films for cell attachment, growth, and proliferation. In addition, the physical properties of this fabricated bioelastomer such as elasticity, stiffness, and pore characteristics were tuned through manipulation of the methacrylation degree and protein concentration. This photocrosslinkable, functional tissue mimetic gel benefits from the innate biological properties of a human elastic protein and opens new opportunities in tissue engineering. PMID:23639533

  18. Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein.

    PubMed

    Terrasse, Rémi; Tacnet-Delorme, Pascale; Moriscot, Christine; Pérard, Julien; Schoehn, Guy; Vernet, Thierry; Thielens, Nicole M; Di Guilmi, Anne Marie; Frachet, Philippe

    2012-12-14

    C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response. PMID:23086952

  19. Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis.

    PubMed

    Murphy, Noelle; Gaynor, Katherine U; Rowan, Simon C; Walsh, Sinead M; Fabre, Aurelie; Boylan, John; Keane, Michael P; McLoughlin, Paul

    2016-03-01

    Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor β1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor β stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury.

  20. Bone morphogenetic protein (BMP) signaling regulates mitotic checkpoint protein levels in human breast cancer cells.

    PubMed

    Yan, Hualong; Zhu, Songcheng; Song, Chenlin; Liu, Naifa; Kang, Jiuhong

    2012-04-01

    Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection. PMID:22234345

  1. Immunohistochemical localization of dentin matrix protein 1 in human dentin.

    PubMed

    Orsini, G; Ruggeri, A; Mazzoni, A; Nato, F; Falconi, M; Putignano, A; Di Lenarda, R; Nanci, A; Breschi, L

    2008-01-01

    Dentin matrix protein 1 (DMP1) is a non-collagenous matrix protein with a recognized role in the formation of mineralized tissues such as dentin. The aim of this study was to analyze the distribution of DMP1 in human dentin by means of immunofluorescence and high-resolution immunogold labeling. Fully developed, sound human dentin specimens were submitted to fluorescence labeling and post-embedding immunolabeling techniques with a rabbit polyclonal antihuman DMP1 antibody followed by corresponding fluorochrome-conjugated or gold-conjugated secondary antibodies. Both immunofluorescence and immunogold labeling showed an intense labeling associated with the peritubular dentin. In addition, at the ultrastructural level, there was also a moderate and diffuse immunoreaction over intertubular dentin, and a weak labeling within predentin which increased in density towards the mineralization front. This study suggests that in adult human teeth, like in rodents, DMP1 is prevalently concentrated at the level of peritubular dentin and this feature is preserved also in fully developed-teeth. These data are consistent with what has been observed in rodents and suggest that DMP1 plays a role in maintenance of the dentin tubular space.

  2. Proteins of human semen. I. Two-dimensional mapping of human seminal fluid.

    PubMed

    Edwards, J J; Tollaksen, S L; Anderson, N G

    1981-08-01

    The proteins in human seminal plasma were mapped by high-resolution two-dimensional electrophoresis (ISO-DALT and BASO-DALT systems). When analyzed under dissociating conditions, samples from normal fertile males revealed a pattern of over 200 proteins, ranging in mass from 10 000 to 100 000 daltons. Comparison of the mapped proteins from these males and those who had undergone vasectomy allowed us to identify one series of glycoproteins as missing from the semen from vasectomized individuals. Glycoproteins isolated by affinity chromatography with use of concanavalin A were also mapped. Some of the protein spots were identified either by co-electrophoresis with purified proteins or by the electrophoretic transfer of proteins to nitrocellulose sheets and subsequent detection by immunological procedures. The proteins identified include a number of serum proteins as well as prostatic acid phosphatase and creatine kinase. Proteolytic events shown to occur during the liquefaction of semen that occurs early after collection indicate the importance of carefully controlled collection and preparation methods for clinical evaluation of seminal plasma. Ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride inhibit this proteolysis. PMID:7273394

  3. Putative protein partners for the human CPI-17 protein revealed by bacterial two-hybrid screening.

    PubMed

    Kim, Kyung-mi; Adyshev, Djanybek M; Kása, Anita; Zemskov, Evgeny A; Kolosova, Irina A; Csortos, Csilla; Verin, Alexander D

    2013-07-01

    We have previously demonstrated that PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI-17) is expressed in lung endothelium. CPI-17, a specific inhibitor of myosin light chain phosphatase (MLCP), is involved in the endothelial cytoskeletal and barrier regulation. In this paper, we report the identification of fourteen putative CPI-17 interacting proteins in the lung using BacterioMatch Two-Hybrid System. Five of them: plectin 1 isoform 1, alpha II spectrin, OK/SW-CL.16, gelsolin isoform a, and junction plakoglobin are involved in actin cytoskeleton organization and cell adhesion, suggesting possible significance of these binding partners in CPI-17-mediated cytoskeletal reorganization of endothelial cells. Furthermore, we confirmed the specific interaction between plakoglobin and CPI-17, which is affected by the phosphorylation status of CPI-17 in human lung microvascular endothelial cells. PMID:23583905

  4. Bone Morphogenetic Protein (BMP) signaling in development and human diseases

    PubMed Central

    Wang, Richard N.; Green, Jordan; Wang, Zhongliang; Deng, Youlin; Qiao, Min; Peabody, Michael; Zhang, Qian; Ye, Jixing; Yan, Zhengjian; Denduluri, Sahitya; Idowu, Olumuyiwa; Li, Melissa; Shen, Christine; Hu, Alan; Haydon, Rex C.; Kang, Richard; Mok, James; Lee, Michael J.; Luu, Hue L.; Shi, Lewis L.

    2014-01-01

    Bone Morphogenetic Proteins (BMPs) are a group of signaling molecules that belongs to the Transforming Growth Factor-β (TGF-β) superfamily of proteins. Initially discovered for their ability to induce bone formation, BMPs are now known to play crucial roles in all organ systems. BMPs are important in embryogenesis and development, and also in maintenance of adult tissue homeostasis. Mouse knockout models of various components of the BMP signaling pathway result in embryonic lethality or marked defects, highlighting the essential functions of BMPs. In this review, we first outline the basic aspects of BMP signaling and then focus on genetically manipulated mouse knockout models that have helped elucidate the role of BMPs in development. A significant portion of this review is devoted to the prominent human pathologies associated with dysregulated BMP signaling. PMID:25401122

  5. RNA-binding protein QKI regulates Glial fibrillary acidic protein expression in human astrocytes.

    PubMed

    Radomska, Katarzyna J; Halvardson, Jonatan; Reinius, Björn; Lindholm Carlström, Eva; Emilsson, Lina; Feuk, Lars; Jazin, Elena

    2013-04-01

    Linkage, association and expression studies previously pointed to the human QKI, KH domain containing, RNA-binding (QKI) as a candidate gene for schizophrenia. Functional studies of the mouse orthologue Qk focused mainly on its role in oligodendrocyte development and myelination, while its function in astroglia remained unexplored. Here, we show that QKI is highly expressed in human primary astrocytes and that its splice forms encode proteins targeting different subcellular localizations. Uncovering the role of QKI in astrocytes is of interest in light of growing evidence implicating astrocyte dysfunction in the pathogenesis of several disorders of the central nervous system. We selectively silenced QKI splice variants in human primary astrocytes and used RNA sequencing to identify differential expression and splice variant composition at the genome-wide level. We found that an mRNA expression of Glial fibrillary acidic protein (GFAP), encoding a major component of astrocyte intermediate filaments, was down-regulated after QKI7 splice variant silencing. Moreover, we identified a potential QKI-binding site within the 3' untranslated region of human GFAP. This sequence was not conserved between mice and humans, raising the possibility that GFAP is a target for QKI in humans but not rodents. Haloperidol treatment of primary astrocytes resulted in coordinated increases in QKI7 and GFAP expression. Taken together, our results provide the first link between QKI and GFAP, two genes with alterations previously observed independently in schizophrenic patients. Our findings for QKI, together with its well-known role in myelination, suggest that QKI is a hub regulator of glia function in humans.

  6. Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins.

    PubMed Central

    Almendral, J M; Huebsch, D; Blundell, P A; Macdonald-Bravo, H; Bravo, R

    1987-01-01

    A full-length cDNA clone for the human nuclear protein cyclin has been isolated by using polyclonal antibodies and sequenced. The sequence predicts a protein of 261 amino acids (Mr 29,261) with a high content of acidic (41, aspartic and glutamic acids) versus basic (24, lysine and arginine) amino acids. The identity of the cDNA clone was confirmed by in vitro hybrid-arrested translation of cyclin mRNA. Blot-hybridization analysis of mouse 3T3 and human MOLT-4 cell RNA revealed a mRNA species of approximately the same size as the cDNA insert. Expression of cyclin mRNA was undetectable or very low in quiescent cells, increasing after 8-10 hr of serum stimulation. Inhibition of DNA synthesis by hydroxyurea in serum-stimulated cells did not affect the increase in cyclin mRNA but inhibited 90% the expression of H3 mRNA. These results suggest that expression of cyclin and histone mRNAs are controlled by different mechanisms. A region of the cyclin sequence shows a significant homology with the putative DNA binding site of several proteins, specially with the transcriptional-regulator cAMP-binding protein of Escherichia coli, suggesting that cyclin could play a similar role in eukaryotic cells. Images PMID:2882507

  7. Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.

    PubMed Central

    Löhr, M.; Trautmann, B.; Göttler, M.; Peters, S.; Zauner, I.; Maillet, B.; Klöppel, G.

    1994-01-01

    Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8286197

  8. Human endogenous retrovirus protein Rec interacts with the testicular zinc-finger protein and androgen receptor.

    PubMed

    Kaufmann, Sabine; Sauter, Marlies; Schmitt, Martina; Baumert, Bianca; Best, Barbara; Boese, Annette; Roemer, Klaus; Mueller-Lantzsch, Nikolaus

    2010-06-01

    More than 2000 human endogenous retrovirus (HERV) sequences are present in the human genome, yet only a few are intact and able to produce proteins. The normal functions of these, if any, are unknown, but some HERV proteins have been implicated in cancers, in particular germ-cell cancers. For instance, it has been documented that (i) patients with germ-cell tumours frequently produce antibodies against HERV proteins; (ii) transgenic mice expressing HERV-K (HML-2) rec are prone to testicular carcinoma in situ; and (iii) Rec can bind and suppress a guardian of germline stem-cell pluripotency, the promyelocytic leukaemia zinc-finger protein (PLZF). This study identified the PLZF-related testicular zinc-finger protein (TZFP) as a binding partner of HERV-K (HML-2) Rec. Interactions occurred via the N- and C-terminal domains of Rec and the C-terminal DNA-binding zinc-finger domain of TZFP (aa 375-450). Not much is known about the function of TZFP. The protein is expressed predominantly in the testis, where it functions as a transcriptional repressor that is active during specific stages of spermatogenesis. The most intensely studied function of TZFP is that of a co-repressor of the activated androgen receptor (AR). Here, it was shown that Rec can form a trimeric complex with TZFP and AR, and can relieve the TZFP-mediated repression of AR-induced transactivation. In addition, Rec was able to overcome the direct transcriptional repression by TZFP of the c-myc gene promoter in reporter assays. Thus, HERV-K (HML-2) Rec may function as an oncoprotein by de-repressing oncogenic transcription factors such as AR.

  9. Human APOBEC3 proteins, retrovirus restriction, and HIV drug resistance.

    PubMed

    Haché, Guylaine; Mansky, Louis M; Harris, Reuben S

    2006-01-01

    Over 40 million people worldwide currently have HIV/AIDS. Many antiretroviral drugs have proven effective, but drug-resistant HIV variants frequently emerge to thwart treatment efforts. Reverse transcription errors undoubtedly contribute to drug resistance, but additional significant sources of viral genetic variation are debatable. The human APOBEC3F and APOBEC3G proteins can potently inhibit retrovirus infection by a mechanism that involves retroviral cDNA cytosine deamination. Here we review the current knowledge on the mechanism of APOBEC3-dependent retrovirus restriction and discuss whether this innate host-defense system actively contributes to HIV genetic variation.

  10. Recombinant human bone morphogenetic protein 2 in lateral ridge augmentation.

    PubMed

    Mehanna, Robert; Koo, Samuel; Kim, David M

    2013-01-01

    This case report describes the augmentation of severe lateral ridge defects in the maxilla and mandible using recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS). The surgical technique used tenting screws and a membrane to maintain space for the ACS. After 7 months of healing, the ridge width increased from 1 to 2 mm to 6 to 9 mm, thus allowing successful placement of dental implants. De novo bone formation through use of the surgical technique for space maintenance of rhBMP-2/ACS was demonstrated without the need for additional particulate bone grafting. PMID:23342352

  11. Genomic structure of the human prion protein gene.

    PubMed Central

    Puckett, C; Concannon, P; Casey, C; Hood, L

    1991-01-01

    Creutzfeld-Jacob disease and Gerstmann-Sträussler syndrome are rare degenerative disorders of the nervous system which have been genetically linked to the prion protein (PrP) gene. The PrP gene encodes a host glycoprotein of unknown function and is located on the short arm of chromosome 20, a region with few known genes or anonymous markers. The complete structure of the PrP gene in man has not been determined despite considerable interest in its relationship to these unusual disorders. We have determined that the human PrP gene has the same simple genomic structure seen in the hamster gene and consists of two exons and a single intron. In contrast to the hamster PrP gene the human gene appears to have a single major transcriptional start site. The region immediately 5' of the transcriptional start site of the human PrP gene demonstrates the GC-rich features commonly seen in housekeeping genes. Curiously, the genomic clone we have isolated contains a 24-bp deletion that removes one of five octameric peptide repeats predicted to form a B-pleated sheet in this region of the PrP. We have also identified 5' of the PrP gene an RFLP which has a high degree of heterozygosity and which should serve as a useful marker for the pter-12 region of human chromosome 20. Images Figure 3 Figure 5 PMID:1678248

  12. Secretion of Human Protein C in Mouse Milk

    PubMed Central

    Park, Chae-Won; Kang, Myung-Hwa; Min, Kwan-Sik

    2015-01-01

    To determine the production of recombinant human protein C (rec-hPC) in milk, we created two homozygous mice lines for the goat β-casein/hPC transgene. Females and males of both lines (#10 and #11) displayed normal growth, fertility, and lactated normally. The copy number of the transgene was about fivefold higher in #10 line as compared to #11 line. mRNA expression of the transgene was only detected in the mammary glands of both lines. Furthermore, mRNA expression was fourfold higher on day 7 than on day 1 during lactation. Northern blot analysis of mRNA expression in the #10 line of transgenic (Tg) mice indicated a strong expression of the transgene in the mammary glands after seven days of lactation. Comparison of rec-hPC protein level with that of mRNA in the mammary glands showed a very similar pattern. A 52-kDa band corresponding to the hPC protein was strongly detected in mammary glands of the #10 line during lactation. We also detected two bands of heavy chain and one weak band of light chain in the milk of the #10 and #11 lines. One single band at 52 kDa was detected from CHO cells transfected with hPC cDNA. hPC was mainly localized in the alveolar epithelial cell of the mammary glands. The protein is strongly expressed in the cytoplasm of the cultured mammary gland tissue. hPC protein produced in milk ranged from 2 to 28 ng/mL. These experiments indicated that rec-hPC can be produced at high levels in mice mammary glands. PMID:25749471

  13. Correlation of protein and gene expression profiles of inflammatory proteins after endotoxin challenge in human subjects.

    PubMed

    Prabhakar, Uma; Conway, Theresa M; Murdock, Paul; Mooney, Jeff L; Clark, Steve; Hedge, Priti; Bond, Brian C; Jazwinska, Elizabeth C; Barnes, Michael R; Tobin, Frank; Damian-Iordachi, Valeriu; Greller, Larry; Hurle, Mark; Stubbs, Andrew P; Li, Zhong; Valoret, Elizabeth I; Erickson-Miller, Connie; Cass, Lisa; Levitt, Blanche; Davis, Hugh M; Jorkasky, Diane K; Williams, William V

    2005-07-01

    Administration of endotoxin (LPS) in humans results in profound physiological responses, including activation of peripheral blood mononuclear cells and the release of inflammatory factors. The time course of the response of selected inflammatory proteins was examined in healthy subjects (n = 6) administered a single intravenous dose of the purified derivative of endotoxin (3.0 ng/kg). Microarray analysis demonstrated changes in the expression of a number of genes, which were confirmed in separate in vitro endotoxin stimulation experiments. Subsequent TaqMan analysis of genes of interest indicated time-dependent changes in the expression of many of these genes. This included pre-B cell enhancing factor, which was identified on microarray analysis as being markedly upregulated following endotoxin stimulation. Protein expression of the genes examined by TaqMan analysis was measured and demonstrated the appearance of tumor necrosis factor (TNF)-alpha and sTNF-R proteins in the plasma beginning within 1 h after dosing, followed by other cytokines/ inflammatory markers (e.g., IL-1ra, G-CSF, IL-6, IL-8, and IL-10) and suppressors of cytokine signaling (SOCS-1 and SOCS-3). In general, cytokine protein expression correlated well with gene expression; however, the temporal profile of expression of some genes did not correlate well with the protein data. For many of these proteins, the lack of correlation was attributable to alternate tissue sources, which were demonstrated on TaqMan analysis. Principal component analysis indicated that cytokines could be grouped according to their temporal pattern of response, with most transcript levels returning to baseline 24 h following endotoxin administration. The combination of cDNA microarray and TaqMan analysis to identify and quantify changes in gene expression, along with the analysis of protein expression, can be useful in investigating inflammatory and other diseases.

  14. Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein.

    PubMed Central

    Alp, N J; Allport, T D; Van Zanten, J; Rodgers, B; Sissons, J G; Borysiewicz, L K

    1991-01-01

    Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known about responses against this protein by CD4+ T cells, which may be important as direct effector cells or helper cells for antibody and CD8+ responses. Proliferative-T-cell responses to HCMV IE1 were studied in normal seropositive subjects. Peripheral blood mononuclear cells from 85% of seropositive subjects proliferated in response to HCMV from infected fibroblasts, and of these, 73% responded to recombinant baculovirus IE1. Responding cells were predominantly CD3+ CD4+. IE1 antigen preparations, including baculovirus recombinant protein, transfected rat cell nuclei, and synthetic peptides, induced IE1-specific T-cell lines which cross-reacted between the preparations. The fine specificity of these IE1-specific T-cell lines was studied by using overlapping synthetic peptides encompassing the entire sequence of the IE1 protein. The regions of the IE1 molecule recognized were identified and these varied between individuals, possibly reflecting differences in major histocompatibility complex (MHC) class II haplotype. In one subject, the peptide specificities of proliferative and MHC class I-restricted cytotoxic determinants on IE1 were spatially distinct. Thus, no single immunodominant T-cell determinant within HCMV IE1 was identified, suggesting that multiple peptides or a region of the 72-kDa IE1 protein would be required to induce specific T-cell responses in humans. PMID:1714519

  15. Effects of C-reactive protein on human lymphocyte responsiveness.

    PubMed

    Vetter, M L; Gewurz, H; Hansen, B; James, K; Baum, L L

    1983-05-01

    C-reactive protein (CRP), a trace serum protein that increases markedly in concentration during inflammatory reactions, was recently shown to bind to a subset of human IgG-FcR-bearing peripheral blood lymphocytes in the presence of a ligand such as pneumococcal C-polysaccharide (CPS). CRP has also been detected on a small percentage of PBL that are associated with NK activity. In the present study, we assessed the effects of CRP and CRP-CPS complexes on a variety of human lymphocyte functions in vitro. CRP and CRP complexes significantly enhanced (generally two to threefold) cell-mediated cytotoxicity, minimally enhanced the MLC reaction, and induced a small but regularly detectable blastogenic response in resting PBL. CRP or CRP-CPS complexes had no effect on mitogen-induced blastogenesis, PWM-induced generation of IgM plaque-forming cells, E-rosette formation, antibody-dependent cell-mediated cytotoxicity, or NK activity. The basis for the preferential ability of CRP to enhance cytotoxicity responses in vitro is under further investigation.

  16. Structure of human Niemann-Pick C1 protein.

    PubMed

    Li, Xiaochun; Wang, Jiawei; Coutavas, Elias; Shi, Hang; Hao, Qi; Blobel, Günter

    2016-07-19

    Niemann-Pick C1 protein (NPC1) is a late-endosomal membrane protein involved in trafficking of LDL-derived cholesterol, Niemann-Pick disease type C, and Ebola virus infection. NPC1 contains 13 transmembrane segments (TMs), five of which are thought to represent a "sterol-sensing domain" (SSD). Although present also in other key regulatory proteins of cholesterol biosynthesis, uptake, and signaling, the structure and mechanism of action of the SSD are unknown. Here we report a crystal structure of a large fragment of human NPC1 at 3.6 Å resolution, which reveals internal twofold pseudosymmetry along TM 2-13 and two structurally homologous domains that protrude 60 Å into the endosomal lumen. Strikingly, NPC1's SSD forms a cavity that is accessible from both the luminal bilayer leaflet and the endosomal lumen; computational modeling suggests that this cavity is large enough to accommodate one cholesterol molecule. We propose a model for NPC1 function in cholesterol sensing and transport. PMID:27307437

  17. Degenerated human intervertebral discs contain autoantibodies against extracellular matrix proteins.

    PubMed

    Capossela, S; Schläfli, P; Bertolo, A; Janner, T; Stadler, B M; Pötzel, T; Baur, M; Stoyanov, J V

    2014-04-04

    Degeneration of intervertebral discs (IVDs) is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP) to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.

  18. Structure of human Niemann–Pick C1 protein

    PubMed Central

    Li, Xiaochun; Wang, Jiawei; Coutavas, Elias; Shi, Hang; Hao, Qi; Blobel, Günter

    2016-01-01

    Niemann–Pick C1 protein (NPC1) is a late-endosomal membrane protein involved in trafficking of LDL-derived cholesterol, Niemann–Pick disease type C, and Ebola virus infection. NPC1 contains 13 transmembrane segments (TMs), five of which are thought to represent a “sterol-sensing domain” (SSD). Although present also in other key regulatory proteins of cholesterol biosynthesis, uptake, and signaling, the structure and mechanism of action of the SSD are unknown. Here we report a crystal structure of a large fragment of human NPC1 at 3.6 Å resolution, which reveals internal twofold pseudosymmetry along TM 2–13 and two structurally homologous domains that protrude 60 Å into the endosomal lumen. Strikingly, NPC1's SSD forms a cavity that is accessible from both the luminal bilayer leaflet and the endosomal lumen; computational modeling suggests that this cavity is large enough to accommodate one cholesterol molecule. We propose a model for NPC1 function in cholesterol sensing and transport. PMID:27307437

  19. Heterochromatin Protein 1 (HP1) Proteins Do Not Drive Pericentromeric Cohesin Enrichment in Human Cells

    PubMed Central

    Serrano, Ángel; Rodríguez-Corsino, Miriam; Losada, Ana

    2009-01-01

    Sister chromatid cohesion mediated by cohesin is essential for accurate chromosome segregation. Classical studies suggest that heterochromatin promotes cohesion, but whether this happens through regulation of cohesin remains to be determined. Heterochromatin protein 1 (HP1) is a major component of heterochromatin. In fission yeast, the HP1 homologue Swi6 interacts with cohesin and is required for proper targeting and/or stabilization of cohesin at the centromeric region. To test whether this pathway is conserved in human cells, we have examined the behavior of cohesin in cells in which the levels of HP1 alpha, beta or gamma (the three HP1 proteins present in mammalian organisms) have been reduced by siRNA. We have also studied the consequences of treating human cells with drugs that change the histone modification profile of heterochromatin and thereby affect HP1 localization. Our results show no evidence for a requirement of HP1 proteins for either loading of bulk cohesin onto chromatin in interphase or retention of cohesin at pericentric heterochromatin in mitosis. However, depletion of HP1gamma leads to defects in mitotic progression. PMID:19352502

  20. Steps towards a repertoire of comprehensive maps of human protein interaction networks: the Human Proteotheque Initiative (HuPI).

    PubMed

    Coulombe, Benoit; Blanchette, Mathieu; Jeronimo, Célia

    2008-04-01

    Defining human protein interaction networks has become essential to develop an overall, systems-based understanding of the molecular events that sustain cell growth in normal and disease conditions. To characterize protein interaction networks from human cells, we have undertaken the development of a systematic, unbiased technology pipeline that couples experimental and computational approaches. This discovery engine is central to the Human Proteotheque Initiative (HuPI), a multidisciplinary project aimed at building a repertoire of comprehensive maps of human protein interaction networks, the Human Proteotheque. The information contained in the Proteotheque is made publicly available through an interactive web site that can be consulted to visualize some of the fundamental molecular connections formed in human cells and to determine putative functions of previously uncharacterized proteins based on guilt by association. The process governing the evolution of HuPI towards becoming a repository of accurate and complete protein interaction maps is described.

  1. Protein Stability and Dynamics Modulation: The Case of Human Frataxin

    PubMed Central

    Gallo, Mariana; Salvay, Andres G.; Ferreiro, Diego U.; Santos, Javier

    2012-01-01

    Frataxin (FXN) is an α/β protein that plays an essential role in iron homeostasis. Apparently, the function of human FXN (hFXN) depends on the cooperative formation of crucial interactions between helix α1, helix α2, and the C-terminal region (CTR) of the protein. In this work we quantitatively explore these relationships using a purified recombinant fragment hFXN90–195. This variant shows the hydrodynamic behavior expected for a monomeric globular domain. Circular dichroism, fluorescence, and NMR spectroscopies show that hFXN90–195 presents native-like secondary and tertiary structure. However, chemical and temperature induced denaturation show that CTR truncation significantly destabilizes the overall hFXN fold. Accordingly, limited proteolysis experiments suggest that the native-state dynamics of hFXN90–195 and hFXN90–210 are indeed different, being the former form much more sensitive to the protease at specific sites. The overall folding dynamics of hFXN fold was further explored with structure-based protein folding simulations. These suggest that the native ensemble of hFXN can be decomposed in at least two substates, one with consolidation of the CTR and the other without consolidation of the CTR. Explicit-solvent all atom simulations identify some of the proteolytic target sites as flexible regions of the protein. We propose that the local unfolding of CTR may be a critical step for the global unfolding of hFXN, and that modulation of the CTR interactions may strongly affect hFXN physiological function. PMID:23049850

  2. Characterization of adenosine binding proteins in human placental membranes

    SciTech Connect

    Hutchison, K.A.

    1989-01-01

    We have characterized two adenosine binding proteins in human placenta. In membranes, one site is detected with ({sup 3}H) -N-ethylcarboxamidoadenosine (({sup 3}H)NECA). This site is similar to the adenosine A{sub 2} receptor. We call this site the adenosine A{sub 2}-like binding site. In detergent extracts, the second site is detected and has the characteristics of an adenosine A{sub 1} receptor. The soluble adenosine A{sub 2}-like binding site cannot be detected without a rapid assay. Binding to the adenosine A{sub 1} receptor with ({sup 3}H)-2-chloroadenosine and ({sup 3}H)NECA is time dependent, saturable, and reversible. Equilibrium displacement analysis with adenosine agonists reveals an A{sub 1} specificity: 2-chloroadenosine > R-phenylisopropyladenosine > 5{prime}-N-ethylcarboxamidoadenosine. The antagonist potency order is 1,3-diethyl-8-phenylxanthine > isobutylmethylxanthine > theophylline. Competition analysis of membranes with the A,-selective ligands ({sup 3}H)-cyclohexyladenosine ({sup 3}H) cylopentylxanthine revealed adenosine A{sub 1} agonist and antagonist potency orders. We have purified the adenosine A{sub 2}-like binding site. The adenosine A{sub 2}-like binding site is an ubiquitous major cellular protein. It is glycosylated, highly asymmetric, and acidic. The native protein is an homodimer with a subunit molecular mass of 98 kDa. The sedimentation coefficient and partial specific volume of the binding complex are 6.9 s and 0.698 ml/g, respectively. The Stokes' radius is 70 {Angstrom}. The native molecular mass of the detergent-protein complex is 230 kDa. The adenosine A{sub 2}-like binding site has an agonist potency order of 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine >> R-phenylisopropyladenosine and an antagonist potency order of isobutylmethylxanthine > theophylline >> 1,3-diethyl-8-phenylxanthine.

  3. C-reactive protein activates complement in infarcted human myocardium.

    PubMed

    Nijmeijer, Remco; Lagrand, Wim K; Lubbers, Yvonne T P; Visser, Cees A; Meijer, Chris J L M; Niessen, Hans W M; Hack, C Erik

    2003-07-01

    Circulating levels of C-reactive protein (CRP) constitute a cardiovascular risk marker. Immunohistochemical studies have revealed co-localization of CRP and activated complement in human infarcted myocardium suggesting CRP to enhance inflammation in ischemic myocardium by inducing local complement activation. The aim was to establish whether CRP activates complement in infarcted human myocardium and to assess the relationship between this activation and the duration of infarction. Myocardial tissue samples from 56 patients that had died from acute myocardial infarction were evaluated. Specimens were taken from infarcted as well as noninfarcted sites of the heart. CRP-mediated complement activation was assessed by immunohistochemistry and by measuring levels of complement, CRP, and CRP-complement complexes, specific markers for CRP-mediated activation, in homogenates of the heart. Infarctions of 12 hours to 5 days had significantly more extensive depositions of complement and CRP and contained significantly more CRP, activated complement, and CRP-complement complexes than infarctions that were less than 12 hours old. Levels of CRP complexes correlated significantly with CRP and complement concentrations in the infarctions, as well as with the extent of complement and CRP depositions as measured via immunohistochemistry. Specific activation products of CRP-mediated activation of complement are increased in infarcts of more than 12 hours in duration and correlate with the extent of complement depositions. Hence, CRP seems to enhance local inflammatory reactions ensuing in human myocardial infarcts of more than 12 hours duration.

  4. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  5. Isotachophoretic Preconcentration of Cardiac Proteins from Human Serum

    NASA Astrophysics Data System (ADS)

    Dutta, Prashanta; Hossan, Mohammad; Jubery, Talukder; Bottenus, Danny; Ivory, Cornelius

    2011-11-01

    Cationic isotachophoresis (ITP) is used to concentrate and detect a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum in a cascade poly(methyl methacrylate) (PMMA) microfluidic channel. PMMA microchannel was formed using solvent imprinting and temperature-assisted bonding. A 100x reduction in cross-sectional area is implemented by gradually decreasing the channel width and height to increase the sensitivity of ITP. The ITP was performed in peak mode with potassium ion as the leading electrolyte and hydronium ions as the terminating electrolyte. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. Experimental results show that ITP can detect cTnI at initial concentrations as low as 46 ng/mL in the presence of human serum proteins, and ITP can obtain cTnI concentrations factors as high as 10,000. This demonstrates the detection of cTnI in depleted human serum at clinically relevant concentrations without the use of antibodies and relying solely on ITP in a cascade microchip.

  6. Darwinian and demographic forces affecting human protein coding genes

    PubMed Central

    Nielsen, Rasmus; Hubisz, Melissa J.; Hellmann, Ines; Torgerson, Dara; Andrés, Aida M.; Albrechtsen, Anders; Gutenkunst, Ryan; Adams, Mark D.; Cargill, Michele; Boyko, Adam; Indap, Amit; Bustamante, Carlos D.; Clark, Andrew G.

    2009-01-01

    Past demographic changes can produce distortions in patterns of genetic variation that can mimic the appearance of natural selection unless the demographic effects are explicitly removed. Here we fit a detailed model of human demography that incorporates divergence, migration, admixture, and changes in population size to directly sequenced data from 13,400 protein coding genes from 20 European-American and 19 African-American individuals. Based on this demographic model, we use several new and established statistical methods for identifying genes with extreme patterns of polymorphism likely to be caused by Darwinian selection, providing the first genome-wide analysis of allele frequency distributions in humans based on directly sequenced data. The tests are based on observations of excesses of high frequency–derived alleles, excesses of low frequency–derived alleles, and excesses of differences in allele frequencies between populations. We detect numerous new genes with strong evidence of selection, including a number of genes related to psychiatric and other diseases. We also show that microRNA controlled genes evolve under extremely high constraints and are more likely to undergo negative selection than other genes. Furthermore, we show that genes involved in muscle development have been subject to positive selection during recent human history. In accordance with previous studies, we find evidence for negative selection against mutations in genes associated with Mendelian disease and positive selection acting on genes associated with several complex diseases. PMID:19279335

  7. Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins.

    PubMed

    Cundiff, Judy K; McConnell, Elizabeth J; Lohe, Kimberly J; Maria, Sarah D; McMahon, Robert J; Zhang, Qiang

    2016-01-01

    Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.

  8. Crystal Structure of the Human Astrovirus Capsid Protein

    PubMed Central

    Toh, Yukimatsu; Harper, Justin; Dryden, Kelly A.; Yeager, Mark; Méndez, Ernesto

    2016-01-01

    ABSTRACT Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ∼34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP9071–415 (amino acids [aa] 71 to 415 of VP90) of human astrovirus serotype 8 at a 2.15-Å resolution. VP9071–415 encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP9071–415 is comprised of two domains: an S domain, which adopts the typical jelly-roll β-barrel fold, and a P1 domain, which forms a squashed β-barrel consisting of six antiparallel β-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP9071–415 structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation. IMPORTANCE Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. As a nonenveloped virus

  9. Novel proteins associated with human dilated cardiomyopathy: selective reduction in α(1A)-adrenergic receptors and increased desensitization proteins.

    PubMed

    Shi, Ting; Moravec, Christine S; Perez, Dianne M

    2013-04-01

    Abstract Therapeutics to treat human heart failure (HF) and the identification of proteins associated with HF are still limited. We analyzed α(1)-adrenergic receptor (AR) subtypes in human HF and performed proteomic analysis on more uniform samples to identify novel proteins associated with human HF. Six failing hearts with end-stage dilated cardiomyopathy (DCM) and four non-failing heart controls were subjected to proteomic analysis. Out of 48 identified proteins, 26 proteins were redundant between samples. Ten of these 26 proteins were previously reported to be associated with HF. Of the newly identified proteins, we found several muscle proteins and mitochondrial/electron transport proteins, while novel were functionally similar to previous reports. However, we also found novel proteins involved in functional classes such as β-oxidation and G-protein coupled receptor signaling and desensitization not previously associated with HF. We also performed radioligand-binding studies on the heart samples and not only confirmed a large loss of β(1)-ARs in end-stage DCM, but also found a selective decrease in the α(1A)-AR subtype not previously reported. We have identified new proteins and functional categories associated with end-stage DCM. We also report that similar to the previously characterized loss of β(1)-AR in HF, there is also a concomitant loss of α(1A)-ARs, which are considered cardioprotective proteins.

  10. RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

    PubMed

    Stefanovic, Branko

    2013-01-01

    Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments.

  11. Bile salt recognition by human liver fatty acid binding protein.

    PubMed

    Favretto, Filippo; Santambrogio, Carlo; D'Onofrio, Mariapina; Molinari, Henriette; Grandori, Rita; Assfalg, Michael

    2015-04-01

    Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 μm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder. PMID:25639618

  12. Bile salt recognition by human liver fatty acid binding protein.

    PubMed

    Favretto, Filippo; Santambrogio, Carlo; D'Onofrio, Mariapina; Molinari, Henriette; Grandori, Rita; Assfalg, Michael

    2015-04-01

    Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 μm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder.

  13. Shedding light on proteins, nucleic acids, cells, humans and fish

    NASA Technical Reports Server (NTRS)

    Setlow, Richard B.

    2002-01-01

    I was trained as a physicist in graduate school. Hence, when I decided to go into the field of biophysics, it was natural that I concentrated on the effects of light on relatively simple biological systems, such as proteins. The wavelengths absorbed by the amino acid subunits of proteins are in the ultraviolet (UV). The wavelengths that affect the biological activities, the action spectra, also are in the UV, but are not necessarily parallel to the absorption spectra. Understanding these differences led me to investigate the action spectra for affecting nucleic acids, and the effects of UV on viruses and cells. The latter studies led me to the discovery of the important molecular nature of the damages affecting DNA (cyclobutane pyrimidine dimers) and to the discovery of nucleotide excision repair. Individuals with the genetic disease xeroderma pigmentosum (XP) are extraordinarily sensitive to sunlight-induced skin cancer. The finding, by James Cleaver, that their skin cells were defective in DNA repair strongly suggested that DNA damage was a key step in carcinogenesis. Such information was important for estimating the wavelengths in sunlight responsible for human skin cancer and for predicting the effects of ozone depletion on the incidence of non-melanoma skin cancer. It took experiments with backcross hybrid fish to call attention to the probable role of the longer UV wavelengths not absorbed by DNA in the induction of melanoma. These reflections trace the biophysicist's path from molecules to melanoma.

  14. Shedding light on proteins, nucleic acids, cells, humans and fish.

    PubMed

    Setlow, Richard B

    2002-03-01

    I was trained as a physicist in graduate school. Hence, when I decided to go into the field of biophysics, it was natural that I concentrated on the effects of light on relatively simple biological systems, such as proteins. The wavelengths absorbed by the amino acid subunits of proteins are in the ultraviolet (UV). The wavelengths that affect the biological activities, the action spectra, also are in the UV, but are not necessarily parallel to the absorption spectra. Understanding these differences led me to investigate the action spectra for affecting nucleic acids, and the effects of UV on viruses and cells. The latter studies led me to the discovery of the important molecular nature of the damages affecting DNA (cyclobutane pyrimidine dimers) and to the discovery of nucleotide excision repair. Individuals with the genetic disease xeroderma pigmentosum (XP) are extraordinarily sensitive to sunlight-induced skin cancer. The finding, by James Cleaver, that their skin cells were defective in DNA repair strongly suggested that DNA damage was a key step in carcinogenesis. Such information was important for estimating the wavelengths in sunlight responsible for human skin cancer and for predicting the effects of ozone depletion on the incidence of non-melanoma skin cancer. It took experiments with backcross hybrid fish to call attention to the probable role of the longer UV wavelengths not absorbed by DNA in the induction of melanoma. These reflections trace the biophysicist's path from molecules to melanoma.

  15. Bacterial Delivery of TALEN Proteins for Human Genome Editing

    PubMed Central

    Bian, Ting; Wu, Donghai; Yang, Lijun; Terada, Naohiro; Wu, Weihui; Jin, Shouguang

    2014-01-01

    Transcription Activator-Like Effector Nucleases (TALENs) are a novel class of sequence-specific nucleases that have recently gained prominence for its ease of production and high efficiency in genome editing. A TALEN pair recognizes specific DNA sequences and introduce double-strand break in the target site, triggering non-homologous end joining and homologous recombination. Current methods of TALEN delivery involves introduction of foreign genetic materials, such as plasmid DNA or mRNA, through transfection. Here, we show an alternative way of TALEN delivery, bacterial type III secretion system (T3SS) mediated direct injection of the TALEN proteins into human cells. Bacterially injected TALEN was shown to efficiently target host cell nucleus where it persists for almost 12 hours. Using a pair of TALENs targeting venus gene, such injected nuclear TALENs were shown functional in introducing DNA mutation in the target site. Interestingly, S-phase cells seem to show greater sensitivity to the TALEN mediated target gene modification. Accordingly, efficiency of such genome editing can easily be manipulated by the infection dose, number of repeated infections as well as enrichment of S phase cells. This work further extends the utility of T3SS in the delivery of functional proteins into mammalian cells to alter their characters for biomedical applications. PMID:24618838

  16. Bacterial delivery of TALEN proteins for human genome editing.

    PubMed

    Jia, Jingyue; Jin, Yongxin; Bian, Ting; Wu, Donghai; Yang, Lijun; Terada, Naohiro; Wu, Weihui; Jin, Shouguang

    2014-01-01

    Transcription Activator-Like Effector Nucleases (TALENs) are a novel class of sequence-specific nucleases that have recently gained prominence for its ease of production and high efficiency in genome editing. A TALEN pair recognizes specific DNA sequences and introduce double-strand break in the target site, triggering non-homologous end joining and homologous recombination. Current methods of TALEN delivery involves introduction of foreign genetic materials, such as plasmid DNA or mRNA, through transfection. Here, we show an alternative way of TALEN delivery, bacterial type III secretion system (T3SS) mediated direct injection of the TALEN proteins into human cells. Bacterially injected TALEN was shown to efficiently target host cell nucleus where it persists for almost 12 hours. Using a pair of TALENs targeting venus gene, such injected nuclear TALENs were shown functional in introducing DNA mutation in the target site. Interestingly, S-phase cells seem to show greater sensitivity to the TALEN mediated target gene modification. Accordingly, efficiency of such genome editing can easily be manipulated by the infection dose, number of repeated infections as well as enrichment of S phase cells. This work further extends the utility of T3SS in the delivery of functional proteins into mammalian cells to alter their characters for biomedical applications. PMID:24618838

  17. Structural and Dynamic Properties of the Human Prion Protein

    PubMed Central

    Chen, Wei; van der Kamp, Marc W.; Daggett, Valerie

    2014-01-01

    Prion diseases involve the conformational conversion of the cellular prion protein (PrPC) to its misfolded pathogenic form (PrPSc). To better understand the structural mechanism of this conversion, we performed extensive all-atom, explicit-solvent molecular-dynamics simulations for three structures of the wild-type human PrP (huPrP) at different pH values and temperatures. Residue 129 is polymorphic, being either Met or Val. Two of the three structures have Met in position 129 and the other has Val. Lowering the pH or raising the temperature induced large conformational changes of the C-terminal globular domain and increased exposure of its hydrophobic core. In some simulations, HA and its preceding S1-HA loop underwent large displacements. The C-terminus of HB was unstable and sometimes partially unfolded. Two hydrophobic residues, Phe-198 and Met-134, frequently became exposed to solvent. These conformational changes became more dramatic at lower pH or higher temperature. Furthermore, Tyr-169 and the S2-HB loop, or the X-loop, were different in the starting structures but converged to common conformations in the simulations for the Met-129, but not the Val-129, protein. α-Strands and β-strands formed in the initially unstructured N-terminus. α-Strand propensity in the N-terminus was different between the Met-129 and Val129 proteins, but β-strand propensity was similar. This study reveals detailed structural and dynamic properties of huPrP, providing insight into the mechanism of the conversion of PrPC to PrPSc. PMID:24606939

  18. Considerations in meeting protein needs of the human milk-fed preterm infant.

    PubMed

    Wagner, Julie; Hanson, Corrine; Anderson-Berry, Ann

    2014-08-01

    Preterm infants provided with sufficient nutrition to achieve intrauterine growth rates have the greatest potential for optimal neurodevelopment. Although human milk is the preferred feeding for preterm infants, unfortified human milk provides insufficient nutrition for the very low-birth-weight infant. Even after fortification with human milk fortifier, human milk often fails to meet the high protein needs of the smallest preterm infants, and additional protein supplementation must be provided. Although substantial evidence exists to support quantitative protein goals for human milk-fed preterm infants, the optimal type of protein for use in human milk fortification remains uncertain. This question was addressed through a PubMed literature search of prospective clinical trials conducted since 1990 in preterm or low-birth-weight infant populations. The following 3 different aspects of protein quality were evaluated: whey-to-casein ratio, hydrolyzed versus intact protein, and bovine milk protein versus human milk protein. Because of a scarcity of current studies conducted with fortified human milk, studies examining protein quality using preterm infant formulas were included to address certain components of the clinical question. Twenty-six studies were included in the review study. No definite advantage was found for any specific whey-to-casein ratio. Protein hydrolyzate products with appropriate formulations can support adequate growth and biochemical indicators of nutrition status and may reduce gastrointestinal transit time, gastroesophageal reflux events, and later incidence of atopic dermatitis in some infants. Plasma amino acid levels similar to those of infants fed exclusive human milk-based diets can be achieved with products composed of a mixture of bovine proteins, peptides, and amino acids formulated to replicate the amino acid composition of human milk. Growth and biochemical indicators of nutrition status are similar for infants fed human milk

  19. Locus heterogeneity disease genes encode proteins with high interconnectivity in the human protein interaction network.

    PubMed

    Keith, Benjamin P; Robertson, David L; Hentges, Kathryn E

    2014-01-01

    Mutations in genes potentially lead to a number of genetic diseases with differing severity. These disease genes have been the focus of research in recent years showing that the disease gene population as a whole is not homogeneous, and can be categorized according to their interactions. Locus heterogeneity describes a single disorder caused by mutations in different genes each acting individually to cause the same disease. Using datasets of experimentally derived human disease genes and protein interactions, we created a protein interaction network to investigate the relationships between the products of genes associated with a disease displaying locus heterogeneity, and use network parameters to suggest properties that distinguish these disease genes from the overall disease gene population. Through the manual curation of known causative genes of 100 diseases displaying locus heterogeneity and 397 single-gene Mendelian disorders, we use network parameters to show that our locus heterogeneity network displays distinct properties from the global disease network and a Mendelian network. Using the global human proteome, through random simulation of the network we show that heterogeneous genes display significant interconnectivity. Further topological analysis of this network revealed clustering of locus heterogeneity genes that cause identical disorders, indicating that these disease genes are involved in similar biological processes. We then use this information to suggest additional genes that may contribute to diseases with locus heterogeneity.

  20. The Protein Architecture of Human Secretory Vesicles Reveals Differential Regulation of Signaling Molecule Secretion by Protein Kinases

    PubMed Central

    Taupenot, Laurent; Ziegler, Michael; O'Connor, Daniel T.; Ma, Qi; Smoot, Michael; Ideker, Trey; Hook, Vivian

    2012-01-01

    Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the ‘human’ dense core secretory vesicles (DCSV) to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimer's disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health. PMID:22916103

  1. Protein kinase C translocation in human blood platelets

    SciTech Connect

    Wang, Hoauyan; Friedman, E. )

    1990-01-01

    Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 {mu}M concentrations did not induce redistribution of platelet PKC. Serotonin and the specific 5-HT{sub 2} receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI) but not the 5-HT{sub 1A} or 5-HT{sub 1B} agonists, ({plus minus}) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT{sub 2} receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT{sub 2} receptors.

  2. Human Articular Chondrocytes Express Multiple Gap Junction Proteins

    PubMed Central

    Mayan, Maria D.; Carpintero-Fernandez, Paula; Gago-Fuentes, Raquel; Martinez-de-Ilarduya, Oskar; Wang, Hong-Zhang; Valiunas, Virginijus; Brink, Peter; Blanco, Francisco J.

    2014-01-01

    Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas. PMID:23416160

  3. A radioimmunoassay for bone Gla protein (BGP) in human plasma.

    PubMed

    Johansen, J S; Mølholm Hansen, J E; Christiansen, C

    1987-03-01

    To study the value of bone Gla protein (BGP) as a biochemical marker of normal bone physiology and metabolic bone disorders, we have developed a radioimmunoassay (RIA) for the detection of BGP in human plasma. Antibodies were generated in rabbits immunized with purified calf BGP conjugated to thyroglobulin. Human plasma BGP reacted identically with the calf BGP standard, thus demonstrating the suitability of the assay to measure plasma BGP levels in man. The RIA is sensitive, accurate, and technically simple. Plasma BGP levels were determined in normal subjects (N = 35) and in patients with hypothyroidism (N = 10), hyperthyroidism (N = 22) and chronic renal failure (N = 35). The mean (+/- 1 SEM) concentration of plasma BGP in normal subjects was 1.27 +/- 0.07 nmol/l. Plasma BGP was significantly increased in patients with hyperthyroidism, 4.04 +/- 0.78 nmol/l (P less than 0.001) and chronic renal failure, 10.17 +/- 2.47 nmol/l (P less than 0.001). Low concentrations were found in patients with hypothyroidism, 0.74 +/- 0.11 nmol/l (P less than 0.01). Our studies indicate that plasma BGP provides a useful technique in the diagnosis of patients with bone disease.

  4. Human immune cell targeting of protein nanoparticles - caveospheres

    NASA Astrophysics Data System (ADS)

    Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

    2016-04-01

    Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

  5. Interaction and localization diversities of global and local hubs in human protein-protein interaction networks.

    PubMed

    Kiran, M; Nagarajaram, H A

    2016-08-16

    Hubs, the highly connected nodes in protein-protein interaction networks (PPINs), are associated with several characteristic properties and are known to perform vital roles in cells. We defined two classes of hubs, global (housekeeping) and local (tissue-specific) hubs. These two categories of hubs are distinct from each other with respect to their abundance, structure and function. However, how distinct are the spatial expression pattern and other characteristics of their interacting partners is still not known. Our investigations revealed that the partners of the local hubs compared with those of global hubs are conserved across the tissues in which they are expressed. Partners of local hubs show diverse subcellular localizations as compared with the partners of global hubs. We examined the nature of interacting domains in both categories of hubs and found that they are promiscuous in global hubs but not so in local hubs. Deletion of some of the local and global hubs has an impact on the characteristic path length of the network indicating that those hubs are inter-modular in nature. Our present study has, therefore, shed further light on the characteristic features of the local and global hubs in human PPIN. This knowledge of different topological aspects of hubs with regard to their types and subtypes is essential as it helps in better understanding of roles of hub proteins in various cellular processes under various conditions including those caused by host-pathogen interactions and therefore useful in prioritizing targets for drug design and repositioning.

  6. Self-similarity of human protein interaction networks: a novel strategy of distinguishing proteins

    PubMed Central

    Fadhal, Emad; Gamieldien, Junaid; Mwambene, Eric C.

    2015-01-01

    The successful determination of reliable protein interaction networks (PINs) in several species in the post-genomic era has hitherto facilitated the quest to understanding systems and structural properties of such networks. It is envisaged that a clearer understanding of their intrinsic topological properties would elucidate evolutionary and biological topography of organisms. This, in turn, may inform the understanding of diseases' aetiology. By analysing sub-networks that are induced in various layers identified by zones defined as distance from central proteins, we show that zones of human PINs display self-similarity patterns. What is observed at a global level is repeated at lower levels of inducement. Furthermore, it is observed that these levels of strength point to refinement and specialisations in these layers. This may point to the fact that various levels of representations in the self-similarity phenomenon offer a way of measuring and distinguishing the importance of proteins in the network. To consolidate our findings, we have also considered a gene co-expression network and a class of gene regulatory networks in the same framework. In all cases, the phenomenon is significantly evident. In particular, the truly unbiased regulatory networks show finer level of articulation of self-similarity. PMID:25720740

  7. Interaction and localization diversities of global and local hubs in human protein-protein interaction networks.

    PubMed

    Kiran, M; Nagarajaram, H A

    2016-08-16

    Hubs, the highly connected nodes in protein-protein interaction networks (PPINs), are associated with several characteristic properties and are known to perform vital roles in cells. We defined two classes of hubs, global (housekeeping) and local (tissue-specific) hubs. These two categories of hubs are distinct from each other with respect to their abundance, structure and function. However, how distinct are the spatial expression pattern and other characteristics of their interacting partners is still not known. Our investigations revealed that the partners of the local hubs compared with those of global hubs are conserved across the tissues in which they are expressed. Partners of local hubs show diverse subcellular localizations as compared with the partners of global hubs. We examined the nature of interacting domains in both categories of hubs and found that they are promiscuous in global hubs but not so in local hubs. Deletion of some of the local and global hubs has an impact on the characteristic path length of the network indicating that those hubs are inter-modular in nature. Our present study has, therefore, shed further light on the characteristic features of the local and global hubs in human PPIN. This knowledge of different topological aspects of hubs with regard to their types and subtypes is essential as it helps in better understanding of roles of hub proteins in various cellular processes under various conditions including those caused by host-pathogen interactions and therefore useful in prioritizing targets for drug design and repositioning. PMID:27400769

  8. Nuclear Localization and DNA Binding Properties of a Protein Expressed by Human c-myc Oncogene

    NASA Astrophysics Data System (ADS)

    Persson, Hakan; Leder, Philip

    1984-08-01

    Antisera to the human cellular myc oncogene product were used to identify a human c-myc specific protein with a molecular weight of 65,000. Subcellular fractionation showed that the human c-myc protein is predominantly found in the cell nucleus. The p65 Kc-myc protein binds to double- and single-stranded DNA as measured by a DNA affinity chromatography assay.

  9. The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

    PubMed

    Mirshafiee, Vahid; Kim, Raehyun; Mahmoudi, Morteza; Kraft, Mary L

    2016-06-01

    Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo.

  10. Functional annotation of the human chromosome 7 "missing" proteins: a bioinformatics approach.

    PubMed

    Ranganathan, Shoba; Khan, Javed M; Garg, Gagan; Baker, Mark S

    2013-06-01

    The chromosome-centric human proteome project aims to systematically map all human proteins, chromosome by chromosome, in a gene-centric manner through dedicated efforts from national and international teams. This mapping will lead to a knowledge-based resource defining the full set of proteins encoded in each chromosome and laying the foundation for the development of a standardized approach to analyze the massive proteomic data sets currently being generated. The neXtProt database lists 946 proteins as the human proteome of chromosome 7. However, 170 (18%) proteins of human chromosome 7 have no evidence at the proteomic, antibody, or structural levels and are considered "missing" in this study as they lack experimental support. We have developed a protocol for the functional annotation of these "missing" proteins by integrating several bioinformatics analysis and annotation tools, sequential BLAST homology searches, protein domain/motif and gene ontology (GO) mapping, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Using the BLAST search strategy, homologues for reviewed non-human mammalian proteins with protein evidence were identified for 90 "missing" proteins while another 38 had reviewed non-human mammalian homologues. Putative functional annotations were assigned to 27 of the remaining 43 novel proteins. Proteotypic peptides have been computationally generated to facilitate rapid identification of these proteins. Four of the "missing" chromosome 7 proteins have been substantiated by the ENCODE proteogenomic peptide data.

  11. HPLC-DAD protein kinase inhibitor analysis in human serum.

    PubMed

    Dziadosz, Marek; Lessig, Rüdiger; Bartels, Heidemarie

    2012-04-15

    We here describe an HPLC-DAD method to analyse different protein kinase inhibitors. Potential applications of this method are pharmacokinetic studies and therapeutic drug monitoring. Optimised chromatography conditions resulted in a very good separation of seven inhibitors (vatalanib, bosutinib, canertinib, tandutinib, pazopanib, dasatinib - internal standard and erlotinib). The good sensitivity makes this method competitive with LC/MS/MS. The separation was performed with a Lichrospher 100-5 RP8, 250 mm × 4 mm column maintained at 30 ± 1 °C, and with a mobile phase of 0.05 M H(3)PO(4)/KH(2)PO(4) (pH=2.3)-acetonitrile (7:3, v/v) at a flow rate of 0.7 mL/min. A simple and fast sample preparation sequence with liquid-liquid extraction led to good recoveries (73-90%) of all analytes. The recovery hardly reached 50% only for pazopanib. This method can also be used for targeted protein kinase inhibitor quantification. A perfect linearity in the validated range (20-10,000 ng/mL) and an LOQ of 20 ng/mL were achieved. The relative standard deviations and accuracies of all examined drug concentrations gave values much lower than 15% both for between- and within-batch calculations. All analysed PKIs were stable for 6 months in a 1mg/mL dimethyl sulfoxide stock solution. Vatalanib, bosutinib and erlotinib were also stable in human serum in the whole examined concentration range. PMID:22425385

  12. A human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus

    PubMed Central

    1989-01-01

    In vitro infection by the human immunodeficiency virus (HIV) of CD4+ H9 lymphoblasts is inhibited by a mannose-binding protein (MBP) purified from human serum. In addition, MBP is able to selectively bind to HIV- infected H9 cells and HIV-infected cells from the monocyte cell line U937. These results indicate MBP most likely recognizes high mannose glycans known to be present on gp120 in the domain that is recognized by CD4 and thereby inhibits viral entry to susceptible cells. In support of this contention, recombinant gp120 binds directly to MBP; the binding is saturable, mannan inhibitable, removed by N-glycanase treatment, and dependent on divalent cations. PMID:2909656

  13. Human Spermatozoa as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project.

    PubMed

    Jumeau, Fanny; Com, Emmanuelle; Lane, Lydie; Duek, Paula; Lagarrigue, Mélanie; Lavigne, Régis; Guillot, Laëtitia; Rondel, Karine; Gateau, Alain; Melaine, Nathalie; Guével, Blandine; Sergeant, Nicolas; Mitchell, Valérie; Pineau, Charles

    2015-09-01

    The Chromosome-Centric Human Proteome Project (C-HPP) aims at cataloguing the proteins as gene products encoded by the human genome in a chromosome-centric manner. The existence of products of about 82% of the genes has been confirmed at the protein level. However, the number of so-called "missing proteins" remains significant. It was recently suggested that the expression of proteins that have been systematically missed might be restricted to particular organs or cell types, for example, the testis. Testicular function, and spermatogenesis in particular, is conditioned by the successive activation or repression of thousands of genes and proteins including numerous germ cell- and testis-specific products. Both the testis and postmeiotic germ cells are thus promising sites at which to search for missing proteins, and ejaculated spermatozoa are a potential source of proteins whose expression is restricted to the germ cell lineage. A trans-chromosome-based data analysis was performed to catalog missing proteins in total protein extracts from isolated human spermatozoa. We have identified and manually validated peptide matches to 89 missing proteins in human spermatozoa. In addition, we carefully validated three proteins that were scored as uncertain in the latest neXtProt release (09.19.2014). A focus was then given to the 12 missing proteins encoded on chromosomes 2 and 14, some of which may putatively play roles in ciliation and flagellum mechanistics. The expression pattern of C2orf57 and TEX37 was confirmed in the adult testis by immunohistochemistry. On the basis of transcript expression during human spermatogenesis, we further consider the potential for discovering additional missing proteins in the testicular postmeiotic germ cell lineage and in ejaculated spermatozoa. This project was conducted as part of the C-HPP initiatives on chromosomes 14 (France) and 2 (Switzerland). The mass spectrometry proteomics data have been deposited with the Proteome

  14. The Hexameric Structures of Human Heat Shock Protein 90

    PubMed Central

    Lee, Cheng-Chung; Lin, Ta-Wei; Ko, Tzu-Ping; Wang, Andrew H.-J.

    2011-01-01

    Background The human 90-kDa heat shock protein (HSP90) functions as a dimeric molecular chaperone. HSP90 identified on the cell surface has been found to play a crucial role in cancer invasion and metastasis, and has become a validated anti-cancer target for drug development. It has been shown to self-assemble into oligomers upon heat shock or divalent cations treatment, but the functional role of the oligomeric states in the chaperone cycle is not fully understood. Principal Findings Here we report the crystal structure of a truncated HSP90 that contains the middle segment and the carboxy-terminal domain, termed MC-HSP90. The structure reveals an architecture with triangular bipyramid geometry, in which the building block of the hexameric assembly is a dimer. In solution, MC-HSP90 exists in three major oligomer states, namely dimer, tetramer and hexamer, which were elucidated by size exclusion chromatography and analytical ultracentrifugation. The newly discovered HSP90 isoform HSP90N that lacks the N-terminal ATPase domain also exhibited similar oligomerization states as did MC-HSP90. Conclusions While lacking the ATPase domain, both MC-HSP90 and HSP90N can self-assemble into a hexameric structure, spontaneously. The crystal structure of MC-HSP90 reveals that, in addition to the C-terminal dimerization domain, the residue W320 in the M domain plays a critical role in its oligomerization. This study not only demonstrates how the human MC-HSP90 forms a hexamer, but also justifies the similar formation of HSP90N by using 3D modeling analysis. PMID:21647436

  15. Conversion of human choriogonadotropin into a follitropin by protein engineering.

    PubMed

    Campbell, R K; Dean-Emig, D M; Moyle, W R

    1991-02-01

    Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the alpha subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their beta subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG beta-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH beta and hFSH beta in receptor recognition and activation. Since the beta subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH beta subunits, coexpressed them with the human alpha subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH beta residues 33-52 for hCG beta residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG beta residues 108-145 or substitution of residues in the "determinant loop" located between hCG beta residues 93 and 100. PMID:1899483

  16. Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

    PubMed Central

    Kampf, Caroline; Olsson, IngMarie; Ryberg, Urban; Sjöstedt, Evelina; Pontén, Fredrik

    2012-01-01

    The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 μm thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (www.proteinatlas.org) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins

  17. Identification of human liver microsomal proteins adducted by a reactive metabolite using shotgun proteomics.

    PubMed

    Yang, Yanou; Xiao, Qing; Humphreys, W Griffith; Dongre, Ashok; Shu, Yue-Zhong

    2014-09-15

    Covalent modification of cellular proteins by chemically reactive compounds/metabolites has the potential to disrupt biological function and elicit serious adverse drug reactions. Information on the nature and binding patterns of protein targets are critical toward understanding the mechanism of drug induced toxicity. Protein covalent binding studies established in liver microsomes can quantitively estimate the extent of protein modification, but they provide little information on the nature of the modified proteins. In this article, we describe a label-free shotgun proteomic workflow for the identification of target proteins modified in situ by reactive metabolites in human liver microsome incubations. First, we developed a shotgun proteomic workflow for the characterization of the human liver microsomal subproteome, which consists of predominately membrane-bound proteins. Human liver microsomes were solubilized with a combination of MS-compatible organic solvents followed by protein reduction, alkylation, and tryptic digestion. The unmodified samples were analyzed by UHPLC-MS/MS, and the proteins were identified by database searching. This workflow led to the successful identification of 329 human liver microsomal subproteome proteins with 1% FDR (false discovery rate). The same method was then applied to identify the modifications of human liver microsomal proteins by a known reactive metabolite 2-(methylsulfonyl)benzo[d]thiazole (2), either after incubation directly with 2 or with its parent compound 2-(methylthio)benzo[d]thiazole (1). A total of 19 modified constituent peptides which could be mapped to 18 proteins were identified in human liver microsomes incubated directly with 2. Among these, 5 modified constituent peptides which could be mapped to 4 proteins were identified in incubation with 1, which is known to generate 2 in human liver microsomal incubations. This label-free workflow is generally applicable to the identification and characterization of

  18. Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria.

    PubMed

    Huang, Jing; Li, Xiangming; Coelho-dos-Reis, Jordana G A; Zhang, Min; Mitchell, Robert; Nogueira, Raquel Tayar; Tsao, Tiffany; Noe, Amy R; Ayala, Ramses; Sahi, Vincent; Gutierrez, Gabriel M; Nussenzweig, Victor; Wilson, James M; Nardin, Elizabeth H; Nussenzweig, Ruth S; Tsuji, Moriya

    2015-12-01

    In this study, we developed human immune system (HIS) mice that possess functional human CD4+ T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4+ T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4+ T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4+ T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4+ T cell responses both specific for a human malaria antigen.

  19. Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria.

    PubMed

    Huang, Jing; Li, Xiangming; Coelho-dos-Reis, Jordana G A; Zhang, Min; Mitchell, Robert; Nogueira, Raquel Tayar; Tsao, Tiffany; Noe, Amy R; Ayala, Ramses; Sahi, Vincent; Gutierrez, Gabriel M; Nussenzweig, Victor; Wilson, James M; Nardin, Elizabeth H; Nussenzweig, Ruth S; Tsuji, Moriya

    2015-12-01

    In this study, we developed human immune system (HIS) mice that possess functional human CD4+ T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4+ T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4+ T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4+ T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4+ T cell responses both specific for a human malaria antigen. PMID:26410104

  20. Sustained release emphasizing recombinant human bone morphogenetic protein-2.

    PubMed

    Hollinger; Uludag; Winn

    1998-05-01

    Bone homeostasis is a dynamic process involving a myriad of cells and substrates modulated by regulatory signals such as hormones, growth and differentiating factors. When this environment is damaged, the regenerative sequalae follows a programmed pattern, and the capacity for successful recovery is often dependent on the extent of the injury. Many bony deficits that are excessively traumatic will not result in complete recovery and require therapeutic intervention(s) such as autografting or grafting from banked bone. However, for numerous reasons, an unacceptably high rate of failure is associated with these conventional therapies. Thus, alternative approaches are under investigation. A class of osteogenic regulatory molecules, the bone morphogenetic proteins (BMPs), have been isolated, cloned and characterized as potent supplements to augment bone regeneration. Optimizing a therapeutic application for BMPs may be dependent upon localized sustained release which in kind relies on a safe and well characterized carrier system. This review will discuss the current status of BMPs in bone regeneration and specifically will present the potential for a clinical therapeutic role of recombinant human BMP-2 sustained release carrier systems. PMID:10837631

  1. C-reactive protein in human atherogenesis: facts and fiction.

    PubMed

    Zimmermann, Oliver; Li, Kefei; Zaczkiewicz, Myron; Graf, Matthias; Liu, Zhongmin; Torzewski, Jan

    2014-01-01

    The role of C-reactive protein (CRP) in atherosclerosis is controversially discussed. Whereas initial experimental studies suggested a pathogenic role for CRP in atherogenesis, more recent genetic data from Mendelian randomization trials failed to provide evidence for a causative role of CRP in cardiovascular disease. Also, experimental results from laboratories all over the world were indeed contradictory, partly because of species differences in CRP biology and partly because data were not accurately evaluated. Here we summarize the published data from experimental work with mainly human material in order to avoid confusion based on species differences in CRP biology. Experimental work needs to be reevaluated after reconsideration of some traditional rules in research: (1) in order to understand a molecule's role in disease it may be helpful to be aware of its role in physiology; (2) it is necessary to define the disease entity that experimental CRP research deals with; (3) the scientific consensus is as follows: do not try to prove your hypothesis. Specific CRP inhibition followed by use of CRP inhibitors in controlled clinical trials may be the only way to prove or disprove a causative role for CRP in cardiovascular disease. PMID:24799767

  2. Expression of bone morphogenetic proteins of human neoplastic epithelial cells.

    PubMed

    Hatakeyama, S; Gao, Y H; Ohara-Nemoto, Y; Kataoka, H; Satoh, M

    1997-07-01

    Bone morphogenetic proteins (BMPs) are crucial factors of osteogenesis. We investigated the expressions of BMP subtypes in human salivary adenocarcinoma cell line (HSG-S8), tongue squamous cell (HSC-4) and gingival squamous cell (Ca9-22) carcinoma cell lines, gastric poorly differentiated adenocarcinoma cell (MNK45) and signet ring cell (KATOIII) carcinoma cell lines, rectal adenocarcinoma (RCM-1, RCM-2, and RCM-3), and thyroid (8505C) and bladder (T24) carcinoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR disclosed that BMP-1 was expressed in all cell lines examined, and BMP-2 was amplified in almost all cells except MKN45. Two squamous cell carcinomas, HSC-4 and Ca9-22, and KATOIII expressed only BMP-1 and BMP-2. MKN45 did not express BMP-2, but expressed BMP-7 and weakly BMP-4 and BMP-5. In addition to the expression BMP-7, and HSG-S8 expressed BMP-6. These findings indicated that the neoplastic epithelial cells possessed a rather great potency to express BMP mRNAs. On the other hand, among these carcinoma cells, HSG-S8 solely induced bone in nude mouse tumors, and HSC-4 and KATOIII contained many calcified masses in tumors while the rest did not induce either. PMID:9247707

  3. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins

    PubMed Central

    Bezawork-Geleta, Ayenachew; Brodie, Erica J.; Dougan, David A.; Truscott, Kaye N.

    2015-01-01

    Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPRmt). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPRmt. Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPRmt, this pathway may preferentially act to promote chaperone mediated refolding of proteins. PMID:26627475

  4. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins.

    PubMed

    Bezawork-Geleta, Ayenachew; Brodie, Erica J; Dougan, David A; Truscott, Kaye N

    2015-12-02

    Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPR(mt)). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPR(mt). Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPR(mt), this pathway may preferentially act to promote chaperone mediated refolding of proteins.

  5. A zinc protein isolated from human parotid saliva.

    PubMed Central

    Henkin, R I; Lippoldt, R E; Bilstad, J; Edelhoch, H

    1975-01-01

    A zinc protein has been isolated and purified to apparent homogeneity from subjects with normal taste acuity by gel filtration and ion-exchange chromatography. The protein has a molecular weight of 37,000 and does not appear to have subunits. It is composed of 8% histidine residues and has 2 moles of zinc per mole of protein. Images PMID:1054831

  6. Development of Human Muscle Protein Measurement with MRI

    NASA Technical Reports Server (NTRS)

    Lin, Chen; Evans, Harlan; Leblanc, Adrian D.

    1997-01-01

    It is known that micro-gravity has a strong influence on the human musculoskeletal system. A number of studies have shown that significant changes in skeletal muscles occur in both space flight and bedrest simulation. In our 5 week bedrest study, the cross-sectional area of soleus-gastrocnemius decreased about 12% while the cross-sectional area of anterior calf muscles decreased about 4%. Using volume measurements, these losses increased after 17 weeks to approximately 30% and 21% respectively. Significant muscle atrophy was also found on the SL-J crew members after only 8 days in space. It is important that these effects are fully understood so that countermeasures can be developed. The same knowledge might also be useful in preventing muscle atrophy related to other medical problems. A major problem with anatomical measurements of muscle during bed rest and microgravity is the influence of fluid shifts and water balance on the measurement of muscle volume, especially when the exposure duration is short and the atrophy is relatively small. Fluid shifts were documented in Skylab by visual observations of blood vessel distention, rapid changes in limb volume, center of mass measurements and subjective descriptions such as puffy faces and head fullness. It has been reported that the muscle water content of biopsied soleus muscles decreased following 8 hours of head down tilt bed rest. Three aspects of fluid shifts that can affect volume measurements are: first, the shift of fluid that occurs whenever there is a change from upright to a recumbent position and vice versa; second, the potential for fluid accumulation in the lower limbs resulting from muscle damage caused by overextending atrophied muscle or swelling caused by deconditioned precapillary sphincter muscles during reambulation; third, the net change of hydration level during and after bed rest or spaceflight. Because of these transitory fluid shifts, muscle protein is expected to represent muscle capacity

  7. Amino acid sequence of the encephalitogenic basic protein from human myelin

    PubMed Central

    Carnegie, P. R.

    1971-01-01

    Myelin from the central nervous system contains an unusual basic protein, which can induce experimental autoimmune encephalomyelitis. The basic protein from human brain was digested with trypsin and other enzymes and the sequence of the 170 amino acids was determined. The localization of the encephalitogenic determinants was described. Possible roles for the protein in the structure and function of myelin are discussed. PMID:4108501

  8. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    SciTech Connect

    Yamakoshi, Takako; Makino, Teruhiko; Ur Rehman, Mati; Yoshihisa, Yoko; Sugimori, Michiya; Shimizu, Tadamichi

    2013-03-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.

  9. The human mitochondrial genome may code for more than 13 proteins.

    PubMed

    Capt, Charlotte; Passamonti, Marco; Breton, Sophie

    2016-09-01

    The human mitochondrial (mt) DNA is commonly described as a small, maternally inherited molecule that encodes 13 protein components of the oxidative phosphorylation system and 24 structural RNAs required for their translation. However, recent studies indicate that the human mtDNA has a larger functional repertoire than previously believed. This paper briefly summarizes these studies, which suggest to reconsider our way to describe the human mitochondrial DNA as it may code for more than 13 proteins.

  10. CD19xCD3 DART protein mediates human B-cell depletion in vivo in humanized BLT mice

    PubMed Central

    Tsai, Perry; Thayer, William O; Liu, Liqin; Silvestri, Guido; Nordstrom, Jeffrey L; Garcia, J Victor

    2016-01-01

    Novel therapeutic strategies are needed for the treatment of hematologic malignancies; and bispecific antibody-derived molecules, such as dual-affinity re-targeting (DART) proteins, are being developed to redirect T cells to kill target cells expressing tumor or viral antigens. Here we present our findings of specific and systemic human B-cell depletion by a CD19xCD3 DART protein in humanized BLT mice. Administration of the CD19xCD3 DART protein resulted in a dramatic sustained depletion of human CD19+ B cells from the peripheral blood, as well as a dramatic systemic reduction of human CD19+ B-cell levels in all tissues (bone marrow, spleen, liver, lung) analyzed. When human CD8+ T cells were depleted from the mice, no significant B-cell depletion was observed in response to CD19xCD3 DART protein treatment, confirming that human CD8+ T cells are the primary effector cells in this in vivo model. These studies validate the use of BLT humanized mice for the in vivo evaluation and preclinical development of bispecific molecules that redirect human T cells to selectively deplete target cells. PMID:27119115

  11. Unconventional actins and actin-binding proteins in human protozoan parasites.

    PubMed

    Gupta, C M; Thiyagarajan, S; Sahasrabuddhe, A A

    2015-06-01

    Actin and its regulatory proteins play a key role in several essential cellular processes such as cell movement, intracellular trafficking and cytokinesis in most eukaryotes. While these proteins are highly conserved in higher eukaryotes, a number of unicellular eukaryotic organisms contain divergent forms of these proteins which have highly unusual biochemical and structural properties. Here, we review the biochemical and structural properties of these unconventional actins and their core binding proteins which are present in commonly occurring human protozoan parasites.

  12. Detection of the human endogenous retrovirus ERV3-encoded Env-protein in human tissues using antibody-based proteomics

    PubMed Central

    Fei, Chen; Atterby, Christina; Edqvist, Per-Henrik; Pontén, Fredrik; Zhang, Wei Wei; Larsson, Erik; Ryan, Frank P

    2014-01-01

    Objectives There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. Design Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. Participants Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. Setting The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. Main Outcome Measures The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. Results We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. Conclusion This technique can now be extended to the study

  13. Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface

    PubMed Central

    Marín, Elvira; Parra-Giraldo, Claudia M.; Hernández-Haro, Carolina; Hernáez, María L.; Nombela, César; Monteoliva, Lucía; Gil, Concha

    2015-01-01

    Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in

  14. Fluorescence energy transfer monitoring of protein-protein interaction in human cells: the Cyclin T1-HIV1 Tat case.

    NASA Astrophysics Data System (ADS)

    Ferrari, Aldo; Cinelli, Riccardo A. G.; Pellegrini, Vittorio; Beltram, Fabio; Marcello, Alessandro; Tyagi, Mudit; Giacca, Mauro

    2001-03-01

    The human immunodeficiency virus type 1 (HIV-1) Tat protein promotes transcriptional elongation of viral RNAs. Here we show that human Cyclin T1 directly binds Tat in cultured cells. By mapping fluorescence resonance energy transfer (FRET) in different cellular compartments we shall present a quantitative analysis of this interaction. The matched tagging pair consists of two optically matched variants of the green fluorescent protein: the enhanced GFP and the blue fluorescent protein. Strong energy transfer was observed between Cyclin T1 and Tat both in the cytoplasm and in specific subnuclear regions. We shall argue that such high-resolution optical studies can provide significant new insight in molecular processes and demonstrate that, for the specific case-study presented, they lead to a model by which Tat recruits Cyclin T1 out of the nuclear compartments where the protein resides to promote transcriptional activation.

  15. Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia.

    PubMed

    Cox, Brian; Sharma, Parveen; Evangelou, Andreas I; Whiteley, Kathie; Ignatchenko, Vladimir; Ignatchenko, Alex; Baczyk, Dora; Czikk, Marie; Kingdom, John; Rossant, Janet; Gramolini, Anthony O; Adamson, S Lee; Kislinger, Thomas

    2011-12-01

    Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent

  16. Toward an understanding of the protein interaction network of the human liver

    PubMed Central

    Wang, Jian; Huo, Keke; Ma, Lixin; Tang, Liujun; Li, Dong; Huang, Xiaobi; Yuan, Yanzhi; Li, Chunhua; Wang, Wei; Guan, Wei; Chen, Hui; Jin, Chaozhi; Wei, Junchen; Zhang, Wanqiao; Yang, Yongsheng; Liu, Qiongming; Zhou, Ying; Zhang, Cuili; Wu, Zhihao; Xu, Wangxiang; Zhang, Ying; Liu, Tao; Yu, Donghui; Zhang, Yaping; Chen, Liang; Zhu, Dewu; Zhong, Xing; Kang, Lixin; Gan, Xiang; Yu, Xiaolan; Ma, Qi; Yan, Jing; Zhou, Li; Liu, Zhongyang; Zhu, Yunping; Zhou, Tao; He, Fuchu; Yang, Xiaoming

    2011-01-01

    Proteome-scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein–protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two-hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver-specific, liver-phenotype and liver-disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver-specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver. PMID:21988832

  17. Regulation of human genome expression and RNA splicing by human papillomavirus 16 E2 protein.

    PubMed

    Gauson, Elaine J; Windle, Brad; Donaldson, Mary M; Caffarel, Maria M; Dornan, Edward S; Coleman, Nicholas; Herzyk, Pawel; Henderson, Scott C; Wang, Xu; Morgan, Iain M

    2014-11-01

    Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome.

  18. The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.

    PubMed Central

    Baker, R T; Board, P G

    1991-01-01

    Complementary DNA clones encoding ubiquitin fused to a 52 amino acid tail protein were isolated from human placental and adrenal gland cDNA libraries. The deduced human 52 amino acid tail protein is very similar to the homologous protein from other species, including the conservation of the putative metal-binding, nucleic acid-binding domain observed in these proteins. Northern blot analysis with a tail-specific probe indicated that the previously identified UbA mRNA species most likely represents comigrating transcripts of the 52 amino acid tail (UbA52) and 80 amino acid tail (UbA80) ubiquitin fusion genes. The UbA52 gene was isolated from a human genomic library and consists of five exons distributed over 3400 base pairs. One intron is in the 5' non-coding region, two interrupt the single ubiquitin coding unit, and the fourth intron is within the tail coding region. Several members of the Alu family of repetitive DNA are associated with the gene. The UbA52 promoter has several features in common with mammalian ribosomal protein genes, including its location in a CpG-rich island, initiation of transcription within a polypyrimidine tract, the lack of a consensus TATA motif, and the presence of Sp1 binding sites, observations that are consistent with the recent identification of the ubiquitin-free tail proteins as ribosomal proteins. Thus, in spite of its unusual feature of being translationally fused to ubiquitin, the 52 amino acid tail ribosomal protein is expressed from a structurally typical ribosomal protein gene. Images PMID:1850507

  19. Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

    SciTech Connect

    Lee, Ji Young; Park, Kyoung sook; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Sang Ki; Lee, Sang Do; Park, Jin Bong; Chang, Seok Jong; Jeon, Byeong Hwa

    2011-07-01

    Highlights: {yields} We have developed an E. coli protein expression vector including human specific gene sequences for protein cellular delivery. {yields} The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence. {yields} HOXA5-APE1/Ref-1 inhibited TNF-alpha-induced monocyte adhesion to endothelial cells. {yields} Human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. -- Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including human specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 {sup o}C was greater than in 4 {sup o}C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-{alpha}-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.

  20. Protein-DNA interactions in the promoter region of the amyloid precursor protein (APP) gene in human neocortex.

    PubMed

    Lukiw, W J; Rogaev, E I; Wong, L; Vaula, G; McLachlan, D R; St George Hyslop, P

    1994-03-01

    We have investigated protein-DNA interactions in the proximal promoter of the human amyloid precursor protein (APP) gene in temporal lobe neocortical nuclei isolated from control and Alzheimer disease (AD) affected brains. We report that the human APP 5' promoter sequence from -203 to +55 bp, which has been previously reported to contain essential regulatory elements for APP gene transcription, lies in a deoxyribonuclease I, micrococcal nuclease- and restriction endonuclease-sensitive, G+C-rich nucleosome-free gap flanked both 5' and 3' by typical nucleosome structures. As analyzed by electrophoretic mobility shift assay, this extended internucleosomal linker DNA is heavily occupied by nuclear protein factors, and interacts differentially with nuclear protein extracts obtained from HeLa and human brain neocortical nuclei. This suggests that the chromatin conformation of the APP gene promoter may vary in different cell types, and may correlate with differences in APP gene expression. Human recombinant transcription factors AP1, SP1 and TFIID (but not AP2 or brain histones H1, H2B and H4) interact with the -203 to +55 bp of the human APP promoter sequence. Only minor differences were observed in the chromatin structure of the immediate APP promoter between non-AD and AD affected neocortical nuclei, suggesting either that post-transcriptional processes, or that regulatory elements lying elsewhere in the APP gene may be important in the aberrant accumulation of the APP gene product.

  1. Expression of Human DNAJ (Heat Shock Protein-40) B3 in Humanized UDP-glucuronosyltransferase 1 Mice

    PubMed Central

    Mitsugi, Ryo; Itoh, Tomoo; Fujiwara, Ryoichi

    2015-01-01

    The human DNAJB3 gene encodes a DNAJ (Heat shock protein 40; Hsp40) homolog, subfamily B, member 3 chaperone protein (DNAJB3), which can be down-regulated in disease conditions, as observed in decreased expression of DNAJB3 mRNA in peripheral blood mononuclear cells (PBMC) of obese patients. Recently, humanized UDP-glucuronosyltransferase (UGT) 1 mice (hUGT1 mice) were developed, in which the introduced human UGT1 gene contained a gene encoding human DNAJB3. In the present study, we analyzed the expression of human DNAJB3 mRNA in hUGT1 mice. Among the examined tissues, the testis had the highest expression of human DNAJB3 mRNA, while the lowest expression was observed in the liver. We found that the pattern of tissue-specific expression of mouse Dnajb3 in hUGT1 mice was very similar to that of human DNAJB3. We further demonstrated that the expression of human DNAJB3 in the liver was significantly reduced in high-fat-diet-fed hUGT1 mice compared to the expression level in the control mice, indicating that the expression of human DNAJB3 in hUGT1 mice could be similarly regulated in disease conditions such as obesity. Humanized UGT1 mice might therefore be useful to investigate the physiological role of human DNAJB3 in vivo. PMID:26147428

  2. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein.

    PubMed

    Buffalo, Cosmo Z; Bahn-Suh, Adrian J; Hirakis, Sophia P; Biswas, Tapan; Amaro, Rommie E; Nizet, Victor; Ghosh, Partho

    2016-01-01

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ∼90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant 'reading head' in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M-C4BP interaction, and also inform a path towards vaccine design. PMID:27595425

  3. Drug screening strategy for human membrane proteins: from NMR protein backbone structure to in silica- and NMR-screened hits

    PubMed Central

    Lindert, Steffen; Maslennikov, Innokentiy; Chiu, Ellis; Pierce, Levi C; McCammon, J. Andrew; Choe, Senyon

    2015-01-01

    About 8,000 genes encode membrane proteins in the human genome. The information about their druggability will be very useful to facilitate drug discovery and development. The main problem, however, consists of limited structural and functional information about these proteins because they are difficult to produce biochemically and to study. In this paper we describe the strategy that combines Cell-free protein expression, NMR spectroscopy, and molecular DYnamics simulation (CNDY) techniques. Results of a pilot CNDY experiment provide us with a guiding light towards expedited identification of the hit compounds against a new uncharacterized membrane protein as a potentially druggable target. These hits can then be further characterized and optimized to develop the initial lead compound quicker. We illustrate such “omics” approach for drug discovery with the CNDY strategy applied to two example proteins: hypoxia-induced genes HIGD1A and HIGD1B. PMID:24525125

  4. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    PubMed

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

  5. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin.

    PubMed

    Yamakoshi, Takako; Makino, Teruhiko; Ur Rehman, Mati; Yoshihisa, Yoko; Sugimori, Michiya; Shimizu, Tadamichi

    2013-03-01

    Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes. PMID:23376073

  6. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  7. Interaction between human BAP31 and respiratory syncytial virus small hydrophobic (SH) protein

    SciTech Connect

    Li, Yan; Jain, Neeraj; Limpanawat, Suweeraya; To, Janet; Quistgaard, Esben M.; Nordlund, Par; Thanabalu, Thirumaran; Torres, Jaume

    2015-08-15

    The small hydrophobic (SH) protein is a short channel-forming polypeptide encoded by the human respiratory syncytial virus (hRSV). Deletion of SH protein leads to the viral attenuation in mice and primates, and delayed apoptosis in infected cells. We have used a membrane-based yeast two-hybrid system (MbY2H) and a library from human lung cDNA to detect proteins that bind SH protein. This led to the identification of a membrane protein, B-cell associated protein 31 (BAP31). Transfected SH protein co-localizes with transfected BAP31 in cells, and pulls down endogenous BAP31. Titration of purified C-terminal endodomain of BAP31 against isotopically labeled SH protein in detergent micelles suggests direct interaction between the two proteins. Given the key role of BAP31 in protein trafficking and its critical involvement in pro- and anti-apoptotic pathways, this novel interaction may constitute a potential drug target. - Highlights: • A yeast two-hybrid system (MbY2H) detected BAP31 as a binder of RSV SH protein. • Transfected SH and BAP31 co-localize in lung epithelial cells. • Endogenous BAP31 is pulled down by RSV SH protein. • BAP31 endodomain interacts with the N-terminal α-helix of SH protein in micelles. • This interaction is proposed to be a potential drug target.

  8. Secretory Expression and Purification of Respiratory Syncytial Virus G and F Proteins in Human Cells.

    PubMed

    Jadhao, Samadhan J; Anderson, Larry J

    2016-01-01

    Respiratory syncytial virus (RSV) is one of the leading causes of range of symptoms from mild upper to serious lower respiratory virus infections in infants, immunocompromised individuals, and the elderly. Despite many decades of research and development, a licensed RSV vaccine is not available for use in human. Since the RSV F and G proteins induce neutralizing antibodies and confer protection from infection, they are important for understanding disease and for developing vaccines and access to purified, expressed proteins is important to RSV research and diagnostics. We describe methods to produce recombinant RSV F and G proteins in human cells and purify these proteins using Ni Sepharose affinity chromatography. PMID:27464687

  9. Identification of proteins sensitive to thermal stress in human neuroblastoma and glioma cell lines.

    PubMed

    Xu, Guilian; Stevens, Stanley M; Kobeissy, Firas; Kobiessy, Firas; Brown, Hilda; McClung, Scott; Gold, Mark S; Borchelt, David R

    2012-01-01

    Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y) and glial (CCF-STTG1) lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48) residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress. PMID:23145051

  10. Structural Bioinformatics Inspection of neXtProt PE5 Proteins in the Human Proteome.

    PubMed

    Dong, Qiwen; Menon, Rajasree; Omenn, Gilbert S; Zhang, Yang

    2015-09-01

    One goal of the Human Proteome Project is to identify at least one protein product for each of the ∼20,000 human protein-coding genes. As of October 2014, however, there are 3564 genes (18%) that have no or insufficient evidence of protein existence (PE), as curated by neXtProt; these comprise 2647 PE2-4 missing proteins and 616 PE5 dubious protein entries. We conducted a systematic examination of the 616 PE5 protein entries using cutting-edge protein structure and function modeling methods. Compared to a random sample of high-confidence PE1 proteins, the putative PE5 proteins were found to be over-represented in the membrane and cell surface proteins and peptides fold families. Detailed functional analyses show that most PE5 proteins, if expressed, would belong to transporters and receptors localized in the plasma membrane compartment. The results suggest that experimental difficulty in identifying membrane-bound proteins and peptides could have precluded their detection in mass spectrometry and that special enrichment techniques with improved sensitivity for membrane proteins could be important for the characterization of the PE5 "dark matter" of the human proteome. Finally, we identify 66 high scoring PE5 protein entries and find that six of them were reported in recent mass spectrometry databases; an illustrative annotation of these six is provided. This work illustrates a new approach to examine the potential folding and function of the dubious proteins comprising PE5, which we will next apply to the far larger group of missing proteins comprising PE2-4.

  11. Purification and functional characterization of a protein: Bombyx mori human growth hormone like protein in silkworm pupa.

    PubMed

    Chen, Jianqing; Shu, Tejun; Lv, Zhengbing; Nie, Zuoming; Chen, Jian; Chen, Hao; Yu, Wei; Gai, Qijing; Zhang, Yaozhou

    2014-01-01

    Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.

  12. Purification and Functional Characterization of a Protein: Bombyx mori Human Growth Hormone Like Protein in Silkworm Pupa

    PubMed Central

    Lv, Zhengbing; Nie, Zuoming; Chen, Jian; Chen, Hao; Yu, Wei; Gai, Qijing; Zhang, Yaozhou

    2014-01-01

    Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people’s interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs. PMID:25469649

  13. Amino acid metabolism, substrate availability and the control of protein dynamics in the human kidney.

    PubMed

    Garibotto, G; Tessari, P; Sacco, P; Deferrari, G

    1999-01-01

    The mechanisms controlling protein metabolism in the human kidney are not well understood. During adult life, kidney protein content and the size of the kidney remain fairly constant, indicating that protein synthesis and degradation within the kidney are tightly regulated. However, kidney protein turnover may change in response to stimuli such as alterations in substrate availability, hormones or growth factors, acid-base balance, renal work or renal injury with a progressive decrease in the number of nephrons. These factors have been evaluated mainly in animals, in vitro or in vivo. Amino acids, the kidneys substrates for protein synthesis, are provided by several routes. Like in other organs, amino acids can reach the kidney cells through the arterial blood flow. However, they may also come from the degradation of reabsorbed low-molecular weight proteins filtered by the glomerulus. The human kidney has high rates of protein turnover and leucine oxidation. The magnitude of the protein turnover across the human kidney suggests that the protein dynamics is partly determined by intrarenal protein catabolism. As evaluated by a steady-state leucine multiple compartment analysis, kidney protein synthesis is dependent to a similar extent on intrarenal generation of amino acids from protein breakdown and from amino acids taken up from the arterial blood. Kidney mass may therefore depend not only on the availability of free amino acids, but also on filtered proteins which are degraded within the kidney. Future studies could define the mechanisms, metabolic pathways and mediators influencing kidney protein turnover in humans, with a view to better comprehension of the mechanisms of disease. PMID:10493563

  14. Identification of Epigenetic Factor Proteins Expressed in Human Embryonic Stem Cell-Derived Trophoblasts and in Human Placental Trophoblasts.

    PubMed

    Sarkar, Prasenjit; Mischler, Adam; Randall, Shan M; Collier, Timothy S; Dorman, Karen F; Boggess, Kim A; Muddiman, David C; Rao, Balaji M

    2016-08-01

    Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development. PMID:27378238

  15. Identification of human protein complexes from local sub-graphs of protein-protein interaction network based on random forest with topological structure features.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Zhou, Xuan; Dai, Zong; Zou, Xiao-Yong

    2012-03-01

    In the post-genomic era, one of the most important and challenging tasks is to identify protein complexes and further elucidate its molecular mechanisms in specific biological processes. Previous computational approaches usually identify protein complexes from protein interaction network based on dense sub-graphs and incomplete priori information. Additionally, the computational approaches have little concern about the biological properties of proteins and there is no a common evaluation metric to evaluate the performance. So, it is necessary to construct novel method for identifying protein complexes and elucidating the function of protein complexes. In this study, a novel approach is proposed to identify protein complexes using random forest and topological structure. Each protein complex is represented by a graph of interactions, where descriptor of the protein primary structure is used to characterize biological properties of protein and vertex is weighted by the descriptor. The topological structure features are developed and used to characterize protein complexes. Random forest algorithm is utilized to build prediction model and identify protein complexes from local sub-graphs instead of dense sub-graphs. As a demonstration, the proposed approach is applied to protein interaction data in human, and the satisfied results are obtained with accuracy of 80.24%, sensitivity of 81.94%, specificity of 80.07%, and Matthew's correlation coefficient of 0.4087 in 10-fold cross-validation test. Some new protein complexes are identified, and analysis based on Gene Ontology shows that the complexes are likely to be true complexes and play important roles in the pathogenesis of some diseases. PCI-RFTS, a corresponding executable program for protein complexes identification, can be acquired freely on request from the authors.

  16. Identification of shed proteins from Chinese hamster ovary cells: Application of statistical confidence using human and mouse protein databases

    SciTech Connect

    Ahram, Mamoun; Strittmatter, Eric F.; Monroe, Matthew E.; Adkins, Joshua N.; Hunter, Joel C.; Miller, John H.; Springer, David L.

    2005-05-01

    The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation in an effort to develop a fundamental understanding of the bystander response. CHO cells were chosen for this study because they have been widely used for radiation studies and since they have been reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and FTICR-mass spectrometry analysis. Since the hamster genome has not been sequenced, mass spectrometry data was searched against the mouse and human proteins databases. Nearly 150 proteins that were identified by tandem mass spectrometry were confirmed by FTICR. When both types of mass spectrometry data were evaluated with a new confidence scoring tool, which is based on discriminant analyses, about 500 protein were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface, hence were likely shed. However, estimates of quantitative changes, based on two independent mass spectrometry approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using mass spectrometry in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.

  17. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation.

    PubMed

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA(Ala) with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of L-serine into human proteins. We also report that this misincorporation can be inhibited by L-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  18. Dengue Virus Type 2: Protein Binding and Active Replication in Human Central Nervous System Cells

    PubMed Central

    Salazar, Ma Isabel; Pérez-García, Marissa; Terreros-Tinoco, Marisol; Castro-Mussot, María Eugenia; Diegopérez-Ramírez, Jaime; Ramírez-Reyes, Alma Griselda; Aguilera, Penélope; Cedillo-Barrón, Leticia; García-Flores, María Martha

    2013-01-01

    An increased number of dengue cases with neurological complications have been reported in recent years. The lack of reliable animal models for dengue has hindered studies on dengue virus (DENV) pathogenesis and cellular tropism in vivo. We further investigate the tropism of DENV for the human central nervous system (CNS), characterizing DENV interactions with cell surface proteins in human CNS cells by virus overlay protein binding assays (VOPBA) and coimmunoprecipitations. In VOPBA, three membrane proteins (60, 70, and 130 kDa) from the gray matter bound the entire virus particle, whereas only a 70 kDa protein bound in white matter. The coimmunoprecipitation assays revealed three proteins from gray matter consistently binding virus particles, one clearly distinguishable protein (~32 kDa) and two less apparent proteins (100 and 130 kDa). Monoclonal anti-NS3 targeted the virus protein in primary cell cultures of human CNS treated with DENV-2, which also stained positive for NeuH, a neuron-specific marker. Thus, our results indicate (1) that DENV-2 exhibited a direct tropism for human neurons and (2) that human neurons sustain an active DENV replication as was demonstrated by the presence of the NS3 viral antigen in primary cultures of these cells treated with DENV-2. PMID:24302878

  19. Effects of Electron Beam and Microwave Irradiation on Human Blood Proteins

    SciTech Connect

    Martin, Diana I.; Craciun, Gabriela D.; Manaila, Elena N.; Ighigeanu, Daniel I.; Iacob, Nicusor I.; Oproiu, Constantin V.; Stan, Dana E.; Radu, Roxana R.; Margaritescu, Irina D.; Chirita, Doru I.

    2007-04-23

    The effects of separated and combined accelerated electron beam (EB) of 6.23 MeV and microwave (MW) of 2.45GHz irradiation on proteins in samples of human serum, human plasma and human integral blood are presented. Also, it was studied the effect of separate and combined EB and MW irradiation on proteins irradiated in samples of human integral blood, without and in the presence of a synthetic compound solution (S.C.S.) which is expected to exhibit various biological actions, such as to diminish or to increase the irradiation effects.

  20. In vitro interaction between coxsackievirus B3 VP1 protein and human pleckstrin homology domain retinal protein (PHR1).

    PubMed

    Zhou, Ying; Zhang, Zhiqin; Wang, Hongluan; Xia, Yanhua; Li, Xiuzhen; Yan, Yan; Zou, Weiwen; Zeng, Lingbing; Huang, Xiaotian

    2015-10-01

    Coxsackievirus B3 (CVB3) infection causes central nervous system diseases including aseptic meningitis and encephalitis. To understand the mechanism of this virus, a yeast two-hybrid system was used to screen cellular proteins from a human heart cDNA library. The results revealed that the human Pleckstrin Homology Domain Retinal protein (PHR1), a PH domain-containing protein with low expression in the heart and high expression in the brain, interacts with CVB3 VP1, a major structural protein of CVB3. Yeast mating assays and in vitro coimmunoprecipitation verified the interaction between CVB3 VP1 and PHR1. An α-galactosidase assay indicated that of α-galactosidase activity was higher in positive clones than in controls suggesting a strong interaction. Furthermore, assay of deletion mutants defined the minimal region of PHR1 required for its interaction with VP1 as amino acids 95-172 and two regions of VP1 required for its interaction with PHR1 as amino acids 729-767 and 811-859. The results revealed multiple binding sites between PHR1 and CVB3 VP1 and suggested that the strong interaction between these two proteins might play an important role in central nervous system disease in the human brain.

  1. Using competitive protein adsorption to measure fibrinogen in undiluted human serum

    NASA Astrophysics Data System (ADS)

    Choi, Seokheun; Wang, Ran; Lajevardi-Khosh, Arad; Chae, Junseok

    2010-12-01

    We report a unique sensing mechanism based on competitive protein adsorption to measure fibrinogen, a cardiovascular biomarker, in undiluted human serum. The method uses physical adsorption of proteins to a surface rather than complex and time-consuming immobilization procedures. Two fibrinogen concentrations were differentiated in spiked in human serum [3.0 mg/ml (normal concentration) versus 3.2 mg/ml (abnormal concentration with heart disease)]. Real-time surface plasmon resonance signals were monitored as fibrinogen displaced a preadsorbed protein, IgM, on a hydrophobic gold surface. The relatively strong-affinity protein, IgM, was displaced primarily by fibrinogen and much less by other proteins in human serum.

  2. Detection and proteomic identification of S-nitrosated proteins in human hepatocytes.

    PubMed

    López-Sánchez, Laura M; Corrales, Fernando J; De La Mata, Manuel; Muntané, Jordi; Rodríguez-Ariza, Antonio

    2008-01-01

    The S-nitrosation of protein thiols is a redox-based posttranslational modification that modulates protein function and cell phenotype. Although the detection of S-nitrosated proteins is problematical because of the lability of S-nitrosothiols, an increasing range of proteins has been shown to undergo S-nitrosation with the improvement of molecular tools. This chapter describes the methodology used to identify potential targets of S-nitrosation in cultured primary human hepatocytes using proteomic approaches. This methodology is based on the biotin switch method, which labels S-nitrosated proteins with an affinity tag, allowing their selective detection and proteomic identification.

  3. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  4. COVALENT BINDING OF TRICHLOROETHYLENE TO PROTEINS IN HUMAN AND RAT HEPATOCYTES. (R826409)

    EPA Science Inventory

    The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte c...

  5. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S.

    PubMed

    Di Scipio, R G; Hermodson, M A; Yates, S G; Davie, E W

    1977-02-22

    Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.

  6. The Contribution of Missense Mutations in Core and Rim Residues of Protein-Protein Interfaces to Human Disease.

    PubMed

    David, Alessia; Sternberg, Michael J E

    2015-08-28

    Missense mutations at protein-protein interaction sites, called interfaces, are important contributors to human disease. Interfaces are non-uniform surface areas characterized by two main regions, "core" and "rim", which differ in terms of evolutionary conservation and physicochemical properties. Moreover, within interfaces, only a small subset of residues ("hot spots") is crucial for the binding free energy of the protein-protein complex. We performed a large-scale structural analysis of human single amino acid variations (SAVs) and demonstrated that disease-causing mutations are preferentially located within the interface core, as opposed to the rim (p<0.01). In contrast, the interface rim is significantly enriched in polymorphisms, similar to the remaining non-interacting surface. Energetic hot spots tend to be enriched in disease-causing mutations compared to non-hot spots (p=0.05), regardless of their occurrence in core or rim residues. For individual amino acids, the frequency of substitution into a polymorphism or disease-causing mutation differed to other amino acids and was related to its structural location, as was the type of physicochemical change introduced by the SAV. In conclusion, this study demonstrated the different distribution and properties of disease-causing SAVs and polymorphisms within different structural regions and in relation to the energetic contribution of amino acid in protein-protein interfaces, thus highlighting the importance of a structural system biology approach for predicting the effect of SAVs. PMID:26173036

  7. The human immunodeficiency virus type 1 Nef protein functions as a protein kinase C substrate in vitro.

    PubMed

    Coates, K; Harris, M

    1995-04-01

    The human immunodeficiency virus type 1 Nef protein was expressed in Escherichia coli as a C-terminal fusion with glutathione S-transferase (GST). The ability of GST-Nef to act as a substrate for cellular kinases in vitro was examined by incubation of purified GST-Nef fusion proteins, immobilized on glutathione-agarose beads, with cytoplasmic extracts from a number of human cell lines. In the presence of [gamma32P]ATP, phosphorylation of Nef occurred predominantly on serine residues. Studies with protein kinase inhibitors suggested that protein kinase C (PKC) was involved in Nef phosphorylation. This was supported further by the demonstration that purified PKC was also able to phosphorylate Nef in the absence of cell extract. Serine/threonine phosphorylation of Nef was also observed in vivo when Nef was expressed with a C-terminal GST or 6-histidine tag in Spodoptera frugiperda insect cells by recombinant baculoviruses. In extracts from Jurkat T cells and U937 monocyte/macrophages Nef also associated with a 57 kDa cellular protein that was itself phosphorylated in vitro. Phosphorylation of this Nef-associated protein was inhibited by heparin and is thus likely to be mediated by casein kinase II. The observation that PKC can phosphorylate Nef in vitro raises the possibility that PKC might play a role in regulating both Nef function and the physical interactions between Nef and cellular components.

  8. Protein and amino acid requirements in human nutrition.

    PubMed

    2007-01-01

    The World Health Organization and the Food and Agriculture Organization have worked to quantify the energy and nutrient needs of populations since 1949. This is the latest in a series of reports that aim to provide: updates on protein and amino acid requirements in health and disease for all age groups and pregnant and lactating mothers; recommendations on protein requirements in health and disease, including their implications for developing countries; recommendations on protein quality and labelling for worldwide use. This report provides the tools to address practical questions on the adequacy of food supplies, targets for food and nutrition policy, and labelling of protein quality. It contains specific recommendations for infant, child and adult nutrition. This report is an essential reference for those who need to determine the adequacy of population food intakes; set national food and nutrition guidelines and regulations on the protein and amino acid content of industrially processed foods; determine nutrient needs, and evaluate and ensure the adequacy of rations for vulnerable groups. The tools in this report can also be used to map and monitor global food shortages and under-nutrition through early warning systems.

  9. Correlation of human sperm centrosomal proteins with fertility

    PubMed Central

    Hinduja, Indira; Baliga, Nishitha B; Zaveri, Kusum

    2010-01-01

    OBJECTIVE: The centrosome is the microtubule organizing center (MTOC) paternally inherited by the zygote during fertilization. As the centrosome is located in the midpiece of the sperm tail, we presume that oligoasthenozoospermic sperm samples should also have abnormal concentrations of centrosomal proteins. This study therefore aims to determine if there is any correlation between sperm centrosomal proteins, centrin, α and γ-tubulin, in sperm samples from normozoospermic and oligoasthenozoospermic men. MATERIALS AND METHODS: Proteins were extracted from the normozoospermic and oligoasthenozoospermic sperm samples and analyzed by Western Blot and ELISA for centrin, α and γ-tubulin. RESULTS: The levels of centrin, α and γ-tubulin are markedly lower in oligoasthenozoospermic sperm samples as compared to the normozoospermic sperm samples. CONCLUSIONS: Lower centrosomal protein expression in sperm samples of oligoasthenozoospermic infertile males may be a possible cause for their reduced fertility status. Further studies on these proteins are warranted to design rational approaches for the diagnosis and treatment of male infertility. PMID:21209754

  10. Coordinated protein and DNA remodeling by human HLTF on stalled replication fork.

    PubMed

    Achar, Yathish Jagadheesh; Balogh, David; Haracska, Lajos

    2011-08-23

    Human helicase-like transcription factor (HLTF) exhibits ubiquitin ligase activity for proliferating cell nuclear antigen (PCNA) polyubiquitylation as well as double-stranded DNA translocase activity for remodeling stalled replication fork by fork reversal, which can support damage bypass by template switching. However, a stalled replication fork is surrounded by various DNA-binding proteins which can inhibit the access of damage bypass players, and it is unknown how these proteins become displaced. Here we reveal that HLTF has an ATP hydrolysis-dependent protein remodeling activity, by which it can remove proteins bound to the replication fork. Moreover, we demonstrate that HLTF can displace a broad spectrum of proteins such as replication protein A (RPA), PCNA, and replication factor C (RFC), thereby providing the first example for a protein clearing activity at the stalled replication fork. Our findings clarify how remodeling of a stalled replication fork can occur if it is engaged in interactions with masses of proteins.

  11. Identification of Posttranslational Modification-Dependent Protein Interactions Using Yeast Surface Displayed Human Proteome Libraries.

    PubMed

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology. PMID:26060076

  12. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

    PubMed

    Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T

    2016-04-01

    Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.

  13. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

    PubMed

    Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T

    2016-04-01

    Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function. PMID:27052166

  14. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  15. [Ectopic bone induction by human fetal enamel proteins].

    PubMed

    Wang, W

    1993-11-01

    Mixture of amelogenin, enamelin and soluble dentin proteins with plaster of Paris and distilled water were implanted in the left thigh-muscle pouch of C57BL/6 T mice, and PLP or BSA/PLP were implanted in the contralateral limbs for controls. The hind limbs were examined by means of microradiographic and histological methods three weeks after the implantation. Implants of PLP, BSA/PLP or E/PLP did not evoke formation of new cartilage or bone. Roentgenography showed highly mineralized tissues in the implantation areas of A/PLP or D/PLP, histological examination confirmed this as induced new bone or cartilage formation. Thus it indicates that amelogenins and soluble dentin proteins have bone induction activity as bone morphogenetic protein, they could induce the differentiation of mesenchymal cell in the muscles into chondrocyte and osteocyte. PMID:8033649

  16. Conserved primary sequences of the DNA terminal proteins of five different human adenovirus groups.

    PubMed

    Green, M; Brackmann, K; Wold, W S; Cartas, M; Thornton, H; Elder, J H

    1979-09-01

    The 31 human adenoviruses (Ad) from five groups (A-E) whose DNAs are <20% homologous by molecular hybridization. Ad5 (group C) DNA contains a 55,000-dalton protein probably covalently bound to each 5' terminus. This covalently bound protein may be analogous to polypeptides found in other viral and nonviral systems that are covalently bound to genomic DNAs or RNAs and that are thought to function in DNA or RNA replication. Because of the importance of proteins linked to nucleic acids, we have investigated whether DNAs from all five groups of human adenoviruses have terminal proteins, as well as the peptide relationships among the different terminal proteins. We show here that DNAs from Ad12, 7, 2, 19, and 4, representing Ad groups A-E, respectively, all contain covalently bound proteins of about 55,000 daltons. To investigate the peptide relatedness among the terminal proteins, we prepared microgram quantities of covalently bound protein from Ads in groups A-E and compared their chymotryptic and tryptic (125)I-labeled peptide maps. We find that the covalently bound protein maps of the five Ad groups are highly related and possibly identical. On the other hand, the tryptic and chymotryptic peptide maps of the major virion protein II and the core proteins V and VII of groups B, C, and E Ads show considerable heterology. Assuming that the covalently bound protein is virally coded, the conserved primary sequence of these proteins suggests a major functional role for the protein in Ad replication. Because the genetic origin of the Ad covalently bound proteins is not established, our data are also consistent with the possibility that the protein is coded by a cellular gene.

  17. Activation of human natural killer cells by the soluble form of cellular prion protein

    SciTech Connect

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  18. A scalable strategy for high-throughput GFP tagging of endogenous human proteins.

    PubMed

    Leonetti, Manuel D; Sekine, Sayaka; Kamiyama, Daichi; Weissman, Jonathan S; Huang, Bo

    2016-06-21

    A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. PMID:27274053

  19. Construction of human LRIG1-TAT fusions and TAT-mediated LRIG1 protein delivery.

    PubMed

    Wang, Yuchun; Fu, Liqi; Liu, Bo; Wang, Xiaomin; Wang, Kai; Ye, Ming

    2015-02-01

    Human leucine-rich repeats and immunoglobulin-like domains (LRIG1) is a tumor suppressor in animals and also functions as an endogenous suppressor in human tumor. The level of LRIG1 expression is highly associated with patient survival in clinic. The exploration of LRIG1 as a protein drug is an important task. HIV-1 transactivator of transcription peptide (TAT) is an excellent candidate for protein transduction. In this study, human LRIG1 was cloned and LRIG1-TAT fusion gene was constructed. The fusion proteins were produced by an Escherichia coli strain and purified by Ni(2+)-resin. Western blot assay and immunofluorescence microscopy were employed for monitoring LRIG1-TAT protein transduction into human neuroblastoma cells. Cell proliferation and invasion were measured for evaluating the effect of LRIG1-TAT on neuroblastoma cell. Our data showed that LRIG1 protein can be delivered into cells or organs in living animals by TAT. One-time transduction of LRIG1 proteins into human neuroblastoma cells enhanced cell proliferation and increased cell invasion. In vivo transduction showed that LRIG1-TAT protein can be presented in living animal organs. Our experiments provide a new vision on LRIG1 applications and also offer a therapy window for revealing the intrinsic function of LRIG1 on cells.

  20. Detection of cow's milk proteins and minor components in human milk using proteomics techniques.

    PubMed

    Coscia, A; Orrù, S; Di Nicola, P; Giuliani, F; Varalda, A; Peila, C; Fabris, C; Conti, A; Bertino, E

    2012-10-01

    Cow's milk proteins (CMPs) are the best characterized food allergens. The aim of this study was to investigate cow's milk allergens in human colostrum of term and preterm newborns' mothers, and other minor protein components by proteomics techniques, more sensitive than other techniques used in the past. Sixty-two term and 11 preterm colostrum samples were collected, subjected to a treatment able to increase the concentration of the most diluted proteins and simultaneously to reduce the concentration of the proteins present at high concentration (Proteominer Treatment), and subsequently subjected to the steps of proteomic techniques. The most relevant finding in this study was the detection of the intact bovine alpha-S1-casein in human colostrum, then bovine alpha-1-casein could be considered the cow's milk allergen that is readily secreted in human milk and could be a cause of sensitization to cow's milk in exclusively breastfed predisposed infants. Another interesting result was the detection, at very low concentrations, of proteins previously not described in human milk (galectin-7, the different isoforms of the 14-3-3 protein and the serum amyloid P-component), probably involved in the regulation of the normal cell growth, in the pro-apoptotic function and in the regulation of tissue homeostasis. Further investigations are needed to understand if these families of proteins have specific biological activity in human milk.

  1. Construction of human LRIG1-TAT fusions and TAT-mediated LRIG1 protein delivery.

    PubMed

    Wang, Yuchun; Fu, Liqi; Liu, Bo; Wang, Xiaomin; Wang, Kai; Ye, Ming

    2015-02-01

    Human leucine-rich repeats and immunoglobulin-like domains (LRIG1) is a tumor suppressor in animals and also functions as an endogenous suppressor in human tumor. The level of LRIG1 expression is highly associated with patient survival in clinic. The exploration of LRIG1 as a protein drug is an important task. HIV-1 transactivator of transcription peptide (TAT) is an excellent candidate for protein transduction. In this study, human LRIG1 was cloned and LRIG1-TAT fusion gene was constructed. The fusion proteins were produced by an Escherichia coli strain and purified by Ni(2+)-resin. Western blot assay and immunofluorescence microscopy were employed for monitoring LRIG1-TAT protein transduction into human neuroblastoma cells. Cell proliferation and invasion were measured for evaluating the effect of LRIG1-TAT on neuroblastoma cell. Our data showed that LRIG1 protein can be delivered into cells or organs in living animals by TAT. One-time transduction of LRIG1 proteins into human neuroblastoma cells enhanced cell proliferation and increased cell invasion. In vivo transduction showed that LRIG1-TAT protein can be presented in living animal organs. Our experiments provide a new vision on LRIG1 applications and also offer a therapy window for revealing the intrinsic function of LRIG1 on cells. PMID:25661388

  2. Immunogenic compositions comprising human immunodeficiency virus (HIV) mosaic Nef proteins

    SciTech Connect

    Korber, Bette T.; Perkins, Simon; Bhattacharya, Tanmoy; Fischer, William M.; Theiler, James; Letvin, Norman; Haynes, Barton F.; Hahn, Beatrice H.; Yusim, Karina; Kuiken, Carla

    2012-02-21

    The present invention relates to mosaic clade M HIV-1 Nef polypeptides and to compositions comprising same. The polypeptides of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  3. Nonstructural Protein NP1 of Human Bocavirus 1 Plays a Critical Role in the Expression of Viral Capsid Proteins

    PubMed Central

    Zou, Wei; Cheng, Fang; Shen, Weiran; Engelhardt, John F.; Yan, Ziying

    2016-01-01

    ABSTRACT A novel chimeric parvoviral vector, rAAV2/HBoV1, in which the recombinant adeno-associated virus 2 (rAAV2) genome is pseudopackaged by the human bocavirus 1 (HBoV1) capsid, has been shown to be highly efficient in gene delivery to human airway epithelia (Z. Yan et al., Mol Ther 21:2181–2194, 2013, http://dx.doi.org/10.1038/mt.2013.92). In this vector production system, we used an HBoV1 packaging plasmid, pHBoV1NSCap, that harbors HBoV1 nonstructural protein (NS) and capsid protein (Cap) genes. In order to simplify this packaging plasmid, we investigated the involvement of the HBoV1 NS proteins in capsid protein expression. We found that NP1, a small NS protein encoded by the middle open reading frame, is required for the expression of the viral capsid proteins (VP1, VP2, and VP3). We also found that the other NS proteins (NS1, NS2, NS3, and NS4) are not required for the expression of VP proteins. We performed systematic analyses of the HBoV1 mRNAs transcribed from the pHBoV1NSCap packaging plasmid and its derivatives in HEK 293 cells. Mechanistically, we found that NP1 is required for both the splicing and the read-through of the proximal polyadenylation site of the HBoV1 precursor mRNA, essential functions for the maturation of capsid protein-encoding mRNA. Thus, our study provides a unique example of how a small viral nonstructural protein facilitates the multifaceted regulation of capsid gene expression. IMPORTANCE A novel chimeric parvoviral vector, rAAV2/HBoV1, expressing a full-length cystic fibrosis transmembrane conductance regulator (CFTR) gene, is capable of correcting CFTR-dependent chloride transport in cystic fibrosis human airway epithelium. Previously, an HBoV1 nonstructural and capsid protein-expressing plasmid, pHBoV1NSCap, was used to package the rAAV2/HBoV1 vector, but yields remained low. In this study, we demonstrated that the nonstructural protein NP1 is required for the expression of capsid proteins. However, we found that the

  4. How much of the human protein interactome remains to be mapped?

    PubMed

    Vidal, Marc

    2016-05-10

    Using systematic approaches, a high-quality reference map of the human protein-protein interactome is within reach. Such a reference will help researchers connect genomic data to cellular phenotypes and enable full exploitation of the output of the genomic revolution for biomedical applications.

  5. Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.

    PubMed

    Boggavarapu, Rajendra; Hirschi, Stephan; Harder, Daniel; Meury, Marcel; Ucurum, Zöhre; Bergeron, Marc J; Fotiadis, Dimitrios

    2016-01-01

    This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail. PMID:27485339

  6. Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes

    PubMed Central

    Koen, Yakov M.; Sarma, Diganta; Williams, Todd D.; Galeva, Nadezhda A.; Obach, R. Scott; Hanzlik, Robert P.

    2012-01-01

    Tienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins. To identify protein targets of TA metabolites, we incubated [14C]-TA with human hepatocytes, separated cellular proteins by 2D gel electrophoresis, and analyzed proteins in 36 radioactive spots by tryptic digestion followed by LC-MS/MS. Thirty one spots contained at least one identifiable protein. Sixteen spots contained only one of 14 non-redundant proteins which were thus considered to be targets of TA metabolites. Six of the 14 were also found in other radioactive spots that contained from 1 to 3 additional proteins. Eight of the 14 had not been reported to be targets for any reactive metabolite other than TA. The other 15 spots each contained from 2–4 identifiable proteins, many of which are known targets of other chemically reactive metabolites, but since adducted peptides were not observed, the identity of the adducted protein(s) in these spots is ambiguous. Interestingly, all the radioactive spots corresponded to proteins of low abundance, while many highly abundant proteins in the mixture showed no radioactivity. Furthermore, of approximately 16 previously reported protein targets of TA in rat liver (Methogo, R., Dansette, P. and Klarskov, K. (2007) Int. J. Mass Spectrom., 268, 284–295), only one (fumarylacetoacetase) is among the 14 targets identified in this work. One reason for this difference may be statistical, given that each study identified a small number of targets from among thousands present in hepatocytes. Another may be the species difference (i.e. rat vs. human), and still another may be the method of detection of adducted

  7. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    PubMed

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H; Burk, Robert D

    2015-06-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  8. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche

    PubMed Central

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H.; Burk, Robert D.

    2015-01-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution. PMID:26086730

  9. New procyanidin B3-human salivary protein complexes by mass spectrometry. Effect of salivary protein profile, tannin concentration, and time stability.

    PubMed

    Perez-Gregorio, Maria Rosa; Mateus, Nuno; De Freitas, Victor

    2014-10-15

    Several factors could influence the tannin-protein interaction such as the human salivary protein profile, the tannin tested, and the tannin/protein ratio. The goal of this study aims to study the effect of different salivas (A, B, and C) and different tannin concentrations (0.5 and 1 mg/mL) on the interaction process as well as the complex's stability over time. This study is focused on the identification of new procyanidin B3-human salivary protein complexes. Thus, 48 major B3-human salivary protein aggregates were identified regardless of the saliva and tannin concentration tested. A higher number of aggregates was found at lower tannin concentration. Moreover, the number of protein moieties involved in the aggregation process was higher when the tannin concentration was also higher. The selectivity of the different groups of proteins to bind tannin was also confirmed. It was also verified that the B3-human salivary protein complexes formed evolved over time.

  10. Effects of atorvastatin on human c reactive protein metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Statins are known to reduce plasma C-reactive protein (CRP) concentrations. Our goals were to define the mechanisms by which CRP was reduced by maximal dose atorvastatin. Eight subjects with combined hyperlipidemia (5 men and 3 postmenopausal women) were enrolled in a randomized, placebo-controlled...

  11. Insulin Regulates the Unfolded Protein Response in Human Adipose Tissue

    PubMed Central

    Boden, Guenther; Cheung, Peter; Salehi, Sajad; Homko, Carol; Loveland-Jones, Catherine; Jayarajan, Senthil; Stein, T. Peter; Williams, Kevin Jon; Liu, Ming-Lin; Barrero, Carlos A.; Merali, Salim

    2014-01-01

    Endoplasmic reticulum (ER) stress is increased in obesity and is postulated to be a major contributor to many obesity-related pathologies. Little is known about what causes ER stress in obese people. Here, we show that insulin upregulated the unfolded protein response (UPR), an adaptive reaction to ER stress, in vitro in 3T3-L1 adipocytes and in vivo, in subcutaneous (sc) adipose tissue of nondiabetic subjects, where it increased the UPR dose dependently over the entire physiologic insulin range (from ∼35 to ∼1,450 pmol/L). The insulin-induced UPR was not due to increased glucose uptake/metabolism and oxidative stress. It was associated, however, with increased protein synthesis, with accumulation of ubiquitination associated proteins, and with multiple posttranslational protein modifications (acetylations, methylations, nitrosylations, succinylation, and ubiquitinations), some of which are potential causes for ER stress. These results reveal a new physiologic role of insulin and provide a putative mechanism for the development of ER stress in obesity. They may also have clinical and therapeutic implications, e.g., in diabetic patients treated with high doses of insulin. PMID:24130338

  12. Plasma proteomics, the Human Proteome Project, and cancer-associated alternative splice variant proteins.

    PubMed

    Omenn, Gilbert S

    2014-05-01

    This article addresses three inter-related subjects: the development of the Human Plasma Proteome Peptide Atlas, the launch of the Human Proteome Project, and the emergence of alternative splice variant transcripts and proteins as important features of evolution and pathogenesis. The current Plasma Peptide Atlas provides evidence on which peptides have been detected for every protein confidently identified in plasma; there are links to their spectra and their estimated abundance, facilitating the planning of targeted proteomics for biomarker studies. The Human Proteome Project (HPP) combines a chromosome-centric C-HPP with a biology and disease-driven B/D-HPP, upon a foundation of mass spectrometry, antibody, and knowledgebase resource pillars. The HPP aims to identify the approximately 7000 "missing proteins" and to characterize all proteins and their many isoforms. Success will enable the larger research community to utilize newly-available peptides, spectra, informative MS transitions, and databases for targeted analyses of priority proteins for each organ and disease. Among the isoforms of proteins, splice variants have the special feature of greatly enlarging protein diversity without enlarging the genome; evidence is accumulating of striking differential expression of splice variants in cancers. In this era of RNA-sequencing and advanced mass spectrometry, it is no longer sufficient to speak simply of increased or decreased expression of genes or proteins without carefully examining the splice variants in the protein mixture produced from each multi-exon gene. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

  13. Meta sequence analysis of human blood peptides and their parent proteins.

    PubMed

    Bowden, Peter; Pendrak, Voitek; Zhu, Peihong; Marshall, John G

    2010-04-18

    Sequence analysis of the blood peptides and their qualities will be key to understanding the mechanisms that contribute to error in LC-ESI-MS/MS. Analysis of peptides and their proteins at the level of sequences is much more direct and informative than the comparison of disparate accession numbers. A portable database of all blood peptide and protein sequences with descriptor fields and gene ontology terms might be useful for designing immunological or MRM assays from human blood. The results of twelve studies of human blood peptides and/or proteins identified by LC-MS/MS and correlated against a disparate array of genetic libraries were parsed and matched to proteins from the human ENSEMBL, SwissProt and RefSeq databases by SQL. The reported peptide and protein sequences were organized into an SQL database with full protein sequences and up to five unique peptides in order of prevalence along with the peptide count for each protein. Structured query language or BLAST was used to acquire descriptive information in current databases. Sampling error at the level of peptides is the largest source of disparity between groups. Chi Square analysis of peptide to protein distributions confirmed the significant agreement between groups on identified proteins.

  14. Human prion protein-induced autophagy flux governs neuron cell damage in primary neuron cells

    PubMed Central

    Moon, Ji-Hong; Lee, Ju-Hee; Nazim, Uddin MD; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-hwa; Park, Sang-Youel

    2016-01-01

    An unusual molecular structure of the prion protein, PrPsc is found only in mammals with transmissible prion diseases. Prion protein stands for either the infectious pathogen itself or a main component of it. Recent studies suggest that autophagy is one of the major functions that keep cells alive and has a protective effect against the neurodegeneration. In this study, we investigated that the effect of human prion protein on autophagy-lysosomal system of primary neuronal cells. The treatment of human prion protein induced primary neuron cell death and decreased both LC3-II and p62 protein amount indicating autophagy flux activation. Electron microscope pictures confirmed the autophagic flux activation in neuron cells treated with prion protein. Inhibition of autophagy flux using pharmacological and genetic tools prevented neuron cell death induced by human prion protein. Autophagy flux induced by prion protein is more activated in prpc expressing cells than in prpc silencing cells. These data demonstrated that prion protein-induced autophagy flux is involved in neuron cell death in prion disease and suggest that autophagy flux might play a critical role in neurodegenerative diseases including prion disease. PMID:27102156

  15. The Broad-Spectrum Antiviral Protein ZAP Restricts Human Retrotransposition

    PubMed Central

    Goodier, John L.; Pereira, Gavin C.; Cheung, Ling E.; Rose, Rebecca J.; Kazazian, Haig H.

    2015-01-01

    Intrinsic immunity describes the set of recently discovered but poorly understood cellular mechanisms that specifically target viral pathogens. Their discovery derives in large part from intensive studies of HIV and SIV that revealed restriction factors acting at various stages of the retroviral life cycle. Recent studies indicate that some factors restrict both retroviruses and retrotransposons but surprisingly in ways that may differ. We screened known interferon-stimulated antiviral proteins previously untested for their effects on cell culture retrotransposition. Several factors, including BST2, ISG20, MAVS, MX2, and ZAP, showed strong L1 inhibition. We focused on ZAP (PARP13/ZC3HAV1), a zinc-finger protein that targets viruses of several families, including Retroviridae, Tiloviridae, and Togaviridae, and show that ZAP expression also strongly restricts retrotransposition in cell culture through loss of L1 RNA and ribonucleoprotein particle integrity. Association of ZAP with the L1 ribonucleoprotein particle is supported by co-immunoprecipitation and co-localization with ORF1p in cytoplasmic stress granules. We also used mass spectrometry to determine the protein components of the ZAP interactome, and identified many proteins that directly interact and colocalize with ZAP, including MOV10, an RNA helicase previously shown to suppress retrotransposons. The detection of a chaperonin complex, RNA degradation proteins, helicases, post-translational modifiers, and components of chromatin modifying complexes suggest mechanisms of ZAP anti-retroelement activity that function in the cytoplasm and perhaps also in the nucleus. The association of the ZAP ribonucleoprotein particle with many interferon-stimulated gene products indicates it may be a key player in the interferon response. PMID:26001115

  16. Proteomic assessment of sulfur mustard-induced protein adducts and other protein modifications in human epidermal keratinocytes

    SciTech Connect

    Mol, Marijke A.E. Berg, Roland M. van den; Benschop, Henk P.

    2008-07-01

    Although some toxicological mechanisms of sulfur mustard (HD) have been uncovered, new knowledge will allow for advanced insight in the pathways that lead towards epidermal-dermal separation in skin. In the present investigation, we aimed to survey events that occur at the protein level in human epidermal keratinocytes (HEK) during 24 h after exposure to HD. By using radiolabeled {sup 14}C-HD, it was found that proteins in cultured HEK are significant targets for alkylation by HD. HD-adducted proteins were visualized by two-dimensional gel electrophoresis and analyzed by mass spectrometry. Several type I and II cytokeratins, actin, stratifin (14-3-3{sigma}) and galectin-7 were identified. These proteins are involved in the maintenance of the cellular cytoskeleton. Their alkylation may cause changes in the cellular architecture and, in direct line with that, be determinative for the onset of vesication. Furthermore, differential proteomic analysis was applied to search for novel features of the cellular response to HD. Partial breakdown of type I cytokeratins K14, K16 and K17 as well as the emergence of new charge variants of the proteins heat shock protein 27 and ribosomal protein P0 were observed. Studies with caspase inhibitors showed that caspase-6 is probably responsible for the breakdown of type I cytokeratins in HEK. The significance of the results is discussed in terms of toxicological relevance and possible clues for therapeutic intervention.

  17. Spectroscopic investigations of the interactions of tramadol hydrochloride and 5-azacytidine drugs with human serum albumin and human hemoglobin proteins.

    PubMed

    Tunç, Sibel; Cetinkaya, Ahmet; Duman, Osman

    2013-03-01

    The interactions of tramadol hydrochloride (THC) and 5-azacytidine (AZA) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins were investigated by fluorescence, UV absorption and circular dichroism (CD) spectroscopy at pH 7.4 and different temperatures. The UV absorption spectra and the fluorescence quenching of HSA and HMG proteins indicated the formation of HSA-THC and HMG-THC complexes via static quenching mechanism. AZA did not interact with HSA and HMG proteins. It was found that the formation of HMG-THC complex was stronger than that of HSA-THC complex. The stability of HSA-THC and HMG-THC complexes decreased with increasing temperature. The number of binding site was found as one for HSA-THC and HMG-THC systems. Negative enthalpy change (ΔH) and Gibbs free energy change (ΔG) and positive entropy change (ΔS) values were obtained for these systems. The binding of THC-HSA and HMG proteins was spontaneous and exothermic. In addition, electrostatic interactions between protein and drug molecules played an important role in the binding processes. The results of CD analysis revealed that the addition of THC led to a significant conformational change in the secondary structure of HSA protein, on the contrary to HMG protein. PMID:23428887

  18. Vaccine for human contraception targeting sperm Izumo protein and YLP12 dodecamer peptide

    PubMed Central

    Naz, Rajesh K

    2014-01-01

    There is an urgent need to develop a better method of contraception which is non-steroidal and reversible to control world population explosion and unintended pregnancies. Contraceptive vaccines (CV), especially targeting sperm-specific proteins, can provide an ideal contraceptive modality. Sperm-specific proteins can induce an immune response in women as well as men, thus can be used for CV development in both sexes. In this article, we will review two sperm-specific proteins, namely Izumo protein and YLP12 dodecamer peptide. Gene-knockout studies indicate that Izumo protein is essential for sperm–egg membrane fusion. Vaccination with Izumo protein or its cDNA causes a significant reduction in fertility of female mice. The antibodies to human Izumo inhibit human sperm penetration assay. Recently, our laboratory found that a significant percentage of infertile women have antibodies to Izumo protein. The second sperm-specific protein is YLP12, a peptide mimetic sequence present on human sperm involved in recognition and binding to the human oocyte zona pellucida. Vaccination with YLP12 or its cDNA causes long-term, reversible contraception, without side effects, in female mice. Infertile, but not fertile, men and women have antibodies to YLP12 peptide. Our laboratory has isolated, cloned, and sequenced cDNA encoding human single chain variable fragment (scFv) antibody from infertile men which reacts with YLP12 peptide. The human YLP12 scFv antibody may provide a novel passive immunocontraceptive, the first of its kind. In conclusion, sperm-specific Izumo protein and YLP12 peptide can provide exciting candidates for antisperm CV development. PMID:24723387

  19. Epigenetic Mechanisms Leading to Overexpression of HMGA Proteins in Human Pituitary Adenomas

    PubMed Central

    D’Angelo, Daniela; Esposito, Francesco; Fusco, Alfredo

    2015-01-01

    Overexpression of the high-mobility group A (HMGA)1 and HMGA2 proteins is a feature of all human pituitary adenoma (PAs) subtypes. However, amplification and/or rearrangement of the HMGA2 have been described in human prolactinomas, but rarely in other pituitary subtypes, and no genomic amplification of HMGA1 was detected in PAs. Here, we summarize the functional role of HMGA proteins in pituitary tumorigenesis and the epigenetic mechanisms contributing to HMGA overexpression in these tumors focusing on recent studies indicating a critical role of non-coding RNAs in modulating HMGA protein levels. PMID:26137461

  20. Network analysis and cross species comparison of protein-protein interaction networks of human, mouse and rat cytochrome P450 proteins that degrade xenobiotics.

    PubMed

    Karthikeyan, Bagavathy Shanmugam; Akbarsha, Mohammad Abdulkader; Parthasarathy, Subbiah

    2016-06-21

    Cytochrome P450 (CYP) enzymes that degrade xenobiotics play a critical role in the metabolism and biotransformation of drugs and xenobiotics in humans as well as experimental animal models such as mouse and rat. These proteins function as a network collectively as well as independently. Though there are several reports on the organization, regulation and functionality of various CYP enzymes at the molecular level, the understanding of organization and functionality of these proteins at the holistic level remain unclear. The objective of this study is to understand the organization and functionality of xenobiotic degrading CYP enzymes of human, mouse and rat using network theory approaches and to study species differences that exist among them at the holistic level. For our analysis, a protein-protein interaction (PPI) network for CYP enzymes of human, mouse and rat was constructed using the STRING database. Topology, centrality, modularity and robustness analyses were performed for our predicted CYP PPI networks that were then validated by comparison with randomly generated network models. Network centrality analyses of CYP PPI networks reveal the central/hub proteins in the network. Modular analysis of the CYP PPI networks of human, mouse and rat resulted in functional clusters. These clusters were subjected to ontology and pathway enrichment analysis. The analyses show that the cluster of the human CYP PPI network is enriched with pathways principally related to xenobiotic/drug metabolism. Endo-xenobiotic crosstalk dominated in mouse and rat CYP PPI networks, and they were highly enriched with endogenous metabolic and signaling pathways. Thus, cross-species comparisons and analyses of human, mouse and rat CYP PPI networks gave insights about species differences that existed at the holistic level. More investigations from both reductionist and holistic perspectives can help understand CYP metabolism and species extrapolation in a much better way. PMID:27194593

  1. Distinct protein classes in human red cell proteome revealed by similarity of phylogenetic profiles.

    PubMed

    Szczesny, Paweł; Mykowiecka, Agnieszka; Pawłowski, Krzysztof; Grynberg, Marcin

    2013-01-01

    The minimal set of proteins necessary to maintain a vertebrate cell forms an interesting core of cellular machinery. The known proteome of human red blood cell consists of about 1400 proteins. We treated this protein complement of one of the simplest human cells as a model and asked the questions on its function and origins. The proteome was mapped onto phylogenetic profiles, i.e. vectors of species possessing homologues of human proteins. A novel clustering approach was devised, utilising similarity in the phylogenetic spread of homologues as distance measure. The clustering based on phylogenetic profiles yielded several distinct protein classes differing in phylogenetic taxonomic spread, presumed evolutionary history and functional properties. Notably, small clusters of proteins common to vertebrates or Metazoa and other multicellular eukaryotes involve biological functions specific to multicellular organisms, such as apoptosis or cell-cell signaling, respectively. Also, a eukaryote-specific cluster is identified, featuring GTP-ase signalling and ubiquitination. Another cluster, made up of proteins found in most organisms, including bacteria and archaea, involves basic molecular functions such as oxidation-reduction and glycolysis. Approximately one third of erythrocyte proteins do not fall in any of the clusters, reflecting the complexity of protein evolution in comparison to our simple model. Basically, the clustering obtained divides the proteome into old and new parts, the former originating from bacterial ancestors, the latter from inventions within multicellular eukaryotes. Thus, the model human cell proteome appears to be made up of protein sets distinct in their history and biological roles. The current work shows that phylogenetic profiles concept allows protein clustering in a way relevant both to biological function and evolutionary history. PMID:23349899

  2. Human chorionic gonadotropin promotes expression of protein absorption factors in the intestine of goldfish (Carassius auratus).

    PubMed

    Zhou, Y; Hao, G; Zhong, H; Wu, Q; Lu, S Q; Zhao, Q; Liu, Z

    2015-07-27

    Protein use is crucial for the ovulation and spawning of fish. Currently, limited information is available regarding the expression of protein absorption factors during the breeding seasons of teleosts and thus how various proteins involved in this process is not well-understood. The expression of CDX2, CREB, gluatamate dehydrogenase, LAT2, aminopeptidase N, PepT1, and SP1 were significantly elevated from the non-breeding season to the breeding season in female goldfish, and all proteins except PepT1 and SP1 were elevated in male goldfish. Injection of human chorionic gonadotropin upregulated the expression of all proteins except for aminopeptidase N in female goldfish and SP1 in male goldfish, suggesting a luteinizing hormone-inductive effect on protein absorption factors. Protein use in the intestine is increased during the breeding seasons as a result of increased luteinizing hormone.

  3. Interaction between human BAP31 and respiratory syncytial virus small hydrophobic (SH) protein.

    PubMed

    Li, Yan; Jain, Neeraj; Limpanawat, Suweeraya; To, Janet; Quistgaard, Esben M; Nordlund, Par; Thanabalu, Thirumaran; Torres, Jaume

    2015-08-01

    The small hydrophobic (SH) protein is a short channel-forming polypeptide encoded by the human respiratory syncytial virus (hRSV). Deletion of SH protein leads to the viral attenuation in mice and primates, and delayed apoptosis in infected cells. We have used a membrane-based yeast two-hybrid system (MbY2H) and a library from human lung cDNA to detect proteins that bind SH protein. This led to the identification of a membrane protein, B-cell associated protein 31 (BAP31). Transfected SH protein co-localizes with transfected BAP31 in cells, and pulls down endogenous BAP31. Titration of purified C-terminal endodomain of BAP31 against isotopically labeled SH protein in detergent micelles suggests direct interaction between the two proteins. Given the key role of BAP31 in protein trafficking and its critical involvement in pro- and anti-apoptotic pathways, this novel interaction may constitute a potential drug target.

  4. Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study.

    PubMed

    Tsai, Irene Y; Tomczyk, Nancy; Eckmann, Joshua I; Composto, Russell J; Eckmann, David M

    2011-05-01

    Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities. PMID:21277175

  5. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    PubMed

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  6. Detection of CFTR protein in human leukocytes by flow cytometry.

    PubMed

    Johansson, Jan; Vezzalini, Marzia; Verzè, Genny; Caldrer, Sara; Bolognin, Silvia; Buffelli, Mario; Bellisola, Giuseppe; Tridello, Gloria; Assael, Baroukh Maurice; Melotti, Paola; Sorio, Claudio

    2014-07-01

    Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 μL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels. © 2014 International Society for Advancement of Cytometry.

  7. Differentially expressed protein markers in human submandibular and sublingual secretions.

    PubMed

    Hu, Shen; Denny, Patricia; Denny, Paul; Xie, Yongming; Loo, Joseph A; Wolinsky, Lawrence E; Li, Yang; McBride, Jim; Ogorzalek Loo, Rachel R; Navazesh, Mavash; Wong, David T

    2004-11-01

    Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

  8. Metaproteomics Reveals Functional Shifts in Microbial and Human Proteins During Infant Gut Colonization Case

    SciTech Connect

    Young, Jacque C.; Pan, Chongle; Adams, Rachel M.; Brooks, Brandon; Banfield, Jillian F.; Morowitz, Michael J.; Robert L. Hettich

    2015-01-01

    The microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. Thus, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity and functions increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data indicating three distinct colonization phases. Overall microbial community functions were established relatively early in development and remained stable. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Moreover, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. Our study provides the first snapshot of coordinated human and microbial protein expression in the infant gut during early development.

  9. Metaproteomics Reveals Functional Shifts in Microbial and Human Proteins During Infant Gut Colonization Case

    DOE PAGESBeta

    Young, Jacque C.; Pan, Chongle; Adams, Rachel M.; Brooks, Brandon; Banfield, Jillian F.; Morowitz, Michael J.; Robert L. Hettich

    2015-01-01

    The microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. Thus, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity and functions increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data indicating three distinct colonization phases. Overall microbial community functions were established relatively early in development andmore » remained stable. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Moreover, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. Our study provides the first snapshot of coordinated human and microbial protein expression in the infant gut during early development.« less

  10. Heart research advances using database search engines, Human Protein Atlas and the Sydney Heart Bank.

    PubMed

    Li, Amy; Estigoy, Colleen; Raftery, Mark; Cameron, Darryl; Odeberg, Jacob; Pontén, Fredrik; Lal, Sean; Dos Remedios, Cristobal G

    2013-10-01

    This Methodological Review is intended as a guide for research students who may have just discovered a human "novel" cardiac protein, but it may also help hard-pressed reviewers of journal submissions on a "novel" protein reported in an animal model of human heart failure. Whether you are an expert or not, you may know little or nothing about this particular protein of interest. In this review we provide a strategic guide on how to proceed. We ask: How do you discover what has been published (even in an abstract or research report) about this protein? Everyone knows how to undertake literature searches using PubMed and Medline but these are usually encyclopaedic, often producing long lists of papers, most of which are either irrelevant or only vaguely relevant to your query. Relatively few will be aware of more advanced search engines such as Google Scholar and even fewer will know about Quertle. Next, we provide a strategy for discovering if your "novel" protein is expressed in the normal, healthy human heart, and if it is, we show you how to investigate its subcellular location. This can usually be achieved by visiting the website "Human Protein Atlas" without doing a single experiment. Finally, we provide a pathway to discovering if your protein of interest changes its expression level with heart failure/disease or with ageing.

  11. Human PIEZO1 Ion Channel Functions as a Split Protein

    PubMed Central

    Bae, Chilman; Suchyna, Thomas M.; Ziegler, Lynn; Sachs, Frederick; Gottlieb, Philip A.

    2016-01-01

    PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the “split protein” is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone. PMID:26963637

  12. Functional Analysis of West Nile Virus Proteins in Human Cells.

    PubMed

    Kaufusi, Pakieli H; Tseng, Alanna; Nerurkar, Vivek R

    2016-01-01

    West Nile Virus (WNV) lineage 2 strains have been responsible for large outbreaks of neuroinvasive disease in the United States and Europe between 1999 and 2012. Different strains in this lineage have previously been shown to produce either severe or mild neuroinvasive disease in mice. Phylogenetic and amino acid comparisons between highly or less virulent lineage 2 strains have demonstrated that the nonstructural (NS) gene(s) were most variable. However, the roles of some of the NS proteins in virus life cycle are unknown. The aim of this chapter is to describe simple computational and experimental approaches that can be used to: (1) explore the possible roles of the NS proteins in virus life cycle and (2) test whether the subtle amino acid changes in WNV NS gene products contributed to the evolution of more virulent strains. The computational approaches include methods based on: (1) sequence similarity, (2) sequence motifs, and (3) protein membrane topology predictions. Highlighted experimental procedures include: (1) isolation of viral RNA from WNV-infected cells, (2) cDNA synthesis and PCR amplification of WNV genes, (3) cloning into GFP expression vector, (4) bacterial transformation, (5) plasmid isolation and purification, (6) transfection using activated dendrimers (Polyfect), and (7) immunofluorescence staining of transfected mammalian cells. PMID:27188549

  13. Human pyruvate kinase M2: a multifunctional protein.

    PubMed

    Gupta, Vibhor; Bamezai, Rameshwar N K

    2010-11-01

    Glycolysis, a central metabolic pathway, harbors evolutionary conserved enzymes that modulate and potentially shift the cellular metabolism on requirement. Pyruvate kinase, which catalyzes the last but rate-limiting step of glycolysis, is expressed in four isozymic forms, depending on the tissue requirement. M2 isoform (PKM2) is exclusively expressed in embryonic and adult dividing/tumor cells. This tetrameric allosterically regulated isoform is intrinsically designed to downregulate its activity by subunit dissociation (into dimer), which results in partial inhibition of glycolysis at the last step. This accumulates all upstream glycolytic intermediates as an anabolic feed for synthesis of lipids and nucleic acids, whereas reassociation of PKM2 into active tetramer replenishes the normal catabolism as a feedback after cell division. In addition, involvement of this enzyme in a variety of pathways, protein-protein interactions, and nuclear transport suggests its potential to perform multiple nonglycolytic functions with diverse implications, although multidimensional role of this protein is as yet not fully explored. This review aims to provide an overview of the involvement of PKM2 in various physiological pathways with possible functional implications. PMID:20857498

  14. Related F-box proteins control cell death in Caenorhabditis elegans and human lymphoma

    PubMed Central

    Chiorazzi, Michael; Rui, Lixin; Yang, Yandan; Ceribelli, Michele; Tishbi, Nima; Maurer, Carine W.; Ranuncolo, Stella M.; Zhao, Hong; Xu, Weihong; Chan, Wing-Chung C.; Jaffe, Elaine S.; Gascoyne, Randy D.; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa M.; Shaham, Shai; Staudt, Louis M.

    2013-01-01

    Cell death is a common metazoan cell fate, and its inactivation is central to human malignancy. In Caenorhabditis elegans, apoptotic cell death occurs via the activation of the caspase CED-3 following binding of the EGL-1/BH3-only protein to the antiapoptotic CED-9/BCL2 protein. Here we report a major alternative mechanism for caspase activation in vivo involving the F-box protein DRE-1. DRE-1 functions in parallel to EGL-1, requires CED-9 for activity, and binds to CED-9, suggesting that DRE-1 promotes apoptosis by inactivating CED-9. FBXO10, a human protein related to DRE-1, binds BCL2 and promotes its degradation, thereby initiating cell death. Moreover, some human diffuse large B-cell lymphomas have inactivating mutations in FBXO10 or express FBXO10 at low levels. Our results suggest that DRE-1/FBXO10 is a conserved regulator of apoptosis. PMID:23431138

  15. Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

    PubMed

    Tjin, Monica Suryana; Chua, Alvin Wen Choong; Ma, Dong Rui; Lee, Seng Teik; Fong, Eileen

    2014-08-01

    Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

  16. Morin, a flavonoid from moraceae, induces apoptosis by induction of BAD protein in human leukemic cells.

    PubMed

    Park, Cheol; Lee, Won Sup; Go, Se-Il; Nagappan, Arulkumar; Han, Min Ho; Hong, Su Hyun; Kim, Gon Sup; Kim, Gi Young; Kwon, Taeg Kyu; Ryu, Chung Ho; Shin, Sung Chul; Choi, Yung Hyun

    2014-12-30

    Evidence suggests that phytochemicals can safely modulate cancer cell biology and induce apoptosis. Here, we investigated the anti-cancer activity of morin, a flavone originally isolated from members of the Moraceae family in human leukemic cells, focusing on apoptosis. An anti-cancer effect of morin was screened with several human leukemic cell lines. U937 cells were most sensitive to morin, where it induced caspase-dependent apoptosis in a dose-dependent manner. It also induced loss of MMP (ΔΨm) along with cytochrome c release, down-regulated Bcl-2 protein, and up-regulated BAX proteins. The apoptotic activity of morin was significantly attenuated by Bcl-2 augmentation. In conclusion, morin induced caspase-dependent apoptosis through an intrinsic pathway by upregulating BAD proteins. In addition, Bcl-2 protein expression is also important in morin-induced apoptosis of U937 cells. This study provides evidence that morin might have anticancer properties in human leukemic cells.

  17. Protein expression profile changes in human fibroblasts induced by low dose energetic protons

    NASA Astrophysics Data System (ADS)

    Zhang, Ye; Clement, Jade Q.; Gridley, Daila S.; Rodhe, Larry H.; Wu, Honglu

    2009-12-01

    Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposures of astronauts in the space radiation environment, relies in part on the mechanistic understanding of radiation induced biological consequences at the molecular level. While some genomic data at the mRNA level are available for cells or animals exposed to radiation, the data at the protein level are still lacking. Here, we studied protein expression profile changes using Panorama antibody microarray chips that contain antibodies to 224 proteins (or their phosphorylated forms) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured until fully confluent and then exposed to 2 cGy of 150 MeV protons at high-dose rate. The proteins were isolated at 2 or 6 h after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loading onto the protein microarray chips. The intensities of the protein spots were analyzed using ScanAlyze software and normalized by the summed fluorescence intensities and the housekeeping proteins. The results showed that low dose protons altered the expression of more than 10% of the proteins listed in the microarray analysis in various protein functional groups. Cell cycle (24%) related proteins were induced by protons and most of them were regulators of G1/S-transition phase. Comparison of the overall protein expression profiles, cell cycle related proteins, cytoskeleton and signal transduction protein groups showed significantly more changes induced by protons compared with other protein functional groups.

  18. Distinguishing protein-coding and noncoding genes in the human genome

    PubMed Central

    Clamp, Michele; Fry, Ben; Kamal, Mike; Xie, Xiaohui; Cuff, James; Lin, Michael F.; Kellis, Manolis; Lindblad-Toh, Kerstin; Lander, Eric S.

    2007-01-01

    Although the Human Genome Project was completed 4 years ago, the catalog of human protein-coding genes remains a matter of controversy. Current catalogs list a total of ≈24,500 putative protein-coding genes. It is broadly suspected that a large fraction of these entries are functionally meaningless ORFs present by chance in RNA transcripts, because they show no evidence of evolutionary conservation with mouse or dog. However, there is currently no scientific justification for excluding ORFs simply because they fail to show evolutionary conservation: the alternative hypothesis is that most of these ORFs are actually valid human genes that reflect gene innovation in the primate lineage or gene loss in the other lineages. Here, we reject this hypothesis by carefully analyzing the nonconserved ORFs—specifically, their properties in other primates. We show that the vast majority of these ORFs are random occurrences. The analysis yields, as a by-product, a major revision of the current human catalogs, cutting the number of protein-coding genes to ≈20,500. Specifically, it suggests that nonconserved ORFs should be added to the human gene catalog only if there is clear evidence of an encoded protein. It also provides a principled methodology for evaluating future proposed additions to the human gene catalog. Finally, the results indicate that there has been relatively little true innovation in mammalian protein-coding genes. PMID:18040051

  19. Protein-losing enteropathy in the human and experimental rat blind-loop syndrome.

    PubMed

    King, C E; Toskes, P P

    1981-03-01

    Protein-losing enteropathy in 2 human subjects with small intestine bacterial overgrowth is reported. Partial improvement in 1 and complete normalization in the 2nd, during long-term antibiotic therapy, demonstrate for the first time antibiotic reversibility of protein-losing enteropathy in the human blind-loop syndrome. Studies in rats with experimental jejunal blind loops revealed depressed serum protein levels and excessive fecal excretion of intravenously administered 51Cr as compared with nonoperated and surgical controls. Comparison of protein loss of rats with blind loops present for varying lengths of time and paired comparison of rats tested for protein loss at two different times revealed a relatively stable degree of protein loss once it was manifest. Reversal of protein loss with antibiotic therapy was accomplished in only a small percentage of rats, and in those only after prolonged therapy. Surgical extirpation of the blind loop from rats with protein-losing enteropathy consistently corrected the protein loss, although this correction was delayed up to 9 wk from the time of corrective surgery. These studies demonstrate (a) significant protein loss as one etiologic factor for protein metabolic disturbances in the human and experimental rat blind-loop syndrome, (b) the occurrence of intestinal protein loss as a manifestation of functionally significant mucosal injury in the contaminated nonstagnant small bowel as well as the stagnant part of the small intestine affected by bacterial overgrowth, and (c) the difficulty of reversing functionally significant mucosal injury in the blind-loop syndrome once it has been manifest.

  20. Utilizing Yeast Surface Human Proteome Display Libraries to Identify Small Molecule-Protein Interactions.

    PubMed

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact with small bioactive molecules is a critical but often difficult and time-consuming step in understanding cellular signaling pathways or molecular mechanisms of drug action. Numerous methods for identifying small molecule-interacting proteins have been developed and utilized, including affinity-based purification followed by mass spectrometry analysis, protein microarrays, phage display, and three-hybrid approaches. Although all these methods have been used successfully, there remains a need for additional techniques for analyzing small molecule-protein interactions. A promising method for identifying small molecule-protein interactions is affinity-based selection of yeast surface-displayed human proteome libraries. Large and diverse libraries displaying human protein fragments on the surface of yeast cells have been constructed and subjected to FACS-based enrichment followed by comprehensive exon microarray-based output analysis to identify protein fragments with affinity for small molecule ligands. In a recent example, a proteome-wide search has been successfully carried out to identify cellular proteins binding to the signaling lipids PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Known phosphatidylinositide-binding proteins such as pleckstrin homology domains were identified, as well as many novel interactions. Intriguingly, many novel nuclear phosphatidylinositide-binding proteins were discovered. Although the existence of an independent pool of nuclear phosphatidylinositides has been known about for some time, their functions and mechanism of action remain obscure. Thus, the identification and subsequent study of nuclear phosphatidylinositide-binding proteins is expected to bring new insights to this important biological question. Based on the success with phosphatidylinositides, it is expected that the screening of yeast surface-displayed human proteome libraries will be of general use for the discovery of novel small

  1. Determination of protein markers in human serum: Analysis of protein expression in toxic oil syndrome studies.

    PubMed

    Quero, Carmen; Colomé, Nuria; Prieto, Maria Rosario; Carrascal, Montserrat; Posada, Manuel; Gelpí, Emilio; Abian, Joaquin

    2004-02-01

    Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS-affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Several problems related with serum analysis by 2-DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty-five of these protein spots were found to be under- or overexpressed in TOS patients (> three-fold increase or decrease). Proteins in the differential spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.

  2. Evidence for keratin proteins in normal and abnormal human meibomian fluids.

    PubMed

    Ong, B L; Hodson, S A; Wigham, T; Miller, F; Larke, J R

    1991-12-01

    Hyperkeratinization of meibomian glands has been postulated to cause gland dysfunction. Recent investigations on rabbits show that keratin proteins are indeed present in the meibomian fluids of these animals. In this report we present our findings on the presence of these water-insoluble proteins in human meibomian secretions. 6 anti-cytokeratin antibodies, CK8, 18, 19, CK7, CK8, CK14, CK19 and AE1/AE3 were used against the keratin proteins expressed from the human meibomian fluids. Using the immunoblotting (dot blot) technique, abnormal waxy meibomian fluids obtained from subjects diagnosed to have meibomian gland dysfunction (MGD) were compared to normal clear meibomian fluids. The results show that keratins are present in a higher concentration (10%) in the abnormal human meibomian excreta as compared to the normals. Even though the presence of protein markers for keratinization in the abnormal meibomian excreta were not shown, the increased presence of keratin proteins in the abnormal meibomian fluids suggests that, in MGD patients, hyperkeratinization of ductal epithelium may have taken place. More keratin proteins (possibly those of higher molecular weights) were produced in addition to the keratin proteins normally produced by the duct epithelium. The increased amount of keratin proteins in the abnormal meibomian fluids may be explained by the susceptibility of duct epithelium to undergo the process of hyperkeratinization as postulated by other researchers.

  3. Evolutionary conservation of mammalian sperm proteins associates with overall, not tyrosine, phosphorylation in human spermatozoa.

    PubMed

    Schumacher, Julia; Ramljak, Sanja; Asif, Abdul R; Schaffrath, Michael; Zischler, Hans; Herlyn, Holger

    2013-12-01

    We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein-protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility.

  4. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation.

    PubMed

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-08-27

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  5. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation

    PubMed Central

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-01-01

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  6. Permanent proteins in the urine of healthy humans during the Mars-500 experiment.

    PubMed

    Larina, Irina M; Pastushkova, Lyudmila Kh; Tiys, Evgeny S; Kireev, Kirill S; Kononikhin, Alexey S; Starodubtseva, Natalia L; Popov, Igor A; Custaud, Marc-Antoine; Dobrokhotov, Igor V; Nikolaev, Evgeny N; Kolchanov, Nikolay A; Ivanisenko, Vladimir A

    2015-02-01

    Urinary proteins serve as indicators of various conditions in human normal physiology and disease pathology. Using mass spectrometry proteome analysis, the permanent constituent of the urine was examined in the Mars-500 experiment (520 days isolation of healthy volunteers in a terrestrial complex with an autonomous life support system). Seven permanent proteins with predominant distribution in the liver and blood plasma as well as extracellular localization were identified. Analysis of the overrepresentation of the molecular functions and biological processes based on Gene Ontology revealed that the functional association among these proteins was low. The results showed that the identified proteins may be independent markers of the various conditions and processes in healthy humans and that they can be used as standards in determination of the concentration of other proteins in the urine.

  7. Silkworms culture as a source of protein for humans in space

    NASA Astrophysics Data System (ADS)

    Yang, Yunan; Tang, Liman; Tong, Ling; Liu, Hong

    2009-04-01

    This paper focuses on the problem about a configuration with complete nutrition for humans in a Controlled Ecological Life Support System (CELSS) applied in the spacebases. The possibility of feeding silkworms to provide edible animal protein with high quality for taikonauts during long-term spaceflights and lunar-based missions was investigated from several aspects, including the nutrition structure of silkworms, feeding method, processing methods, feeding equipment, growing conditions and the influences on the space environmental condition changes caused by the silkworms. The originally inedible silk is also regarded as a protein source. A possible process of edible silk protein was brought forward in this paper. After being processed, the silk can be converted to edible protein for humans. The conclusion provides a promising approach to solving the protein supply problem for the taikonauts living in space during an extended exploration period.

  8. Permanent proteins in the urine of healthy humans during the Mars-500 experiment.

    PubMed

    Larina, Irina M; Pastushkova, Lyudmila Kh; Tiys, Evgeny S; Kireev, Kirill S; Kononikhin, Alexey S; Starodubtseva, Natalia L; Popov, Igor A; Custaud, Marc-Antoine; Dobrokhotov, Igor V; Nikolaev, Evgeny N; Kolchanov, Nikolay A; Ivanisenko, Vladimir A

    2015-02-01

    Urinary proteins serve as indicators of various conditions in human normal physiology and disease pathology. Using mass spectrometry proteome analysis, the permanent constituent of the urine was examined in the Mars-500 experiment (520 days isolation of healthy volunteers in a terrestrial complex with an autonomous life support system). Seven permanent proteins with predominant distribution in the liver and blood plasma as well as extracellular localization were identified. Analysis of the overrepresentation of the molecular functions and biological processes based on Gene Ontology revealed that the functional association among these proteins was low. The results showed that the identified proteins may be independent markers of the various conditions and processes in healthy humans and that they can be used as standards in determination of the concentration of other proteins in the urine. PMID:25572715

  9. Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin beta subunit.

    PubMed Central

    Ruddon, R. W.; Sherman, S. A.; Bedows, E.

    1996-01-01

    There have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin beta subunit (hCG-beta), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG-beta fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the beta subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects. PMID:8844836

  10. Human fallopian tube proteome shows high coverage of mesenchymal stem cells associated proteins.

    PubMed

    Wang, Chenyuan; Liu, Yang; Chang, Cheng; Wu, Songfeng; Gao, Jie; Zhang, Yang; Chen, Yingjie; Zhong, Fan; Deng, Gaopi

    2016-01-01

    The object of this research was to report a draft proteome of human fallopian tube (hFT) comprises 5416 identified proteins, which could be considered as a physiological reference to complement Human Proteome Draft. The proteomic raw data and metadata were stored in an integrated proteome resources centre iProX (IPX00034300). This hFT proteome contains many hFT markers newly identified by mass spectrum. This hFT proteome comprises 660 high-, 3605 medium- and 1181 low-abundant proteins. Ribosome, cytoskeleton, vesicle and protein folding associated proteins showed obvious tendency to be higher abundance in hFT. The extraordinary high coverage of mesenchymal stem cells (MSCs)-associated proteins were identified in this hFT proteome, which highly supported that hFT should contain a plenty of MSCs. PMID:26759384

  11. Crystallization and preliminary X-ray diffraction analysis of human phosphate-binding protein

    SciTech Connect

    Contreras-Martel, Carlos; Carpentier, Philippe; Morales, Renaud; Renault, Frédérique; Chesne-Seck, Marie-Laure; Rochu, Daniel; Masson, Patrick; Fontecilla-Camps, Juan Carlos; Chabrière, Eric

    2006-01-01

    The purification, detergent-exchange protocol and crystallization conditions that led to the discovery of HPBP are reported. HPBP is a new human apoprotein that is absent from the genomic database and is the first phosphate transporter characterized in human plasma. Human phosphate-binding protein (HPBP) was serendipitously discovered by crystallization and X-ray crystallography. HPBP belongs to a eukaryotic protein family named DING that is systematically absent from the genomic database. This apoprotein of 38 kDa copurifies with the HDL-associated apoprotein paraoxonase (PON1) and binds inorganic phosphate. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus, it may be regarded as a predictor of phosphate-related diseases such as atherosclerosis. In addition, HPBP may be a potential therapeutic protein for the treatment of such diseases. Here, the purification, detergent-exchange protocol and crystallization conditions that led to the discovery of HPBP are reported.

  12. Molecular cloning, genomic organization, and chromosomal localization of the human pancreatitis-associated protein (PAP) gene

    SciTech Connect

    Dusetti, N.J.; Frigerio, J.M.; Dagorn, J.C.; Iovanna, J.L. ); Fox, M.F.; Swallow, D.M. )

    1994-01-01

    Pancreatitis-associated protein (PAP) is a secretory pancreatic protein present in small amounts in normal pancreas and overexpressed during the acute phase of pancreatitis. In this paper, the authors describe the cloning, characterization, and chromosomal mapping of the human PAP gene. The gene spans 2748 bp and contains six exons interrupted by five introns. The gene has a typical promoter containing the sequences TATAAA and CCAAT 28 and 52 bp upstream of the cap site, respectively. They found striking similarities in genomic organization as well as in the promoter sequences between the human and rat PAP genes. The human PAP gene was mapped to chromosome 2p12 using rodent-human hybrid cells and in situ chromosomal hybridization. This localization coincides with that of the reg/lithostathine gene, which encodes a pancreatic secretory protein structurally related to PAP, suggesting that both genes derived from the same ancestral gene by duplication. 35 refs., 4 figs., 1 tab.

  13. Yeast prions and human prion-like proteins: sequence features and prediction methods.

    PubMed

    Cascarina, Sean M; Ross, Eric D

    2014-06-01

    Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis. This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms.

  14. Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs

    PubMed Central

    Wiemann, Stefan; Weil, Bernd; Wellenreuther, Ruth; Gassenhuber, Johannes; Glassl, Sabine; Ansorge, Wilhelm; Böcher, Michael; Blöcker, Helmut; Bauersachs, Stefan; Blum, Helmut; Lauber, Jürgen; Düsterhöft, Andreas; Beyer, Andreas; Köhrer, Karl; Strack, Normann; Mewes, Hans-Werner; Ottenwälder, Birgit; Obermaier, Brigitte; Tampe, Jens; Heubner, Dagmar; Wambutt, Rolf; Korn, Bernhard; Klein, Michaela; Poustka, Annemarie

    2001-01-01

    With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2%–5 % of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies. [The sequence data described in this paper have been submitted to the EMBL database under the accession nos. given in Table 2.] PMID:11230166

  15. The Human Antimicrobial Protein Bactericidal/Permeability-Increasing Protein (BPI) Inhibits the Infectivity of Influenza A Virus

    PubMed Central

    Pinkenburg, Olaf; Meyer, Torben; Bannert, Norbert; Norley, Steven; Bolte, Kathrin; Czudai-Matwich, Volker; Herold, Susanne; Gessner, André; Schnare, Markus

    2016-01-01

    In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPI-peptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely. PMID:27273104

  16. Isoelectric point differentiates PHF-tau from biopsy-derived human brain tau proteins.

    PubMed

    Sergeant, N; Bussière, T; Vermersch, P; Lejeune, J P; Delacourte, A

    1995-11-13

    In the present study, Tau proteins were detected by two monoclonal antibodies AD2 and Tau-1 raised against PHF-tau and normal Tau proteins respectively using single- and two-dimensional immunoblotting. We demonstrate here the presence of a Tau triplet in brain homogenates from patients with Alzheimer's disease (AD) processed human brain biopsies from controls. However PHF-tau proteins have a slight but significantly higher mol. wt and a much more acidic isoelectric point. Therefore, Tau proteins are more phosphorylated in AD.

  17. P. falciparum: merozoite surface protein-8 peptides bind specifically to human erythrocytes.

    PubMed

    Puentes, Alvaro; García, Javier; Ocampo, Marisol; Rodríguez, Luis; Vera, Ricardo; Curtidor, Hernando; López, Ramsés; Suarez, Jorge; Valbuena, John; Vanegas, Magnolia; Guzman, Fanny; Tovar, Diana; Patarroyo, Manuel E

    2003-07-01

    This work determined Plasmodium falciparum merozoite surface protein-8 (MSP-8) regions specifically binding to membrane surface receptors on human erythrocytes. Five high activity binding peptides (HABPs), whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase and chymotrypsin were identified from the MSP-8 protein. Those amino acids directly involved in interaction with erythrocytes were also determined for each one of the HABPs. Some of them specifically recognized 28, 46, and 73 kDa erythrocyte membrane proteins. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by up to 98%, suggesting the MSP-8 protein's possible role in the invasion process.

  18. Proteins, peptides, polysaccharides, and nucleotides with inhibitory activity on human immunodeficiency virus and its enzymes.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Chan, Wai Yee

    2015-12-01

    Human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome, has claimed innumerable lives in the past. Many biomolecules which suppress HIV replication and also other biomolecules that inhibit enzymes essential to HIV replication have been reported. Proteins including a variety of milk proteins, ribosome-inactivating proteins, ribonucleases, antifungal proteins, and trypsin inhibitors; peptides comprising cathelicidins, defensins, synthetic peptides, and others; polysaccharides and polysaccharopeptides; nucleosides, nucleotides, and ribozymes, demonstrated anti-HIV activity. In many cases, the mechanism of anti-HIV action has been elucidated. Strategies have been devised to augment the anti-HIV potency of these compounds.

  19. Non-collagenous protein screening in the human chondrodysplasias: link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin.

    PubMed

    Stanescu, V; Do, T P; Chaminade, F; Maroteaux, P; Stanescu, R

    1994-05-15

    A gel-electrophoretic screening for link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin abnormalities was performed in fetuses, newborn infants, and children with various types of chondrodysplasia. Microdissected freeze-dried sections of upper tibial growth cartilage were extracted with 4M guanidinium chloride in the presence of proteolysis inhibitors. After dialysis against 8M urea, the extracts were submitted to stepwise ion-exchange chromatography to separate the large proteoglycans (aggrecans) from the other components. The latter were analyzed by gel electrophoresis, electrotransferred onto nitrocellulose membranes, and reacted with specific antibodies. Control samples from individuals with apparently normal growth were analyzed in the same runs. Two link protein bands with abnormal electrophoretic migration were found in a sporadic case of spondylometaphyseal dysplasia, Kozlowski type. Three link protein bands with the same migration as in the control samples were found in thanatophoric dysplasia, homozygous achondroplasia, achondrogenesis type II, hypochondrogenesis, Goldblatt syndrome, Desbuquois dysplasia, pseudoachondroplasia, and diastrophic dysplasia. In several pathologic cases with normal electrophoretic pattern of the link proteins, small link protein fragments appeared after reduction. The gel electrophoretic pattern of COMP was studied in thanatophoric dysplasia, diastrophic dysplasia, homozygous achondroplasia, fibrochondrogenesis, hypochondrogenesis, Goldblatt syndrome, and Kniest dysplasia. In all these cases the pattern was the same as in the control samples. The main band of fibromodulin had a normal migration rate in fibrochondrogenesis, Desbuquois dysplasia, Kniest dysplasia, and pseudoachondroplasia. It was delayed in diastrophic dysplasia. PMID:8030664

  20. Comparative Analysis of Human, Mouse, and Pig Glial Fibrillary Acidic Protein Gene Structures.

    PubMed

    Eun, Kiyoung; Hwang, Seon-Ung; Jeon, Hye-Min; Hyun, Sang-Hwan; Kim, Hyunggee

    2016-01-01

    Comparing the coding and regulatory sequences of genes in different species provides information on whether proteins translated from genes have conserved functions or gene expressions are regulated by analogical mechanisms. Herein, we compared the coding and regulatory sequences of glial fibrillary acidic protein (GFAP) from humans, mice, and pigs. The GFAP gene encodes a class III intermediate filament protein expressed specifically in astrocytes of the central nervous system. On comparing the mRNA, regulatory region (promoter), and protein sequences of GFAP gene in silico, we found that GFAP mRNA 3'-untranslated region (3'-UTR), promoter, and amino acid sequences showed higher similarities between humans and pigs than between humans and mice. In addition, the promoter-luciferase reporter gene assay revealed that the pig GFAP promoter functioned in human astrocytes. Notably, the 1.8-kb promoter fragment upstream from transcription initiation site showed strongest transcriptional activity compared to 5.2-kb DNA fragment or other regions of GFAP promoter. We also found that pig GFAP mRNA and promoter activity increased in pig fibroblasts by human IL-1β treatment. Taken together, these results suggest that the regulatory mechanisms and functions of pig genes might be more similar to those of humans than mice, indicating that pigs, particularly miniature pigs, are a useful model for studying human biological and pathological events. PMID:26913554

  1. Comparative Analysis of Human, Mouse, and Pig Glial Fibrillary Acidic Protein Gene Structures.

    PubMed

    Eun, Kiyoung; Hwang, Seon-Ung; Jeon, Hye-Min; Hyun, Sang-Hwan; Kim, Hyunggee

    2016-01-01

    Comparing the coding and regulatory sequences of genes in different species provides information on whether proteins translated from genes have conserved functions or gene expressions are regulated by analogical mechanisms. Herein, we compared the coding and regulatory sequences of glial fibrillary acidic protein (GFAP) from humans, mice, and pigs. The GFAP gene encodes a class III intermediate filament protein expressed specifically in astrocytes of the central nervous system. On comparing the mRNA, regulatory region (promoter), and protein sequences of GFAP gene in silico, we found that GFAP mRNA 3'-untranslated region (3'-UTR), promoter, and amino acid sequences showed higher similarities between humans and pigs than between humans and mice. In addition, the promoter-luciferase reporter gene assay revealed that the pig GFAP promoter functioned in human astrocytes. Notably, the 1.8-kb promoter fragment upstream from transcription initiation site showed strongest transcriptional activity compared to 5.2-kb DNA fragment or other regions of GFAP promoter. We also found that pig GFAP mRNA and promoter activity increased in pig fibroblasts by human IL-1β treatment. Taken together, these results suggest that the regulatory mechanisms and functions of pig genes might be more similar to those of humans than mice, indicating that pigs, particularly miniature pigs, are a useful model for studying human biological and pathological events.

  2. The human homolog of the JE gene encodes a monocyte secretory protein.

    PubMed Central

    Rollins, B J; Stier, P; Ernst, T; Wong, G G

    1989-01-01

    The mouse fibroblast gene, JE, was one of the first platelet-derived growth factor-inducible genes to be described as such. The protein encoded by JE (mJE) is the prototype of a large family of secreted, cytokinelike glycoproteins, all of whose members are induced by a mitogenic or activation signal in monocytes macrophages, and T lymphocytes; JE is the only member to have been identified in fibroblasts. We report the identification of a human homolog for murine JE, cloned from human fibroblasts. The protein predicted by the coding sequence of human JE (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to that of a recently purified monocyte chemoattractant. When expressed in COS cells, the human JE cDNA directed the secretion of N-glycosylated proteins of Mr 16,000 to 18,000 as well as proteins of Mr 15,500, 15,000, and 13,000. Antibodies raised against mJE recognized these hJE species, all of which were secreted by human fibroblasts. hJE expression was stimulated in HL60 cells during phorbol myristate acetate-induced monocytoid differentiation. However, resting human monocytes constitutively secreted hJE; treatment with gamma interferon did not enhance hJE expression in monocytes, and treatment with phorbol myristate acetate or lipopolysaccharide inhibited its expression. Thus, human JE encodes yet another member of the large family of JE-related cytokinelike proteins, in this case a novel human monocyte and fibroblast secretory protein. Images PMID:2513477

  3. Antiproliferative effects of galectin-1 from Rana catesbeiana eggs on human leukemia cells and its binding proteins in human cells.

    PubMed

    Yasumitsu, Hidetaro; Mochida, Keiichi; Yasuda, Chie; Isobe, Masaharu; Kawsar, Sarkar M A; Fujii, Yuki; Matsumoto, Ryo; Kanaly, Robert A; Ozeki, Yasuhiro

    2011-12-01

    Galectin-1 from American bullfrog, RCG1, was isolated to high purity, and its growth inhibitory properties against human cells were examined. The results demonstrated that highly purified RCG1 induced large cell aggregates and revealed cell-type-specific growth inhibition. It significantly inhibited all human leukemia cell lines tested such as HL-60, U937, and K562 cells but did not inhibit human colon cancer cell line, Colo 201, or mouse mammary tumor cell line FM3A cells. Although most of the galectin-induced growth inhibitions are known to be apoptic, RCG1 induced growth arrest and neither apoptosis nor necrosis. RCG1-mediated growth inhibition was specifically suppressed by the corresponding sugar, lactose, but not by sucrose or even the structurally similar sugar, melibiose. Several studies have reported that galectin-mediated biological functions were modulated by charge modification. Since the high purity of RCG1 was demonstrated but a moderate degree of growth inhibition occurred, it is possible protein charge modification was examined by isoelectric focusing, and it was found to be highly heterogeneous in charge. RCG1 binding proteins in human cells were analyzed by lectin blotting using biotinylated RCG1, and lectin blotting revealed that in human cell extracts the specific proteins at molecular weight 37 and 50 kDa possessed the responsive features of RCG1 binding and lactose competition. PMID:22012416

  4. Antiproliferative effects of galectin-1 from Rana catesbeiana eggs on human leukemia cells and its binding proteins in human cells.

    PubMed

    Yasumitsu, Hidetaro; Mochida, Keiichi; Yasuda, Chie; Isobe, Masaharu; Kawsar, Sarkar M A; Fujii, Yuki; Matsumoto, Ryo; Kanaly, Robert A; Ozeki, Yasuhiro

    2011-12-01

    Galectin-1 from American bullfrog, RCG1, was isolated to high purity, and its growth inhibitory properties against human cells were examined. The results demonstrated that highly purified RCG1 induced large cell aggregates and revealed cell-type-specific growth inhibition. It significantly inhibited all human leukemia cell lines tested such as HL-60, U937, and K562 cells but did not inhibit human colon cancer cell line, Colo 201, or mouse mammary tumor cell line FM3A cells. Although most of the galectin-induced growth inhibitions are known to be apoptic, RCG1 induced growth arrest and neither apoptosis nor necrosis. RCG1-mediated growth inhibition was specifically suppressed by the corresponding sugar, lactose, but not by sucrose or even the structurally similar sugar, melibiose. Several studies have reported that galectin-mediated biological functions were modulated by charge modification. Since the high purity of RCG1 was demonstrated but a moderate degree of growth inhibition occurred, it is possible protein charge modification was examined by isoelectric focusing, and it was found to be highly heterogeneous in charge. RCG1 binding proteins in human cells were analyzed by lectin blotting using biotinylated RCG1, and lectin blotting revealed that in human cell extracts the specific proteins at molecular weight 37 and 50 kDa possessed the responsive features of RCG1 binding and lactose competition.

  5. Highly Elastic and Conductive Human-Based Protein Hybrid Hydrogels.

    PubMed

    Annabi, Nasim; Shin, Su Ryon; Tamayol, Ali; Miscuglio, Mario; Bakooshli, Mohsen Afshar; Assmann, Alexander; Mostafalu, Pooria; Sun, Jeong-Yun; Mithieux, Suzanne; Cheung, Louis; Tang, Xiaowu Shirley; Weiss, Anthony S; Khademhosseini, Ali

    2016-01-01

    A highly elastic hybrid hydrogel of methacryloyl-substituted recombinant human tropoelastin (MeTro) and graphene oxide (GO) nanoparticles are developed. The synergistic effect of these two materials significantly enhances both ultimate strain (250%), reversible rotation (9700°), and the fracture energy (38.8 ± 0.8 J m(-2) ) in the hybrid network. Furthermore, improved electrical signal propagation and subsequent contraction of the muscles connected by hybrid hydrogels are observed in ex vivo tests.

  6. Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.

    PubMed Central

    Guénette, S Y; Chen, J; Jondro, P D; Tanzi, R E

    1996-01-01

    We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855266

  7. Protein western array analysis in human pituitary tumours: insights and limitations.

    PubMed

    Ribeiro-Oliveira, Antônio; Franchi, Giulia; Kola, Blerina; Dalino, Paolo; Pinheiro, Sérgio Veloso Brant; Salahuddin, Nabila; Musat, Madalina; Góth, Miklós I; Czirják, Sándor; Hanzély, Zoltán; da Silva, Deivid Augusto; Paulino, Eduardo; Grossman, Ashley B; Korbonits, Márta

    2008-12-01

    The molecular analysis of pituitary tumours has received a great deal of attention, although the majority of studies have concentrated on the genome and the transcriptome. We aimed to study the proteome of human pituitary adenomas. A protein array using 1005 monoclonal antibodies was used to study GH-, corticotrophin- and prolactin-secreting as well as non-functioning pituitary adenomas (NFPAs). Individual protein expression levels in the tumours were compared with the expression profile of normal pituitary tissue. Out of 316 proteins that were detected in the pituitary tissue samples, 116 proteins had not previously been described in human pituitary tissue. Four prominent differentially expressed proteins with potential importance to tumorigenesis were chosen for validation by immunohistochemistry and western blotting. In the protein array analysis heat shock protein 110 (HSP110), a chaperone associated with protein folding, and B2 bradykinin receptor, a potential regulator of prolactin secretion, were significantly overexpressed in all adenoma subtypes, while C-terminal Src kinase (CSK), an inhibitor of proto-oncogenic enzymes, and annexin II, a calcium-dependent binding protein, were significantly underexpressed in all adenoma subtypes. The immunohistochemical analysis confirmed the overexpression of HSP110 and B2 bradykinin receptor and underexpression of CSK and annexin II in pituitary adenoma cells when compared with their corresponding normal pituitary cells. Western blotting only partially confirmed the proteomics data: HSP110 was significantly overexpressed in prolactinomas and NFPAs, the B2 bradykinin receptor was significantly overexpressed in prolactinomas, annexin II was significantly underexpressed in somatotrophinomas, while CSK did not show significant underexpression in any tumour. Protein expression analysis of pituitary samples disclosed both novel proteins and putative protein candidates for pituitary tumorigenesis, though validation using

  8. Effects of Salvianolic Acid B on Protein Expression in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Chang, Tsong-Min; Shi, Guey-Yueh; Wu, Hua-Lin; Wu, Chieh-Hsi; Su, Yan-Di; Wang, Hui-Lin; Wen, Hsin-Yun; Huang, Huey-Chun

    2011-01-01

    Salvianolic acid B (Sal B), a pure water-soluble compound extracted from Radix Salviae miltiorrhizae, has been reported to possess potential cardioprotective efficacy. To identify proteins or pathways by which Sal B might exert its protective activities on the cardiovascular system, two-dimensional gel electrophoresis-based comparative proteomics was performed, and proteins altered in their expression level after Sal B treatment were identified by MALDI-TOF MS/MS. Human umbilical vein endothelial cells (HUVECs) were incubated at Sal B concentrations that can be reached in human plasma by pharmacological intervention. Results indicated that caldesmon, an actin-stabilizing protein, was downregulated in Sal B-exposed HUVECs. Proteins that showed increased expression levels upon Sal B treatment were vimentin, T-complex protein 1, protein disulfide isomerase, tropomyosin alpha, heat shock protein beta-1, UBX domain-containing protein 1, alpha enolase, and peroxiredoxin-2. Additionally, Sal B leads to increased phosphorylation of nucleophosmin in a dose-dependent manner and promotes proliferation of HUVECs. We found that Sal B exhibited a coordinated regulation of enzymes and proteins involved in cytoskeletal reorganization, oxidative stress, and cell growth. Our investigation would provide understanding to the endothelium protection information of Sal B. PMID:21423689

  9. Induction of a putative laminin-binding protein of Streptococcus gordonii in human infective endocarditis.

    PubMed Central

    Sommer, P; Gleyzal, C; Guerret, S; Etienne, J; Grimaud, J A

    1992-01-01

    There is evidence to suggest that the virulence of Streptococcus strains in infective endocarditis might be due to the expression of binding sites for the extracellular matrix proteins of damaged valves. In this communication, we draw attention to one laminin-binding protein from a strain of Streptococcus gordonii isolated from a patient with human endocarditis. This 145-kDa protein was found on the cell wall of the bacterium. The level of expression of this binding protein might be regulated by the presence of extracellular matrix proteins: the protein was lacking after in vitro selection of laminin, collagen I, and fibronectin nonbinding variants, and it was recovered after growth of the variants when laminin or collagen I was added to the growth medium. It was also missing after 10 subcultures in minimal medium, indicating some positive control. Furthermore, the 145-kDa protein was recognized as a major antigen by sera from patients treated for streptococcal infective endocarditis, while sera from patients with valvulopathies gave only slight recognition, suggesting an increase of the expression of this protein during infective endocarditis. It was also shown that the 145-kDa protein carried a collagen I-like determinant detected with anti-human collagen I antibodies. Images PMID:1530927

  10. Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

    PubMed Central

    Candau, R; Moore, P A; Wang, L; Barlev, N; Ying, C Y; Rosen, C A; Berger, S L

    1996-01-01

    Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation. PMID:8552087

  11. Meibomian gland dysfunction. I. Keratin protein expression in normal human and rabbit meibomian glands.

    PubMed

    Jester, J V; Nicolaides, N; Smith, R E

    1989-05-01

    The expression of keratin proteins from meibomian glands and their correlation with skin epidermal keratins were determined. Keratin proteins were localized in both human and rabbit meibomian glands by indirect immunofluorescence using mouse monoclonal antibodies AE1, AE2 and AE3, which are known to react with human epidermal keratins as well as with keratins from other sources. Keratin proteins from rabbit meibomian glands were further isolated and characterized by SDS-PAGE and immunoblot using mouse monoclonal antibodies AE1 and AE3. Meibomian glands from human and rabbit showed similar immunofluorescent staining with each monoclonal antibody. AE1 antibody, which stains human basal epithelial cells of skin, stains all duct epithelial cells in the human but only the superficial duct epithelial cells in the rabbit meibomian gland. AE2 antibody, which stains human suprabasal epithelial cells of skin and is a marker for fully keratinized epithelia, stains the suprabasal epithelial cells of the central duct and ductules in both the human and rabbit meibomian gland. AE3 antibody, which stains all human epithelial cells of skin, stains all epithelial cells of the duct and ductules, as well as the basal epithelial cells of the acinus in both the human and rabbit meibomian gland. Keratins isolated from whole rabbit meibomian glands contained a 65-67 kD and 58 kD AE3-positive, and a 56.5 kD and 50 kD AE1-positive keratin protein. Expression of 65-67 kD/56.5 kD keratin proteins, and the immunofluorescent staining of the duct epithelium by the AE2 antibody, indicate that the meibomian gland duct epithelium is committed to the process of keratinization.

  12. Rate of spontaneous mutation at human loci encoding protein structure.

    PubMed Central

    Neel, J V; Mohrenweiser, H W; Meisler, M H

    1980-01-01

    The techniques of electrophoresis were used in a search for evidence of mutation affecting protein structure, the indicators being hemoglobin and a set of serum proteins and erythrocyte enzymes. Among 94,796 locus tests on Amerindians from Central and South America, there was no evidence for mutation. Among 105,649 locus tests on newborn infants in Ann Arbor, Michigan, there was also no evidence for mutation. We have previously failed to encounter any mutations in a series of 208,196 locus tests involving Japanese children [Neel, J. V., Satoh, C., Hamilton, H. B., Otake, M., Goriki, K., Kageoka, T., Fugita, M., Neriishi, S & Asakawa,J. (1980) Proc. Natl. Acad. Sci. USA 77, 4221-4225], and H. Harris, D. A. Hopkinson, and E. B. Robson [(1974) Ann. Hum. Genet. 37, 237-253] found no mutations in 113,478 locus tests on inhabitants of the United Kingdom. This failure to demonstrate any mutations of this type in a total of 522,119 locus tests excludes, at the 95% level of probability, a mutation rate greater than 0.6 X 10(-5)/locus per generation in this combination of populations. PMID:6934530

  13. Biochemical analysis of the stress protein response in human oesophageal epithelium

    PubMed Central

    Hopwood, D; Moitra, S; Vojtesek, B; Johnston, D; Dillon, J; Hupp, T

    1997-01-01

    Background—The oesophageal epithelium is exposed routinely to noxious agents in the environment, including gastric acid, thermal stress, and chemical toxins. These epithelial cells have presumably evolved effective protective mechanisms to withstand tissue damage and repair injured cells. Heat shock protein or stress protein responses play a central role in protecting distinct cell types from different types of injury. 
Aim—To determine (i) whether biochemical analysis of stress protein responses in pinch biopsy specimens from human oesophageal epithelium is feasible; (ii) whether undue stresses are imposed on cells by the act of sample collection, thus precluding analysis of stress responses; and (iii) if amenable to experimentation, the type of heat shock protein (Hsp) response that operates in the human oesophageal epithelium. 
Methods—Tissue from the human oesophagus comprised predominantly of squamous epithelium was acquired within two hours of biopsy and subjected to an in vitro heat shock. Soluble tissue cell lysates derived from untreated or heat shocked samples were examined using denaturing polyacrylamide gel electrophoresis for changes in: (i) the pattern of general protein synthesis by labelling epithelial cells with 35S-methionine and (ii) the levels of soluble Hsp70 protein and related isoforms using immunochemical protein blots. 
Results—A single pinch biopsy specimen is sufficient to extract and analyse specific sets of polypeptides in the oesophageal epithelium. After ex vivo heat shock, a classic inhibition of general protein synthesis is observed and correlates with the increased synthesis of two major proteins of molecular weight of 60 and 70 kDa. Notably, cells from unheated controls exhibit a "stressed" biochemical state 22 hours after incubation at 37°C, as shown by inhibition of general protein synthesis and increased synthesis of the 70 kDa protein. These data indicate that only freshly acquired specimens are suitable for

  14. Protein kinase C phosphorylates a recently identified membrane skeleton-associated calmodulin-binding protein in human erythrocytes.

    PubMed

    Ling, E; Gardner, K; Bennett, V

    1986-10-25

    A membrane skeleton-associated protein with calmodulin-binding activity recently has been purified and characterized from human erythrocytes (Gardner, K. and Bennett, V. (1986) J. Biol. Chem. 261, 1339-1348). This new protein (CaM-BP103/97) has now been identified as a major substrate for protein kinase C in erythrocytes since phosphorylation of both of its subunits (Mr = 103,000 and 97,000) is elevated 3-15-fold in the presence of the phorbol ester, 12-O-tetradecanoylphorbol beta-acetate (TPA), under the following conditions: ghost membranes incubated with protein kinase C purified from rat brain, ghost membranes from erythrocytes pretreated with TPA, and intact erythrocytes metabolically labeled with 32PO4 and stimulated by TPA. The sites of phosphorylation of this protein by exogenous and endogenous protein kinase C are identical since two-dimensional 32P-peptide maps of both subunits labeled by either endogenous or exogenous enzyme are indistinguishable. Each subunit of CaM-BP103/97 accepts up to 3 mol of phosphate/polypeptide chain. In the presence of low calcium concentrations and in the absence of cytosol, the phosphorylation of CaM-BP103/97 is, on a molar basis, equal to or greater than that of proteins 4.1 and 4.9. As a target for both calmodulin and protein kinase C, CaM-BP103/97 is likely to play a key role in the effect of calcium on erythrocyte membrane shape and stability.

  15. Aqueous two-phase partitioning of milk proteins. Application to human protein C secreted in pig milk.

    PubMed

    Cole, K D; Lee, T K; Lubon, H

    1997-01-01

    Milk of transgenic pigs secreting recombinant human Protein C (rHPC) was used as a model system to determine the utility of aqueous two-phase extraction systems (ATPS) for the initial step in the purification of proteins from milk. The major challenges in purification of recombinant proteins from milk are removal of casein micelles (that foul processing equipment) and elimination of the host milk proteins from the final product. When milk was partitioned in ATPS composed of polyethylene glycol (PEG) and ammonium sulfate (AS), the phases were clarified and most of the caseins precipitated at the interphase. The partition coefficients of the major milk proteins and rHPC were dependent upon the molecular weight of the PEG used in the ATPS. Higher-partition coefficients of the major whey proteins, beta-lactoglobulin, and alpha-lactalbumin were observed in ATPS made up of lower molecular-weight PEG (1000 or 1450) as compared to systems using higher molecular-weight PEG. Lowering the pH of the ATPS from 7.5 to 6.0 resulted in increased precipitation of the caseins and decreased their concentration in both phases. rHPC had a partition coefficient of 0.04 in a system composed of AS and PEG 1450. The rHPC in pig milk was shown to be highly heterogenous by two-dimensional gel electrophoresis. The heterogeneity was owing to inefficient proteolytic processing of the single chain to the heterodimeric form and differences in glycosylation and other post-translational processing. Differential partitioning of the multiple forms of purified rHPC in the ATPS was not observed. rHPC after processing in ATPS was recovered in a clear phase free of most major milk proteins. ATPS are useful as the initial processing step in the purification of recombinant proteins from milk because clarification and enrichment in combined in a single step. PMID:9382491

  16. RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome.

    PubMed

    Silverman, Ian M; Li, Fan; Alexander, Anissa; Goff, Loyal; Trapnell, Cole; Rinn, John L; Gregory, Brian D

    2014-01-07

    Although numerous approaches have been developed to map RNA-binding sites of individual RNA-binding proteins (RBPs), few methods exist that allow assessment of global RBP-RNA interactions. Here, we describe PIP-seq, a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply PIP-seq to the HeLa transcriptome and compare binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP-binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites.

  17. Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein.

    PubMed

    Denne, Miriam; Sauter, Marlies; Armbruester, Vivienne; Licht, Jonathan D; Roemer, Klaus; Mueller-Lantzsch, Nikolaus

    2007-06-01

    Only few of the human endogenous retrovirus (HERV) sequences in the human genome can produce proteins. We have previously reported that (i) patients with germ cell tumors often make antibodies against proteins encoded by HERV-K elements, (ii) expression of the HERV-K rec gene in transgenic mice can interfere with germ cell development and induce carcinoma in situ, and (iii) HERV-K np9 transcript is overproduced in many tumors including breast cancers. Here we document that both Np9 and Rec physically and functionally interact with the promyelocytic leukemia zinc finger (PLZF) tumor suppressor, a transcriptional repressor and chromatin remodeler implicated in cancer and the self-renewal of spermatogonial stem cells. Interaction is mediated via two different central and C-terminal domains of Np9 and Rec and the C-terminal zinc fingers of PLZF. One major target of PLZF is the c-myc proto-oncogene. Coexpression of Np9 and Rec with PLZF abrogates the transcriptional repression of the c-myc gene promoter by PLZF and results in c-Myc overproduction, altered expression of c-Myc-regulated genes, and corresponding effects on cell proliferation and survival. Thus, the human endogenous retrovirus proteins Np9 and Rec may act oncogenically by derepressing c-myc through the inhibition of PLZF.

  18. Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets

    SciTech Connect

    Bruene, B.; Molina Y Vedia, L.; Lapetina, E.G. )

    1990-05-01

    {alpha}-Thrombin and phorbol 12,13-dibutyrate stimulated the mono(ADP-ribosyl)ation of a 42-kDa cytosolic protein of human platelets. This effect was mediated by protein kinase C activation and was inhibited by protein kinase C inhibitor staurosporine. It also was prevented by prostacyclin, which is known to inhibit the phospholipase C-induced formation of 1,2-diacylglycerol, which is one of the endogenous activators of protein kinase C. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the 42-kDa protein that is ADP-ribosylated by {alpha}-thrombin was clearly distinct from the {alpha} subunits of membrane-bound inhibitory and stimulatory guanine nucleotide-binding regulatory proteins, respectively G{sub i{alpha}} and G{sub s{alpha}}; the 47-kDa protein that is phosphorylated by protein kinase C in platelets; and the 39-kDa protein that has been shown to be endogenously ADP-ribosylated by agents that release nitric oxide. This information shows that agonist-induced activation of protein kinase leads to the ADP-ribosylation of a specific protein. This covalent modification might have a functional role in platelet activation.

  19. Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

    PubMed Central

    Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; Yano, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

    2015-01-01

    To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources. PMID:26308446

  20. Regulation of human ornithine decarboxylase expression by the c-Myc.Max protein complex.

    PubMed

    Peña, A; Reddy, C D; Wu, S; Hickok, N J; Reddy, E P; Yumet, G; Soprano, D R; Soprano, K J

    1993-12-25

    The presence of a CACGTG element within a region of the human ornithine decarboxylase (ODC) promoter located at -491 to -474 base pairs 5' to the start site of transcription suggested that the c-Myc.Max protein complex may play a role in the regulation of ODC expression during growth. Electrophoretic mobility shift assays and methylation interference analysis showed that the nuclei of WI-38 cells expressing ODC contained proteins that bound to this region of the ODC gene in a manner that correlated with growth-associated ODC expression. Also, use of antibodies against c-Myc and Max and purified recombinant c-Myc and Max protein in the electrophoretic mobility shift assay confirmed that these proteins can specifically bind this portion of the human ODC promoter. Transient transfection studies showed that increase in the level of c-Myc and/or Max led to a significant enhancement of expression of a human ODC promoter-CAT reporter construct. Moreover, treatment of actively growing WI-38 cells with an antisense oligomer to c-Myc reduced the amount of endogenous protein complex formed and the amount of endogenous ODC mRNA expressed. These studies show that the c-Myc.Max protein complex plays a role in the transcriptional regulation of human ODC in vivo.

  1. Host-Pathogen Interaction Profiling Using Self-Assembling Human Protein Arrays

    PubMed Central

    Yu, Xiaobo; Decker, Kimberly B.; Barker, Kristi; Neunuebel, M. Ramona; Saul, Justin; Graves, Morgan; Westcott, Nathan; Hang, Howard; LaBaer, Joshua; Qiu, Ji; Machner, Matthias P.

    2015-01-01

    Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10,000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or pre-labeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome. PMID:25739981

  2. Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins.

    PubMed

    Kudryashova, Elena; Koneru, Pratibha C; Kvaratskhelia, Mamuka; Strömstedt, Adam A; Lu, Wuyuan; Kudryashov, Dmitri S

    2016-01-01

    Human defensins are innate immune defense peptides with a remarkably broad repertoire of anti-pathogen activities. In addition to modulating immune response, inflammation, and angiogenesis, disintegrating bacterial membranes, and inactivating bacterial toxins, defensins are known to intercept various viruses at different stages of their life cycles, while remaining relatively benign towards human cells and proteins. Recently we have found that human defensins inactivate proteinaceous bacterial toxins by taking advantage of their low thermodynamic stability and acting as natural "anti-chaperones", i.e. destabilizing the native conformation of the toxins. In the present study we tested various proteins produced by several viruses (HIV-1, PFV, and TEV) and found them to be susceptible to destabilizing effects of human α-defensins HNP-1 and HD-5 and the synthetic θ-defensin RC-101, but not β-defensins hBD-1 and hBD-2 or structurally related plant-derived peptides. Defensin-induced unfolding promoted exposure of hydrophobic groups otherwise confined to the core of the viral proteins. This resulted in precipitation, an enhanced susceptibility to proteolytic cleavage, and a loss of viral protein activities. We propose, that defensins recognize and target a common and essential physico-chemical property shared by many bacterial toxins and viral proteins - the intrinsically low thermodynamic protein stability. PMID:27581352

  3. Protein profile changes in the frontotemporal lobes in human severe traumatic brain injury.

    PubMed

    Xu, Benhong; Tian, Rui; Wang, Xia; Zhan, Shaohua; Wang, Renzhi; Guo, Yi; Ge, Wei

    2016-07-01

    Severe traumatic brain injury (sTBI) is a serious public health issue with high morbidity and mortality rates. Previous proteomic studies on sTBI have mainly focused on human cerebrospinal fluid and serum, as well as on brain protein changes in murine models. However, human proteomic data in sTBI brain is still scarce. We used proteomic and bioinformatic strategies to investigate variations in protein expression levels in human brains after sTBI, using samples from the Department of Neurosurgery, Affiliated Hospital of Hebei University (Hebei, China). Our proteomic data identified 4031 proteins, of which 160 proteins were overexpressed and 5 proteins were downregulated. Bioinformatics analysis showed significant changes in biological pathways including glial cell differentiation, complement activation and apolipoprotein catalysis in the statin pathway. Western blot verification of protein changes in a subset of the available tissue samples showed results that were consistent with the proteomic data. This study is one of the first to investigate the whole proteome of human sTBI brains, and provide a characteristic signature and overall landscape of the sTBI brain proteome.

  4. Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins

    PubMed Central

    Kudryashova, Elena; Koneru, Pratibha C.; Kvaratskhelia, Mamuka; Strömstedt, Adam A.; Lu, Wuyuan; Kudryashov, Dmitri S.

    2016-01-01

    Human defensins are innate immune defense peptides with a remarkably broad repertoire of anti-pathogen activities. In addition to modulating immune response, inflammation, and angiogenesis, disintegrating bacterial membranes, and inactivating bacterial toxins, defensins are known to intercept various viruses at different stages of their life cycles, while remaining relatively benign towards human cells and proteins. Recently we have found that human defensins inactivate proteinaceous bacterial toxins by taking advantage of their low thermodynamic stability and acting as natural “anti-chaperones”, i.e. destabilizing the native conformation of the toxins. In the present study we tested various proteins produced by several viruses (HIV-1, PFV, and TEV) and found them to be susceptible to destabilizing effects of human α-defensins HNP-1 and HD-5 and the synthetic θ-defensin RC-101, but not β-defensins hBD-1 and hBD-2 or structurally related plant-derived peptides. Defensin-induced unfolding promoted exposure of hydrophobic groups otherwise confined to the core of the viral proteins. This resulted in precipitation, an enhanced susceptibility to proteolytic cleavage, and a loss of viral protein activities. We propose, that defensins recognize and target a common and essential physico-chemical property shared by many bacterial toxins and viral proteins – the intrinsically low thermodynamic protein stability. PMID:27581352

  5. High-mobility group A1 proteins are overexpressed in human leukaemias.

    PubMed Central

    Pierantoni, Giovanna Maria; Agosti, Valter; Fedele, Monica; Bond, Heather; Caliendo, Irene; Chiappetta, Gennaro; Lo Coco, Francesco; Pane, Fabrizio; Turco, Maria Caterina; Morrone, Giovanni; Venuta, Salvatore; Fusco, Alfredo

    2003-01-01

    High-mobility group A (HMGA) proteins are non-histone nuclear proteins that bind DNA and several transcription factors. They are involved in the regulation of chromatin structure and function. HMGA protein expression is low in normal adult tissues, but abundant during embryonic development and in several human tumours. Rearrangements of the HMGA genes have been frequently detected in human benign tumours of mesenchymal origin, e.g. lipomas, lung hamartomas and uterine leiomiomas. HMGA proteins have been implicated in the control of cell growth and differentiation of the pre-adipocytic cell line 3T3-L1. In an attempt to better understand the role of HMGA1 proteins in haematological neoplasias and in the differentiation of haematopietic cells, we have investigated their expression in human leukaemias and in leukaemic cell lines induced to terminal differentiation. Here we report HMGA1 overexpression in most fresh human leukaemias of different origin and in several leukaemic cell lines. Moreover, differentiation of three cell lines towards the megakaryocytic phenotype was associated with HMGA1 protein induction, whereas induction of erythroid and monocytic differentiation generally resulted in reduced HMGA1 expression. PMID:12573034

  6. Immunolocalization of a human cementoblastoma-conditioned medium-derived protein.

    PubMed

    Arzate, H; Jiménez-García, L F; Alvarez-Pérez, M A; Landa, A; Bar-Kana, I; Pitaru, S

    2002-08-01

    Little is known about the molecular mechanisms that regulate the cementogenesis process, because specific cementum markers are not yet available. To investigate whether a cementoblastoma-conditioned medium-derived protein (CP) could be useful as a cementum biological marker, we studied its expression and distribution in human periodontal tissues, human periodontal ligament, alveolar bone, and cementoblastoma-derived cells. In human periodontal tissues, immunoreactivity to anti-CP was observed throughout the cementoid phase of acellular and cellular cementum, cementoblasts, cementocytes, cells located in the endosteal spaces of human alveolar bone, and in cells in the periodontal ligament located near the blood vessels. Immunopurified CP promoted cell attachment on human periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts. A monoclonal antibody against bovine cementum attachment protein (CAP) cross-reacted with CP. These findings indicate that CP identifies potential cementoblast progenitor cells, is immunologically related to CAP species, and serves as a biological marker for cementum. PMID:12147744

  7. Human protein C: new preparations. Effective replacement therapy for some clotting disorders.

    PubMed

    2003-02-01

    (1) Depending on its severity, congenital protein C deficiency can cause a variety of problems, such as increasing the frequency of venous thrombosis in high risk situations; recurrent venous thrombosis; skin necrosis at the start of treatment with a vitamin K antagonist; and severe thrombotic events in neonates. For many years the only available replacement treatment consisted of fresh frozen plasma which, among other adverse effects, carries a risk of hypervolemia. (2) Two human protein C concentrates prepared from donated blood have been given marketing authorisation in Europe for intravenous replacement therapy (Ceprotin from Baxter, and Protexel from LFB). (3) Their clinical files contain only retrospective case series (22 children with severe deficiency treated with Ceprotin; and 10 patients of various ages and with different degrees of severity treated with Protexel). The two preparations have not been compared with each other. (4) In patients with severe protein C deficiency, including neonates, replacement therapy with human protein C is effective, especially for treating cutaneous thrombosis and preventing thrombosis in high risk situations. (5) In patients with moderate deficiency, a short-course of human protein C prophylaxis reduces the frequency of thrombosis in high risk situations. (6) In long-term prophylaxis, human protein C replacement therapy, added to ongoing (but inadequately effective) vitamin K antagonist therapy, seems to reduce the risk of recurrent venous thrombosis even though it has some constraints. (7) The adverse effects of the two preparations are poorly documented. Allergic reactions and bleeding have been reported. Human protein C is a blood product, and therefore carries a risk of infection. (8) Ceprotin offers a small advantage, being available in two dose strengths: for a given dose the volume injected is halved. (9) In practice, Ceprotin and Protexel are the reference drugs for replacement therapy of constitutional protein C

  8. Human Neutrophils Secrete Bioactive Paucimannosidic Proteins from Azurophilic Granules into Pathogen-Infected Sputum*

    PubMed Central

    Thaysen-Andersen, Morten; Venkatakrishnan, Vignesh; Loke, Ian; Laurini, Christine; Diestel, Simone; Parker, Benjamin L.; Packer, Nicolle H.

    2015-01-01

    Unlike plants and invertebrates, mammals reportedly lack proteins displaying asparagine (N)-linked paucimannosylation (mannose1–3fucose0–1N-acetylglucosamine2Asn). Enabled by technology advancements in system-wide biomolecular characterization, we document that protein paucimannosylation is a significant host-derived molecular signature of neutrophil-rich sputum from pathogen-infected human lungs and is negligible in pathogen-free sputum. Five types of paucimannosidic N-glycans were carried by compartment-specific and inflammation-associated proteins of the azurophilic granules of human neutrophils including myeloperoxidase (MPO), azurocidin, and neutrophil elastase. The timely expressed human azurophilic granule-resident β-hexosaminidase A displayed the capacity to generate paucimannosidic N-glycans by trimming hybrid/complex type N-glycan intermediates with relative broad substrate specificity. Paucimannosidic N-glycoepitopes showed significant co-localization with β-hexosaminidase A and the azurophilic marker MPO in human neutrophils using immunocytochemistry. Furthermore, promyelocyte stage-specific expression of genes coding for paucimannosidic proteins and biosynthetic enzymes indicated a novel spatio-temporal biosynthetic route in early neutrophil maturation. The absence of bacterial exoglycosidase activities and paucimannosidic N-glycans excluded exogenous origins of paucimannosylation. Paucimannosidic proteins from isolated and sputum neutrophils were preferentially secreted upon inoculation with virulent Pseudomonas aeruginosa. Finally, paucimannosidic proteins displayed affinities to mannose-binding lectin, suggesting immune-related functions of paucimannosylation in activated human neutrophils. In conclusion, we are the first to document that human neutrophils produce, store and, upon activation, selectively secrete bioactive paucimannosidic proteins into sputum of lungs undergoing pathogen-based inflammation. PMID:25645918

  9. Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.

    PubMed

    Thaysen-Andersen, Morten; Venkatakrishnan, Vignesh; Loke, Ian; Laurini, Christine; Diestel, Simone; Parker, Benjamin L; Packer, Nicolle H

    2015-04-01

    Unlike plants and invertebrates, mammals reportedly lack proteins displaying asparagine (N)-linked paucimannosylation (mannose(1-3)fucose(0-1)N-acetylglucosamine(2)Asn). Enabled by technology advancements in system-wide biomolecular characterization, we document that protein paucimannosylation is a significant host-derived molecular signature of neutrophil-rich sputum from pathogen-infected human lungs and is negligible in pathogen-free sputum. Five types of paucimannosidic N-glycans were carried by compartment-specific and inflammation-associated proteins of the azurophilic granules of human neutrophils including myeloperoxidase (MPO), azurocidin, and neutrophil elastase. The timely expressed human azurophilic granule-resident β-hexosaminidase A displayed the capacity to generate paucimannosidic N-glycans by trimming hybrid/complex type N-glycan intermediates with relative broad substrate specificity. Paucimannosidic N-glycoepitopes showed significant co-localization with β-hexosaminidase A and the azurophilic marker MPO in human neutrophils using immunocytochemistry. Furthermore, promyelocyte stage-specific expression of genes coding for paucimannosidic proteins and biosynthetic enzymes indicated a novel spatio-temporal biosynthetic route in early neutrophil maturation. The absence of bacterial exoglycosidase activities and paucimannosidic N-glycans excluded exogenous origins of paucimannosylation. Paucimannosidic proteins from isolated and sputum neutrophils were preferentially secreted upon inoculation with virulent Pseudomonas aeruginosa. Finally, paucimannosidic proteins displayed affinities to mannose-binding lectin, suggesting immune-related functions of paucimannosylation in activated human neutrophils. In conclusion, we are the first to document that human neutrophils produce, store and, upon activation, selectively secrete bioactive paucimannosidic proteins into sputum of lungs undergoing pathogen-based inflammation. PMID:25645918

  10. Identification of Proteins in Human Cytomegalovirus (HCMV) Particles: the HCMV Proteome

    SciTech Connect

    Varnum, Susan M.; Streblow, Daniel N.; Monroe, Matthew E.; Smith, Patricia; Auberry, Kenneth J.; Pasa-Tolic, Liljiana; Wang, Dai; Camp, David G.; Rodland, Karin D.; Wiley, H S.; Britt, William; Shenk, Thomas; Smith, Richard D.; Nelson, Jay

    2004-10-15

    Human cytomegalovirus (HCMV), a member of the herpes virus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.

  11. Association of filamin A and vimentin with hepatitis C virus proteins in infected human hepatocytes.

    PubMed

    Ghosh, S; Ahrens, W A; Phatak, S U; Hwang, S; Schrum, L W; Bonkovsky, H L

    2011-10-01

    Chronic hepatitis C (CHC) infection caused by hepatitis C virus (HCV) is a major cause of liver disease and remains a major therapeutic challenge. A variety of host proteins interact with HCV proteins. The definitive role of cytoskeletal (CS) proteins in HCV infection remains to be determined. In this study, our aim was to determine the expression profile of differentially regulated and expressed selected CS proteins and their association with HCV proteins in infected hepatocytes as possible therapeutic targets. Using proteomics, qRT-PCR, Western blot and immunofluorescence techniques, we revealed that filamin A (fila) and vimentin (vim) were prominently increased proteins in HCV-expressing human hepatoma cells compared with parental cells and in liver biopsies from patients with CHC vs controls. HCV nonstructural (NS) 3 and NS5A proteins were associated with fila, while core protein partially with fila and vim. Immunoprecipitation showed interactions among fila and NS3 and NS5A proteins. Cells treated with interferon-α showed a dose- and time-dependent decrease in CS and HCV proteins. NS proteins clustered at the perinuclear region following cytochalasin b treatment, whereas disperse cytoplasmic and perinuclear distribution was observed in the no-treatment group. This study demonstrates and signifies that changes occur in the expression of CS proteins in HCV-infected hepatocytes and, for the first time, shows the up-regulation and interaction of fila with HCV proteins. Association between CS and HCV proteins may have implications in future design of CS protein-targeted therapy for the treatment for HCV infection.

  12. A High-Yield Two-Hour Protocol for Extraction of Human Hair Shaft Proteins

    PubMed Central

    Wong, Sing Ying; Lee, Ching Chin; Ashrafzadeh, Ali; Junit, Sarni Mat; Abrahim, Nazirahanie

    2016-01-01

    Proteome analysis of the human hair remains challenging due to the poor solubility of hair proteins and the difficulty in their extraction. In the present study, we have developed a rapid extraction protocol for hair shaft protein using alkaline-based buffer. The new protocol accelerated the procedure by reducing the extraction time from at least a day to less than two hours and showed a protein recovery of 47.3 ± 3.72%. Further analyses of the extracted protein sample through sodium dodecyl sulfate polyacrylamide gel electrophoresis and Quadrupole-time-of-flight mass spectrometry analysis unveiled a total of 60 proteins, including 25 that were not previously reported. Identification of these proteins is anticipated to be crucial in helping to understand the molecular basis of hair for potential applications in the future. PMID:27741315

  13. Cytoplasmic Ig-Domain Proteins: Cytoskeletal Regulators with a Role in Human Disease

    PubMed Central

    Otey, Carol A.; Dixon, Richard; Stack, Christianna; Goicoechea, Silvia M.

    2009-01-01

    Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain proteins are more than structural elements of the cell, and may have evolved to fill different needs in different cellular compartments. PMID:19466753

  14. Identification of nuclear localization signals within the human BCOR protein.

    PubMed

    Surapornsawasd, Thunyaporn; Ogawa, Takuya; Moriyama, Keiji

    2015-10-24

    Mutations in the BCL-6 corepressor (BCOR) gene, which encodes a transcriptional corepressor, were described to cause oculofaciocardiodental syndrome (MIM 300166). The purpose of this study was to localize the classical nuclear localization signals (NLSs) of the BCOR using reported human BCOR mutations with comparable phenotypes. The genotype-phenotype correlation among the mutations could not be clearly explained; however, the classical NLSs were identified at two possible sites; RVDRKRKVSGD at aa1131-1141 (NLS1) and LKAKRRRVSK at aa1158-1167 (NLS2). In addition, according to our results, NLS2 displayed a more efficient nuclear import function than NLS1. PMID:26054978

  15. The human cut homeodomain protein represses transcription from the c-myc promoter.

    PubMed Central

    Dufort, D; Nepveu, A

    1994-01-01

    Studies of the c-myc promoter have shown that efficient transcription initiation at the P2 start site as well as the block to elongation of transcription require the presence of the ME1a1 protein binding site upstream of the P2 TATA box. Following fractionation by size exclusion chromatography, three protein-ME1a1 DNA complexes, a, b, and c, were detected by electrophoretic mobility shift assay. A cDNA encoding a protein present in complex c was isolated by screening of an expression library with an ME1a1 DNA probe. This cDNA was found to encode the human homolog of the Drosophila Cut homeodomain protein. The bacterially expressed human Cut (hu-Cut) protein bound to the ME1a1 site, and antibodies against hu-Cut inhibited the ME1a1 binding activity c in nuclear extracts. In cotransfection experiments, the hu-Cut protein repressed transcription from the c-myc promoter, and this repression was shown to be dependent on the presence of the ME1a1 site. Using a reporter construct with a heterologous promoter, we found that c-myc exon 1 sequences were also necessary, in addition to the ME1a1 site, for repression by Cut. Taken together, these results suggest that the human homolog of the Drosophila Cut homeodomain protein is involved in regulation of the c-myc gene. Images PMID:8196661

  16. Comprehensive prediction of drug-protein interactions and side effects for the human proteome

    PubMed Central

    Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

    2015-01-01

    Identifying unexpected drug-protein interactions is crucial for drug repurposing. We develop a comprehensive proteome scale approach that predicts human protein targets and side effects of drugs. For drug-protein interaction prediction, FINDSITEcomb, whose average precision is ~30% and recall ~27%, is employed. For side effect prediction, a new method is developed with a precision of ~57% and a recall of ~24%. Our predictions show that drugs are quite promiscuous, with the average (median) number of human targets per drug of 329 (38), while a given protein interacts with 57 drugs. The result implies that drug side effects are inevitable and existing drugs may be useful for repurposing, with only ~1,000 human proteins likely causing serious side effects. A killing index derived from serious side effects has a strong correlation with FDA approved drugs being withdrawn. Therefore, it provides a pre-filter for new drug development. The methodology is free to the academic community on the DR. PRODIS (DRugome, PROteome, and DISeasome) webserver at http://cssb.biology.gatech.edu/dr.prodis/. DR. PRODIS provides protein targets of drugs, drugs for a given protein target, associated diseases and side effects of drugs, as well as an interface for the virtual target screening of new compounds. PMID:26057345

  17. A comprehensive analysis of the Streptococcus pyogenes and human plasma protein interaction network.

    PubMed

    Sjöholm, Kristoffer; Karlsson, Christofer; Linder, Adam; Malmström, Johan

    2014-07-01

    Streptococcus pyogenes is a major human bacterial pathogen responsible for severe and invasive disease associated with high mortality rates. The bacterium interacts with several human blood plasma proteins and clarifying these interactions and their biological consequences will help to explain the progression from mild to severe infections. In this study, we used a combination of mass spectrometry (MS) based techniques to comprehensively quantify the components of the S. pyogenes-plasma protein interaction network. From an initial list of 181 interacting human plasma proteins defined using liquid chromatography (LC)-MS/MS analysis we further subdivided the interacting protein list using selected reaction monitoring (SRM) depending on the level of enrichment and protein concentration on the bacterial surface. The combination of MS methods revealed several previously characterized interactions between the S. pyogenes surface and human plasma along with many more, so far uncharacterised, possible plasma protein interactions with S. pyogenes. In follow-up experiments, the combination of MS techniques was applied to study differences in protein binding to a S. pyogenes wild type strain and an isogenic mutant lacking several important virulence factors, and a unique pair of invasive and non-invasive S. pyogenes isolates from the same patient. Comparing the plasma protein-binding properties of the wild type and the mutant and the invasive and non-invasive S. pyogenes bacteria revealed considerable differences, underlining the significance of these protein interactions. The results also demonstrate the power of the developed mass spectrometry method to investigate host-microbial relationships with a large proteomics depth and high quantitative accuracy.

  18. Ewing Sarcoma Protein: A Key Player in Human Cancer

    PubMed Central

    2013-01-01

    The Ewing sarcoma protein (EWS) is a well-known player in cancer biology for the specific translocations occurring in sarcomas. The EWS-FLI1 gene fusion is the prototypical translocation that encodes the aberrant, chimeric transcription factor, which is a landmark of Ewing tumors. In all described Ewing sarcoma oncogenes, the EWS RNA binding domains are completely missing; thus RNA binding properties are not retained in the hybrid proteins. However, it is currently unknown whether the absence of EWS function in RNA metabolism plays a role in oncogenic transformation or if EWS plays a role by itself in cancer development besides its contribution to the translocation. In this regard, recent reports have highlighted an essential role for EWS in the regulation of DNA damage response (DDR), a process that counteracts genome stability and is often deregulated in cancer cells. The first part of this review will describe the structural features of EWS and its multiple roles in the regulation of gene expression, which are exerted by coordinating different steps in the synthesis and processing of pre-mRNAs. The second part will examine the role of EWS in the regulation of DDR- and cancer-related genes, with potential implications in cancer therapies. Finally, recent advances on the involvement of EWS in neuromuscular disorders will be discussed. Collectively, the information reviewed herein highlights the broad role of EWS in bridging different cellular processes and underlines the contribution of EWS to genome stability and proper cell-cycle progression in higher eukaryotic cells. PMID:24082883

  19. Iron-sulfur cluster exchange reactions mediated by the human Nfu protein.

    PubMed

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

    2016-10-01

    Human Nfu is an iron-sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron-sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe-2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe-2S](GS)4 cluster complex, and extraction of the Nfu-bound iron-sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe-2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe-2S] cluster trafficking and delivery. PMID:27538573

  20. Conserved differences in protein sequence determine the human pathogenicity of Ebolaviruses

    PubMed Central

    Pappalardo, Morena; Juliá, Miguel; Howard, Mark J.; Rossman, Jeremy S.; Michaelis, Martin; Wass, Mark N.

    2016-01-01

    Reston viruses are the only Ebolaviruses that are not pathogenic in humans. We analyzed 196 Ebolavirus genomes and identified specificity determining positions (SDPs) in all nine Ebolavirus proteins that distinguish Reston viruses from the four human pathogenic Ebolaviruses. A subset of these SDPs will explain the differences in human pathogenicity between Reston and the other four ebolavirus species. Structural analysis was performed to identify those SDPs that are likely to have a functional effect. This analysis revealed novel functional insights in particular for Ebolavirus proteins VP40 and VP24. The VP40 SDP P85T interferes with VP40 function by altering octamer formation. The VP40 SDP Q245P affects the structure and hydrophobic core of the protein and consequently protein function. Three VP24 SDPs (T131S, M136L, Q139R) are likely to impair VP24 binding to human karyopherin alpha5 (KPNA5) and therefore inhibition of interferon signaling. Since VP24 is critical for Ebolavirus adaptation to novel hosts, and only a few SDPs distinguish Reston virus VP24 from VP24 of other Ebolaviruses, human pathogenic Reston viruses may emerge. This is of concern since Reston viruses circulate in domestic pigs and can infect humans, possibly via airborne transmission. PMID:27009368

  1. Iron-sulfur cluster exchange reactions mediated by the human Nfu protein.

    PubMed

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

    2016-10-01

    Human Nfu is an iron-sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron-sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe-2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe-2S](GS)4 cluster complex, and extraction of the Nfu-bound iron-sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe-2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe-2S] cluster trafficking and delivery.

  2. Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

    PubMed Central

    Bandaru, Viswanath; Zhao, Xiaobei; Newton, Michael R.; Burrows, Cynthia J.; Wallace, Susan S.

    2007-01-01

    Endonuclease VIII (Nei), which recognizes and repairs oxidized pyrimidines in the Base Excision Repair (BER) pathway, is sparsely distributed among both the prokaryotes and eukaryotes. Recently, we and others identified three homologs of E. coli endonuclease VIII-like (NEIL) proteins in humans. Here, we report identification of human NEIL homologs in Mimivirus, a giant DNA virus that infects Acanthamoeba. Characterization of the two mimiviral homologs, MvNei1 and MvNei2, showed that they share not only sequence homology but also substrate specificity to the human NEIL proteins, that is, they recognize oxidized pyrimidines in duplex DNA and in bubble substrates and as well show 5′2-deoxyribose-5-phosphate lyase (dRP lyase) activity. However, unlike MvNei1 and the human NEIL proteins, MvNei2 preferentially cleaves oxidized pyrimidines in single stranded DNA forming products with a different end chemistry. Interestingly, opposite base specificity of MvNei1 resembles human NEIL proteins for pyrimidine base damages whereas it resembles E. coli formamidopyrimidine DNA glycosylase (Fpg) for guanidinohydantoin (Gh), an oxidation product of 8-oxoguanine. Finally, a conserved arginine residue in the “zincless finger” motif, previously identified in human NEIL1, is required for the DNA glycosylase activity of MvNeil. Thus, Mimivirus represents the first example of a virus to carry oxidative DNA glycosylases with substrate specificities that resemble human NEIL proteins. Based on the sequence homology to the human NEIL homologs and novel bacterial NEIL homologs identified here, we predict that Mimivirus may have acquired the DNA glycosylases through the host-mediated lateral transfer from either a bacterium or from vertebrates. PMID:17627905

  3. Characterization of Chlamydia pneumoniae species-specific proteins immunodominant in humans.

    PubMed Central

    Iijima, Y; Miyashita, N; Kishimoto, T; Kanamoto, Y; Soejima, R; Matsumoto, A

    1994-01-01

    Proteins of Chlamydia pneumoniae immunodominant in humans were characterized with the sera of 13 patients who were not likely to have been exposed to C. trachomatis or C. psittaci. The serological responses among these patients were similar on a qualitative basis, but some differences were found quantitatively. However, the serological responses of the patients who were infected with C. pneumoniae differed markedly from those of two patients who were infected with C. trachomatis and two who were infected with C. psittaci and those of mice that were transtracheally infected with C. pneumoniae. Among proteins immunodominant in the patients who were infected with C. pneumoniae, a 40-kDa major outer membrane protein was genus specific and 53-, 46-, and 43-kDa proteins were species specific in their reactions with the majority of the human sera used. A few sera reacted strongly with a 73-kDa protein genus specifically. Some proteins with weak immunogenicity exhibited species specificity. An antigenic analysis with human sera and murine monoclonal antibodies against the 53-kDa protein showed that hte antigenicities were strictly conserved among the seven strains of C. pneumoniae tested. The genus-specific 73-kDa protein was solubilized with octylglucoside. All of the species-specific immunodominant proteins were solubilized with sodium dodecyl sulfate, but the genus-specific major outer membrane protein was not. These results suggest that a serological diagnosis of C. pneumoniae infection could be achieved species specifically by comparison of the serum responses to sodium dodecyl sulfate- and octylglucoside-soluble fractions. Images PMID:8195362

  4. Sequence-related human proteins cluster by degree of evolutionary conservation.

    PubMed

    Mrowka, Ralf; Patzak, Andreas; Herzel, Hanspeter; Holste, Dirk

    2004-11-01

    Gene duplication followed by adaptive evolution is thought to be a central mechanism for the emergence of novel genes. To illuminate the contribution of duplicated protein-coding sequences to the complexity of the human genome, we study the connectivity of pairwise sequence-related human proteins and construct a network (N) of linked protein sequences with shared similarities. We find that (i) the connectivity distribution P (k) for k sequence-related proteins decays as a power law P (k) approximately k(-gamma) with gamma approximately 1.2 , (ii) the top rank of N consists of a single large cluster of proteins ( approximately 70%) , while bottom ranks consist of multiple isolated clusters, and (iii) structural characteristics of N show both a high degree of clustering and an intermediate connectivity ("small-world" features). We gain further insight into structural properties of N by studying the relationship between the connectivity distribution and the phylogenetic conservation of proteins in bacteria, plants, invertebrates, and vertebrates. We find that (iv) the proportion of sequence-related proteins increases with increasing extent of evolutionary conservation. Our results support that small-world network properties constitute a footprint of an evolutionary mechanism and extend the traditional interpretation of protein families.

  5. Sequence-related human proteins cluster by degree of evolutionary conservation

    NASA Astrophysics Data System (ADS)

    Mrowka, Ralf; Patzak, Andreas; Herzel, Hanspeter; Holste, Dirk

    2004-11-01

    Gene duplication followed by adaptive evolution is thought to be a central mechanism for the emergence of novel genes. To illuminate the contribution of duplicated protein-coding sequences to the complexity of the human genome, we study the connectivity of pairwise sequence-related human proteins and construct a network (N) of linked protein sequences with shared similarities. We find that (i) the connectivity distribution P(k) for k sequence-related proteins decays as a power law P(k)˜k-γ with γ≈1.2 , (ii) the top rank of N consists of a single large cluster of proteins (≈70%) , while bottom ranks consist of multiple isolated clusters, and (iii) structural characteristics of N show both a high degree of clustering and an intermediate connectivity (“small-world” features). We gain further insight into structural properties of N by studying the relationship between the connectivity distribution and the phylogenetic conservation of proteins in bacteria, plants, invertebrates, and vertebrates. We find that (iv) the proportion of sequence-related proteins increases with increasing extent of evolutionary conservation. Our results support that small-world network properties constitute a footprint of an evolutionary mechanism and extend the traditional interpretation of protein families.

  6. A 92-kDa human immunostimulatory protein.

    PubMed Central

    Fontan, E; Briend, E; Saklani-Jusforgues, H; d'Alayer, J; Vandekerckhove, J; Fauve, R M

    1994-01-01

    We purified to apparent homogeneity a human urinary glycoprotein of 92 kDa (HGP.92) that, administered intravenously at 250 micrograms/kg, fully protected mice against a lethal inoculum of Listeria monocytogenes. Since HGP.92 protected scid mice, which lack B and T lymphocytes, this increased resistance to Listeria did not appear to be lymphocyte mediated. Furthermore, inflammatory macrophages incubated with 6 nM HGP.92 inhibited the growth of Lewis carcinoma cells in vitro. These two activities appeared to depend on an oligosaccharide moiety, as they were lost after N-Glycanase treatment of HGP.92. Thus, the biological activity of HGP.92 was in some way related to a glycan moiety. Images PMID:8078887

  7. Mycoplasmal lipoprotein p37 binds human protein HER2.

    PubMed

    Wu, Jun; Wu, Lijuan; Fang, Cheng; Nie, Rong; Wang, Jiamou; Wang, Xuan; Liu, Wenbin

    2016-11-01

    Mycoplasmas are a group of microbes that can cause human diseases. The mycoplasmal lipoprotein p37 promotes cancer metastasis, at least in part, by interacting with EGFR. In this study, we show that the p37 lipoprotein binds another member of the EGFR family, HER2, through the HER2 extracellular domain. The binding of p37-HER2 promotes phosphorylation of HER2 and activates the downstream signaling molecule Erk1/2. Because the HER2 signaling pathway contributes to breast tumor metastasis, our results imply that the mycoplasmal lipoprotein p37 may also be involved in breast cancer metastasis. This study contributes to our understanding of mycoplasmal lipoprotein p37 function and its potential involvement in tumorigenesis. PMID:27664744

  8. Identification of Human Proteins That Modify Misfolding and Proteotoxicity of Pathogenic Ataxin-1

    PubMed Central

    Riechers, Sean-Patrick; Muehlenberg, Katja; Möller, Angeli; Reinhardt, Anita; Vinayagam, Arunachalam; Schaefer, Martin H.; Boutros, Michael; Tricoire, Hervé; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2012-01-01

    Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo. PMID:22916034

  9. Quantitative Analysis of Age Specific Variation in the Abundance of Human Female Parotid Salivary Proteins

    PubMed Central

    Ambatipudi, Kiran S.; Lu, Bingwen; Hagen, Fred K; Melvin, James E.; Yates, John R.

    2010-01-01

    Summary Human saliva is a protein-rich, easily accessible source of potential local and systemic biomarkers to monitor changes that occur under pathological conditions; however little is known about the changes in abundance associated with normal aging. In this study, we performed a comprehensive proteomic profiling of pooled saliva collected from the parotid glands of healthy female subjects, divided into two age groups 1 and 2 (20–30 and 55–65 years old, respectively). Hydrophobic charge interaction chromatography was used to separate high from low abundant proteins prior to characterization of the parotid saliva using multidimensional protein identification technology (MudPIT). Collectively, 532 proteins were identified in the two age groups. Of these proteins, 266 were identified exclusively in one age group, while 266 proteins were common to both groups. The majority of the proteins identified in the two age groups belonged to the defense and immune response category. Of note, several defense related proteins (e.g. lysozyme, lactoferrin and histatin-1) were significantly more abundant in group 2 as determined by G-test. Selected representative mass spectrometric findings were validated by western blot analysis. Our study reports the first quantitative analysis of differentially regulated proteins in ductal saliva collected from young and older female subjects. This study supports the use of high-throughput proteomics as a robust discovery tool. Such results provide a foundation for future studies to identify specific salivary proteins which may be linked to age-related diseases specific to women. PMID:19764810

  10. Human Genome Encodes Many Proteins with Charge Periodicity of 28 Residues

    NASA Astrophysics Data System (ADS)

    Ke, Runcong; Sakiyama, Noriyuki; Sawada, Ryusuke; Sonoyama, Masashi; Mitaku, Shigeki

    2007-09-01

    The human genome includes more than 36,000 open reading frames that are translated to amino acid sequences of proteins. When the charge distribution in amino acid sequences from the total human genome was analyzed by the autocorrelation function, a surprisingly sharp periodicity of 28 residues was observed. Every protein with the charge periodicity of 28 residues (PCP28) could be discriminated by a simple algorithm, and the number of PCP28 amounted to about 3% of the total open reading frames of the human genome. The net charge of most PCP28 was highly positive. The possible structural and functional features of this type of protein were discussed in terms of the electric repulsion within molecules.

  11. Multiple Plasmodium falciparum Merozoite Surface Protein 1 Complexes Mediate Merozoite Binding to Human Erythrocytes.

    PubMed

    Lin, Clara S; Uboldi, Alessandro D; Epp, Christian; Bujard, Hermann; Tsuboi, Takafumi; Czabotar, Peter E; Cowman, Alan F

    2016-04-01

    Successful invasion of human erythrocytes byPlasmodium falciparummerozoites is required for infection of the host and parasite survival. The early stages of invasion are mediated via merozoite surface proteins that interact with human erythrocytes. The nature of these interactions are currently not well understood, but it is known that merozoite surface protein 1 (MSP1) is critical for successful erythrocyte invasion. Here we show that the peripheral merozoite surface proteins MSP3, MSP6, MSPDBL1, MSPDBL2, and MSP7 bind directly to MSP1, but independently of each other, to form multiple forms of the MSP1 complex on the parasite surface. These complexes have overlapping functions that interact directly with human erythrocytes. We also show that targeting the p83 fragment of MSP1 using inhibitory antibodies inhibits all forms of MSP1 complexes and disrupts parasite growthin vitro.

  12. Deorphanizing the human transmembrane genome: A landscape of uncharacterized membrane proteins

    PubMed Central

    Babcock, Joseph J; Li, Min

    2014-01-01

    The sequencing of the human genome has fueled the last decade of work to functionally characterize genome content. An important subset of genes encodes membrane proteins, which are the targets of many drugs. They reside in lipid bilayers, restricting their endogenous activity to a relatively specialized biochemical environment. Without a reference phenotype, the application of systematic screens to profile candidate membrane proteins is not immediately possible. Bioinformatics has begun to show its effectiveness in focusing the functional characterization of orphan proteins of a particular functional class, such as channels or receptors. Here we discuss integration of experimental and bioinformatics approaches for characterizing the orphan membrane proteome. By analyzing the human genome, a landscape reference for the human transmembrane genome is provided. PMID:24241348

  13. Bioactive peptides derived from human milk proteins--mechanisms of action.

    PubMed

    Wada, Yasuaki; Lönnerdal, Bo

    2014-05-01

    Human milk contains a multitude of bioactive proteins with very diverse functions, which are beneficial for the rapidly growing neonate. The large variety of bioactivities is accomplished by the combination of bioactive proteins per se and gastrointestinal release of bioactive peptides derived from them. The bioactivities exerted by these peptides include enhancement of mineral absorption, immunomodulation, opioid, antihypertensive and antimicrobial activities. Notably, several of the activities are not attributed to the parental proteins, but exclusively to released bioactive peptides. This article reviews studies on bioactive peptides derived from major human milk proteins, such as caseins, α-lactalbumin and lactoferrin, during gastrointestinal digestion. Studies of bovine milk counterparts are also cited as a comparison.

  14. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2.

    PubMed

    Gyuris, J; Golemis, E; Chertkov, H; Brent, R

    1993-11-19

    We used the interaction trap, a yeast genetic selection for interacting proteins, to isolate human cyclin-dependent kinase interactor 1 (Cdi1). In yeast, Cdi1 interacts with cyclin-dependent kinases, including human Cdc2, Cdk2, and Cdk3, but not with Ckd4. In HeLa cells, Cdi1 is expressed at the G1 to S transition, and the protein forms stable complexes with Cdk2. Cdi1 bears weak sequence similarity to known tyrosine and dual specificity phosphatases. In vitro, Cdi1 removes phosphate from tyrosine residues in model substrates, but a mutant protein that bears a lesion in the putative active site cysteine does not. Overexpression of wild-type Cdi1 delays progression through the cell cycle in yeast and HeLa cells; delay is dependent on Cdi1 phosphatase activity. These experiments identify Cdi1 as a novel type of protein phosphatase that forms complexes with cyclin-dependent kinases. PMID:8242750

  15. Human kidney amiloride-binding protein: cDNA structure and functional expression

    SciTech Connect

    Barbry, P.; Chassande, O.; Champigny, G.; Lingueglia, E.; Frelin, C.; Lazdunski, M. ); Champe, M.; Munemitsu, S.; Ullrich, A. ); Maes, P.; Tartar, A. Institut Pasteur de Lille )

    1990-10-01

    Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na{sup plus} channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Na{sup plus} channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein.

  16. Human kidney amiloride-binding protein: cDNA structure and functional expression.

    PubMed Central

    Barbry, P; Champe, M; Chassande, O; Munemitsu, S; Champigny, G; Lingueglia, E; Maes, P; Frelin, C; Tartar, A; Ullrich, A

    1990-01-01

    Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na+ channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Na+ channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein. Images PMID:2217167

  17. Oncogenic viral protein HPV E7 up-regulates the SIRT1 longevity protein in human cervical cancer cells.

    PubMed

    Allison, Simon J; Jiang, Ming; Milner, Jo

    2009-03-02

    Senescence is blocked in human cervical keratinocytes infected with high risk human papillomavirus (e.g. HPV type16). Viral oncoproteins HPV E6 and HPV E7 access the cell cycle via cellular p53 and retinoblastoma proteins respectively. Previously we have shown that HPV E7, not HPV E6, is also responsible for cervical cancer cell survival (SiHa cells; HPV type16). We now present evidence that SIRT1, an aging-related NAD-dependent deacetylase, mediates HPV E7 survival function in SiHa cervical cancer cells. Moreover, HPV E7 up-regulates SIRT1 protein when expressed in primary human keratinocytes. Conversely, SIRT1 levels decrease following RNAi-mediated silencing of HPV E7 in SiHa cells. Silencing HPV E6 has no effect on SIRT1 but, as expected, causes marked accumulation of p53 protein accompanied by p53-mediated up-regulation of p21. However, p53 acetylation (K382Ac) was barely detectable. Since p53 is a known SIRT1 substrate we propose that elevated SIRT1 levels (induced by HPV E7) attenuate p53 pro-apoptotic capacity via its de-acetylation. Our discovery that HPV E7 up-regulates SIRT1 links a clinically important oncogenic virus with the multi-functional SIRT1 protein. This link may open the way for a more in-depth understanding of the process of HPV-induced malignant transformation and also of the inter-relationships between aging and cancer.

  18. Humoral and Cell-mediated Autoimmune Reactions to Human Acidic Ribosomal P2 Protein in Individuals Sensitized to Aspergillus fumigatus P2 Protein

    PubMed Central

    Mayer, Christina; Appenzeller, Ulrich; Seelbach, Heike; Achatz, Gernot; Oberkofler, Hannes; Breitenbach, Michael; Blaser, Kurt; Crameri, Reto

    1999-01-01

    A panel of cDNAs encoding allergenic proteins was isolated from an Aspergillus fumigatus cDNA library displayed on the surface of filamentous phage. Solid phase–immobilized serum immunoglobulin E (IgE) from A. fumigatus–allergic individuals was used to enrich phage displaying IgE-binding molecules. One of the cDNAs encoded a 11.1-kD protein that was identified as acidic ribosomal phosphoprotein type 2 (P2 protein). The allergen, formally termed rAsp f 8, shares >62% sequence identity and >84% sequence homology to corresponding eukaryotic P2 proteins, including human P2 protein. The sequences encoding human and fungal P2 protein were subcloned, expressed in Escherichia coli as His6-tagged fusion proteins, and purified by Ni2+–chelate affinity chromatography. Both recombinant P2 proteins were recognized by IgE antibodies from allergic individuals sensitized to the A. fumigatus P2 protein and elicited strong type 1–specific skin reactions in these individuals. Moreover, human and fungal P2 proteins induced proliferative responses in peripheral blood mononuclear cells of A. fumigatus– allergic subjects sensitized to the fungal P2 protein. These data provide strong evidence for in vitro and in vivo humoral and cell-mediated autoreactivity to human P2 protein in patients suffering from chronic A. fumigatus allergy. PMID:10224291

  19. Establishment and characterization of human engineered cells stably expressing large extracellular matrix proteins.

    PubMed

    Kwon, Daekee; Kang, Gwang-Sik; Han, Dong Keun; Park, Kwideok; Kim, Jae-Hwan; Lee, Soo-Hong

    2014-01-01

    Commercially available extracellular matrix (ECM) hydrogel-coated culture plates have been used to study the relationship between the ECM microenvironment and stem cell behavior. However, it is unclear whether ECM-coated dishes mimic the natural ECM microenvironment because the architecture of the ECM is constructed of randomly distributed fibers. The purpose of this study was the production and confirmation of human engineered cell lines stably expressing large ECM proteins such as collagen I/II and fibronectin. First, large (over 10 kb) ECM vectors encoding human collagen I/II and fibronectin were constructed and the circular vectors were linearized. Second, the linear ECM vectors were introduced into immortalized human embryonic kidney cells using various transfection methods. The polyethylenimine and liposome methods showed higher efficiencies than electroporation for transfection of these large vectors. Third, human ECM engineered cells were established by stable integration of the vector into the genomic DNA and resulted in stable overexpression of mRNA and proteins. In summary, human engineered cell lines stably expressing large ECM proteins such as human collagen I/II and fibronectin were successfully prepared, and secretion of the ECM components into the surrounding environment was confirmed by immunocytochemistry. Thus, human ECM engineered cells naturally secreting ECM components could be valuable for studying the relationship between the native ECM microenvironment and stem cell behavior.

  20. A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human Disease.

    PubMed

    Gilmore, Brian L; Winton, Carly E; Demmert, Andrew C; Tanner, Justin R; Bowman, Sam; Karageorge, Vasilea; Patel, Kaya; Sheng, Zhi; Kelly, Deborah F

    2015-01-01

    We present a new molecular toolkit to investigate protein assemblies natively formed in the context of human disease. The system employs tunable microchips that can be decorated with switchable adaptor molecules to select for target proteins of interest and analyze them using molecular microscopy. Implementing our new streamlined microchip approach, we could directly visualize BRCA1 gene regulatory complexes from patient-derived cancer cells for the first time. PMID:26395823

  1. Novel characterization of the HSPA2-stabilizing protein BAG6 in human spermatozoa.

    PubMed

    Bromfield, Elizabeth; Aitken, R John; Nixon, Brett

    2015-10-01

    While a large cohort of sperm surface receptors underpin sperm-oocyte adhesion processes, our recent work has revealed that the molecular chaperone Heat Shock Protein A2 (HSPA2) is a key regulator of zona pellucida-receptor complex assembly in our own species. Indeed, in the infertile population, spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. While the mechanisms behind this protein deficiency are under consideration, BCL2-associated athanogene 6 (BAG6) has been identified as a key regulator of HSPA2 stability in mouse germ cells. However, in the human, the presence of BAG family proteins remains completely uncharacterized. Consequently, this study aimed to determine the presence of BAG6 in human sperm cells and to characterize its putative interaction with HSPA2 throughout sperm cell development. BAG6 was shown to co-localize with HSPA2 in human testicular germ cells and epididymal spermatozoa. Similarly, BAG6 was identified in the equatorial region of non-capacitated spermatozoa but underwent a marked relocation to the anterior region of the head upon the induction of capacitation in these cells. Protein-protein interaction assays revealed the stable interaction of BAG6 and HSPA2 proteins in mature spermatozoa. Furthermore, examination of the spermatozoa of infertile men with zona pellucida binding defects, related to a lack of HSPA2, revealed a concomitant deficiency in BAG6 protein expression. In view of the findings described in this study, we propose that BAG6 is likely a key regulator of HSPA2 stability/function in human germ cells. Moreover, its under-representation in spermatozoa with zona pellucida binding deficiency suggests that BAG6 may be an important candidate to study for a further understanding of male idiopathic infertility.

  2. Human MHC class I antigens are associated with a 90-kDa cell surface protein.

    PubMed

    Ferm, M T; Grönberg, A

    1991-08-01

    Human MHC class I proteins are expressed on almost all nucleated cells as a heavy chain (about 45 kDa) non-covalently associated with beta 2-microglobulin (12 kDa). In this report we show that MHC class I (MHC-I) proteins can also be associated with a 90-kDa protein in the cell membrane. Surface-radiolabelled cells were treated with dithiobis succinimidyl propionate (DSP) in order to preserve multimer protein complexes during cell lysis. The lysates were immunoprecipitated and analysed by SDS-PAGE and autoradiography. Immunoprecipitation of human MHC-I proteins co-precipitated another protein of about 90 kDa in molecular weight-p90. p90 was coprecipitated from all the MHC-I expressing cells tested: U937, Raji, Molt-4 and IFN-gamma treated K562, but not from untreated, MHC-I negative K562. A 90-kDa protein was also co-precipitated with MHC-I from fresh peripheral blood mononuclear cells (PBMC). Furthermore, p90 was coprecipitated by different MoAbs to the MHC-I heavy chain or beta 2-microglobulin, but not by control antibodies. Two additional co-precipitating proteins at 34 kDa and 28 kDa were seen in MHC-I precipitates from Raji cells. Our results suggest that MHC-I proteins and the 90-kDa protein are associated in the cell membrane, probably by a close but weak, non-covalent interaction. Two additional cell surface proteins at 34 kDa and 28 kDa seem to be MHC-I associated on Raji Burkitt's lymphoma cells.

  3. Separation of recombinant human protein C from transgenic animal milk using immobilized metal affinity chromatography.

    PubMed

    Dalton, J C; Bruley, D F; Kang, K A; Drohan, W N

    1997-01-01

    Protein C is an important serine protease due to its ability to proteolytically cleave activated Factors V and VIII. Excess coagulation and blood agglutination can lead to plugged capillaries, thereby reducing oxygen transport to interstitial tissues. To treat patients with hereditary and acquired protein C deficiency would require a greater amount of Protein C than that available from human plasma. However, the potential demand for this protein could be met by the production of human protein C from transgenic animal mammary glands. Thus, research into inexpensive, efficient methods to purify proteins from transgenic animal milk will be a critical area of study for the large scale production of protein C. Immobilized metal affinity chromatography (IMAC) is a novel method for the purification of protein C. A proposed method of purification is to take advantage of protein C's strong metal ion binding characteristics with IMAC to assist in the separation from transgenic animal milk. The separation procedure is benchmarked against current systems in use by the American Red Cross for purification of Protein C from transgenic porcine milk. Common problems in developing separation schemes for new therapeutics are the initial availability of the product (protein), and time-to-market concerns. Extensive experimental tests for scaleable purification schemes are often cost and time prohibitive. In order to optimize an IMAC protocol with minimal waste of time and resources, total quality management tools have been adopted. Initial experiments were designed to choose buffer conditions, eluents, immobilized valence metals, and flow rates using Taguchi experimental design, which is a total quality management (TQM) tool. One of the values of Taguchi methods lies in the use of Latin orthogonal sets. Through the use of the orthogonal sets, the total number of experiments may be reduced, shortening the focus time on optimal conditions.

  4. Long interspersed element-1 protein expression is a hallmark of many human cancers.

    PubMed

    Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S; Hruban, Ralph H; Iacobuzio-Donahue, Christine A; Maitra, Anirban; Torbenson, Michael S; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S; Montgomery, Elizabeth A; Gabrielson, Edward; Netto, George J; Lotan, Tamara L; De Marzo, Angelo M; Westra, William; Binder, Zev A; Orr, Brent A; Gallia, Gary L; Eberhart, Charles G; Boeke, Jef D; Harris, Chris R; Burns, Kathleen H

    2014-05-01

    Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen.

  5. Chromosomal localization of murine and human oligodendrocyte-specific protein genes

    SciTech Connect

    Bronstein, J.M.; Wu, S.; Korenberg, J.R.

    1996-06-01

    Oligodendrocyte-specific protein (OSP) is a recently described protein present only in myelin of the central nervous system. Several inherited disorders of myelin are caused by mutations in myelin genes but the etiology of many remain unknown. We mapped the location of the mouse OSP gene to the proximal region of chromosome 3 using two sets of multilocus crosses and to human chromosome 3 using somatic cell hybrids. Fine mapping with fluorescence in situ hybridization placed the OSP gene at human chromosome 3q26.2-q26.3. To date, there are no known inherited neurological disorders that localize to these regions. 24 refs., 2 figs.

  6. Pez: a novel human cDNA encoding protein tyrosine phosphatase- and ezrin-like domains.

    PubMed

    Smith, A L; Mitchell, P J; Shipley, J; Gusterson, B A; Rogers, M V; Crompton, M R

    1995-04-26

    We have isolated cDNAs from normal human breast tissue and breast tumour cells that encode a protein (pez) with features of a novel non-receptor tyrosine phosphatase possessing N-terminal sequence homology to the ezrin-band 4.1-merlin-radixin protein family. Northern blot analysis indicates that pez is expressed in a variety of human tissues including kidney, skeletal muscle, lung and placenta. Fluorescence in situ hybridization has mapped pez to chromosome 1 region q32.2-41. Sequence identity to a characterized polymorphic marker confirms this localization. PMID:7733990

  7. Atrazine Affects Phosphoprotein and Protein Expression in MCF-10A Human Breast Epithelial Cells

    PubMed Central

    Huang, Peixin; Yang, John; Song, Qisheng; Sheehan, David

    2014-01-01

    Atrazine, a member of the 2-chloro-s-triazine family of herbicides, is the most widely used pesticide in the world and often detected in agriculture watersheds. Although it was generally considered as an endocrine disruptor, posing a potential threat to human health, the molecular mechanisms of atrazine effects remain unclear. Using two-dimensional gel electrophoresis, we identified a panel of differentially expressed phosphoproteins and total proteins in human breast epithelial MCF-10A cells after being exposed to environmentally relevant concentrations of atrazine. Atrazine treatments for 6 h resulted in differential expression of 4 phosphoproteins and 8 total-proteins as compared to the control cells (>1.5-fold, p < 0.05). MALDI-TOF MS/MS analysis revealed that the differentially expressed proteins belong to various cellular compartments (nucleus, cytosol, membrane) and varied in function, including those regulating the stress response such as peroxiredoxin I, HSP70 and HSP27; structural proteins such as tropomyosin and profilin 1; and oncogenesis proteins such as ANP32A. Six of the 12 identified proteins were verified by quantitative PCR for their transcript levels. The most up-regulated phosphoprotein by atrazine treatment, ANP32A, was further analyzed for its expression, distribution and cellular localization using Western blot and immunocytochemical approaches. The results revealed that ANP32 expression after atrazine treatment increased dose and time dependently and was primarily located in the nucleus. This study may provide new evidence on the potential toxicity of atrazine in human cells. PMID:25275270

  8. Atrazine affects phosphoprotein and protein expression in MCF-10A human breast epithelial cells.

    PubMed

    Huang, Peixin; Yang, John; Song, Qisheng

    2014-10-01

    Atrazine, a member of the 2-chloro-s-triazine family of herbicides, is the most widely used pesticide in the world and often detected in agriculture watersheds. Although it was generally considered as an endocrine disruptor, posing a potential threat to human health, the molecular mechanisms of atrazine effects remain unclear. Using two-dimensional gel electrophoresis, we identified a panel of differentially expressed phosphoproteins and total proteins in human breast epithelial MCF-10A cells after being exposed to environmentally relevant concentrations of atrazine. Atrazine treatments for 6 h resulted in differential expression of 4 phosphoproteins and 8 total-proteins as compared to the control cells (>1.5-fold, p<0.05). MALDI-TOF MS/MS analysis revealed that the differentially expressed proteins belong to various cellular compartments (nucleus, cytosol, membrane) and varied in function, including those regulating the stress response such as peroxiredoxin I, HSP70 and HSP27; structural proteins such as tropomyosin and profilin 1; and oncogenesis proteins such as ANP32A. Six of the 12 identified proteins were verified by quantitative PCR for their transcript levels. The most up-regulated phosphoprotein by atrazine treatment, ANP32A, was further analyzed for its expression, distribution and cellular localization using Western blot and immunocytochemical approaches. The results revealed that ANP32 expression after atrazine treatment increased dose and time dependently and was primarily located in the nucleus. This study may provide new evidence on the potential toxicity of atrazine in human cells.

  9. Evolutionary Divergence of Gene and Protein Expression in the Brains of Humans and Chimpanzees.

    PubMed

    Bauernfeind, Amy L; Soderblom, Erik J; Turner, Meredith E; Moseley, M Arthur; Ely, John J; Hof, Patrick R; Sherwood, Chet C; Wray, Gregory A; Babbitt, Courtney C

    2015-08-01

    Although transcriptomic profiling has become the standard approach for exploring molecular differences in the primate brain, very little is known about how the expression levels of gene transcripts relate to downstream protein abundance. Moreover, it is unknown whether the relationship changes depending on the brain region or species under investigation. We performed high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses on two regions of the human and chimpanzee brain: The anterior cingulate cortex and caudate nucleus. In both brain regions, we found a lower correlation between mRNA and protein expression levels in humans and chimpanzees than has been reported for other tissues and cell types, suggesting that the brain may engage extensive tissue-specific regulation affecting protein abundance. In both species, only a few categories of biological function exhibited strong correlations between mRNA and protein expression levels. These categories included oxidative metabolism and protein synthesis and modification, indicating that the expression levels of mRNA transcripts supporting these biological functions are more predictive of protein expression compared with other functional categories. More generally, however, the two measures of molecular expression provided strikingly divergent perspectives into differential expression between human and chimpanzee brains: mRNA comparisons revealed significant differences in neuronal communication, ion transport, and regulatory processes, whereas protein comparisons indicated differences in perception and cognition, metabolic processes, and organization of the cytoskeleton. Our results highlight the importance of examining protein expression in evolutionary analyses and call for a more thorough understanding of tissue-specific protein expression levels. PMID:26163674

  10. Evolutionary Divergence of Gene and Protein Expression in the Brains of Humans and Chimpanzees

    PubMed Central

    Bauernfeind, Amy L.; Soderblom, Erik J.; Turner, Meredith E.; Moseley, M. Arthur; Ely, John J.; Hof, Patrick R.; Sherwood, Chet C.; Wray, Gregory A.; Babbitt, Courtney C.

    2015-01-01

    Although transcriptomic profiling has become the standard approach for exploring molecular differences in the primate brain, very little is known about how the expression levels of gene transcripts relate to downstream protein abundance. Moreover, it is unknown whether the relationship changes depending on the brain region or species under investigation. We performed high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses on two regions of the human and chimpanzee brain: The anterior cingulate cortex and caudate nucleus. In both brain regions, we found a lower correlation between mRNA and protein expression levels in humans and chimpanzees than has been reported for other tissues and cell types, suggesting that the brain may engage extensive tissue-specific regulation affecting protein abundance. In both species, only a few categories of biological function exhibited strong correlations between mRNA and protein expression levels. These categories included oxidative metabolism and protein synthesis and modification, indicating that the expression levels of mRNA transcripts supporting these biological functions are more predictive of protein expression compared with other functional categories. More generally, however, the two measures of molecular expression provided strikingly divergent perspectives into differential expression between human and chimpanzee brains: mRNA comparisons revealed significant differences in neuronal communication, ion transport, and regulatory processes, whereas protein comparisons indicated differences in perception and cognition, metabolic processes, and organization of the cytoskeleton. Our results highlight the importance of examining protein expression in evolutionary analyses and call for a more thorough understanding of tissue-specific protein expression levels. PMID:26163674

  11. Mapping the Homodimer Interface of an Optimized, Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor

    PubMed Central

    Bears, Zachary; Barrera, Francisco N.; Alonso, Miriam; Engelman, Donald M.; DiMaio, Daniel

    2014-01-01

    Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR) in trans. This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells in vitro, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation. PMID:24788775

  12. The apoptosis-1/Fas protein in human systemic lupus erythematosus.

    PubMed Central

    Mysler, E; Bini, P; Drappa, J; Ramos, P; Friedman, S M; Krammer, P H; Elkon, K B

    1994-01-01

    Three independent mutations involving the apoptosis-1 (APO-1)/Fas receptor or its putative ligand have led to lupuslike diseases associated with lymphadenopathy in different strains of mice. To determine whether humans with SLE also have a defect in this apotosis pathway, we analyzed the expression of APO-1 on freshly isolated blood mononuclear cells and on lymphocytes activated in vitro using flow cytometry and the monoclonal antibody anti-APO-1. Significantly higher level of APO-1 expression were detected on freshly isolated peripheral B cells and both CD4+ and CD8+ T lymphocyte populations obtained from lupus patients when compared with normal controls (P < 0.001). Almost 90% of the cells that stained positive for APO-1 also expressed the CD29 antigen, suggesting that APO-1 was upregulated after lymphocyte activation in vivo. No defect in APO-1 regulation was detected after activation of SLE T (with anti-CD3) or B (with Staphylococcus aureus Cowan 1) lymphocytes in the presence of IL-2 in vitro. Similarly, the anti-APO-1 antibody induced apoptosis in 74 +/- 5% of activated SLE T cells in vitro compared with 79 +/- 6% of the normal controls (P > 0.05). These results reveal that, while APO-1/Fas may play an important role in the regulation of lymphocyte survival in SLE, no consistent defect in the expression or function of the receptor could be detected in these studies. Images PMID:7510716

  13. Differential protein expression in human corneal endothelial cells cultured from young and older donors

    PubMed Central

    Zhu, Cheng; Rawe, Ian

    2008-01-01

    Purpose To establish a baseline protein fingerprint of cultured human corneal endothelial cells (HCEC), to determine whether the protein profiles exhibit age-related differences, and to identify proteins differentially expressed in HCEC cultured from young and older donors. Methods Corneas were obtained from five young (<30 years old) and five older donors (>50 years old). HCEC were cultured, and protein was extracted from confluent passage 3 cells. Extracts from each age group were pooled to form two samples. Proteins were separated on two-dimensional (2-D) gels and stained with SyproRuby. Resultant images were compared to identify protein spots that were either similarly expressed or differentially expressed by at least twofold. Protein spots were then identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Results Protein spots were well resolved, and patterns were reproducible on 2-D gels using either pH 3–10 or pH 4–7 IPG strips. Two-dimensional gels prepared with pH 4–7 IPG strips were used for differential display analysis, which was reproduced on three separate pairs of gels. MALDI-TOF identified 58 proteins with similar expression; 30 proteins were expressed twofold higher in HCEC from young donors; five proteins were expressed twofold higher in cells from older donors; and 10 proteins were identified in gels from young donors that did not match in gels from older donors. Several proteins expressed at higher levels in younger donors support metabolic activity, protect against oxidative damage, or mediate protein folding or degradation. Conclusions This is the first proteomic comparison of proteins expressed in HCEC cultured from young and older donors. Although restricted to proteins with isoelectric points between pH 4.0 and pH 7.0, the data obtained represent an initial step in the investigation of molecular mechanisms that underlie physiologically important age-related differences in cultured HCEC

  14. Repair of UV damaged DNA, genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1991-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, as well as studying the human homologs of yeast excision repair and postreplication repair proteins. In addition to its single-stranded DNA-dependent A TPase and DNA helicase activities, we have found that RAD3 protein also possesses DNA-RNA helicase activity, and that like RAD3, the Schizosaccharomyces pombe RAD3 homolog, rhp3{sup +}, is an essential gene. We have overexpressed the human RAD3 homolog, ERCC2, in yeast to facilitate its purification. The RAD10 protein was purified to homogeneity and shown to bind DNA. ERCC3y, the yeast homolog of the human ERCC-3/XP-B gene, has been sequenced and shown to be essential for viability. The Drosophila and human homologs of RAD6, required for postreplication repair and UV induced mutagenesis, were shown to complement the rad6 {Delta} mutation of yeast. Since defective DNA repair and enhanced neoplasia characterize several human genetic diseases, and repair proteins are highly conserved between yeast and man, a thorough understanding of the molecular mechanisms of DNA repir in yeast should provide a better understanding of the causes of carcinogenesis.

  15. Identification and Functional Analysis of Novel Nonstructural Proteins of Human Bocavirus 1

    PubMed Central

    Shen, Weiran; Deng, Xuefeng; Zou, Wei; Cheng, Fang; Engelhardt, John F.; Yan, Ziying

    2015-01-01

    ABSTRACT Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study, we identified novel splice acceptor and donor sites, namely, A1′ and D1′, in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2, NS3, and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1′ and smD1′ at the A1′ and D1′ splice sites, respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly, the smA1′ mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however, the smD1′ mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus, our study identified three novel NS proteins, NS2, NS3, and NS4, and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI. IMPORTANCE Human bocavirus 1 infection causes respiratory diseases, including acute wheezing in infants, of which life-threatening cases have been reported. In vitro, human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report, we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them, we proved that one nonstructural protein is critical to the replication of the

  16. Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein.

    PubMed

    Alexander, Crispin G; Jürgens, Maike C; Shepherd, Dale A; Freund, Stefan M V; Ashcroft, Alison E; Ferguson, Neil

    2013-07-23

    HBc, the capsid-forming "core protein" of human hepatitis B virus (HBV), is a multidomain, α-helical homodimer that aggressively forms human HBV capsids. Structural plasticity has been proposed to be important to the myriad functions HBc mediates during viral replication. Here, we report detailed thermodynamic analyses of the folding of the dimeric HBc protomer under conditions that prevented capsid formation. Central to our success was the use of ion mobility spectrometry-mass spectrometry and microscale thermophoresis, which allowed folding mechanisms to be characterized using just micrograms of protein. HBc folds in a three-state transition with a stable, dimeric, α-helical intermediate. Extensive protein engineering showed thermodynamic linkage between different structural domains. Unusual effects associated with mutating some residues suggest structural strain, arising from frustrated contacts, is present in the native dimer. We found evidence of structural gatekeepers that, when mutated, alleviated native strain and prevented (or significantly attenuated) capsid formation by tuning the population of alternative native conformations. This strain is likely an evolved feature that helps HBc access the different structures associated with its diverse essential functions. The subtle balance between native and strained contacts may provide the means to tune conformational properties of HBc by molecular interactions or mutations, thereby conferring allosteric regulation of structure and function. The ability to trap HBc conformers thermodynamically by mutation, and thereby ablate HBV capsid formation, provides proof of principle for designing antivirals that elicit similar effects.

  17. Inhibition of platelet (/sup 3/H)- imipramine binding by human plasma protein fractions

    SciTech Connect

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity (/sup 3/H)-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha/sub 1/ acid glycoprotein, high density and low density lipoprotein, IgG and ..cap alpha../sub 1/-antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of (/sup 3/H)-imipramine binding site.

  18. Human pentatricopeptide proteins: only a few and what do they do?

    PubMed

    Lightowlers, Robert N; Chrzanowska-Lightowlers, Zofia M A

    2013-01-01

    Pentatricopeptide repeat (PPR) proteins constitute a large family of RNA-binding proteins that contain a canonical 35 residue repeat motif. Originally identified in Arabidopsis thaliana, family members are found in protists, fungi, and metazoan but are by far most abundant in plant organelles. Seven examples have been identified in human mitochondria and roles have been tentatively ascribed to each. In this review, we briefly outline each of these PPR proteins and discuss the role each is believed to play in facilitating mitochondrial gene expression.

  19. Cataract-related changes in protein aggregates of human lens studied by ultracentrifugation.

    PubMed

    Twardowski, J; Hoja, D

    1990-01-01

    In human lens, cataract development causes an increase in the amount of protein aggregates. Their specific density of 1.25 g/ml is much higher than that of 1.14 g/ml and 1.06 g/ml obtained for water-soluble proteins. During the formation of water-insoluble aggregates their size varies up to some micrometers. Infrared spectra of water-soluble protein and in the aggregates confirm that the content of bound water in aggregates decreases. PMID:2279587

  20. An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii

    PubMed Central

    Liu, Jun; He, Yudou; Benmerzouga, Imaan; Sullivan, William J.; Morrissette, Naomi S.; Murray, John M.; Hu, Ke

    2016-01-01

    Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy. PMID:26680740

  1. A method to determine the kinetics of multiple proteins in human infants with respiratory distress syndrome.

    PubMed

    Bereman, Michael S; Tomazela, Daniela M; Heins, Hillary S; Simonato, Manuela; Cogo, Paola E; Hamvas, Aaron; Patterson, Bruce W; Cole, F Sessions; MacCoss, Michael J

    2012-06-01

    We report a method to measure in vivo turnover of four proteins from sequential tracheal aspirates obtained from human newborn infants with respiratory distress syndrome using targeted proteomics. We detected enrichment for all targeted proteins approximately 3 h from the start of infusion of [5,5,5-(2)H(3)] leucine, secretion times that varied from 1.2 to 2.5 h, and half lives that ranged between 10 and 21 h. Complement factor B, a component of the alternative pathway of complement activation, had an approximately twofold-longer half-life than the other three proteins. In addition, the kinetics of mature and carboxy-terminal tryptic peptides from the same protein (surfactant protein B) were not statistically different (p = 0.49).

  2. Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

    PubMed Central

    Josic, Djuro; Breen, Lucas; Clifton, James; Gajdosik, Martina Srajer; Gaso-Sokac, Dajana; Rucevic, Marijana; Müller, Egbert

    2013-01-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS. PMID:22740472

  3. Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode.

    PubMed

    Josic, Djuro; Breen, Lucas; Clifton, James; Gajdosik, Martina Srajer; Gaso-Sokac, Dajana; Rucevic, Marijana; Müller, Egbert

    2012-07-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low-abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS.

  4. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease.

    PubMed

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T

    2016-02-22

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

  5. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease

    PubMed Central

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T.

    2016-01-01

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects. PMID:26907261

  6. An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii.

    PubMed

    Liu, Jun; He, Yudou; Benmerzouga, Imaan; Sullivan, William J; Morrissette, Naomi S; Murray, John M; Hu, Ke

    2016-02-01

    Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy. PMID:26680740

  7. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi.

    PubMed

    Moon, Robert W; Sharaf, Hazem; Hastings, Claire H; Ho, Yung Shwen; Nair, Mridul B; Rchiad, Zineb; Knuepfer, Ellen; Ramaprasad, Abhinay; Mohring, Franziska; Amir, Amirah; Yusuf, Noor A; Hall, Joanna; Almond, Neil; Lau, Yee Ling; Pain, Arnab; Blackman, Michael J; Holder, Anthony A

    2016-06-28

    The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen. PMID:27303038

  8. Production and supply of high-quality food protein for human consumption: sustainability, challenges, and innovations.

    PubMed

    Wu, Guoyao; Fanzo, Jessica; Miller, Dennis D; Pingali, Prabhu; Post, Mark; Steiner, Jean L; Thalacker-Mercer, Anna E

    2014-08-01

    The Food and Agriculture Organization of the United Nations estimates that 843 million people worldwide are hungry and a greater number suffer from nutrient deficiencies. Approximately one billion people have inadequate protein intake. The challenge of preventing hunger and malnutrition will become even greater as the global population grows from the current 7.2 billion people to 9.6 billion by 2050. With increases in income, population, and demand for more nutrient-dense foods, global meat production is projected to increase by 206 million tons per year during the next 35 years. These changes in population and dietary practices have led to a tremendous rise in the demand for food protein, especially animal-source protein. Consuming the required amounts of protein is fundamental to human growth and health. Protein needs can be met through intakes of animal and plant-source foods. Increased consumption of food proteins is associated with increased greenhouse gas emissions and overutilization of water. Consequently, concerns exist regarding impacts of agricultural production, processing and distribution of food protein on the environment, ecosystem, and sustainability. To address these challenging issues, the New York Academy of Sciences organized the conference "Frontiers in Agricultural Sustainability: Studying the Protein Supply Chain to Improve Dietary Quality" to explore sustainable innovations in food science and programming aimed at producing the required quality and quantity of protein through improved supply chains worldwide. This report provides an extensive discussion of these issues and summaries of the presentations from the conference. PMID:25123207

  9. Activation of protein kinase C in permeabilized human neuroblastoma SH-SY5Y cells.

    PubMed

    Larsson, C; Saermark, T; Mau, S; Simonsson, P

    1992-08-01

    The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.

  10. Human, vector and parasite Hsp90 proteins: A comparative bioinformatics analysis

    PubMed Central

    Faya, Ngonidzashe; Penkler, David L.; Tastan Bishop, Özlem

    2015-01-01

    The treatment of protozoan parasitic diseases is challenging, and thus identification and analysis of new drug targets is important. Parasites survive within host organisms, and some need intermediate hosts to complete their life cycle. Changing host environment puts stress on parasites, and often adaptation is accompanied by the expression of large amounts of heat shock proteins (Hsps). Among Hsps, Hsp90 proteins play an important role in stress environments. Yet, there has been little computational research on Hsp90 proteins to analyze them comparatively as potential parasitic drug targets. Here, an attempt was made to gain detailed insights into the differences between host, vector and parasitic Hsp90 proteins by large-scale bioinformatics analysis. A total of 104 Hsp90 sequences were divided into three groups based on their cellular localizations; namely cytosolic, mitochondrial and endoplasmic reticulum (ER). Further, the parasitic proteins were divided according to the type of parasite (protozoa, helminth and ectoparasite). Primary sequence analysis, phylogenetic tree calculations, motif analysis and physicochemical properties of Hsp90 proteins suggested that despite the overall structural conservation of these proteins, parasitic Hsp90 proteins have unique features which differentiate them from human ones, thus encouraging the idea that protozoan Hsp90 proteins should be further analyzed as potential drug targets. PMID:26793431

  11. Polycomb Group (PcG) Proteins and Human Cancers: Multifaceted Functions and Therapeutic Implications

    PubMed Central

    Wang, Wei; Qin, Jiang-Jiang; Voruganti, Sukesh; Nag, Subhasree; Zhou, Jianwei; Zhang, Ruiwen

    2016-01-01

    Polycomb group (PcG) proteins are transcriptional repressors that regulate several crucial developmental and physiological processes in the cell. More recently, they have been found to play important roles in human carcinogenesis and cancer development and progression. The deregulation and dysfunction of PcG proteins often lead to blocking or inappropriate activation of developmental pathways, enhancing cellular proliferation, inhibiting apoptosis, and increasing the cancer stem cell population. Genetic and molecular investigations of PcG proteins have long been focused on their PcG functions. However, PcG proteins have recently been shown to exert non-polycomb functions, contributing to the regulation of diverse cellular functions. We and others have demonstrated that PcG proteins regulate the expression and function of several oncogenes and tumor suppressor genes in a PcG-independent manner, and PcG proteins are associated with the survival of patients with cancer. In this review, we summarize the recent advances in the research on PcG proteins, including both the polycomb-repressive and non-polycomb functions. We specifically focus on the mechanisms by which PcG proteins play roles in cancer initiation, development, and progression. Finally, we discuss the potential value of PcG proteins as molecular biomarkers for the diagnosis and prognosis of cancer, and as molecular targets for cancer therapy. PMID:26227500

  12. Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress

    PubMed Central

    Baqader, Noor O.; Radulovic, Marko; Crawford, Mark; Stoeber, Kai; Godovac-Zimmermann, Jasminka

    2014-01-01

    We have used a subcellular spatial razor approach based on LC–MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function. PMID:25133973

  13. Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells

    PubMed Central

    Barjaktarovic, Zarko; Kempf, Stefan J.; Sriharshan, Arundhathi; Merl-Pham, Juliane; Atkinson, Michael J.; Tapio, Soile

    2015-01-01

    Reversible lysine acetylation is a highly regulated post-translational protein modification that is known to regulate several signaling pathways. However, little is known about the radiation-induced changes in the acetylome. In this study, we analyzed the acute post-translational acetylation changes in primary human cardiac microvascular endothelial cells 4 h after a gamma radiation dose of 2 Gy. The acetylated peptides were enriched using anti-acetyl conjugated agarose beads. A total of 54 proteins were found to be altered in their acetylation status, 23 of which were deacetylated and 31 acetylated. Pathway analyses showed three protein categories particularly affected by radiation-induced changes in the acetylation status: the proteins involved in the translation process, the proteins of stress response, and mitochondrial proteins. The activation of the canonical and non-canonical Wnt signaling pathways affecting actin cytoskeleton signaling and cell cycle progression was predicted. The protein expression levels of two nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 1 and sirtuin 3, were significantly but transiently upregulated 4 but not 24 h after irradiation. The status of the p53 protein, a target of sirtuin 1, was found to be rapidly stabilized by acetylation after radiation exposure. These findings indicate that post-translational modification of proteins by acetylation and deacetylation is essentially affecting the radiation response of the endothelium. PMID:25840449

  14. Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

    SciTech Connect

    Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta; Yu, Carol; Abraham, Thomas; Borchers, Christoph; Bernatchez, Pascal

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. Black-Right-Pointing-Pointer Dysferlin interacts with key signaling proteins for transcytosis in EC. Black-Right-Pointing-Pointer Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.

  15. Production and supply of high-quality food protein for human consumption: sustainability, challenges, and innovations.

    PubMed

    Wu, Guoyao; Fanzo, Jessica; Miller, Dennis D; Pingali, Prabhu; Post, Mark; Steiner, Jean L; Thalacker-Mercer, Anna E

    2014-08-01

    The Food and Agriculture Organization of the United Nations estimates that 843 million people worldwide are hungry and a greater number suffer from nutrient deficiencies. Approximately one billion people have inadequate protein intake. The challenge of preventing hunger and malnutrition will become even greater as the global population grows from the current 7.2 billion people to 9.6 billion by 2050. With increases in income, population, and demand for more nutrient-dense foods, global meat production is projected to increase by 206 million tons per year during the next 35 years. These changes in population and dietary practices have led to a tremendous rise in the demand for food protein, especially animal-source protein. Consuming the required amounts of protein is fundamental to human growth and health. Protein needs can be met through intakes of animal and plant-source foods. Increased consumption of food proteins is associated with increased greenhouse gas emissions and overutilization of water. Consequently, concerns exist regarding impacts of agricultural production, processing and distribution of food protein on the environment, ecosystem, and sustainability. To address these challenging issues, the New York Academy of Sciences organized the conference "Frontiers in Agricultural Sustainability: Studying the Protein Supply Chain to Improve Dietary Quality" to explore sustainable innovations in food science and programming aimed at producing the required quality and quantity of protein through improved supply chains worldwide. This report provides an extensive discussion of these issues and summaries of the presentations from the conference.

  16. Biocompatibility study of protein capped and uncapped silver nanoparticles on human hemoglobin

    NASA Astrophysics Data System (ADS)

    Bhunia, Amit Kumar; Kanti Samanta, Pijus; Aich, Debasish; Saha, Satyajit; Kamilya, Tapanendu

    2015-06-01

    The interactions of human hemoglobin with protein capped silver nanoparticles and bare silver nanoparticles were studied to understand fundamental perspectives about the biocompatibility of protein capped silver nanoparticles compared with bare silver nanoparticles. Bare silver (Ag) nanoparticles (NPs) were prepared by the chemical reduction method. High resolution transmission electron microscopy (HRTEM) analysis along with absorption at ~390 nm indicated the formation of bare Ag NPs. Protein coated Ag NPs were prepared by a green synthesis method. Absorption at ~440 nm along with ~280 nm indicated the formation of protein coated Ag NPs. The biocompatibility of the above mentioned Ag NPs was studied by interaction with human hemoglobin (Hb) protein. In presence of bare Ag NPs, the Soret band of Hb was red shifted. This revealed the distortion of iron from the heme pockets of Hb. Also, the fluorescence peak of Hb was quenched and red shifted which indicated that Hb became unfolded in the presence of bare Ag NPs. No red shift of the absorption of Soret, along with no shift and quenching of the fluorescence peak of Hb were observed in the presence of protein coated Ag NPs. A hemolysis assay suggested that protein coated Ag NPs were more biocompatible than bare one.

  17. Analyzing the functions and structure of the human lipodystrophy protein seipin.

    PubMed

    Sim, M F Michelle; Talukder, Mesbah Uddin; Dennis, Rowena J; Edwardson, J Michael; Rochford, Justin J

    2014-01-01

    Disruption of the gene BSCL2, which encodes the protein seipin, causes severe generalized lipodystrophy in humans with a near complete absence of adipose tissue. Moreover, cell culture studies have demonstrated that seipin plays a critical cell-autonomous role in adipocyte differentiation. These observations reveal seipin as a critical regulator of human adipose tissue development; however, until recently very little has been known about the potential molecular functions of this intriguing protein. Despite significant recent interest in the function of seipin, our understanding of its molecular role(s) remains limited. The topology of seipin and lack of evidence for any enzymatic domains or activity indicate that it may act principally as a scaffold for other proteins or play a structural role in altering membrane curvature and/or budding. Work in this area has been hampered by several factors, including the lack of homology that might imply testable functions, the poor availability of antibodies to the endogenous protein and the observation that this hydrophobic ER membrane-resident protein is difficult to analyze by standard Western blotting techniques. Here we summarize some of the techniques we have applied to investigate the association of seipin with a recently identified binding partner, lipin 1. In addition, we describe the use of atomic force microscopy (AFM) to image oligomers of the seipin protein. We believe that AFM will offer a valuable tool to examine the association of candidate binding proteins with the seipin oligomer.

  18. Cardiovascular-related proteins identified in human plasma by the HUPO Plasma Proteome Project pilot phase.

    PubMed

    Berhane, Beniam T; Zong, Chenggong; Liem, David A; Huang, Aaron; Le, Steven; Edmondson, Ricky D; Jones, Richard C; Qiao, Xin; Whitelegge, Julian P; Ping, Peipei; Vondriska, Thomas M

    2005-08-01

    Proteomic profiling of accessible bodily fluids, such as plasma, has the potential to accelerate biomarker/biosignature development for human diseases. The HUPO Plasma Proteome Project pilot phase examined human plasma with distinct proteomic approaches across multiple laboratories worldwide. Through this effort, we confidently identified 3020 proteins, each requiring a minimum of two high-scoring MS/MS spectra. A critical step subsequent to protein identification is functional annotation, in particular with regard to organ systems and disease. Performing exhaustive literature searches, we have manually annotated a subset of these 3020 proteins that have cardiovascular-related functions on the basis of an existing body of published information. These cardiovascular-related proteins can be organized into eight groups: markers of inflammation and/or cardiovascular disease, vascular and coagulation, signaling, growth and differentiation, cytoskeletal, transcription factors, channels/receptors and heart failure and remodeling. In addition, analysis of the peptide per protein ratio for MS/MS identification reveals group-specific trends. These findings serve as a resource to interrogate the functions of plasma proteins, and moreover, the list of cardiovascular-related proteins in plasma constitutes a baseline proteomic blueprint for the future development of biosignatures for diseases such as myocardial ischemia and atherosclerosis. PMID:16052623

  19. RAID: a comprehensive resource for human RNA-associated (RNA–RNA/RNA–protein) interaction

    PubMed Central

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-01-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA–RNA/RNA–protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA–RNA interactions and 1619 RNA–protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA–RNA/RNA–protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA–RNA/RNA–protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

  20. RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction.

    PubMed

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-07-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network.

  1. Human prion protein sequence elements impede cross-species chronic wasting disease transmission.

    PubMed

    Kurt, Timothy D; Jiang, Lin; Fernández-Borges, Natalia; Bett, Cyrus; Liu, Jun; Yang, Tom; Spraker, Terry R; Castilla, Joaquín; Eisenberg, David; Kong, Qingzhong; Sigurdson, Christina J

    2015-04-01

    Chronic wasting disease (CWD) is a fatal prion disease of North American deer and elk and poses an unclear risk for transmission to humans. Human exposure to CWD occurs through hunting activities and consumption of venison from prion-infected animals. Although the amino acid residues of the prion protein (PrP) that prevent or permit human CWD infection are unknown, NMR-based structural studies suggest that the β2-α2 loop (residues 165-175) may impact species barriers. Here we sought to define PrP sequence determinants that affect CWD transmission to humans. We engineered transgenic mice that express human PrP with four amino acid substitutions that result in expression of PrP with a β2-α2 loop (residues 165-175) that exactly matches that of elk PrP. Compared with transgenic mice expressing unaltered human PrP, mice expressing the human-elk chimeric PrP were highly susceptible to elk and deer CWD prions but were concurrently less susceptible to human Creutzfeldt-Jakob disease prions. A systematic in vitro survey of amino acid differences between humans and cervids identified two additional residues that impacted CWD conversion of human PrP. This work identifies amino acids that constitute a substantial structural barrier for CWD transmission to humans and helps illuminate the molecular requirements for cross-species prion transmission. PMID:25705888

  2. Human prion protein sequence elements impede cross-species chronic wasting disease transmission

    PubMed Central

    Kurt, Timothy D.; Jiang, Lin; Fernández-Borges, Natalia; Bett, Cyrus; Liu, Jun; Yang, Tom; Spraker, Terry R.; Castilla, Joaquín; Eisenberg, David; Kong, Qingzhong; Sigurdson, Christina J.

    2015-01-01

    Chronic wasting disease (CWD) is a fatal prion disease of North American deer and elk and poses an unclear risk for transmission to humans. Human exposure to CWD occurs through hunting activities and consumption of venison from prion-infected animals. Although the amino acid residues of the prion protein (PrP) that prevent or permit human CWD infection are unknown, NMR-based structural studies suggest that the β2-α2 loop (residues 165–175) may impact species barriers. Here we sought to define PrP sequence determinants that affect CWD transmission to humans. We engineered transgenic mice that express human PrP with four amino acid substitutions that result in expression of PrP with a β2-α2 loop (residues 165–175) that exactly matches that of elk PrP. Compared with transgenic mice expressing unaltered human PrP, mice expressing the human-elk chimeric PrP were highly susceptible to elk and deer CWD prions but were concurrently less susceptible to human Creutzfeldt-Jakob disease prions. A systematic in vitro survey of amino acid differences between humans and cervids identified two additional residues that impacted CWD conversion of human PrP. This work identifies amino acids that constitute a substantial structural barrier for CWD transmission to humans and helps illuminate the molecular requirements for cross-species prion transmission. PMID:25705888

  3. Heroin-Induces Differential Protein Expression by Normal Human Astrocytes (NHA).

    PubMed

    Reynolds, Jessica L; Mahajan, Supriya D; Sykes, Donald; Nair, Madhavan P N

    2006-01-01

    Heroin use is postulated to act as a cofactor in the neuropathogenesis of human immunodeficiency virus (HIV-1) infection. Astrocytes, integral components of the CNS, are reported to be susceptible to HIV-1 infection. Upon activation, astrocytes release a number of immunoregulatory products or modulate the expression of a number of proteins that foster the immunopathogenesis of HIV-1 infection. However, the role of heroin on HIV-1 infectivity and the expression of the proteome of normal human astrocytes (NHA) have not been elucidated. We hypothesize that heroin modulates the expression of a number of proteins by NHA that foster the neuoropathogenesis of HIV-1 infection. We utilized LTR amplification and the p24 antigen assay to quantitate the effect of heroin on HIV-1 infectivity while difference gel electrophoresis (DIGE) combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to analyze the effects of heroin on the proteomic profile of NHA. Results demonstrate that heroin potentiates HIV-1 replication in NHA. Furthermore, heroin significantly increased protein expression levels for protein kinase C (PKC), reticulocalbin 1 precursor, reticulocalbin 1, tyrosine 3-monooxgenase/tryptophan 5-monooxgenase activation protein, chloride intracellular channel 1, cathepsin D preproprotein, galectin 1 and myosin light chain alkali. Heroin also significantly decreased protein expression for proliferating cell nuclear antigen, proteasome beta 6 subunit, tropomyosin 3, laminin receptor 1, tubulin alpha 6, vimentin, EF hand domain family member D2, Tumor protein D54 (hD54), ATP synthase, H+ transporting, mitochondrial F1 complex and ribosomal protein S14. Identification of unique, heroin-induced proteins may help to develop novel markers for diagnostic, preventative and therapeutic targeting in heroin using subjects. PMID:17235376

  4. In vitro binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase.

    PubMed

    Bodéus, M; Marie-Cardine, A; Bougeret, C; Ramos-Morales, F; Benarous, R

    1995-06-01

    Although the human immunodeficiency virus type 1 (HIV-1) nef gene still has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1LAI nef gene product was expressed in the bacterial vector pGEX-2T as a glutathione S-transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites in vitro: one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef in vitro. The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis.

  5. Nonstructural Protein 1 of Influenza A Virus Interacts with Human Guanylate-Binding Protein 1 to Antagonize Antiviral Activity

    PubMed Central

    Yan, Wenjun; Wei, Jianchao; Shao, Donghua; Deng, Xufang; Wang, Shaohui; Li, Beibei; Tong, Guangzhi; Ma, Zhiyong

    2013-01-01

    Human guanylate-binding protein 1 (hGBP1) is an interferon-inducible protein involved in the host immune response against viral infection. In response to infection by influenza A virus (IAV), hGBP1 transcript and protein were significantly upregulated. Overexpression of hGBP1 inhibited IAV replication in a dose-dependent manner in vitro. The lysine residue at position 51 (K51) of hGBP1 was essential for inhibition of IAV replication. Mutation of K51 resulted in an hGBP1 that was unable to inhibit IAV replication. The viral nonstructural protein 1 (NS1) was found to interact directly with hGBP1. K51 of hGBP1 and a region between residues 123 and 144 in NS1 were demonstrated to be essential for the interaction between NS1 and hGBP1. Binding of NS1 to hGBP1 resulted in a significant reduction in both GTPase activity and the anti-IAV activity of hGBP1. These findings indicated that hGBP1 contributed to the host immune response against IAV replication and that hGBP1-mediated antiviral activity was antagonized by NS1 via binding to hGBP1. PMID:23405236

  6. Nonstructural protein 1 of influenza A virus interacts with human guanylate-binding protein 1 to antagonize antiviral activity.

    PubMed

    Zhu, Zixiang; Shi, Zixue; Yan, Wenjun; Wei, Jianchao; Shao, Donghua; Deng, Xufang; Wang, Shaohui; Li, Beibei; Tong, Guangzhi; Ma, Zhiyong

    2013-01-01

    Human guanylate-binding protein 1 (hGBP1) is an interferon-inducible protein involved in the host immune response against viral infection. In response to infection by influenza A virus (IAV), hGBP1 transcript and protein were significantly upregulated. Overexpression of hGBP1 inhibited IAV replication in a dose-dependent manner in vitro. The lysine residue at position 51 (K51) of hGBP1 was essential for inhibition of IAV replication. Mutation of K51 resulted in an hGBP1 that was unable to inhibit IAV replication. The viral nonstructural protein 1 (NS1) was found to interact directly with hGBP1. K51 of hGBP1 and a region between residues 123 and 144 in NS1 were demonstrated to be essential for the interaction between NS1 and hGBP1. Binding of NS1 to hGBP1 resulted in a significant reduction in both GTPase activity and the anti-IAV activity of hGBP1. These findings indicated that hGBP1 contributed to the host immune response against IAV replication and that hGBP1-mediated antiviral activity was antagonized by NS1 via binding to hGBP1.

  7. The poor digestibility of rapeseed protein is balanced by its very high metabolic utilization in humans.

    PubMed

    Bos, Cécile; Airinei, Gheorghe; Mariotti, François; Benamouzig, Robert; Bérot, Serge; Evrard, Jacques; Fénart, Evelyne; Tomé, Daniel; Gaudichon, Claire

    2007-03-01

    Rapeseed protein (RP, Brassica napus) is used in only animal feed despite its high nutritional potential for human nutrition. We sought to assess the nutritional quality of rapeseed by measuring its real ileal digestibility (RID) and net postprandial protein utilization (NPPU) in humans fed (15)N-RP. Volunteers equipped with an intestinal tube at the jejunal (n = 5) or ileal level (n = 7) ingested a mixed meal containing 27.3 g (15)N-RP and a total energy content of 700 kcal (2.93 MJ). Dietary N kinetics was quantified in intestinal fluid, urine, and blood sampled at regular intervals during the postprandial period. The RID of RP was 84.0 +/- 8.8%. Dietary N at the ileal level was mostly in the form of undigested protein from both 12S and 2S rapeseed fractions. Aminoacidemia was not significantly increased by meal ingestion. The postprandial distribution of dietary N was 5.4 +/- 1.8% in urinary urea and ammonia, 8.2 +/- 3.4% in body urea, and 7.7 +/- 2.0% in plasma protein 8 h after the meal. The NPPU of RP amounted to 70.5 +/- 9.6% and the postprandial biological value (PBV) was high at 83.8 +/- 4.6%. RP has a low RID in humans compared with other plant proteins but also exhibits a very low deamination rate. Thus, the PBV of RP is excellent in humans, being as high as that of milk protein. We conclude that RP has a high nutritional potential for human nutrition.

  8. Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

    PubMed

    Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

    2016-06-01

    Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. PMID:27167364

  9. Finding Missing Proteins from the Epigenetically Manipulated Human Cell with Stringent Quality Criteria.

    PubMed

    Yang, Lijuan; Lian, Xinlei; Zhang, Wanling; Guo, Jie; Wang, Qing; Li, Yaxing; Chen, Yang; Yin, Xingfeng; Yang, Pengyuan; Lan, Fei; He, Qing-Yu; Zhang, Gong; Wang, Tong

    2015-09-01

    The chromosome-centric human proteome project (C-HPP) has made great progress of finding protein evidence (PE) for missing proteins (PE2-4 proteins defined by the neXtProt), which now becomes an increasingly challenging field. As a majority of samples tested in this field were from adult tissues/cells, the developmental stage specific or relevant proteins could be missed due to biological source availability. We posit that epigenetic interventions may help to partially bypass such a limitation by stimulating the expression of the "silenced" genes in adult cells, leading to the increased chance of finding missing proteins. In this study, we established in vitro human cell models to modify the histone acetylation, demethylation, and methylation with near physiological conditions. With mRNA-seq analysis, we found that histone modifications resulted in overall increases of expressed genes in an even distribution manner across different chromosomes. We identified 64 PE2-4 and six PE5 proteins by MaxQuant (FDR < 1% at both protein and peptide levels) and 44 PE2-4 and 7 PE5 proteins by Mascot (FDR < 1% at peptide level) searches, respectively. However, only 24 PE2-4 and five PE5 proteins in Mascot, and 12 PE2-4 and one PE5 proteins in MaxQuant searches could, respectively, pass our stringently manual spectrum inspections. Collectively, 27 PE2-4 and five PE5 proteins were identified from the epigenetically modified cells; among them, 19 PE2-4 and three PE5 proteins passed FDR < 1% at both peptide and protein levels. Gene ontology analyses revealed that the PE2-4 proteins were significantly involved in development and spermatogenesis, although their chemical-physical features had no statistical difference from the background. In addition, we presented an example of suspicious PE5 peptide spectrum matched with unusual AA substitutions related to post-translational modification. In conclusion, the epigenetically manipulated cell models should be a useful tool for finding

  10. Detection of vitamin D binding protein on the surface of cytotrophoblasts isolated from human placentae

    SciTech Connect

    Nestler, J.E.; McLeod, J.F.; Kowalski, M.A.; Strauss, J.F. 3d.; Haddad, J.G. Jr.

    1987-05-01

    Vitamin D binding protein (DBP), a Mr 56,000-58,000 alpha 2-glycoprotein, is the major serum protein involved in the transport of vitamin D sterols. Recently it has been suggested that DBP may also be involved in immunoglobulin G binding to cells. Because the trophoblast is involved in the transport of molecules such as vitamin D and immunoglobulin G to the fetus, we asked whether DBP could be detected on the surface of human placental trophoblast cells. Cytotrophoblasts purified from human term placentae were fixed and made permeant with Triton X-100 and examined by indirect immunofluorescence after incubation with a monoclonal antibody to DBP. Greater than 90% of these cells stained positively, whereas no staining was observed with nonimmune antiserum. The presence of DBP on/in the surface of cytotrophoblasts could also be demonstrated by fluorescent cytometry. When cell surface-associated proteins of cytotrophoblasts were radioiodinated, a Mr 57,000 radiolabeled protein could be immunoisolated from the cell lysate with a purified monospecific polyclonal antibody to DBP. Immunoisolation of this radiolabeled protein was prevented by the addition of excess unlabeled human DBP to the cell lysate before incubation with antibody. This Mr 57,000 radiolabeled protein could also be isolated by affinity chromatography selecting for proteins that bind to globular actin. When cytotrophoblasts were incubated with (/sup 35/S)methionine for 3 or 18 h, active synthesis of DBP could not be demonstrated by immunoisolation techniques. These studies demonstrate the presence of DBP on the surface of well washed, human cytotrophoblasts. This DBP may be maternally derived, since active synthesis of DBP could not be demonstrated.

  11. Proteinaceous precursors of human axillary odor: isolation of two novel odor-binding proteins.

    PubMed

    Spielman, A I; Zeng, X N; Leyden, J J; Preti, G

    1995-01-15

    The characteristic odor which arises in the human axillary region consists of volatile C6-C11 acids with the most abundant being (E)-3-methyl-2-hexenoic acid (E-3M2H). This acid, as well as several other components of the characteristic axillary odor, can be liberated from the odorless, aqueous soluble components of apocrine secretion by either saponification or bacteriolysis. It is therefore likely that a major characteristic odor is being carried to the skin surface bound to a water soluble precursor where it is liberated by axillary bacteria. The individual proteins found in apocrine secretions were separated, isolated and hydrolyzed with the resultant hydrolyzates analyzed by gas chromatography/mass spectrometry. These studies demonstrated that 3M2H was liberated from 2 proteins with apparent molecular mass of 26 and 45 kilodaltons: Apocrine Secretion Odor-Binding Protein 1 and 2, respectively (ASOB1 and ASOB2). Antisera to these proteins were prepared and used to examine a variety of other body fluids. Several fluids contained an immunoreactive protein with the same electropheretic migration pattern as the 45 KDa protein. Three of these body fluids (tears, nasal secretions and saliva) were separated into aqueous and organic soluble fractions and hydrolyzed to demonstrate that 3M2H could be liberated from the aqueous soluble materials. These results suggest interesting parallels between non-human mammalian odors used as chemical signals and human axillary odor. Previous studies have suggested the axillae as a source of human primer-type pheromones; consequently, if the odors which characterize the underarm are responsible for the pheromonal activity, then the chemistry involved may be similar to that in other mammalian chemical communication systems where proteins act as carriers of one or more chemical signals.

  12. Molecular characterization of a human gene for S28 ribosomal binding protein

    SciTech Connect

    Wong, P.; Borst, D.E.; Chader, G.J.

    1994-09-01

    The mechanism of ribosome action and the ribosomal binding proteins which cooperatively interact in the working of this structure are not completely understood. Theoretically, mutations in genes that encode these proteins may compromise the efficiency of protein synthesis and therefore lead to a functional disorder. In the course of our search for human genes which show homology to the C. elegans CED-4 death gene, we have serendipitously identified one of the human S28 ribosomal binding protein genes as a random fragment fused to the end of one of our putative CED-4 positive homologue clones. The cloned S28 fragment consists of 381 nucleotides with a putative open reading frame of 113 amino acids. Sequence comparisons to GenBank revealed significant homologies to ribosomal binding protein genes in other species (including the rat S28 ribosomal binding protein gene) indicating that the S28 gene sequence is highly conserved. This finding is confirmed by zooblot analysis. Significant homologies also exist to two human expressed tagged sites (HUMRIBPROB; L05091 and HSAFIF072; Z21908). Analysis of the putative S28 peptide sequence allows insights into possible functional regions of the protein. The identification of 8 distinct bands upon Southern analysis of the S28 fragments suggests that there are multiple copies of the S28 gene in the human genome. Mapping of the S28 fragment on somatic cell hybrid panels identified distinct S28 gene loci on chromosomes 1, 2, 7, 10, 11, 12, 17 expression in adult tissues (pancreas, kidney, muscle, liver, lung, placenta, brain, heart, and retina) as well as in fetal tissues (kidney, liver, lung, brain, and heart).

  13. JAB1 regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cells.

    PubMed

    Nishimoto, Arata; Kugimiya, Naruji; Hosoyama, Toru; Enoki, Tadahiko; Li, Tao-Sheng; Hamano, Kimikazu

    2013-08-30

    Recent studies have revealed that unphosphorylated STAT3 forms a dimer, translocates to the nucleus, binds to the STAT3 binding site, and activates the transcription of STAT3 target genes, thereby playing an important role in oncogenesis in addition to phosphorylated STAT3. Among signaling steps of unphosphorylated STAT3, nuclear translocation and target DNA-binding are the critical steps for its activation. Therefore, elucidating the regulatory mechanism of these signaling steps of unphosphorylated STAT3 is a potential step in the discovery of a novel cancer drug. However, the mechanism of unphosphorylated STAT3 binding to the promoter of target genes remains unclear. In this study, we focused on Jun activation domain-binding protein 1 (JAB1) as a candidate protein that regulates unphosphorylated STAT3 DNA-binding activity. Initially, we observed that both unphosphorylated STAT3 and JAB1 existed in the nucleus of human colon cancer cell line COLO205 at the basal state (no cytokine stimulation). On the other hand, phosphorylated STAT3 did not exist in the nucleus of COLO205 cells at the basal state. Immunoprecipitation using nuclear extract of COLO205 cells revealed that JAB1 interacted with unphosphorylated STAT3. To investigate the effect of JAB1 on unphosphorylated STAT3 activity, RNAi studies were performed. Although JAB1 knockdown tended to increase nuclear STAT3 expression, it significantly decreased unphosphorylated STAT3 DNA-binding activity. Subsequently, JAB1 knockdown significantly decreased the expression levels of MDR1, NANOG, and VEGF, which are STAT3 target genes. Furthermore, the expression level of nuclear JAB1, but not nuclear STAT3, correlated with unphosphorylated STAT3 DNA-binding activity between COLO205 and LoVo cells. Taken together, these results suggest that nuclear JAB1 positively regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cell line COLO205.

  14. Functional features and protein network of human sperm-egg interaction.

    PubMed

    Sabetian, Soudabeh; Shamsir, Mohd Shahir; Abu Naser, Mohammed

    2014-12-01

    Elucidation of the sperm-egg interaction at the molecular level is one of the unresolved problems in sexual reproduction, and understanding the molecular mechanism is crucial in solving problems in infertility and failed in vitro fertilization (IVF). Many molecular interactions in the form of protein-protein interactions (PPIs) mediate the sperm-egg membrane interaction. Due to the complexity of the problem such as difficulties in analyzing in vivo membrane PPIs, many efforts have failed to comprehensively elucidate the fusion mechanism and the molecular interactions that mediate sperm-egg membrane fusion. The main purpose of this study was to reveal possible protein interactions and associated molecular function during sperm-egg interaction using a protein interaction network approach. Different databases have been used to construct the human sperm-egg interaction network. The constructed network revealed new interactions. These included CD151 and CD9 in human oocyte that interact with CD49 in sperm, and CD49 and ITGA4 in sperm that interact with CD63 and CD81, respectively, in the oocyte. These results showed that the different integrins in sperm may be involved in human sperm-egg interaction. It was also suggested that sperm ADAM2 plays a role as a protein candidate involved in sperm-egg membrane interaction by interacting with CD9 in the oocyte. Interleukin-4 receptor activity, receptor signaling protein tyrosine kinase activity, and manganese ion transmembrane transport activity are the major molecular functions in sperm-egg interaction protein network. The disease association analysis indicated that sperm-egg interaction defects are also reflected in other disease networks such as cardiovascular, hematological, and breast cancer diseases. By analyzing the network, we identified the major molecular functions and disease association genes in sperm-egg interaction protein. Further experimental studies will be required to confirm the significance of these new

  15. Functional features and protein network of human sperm-egg interaction.

    PubMed

    Sabetian, Soudabeh; Shamsir, Mohd Shahir; Abu Naser, Mohammed

    2014-12-01

    Elucidation of the sperm-egg interaction at the molecular level is one of the unresolved problems in sexual reproduction, and understanding the molecular mechanism is crucial in solving problems in infertility and failed in vitro fertilization (IVF). Many molecular interactions in the form of protein-protein interactions (PPIs) mediate the sperm-egg membrane interaction. Due to the complexity of the problem such as difficulties in analyzing in vivo membrane PPIs, many efforts have failed to comprehensively elucidate the fusion mechanism and the molecular interactions that mediate sperm-egg membrane fusion. The main purpose of this study was to reveal possible protein interactions and associated molecular function during sperm-egg interaction using a protein interaction network approach. Different databases have been used to construct the human sperm-egg interaction network. The constructed network revealed new interactions. These included CD151 and CD9 in human oocyte that interact with CD49 in sperm, and CD49 and ITGA4 in sperm that interact with CD63 and CD81, respectively, in the oocyte. These results showed that the different integrins in sperm may be involved in human sperm-egg interaction. It was also suggested that sperm ADAM2 plays a role as a protein candidate involved in sperm-egg membrane interaction by interacting with CD9 in the oocyte. Interleukin-4 receptor activity, receptor signaling protein tyrosine kinase activity, and manganese ion transmembrane transport activity are the major molecular functions in sperm-egg interaction protein network. The disease association analysis indicated that sperm-egg interaction defects are also reflected in other disease networks such as cardiovascular, hematological, and breast cancer diseases. By analyzing the network, we identified the major molecular functions and disease association genes in sperm-egg interaction protein. Further experimental studies will be required to confirm the significance of these new

  16. Identification and purification of a protein encoded by the human adenovirus type 2 transforming region.

    PubMed Central

    Green, M; Brackmann, K H; Cartas, M A; Matsuo, T

    1982-01-01

    The human adenovirus type 2 (Ad2) transforming genes are located in early regions E1a (map position 1.3 to 4.5) and E1b (map position 4.6 to 11.2). We have identified and purified to near homogeneity a major 20,000-molecular-weight (20K) protein and have shown that it is coded by E1b. Using an Ad2-transformed cell antiserum which contained antibody to E1b-coded proteins, we immunoprecipitated 53K and 19K proteins from the nucleoplasm and 53K, 19K, and 20K proteins from the cytoplasmic S-100 fraction of Ad2 productively infected and Ad2-transformed cells. The 19K protein was present in both the nucleoplasm and the cytoplasm, whereas the 20K protein was found only in the cytoplasm. The 53K and 19K proteins are known Ad2 E1b-coded proteins. The 20K protein was purified to near homogeneity in 20 to 50% yields by sequential DEAE-Sephacel chromatography and reverse-phase high-performance liquid chromatography. Purified 20K protein shares most of its methionine-labeled tryptic peptides with E1b-53K, as shown by reverse-phase high-performance liquid chromatography, and therefore is closely related to the 53K protein. The 19K protein does not appear to share tryptic peptides with either 20K or 53K protein. To provide more direct evidence that 20K protein is virus-coded, we translated E1b-specific mRNA in vitro. Both immunoprecipitation analysis and high-performance liquid chromatography purification of the translated product identified a 20K protein that has the same tryptic peptides as the 20K protein isolated from infected and from transformed cells. These findings suggest that the Ad2 20K protein is a primary translation product of an Ad2 E1b mRNA. Images PMID:7045392

  17. Identification and purification of a protein encoded by the human adenovirus type 2 transforming region.

    PubMed

    Green, M; Brackmann, K H; Cartas, M A; Matsuo, T

    1982-04-01

    The human adenovirus type 2 (Ad2) transforming genes are located in early regions E1a (map position 1.3 to 4.5) and E1b (map position 4.6 to 11.2). We have identified and purified to near homogeneity a major 20,000-molecular-weight (20K) protein and have shown that it is coded by E1b. Using an Ad2-transformed cell antiserum which contained antibody to E1b-coded proteins, we immunoprecipitated 53K and 19K proteins from the nucleoplasm and 53K, 19K, and 20K proteins from the cytoplasmic S-100 fraction of Ad2 productively infected and Ad2-transformed cells. The 19K protein was present in both the nucleoplasm and the cytoplasm, whereas the 20K protein was found only in the cytoplasm. The 53K and 19K proteins are known Ad2 E1b-coded proteins. The 20K protein was purified to near homogeneity in 20 to 50% yields by sequential DEAE-Sephacel chromatography and reverse-phase high-performance liquid chromatography. Purified 20K protein shares most of its methionine-labeled tryptic peptides with E1b-53K, as shown by reverse-phase high-performance liquid chromatography, and therefore is closely related to the 53K protein. The 19K protein does not appear to share tryptic peptides with either 20K or 53K protein. To provide more direct evidence that 20K protein is virus-coded, we translated E1b-specific mRNA in vitro. Both immunoprecipitation analysis and high-performance liquid chromatography purification of the translated product identified a 20K protein that has the same tryptic peptides as the 20K protein isolated from infected and from transformed cells. These findings suggest that the Ad2 20K protein is a primary translation product of an Ad2 E1b mRNA.

  18. Different digestion of caprine whey proteins by human and porcine gastrointestinal enzymes.

    PubMed

    Eriksen, Ellen K; Holm, Halvor; Jensen, Einar; Aaboe, Ragnhild; Devold, Tove G; Jacobsen, Morten; Vegarud, Gerd E

    2010-08-01

    The objective of the present study was twofold: first to compare the degradation patterns of caprine whey proteins digested with either human digestive juices (gastric or duodenal) or commercial porcine enzymes (pepsin or pancreatic enzymes) and second to observe the effect of gastric pH on digestion. An in vitro two-step assay was performed at 37 degrees C to simulate digestion in the stomach (pH 2, 4 or 6) and the duodenum (pH 8). The whey proteins were degraded more efficiently by porcine pepsin than by human gastric juice at all pH values. Irrespective of the enzyme source, gastric digestion at pH 2 followed by duodenal digestion resulted in the most efficient degradation. Lactoferrin, serum albumin and the Ig heavy chains were highly degraded with less than 6 % remaining after digestion. About 15, 56 and 50 % Ig light chains, beta-lactoglobulin (beta-LG) and alpha-lactalbumin remained intact, respectively, when digested with porcine enzymes compared with 25, 74 and 81 % with human digestive juices. For comparison, purified bovine beta-LG was digested and the peptide profiles obtained were compared with those of the caprine beta-LG in the digested whey. The bovine beta-LG seemed to be more extensively cleaved than the caprine beta-LG in the whey. Commercial enzymes appear to digest whey proteins more efficiently compared with human digestive juices when used at similar enzyme activities. This could lead to conflicting results when comparing human in vivo protein digestion with digestion using purified enzymes of non-human species. Consequently the use of human digestive juices might be preferred.

  19. Different digestion of caprine whey proteins by human and porcine gastrointestinal enzymes.

    PubMed

    Eriksen, Ellen K; Holm, Halvor; Jensen, Einar; Aaboe, Ragnhild; Devold, Tove G; Jacobsen, Morten; Vegarud, Gerd E

    2010-08-01

    The objective of the present study was twofold: first to compare the degradation patterns of caprine whey proteins digested with either human digestive juices (gastric or duodenal) or commercial porcine enzymes (pepsin or pancreatic enzymes) and second to observe the effect of gastric pH on digestion. An in vitro two-step assay was performed at 37 degrees C to simulate digestion in the stomach (pH 2, 4 or 6) and the duodenum (pH 8). The whey proteins were degraded more efficiently by porcine pepsin than by human gastric juice at all pH values. Irrespective of the enzyme source, gastric digestion at pH 2 followed by duodenal digestion resulted in the most efficient degradation. Lactoferrin, serum albumin and the Ig heavy chains were highly degraded with less than 6 % remaining after digestion. About 15, 56 and 50 % Ig light chains, beta-lactoglobulin (beta-LG) and alpha-lactalbumin remained intact, respectively, when digested with porcine enzymes compared with 25, 74 and 81 % with human digestive juices. For comparison, purified bovine beta-LG was digested and the peptide profiles obtained were compared with those of the caprine beta-LG in the digested whey. The bovine beta-LG seemed to be more extensively cleaved than the caprine beta-LG in the whey. Commercial enzymes appear to digest whey proteins more efficiently compared with human digestive juices when used at similar enzyme activities. This could lead to conflicting results when comparing human in vivo protein digestion with digestion using purified enzymes of non-human species. Consequently the use of human digestive juices might be preferred. PMID:20307348

  20. Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

    PubMed Central

    1994-01-01

    Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti- Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12- myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins

  1. Probing thyroglobulin in undiluted human serum based on pattern recognition and competitive adsorption of proteins

    NASA Astrophysics Data System (ADS)

    Wang, Ran; Huang, Shuai; Li, Jing; Chae, Junseok

    2014-10-01

    Thyroglobulin (Tg) is a sensitive indicator of persistent or recurrent differentiated thyroid cancer of follicular cell origin. Detection of Tg in human serum is challenging as bio-receptors, such as anti-Tg, used in immunoassay have relatively weak binding affinity. We engineer sensing surfaces using the competitive adsorption of proteins, termed the Vroman Effect. Coupled with Surface Plasmon Resonance, the "cross-responsive" interactions of Tg on the engineered surfaces produce uniquely distinguishable multiple signature patterns, which are discriminated using Linear Discriminant Analysis. Tg-spiked samples, down to 2 ng/ml Tg in undiluted human serum, are sensitively and selectively discriminated from the control (undiluted human serum).

  2. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    SciTech Connect

    Smiley, R.M. Columbia Univ College of Physicians and Surgeons, New York, NY ); Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J. )

    1990-01-01

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with {sup 32}PO{sub 4} in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the {beta}-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M{sub r} 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the {alpha}-2 agonist clonidine. Epinephrine, a combined {alpha} and {beta} agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the {alpha}-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

  3. Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes

    PubMed Central

    Kim, Cy Hyun; Shin, Jin-Hong; Hwang, Sung Jun; Choi, Yung Hyun; Kim, Dae-Seong; Kim, Cheol Min

    2016-01-01

    Schisandrae fructus (SF) has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 μg/mL) of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 μg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged with sarcopenia and the patients with disuse atrophy. PMID:27330287

  4. Human polypyrimidine tract-binding protein interacts with mitochondrial tRNAThr in the cytosol

    PubMed Central

    Marnef, Aline; Jády, Beáta E.; Kiss, Tamás

    2016-01-01

    Human polypyrimidine tract-binding protein PTB is a multifunctional RNA-binding protein with four RNA recognition motifs (RRM1 to RRM4). PTB is a nucleocytoplasmic shuttle protein that functions as a key regulator of alternative pre-mRNA splicing in the nucleoplasm and promotes internal ribosome entry site-mediated translation initiation of viral and cellular mRNAs in the cytoplasm. Here, we demonstrate that PTB and its paralogs, nPTB and ROD1, specifically interact with mitochondrial (mt) tRNAThr both in human and mouse cells. In vivo and in vitro RNA-binding experiments demonstrate that PTB forms a direct interaction with the T-loop and the D-stem-loop of mt tRNAThr using its N-terminal RRM1 and RRM2 motifs. RNA sequencing and cell fractionation experiments show that PTB associates with correctly processed and internally modified, mature mt tRNAThr in the cytoplasm outside of mitochondria. Consistent with this, PTB activity is not required for mt tRNAThr biogenesis or for correct mitochondrial protein synthesis. PTB association with mt tRNAThr is largely increased upon induction of apoptosis, arguing for a potential role of the mt tRNAThr/PTB complex in apoptosis. Our results lend strong support to the recently emerging conception that human mt tRNAs can participate in novel cytoplasmic processes independent from mitochondrial protein synthesis. PMID:26657638

  5. Boric acid gel enrichment of glycosylated proteins in human wound fluids.

    PubMed

    Krisp, Christoph; Kubutat, Caroline; Kyas, Andreas; Steinsträsser, Lars; Jacobsen, Frank; Wolters, Dirk

    2011-04-01

    The enrichment of glycosylated proteins by glycocapturing materials plays a pivotal role for the investigation of polysaccharide containing proteins in disease pathogenesis. Hence, we investigated a boric acid gel as a binding material for glycoprotein enrichment. The bovine proteins alpha-1-acid-glycoprotein (A1AG) and alpha-2-HS-glycoprotein (fetuin A) were spiked in human chronic wound fluids and were subsequently enriched by a boric acid gel affinity chromatography (BAGAC). The enrichment efficiency was evaluated by western blot analysis and mass spectrometry. Additionally, glycoproteins of human wound fluids from diabetes mellitus patients with chronic foot ulcers were analyzed after BAGAC enrichments. In total 104 glycoproteins were identified, with reported glycosylation sites. 60 proteins were detected in at least 2 out of 3 biological replicates and were used for quantitative analysis between the bound and unbound fractions. Almost 80% of these glycoproteins were more prominent in the bound fraction. Only 2 glycoproteins revealed higher spectral counts in the flow through fraction compared to the bound fraction. These findings demonstrate the capability of the BAGAC material to enrich glycosylated proteins from complex human wound fluids.

  6. Quantitative variability of 342 plasma proteins in a human twin population

    PubMed Central

    Liu, Yansheng; Buil, Alfonso; Collins, Ben C; Gillet, Ludovic CJ; Blum, Lorenz C; Cheng, Lin-Yang; Vitek, Olga; Mouritsen, Jeppe; Lachance, Genevieve; Spector, Tim D; Dermitzakis, Emmanouil T; Aebersold, Ruedi

    2015-01-01

    The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis-SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood-based biomarker studies. PMID:25652787

  7. Human polypyrimidine tract-binding protein interacts with mitochondrial tRNA(Thr) in the cytosol.

    PubMed

    Marnef, Aline; Jády, Beáta E; Kiss, Tamás

    2016-02-18

    Human polypyrimidine tract-binding protein PTB is a multifunctional RNA-binding protein with four RNA recognition motifs (RRM1 to RRM4). PTB is a nucleocytoplasmic shuttle protein that functions as a key regulator of alternative pre-mRNA splicing in the nucleoplasm and promotes internal ribosome entry site-mediated translation initiation of viral and cellular mRNAs in the cytoplasm. Here, we demonstrate that PTB and its paralogs, nPTB and ROD1, specifically interact with mitochondrial (mt) tRNA(Thr) both in human and mouse cells. In vivo and in vitro RNA-binding experiments demonstrate that PTB forms a direct interaction with the T-loop and the D-stem-loop of mt tRNA(Thr) using its N-terminal RRM1 and RRM2 motifs. RNA sequencing and cell fractionation experiments show that PTB associates with correctly processed and internally modified, mature mt tRNA(Thr) in the cytoplasm outside of mitochondria. Consistent with this, PTB activity is not required for mt tRNA(Thr) biogenesis or for correct mitochondrial protein synthesis. PTB association with mt tRNA(Thr) is largely increased upon induction of apoptosis, arguing for a potential role of the mt tRNA(Thr)/PTB complex in apoptosis. Our results lend strong support to the recently emerging conception that human mt tRNAs can participate in novel cytoplasmic processes independent from mitochondrial protein synthesis. PMID:26657638

  8. Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins.

    PubMed

    Datskevich, Petr N; Mymrikov, Evgeny V; Sluchanko, Nikolai N; Shemetov, Anton A; Sudnitsyna, Maria V; Gusev, Nikolai B

    2012-03-01

    Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may

  9. Identification and baculovirus expression of the VP4 protein of the human group B rotavirus ADRV.

    PubMed Central

    Mackow, E R; Werner-Eckert, R; Fay, M E; Tao, H; Chen, G

    1993-01-01

    A complete cDNA copy of the fourth RNA segment of the human group B rotavirus adult diarrheal rotavirus (ADRV) has been cloned into lambda phage and excised into plasmid pSK Bluescript. Gene segment 4 contains 2,303 bases and encodes one long open reading frame beginning at base 16 and terminating at base 2263. The encoded protein contains 749 amino acids, with a calculated molecular mass of 84.4 kDa and a pI of 6.1. Gene 4 cDNA was inserted into a recombinant baculovirus via homologous recombination. The gene 4 polypeptide migrates at 84 kDa when expressed either by a recombinant baculovirus or in vitro in a rabbit reticulocyte lysate. The gene 4 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent-phase serum, a porcine group B rotavirus infection serum, and a monoclonal antibody made to ADRV virion. Guinea pig hyperimmune serum to the baculovirus-expressed ADRV VP4 protein recognizes virus and immunoprecipitates an 84-kDa protein from in vitro translations of total ADRV mRNA. In addition, the gene 4-encoded protein shares significant amino acid identity and similarity with the group A rotavirus VP4 protein. This information, together with our previous identification of an 84-kDa protein present on iodinated intact virion but not EDTA-treated ADRV, suggests that gene 4 encodes the VP4 protein equivalent present on the outer capsid of ADRV. The ADRV VP4 protein is also 58% identical to the IDIR rat group B rotavirus gene segment 3 protein. The substantial differences between these two group B VP4 proteins suggests that they are distantly related and likely to define two different group B rotavirus VP4 serotypes. The baculovirus-expressed VP4 protein should be useful for developing serotyping reagents and tests for human and animal group B rotaviruses as well as for addressing the role of VP4 in ADRV neutralization. Images PMID:8386274

  10. Repair of uv damaged DNA: Genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  11. Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis.

    PubMed

    Adly, Mohamed A; Assaf, Hanan A; Hussein, Mahmoud Rezk A

    2008-10-01

    Heat shock proteins (HSPs) are molecular chaperones involved in protein folding, assembly and transport, and which play critical roles in the regulation of cell growth, survival and differentiation. We set out to test the hypothesis that HSP27 protein is expressed in the human testes and its expression varies with the state of spermatogenesis. HSP27 expression was examined in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) using immunofluorescent methods. The biopsies were obtained from patients undergoing investigations for infertility. The seminiferous epithelium of the human testes showing normal spermatogenesis had a cell type-specific expression of HSP27. HSP27 expression was strong in the cytoplasm of the Sertoli cells, spermatogonia, and Leydig cells. Alternatively, the expression was moderate in the spermatocytes, weak in the spermatids and absent in the spermatozoa. In testes showing maturation arrest, HSP27 expression was strong in the Sertoli cells, weak in the spermatogonia, and spermatocytes. It was absent in the spermatids and Leydig cells. In Sertoli cell only syndrome, HSP27 expression was strong in the Sertoli cells and absent in the Leydig cells. We report for the first time the expression patterns of HSP27 in the human testes and show differential expression during normal spermatogenesis, indicating a possible role in this process. The altered expression of this protein in testes showing abnormal spermatogenesis may be related to the pathogenesis of male infertility.

  12. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  13. The presynaptic cytomatrix protein Bassoon: sequence and chromosomal localization of the human BSN gene.

    PubMed

    Winter, C; tom Dieck, S; Boeckers, T M; Bockmann, J; Kämpf, U; Sanmartí-Vila, L; Langnaese, K; Altrock, W; Stumm, M; Soyke, A; Wieacker, P; Garner, C C; Gundelfinger, E D

    1999-05-01

    Bassoon is a novel 420-kDa protein recently identified as a component of the cytoskeleton at presynaptic neurotransmitter release sites. Analysis of the rat and mouse sequences revealed a polyglutamine stretch in the C-terminal part of the protein. Since it is known for some proteins that abnormal amplification of such polyglutamine regions can cause late-onset neurodegeneration, we cloned and localized the human BASSOON gene (BSN). Phage clones spanning most of the open reading frame and the 3' untranslated region were isolated from a human genomic library and used for chromosomal localization of BSN to chromosome 3p21 by FISH. The localization was confirmed by PCR on rodent/human somatic cell hybrids; it is consistent with the localization of the murine Bsn gene at chromosome 9F. Sequencing revealed a polyglutamine stretch of only five residues in human, and PCR amplifications from 50 individuals showed no obvious length polymorphism in this region. Analysis of the primary structure of Bassoon and comparison to previous database entries provide evidence for a newly emerging protein family.

  14. In vitro thermodynamic dissection of human copper transfer from chaperone to target protein.

    PubMed

    Niemiec, Moritz S; Weise, Christoph F; Wittung-Stafshede, Pernilla

    2012-01-01

    Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer. PMID:22574136

  15. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity

    PubMed Central

    Bugreev, Dmitry V.; Mazin, Alexander V.

    2004-01-01

    Human Rad51 (hRad51) protein plays a key role in homologous recombination and DNA repair. hRad51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange. hRad51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood. Our current results show that Ca2+ greatly stimulates DNA strand exchange activity of hRad51 protein. We found that Ca2+ exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein. Our data demonstrate that, in the presence of Mg2+, the hRad51-ATP-ssDNA filament is quickly converted to an inactive hRad51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP. Ca2+ maintains the active hRad51-ATP-ssDNA filament by reducing the ATP hydrolysis rate. These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRad51 protein. This mechanism of Rad51 protein regulation by modulating its ATPase activity is evolutionarily recent; we found no such mechanism for yeast Rad51 (yRad51) protein. PMID:15226506

  16. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.

    PubMed

    Loo, T W; Clarke, D M

    1997-01-10

    There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

  17. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    SciTech Connect

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A.; Lees-Miller, S.P.; Lintott, L.G.; Sakaguchi, Kazuyasu; Appella, E.

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequen