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E Appella

Publications and source records attributed to E Appella.

At least 199 records · Page 11Linked to original sources
PubMed1990

High-resolution three-dimensional structure of a single zinc finger from a human enhancer binding protein in solution.

The three-dimensional structure of a 30-residue synthetic peptide containing the carboxy-terminal "zinc finger" motif of a human enhancer binding protein has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure determination is based on 487 approximate interproton distance and 63 torsion angle (phi, psi, and chi 1) restraints. A total of 40 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions (excluding residues 29 and 30 which are ill-defined) is 0.4 A for the backbone atoms, 0.8 A for all atoms, and 0.41 A for all atoms excluding the lysine and arginine side chains, which are disordered. The solution structure of the zinc finger consists of two irregular antiparallel beta-strands connected by an atypical turn (residues 3-12) and a classical alpha-helix (residues 14-24). The zinc is tetrahedrally coordinated to the sulfur atoms of two cysteines (Cys-5 and Cys-8) and to the N epsilon 2 atoms of two histidines (His-21 and His-27). The two cysteine residues are located in the turn connecting the two beta-strands (residues 5-8); one of the histidine ligands (His-21) is in the alpha-helix, while the second histidine (His-27) is at the end of a looplike structure (formed by the end of the alpha-helix and a turn). The general architecture is qualitatively similar to two previously determined low-resolution Cys2-His2 zinc finger structures, although distinct differences can be observed in the beta-strands and turn and in the region around the two histidines coordinated to zinc. Comparison of the overall polypeptide fold of the enhancer binding protein zinc finger with known structures in the crystallographic data base reveals a striking similarity to one region (residues 23-44) of the X-ray structure of proteinase inhibitor domain III of Japanese quail ovomucoid [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I., & Laskowski, M. (1982) J. Mol. Biol. 158, 515-537], which could be superimposed with a backbone atomic rms difference of 0.95 A on residues 3-25 (excluding residue 6) of the zinc finger from the enhancer binding protein. The presence of structural homology between two proteins of very different function may indicate that the so-called zinc finger motif is not unique for a class of DNA binding proteins but may represent a general folding motif found in a variety of proteins irrespective of their function.

Amino Acid Sequence↗
PubMed1990

Characterization of the specificity of peptide binding to four DR haplotypes.

This study describes the establishment of a peptide-binding assay for purified, detergent-solubilized DR molecules. For each of the DR specificities and peptides studied, a unique pattern of interaction was observed. Excellent correlation was detected between the DR1-, 2-, 5-, and 52a-binding capacities and the known DR restrictions of a panel of synthetic peptides. This supports the immunologic relevance of the binding assay, and emphasizes the importance of determinant selection in defining the immune response of individuals. We have also examined the capacity of a panel of DR-restricted peptides to compete with one another for binding to DR1. The results obtained are compatible with a single peptide-binding site on DR molecules. The peptide-binding capacity of the four different DR types (DR1, DR2, DR5, and DR52a) has been further examined by testing a collection of 133 different peptides. This collection is unbiased with respect to previously known DR binding and restrictions, and includes peptides of eukaryotic, bacterial, and viral origins. It was found that: 1) approximately 15 to 35% of the peptides tested bound any given DR type; 2) DR-binding capacities appeared to correlate with each other, suggesting that different alleles of the DR isotype may recognize related structures on an Ag molecule; and 3) despite the statistical correlation between binding capacity of different DR types, approximately 50% of the peptides that were positive binders still were specific in that they could bind only one of the four DR molecules tested. Degenerate binding (i.e., binding to most or all the DR molecules tested) was detected in only a minority of the cases analyzed (approximately 25%).

Amino Acid Sequence↗
PubMed1990

Inhibition by brefeldin A of presentation of exogenous protein antigens to MHC class II-restricted T cells.

Peptides bound to class I or class II major histocompatibility complex (MHC)-encoded molecules are ligands for the antigen-specific T-cell receptor of T-cells carrying the CD8 and CD4 antigens, respectively. MHC class I-restricted T cells generally recognize peptides derived from processing of endogenously synthesized cellular antigens, whereas class II-restricted T cells usually recognize peptides derived from exogenous antigens entering antigen presenting cells. Accordingly, two separate pathways of antigen processing and presentation have been proposed. The fungal metabolite brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum, inhibits presentation of endogenous antigens for MHC-restricted T-cell recognition. The selectivity of BFA activity has been inferred to reflect presentation of a given antigen processed through the cytosolic or the endocytic route. Here we show that BFA also greatly inhibits the presentation of exogenous protein antigens by MHC class II molecules to T cells, indicating a broader effect of this drug on antigen presentation and an additional similarity between the two processing pathways. As cycloheximide, a protein synthesis inhibitor, also inhibits presentation of protein antigens to class II-restricted T cells, the data indicate that peptides generated by processing of exogenous proteins binds to newly synthesized class II molecules for presentation to T cells.

Animals↗
PubMed1990

Properties of monocyte chemotactic and activating factor (MCAF) purified from a human fibrosarcoma cell line.

A monocyte chemotactic and activating factor (MCAF) has been purified from TNF-stimulated 8387 human fibrosarcoma cell line-conditioned media. The purified MCAF showed microheterogeneity yielding two bands on SDS-PAGE analysis. Fibrosarcoma-derived MCAF specifically competed with THP-1 (a human monocytic cell line)-derived 125I-labeled MCAF in binding to human PBMC, whereas a similar basic heparin-binding leukocyte chemoattractant, IL-8, did not. The purified MCAF stimulated superoxide anion and N-acetyl beta-D glucosaminidase-releasing activity in human monocytes, as well as monocyte cytostatic augmenting activity against tumor cells and chemotactic activity for monocytes. When injected subcutaneously into Lewis rat ears, the purified human MCAF also induced considerable in vivo local monocyte infiltration beginning at 3 h and becoming maximal at 18 h. In conclusion, the data presented in this paper indicate that MCAF is a potent activator of monocytes as well as a monocyte recruitment factor that acts through receptors that are specific for this novel molecule. This novel cytokine might have an important role in tumor growth control due to its ability to attract and activate monocytes.

Acetylglucosaminidase↗
PubMed1990

The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants.

The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported. The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein to that predicted for the u-PA receptor in the same cross-linking studies; 4) the identical extent of glycosylation of the purified protein and of the u-PA receptor in crude membrane fractions, as detected after cross-linking; 5) the ability of antibodies raised against the purified protein to inhibit cellular binding of the amino-terminal fragment of u-PA.

Amino Acid Sequence↗
PubMed1990

Affinity purification, peptide analysis, and cDNA sequence of the mouse interferon gamma receptor.

The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.

Amino Acid Sequence↗
PubMed1990

Three-dimensional structure of interleukin 8 in solution.

The solution structure of the interleukin 8 (IL-8) dimer has been solved by nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure determination is based on a total of 1880 experimental distance restraints (of which 82 are intersubunit) and 362 torsion angle restraints (comprising phi, psi, and chi 1 torsion angles). A total of 30 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions (excluding residues 1-5 of each subunit) is 0.41 +/- 0.08 A for the backbone atoms and 0.90 +/- 0.08 A for all atoms. The three-dimensional solution structure of the IL-8 dimer reveals a structural motif in which two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by about 14 A, lie on top of a six-stranded antiparallel beta-sheet platform derived from two three-stranded Greek keys, one from each monomer unit. The general architecture is similar to that of the alpha 1/alpha 2 domains of the human class I histocompatibility antigen HLA-A2. It is suggested that the two alpha-helices form the binding site for the cellular receptor and that the specificity of IL-8, as well as that of a number of related proteins involved in cell-specific chemotaxis, mediation of cell growth, and the inflammatory response, is achieved by the distinct distribution of charged and polar residues at the surface of the helices.

Amino Acid Sequence↗
PubMed1990

Identification and immunogenic properties of an 80-kDa surface antigen on a drug-treated tumor variant: relationship to MuLV gp70.

Highly immunogenic tumor variants are generated by in vitro or in vivo treatment of L5178Y murine lymphoma cells with triazene derivatives. Most of these variants express new transplantation antigens which are not present on the original L5178Y tumor cells. In this study, a polyclonal syngeneic antiserum raised to one such variant (L5178Y/DTIC) was employed in immunoprecipitation studies of cell surface and metabolically labeled L5178Y/DTIC cells. One- and two-dimensional electrophoretic analyses of the immunoprecipitates detected a surface antigen of approximately 80 kDa. Additionally, a 45-kDa component was detected in the lysate of [35S]methionine-labeled cells. Anti-xenotropic MuLV gp70 serum precipitated material whose electrophoretic pattern was similar to that of the 80-kDa surface antigen. Sequential immunoprecipitation analysis revealed that the molecules reactive with the variant-specific antiserum were removed by the anti-xenotropic gp70 antibodies, whereas immunodepletion was only partial when the cell extract was first treated with the variant-specific antibodies. After Western blotting, the 80- and 45-kDa antigens precipitated by the variant-specific antibodies were injected intrasplenically into recipient mice. Only the animals sensitized with the 80-kDa antigen developed specific immunity to L5178Y/DTIC cells in that they displayed an increased frequency in CTL precursors (CTLp) to the variant cells. Sera from mice sensitized to the 80-kDa protein specifically inhibited the development of a primary CTL response to L5178Y/DTIC cells.

Animals↗
PubMed1990

Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis.

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.

Amino Acid Sequence↗
PubMed1990

Structural requirements for the interaction between class II MHC molecules and peptide antigens.

Previous work from our and other laboratories indicates that T cells recognize a complex between the MHC restriction element and peptide antigen fragments. This paper reviews the structural characteristics of the formation of such a complex. By analyzing in detail the interactions between purified IA(d) and IE(d) molecules and their peptide ligands, we found that some structural characteristics apply to both antigen-MHC interactions. In particular, we found: 1) each MHC molecule is capable of binding many unrelated peptides through the same peptide-binding site; 2) despite this permissiveness of binding, it is possible to define certain structural features of peptides that are associated with the capacity to bind to a particular MHC specificity (IA(d) or IE(d)); 3) IA(d) and IE(d) molecules recognize different and independent structures on the antigen molecule; 4) only about 10% of the single amino acid substitutions tested on two IA(d)- and IE(d)-binding peptides had significant effect on their MHC-binding capacities, while over 80% of these substitutions significantly impaired T cell recognition of the Ia-peptide complex; 5) based on the segregation between residues that are crucial for T cell activation and Ia binding, the easiest model for the antigen-Ia-T-cell-receptor complex pictures the antigen molecule sandwiched in a planar conformation between the MHC and the T cell.

Amino Acid Sequence↗
PubMed1990

An interferon gamma-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes.

Interferons (IFNs) induce transcription of major histocompatibility complex (MHC) class I genes through the conserved IFN consensus sequence (ICS) that contains an IFN response motif shared by many IFN-regulated genes. By screening mouse lambda ZAP expression libraries with the ICS as a probe, we isolated a cDNA clone encoding a protein that binds the ICS, designated ICSBP. Protein blot analysis with labeled oligonucleotide probes showed that ICSBP binds not only the MHC class I ICS but also IFN response motifs of many IFN-regulated genes, as well as a virus-inducible element of the IFN-beta gene. The ICSBP cDNA encodes 424 amino acids and a long 3' untranslated sequence. The N-terminal 115 amino acids correspond to a putative DNA-binding domain and show significant sequence similarity with other cloned IFN response factors (IRF-1 and IRF-2). Because of the structural similarity and shared binding specificity, we conclude that ICSBP is a third member of the IRF gene family, presumably playing a role in IFN- and virus-mediated regulation of many genes. Although IRF-1 and IRF-2 share some similarity in their C-terminal regions, ICSBP shows no similarity to IRF-1 or IRF-2 in this region, suggesting that it is more distantly related. We show that ICSBP mRNA is expressed predominantly in lymphoid tissues and is inducible preferentially by IFN-gamma. The induction by IFN-gamma appears to be predominant in lymphocytes and macrophages, implying that ICSBP plays a regulatory role in cells of the immune system. The presence of multiple factors that bind common IFN response motifs may partly account for the complexity and diversity of IFN action as well as IFN-regulated gene expression.

Amino Acid Sequence↗
PubMed1990

Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53.

To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation we constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastoma multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. We show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits G0/G1 progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the G0/G1 phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.

Blotting, Northern↗
PubMed1990

Wild type human p53 is antiproliferative in SV40-transformed hamster cells.

The transformation related protein p53 has been implicated in the process of normal cell proliferation and neoplastic transformation. In this study, the influence of wild type human p53 on cell proliferation was examined. Plasmid constructs encoding the wild type human p53 and various mutant p53 cDNAs, driven by the mouse mammary tumor virus (MMTV) promoter linked to the dominant biochemical selection marker gpt, were used in a colony forming assay employing SV40 transformed HR8 hamster cells. Plasmids encoding wild type p53 drastically reduced the number of gpt+ colonies obtained after transfection, whereas the mutant forms of p53 had no effect. Stable clonal hamster cell lines that constitutively express wild type p53 were isolated and found to have altered growth characteristics (i.e. lower saturation densities, increased doubling times). These findings are consistent with the notion that wild type p53 protein could function as a growth suppressor. The potential role of p53 in the normal cell cycle and in the transformation process is discussed.

Blotting, Northern↗
PubMed1989

Competition for antigen presentation in living cells involves exchange of peptides bound by class II MHC molecules.

T cells recognize foreign proteins as peptides bound to self molecules encoded by the major histocompatibility complex (MHC). The kinetics of interaction between purified class II MHC molecules and peptides is unusual, in that the rate of association is very slow, but once formed, the complexes are extremely stable. This raises the question of how the antigen-presenting cell provides a sufficient number of free MHC binding sites to ensure T cell immunity. We present results suggesting that an exchange of peptide in MHC binding sites may take place under physiological conditions.

Ammonium Chloride↗
PubMed1989

Characterization of a naturally processed MHC class II-restricted T-cell determinant of hen egg lysozyme.

Compelling evidence indicates that T cells recognize complexes formed by major histocompatibility complex-encoded molecules and antigenic peptide fragments. This is based largely on the ability of small synthetic peptides to substitute for naturally processed antigen in stimulating T cells. Naturally processed fragments of exogenous antigen are thought to arise by limited proteolytic degradation of native antigen inside acidic compartments of antigen-presenting cells, but until now no physiologically processed antigen has been directly analysed. Here we report the characterization of physiologically processed antigen eluted from mouse class II major histocompatibility complex I-Ed molecules. The antigenic material corresponds to a previously described antigenic determinant of hen egg lysozyme (HEL 107-116) and has a relative molecular mass Mr of about 2,000. HPLC analysis identified at least two or three separate molecular species, suggesting limited, albeit significant, heterogeneity of naturally processed peptides. Finally, under our experimental conditions, it was calculated that a substantial proportion (10-40%) of I-Ed molecules were occupied by these HEL-derived antigenic determinants.

Animals↗
PubMed1989

Determination of the secondary structure of interleukin-8 by nuclear magnetic resonance spectroscopy.

The solution conformation of interleukin-8 (IL-8), a small protein of 72 residues with a wide range of proinflammatory activities, has been investigated by two-dimensional NMR spectroscopy. The 1H-NMR spectrum of IL-8 is assigned in a sequential manner and regular elements of secondary structure are identified on the basis of a qualitative interpretation of the nuclear Overhauser, coupling constant and amide exchange data. The IL-8 monomer contains a triple stranded anti-parallel beta-sheet arranged in a Greek key and a long C-terminal helix (residues 57-72). It is shown that IL-8 is a dimer in solution in which the interface is principally formed by six backbone hydrogen bonds between residues 25, 27, and 29 of one monomer and residues 29, 27, and 25, respectively, of the other. As a result, the two units of the dimer form a contiguous six-stranded anti-parallel beta-sheet. The secondary structure of IL-8 is similar to that found in the crystal structure of the sequence related protein platelet factor 4.

Amino Acid Sequence↗