Biomedical subjects

E Appella

Publications and source records attributed to E Appella.

At least 145 records · Page 8Linked to original sources

PubMed1994

Negative selection of CD4+ CD8+ thymocytes by T-cell receptor peptide antagonists.

Antigen-induced activation of T cells can be specifically inhibited by antigen analogs that have been termed T-cell receptor peptide antagonists. These antagonists appear to act by inducing the formation of nonstimulatory or partially stimulatory complexes between T-cell receptors and the major histocompatibility complex molecules presenting the peptides. Herein, we have investigated the effect of T-cell receptor peptide antagonists on thymocyte negative selection. First, peptide antagonists were identified for the cytochrome c-specific T-cell clone AD10. These peptides were then tested for their ability to induce negative selection in an in vitro model system using thymocytes from mice transgenic for the AD10 T-cell receptor. Though unable to induce mature T-cell activation, the T-cell receptor peptide antagonists induced deletion of CD4+ CD8+ thymocytes. These results suggest that negative selection of CD4+ CD8+ thymocytes can be induced by T-cell receptor interactions of a lower affinity than those required for mature T-cell activation.

Animals↗

PubMed1994

Definition of specific peptide motifs for four major HLA-A alleles.

Allele-specific motifs for the human MHC class I molecules, HLA-A1, A3, A11, and A24 were characterized by three complementary approaches. First, amino acid sequence analysis of acid eluted peptide pools from affinity purified class I molecules defined putative motifs 9 or 10 amino acids in length and bearing critical anchor residues at position 2 and at the COOH-terminal. These motifs were distinct, with the exception of the HLA-A3 and A11 motifs that were very similar to each other. Second, the correctness of these putative motifs was verified by analyzing the binding capacity of polyalanine peptide analogues to purified HLA-A molecules. Several alternative anchor residues that were not obvious from the pooled peptide sequencing analysis were identified. Third, sequences of individual peptides eluted from HLA-A1, A11, and A24 were determined by tandem mass spectrometry. Nonamers were the predominant species, although peptides of 8, 10, 11, and 12 amino acids in length were also identified. These peptides displayed anchor residues predicted by the specific motifs at position 2 and at the COOH-terminal, regardless of peptide length. Synthetic versions of the naturally processed peptides were shown to bind to the appropriate HLA-A alleles with IC50 values in the 0.3- to 200-nM range. A rational approach to search Ags with known amino acid sequences for epitopes restricted by some of the most common HLA-A types and of potential clinical importance is now feasible.

Alleles↗

PubMed1994

Naturally processed peptides longer than nine amino acid residues bind to the class I MHC molecule HLA-A2.1 with high affinity and in different conformations.

An equilibrium binding assay was used to directly measure the relative affinities of naturally processed 9-mer, 10-mer, and 12-mer peptides for the human class I MHC molecule HLA-A2.1. The peptides exhibited a range of affinities with IC50 values of 11 to 214 nM. The mode of interaction between these peptides and HLA-A2.1 was examined using peptides in which Asp had been substituted for suspected anchor residues. Regardless of length, the previously identified Leu at position 2 relative to the amino terminus was critical for peptide binding. While the carboxyl terminal residue was also critical for the binding of a 9-mer peptide, it was much less important in the binding of longer peptides. Additional residues close to the carboxyl terminus that contained aliphatic hydrocarbon side chains were of similar or greater importance in peptide binding. In addition, residue at position 3 also appeared to be important for the binding of longer peptides. The data suggest that different naturally occurring longer peptides can bind in different conformations to class I MHC molecules. While one of these is similar to the kinked conformation described by others, another conformation would involve an extension of the carboxyl terminus out of the class I binding site. The ability of MHC molecules to accommodate the same peptide in different conformations would appear to have distinct advantages to the immune system.

Amino Acid Sequence↗

PubMed1994

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component.

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.

Amino Acid Sequence↗

PubMed1994

Peripheral T cell response to A-gliadin in celiac disease: differential processing and presentation capacities of Epstein-Barr-transformed B cells and fibroblasts.

Celiac disease (CD) is a small intestinal disorder characterized by the malabsorption of most nutrients. Disease pathogenesis appears to be associated with immune-mediated pathology. Susceptibility is associated with genes coding for DQw2 class II molecules. In the present report we investigated T cell responses to A-gliadin (AGL), a major alpha-gliadin component known to activate disease. Gliadin-specific lines were generated from a CD patient and a normal donor. Three major points were revealed by the analysis of these T cells: (1) On the basis of mapping experiments using Epstein-Barr virus (EBV) lines and DR-transfected fibroblasts and DR-, DP-, and DQ-specific monoclonal antibodies (mAb), all responses appeared to be DR-restricted. Thus, in contrast to the strong association of disease susceptibility with DQ molecules, no DQ-restricted, gliadin-specific response was detectable. (2) Fine specificity analysis, using a panel of synthetic peptides spanning the entire alpha-gliadin component molecule, revealed that the clones derived from the normal donor were DR53-restricted and AGL 21-40-specific, while clones derived from the CD patient were DR7-restricted and peptide 1-20-specific. (3) Both whole AGL and AGL 1-20 were presented to the patient-derived clones with much higher efficiency by DDR-transfected fibroblasts than by EBV lines. These data suggested that fibroblasts processed this determinant efficiently, while EBV lines were unable to do so. Indeed, analysis of a panel of truncated AGL 1-20 analogs revealed that peptide AGL 1-8, which contained the minimal T cell epitope, was presented with equal efficiency by fixed or irradiated EBV and irradiated DR7-transfected fibroblasts.

Adult↗

PubMed1994

Structure of three-way DNA junctions. 1. Non-planar DNA geometry.

Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20-21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60-90 degrees. Rotation of the hairpin position in the three-way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60-90 degrees between the arms. Computer modeling of the three-way junction with 60 degrees pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.

Base Sequence↗

PubMed1994

Structure of three-way DNA junctions. 2. Effects of extra bases and mismatches.

The structure of three-way DNA junctions, containing two linear double helices (arms) and a hairpin as a third arm, was studied by means of a cyclization technique. In addition to branched molecules containing perfect base-pairing in helical parts, three-way junctions with mismatches and extra non-complementary nucleotides (bulges) at junction points were studied. Molecules thus designed were ligated at identical conditions and their geometry was compared through the analysis of the efficiency of circle formation. The analysis showed that irregularities in base pairing listed above dramatically change the static and dynamic structural characteristics of the three-way junctions. All mismatches facilitate the kink between linear arms, but quantitatively, the effect depends on the position of the mismatch. The effect is maximal for GG-mismatch placed at the hairpin junction point. The results for bulges are of different kind, and they lead us to conclude that the three-way DNA junction with unpaired nucleotides adopts a T-like geometry with an angle around 90 degrees between arms containing the bulge and two other arms coaxially stacked. Broad distribution of circles indicates that this T-form geometry of bulge-containing junction is more flexible than initial pyramidal structure predominantly due to high mobility of the third arm.

Base Composition↗

PubMed1994

The carboxy-terminal serine 392 phosphorylation site of human p53 is not required for wild-type activities.

Wild-type p53 functions in the G1 DNA damage checkpoint pathway by activating gene transcription and preventing cell cycle progression. Others reported that mutation of the serine 386 codon in mouse p53 abolished its ability to suppress growth. Serine 386 of murine p53 and the homologous residue of human p53, serine 392, are phosphorylated in vivo and can be phosphorylated in vitro by casein kinase II (CKII). We constructed mutants that changed serine 392 of human p53 to alanine (p53-S392A) or aspartic acid (p53-S392D); cotransfection of both these mutants with a reporter gene carrying a p53-responsive element into the p53-null Saos-2 cell line activated transcription as well as did wild-type p53. Furthermore, both mutants blocked cell cycle progression after transient transfection in these cells. A stable derivative of the T98G human glioblastoma cell line was established that expressed p53-S392A in response to dexamethasone. Overexpression of this mutant activated transcription of the endogenous waf1 (also called cip1) and mdm2 genes to the same extent as wild-type p53 and also produced growth arrest. Finally, p53-S392A and p53-S392D suppressed foci formation by activated ras and adenovirus E1A oncogenes as efficiently as did wild-type p53. Thus, unlike mutants that altered the serine 15 phosphorylation site, elimination of the serine 392 phosphorylation site had no discernible effect on p53 function. We conclude that neither phosphorylation nor RNA attachment to serine 392 are required for human p53's ability to suppress cell growth or to activate transcription in vivo.

Adenovirus E1A Proteins↗

PubMed1993

Direct identification of an endogenous peptide recognized by multiple HLA-A2.1-specific cytotoxic T cells.

An endogenous peptide recognized by a murine T-cell clone specific for the human class I major histocompatibility complex-encoded molecule HLA-A2.1 was identified through the use of microcapillary high-performance liquid chromatography coupled with electrospray-ionization tandem mass spectrometry. The peptide was associated with HLA-A2.1 on both normal cells and the antigen-processing-mutant cell line T2. This observation demonstrates that a processing mechanism other than that involving the transporter associated with antigen processing (TAP) proteins 1 and 2 can produce peptides that can be recognized by T cells. The peptide was also recognized by four other independently derived murine HLA-A2.1-specific murine T-cell clones. This suggests that xenogeneic responses are directed at a restricted subset of major histocompatibility complex product-associated peptides. Finally, quantitation of this peptide in cell extracts using mass spectrometry showed it to be among the most dominant HLA-A2.1 associated species on human lymphoid cells. The potential relevance of this observation to models of alloreactivity will be discussed. The methodology described should be generally useful for the identification of peptide epitopes recognized by alloreactive, tumor-specific, and autoimmune T cells.

Amino Acid Sequence↗

PubMed1993

Structure of hydrated oligonucleotides studied by in situ scanning tunneling microscopy.

We have used the scanning tunneling microscope (STM) to image several synthetic oligonucleotides adsorbed onto a positively charged Au(111) electrode. The molecules were deposited and imaged in aqueous electrolyte under potential control, a procedure that eliminated the problem of the substrate artifacts that had limited some previous STM studies. Experiments were carried out with two types of single-stranded molecules (11 and 20 bases long) and three types of double-stranded molecules (20 and 61 base pairs and 31 bases with 25 bases paired and 6-base "sticky" ends). The molecules lie along symmetry directions on the reconstructed (23 x square root of 3) gold surface, and length measurements indicate that they adopt simple base-stacked structures. The base stacking distances are, within experimental uncertainty, equal to the 0.33 nm measured for polymeric aggregates of stacked purines by direct imaging in identical conditions. The images show features consistent with helical structures. Double helices have a major-groove periodicity that is consistent with a 36 degrees twist. The single helices appear to be more tightly twisted. A simple tunneling model of STM contrast generates good agreement between measured and calculated images.

Base Sequence↗

PubMed1993

Characteristics of endogenous peptides eluted from the class I MHC molecule HLA-B7 determined by mass spectrometry and computer modeling.

Microcapillary HPLC electrospray ionization tandem mass spectrometry was used to sequence 15 peptides eluted from HLA-B7. Sequence alignment implicated four peptide positions in specific interactions with the class I molecule, and their importance was confirmed using synthetic peptides. Because no crystal structure for HLA-B7 was available, computer-assisted modeling was used to understand novel aspects of peptide binding specificity and to accurately predict the effect of defined changes in peptide structure. The results demonstrate that mass-spectrometric sequencing coupled with computer-assisted modeling can be used in the absence of a crystal structure to make accurate predictions concerning requirements for peptide binding to class I molecules. These techniques may be valuable to predict or engineer T cell epitopes.

Amino Acid Sequence↗

PubMed1993

NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1.

The three-dimensional solution structure of a complex between the DNA binding domain of the chicken erythroid transcription factor GATA-1 and its cognate DNA site has been determined with multidimensional heteronuclear magnetic resonance spectroscopy. The DNA binding domain consists of a core which contains a zinc coordinated by four cysteines and a carboxyl-terminal tail. The core is composed of two irregular antiparallel beta sheets and an alpha helix, followed by a long loop that leads into the carboxyl-terminal tail. The amino-terminal part of the core, including the helix, is similar in structure, although not in sequence, to the amino-terminal zinc module of the glucocorticoid receptor DNA binding domain. In the other regions, the structures of these two DNA binding domains are entirely different. The DNA target site in contact with the protein spans eight base pairs. The helix and the loop connecting the two antiparallel beta sheets interact with the major groove of the DNA. The carboxyl-terminal tail, which is an essential determinant of specific binding, wraps around into the minor groove. The complex resembles a hand holding a rope with the palm and fingers representing the protein core and the thumb, the carboxyl-terminal tail. The specific interactions between GATA-1 and DNA in the major groove are mainly hydrophobic in nature, which accounts for the preponderance of thymines in the target site. A large number of interactions are observed with the phosphate backbone.

Amino Acid Sequence↗

PubMed1993

Two-dimensional nuclear magnetic resonance analysis of a labeled peptide bound to a class II major histocompatibility complex molecule.

The formation of peptide/major histocompatibility complex (MHC) complexes and their subsequent recognition by T cells is a pivotal event in the initiation of an immune response. While X-ray crystal structures are now available for class I MHC/peptide complexes, little detailed structural information is known about the class II MHC equivalent, and there are no solution structure data for either. A 16 amino acid residue moth cytochrome c peptide (residues 88 to 103) was 13C-labeled for two-dimensional isotope-edited NMR analysis. The peptide was labeled either selectively in the methyl groups of alanine residues or uniformly at every carbon position, and bound to unlabeled soluble mouse I-Ek class II MHC molecules. Although alpha-helical in the native cytochrome c protein and with no uniform structure in solution, the peptide is bound to the I-Ek molecule with the alpha-carbon atoms of the 11 C-terminal residues held in the binding groove. This indicates that the class II MHC peptide binding site is somewhat larger than that of class I MHC molecules (> or = 11 amino acid residues versus 8 to 10 amino acid residues), consistent with recent data on eluted peptides. Despite the large size of the complex (approximately 70 kDa), nuclear Overhauser effects are clearly detectable between peptide side-chains and the MHC molecule. Indications of the buried or exposed nature of particular side-chains within the bound peptide are derived from the NMR data and these are used together with information from previous biological studies to propose a crude model of the interaction of the peptide with the groove of the MHC molecule. We find no evidence for a conformational change in the peptide/MHC complex in the spectra at pH 5.0 versus pH 7.0, despite a 40-fold faster on-rate for the peptide at the lower pH value.

Amino Acid Sequence↗

PubMed1993

Phosphorylation at Ser-15 and Ser-392 in mutant p53 molecules from human tumors is altered compared to wild-type p53.

The product of the p53 gene suppresses cell growth and plays a critical role in suppressing development of human tumors. p53 protein binds DNA, activates transcription, and can be phosphorylated at N- and C-terminal sites. Previously, wild-type p53 was shown to be hyperphosphorylated compared to mutant p53 during p53-mediated growth arrest in vivo. Here we show that Ser-15 and Ser-9 in the N-terminal transactivation domain of wild-type human p53 are phosphorylated in vivo in cells derived from the human glioblastoma line T98G. In [Ile237]p53 and [Ala143]p53, two natural p53 mutants from human tumors that are defective for activation of transcription, phosphorylation at Ser-15 was reduced and phosphorylation at Ser-392 was increased compared to wild-type p53. No change was observed at Ser-9. [His273]p53, a third mutant, had a phosphorylation state similar to that of wild-type p53. We suggest that phosphorylation of Ser-15 may depend on the ability of p53 to adopt a wild-type conformation and may contribute to p53's ability to block cell growth.

Amino Acid Sequence↗

PubMed1993

Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein.

The nucleocapsid (NC) protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is important for encapsidation of the virus genome, RNA dimerization, and primer tRNA annealing in vitro. Here we present evidence from gel mobility-shift experiments indicating that NCp7 binds specifically to an RNA sequence. Two complexes were identified in native gels. The more slowly migrating complex contained two RNA molecules and one peptide, while the more rapidly migrating one is composed of one RNA and one peptide. Further, mutational analysis of the RNA shows that the predicted stem and loop structure of stem-loop 1 plays a critical role. Our results show that NCp7 binds to a unique RNA structure within the psi region; in addition, this structure is necessary for RNA dimerization. We propose that NCp7 binds to the RNA via a direct interaction of one zinc-binding motif to stem-loop 1 followed by binding of the other zinc-binding motif to stem-loop 1, stem-loop 2, or the linker region of the second RNA molecule, forming a bridge between the two RNAs.

Base Sequence↗

PubMed1993

Sequence of a cDNA encoding bovine apolipoprotein H.

The nucleotide sequence of the ApoH cDNA encoding the bovine apolipoprotein H (ApoH) has been determined. The deduced protein, which contains a 19-amino-acid (aa) signal peptide and the 326-aa mature ApoH, shows 89% and 86% homology with human and rat ApoH, respectively.

Amino Acid Sequence↗

PubMed1993

CA runs increase DNA flexibility in the complex of lambda Cro protein with the OR3 site.

The alternating pyrimidine-purine elements CA, CAC, and CACA are anisotropically flexible, as deduced from gel circularization assays on point mutations and single-base mismatches in the OR3 site of lambda phage alone and in the specific complex with the Cro protein. These sequences evidently promote DNA bending in the specific binding region of the complex and may also facilitate overwinding in the central nonbinding region. Effects for CACA are exceptionally large and suggest that an alternative DNA structure may occur in this element.

Adenine↗

PubMed1993

The high-resolution structure of the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The three-dimensional structure of a 43-residue active, synthetic peptide encompassing the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of a multi-cooling dynamical simulated annealing protocol using restraints derived from 1H nuclear magnetic resonance spectroscopy. A total of 442 experimentally derived restraints including 13 dihedral angle (phi, chi 1) restraints were used. A final set of 35 structures was calculated with a root-mean-square deviation from the mean co-ordinates of 0.36 A for the backbone atoms and 0.96 A when side-chain heavy atoms were included for the well-defined region comprising residues Val7 to Leu39. Although assignments were made and sequential connectivities observed for the N-terminal six and C-terminal four residues, the absence of long-range NOEs suggests that the terminal regions are largely unstructured. The binding domain contains two short parallel alpha-helices (residues Val7 to Lys14 and Lys32 to Leu39), a3(10)-helix (residues Asp17 to Val21) and a structured loop made up of overlapping beta-turns (residues Gln22 to Leu31), which enclose a close-packed hydrophobic core. The loop is stabilized to a large extent by Asp34. This residue is conserved in all peripheral subunit-binding domains and its carboxylate side-chain forms a set of side-chain-main-chain hydrogen bonds with the main-chain amide protons of Gly23, Thr24, Gly25 and Leu31 and a side-chain-side-chain hydrogen bond with the hydroxyl group of Thr24. We propose that a peripheral subunit-binding site may be located in the loop region, which contains a series of highly conserved residues and provides a number of potential recognition sites. The structured region of the binding domain, comprising 33 residues, represents an exceptionally short amino acid sequence with defined tertiary structure that has no disulphide bond, ligand or cofactor to stabilize the fold. It may be approaching the lower size limit for a three-dimensional structure possessing features characteristic of larger structures, including a close-packed, non-polar interior. The organization of the side-chains in the hydrophobic core may have implications for de novo protein design.

Acetyltransferases↗