Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, is responsible for the removal of a wide variety of alkylated base lesions in DNA, e.g., N-alkylpurines and cyclic ethenoadducts of adenine, guanine, and cytosine. These lesions, some of which are mutagenic and toxic, are generated endogenously or by genotoxic agents such as N-alkylnitrosamines and vinyl chloride. Wild-type mouse MPG, expressed from recombinant baculovirus, was purified to near homogeneity for studying its specific interaction with substrate, 1,N6-ethenoadenine- (epsilonA-) containing DNA. Electrophoretic mobility shift assays (EMSA) indicated that MPG formed a specific complex with a 50-mer epsilonA-containing duplex oligonucleotide. This complex was shown to be a transient reaction intermediate, because it could be formed only with the unreacted substrate and contained active enzyme molecules. DNA footprinting studies confirmed the specific binding of the protein to the epsilonA-containing duplex oligonucleotide; eight nucleotides on the epsilonA-containing strand and 16-17 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion. A systematic deletion analysis of MPG was carried out in order to determine the minimally sized polypeptide capable of forming a stable substrate complex that is also suitable for characterization by NMR spectroscopy and X-ray crystallography. A truncated polypeptide (NDelta100CDelta18) lacking 100 and 18 amino acid residues from the amino and carboxyl termini, respectively, was found to be the minimal size that retained activity. The truncated and wild-type enzymes have similar kinetic properties. Moreover, both EMSA and DNase I footprinting studies indicated identical pattern of specific binding by the truncated and full-length polypeptides. Removal of five and nine additional residues from the amino- and carboxyl-termini of this polypeptide, respectively, resulted in a complete loss of activity. These results suggest that minimal structural change occured as a result of truncation in the NDelta100CDelta18 mutant, which may thus be suitable for elucidating the structure and mechanism of MPG.
 ...
PMID:Specific interaction of wild-type and truncated mouse N-methylpurine-DNA glycosylase with ethenoadenine-containing DNA. 942 80

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.
 ...
PMID:B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis. 945 32

The chaperonin-containing TCP-1 (CCT) assists in the folding of actins and tubulins in eukaryotic cells. CCT is composed of 8 subunit species encoded by separate genes. CCT purifies as a single hetero-oligomeric protein complex of 950 kDa through multiple chromatographic and antibody affinity procedures. The CCT 16-mer contains 7 polypeptide species in equimolar amounts (CCTalpha, beta, gamma, delta, epsilon, zeta, eta), together with another subunit (CCTtheta) which is around half-molar. Here we show, by in vitro translation of CCT subunit mRNAs in rabbit reticulocyte lysate, that none of the CCT subunit proteins are themselves folded by CCT. However, the newly translated CCT subunits can incorporate into the endogenous CCT complex present in the lysate via a mechanism involving a nucleotide-dependent disassembly reaction to produce single-rings and then a reassembly reaction whereby free CCT subunits assemble onto these single-rings. This cycling behaviour is an inherent property of the CCT chaperonin complex and provides a powerful method for introducing single amino acid residue changes into this 8578 residue protein complex.
 ...
PMID:The chaperonin containing TCP-1 (CCT) displays a single-ring mediated disassembly and reassembly cycle. 956 27

Murine peritoneal macrophages bind to Salmonella typhimurium in vitro in the absence of exogenous opsonins. We have identified an outer membrane protein of S. typhimurium that mediates this adhesion. Biotin-labeled macrophages were used to probe electroblotted envelope proteins of S. typhimurium that had been previously resolved by polyacrylamide electrophoresis under denaturing and reducing conditions. Macrophages bound to an outer membrane protein with an apparent molecular mass of 44 kDa. The protein was purified to homogeneity and free of detectable lipopolysaccharide. Limited microsequencing of this protein resulted in a 15-amino acid query sequence of A-E-V-Y-N-K-D-G-N-K-L-D-L-Y-G, which shares complete identity with a 15-mer of both the OmpD of S. typhimurium SH 7454 and the OmpC polypeptide of Escherichia coli K-12. Picomolar concentrations of this purified protein significantly inhibited the subsequent adherence of 35S-labeled S. typhimurium to macrophages in monolayers. We propose that this 44-kDa protein is involved in the recognition of S. typhimurium by macrophage during the initial stages of infection.
 ...
PMID:Macrophages recognize and adhere to an OmpD-like protein of Salmonella typhimurium. 956 90

The SmD1 protein is a specific target for the autoantibody response in SLE. To further analyze this reactivity epitope, mapping was performed with cellulose-bound 13-mer peptides overlapping 10 amino acids (aa). In this initial approach, 4 out of 15 SLE sera recognized more than five overlapping peptides of the SmD1 C-terminus. Therefore, longer oligopeptides of up to 37 aa of this region were generated and probed for as antigens by ELISA. For the SmD1 aa 83-119 polypeptide, there was a striking increase of reactivity with 70.0% positive reactions out of 167 SLE sera. In contrast, 105 healthy control sera were negative, and only 8.3% of sera from patients with other inflammatory diseases (n = 267) exhibited a response, which was of low level only. The anti-SmD183-119 reactivity was significantly higher in anti-dsDNA antibody positive vs. negative sera (P < 0.001) and correlated with disease activity. Four of five human monoclonal anti-dsDNA antibodies also reacted with SmD183-119. The specificity for SmD1 was demonstrated by inhibition experiments and immunization of rabbits with SmD183-119 inducing SmD1-specific antibodies. In conclusion, the SmD183-119 peptide was demonstrated to be an important and highly specific target of the autoimmune response in SLE. The high sensitivity of this ELISA probably depends on a conformational epitope, which appears not to be accessible in the full-size SmD1 protein.
 ...
PMID:A novel epitope on the C-terminus of SmD1 is recognized by the majority of sera from patients with systemic lupus erythematosus. 971 Apr 44

The nature of the nucleation-collapse mechanism in protein folding is probed using 27-mer and 36-mer lattice models. Three different forms for the interaction potentials are used. Three of the four 27-mer sequences have maximally compact and identical native state while the other has a non-compact native conformation. All the sequences fold thermodynamically and kinetically by a two-state process. Analysis of individual trajectories for each sequence using a self-organizing neural net algorithm shows that upon formation of a critical set of contacts the polypeptide chain rapidly reaches the native conformation which is consistent with a nucleation-collapse mechanism. The algorithm, which reduces the identification of the folding nucleus for each trajectory to one of pattern recognition, is used to show that there are multiple folding nuclei. There is a distribution of nucleation contacts in the transition states with some of them occurring with more probability (when averaged over the denatured ensemble) than others. We also show that there is a distribution in the size of the nuclei with the average number of residues in the folding nuclei being less than about one-third of the chain size. The fluctuations in the sizes of the nuclei are large, suggestive of a broad transition region. The folding nuclei, the structures of each are the corresponding transition states, have varying degree of overlap with the native conformation. The distribution of the radius of gyration of the transition states shows that these structures are an expanded form (by about 25% in the radius of gyration) of the native conformation. Local contacts are most dominant in the folding nuclei while a certain fraction of non-local contacts is necessary to stabilize the transition states. The search for the critical nuclei initially involves the formation of local contacts, while non-local contacts are formed later. The fractional values of PhiF for the two 27-mer mutants found by using the protein engineering protocol are consistent with the microscopic picture of partial formation of structures involving these residues in the transition state. These observations lead to a multiple folding nuclei (MFN) model for nucleation-collapse mechanism in protein folding. The major implication of the MFN model is that, even if the residues whose tertiary interactions are formed nearly completely in the transition state are mutated, it does not disrupt the nature of the nucleation-collapse mechanism. We analyze the experiments on chymotrypsin inhibitor 2 and alpha-spectrin SH3 domain and two circular permutants in light of the MFN model. It is shown that the PhiF-value analysis for these proteins gives considerable support to the MFN model. The theoretical and experimental studies give a coherent picture of the nucleation-collapse mechanism in which there is a distribution of folding nuclei with some more probable than others. The formation of any specific nucleus is not necessary for efficient two-state folding.
 ...
PMID:Lattice models for proteins reveal multiple folding nuclei for nucleation-collapse mechanism. 973 20

A high proportion of tumors arise due to mutation of the p53 tumor suppressor protein. A p53 hotspot mutation at amino acid position 273 from R to H, flanking a peptide epitope that spans residues 264-272, renders cells resistant to killing by human histocompatibility leukocyte antigen (HLA)-A*0201-restricted cytotoxic T lymphocytes (CTLs) specific for this epitope. Acquisition of the R to H mutation at residue 273 of the human p53 protein promotes tumor growth in vivo by selective escape from recognition by p53.264-272 peptide-specific CTLs. Synthetic 27-mer p53 polypeptides covering the antigenic nonamer region 264-272 of p53 were used as proteasome substrates to investigate whether the R to H mutation at the P1' position of the COOH terminus of the epitope affects proteasome-mediated processing of the protein. Analysis of the generated products by tandem mass spectrometry and the kinetics of polypeptide processing in conjunction with CTL assays demonstrate that the R to H mutation alters proteasomal processing of the p53 protein by inhibiting proteolytic cleavage between residues 272 and 273. This prevents the release of the natural CTL epitope that spans flanking residues 264-272 as well as a putative precursor peptide. These results demonstrate that mutation of p53 not only leads to malignant transformation but may also, in some instances, affect immune surveillance and should be considered in the design of cancer vaccines.
 ...
PMID:The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope. 974 20

The neurotoxins from Clostridium botulinum (BoNT serotypes A-G) exert their lethal effect by preventing the release of acetylcholine at the neuromuscular junction. As with tetanus toxin, immunization with a non-toxic fragment, the 50 kDa C-terminal portion of BoNT/A (Hc; residues 861-1296), protects mice against lethal challenges with the intact toxin. To locate the neutralizing epitopes, several protective monoclonal antibodies (mAbs) against BoNT/A-Hc were isolated and cloned. Specific binding of the mAbs to BoNT/A-Hc was demonstrated by surface plasmon resonance, with Kas in the range of 10(-10) to 10(-11) M. These antibodies recognized a genetically engineered polypeptide (1150-1289) that was previously shown to induce protective immunity. Prior to the determination of the X-ray crystal structure of the tetanus neurotoxin Hc fragment, molecular modelling studies indicated that it contained two highly solvent-exposed loops. Based on these predictions, two 25-mer Hc-peptides corresponding to these two regions were synthesized and were demonstrated to bind the neutralizing mAbs. Mice immunized with the Hc-peptides had high levels of antibodies that recognized BoNT/A-Hc. However, immunizations with only one of the Hc peptides protected when mice were challenged with BoNT/A. On the basis of these analyses, it should be possible to develop small peptides that could be useful in the design of future vaccines against these neurotoxins.
 ...
PMID:Identifying the principal protective antigenic determinants of type A botulinum neurotoxin. 979 91

Interleukin-5 (IL-5) is the predominant cytokine associated with antigen-induced eosinophilic inflammation in the lung. The activation of TH2 cells leads to the production of IL-5. The proeosinophilic effects of IL-5 include 1) enhanced replication and differentiation of eosinophilic myelocytes; 2) enhanced degranulation of eosinophils; 3) prolonged survival time of eosinophils; and 4) enhanced adhesion of eosinophils. The effects of IL-5 are mediated via the interaction of IL-5 with receptors (Il-5R) expressed on the eosinophil cell membrane. Intracellular signaling produced by occupation of the IL-5R by IL-5 occurs via the JAK-STAT system. IL-5 is a 45kD glycoprotein that consists of two identical polypeptide chains. The 5'-promoter region of the IL-5 gene contains elements that are down-regulated by glucocorticoids. A 16-mer deoxyoligonucleotide, antisense to IL-5 mRNA and with two phosphorothioate modifications, produced, at 20 micromolar concentration, complete inhibition of IL-5 secretion by human peripheral blood mononuclear cells. The targeted 16-mer sequence of the IL-5 mRNA did not display complete homology with any other known human gene sequences. These results suggest that the 16-mer phosphorothioate antisense IL-5 provides the basis for a non-glucocorticoid, sequence-specific inhibitor of IL-5.
 ...
PMID:Interleukin-5: a proeosinophil cytokine mediator of inflammation in asthma and a target for antisense therapy. 980 38

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.
 ...
PMID:Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro. 987 16


<< Previous 1 2 3 4 5 6 7 8 9 10