Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Previous structural and kinetic characterization of mutations within the active site of alpha-lytic protease have demonstrated that amino acid residues in direct contact with the substrate are major substrate specificity determinants. The experiments described here identify residues 216-226 of alpha-lytic protease as a region of structure peripheral to the active site that also plays an important role in establishing the substrate specificity of the enzyme. Alanine substitution mutations within this surface loop of 19 amino acid residues significantly perturb the enzyme's specificity profile, despite being as far as 21 A from the hydroxyl group of Ser195. The kinetic consequences of the mutations are remarkably independent of position within the loop and suggest that active site plasticity is affected more than static structure. Kinetic characterization of double mutants with the Met190-->Ala broad-specificity active site mutation reveals varying degrees of non-additivity and indicates that active site plasticity can be influenced through multiple sets of interactions. Although these results clearly demonstrate that tuning of serine protease activity is possible through remodelling of structure surrounding the active site, practical issues such as retaining compatibility with the folding mechanism and stability of the mature enzyme present significant obstacles to general application of the technique.
J Mol Biol 1995 Aug 04
PMID:Functional linkage between the active site of alpha-lytic protease and distant regions of structure: scanning alanine mutagenesis of a surface loop affects activity and substrate specificity. 764 81

A set of 58 overlapping rod-bound peptides was used to map the antigenic reactivity pattern of a 64-residue neurotoxin (AaH II) from the venom of the scorpion Androctonus australis hector. Five anti-toxin rabbit antisera were assayed serially for their capacity to bind to each peptide in the set. Six regions of antigenic reactivity were thus identified (sequences: 1-8, 4-12, 27-35, 39-45, 52-58 and 55-61). When positioned on a 3-D model of the toxin, these regions appeared to correspond to either beta-turn or extended parts of the molecule. The antigenic regions revealed by this technique agree fairly well with those previously mapped on the same toxin by different methods. One discrepancy was, however, that the present study shows the N-terminus to be strongly reactive with anti-toxin antibodies. The antigenicity of this region was confirmed, since rabbit antibodies raised against a synthetic peptide mimicking the sequence 1-8 of the toxin were found to bind the toxin with high efficiency. A fine analysis of the recognition of this region was performed. Alanine-containing analogs of the sequence 1-7 and peptides mimicking the N-terminal of the four main toxins of AaH were probed with anti-toxin and anti-peptide antibodies. Lysine 2, aspartic acid 3 and glycine 4 were shown to be key residues in the recognition of the N-terminal region of the AaH II toxin by anti-toxin antibodies. In contrast, a loose specificity of recognition was shown by one anti-peptide serum which was, in addition, able to recognize the N-termini of all four AaH toxins.
Mol Immunol 1993 Aug
PMID:Fine molecular analysis of the antigenicity of the Androctonus australis hector scorpion neurotoxin II: a new antigenic epitope disclosed by the Pepscan method. 769 Jan 10

The eukaryotic transcription factor NF-kappa B plays a central role in the induced expression of human immunodeficiency virus type 1 and in many aspects of the genetic program mediating normal T-cell activation and growth. The nuclear activity of NF-kappa B is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called I kappa B alpha. This cytoplasmic inhibitor is rapidly phosphorylated and degraded in response to a diverse set of NF-kappa B-inducing agents, including T-cell mitogens, proinflammatory cytokines, and viral transactivators such as the Tax protein of human T-cell leukemia virus type 1. To explore these I kappa B alpha-dependent mechanisms for NF-kappa B induction, we identified novel mutants of I kappa B alpha that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of I kappa B alpha failed to disrupt its ability to form latent complexes with NF-kappa B in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of I kappa B alpha from NF-kappa B in Tax-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of I kappa B alpha also yielded constitutive repressors that escaped from Tax-induced turnover and that potently inhibited immune activation pathways for NF-kappa B induction, including those initiated from antigen and cytokine receptors. In contrast, introduction of a phosphoserine mimetic at these sites rectified this functional defect, a finding consistent with a causal linkage between the phosphorylation status and proteolytic stability of this cytoplasmic inhibitor. Together, these in vivo studies define a critical signal response domain in I kappa B alpha that coordinately controls the biologic activities of I kappa B alpha and NF-kappa B in response to viral and immune stimuli.
Mol Cell Biol 1995 May
PMID:Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation. 773 62

When Leishmania donovani promastigote forms, were cultured in TC-199 medium at 28 degrees C and subsequently incubated at 38 degrees C, they turned into aflagellate (amastigote-like) forms. A return of the incubation-culture temperature to 28 degrees C these amastigote-like forms to revert to promastigotes. The amastigotes obtained by heat-shock, were viable and retained antigenic capacity being recognized by the sera of naturally infected patients. These forms, remained also capable of multiplying inside the J-774A.1 macrophages. When the amastigote-like forms are kept in culture at 38 degrees C retained their rounded appearance and their biological characteristics for more than 3 months subculturing every 6 days. These amastigote-like forms, when used for subcultures at 28 degrees C, transformed into promastigotes capable of multiplying as flagellate forms. The amastigote-like forms obtained in vitro can be used in biochemical studies related to chemotherapy and immunology studies, as part of an effort to combat this parasite. The end-products of of glycolysis were studied in both the amastigote-like and promastigote forms of L. donovani, by proton magnetic resonance analysis of the culture media. Alanine, succinate, and acetate, were predominant, and to a lesser extent pyruvate, glycine and D-lactate. Our results suggest that both forms of Leishmania use different biochemical strategies to obtain their energy.
Mol Cell Biochem 1995 Jan 26
PMID:Leishmania donovani: in vitro culture and [1H] NMR characterization of amastigote-like forms. 777 70

To probe the structure and function of the Saccharomyces cerevisiae general transcription factor TFIIA, we have systematically mutagenized the genes encoding both subunits and analyzed the effects of the mutations both in vivo and in vitro. We found that the central nonconserved region of the large subunit is not essential for function and likely acts as a spacer between the conserved N- and C-terminal regions. Deletion mutagenesis of the large subunit defined a region which is required for TATA binding protein (TBP) interaction. Alanine scanning mutagenesis defined a cluster of four basic residues which are likely required for interaction with DNA in the TBP-DNA complex. Much of the conserved regions of both subunits is required for subunit association, suggesting that these conserved regions fold into compact domains which extensively interact. In vitro transcription performed with extracts from yeast strains with mutations in either the large or the small TFIIA subunit demonstrated that TFIIA stimulates both basal and activated polymerase II (Pol II) transcription. The TFIIA-depleted extracts have normal Pol I and Pol III transcription activity, showing that TFIIA is a Pol II-specific factor. In vivo depletion of TFIIA activity reduced transcription from four different Pol II promoters. Finally, alanine scanning mutagenesis of TFIIA's small subunit has identified at least one mutation which is defective in transcription but which is not defective in subunit association or binding to TBP or TBP-DNA complexes.
Mol Cell Biol 1995 Mar
PMID:Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription. 786 17

TSH and immunoglobulin G (IgG) preparations from patients with Graves' disease increase inositol phosphate as well as cAMP formation in Cos-7 cells transfected with rat TSH receptor (TSHR) cDNA. In a previous report, we mutated alanine 623 of the third cytoplasmic loop (residues 605-625) of the TSHR and showed it was critical for TSH and Graves' IgG initiation of phosphatidylinositol bisphosphate (PIP2) but not cAMP signaling. In this report, we substituted residues in the third loop of the TSHR with sequences from the N- and C-termini of the third loop of the alpha 1- and beta 2-adrenergic receptors (ARs), which computer analysis has identified as homologous to those in the TSHR. Alanine 623 is conserved in most ARs as well as in glycoprotein hormone receptors; there is, therefore, no change in alanine 623. After transfection of the mutant TSHR cDNAs into Cos-7 cells, we show that the mutant proteins are normally synthesized, processed, and incorporated into the membrane bilayer by Western blotting with a specific receptor antibody. We also show that the dissociation constant for TSH binding in all mutants is the same or lower than wild type TSHR. We then evaluated the ability of TSH or Graves' IgG to increase PIP2 and cAMP signals in each transfectant. Mutants A622 and B621 replace, respectively, residues 622-625 and 621-625 of the TSHR with alpha 1- and beta 2-AR residues from the C-terminus of the third cytoplasmic loop; mutants A607 and B605 replace, respectively, TSHR residues 607-609 and 605-609 with N-terminus residues from alpha 1- and beta 2-AR. All four mutants, like the alanine 623 mutant, result in transfected cells which lose TSH and Graves' IgG initiation of PIP2 but not cAMP signalling. Like the alanine 623 mutation to glutamic acid, the A607, B605, A622, and B621 mutants also result in decreased basal cAMP, but not inositol phosphate levels, relative to wild type receptor. In contrast to these results, mutants A610, B610, A617, and B617, which replace residues 610-613 or 617-620 of the TSHR with corresponding residues of the alpha 1- and beta 2-AR, retain TSH and Graves' IgG responsiveness in both inositol phosphate and cAMP assays. Mutation of residues 610-613, in fact, potentiates TSH-increased inositol phosphate production, despite having no effect on TSH-increased cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1993 Aug
PMID:Substitutions of different regions of the third cytoplasmic loop of the thyrotropin (TSH) receptor have selective effects on constitutive, TSH-, and TSH receptor autoantibody-stimulated phosphoinositide and 3',5'-cyclic adenosine monophosphate signal generation. 790 57

It has been proposed that the surface loop consisting of amino acid residues 152 to 166 of the catabolite gene activator protein (CAP) of Escherichia coli makes direct protein-protein contact with RNA polymerase at the lac promoter. In this work, we have used targeted saturation mutagenesis of codons 152 to 166 of the gene encoding CAP, followed by a screen, to isolate more than 200 independent mutants of CAP defective in transcription activation but not defective in DNA binding. All isolated single-substitution mutants map to just eight amino acid residues; 156, 157, 158, 159, 160, 162, 163 and 164. We propose that these residues define the full extent of the epitope on CAP for the proposed CAP-RNA polymerase interaction. In addition, we have constructed alanine substitutions at each position from residue 152 to 166 of CAP, and we have analyzed the effects on transcription activation at the lac promoter and on DNA binding. Alanine substitution of Thr158 results in an approximately eightfold specific defect in transcription activation. In contrast, alanine substitution of no other residue tested results in a more than twofold specific defect in transcription activation. We conclude that, for Thr158, side-chain atoms beyond C beta are essential for transcription activation at the lac promoter, and we propose that Thr158 OH7 gamma makes direct contact with RNA polymerase in the ternary complex of lac promoter, CAP and RNA polymerase. We conclude further that for no residue other than Thr158 are side-chain atoms beyond C beta essential for transcription activation at the lac promoter.
J Mol Biol 1994 Nov 04
PMID:Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). I. Saturation and alanine-scanning mutagenesis. 796 84

We identified a minimal domain of human p53 required for the transactivation of a p53 response element in Saccharomyces cerevisiae. This domain contains the central region of p53 sufficient for specific DNA binding, which colocalizes with the region responsible for binding simian virus 40 large T antigen, 53BP1, and 53BP2. Thirty amino acid positions, including natural mutational hot spots (R175, R213, R248, R249, and R273), in the minimal DNA-binding domain were mutated by alanine substitution. Alanine substitutions at positions R213, R248, R249, D281, R282, R283, E286, and N288 affected transactivation but allowed binding to at least one of the three interacting proteins; these amino acids may be involved in amino acid-base pair contacts. Surprisingly, alanine substitution at the mutational hot spot R175 did not affect DNA binding, transactivation, or T-antigen binding, although it nearly eliminated binding to 53BP1 and 53BP2. Mutation of H168 significantly affected only T-antigen binding, and mutation of E285 affected only 53BP1 binding. Thus, we implicate specific residues of p53 in different DNA and protein interactions.
Mol Cell Biol 1994 Dec
PMID:Distinct residues of human p53 implicated in binding to DNA, simian virus 40 large T antigen, 53BP1, and 53BP2. 796 67

Site-directed mutagenesis and mammalian cell expression was used to analyze the function of each of the 13 cysteine residues in the human activin A beta-subunit precursor. Substitution of the four cysteine residues in the proregion with alanine residues did not affect the function of the proregion in facilitating the dimerization and secretion of activin A homodimers. A series of activin mutants were constructed in which the nine cysteine residues (amino acids 4, 11, 12, 40, 44, 80, 81, 113, and 115) in the mature 116-amino acid beta-subunit were individually altered to alanine residues. Alanine substitution at either cysteine residues 4 or 12 did not interfere with homodimer formation, but the mutant activin A molecules had reduced biological and receptor binding activity (2- to 3-fold). Activin A monomers were produced when cysteine mutants 44, 80, and 113 were expressed in tissue culture cells. Monomers of cys mutants 44 and 80 had approximately 2% of the biological and receptor binding activity of wild type activin A. Cys 113 monomers had undetectable levels of biological activity. No detectable activin monomers or dimers were secreted from cells transfected with plasmids containing cys mutants 11, 40, 81, and 115. The data presented here suggest that a low level of noncovalent dimer formation of cysteine mutant monomers 44 and 80 may explain their low level of biological activity. Therefore, dimer formation is suggested to be an essential prerequisite for high affinity receptor binding and biological potency.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1994 Mar
PMID:Functional analysis of the cysteine residues of activin A. 801 50

Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.
Mol Cell Biol 1994 Jun
PMID:Activation and inhibition of erythropoietin receptor function: role of receptor dimerization. 819


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