RÉSUMÉ
Fluorescence resonance energy transfer (FRET) data, in accordance with lateral mobility measurements, suggested the existence of class I HLA dimers and oligomers at the surface of live human cells, including the B lymphoblast cell line (JY) used in the present study. Intra- and intermolecular class I HLA epitope distances were measured on JY B cells by FRET using fluorophore-conjugated Ag-binding fragments of mAbs W6/32 and L368 directed against structurally well-characterized heavy and light chain epitopes, respectively. Out-of-plane location of these epitopes relative to the membrane-bound BODIPY-PC (2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) was also determined by FRET. Computer-simulated docking of crystallographic structures of class I HLA and epitope-specific Ag-binding fragments, with experimentally determined interepitope and epitope to cell surface distances as constraints, revealed several sterically allowed and FRET-compatible class I HLA dimeric and tetrameric arrangements. Extension of the tetrameric class I HLA model with interacting TCR and CD8 resulted in a model of a supramolecular cluster that may exist physiologically and serve as a functionally significant unit for a network of CD8-HLA-I complexes providing enhanced signaling efficiency even at low MHC-peptide concentrations at the interface of effector and APCs.
Sujet(s)
Antigènes CD8/composition chimique , Transfert d'énergie/immunologie , Antigènes HLA/composition chimique , Antigènes d'histocompatibilité de classe I/composition chimique , Modèles moléculaires , Récepteurs aux antigènes des cellules T/composition chimique , Cellules présentatrices d'antigène/composition chimique , Cellules présentatrices d'antigène/immunologie , Lymphocytes B/composition chimique , Lymphocytes B/immunologie , Lignée de cellules transformées , Membrane cellulaire/composition chimique , Membrane cellulaire/immunologie , Simulation numérique , Cristallographie aux rayons X/méthodes , Déterminants antigéniques des lymphocytes B/composition chimique , Antigène HLA-A2/composition chimique , Humains , Fragments Fab d'immunoglobuline/composition chimique , Cartographie peptidique , Spectrométrie de fluorescence/méthodes , bêta-2-Microglobuline/composition chimiqueRÉSUMÉ
Human T-cell leukemia virus type-1 (HTLV-1) is associated with a number of human diseases. Based on the therapeutic success of human immunodeficiency virus type 1 (HIV-1) PR inhibitors, the proteinase (PR) of HTLV-1 is a potential target for chemotherapy. To facilitate the design of potent inhibitors, the subsite specificity of HTLV-1 PR was characterized and compared to that of HIV-1 PR. Two sets of substrates were used that contained single amino-acid substitutions in peptides representing naturally occurring cleavage sites in HIV-1 and HTLV-1. The original HIV-1 matrix/capsid cleavage site substrate and most of its substituted peptides were not hydrolyzed by the HTLV-1 enzyme, except for those with hydrophobic residues at the P4 and P2 positions. On the other hand, most of the peptides representing the HTLV-1 capsid/nucleocapsid cleavage site were substrates of both enzymes. A large difference in the specificity of HTLV-1 and HIV-1 proteinases was demonstrated by kinetic measurements, particularly with regard to the S4 and S2 subsites, whereas the S1 subsite appeared to be more conserved. A molecular model of the HTLV-1 PR in complex with this substrate was built, based on the crystal structure of the S9 mutant of Rous sarcoma virus PR, in order to understand the molecular basis of the enzyme specificity. Based on the kinetics of shortened analogs of the HTLV-1 substrate and on analysis of the modeled complex of HTLV-1 PR with substrate, the substrate binding site of the HTLV-1 PR appeared to be more extended than that of HIV-1 PR. Kinetic results also suggested that the cleavage site between the capsid and nucleocapsid protein of HTLV-1 is evolutionarily optimized for rapid hydrolysis.
Sujet(s)
Aspartic acid endopeptidases/composition chimique , Aspartic acid endopeptidases/métabolisme , Protéase du VIH/composition chimique , Protéase du VIH/métabolisme , Séquence d'acides aminés , Virus du sarcome aviaire/enzymologie , Humains , Cinétique , Modèles moléculaires , Données de séquences moléculaires , Oligopeptides/composition chimique , Oligopeptides/métabolisme , Inhibiteurs de protéases/pharmacologie , Structure secondaire des protéines , Alignement de séquences , Spécificité du substratRÉSUMÉ
The proteinase of bovine leukemia virus (BLV) was cloned into pMal-c2 vector with N-terminal or with N- as well as C-terminal flanking sequences, and expressed in fusion with maltose binding protein. The proteinase self-processed itself from the fusion protein during expression and formed inclusion bodies. The enzyme was purified from inclusion bodies by cation-exchange chromatography followed by gel filtration. Specificity of the enzyme was compared to that of human T-cell leukemia proteinase type 1. Although the two viruses belong to the same subfamily of retroviruses, the differences in their proteinase specificity, based on kinetics with oligopeptide substrates representing naturally occurring cleavage sites as well as on inhibition pattern, appear to be pronounced.
Sujet(s)
Endopeptidases/génétique , Virus de la leucémie bovine/enzymologie , Séquence d'acides aminés , Aspartic acid endopeptidases/métabolisme , Sites de fixation , Clonage moléculaire , Endopeptidases/isolement et purification , Endopeptidases/métabolisme , Antienzymes/pharmacologie , Escherichia coli/génétique , Cinétique , Données de séquences moléculaires , Protéines de fusion recombinantes/métabolisme , Alignement de séquences , Spécificité du substratRÉSUMÉ
The substrate sequence requirements for preference toward P2' Glu residue by human immunodeficiency virus type 1 (HIV-1) proteinase were studied in both the matrix protein/ capsid protein (MA/CA) and CA/p2 cleavage site sequence contexts. These sequences represent typical type 1 (-aromatic*Pro-) and type 2 (-hydrophobic* hydrophobic-) cleavage site sequences, respectively. While in the type 1 sequence context, the preference for P2' Glu over Ile or Gln was found to be strongly dependent on the ionic strength and the residues being outside the P2-P2' region of the substrate, it remained preferable in the type 2 substrates when typical type 1 substrate sequence residues were substituted into the outside regions. The pH profile of the specificity constants suggested a lower pH optimum for substrates having P2' Glu in contrast to those having uncharged residues, in both sequence contexts. The very low frequency of P2' Glu in naturally occurring retroviral cleavage sites of various retroviruses including equine infectious anemia virus (EIAV) and murine leukemia virus (MuLV) suggests that such a residue may not have a general regulatory role in the retroviral life cycle. In fact, unlike HIV-1 and HIV-2, EIAV and MuLV proteinases do not favor P2' Glu in either the MA/CA or CA/p2 sequence contexts.
Sujet(s)
Endopeptidases/composition chimique , Endopeptidases/métabolisme , Protéase du VIH/composition chimique , Protéase du VIH/métabolisme , Retroviridae/enzymologie , Séquence d'acides aminés , Animaux , Sites de fixation , Acide glutamique/composition chimique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , VIH-2 (Virus de l'Immunodéficience Humaine de type 2)/enzymologie , Humains , Concentration en ions d'hydrogène , Virus de l'anémie infectieuse équine/enzymologie , Virus de la leucémie murine/enzymologie , Souris , Modèles moléculaires , Oligopeptides/composition chimique , Oligopeptides/métabolisme , Conformation des protéines , Spécificité du substrat , ThermodynamiqueRÉSUMÉ
Vimentin, a cellular substrate of HIV type 1 (HIV-1) proteinase, contains a protein kinase C (PKC) phosphorylation site at one of its cleavage sites. Peptides representing this site were synthesized in P2 Ser-phosphorylated and nonphosphorylated forms. While the nonphosphorylated peptide was a fairly good substrate of the enzyme, phosphorylation prevented hydrolysis. Phosphorylation of human recombinant vimentin by PKC prevented its processing within the head domain, where the phosphorylation occurred. Oligopeptides representing naturally occurring cleavage sites at the C-terminus of the Rous sarcoma virus integrase were assayed as substrates of the avian proteinase. Unlike the nonphosphorylated peptides, a Ser-phosphorylated peptide was not hydrolyzed by the enzyme at the Ser-Pro bond, suggesting the role of previously established phosphorylation in processing at this site. Ser-phosphorylated and Tyr-phosphorylated forms of model substrates were also tested as substrates of the HIV-1 and the avian retroviral proteinases. In contrast to the moderate effect of P4 Ser phosphorylation, phosphorylation of P1 Tyr prevented substrate hydrolysis by HIV-1 proteinase. Substrate phosphorylation had substantially smaller effects on the hydrolysis by the avian retroviral proteinase. As the active retroviral proteinase as well as various protein kinases are incorporated into mature virions, substrate phosphorylation resulting in attenuation or prevention of proteolytic processing may have important consequences in the regulation of the retroviral life cycle as well as in virus-host cell interactions.
Sujet(s)
Aspartic acid endopeptidases/métabolisme , Phosphoprotéines/métabolisme , Maturation post-traductionnelle des protéines , Retroviridae/enzymologie , Vimentine/métabolisme , Domaine catalytique , Protéase du VIH/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , Modèles moléculaires , Phosphosérine , Phosphotyrosine , Spécificité du substratRÉSUMÉ
The human foamy virus proteinase was expressed in fusion with maltose binding protein in Escherichia coli and purified. The specific activity of the fusion protein was similar to that of the processed enzyme. The kinetic constants on foamy virus cleavage site substrates were very low but comparable to those obtained with the gag-encoded avian proteinase on its own substrates. The proteinase showed preference for high ionic strength and a pH optimum of 6.6. None of the tested retroviral cleavage site peptides were substrates, however, some peptides representing cleavage sites in retrotransposons were properly processed by the enzyme.
Sujet(s)
Transporteurs ABC , Aspartic acid endopeptidases/biosynthèse , Aspartic acid endopeptidases/composition chimique , Protéines Escherichia coli , Transporteurs de monosaccharides , Séquence d'acides aminés , Aspartic acid endopeptidases/isolement et purification , Protéines de transport/métabolisme , Clonage moléculaire , Humains , Concentration en ions d'hydrogène , Cinétique , Protéines de liaison au maltose , Données de séquences moléculaires , Oligopeptides/métabolisme , Protéines de fusion recombinantes/biosynthèse , Protéines de fusion recombinantes/composition chimique , Protéines de fusion recombinantes/isolement et purification , Rétroéléments , Retroviridae/métabolisme , Similitude de séquences d'acides aminés , Facteurs tempsRÉSUMÉ
The catalytic subunit of protein phosphatase 1 (PP1c) was purified from an alfalfa (Medicago sativa) microcallus cell culture. The preparation was inhibited by rabbit muscle inhibitor-2 and okadaic acid and had a molecular mass of 35 kDa. Five distinct cDNAs termed MsPP1alpha, -beta, -gamma, -delta, and -epsilon were cloned from a M. sativa somatic embryo library. MsPP1alpha was identical to a cDNA reported earlier [A. Páy, M. Pirck, L. Bögre, H. Hirt, and E. Heberle-Bors Mol. Gen. Genet. 244, 176-182, 1994], while the others represented novel isoforms encoded by separate genes. The predicted amino acid sequences of MsPP1alpha, -beta, -gamma, -delta, and -epsilon were highly similar to each other and to other known PP1c sequences. The GST-MsPP1ss fusion protein expressed in Escherichia coli was catalytically active and was inhibited by inhibitor-2 and okadaic acid. Affinity-purified polyclonal MsPP1antipeptide antibody detected a protein of 36 kDa in crude cell extracts. These results proved that the cDNA clone encoded an active PP1c which was very similar to the purified enzyme. The mRNA and protein concentrations of PP1c as well as the specific activity of protein phosphatase 1 did not change during the cell cycle in a synchronized alfalfa cell culture. On the other hand, the isoforms exhibited different steady-state mRNA levels in different plant organs suggesting tissue-specific functions.
Sujet(s)
Domaine catalytique , Clonage moléculaire , Isoenzymes/métabolisme , Medicago sativa/enzymologie , Phosphoprotein Phosphatases/métabolisme , Séquence d'acides aminés , Anticorps , Aphidicoline/pharmacologie , Technique de Northern , Technique de Southern , Cycle cellulaire/effets des médicaments et des substances chimiques , Séquence conservée , Escherichia coli , Expression des gènes/effets des médicaments et des substances chimiques , Banque de gènes , Concentration inhibitrice 50 , Isoenzymes/composition chimique , Isoenzymes/génétique , Isoenzymes/immunologie , Medicago sativa/génétique , Données de séquences moléculaires , Spécificité d'organe , Phosphoprotein Phosphatases/antagonistes et inhibiteurs , Phosphoprotein Phosphatases/composition chimique , Phosphoprotein Phosphatases/génétique , Structures de plante/génétique , Protein Phosphatase 1 , Protéines de fusion recombinantes/antagonistes et inhibiteurs , Protéines de fusion recombinantes/immunologie , Protéines de fusion recombinantes/isolement et purification , Protéines de fusion recombinantes/métabolismeRÉSUMÉ
The specificity of linked homo- and heterodimeric HIV-1 and HIV-2 proteinases was characterized by using oligopeptide substrates. For two substrates the k(cat)/Km values for the heterodimers were the mean values for those of the homodimers, suggesting that these substrates could productively bind into the heterodimers in both directions. However, for two other substrates the k(cat)/Km values for the heterodimers were higher than those of the homodimers, suggesting that these substrates could productively bind into the enzymes in a preferable direction. However, the mode of binding does not seem to depend on the sequential position of the subunits. The studied linked homo- and heterodimers may represent intermediate stages in the evolution of bilobal aspartic proteinases. As divergence in sequence of the two halves of such a proteinase increases, the possibility of bidirectional binding is likely lost at the expense of the optimized side-chain subsite interactions. The differences in observed and calculated k(cat)/Km values revealed dependence of the substrate specificity at one subsite of the enzyme from the next residue in sequence of substrate. These findings were also supported by molecular modeling studies.
Sujet(s)
Aspartic acid endopeptidases/métabolisme , Dimérisation , Protéase du VIH/métabolisme , Oligopeptides/métabolisme , Aspartic acid endopeptidases/composition chimique , Capside/métabolisme , Protéase du VIH/composition chimique , Cinétique , Oligopeptides/composition chimique , Relation structure-activité , Spécificité du substrat , Thermodynamique , Protéines de la matrice virale/métabolismeRÉSUMÉ
Two major types of cleavage sites with different sequence preferences have been proposed for the human immunodeficiency virus type 1 (HIV-1) proteinase. To understand the nature of these sequence preferences better, single and multiple amino acid substitutions were introduced into a type 1 cleavage site peptide, thus changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a residue at a particular position in the substrate depends strongly on the neighboring residues, including both those at the same side and at the opposite side of the peptide backbone of the substrate. Based on these results, pseudosymmetric (palindromic) substrates were designed. The retroviral proteinases are symmetrical dimers of two identical subunits; however, the residues of naturally occurring cleavage sites do not show symmetrical arrangements, and no obvious symmetrical substrate preference has been observed for the specificity of HIV proteinase. To examine the role of the asymmetry created by the peptide bonds on the specificity of the respective primed and nonprimed halves of the binding site, amino acid substitutions were introduced into a palindromic sequence. In general, the results suggested that the asymmetry does not result in substantial differences in specificity of the S3 and S3' subsites, whereas its effect is more pronounced for the S2 and S2' subsites. Although it was possible to design several good palindromic substrates, asymmetrical arrangements may be preferred by the HIV proteinase.
Sujet(s)
Protéase du VIH/métabolisme , Humains , Hydrolyse , Cinétique , Modèles moléculaires , Peptides/synthèse chimique , Peptides/métabolisme , Relation structure-activité , Spécificité du substratRÉSUMÉ
The tethered-dimer protease of human immunodeficiency virus 1 (HIV-1) [Cheng Y.-S. E., Yin, F.H., Foundling, S., Blomstrom, D. & Kettner, C. A. (1990) Proc. Natl Acad. Sci. USA 87, 9660-9664] and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Escherichia coli. These mutant enzymes showed various degrees of self-processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type. Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in K(m) and decrease in Kcat values. Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap. Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P2 and P2' substrate positions showed only moderate changes in the substrate specificity of the mutant proteases. Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements. In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen-bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis.
Sujet(s)
Protéase du VIH/génétique , Protéase du VIH/métabolisme , Pliage des protéines , Séquence d'acides aminés/génétique , Produits du gène gag/métabolisme , Produits du gène pol/métabolisme , Protéase du VIH/composition chimique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , Hydrolyse , Cinétique , Modèles moléculaires , Données de séquences moléculaires , Mutagenèse , Oligopeptides/métabolisme , Concentration osmolaire , Peptides/génétique , Spécificité du substratRÉSUMÉ
Mutations were introduced into the active site triplet (Asp-Thr-Gly) of one or both subunits of a linked dimer of human immunodeficiency virus type 1 proteinase. Mutation of Thr to Ser in one or both subunits did not alter the activity of the enzyme substantially, whereas its mutation to Asn in one subunit caused a dramatic decrease in catalytic efficiency. Mutation of Gly to Val in one subunit also yielded an enzyme with very low activity. The enzymes containing Thr-->Asn and Gly-->Val mutations in both subunits resulted in inactive enzymes, based on their inability to self-process and on assay with an oligopeptide substrate. The dramatic decrease in enzyme efficiency of the mutants was interpreted using molecular models of the enzymes.
Sujet(s)
Protéase du VIH/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , Acide aspartique/génétique , Sites de fixation/génétique , Dimérisation , Gènes de synthèse , Glycine/génétique , Protéase du VIH/génétique , Cinétique , Modèles moléculaires , Mutagenèse , Oligopeptides/métabolisme , Maturation post-traductionnelle des protéines , Thréonine/génétiqueRÉSUMÉ
The retroviral proteinase (PR) seems to play crucial roles in the viral life cycle, therefore it is an attractive target for chemotherapy. Previously we studied the specificity of human immunodeficiency virus (HIV) type 1 and type 2 as well as equine infectious anemia virus PRs using oligopeptide substrates. Here a similar approach is used to characterize the specificity of avian myeloblastosis virus (AMV) PR and to compare it with those of the previously characterized lentiviral PRs. All peptides representing naturally occurring Gag and Gag-Pol cleavage sites were substrates of the AMV PR. Only half of these peptides were substrates of HIV-1 PR. The Km values for AMV PR were in a micromolar range previously found for the lentiviral PRs; however, the kcat values were in a 10 30-fold lower range. A series of peptides containing single amino acid substitutions in a sequence representing a naturally occurring HIV cleavage site was used to characterize the seven substrate binding subsites of the AMV PR. The largest differences were found at the P4 and P2 positions of the substrate. Detailed analysis of the results by molecular modeling and comparison with previously reported data revealed the common characteristics of the specificity of the retroviral PRs as well as its strong dependence on the sequence context of the substrate.