Hydroxyethylamine analogues of the p17/p24 substrate cleavage site are tight-binding inhibitors of HIV protease.
Explore the source record for details and available documents.
Endopeptidases↗
Biomedical subjects
S B Kent
Publications and source records attributed to S B Kent.
X-ray crystallographic structure of a complex between a synthetic protease of human immunodeficiency virus 1 and a substrate-based hydroxyethylamine inhibitor.
The structure of a crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site has been determined. The sequence of the inhibitor JG-365 is Ac-Ser-Leu-Asn-Phe-psi[CH(OH)CH2N]-Pro-Ile-Val-OMe; the Ki is 0.24 nM. The hydroxyethylamine moiety, in place of the normal scissile bond of the substrate, is believed to mimic a tetrahedral reaction intermediate. The structure of the complex has been refined to an R factor of 0.146 at 2.4-A resolution by using restrained least squares with rms deviations in bond lengths of 0.02 A and bond angles of 4. The bound inhibitor diastereomer has the S configuration at the hydroxyethylamine chiral carbon, and the hydroxyl group is positioned between the active site aspartate carboxyl groups within hydrogen bonding distance. Comparison of this structure with a reduced peptide bond inhibitor-protease complex indicates that these contacts confer the exceptional binding strength of JG-365.
Amino Acid Sequence↗
Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution.
The structure of a complex between a peptide inhibitor with the sequence N-acetyl-Thr-Ile-Nle-psi[CH2-NH]-Nle-Gln-Arg.amide (Nle, norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 A resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substantial changes, particularly in an extended region corresponding to the "flaps" (residues 35 to 57 in each chain), where backbone movements as large as 7 A are observed.
Amino Acid Sequence↗
Substitutions engineered by chemical synthesis at three conserved sites in mitochondrial cytochrome c. Thermodynamic and functional consequences.
Analogues of the 39-residue CNBr fragment of horse cytochrome c (66-104) have been prepared by total chemical synthesis. Conformationally assisted ligation of these peptides with the native cytochrome c fragment 1-65 (homoserine lactone form) occurred in high yield. Semisynthetic protein molecules of the expected molecular weight were obtained that had folded structures similar to the native molecule as shown by spectral properties and by cross-reactivity with a panel of monoclonal antibodies sensitive to the three-dimensional integrity of cytochrome c. Point mutations were introduced into the horse sequence at three strongly conserved sites: Tyr67, Thr78, and Ala83. The contributions of these 3 residues to the stability of the heme crevice were estimated by titration of the 695 nm absorption due to coordination of ferric iron by the sixth ligand methionine sulfur. The roles of these residues in catalysis of electron transfer and in establishing the value of the redox potential of cytochrome c were also investigated. The hydroxyl group of Tyr67 modulates the spectral properties of the heme and has a profound influence on its redox properties, but hydrogen bonding involving this phenolic hydroxyl does not stabilize the heme crevice. In contrast, we find that Thr78 is strongly stabilizing and that asparagine is not an adequate substitute for this residue because of the greater entropic cost of burying its side chain. The low biological activity of analogues modified at this position, despite normal redox potentials, imply a role for Thr78 in the electron transfer mechanism. The replacement of Ala83 by proline induces a similar phenomenon. An involvement of this residue in the catalysis of electron transfer provides an explanation of the low reactivity of plant mitochondrial cytochromes c in mammalian redox systems.
Amino Acid Sequence↗
Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease.
The rational design of drugs that can inhibit the action of viral proteases depends on obtaining accurate structures of these enzymes. The crystal structure of chemically synthesized HIV-1 protease has been determined at 2.8 angstrom resolution (R factor of 0.184) with the use of a model based on the Rous sarcoma virus protease structure. In this enzymatically active protein, the cysteines were replaced by alpha-amino-n-butyric acid, a nongenetically coded amino acid. This structure, in which all 99 amino acids were located, differs in several important details from that reported previously by others. The interface between the identical subunits forming the active protease dimer is composed of four well-ordered beta strands from both the amino and carboxyl termini and residues 86 to 94 have a helical conformation. The observed arrangement of the dimer interface suggests possible designs for dimerization inhibitors.
Amino Acid Sequence↗
Chemical synthesis in protein engineering: total synthesis, purification and covalent structural characterization of a mitogenic protein, human transforming growth factor-alpha.
Successful approaches to protein engineering required that the desired analogs be easily and rapidly obtained in sufficient quantities and purities for unambiguous structural and functional characterizations. Chemical synthesis is the method of choice for engineering small peptides. We now demonstrate that with improved methodologies and instrumentation, total chemical synthesis can be used to produce a small protein in a form suitable for engineering studies. Active human transforming growth factor-alpha (TGF-alpha), a 50 amino acid long protein with three disulfide bonds, has been synthesized and purified in multiple tens of mg amounts in less than 7 days. The purified human TGF-alpha migrated as a single band on SDS-polyacrylamide gels, ran as a single sharp major band at pI = 6.2 on isoelectric focusing gels, displayed an MW = 5546.2 (Th.5546.3) by mass spectrometry, contained three disulfide bonds and had EGF receptor binding, mitogenic and soft agar colony formation activities. The locations of disulfide bonds were found to be analogous to those found in epidermal growth factor (EGF) and in human TGF-alpha expressed in bacteria.
Amino Acid Sequence↗
Covalent immobilization of proteins for high-sensitivity sequence analysis: electroblotting onto chemically activated glass from sodium dodecyl sulfate-polyacrylamide gels.
We report a new method for the preparation of proteins in a form suitable for high-sensitivity N-terminal amino acid sequence analysis. Proteins separated by polyacrylamide gel electrophoresis were electrophoretically transferred onto glass fiber filter paper chemically activated by the introduction of phenyl isothiocyanate functional groups. The proteins became covalently coupled to the matrix during the electrotransfer process. Bands containing transferred proteins were detected by fluorescent staining or autoradiography, cut out from the glass fiber filter, and directly loaded into the cartridge of a gas-phase sequenator. The covalent nature of the interactions between protein and glass fiber support permitted the use of more vigorous solid-phase sequencing protocols and of alternative sequencing reagents. This high-efficiency isolation and covalent coupling method provides the essential first step toward enhanced-sensitivity protein sequence analysis. The method has been successfully applied to the isolation of a wide variety of proteins from SDS-polyacrylamide gels, and was shown to be compatible with both the standard Edman reagent phenyl isothiocyanate and alternative sequencing reagents such as 4-(N,N'-dimethylamino)azobenzene-4'-isothiocyanate (DABITC).
Amino Acid Sequence↗
Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity.
Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected.
Amino Acid Sequence↗
Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease.
A protein corresponding to the putative protease of the human immunodeficiency virus 1 (HIV-1) has been prepared by total chemical synthesis. This 99 residue synthetic enzyme showed specific proteolytic activity on fragments of the natural gag precursor and on synthetic peptide substrates, two of which released fragments corresponding to the N terminus and C terminus of the protease molecule itself. The observed substrate specificity was not restricted to cleavage at Phe/Tyr-Pro bonds. Inhibition studies provided direct evidence that the HIV-1 protease belongs to the family of aspartic proteases. The availability of the HIV-1 protease as a defined molecular species has important implications for the design of specific inhibitors that do not interfere with the host cell metabolism as a possible route to antiviral agents against acquired immunodeficiency syndrome (AIDS).
Amino Acid Sequence↗
Location and chemical synthesis of a binding site for HIV-1 on the CD4 protein.
The human immunodeficiency virus type 1 (HIV-1) uses the CD4 protein as a receptor for infection of susceptible cells. A candidate structure for the HIV-1 binding site on the CD4 protein was identified by epitope mapping with a family of eight functionally distinct CD4-specific monoclonal antibodies in conjunction with a panel of large CD4-derived synthetic peptides. All of the seven epitopes that were located reside within two immunoglobulin-like disulfide loops situated between residues 1 and 168 of the CD4 protein. The CD4-specific monoclonal antibody OKT4A, a potent inhibitor of HIV-1 binding, recognized a site between residues 32 and 47 on the CD4 protein. By analogy to other members of the immunoglobulin superfamily of proteins, this particular region has been predicted to exist as a protruding loop. A synthetic analog of this loop (residues 25 to 58) showed a concentration-dependent inhibition of HIV-1-induced cell fusion. It is proposed that a loop extending from residues 37 to 53 of the CD4 protein is a binding site for the AIDS virus.
Antibodies, Monoclonal↗
Characterization of prion proteins with monospecific antisera to synthetic peptides.
The prion protein (PrP) 27-30 is the major macromolecular component in highly purified preparations of prions derived from scrapie-infected hamster brain. Immunoblotting studies demonstrated that this protein is generated by partial protease digestion of a larger precursor (PrPSc) with an apparent Mr of 33 to 35 kDa, and that a protease-sensitive cellular PrP isoform, designated PrPC, is present in normal hamster brain. To characterize the relationships among these proteins, ELISA and immunoblotting studies were undertaken with rabbit antisera raised against three synthetic PrP peptides. All three antisera were found to specifically react with the prion proteins, and failed to identify other lower or higher m.w. PrP proteins. Our results provide evidence that the primary structures of PrP 27-30, PrPSc, and PrPC are related; this conclusion supports molecular cloning studies indicating that these proteins are encoded by the same chromosomal gene.
Animals↗
Monoclonal antipeptide antibodies recognize IL-3 and neutralize its bioactivity in vivo.
Mixtures of synthetic peptides corresponding to segments of murine IL-3 or synthetic IL-3 were used to raise murine mAb. Several mAb able to recognize synthetic IL-3 were obtained, two of which exhibited significant cross-reactivity with native IL-3 as shown by precipitation of biosynthetically 35S-labeled IL-3 and their effectiveness as affinity reagents for the purification of IL-3 from conditioned medium. The amino acid sequence recognized by the two mAb was determined by using synthetic peptide segments of IL-3. In both cases binding of the mAb to synthetic IL-3 was inhibited best with a hexapeptide corresponding to the amino acid residues 130-135 of IL-3, although the mAb differed in other characteristics. Neither mAb neutralized IL-3 bioactivity in vitro. However, we observed that in vivo administration of one mAb abrogated the increase in splenic mast cells and their precursors that normally occurred in mice bearing a s.c. IL-3-producing tumor, WEHI-3B.
Animals↗
N-terminal and internal sequence determination of microgram amounts of proteins separated by isoelectric focusing in immobilized pH gradients.
Isolation of microgram amounts of proteins and submicrogram quantities of peptides in a form suitable for sequence analysis is a key step in high sensitivity protein sequencing technology. Recently, methods have been developed which allow the direct, high yield, recovery of microgram amounts of sequenceable proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis. In the present publication, we describe an extension of these methods to obtain N-terminal or internal sequence information from proteins separated by isoelectric focusing in polyacrylamide gels containing immobilized pH gradients. For N-terminal sequence analysis, separated proteins were electrophoretically transferred (electroblotted) onto chemically-modified glass fiber sheets, a support compatible with Edman degradation chemistry. Transferred protein bands were detected on the support, cut out and directly inserted into the cartridge of a gas-phase protein sequenator. For internal sequence analysis, separated proteins were electrophoretically transferred onto nitrocellulose. Protein bands were detected by staining, cut out and the proteins subjected to enzymatic digestion directly on the support. The resulting cleavage fragments (peptides) were released into the supernatant where they were recovered and separated by narrow-bore reversed-phase high performance liquid chromatography for sequence analysis. The potential of this methodology is illustrated by the comparative peptide mapping of isoforms of bovine carbonic anhydrase.
Amino Acid Sequence↗
Antibodies recognizing human serum albumin are not elicited by immunization with preS2 sequences of the hepatitis B virus envelope protein.
Antibodies to the preS2 region of the hepatitis B virus (HBV) envelope protein and to human serum albumin (HSA) were allegedly detected at about the same level in sera of humans with acute or chronic hepatitis B [Hellström et al., 1986]. It was claimed that anti-HSA arises as a result of an immune response to the preS2 sequence and that it was involved in hepatocellular damage. Over 100 sera from animals and humans immunized with HBsAg containing preS2 sequences, or with synthetic peptides from the preS1, preS2, and S regions of the HBV env protein were assayed for anti-HSA. The results revealed the following: 1) Immunization with the native preS2 sequence or with unconjugated synthetic peptides derived from that sequence does not result in elicitation of anti-HSA. Therefore the alleged appearance of anti-HSA during hepatitis B cannot be directly related to an anti-preS2-specific immune response. 2) Some synthetic peptides, whether or not they were derived from the preS2 sequence, when linked to certain carriers, but not to others, elicited in rabbits an anti-HSA response, which was markedly lower than the response to the homologous peptide. These anti-HSA antibodies could be separated from anti-preS2-specific antibodies by affinity chromatography and did not recognize the synthetic peptide used for immunization. The use in active immunoprophylaxis of hepatitis B of unconjugated peptides from the preS2 sequence with proven high immunogenicity will avoid carrier/linker-mediated induction of antibodies not relevant to protection against HBV.
Animals↗
The pre-S region of hepadnavirus envelope proteins.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Role of disulfide bridges in determining the biological activity of interleukin 3.
Total chemical synthesis of analog proteins was used to examine the requirement for specific disulfide bridges for the biological activity of interleukin 3 (IL-3), a growth factor that stimulates multiple lineages of hemopoietic cells. Four structural analogs of the mature, 140 amino acid murine IL-3 molecule were synthesized in which specific cysteine residues were replaced by alanines. In a quantitative IL-3 assay, based on [3H]thymidine incorporation into factor-dependent cells, the IL-3 analog with alanines substituted for all four cysteines--i.e., [Ala17,79,80,140]IL-3--had 1/500th as much activity as the molecule synthesized according to the native sequence. The two analogs [Cys17,79,Ala80,140]IL-3 and [Cys17,140,Ala79,80]IL-3 had similarly low activity, whereas the [Cys17,80,Ala79,140]IL-3 analog had 2000-fold higher activity than these three analogs, and 3-fold higher than the molecule with the native sequence. This shows that in IL-3 a single disulfide bridge, between cysteines 17 and 80, is required for biological activity that approximates physiological levels. This disulfide probably stabilizes the tertiary structure of the protein to give a conformation that is optimal for function.
Alanine↗
Identification of a novel retroviral gene unique to human immunodeficiency virus type 2 and simian immunodeficiency virus SIVMAC.
Human and simian immunodeficiency-associated retroviruses are extraordinarily complex, containing at least five genes, tat, art, sor, R, and 3' orf, in addition to the structural genes gag, pol, and env. Recently, nucleotide sequence analysis of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus SIVMAC revealed the existence of still another open reading frame, termed X, which is highly conserved between these two viruses but absent from HIV-1. In this report, we demonstrate for the first time that the X open reading frame represents a functional retroviral gene in both HIV-2 and SIVMAC and that it encodes a virion-associated protein of 14 and 12 kilodaltons, respectively. We also describe the production of recombinant TrpE/X fusion proteins in Escherichia coli and show that sera from some HIV-2-infected individuals specifically recognize these proteins.
Amino Acid Sequence↗
Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts.
The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors.
Amino Acid Sequence↗