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R L Heinrikson

Publications and source records attributed to R L Heinrikson.

At least 37 records · Page 2Linked to original sources
PubMed1996

Identification of signal-induced IkappaB-alpha kinases in human endothelial cells.

Activation of the nuclear transcription factor-kappaB is an early event in endothelial activation. NF-kappaB activation is regulated by the inducible phosphorylation and subsequent degradation of the inhibitory subunit IkappaB-alpha. We identified two discrete kinases of approximately 36 and 41 kDa in the cytoplasm of human umbilical vein endothelial cells that specifically bind to and phosphorylate the IkappaB-alpha subunit. IkappaB-alpha kinase activity is transiently elevated following treatment with either tumor necrosis factor alpha, interleukin-1beta, or bacterial lipopolysaccharides and precedes activation of either mitogen-activated kinase or Jun kinase. Furthermore, activation of the IkappaB-alpha kinases precedes both the appearance of hyperphosphorylated IkappaB-alpha and its subsequent degradation, as well as the translocation of NF-kappaB to the nucleus. Deletion mutagenesis of the IkappaB-alpha polypeptide revealed that these kinases bind in or around the ankyrin repeat domains and phosphorylate residues within the C terminus. These kinases, however, were not identical to casein kinase II and displayed a pharmacologic profile distinct from other known kinases. These kinases may represent components of a signal transduction pathway regulating IkappaB-alpha levels in vascular endothelium.

Ankyrins↗
PubMed1996

Predicting human immunodeficiency virus protease cleavage sites in proteins by a discriminant function method.

Based on the sequence-coupled (Markov chain) model and vector-projection principle, a discriminant function method is proposed to predict sites in protein substrates that should be susceptible to cleavage by the HIV-1 protease. The discriminant function is defined by delta = phi+ - phi-, where phi+ and phi- are the cleavable and noncleavable attributes for a given peptide, and they can be derived from two complementary sets of peptides, S+ and S-, known to be cleavable and noncleavable, respectively, by the enzyme. The rate of correct prediction by the method for the 62 cleavable peptides and 239 noncleavable peptides in the training set are 100 and 96.7%, respectively. Application of the method to the 55 sequences which are outside the training set and known to be cleaved by the HIV-1 protease accurately predicted 100% of the peptides as substrates of the enzyme. The method also predicted all but one of the sites hydrolyzed by the protease in native HIV-1 and HIV-2 reverse transcriptases, where the HIV-1 protease discriminates between nearly identical sequences in a very subtle fashion. Finally, the algorithm predicts correctly all of the HIV-1 protease processing sites in the native gag and gag/pol HIV-1 polyproteins, and all of the cleavage sites identified in denatured protease and reverse transcriptase. The new predictive algorithm provides a novel route toward understanding the specificity of this important therapeutic target.

Algorithms↗
PubMed1995

Purification and structural characterization of the CD11b/CD18 integrin alpha subunit I domain reveals a folded conformation in solution.

The alpha subunits of the leukocyte CD11/CD18 integrins contain an approximately 200 amino acid 'inserted' or I domain. The I domain of the cell-surface Mac-1 (CD11b/CD18) integrin has been shown to be the major recognition site for several adhesion ligands, including iC3b, fibrinogen, factor X, and ICAM-1. The I domain from the Mac-1 alpha subunit has been expressed in Escherichia coli as a soluble GST-fusion protein containing a factor Xa sensitive cleavage site. Analytical characterization of the purified I domain reveals that it is obtained in very high quality at high yields. CD and NMR spectra indicate that I domain adopts a predominantly folded structure in solution, independent of the remainder of the alpha subunit. Addition of Ca2+ and Mg2+ did not significantly perturb the structural conformation.

Amino Acid Sequence↗
PubMed1995

The differential processing of homodimers of reverse transcriptases from human immunodeficiency viruses type 1 and 2 is a consequence of the distinct specificities of the viral proteases.

Active, recombinant p68 reverse transcriptase (RT) from human immunodeficiency virus type 2 (HIV-2), with an NH2-terminal extension containing a hexahistidine sequence was isolated from extracts of Escherichia coli by immobilized metal affinity chromatography. Treatment of the purified p68/p68 homodimer of HIV-2 RT with recombinant HIV-2 protease generates stable, active heterodimer (p68/p58) that is resistant to further hydrolysis. Analysis of this p68/p58 HIV-2 RT heterodimer revealed that while one subunit is intact p68, the p58 subunit is COOH-terminally truncated by cleavage, not at Phe440 as is seen in processing of the p66/p66 HIV-1 RT homodimer by HIV-1 protease, but at Met484. The expected COOH-terminal p10 fragment resulting from hydrolysis of p68 at Met484 is not released intact, but undergoes further cleavage at Asn494, Met503, and Tyr532. Processing of p68/p68 HIV-2 RT with the HIV-1 protease led to cleavage of the Phe440-Tyr441 bond, exactly as is seen with p66/p66 HIV-1 RT, to give the analogous p53 subunit. Studies of a peptide substrate modeled after residues 437-444 in HIV-2 RT showed that while the HIV-1 protease was able to cleave the Phe440 bond, this bond was resistant to cleavage by the HIV-2 enzyme. Our findings provide a rationale for the previous observation that the RT heterodimer isolated from HIV-2 lysates is larger than that from HIV-1. We conclude that the p68/p58 HIV-2 RT heterodimer, containing the Met484 truncated p58 subunit, is a biologically relevant form of the enzyme in vivo.

Amino Acid Sequence↗
PubMed1995

CXC chemokines connective tissue activating peptide-III and neutrophil activating peptide-2 are heparin/heparan sulfate-degrading enzymes.

Heparan sulfate proteoglycans at cell surfaces or in extracellular matrices bind diverse molecules, including growth factors and cytokines, and it is believed that the activities of these molecules may be regulated by the metabolism of heparan sulfate. In this study, purification of a heparan sulfate-degrading enzyme from human platelets led to the discovery that the enzymatic activity residues in at least two members of the platelet basic protein (PBP) family known as connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2. PBP and its N-truncated derivatives, CTAP-III and neutrophil activating peptide-2, are CXC chemokines, a group of molecules involved in inflammation and wound healing. SDS-polyacrylamide gel electrophoresis analysis of the purified heparanase resulted in a single broad band at 8-10 kDa, the known molecular weight of PBP and its truncated derivatives. Gel filtration chromatography of heparanase resulted in peaks of activity corresponding to monomers, dimers, and tetramers; these higher order aggregates are known to form among the chemokines. N-terminal sequence analysis of the same preparation indicated that only PBP and truncated derivatives were present, and commercial CTAP-III from three suppliers had heparanase activity. Antisera produced in animals immunized with a C-terminal synthetic peptide of PBP inhibited heparanase activity by 95%, compared with activity of the purified enzyme in the presence of the preimmune sera. The synthetic peptide also inhibited heparanase by 95% at 250 microM, compared to the 33% inhibition of heparanase activity by two other peptides. The enzyme was determined to be an endoglucosaminidase, and it degraded both heparin and heparan sulfate with optimal activity at pH 5.8. Chromatofocusing of the purified heparanase resulted in two protein peaks: an inactive peak at pI7.3, and an active peak at pI 4.8-5.1. Sequence analysis showed that the two peaks contained identical protein, suggesting that a post-translational modification activates the enzyme.

Amino Acid Sequence↗
PubMed1995

Effect of point mutations on the kinetics and the inhibition of human immunodeficiency virus type 1 protease: relationship to drug resistance.

Mutations of human immunodeficiency virus type 1 (HIV-1) protease at four positions, Val82, Asp30, Gly48, and Lys45 were analyzed for the resulting effects on kinetics and inhibition. In these mutants, Val82 was substituted separately by Asn, Glu, Ala, Ser, Asp, and Gln; Asp30 was individually substituted by Phe or Trp; Gly48 by His, Asp, and Tyr, respectively; and Lys45 by Glu. By examination of the inhibition of a single inhibitor, the differences in Ki values between the native and mutant enzymes can range from very large to insignificant even for the mutants with substitutions at the same position. By examination of a single mutant enzyme, the same broad range of Ki changes was observed for a group of inhibitors: Thus, how much the inhibition changes from the wild-type enzyme to a mutant is dependent on both the mutation and the inhibitor. The examination of Ki changes of inhibitors with closely related structures binding to Val82 mutants also reveals that the change of inhibition involves subsites in which Val82 is not in direct contact, indicating a considerable flexibility of the conformation of HIV protease. For the catalytic activities of the mutants, the kcat and Km values of many Val82 mutants and a Lys45 mutant are comparable to the native enzyme. Surprisingly, Gly48 mutations produce enzymes with catalytic efficiency superior to that of the wild-type enzyme by as much as 10-fold. Modeling of the structure of the mutants suggests that the high catalytic efficiency of some substrates is related to an increase of rigidity of the flap region of the mutants. The examination of the relative changes of inhibition and catalysis of mutants suggests that some of the Val82 and Gly48 mutants are potential resistance mutants. However, the resistance is specific with respect to individual inhibitors.

Amino Acid Sequence↗
PubMed1993

An active FK506-binding domain of 17,000 daltons is isolated following limited proteolysis of chicken thymus hsp56.

We have previously identified hsp56, a protein component of steroid receptor complexes, as an FK506 binding protein [Yem et al. (1992) J. Biol. Chem. 267, 2868-2871]. We now report that hsp56 is also found to be a major immunophilin in chicken thymus, by virtue of binding to FK506-Affi-Gel-10 as well as positive cross-reactivity with a polyclonal antiserum directed against human hsp56. Limited digests of purified chicken hsp56 with endoproteinase Lys C result in the production of a unique polypeptide having a mass of about 17 kDa (p17), as judged by Western blotting. Peptide mapping provided additional proof that p17 is a fragment which comprises the entire FK506 binding domain I of chicken hsp56, terminating with an Arg-Lys which might represent a processing site. Binding of radiolabeled dihydro FK506 to p17 is saturable with a calculated KD of 42 nM. Since size exclusion chromatography of drug-p17 complexes indicates that the active species is a homodimer with a mass of 30-40 kDa, the stoichiometry calculated for the drug-protein complex is approximately 1:1. Furthermore, unlike FKBP-12, chicken p17 bound to FK506 does not bind to calcineurin-calmodulin complexes. This work demonstrates the excision of a domain from an hsp56 protein that is active in binding FK506 and functionally distinct from FKBP-12, a protein of similar size and structure.

Amino Acid Sequence↗
PubMed1993

The crystallographic structure of the protease from human immunodeficiency virus type 2 with two synthetic peptidic transition state analog inhibitors.

The crystal structure of human immunodeficiency virus (HIV) type 2 protease has been determined in complexes with peptidic inhibitors Noa-His-Cha psi [CH(OH)CH(OH)]Val-Ile-Amp (U75875) and Qnc-Asn-Cha psi [CH(OH)CH2]Val-Npt(U92163) (where Noa is naphthyloxyacetyl, Cha is cyclohexylalanine, Amp is 2-aminomethylpyridine, Qnc is quinoline-2-carbonyl, and Npt is neopentylamine), which have dihydroxyethylene and hydroxyethylene moieties, respectively, in place of the normal scissile bond of the natural ligand. The complexes crystallize in space group P2(1)2(1)2(1), with one dimer-inhibitor complex per asymmetric unit and average cell dimensions of a = 33.28 A, b = 45.35 A, c = 135.84 A. Data were collected to approximately 2.5-A resolution. The model structures were refined with resulting R-factors of around 0.19. As expected, the HIV-2 protease structure is approximately C2-symmetric with a gross structure very similar to that of the HIV-1 enzyme. The inhibitors bind in an extended conformation positioned lengthwise in the binding cleft in a manner similar to that found in the HIV-1 protease-inhibitor complexes previously reported. The substitution of the bulkier Ile82 side chain in the HIV-2 protease may help explain the better ability of HIV-2 protease to bind and hydrolyze ligands with small P1 and P1' side groups. It appears that differences in specificity between the proteases of HIV-1 and HIV-2 are not merely a result of simple side chain substitutions, but may be complicated by differences in main chain flexibility as well.

Aspartic Acid Endopeptidases↗
PubMed1993

Inhibitors of the protease from human immunodeficiency virus: synthesis, enzyme inhibition, and antiviral activity of a series of compounds containing the dihydroxyethylene transition-state isostere.

A number of potential HIV protease inhibitory peptides that contain the dihydroxyethylene isostere were prepared and evaluated for their enzyme binding affinity and antiviral activity in cell cultures. From the template of a previously reported active peptide A, modifications at the N- and C-terminal groups were assessed for potential maintenance of good inhibitory activity of the resulting peptides. Among the active peptides found, peptide XVIII exhibited potent enzyme inhibitory activity. Interestingly, the previously reported, effective 1(S)-amino-2(R)-hydroxyindan C-terminal group for the preparation of very active HIV protease inhibitory peptides could not be applied to the template of peptide XVIII. Molecular modeling of peptide XVIII was studied using the X-ray crystal structure of peptide A as a starting point in order to study the likely conformation of peptide XVIII in the active-site cleft. Relative binding conformations of peptide A and XVIII were obtained, although the reason for poor binding affinity for a number of congeneric peptides in this report was not straightforwardly apparent. More importantly, however, peptide XVIII was found to exhibit more effective antiviral activity in the HIV-1/PBMC assay than the reference peptide A which was previously reported to be approximately equal in efficacy to the reverse transcriptase inhibitor AZT in this assay.

Animals↗
PubMed1993

Structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in Alzheimer's disease.

The structure of beta-amyloid (beta A) from Alzheimer disease brains was examined to determine if post-translational modifications might be linked to the abnormal deposition of this peptide in the diseased tissue. The beta A peptides were isolated from the compact amyloid cores of neuritic plaques and separated from minor glycoprotein components by size-exclusion high-pressure liquid chromatography (HPLC). This parenchymal beta A has a maximal length of 42 residues, but shorter forms with "ragged" NH2 termini are also present. Tryptic peptide analysis revealed heterogeneity in the beta A1-5 and beta A6-16 peptides, each of which eluted as four peaks on reverse phase HPLC. Amino acid composition and sequence analyses, mass spectrometry, enzymatic methylation, and stereoisomer determinations revealed that these multiple peptide forms resulted from structural rearrangements of the aspartyl residues at beta A positions 1 and 7. The L-isoaspartyl form predominates at each of these positions, whereas the D-isoaspartyl, L-aspartyl, and D-aspartyl forms are present in lesser amounts. beta A purified from the leptomeningeal microvasculature contains the same structural alterations as parenchymal beta A, but is 2 residues shorter at its COOH terminus. Using two different purification protocols, and using a synthetic beta A1-42 peptide as a control, we show that these modifications arose endogenously and were not caused by the experimental manipulations. The abundance of structurally altered aspartyl residues may profoundly affect the conformation of the beta A protein within plaque cores and thus significantly impact normal catabolic processes designed to limit its deposition. These alterations may therefore contribute to the production and stability of beta-amyloid deposits in Alzheimer brain tissue.

Alzheimer Disease↗
PubMed1993

Human immunodeficiency virus type-1 reverse transcriptase and ribonuclease H as substrates of the viral protease.

A study has been made of the susceptibility of recombinant constructs of reverse transcriptase (RT) and ribonuclease H (RNase H) from human immunodeficiency virus type 1 (HIV-1) to digestion by the HIV-1 protease. At neutral pH, the protease attacks a single peptide bond, Phe440-Tyr441, in one of the protomers of the folded, active RT/RNase H (p66/p66) homodimer to give a stable, active heterodimer (p66/p51) that is resistant to further hydrolysis (Chattopadhyay, D., et al., 1992, J. Biol. Chem. 267, 14227-14232). The COOH-terminal p15 fragment released in the process, however, is rapidly degraded by the protease by cleavage at Tyr483-Leu484 and Tyr532-Leu533. In marked contrast to this p15 segment, both p66/p51 and a folded RNase H construct are stable to breakdown by the protease at neutral pH. It is only at pH values around 4 that these latter proteins appear to unfold and, under these conditions, the heterodimer undergoes extensive proteolysis. RNase H is also hydrolyzed at low pH, but cleavage takes place primarily at Gly436-Ala437 and at Phe440-Tyr441, and only much more slowly at residues 483, 494, and 532. This observation can be reconciled by inspection of crystallographic models of RNase H, which show that residues 483, 494, and 532 are relatively inaccessible in comparison to Gly436 and Phe440. Our results fit a model in which the p66/p66 homodimer exists in a conformation that mirrors that of the heterodimer, but with a p15 segment on one of the protomers that is structurally disordered to the extent that all of its potential HIV protease cleavage sites are accessible for hydrolysis.

Amino Acid Sequence↗
PubMed1993

An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom of Agkistrodon p. piscivorus.

Phospholipases A2 may exist in solution both as monomers and dimers, but enzymes that form strong dimers (KD approximately 10(-9) M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A2 from Agkistrodon piscivorus piscivorus (American cottonmouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A2 are highly homologous to the dimeric phospholipases A2 from Crotalus atrox and Crotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phospholipases A2 that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116-122). The other is a low isoelectric point; only the acidic phospholipases A2 have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A2 dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A2 from A.p. piscivorus venom.

Amino Acid Sequence↗
PubMed1993

Large scale purification and refolding of HIV-1 protease from Escherichia coli inclusion bodies.

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered in Escherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-1 protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band of M(r) approximately 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.

Acetates↗
PubMed1993

Sequence analysis and expression of the cDNA for the phenol-sulfating form of human liver phenol sulfotransferase.

A cDNA encoding the human liver phenol-sulfating form of phenol sulfotransferase (P-PST) has been isolated and characterized from a lambda Uni-Zap XR human liver cDNA library. P-PST is the major form of phenol sulfotransferase involved in drug and xenobiotic metabolism in human liver. P-PST is also responsible for the sulfation and activation of minoxidil to its therapeutically active sulfate ester. The full length cDNA, P-PST-1, is 1206 base pairs in length and encodes a 295-amino acid protein with a molecular mass of 34,097 Da. The translation sequence of P-PST-1 is 96% similar to the amino acid sequences of five peptides derived from the purified protein. In vitro transcription and translation of P-PST-1 generated a protein that comigrates with immunoreactive P-PST from human liver. Significant increases in sulfotransferase activity toward two P-PST-specific substrates, minoxidil and 4-nitrophenol, were detected in cytosol prepared from COS-7 cells transfected with P-PST-1 in the expression vector p-SV-SPORT-1. Northern blot analysis of human liver RNA detected a transcript of approximately 1300 nucleotides in length. Characterization of P-PST at the molecular level provides insight into the structure and heterogeneity of this major class of drug-metabolizing enzymes.

Amino Acid Sequence↗
PubMed1992

Alzheimer's disease and control brain contain soluble derivatives of the amyloid protein precursor that end within the beta amyloid protein region.

The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta. Our study was designed to determine whether the soluble APP derivatives in human brain end within the A beta as described in cell culture or whether AD brain produces potentially amyloidogenic soluble derivatives that contain the entire A beta. We find that both AD and control brain contain nonamyloidogenic soluble derivatives that end at position 15 of the A beta. We have been unable to detect any soluble derivatives that contain the entire A beta in either the AD or control brain.

Alzheimer Disease↗