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Biomedical subjects

M H Lambert

Publications and source records attributed to M H Lambert.

31 records · Page 2Linked to original sources
PubMed1999

Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.

ADAM Proteins↗
PubMed1999

Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids (FAs) that regulate glucose and lipid homeostasis. We report the crystal structure of the PPAR delta ligand-binding domain (LBD) bound to either the FA eicosapentaenoic acid (EPA) or the synthetic fibrate GW2433. The carboxylic acids of EPA and GW2433 interact directly with the activation function 2 (AF-2) helix. The hydrophobic tail of EPA adopts two distinct conformations within the large hydrophobic cavity. GW2433 occupies essentially the same space as EPA bound in both conformations. These structures provide molecular insight into the propensity for PPARs to interact with a variety of synthetic and natural compounds, including FAs that vary in both chain length and degree of saturation.

Animals↗
PubMed1998

Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-gamma ligand-binding domain (LBD), at 2.2 A resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-gamma. We also describe the ternary complex containing the PPAR-gamma LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 A resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a 'charge clamp' that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-gamma homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.

Amino Acid Sequence↗
PubMed1997

Reciprocal size-effect relationship of the key residues in determining regio- and stereospecificities of DHEA hydroxylase activity in P450 2a5.

Collectively, the P450 2a4/2a5 system hyrdoxylates DHEA in at least three positions (7alpha, 7beta, and 2alpha). An individual P450, however, exhibits high specificity to one of these products. Using site-directed mutagenesis of mP450 2a5 from the wild mouse Mus minutoides and bacterial expression, we have associated the function of residues 117, 209, and 481 with the respective specificity observed in each P450. Ala at position 117 determines the 7beta-hydroxylase activity, whereas Val at this position defines the 2alpha-hydroxylase activity. Leu at position 209 is essential for high DHEA 7alpha-hydroxylase activity. The substitutions of residue 481 with various hydrophobic amino acids elicited a profound alteration of the specific hydroxylation rates, but did not influence the regio- and stereospecificities at either of the three positions of DHEA. The alterations caused by residue 481 also depended on the residue identity at position 117 or 209. The results indicate that the sizes of several key residues obey a concerted reciprocal relationship whereby the substrate pocket of the P450s adjusts to accommodate DHEA. A limited molecular modeling study successfully correlates DHEA binding to experimental DHEA hydroxylase activities for a series of mutants at key positions.

Amino Acid Sequence↗
PubMed1997

Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.

Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.

ADAM Proteins↗
PubMed1994

Substrate specificity in short-chain phospholipid analogs at the active site of human synovial phospholipase A2.

The substrate specificity at the active site of recombinant human synovial fluid phospholipase A2 (hs-PLA2) was investigated by the preparation of a series of short-chain phospholipid analogs and measurement of their enzymatic hydrolysis at concentrations well below the critical micelle concentration. Substrates used in the study included 1,2-dihexanoylglycerophospholipids, 1,2-bis(alkanoylthio)glycerophospholipids, and 1-O-alkyl-2-(alkanoylthio)phospholipids. Turnover was observed for only a few of the 1,2-dihexanoylglycerophospholipids, and the rate of hydrolysis was very low, near the limit of detection of the assay. In contrast, selected 2-(alkanoylthio)-glycerophospholipids were hydrolyzed by hs-PLA2 at much higher rates at concentrations well below their critical micelle concentration (cmc). Thus, the 1,2-bis(hexanoylthio)glycerophosphatidylmethanol exhibits a k(cat)/K(M) = 1800 L mol-1 s-1. Over the calculated log P (cLogP) range of 3-9, cLogP and log(k(cat)/K(M) were linearly related for compounds with straight-chain sn-1 and sn-2 substituents. At comparable cLogP's, the sn-1 ethers and thioesters were hydrolyzed at comparable rates. A negative charge in the phosphate head group was required for enzyme activity. Unsaturation, aromaticity, and branching in the sn-2 substituent reduce turnover dramatically. The same structural modifications in the sn-1 substituent have less effect on turnover. Certain of these substrates, e.g., 1,2-bis(hexanoylthio)glycerophosphatidylmethanol, may be useful in assaying for active site inhibitors of PLA2. The structure--activity relationships established here for substrates should serve as a reference for the structure--activity relationships of substrate-based inhibitors.

Binding Sites↗
PubMed1994

Structure of the catalytic domain of fibroblast collagenase complexed with an inhibitor.

Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.

Amino Acid Sequence↗
PubMed1993

A novel dimer configuration revealed by the crystal structure at 2.4 A resolution of human interleukin-5.

Interleukin-5 (IL-5) is a lineage-specific cytokine for eosinophilpoiesis and plays an important part in diseases associated with increased eosinophils, such as asthma. Human IL-5 is a disulphide-linked homodimer with 115 amino-acid residues in each chain. The crystal structure at 2.4 A resolution reveals a novel two-domain structure, with each domain showing a striking similarity to the cytokine fold found in granulocyte macrophage and macrophage colony-stimulating factors, IL-2 (ref. 5), IL-4 (ref. 6), and human and porcine growth hormones. IL-5 is unique in that each domain requires the participation of two chains. The IL-5 structure consists of two left-handed bundles of four helices laid end to end and two short beta-sheets on opposite sides of the molecule. Surprisingly, the C-terminal strand and helix of one chain complete a bundle of four helices and a beta-sheet with the N-terminal three helices and one strand of the other chain. The structure of IL-5 provides a molecular basis for the design of antagonists and agonists that would delineate receptor recognition determinants critical in signal transduction. This structure determination extends the family of the cytokine bundle of four helices and emphasizes its fundamental significance and versatility in recognizing its receptor.

Animals↗
PubMed1989

Cardiac arrhythmias modelled by Cai-inactivated Ca2+ channels.

A model of cardiac cells incorporating the membrane potential and the intracellular calcium concentration as the two dynamical variables is developed. This model is applied to simple systems of cells to investigate its behavior as a function of the model parameters. Rational entrainment is observed in systems of two coupled pacemaker cells. The propagation of the membrane potential and intracellular calcium concentration through a sheet is simulated. Behavior suggestive of circus movement tachycardias is observed.

Arrhythmias, Cardiac↗
PubMed1989

A comparison of the CHARMM, AMBER and ECEPP potentials for peptides. II. Phi-psi maps for N-acetyl alanine N'-methyl amide: comparisons, contrasts and simple experimental tests.

phi-psi maps of N-acetyl alanine N'-methyl amide have been computed using the CHARMM potential, the all-atom AMBER potential, and the ECEPP/2 potential, before and after adiabatic relaxation. Maps using the CHARMM and AMBER potentials were determined with values of 1.0 and 4.0 for the dielectric constant epsilon, and with a distance dependent dielectric constant. Adiabatic relaxation was carried out using flexible geometry for the CHARMM and AMBER potentials, and using rigid geometry for the AMBER and ECEPP potentials. In all cases, the lowest energy was found in the C7eq region (phi approximately -70 degrees, psi approximately 70 degrees). The maps with CHARMM and AMBER with epsilon = 4.0 and with ECEPP, without adiabatic relaxation, were broadly similar but differed in the relative energies allotted to high-energy regions of the map. After adiabatic relaxation with rigid geometry, the map with ECEPP, and the map with AMBER using a distance-dependent dielectric constant, agreed fairly well apart from differences in the relative energies of the alpha R, alpha L, and C7ax regions. After adiabatic relaxation with flexible geometry, the maps with CHARMM and AMBER became very similar; the lowest energies were observed in the C7eq region, the C5 region (phi approximately -150 degrees, psi approximately 150 degrees) and the C7ax region (phi approximately 70 degrees, psi approximately -70 degrees). Breakdown of the energies, after adiabatic relaxation, into electrostatic, nonbonded, and geometric (including torsional) contributions, showed that (1) with fixed geometry, the nonbonded and torsional contribution to the ECEPP and AMBER potentials were very similar, but the electrostatic contributions were markedly different; (2) with flexible geometry, the nonbonded contribution to the CHARMM and AMBER potentials did not vary greatly over the whole map. The phi-psi maps were subjected to three simple comparisons with experiment. (1) The maps were used to predict the characteristic ratio for poly-L-alanine, and the results were compared with experimental findings (D.A. Brant and P.J. Flory, J. Amer. Chem. Soc. 87, 2788-2791, 1965). The agreement with experiment was acceptable for ECEPP, and for CHARMM after adiabatic relaxation, marginal for AMBER after adiabatic relaxation, and unsatisfactory for CHARMM or AMBER without adiabatic relaxation. (2) Deviations of bond angles from their equilibrium values, in energy-minimized conformations, were compared with values deduced from crystals of terminally-blocked amino acids. With both the CHARMM and AMBER potentials using flexible geometry, one or more excessive deviations was observed in the C7ax local minimum.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗