Localisation of gelatinase activity in epidermal hoof lamellae by in situ zymography

In situ gelatin zymography is a technique, which utilises a gelatin-based emulsion overlay to detect and, more importantly, localise the gelatinase activity in underlying tissue. Gelatinase A [matrix metalloproteinase-2 (MMP-2)] and gelatinase B [matrix metalloproteinase-9 (MMP-9)] are present in equine hoof homogenates and supernatants from cultured hoof explants by SDS-PAGE gelatin zymography, and it has been assumed that the enzymes are derived solely from matrix and epithelia and not from other sources such as leucocytes. Using in situ zymography, gelatinases are shown to be localised within the equine epidermal hoof lamellae and, more specifically, are apparently produced by epidermal basal and/or parabasal cells. The pattern of expression correlates with that expected based on the progression of pathological changes observed during the onset of laminitis, thus providing further evidence that laminitis pathology probably arises as a result of inadequate local MMP regulation.

NMR structure of C-terminally tagged gramicidin channels

A biotin group was covalently attached to the C terminus of gramicidin A (gA) through a linker arm comprising a glycine residue with either one (gAXB) or two caproyl groups (gAXXB). High-resolution two-dimensional NMR spectroscopy was used to determine the structure of these modified gA analogues and [Lys(16)]gramicidin A (gA-Lys) in sodium dodecyl-d(25) sulphate micelles. Gated gA ion channels based on linking a receptor group to these gA analogues have been used recently as a component in a sensing device. The conformations of the gA backbones and amino acid side chains of lysinated gA and biotinylated gA in detergent micelles were found to be almost identical to that of native gA, i.e. that of an N-terminal to N-terminal (head to head) dimer formed by two right-handed, single-stranded beta(6.3) helices. The biotin tail of the gAXB and gAXXB and the lysine extremity of gA-Lys appeared to lie outside the micelle. Thus it appears that the covalent attachment of functional groups to the C terminus of gA does not disrupt the peptide's helical configuration. Further, single channel measurements of all three gA analogues showed that functioning ion channels were preserved within a membrane environment. (C) 1999 Elsevier Science B.V. All rights reserved.

Temperature-induced bleaching of corals begins with impairment of the CO2 fixation mechanism in zooxanthellae

The early effects of heat stress on the photosynthesis of symbiotic dinoflagellates (zooxanthellae) within the tissues of a reef-building coral were examined using pulse-amplitude-modulated (PAM) chlorophyll fluorescence and photorespirometry. Exposure of Stylophora pistillata to 33 and 34 degrees C for 4 h resulted in (1) the development of strong non-photochemical quenching (qN) of the chlorophyll fluorescence signal, (2) marked decreases in photosynthetic oxygen evolution, and (3) decreases in optimal quantum yield (F-v/F-m) of photosystern II (PSII), Quantum yield decreased to a greater extent on the illuminated surfaces of coral branches than on lower (shaded) surfaces, and also when high irradiance intensities were combined with elevated temperature (33 degrees C as opposed to 28 degrees C), qN collapsed in heat-stressed samples when quenching analysis was conducted in the absence of oxygen, Collectively, these observations are interpreted as the initiation of photoprotective dissipation of excess absorbed energy as heat (qN) and O-2-dependent electron flow through the Mehler-Ascorbate-Peroxidase cycle (MAP-cycle) following the point at which the rate of light-driven electron transport exceeds the capacity of the Calvin cycle. A model for coral bleaching is proposed whereby the primary site of heat damage in S, pistillata is carboxylation within the Calvin cycle, as has been observed during heat damage in higher plants, Damage to PSII and a reduction in F-v/F-m (i.e. photoinhibition) are secondary effects following the overwhelming of photoprotective mechanisms by light. This secondary factor increases the effect of the primary variable, temperature. Potential restrictions of electron flow in heat-stressed zooxanthellae are discussed with respect to Calvin cycle enzymes and the unusual status of the dinoflagellate Rubisco, Significant features of our model are that (1) damage to PSII is not the initial step in the sequence of heat stress in zooxanthellae, acid (2) light plays a key secondary role in the initiation of the bleaching phenomena.

The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain

Correct placement of the division septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles, Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88, Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE(1-22) indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations, This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.

The influence of Lys68 in decapeptide agonists of C5a on C5a receptor binding, activation and selectivity

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a(65-74), Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gin (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Interaction of zinc(II) with the cyclic octapeptides, cyclo[Ile(Oxn)-D-Val(Thz)](2) and ascidiacyclamide, a cyclic peptide from Lissoclinum patella

The interactions between zinc salts and the naturally occurring cyclic octapeptide ascidiacyclamide in methanol, as well as a synthetic analogue cyclo[Ile(Oxn)-D-Val(Thz)](2), were monitored by H-1 NMR and CD spectroscopy. Three zinc complexes were identified, their relative amounts depending on the nature of the anion (perchlorate, triflate or chloride) and the presence or absence of base. Binding constants for two of the zinc species were calculated from CD or H-1 NMR spectra, [Zn(L - H)](+) (KZn(L-H) = [Zn(L - H)(+)]/[Zn2+][(L - H)(-)] = 10(7 +/- 2) M-1; 95% methanol/5% water, 298.0 K, NEt3/HClO4 buffer 0.04 M) and [ZnLCl](+) (K-ZnCIL = [ZnCIL+]/[Zn2+][Cl-][L] = 10(7.2) (+/-) (0.1) M-2; d(3)-methanol, 301 K).

Structural basis of autoregulation of phenylalanine hydroxylase

Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an alpha-beta sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.

Molecular characterization of Drosophila C virus isolates

Reverse transcription coupled with polymerase chain reaction and restriction enzyme analysis was used to characterize 12 Drosophila C virus isolates from geographically different regions. A 1.2-kb fragment was amplified from cDNA and profiles from digestion with 20 restriction enzymes were generated. Analysis of the restriction fragment data gave estimates of nucleotide divergence of 0-10% between isolates. The isolates were grouped on the basis of genetic distance estimates derived from the restriction data. For the isolates from which a single genotype could be purified, a geographical pattern in the distribution of viral genotypes was identified. The 4 Moroccan isolates were very closely related to each other, differing in only 1 restriction profile. The 2 Australian isolates were each other's closest relatives, as were the 2 isolates first recovered in France. The PCR-RFLP technique used in this study has provided us with a simple procedure which can be used to characterize DCV isolates. A single enzyme, Tag I, generated 5 distinct and diagnostic restriction fragment patterns, which allowed easy assignment of isolates to one of the five viral genotypes identified in this study. (C) 1999 Academic Press.

A novel two-chain proteinase inhibitor generated by circularization of a multidomain precursor protein

Female reproductive tissues of the ornamental tobacco amass high levels of serine proteinase inhibitors (PIs) for protection against pests and pathogens. These PIs are produced from a precursor protein composed of six repeats each with a protease reactive site. Here we show that proteolytic processing of the precursor generates five single-chain PIs and a remarkable two-chain inhibitor formed by disulfide-bond Linkage of Nand C-terminal peptide fragments. Surprisingly, PI precursors adopt this circular structure regardless of the number of inhibitor domains, suggesting this bracelet-like conformation is characteristic of the widespread potato inhibitor II (Pot II) protein family.

H-1-NMR structural studies of a cystine-linked peptide containing residues 71-93 of transthyretin and effects of a Ser84 substitution implicated in familial amyloidotic polyneuropathy

The Ile-->Ser84 substitution in the thyroid hormone transport protein transthyretin is one of over 50 variations found to be associated with familial amyloid polyneuropathy, a hereditary type of lethal amyloidosis. Using a peptide analogue of the loop containing residue 84 in transthyretin, we have examined the putative local structural effects of this substitution using H-1-NMR spectroscopy. The peptide, containing residues 71-93 of transthyretin with its termini linked via a disulfide bond, was found to possess the same helix-turn motif as in the corresponding region of the crystallographically derived structure of transthyretin in 20% trifluoroethanol (TFE) solution. It therefore, represents a useful model with which to examine the effects of amyloidogenic substitutions. In a peptide analogue containing the Ile84-->Ser substitution it was found that the substitution does not greatly disrupt the overall three-dimensional structure, but leads to minor local differences at the turn in which residue 84 is involved. Coupling constant and NOE measurements indicate that the helix-turn motif is still present, but differences in chemical shifts and amide-exchange rates reflect a small distortion. This is in keeping with observations that several other mutant forms of transthyretin display similar subunit interactions and those that have been structurally analysed possess a near native structure. We propose that the Ser84 mutation induces only subtle perturbations to the transthyretin structure which predisposes the protein to amyloid formation.

Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a

Activation of the human complement system of plasma proteins during immunological host defense can result in overproduction of potent proinflammatory peptides such as the anaphylatoxin C5a. Excessive levels of C5a are associated with numerous immunoinflammatory diseases, but there is as yet no clinically available antagonist to regulate the effects of C5a. We now describe a series of small molecules derived from the C-terminus of C5a, some of which are the most potent low-molecular-weight C5a receptor antagonists reported to date for the human polymorphonuclear leukocyte (PMN) C5a receptor. H-1 NMR spectroscopy was used to determine solution structures for two cyclic antagonists and to indicate that antagonism is related to a turn conformation, which can be stabilized in cyclic molecules that are preorganized for receptor binding. While several cyclic derivatives were of similar antagonistic potency, the most potent antagonist was a hexapeptide-derived macrocycle AcF[OPdChaWR] with an IC50 = 20 nM against a maximal concentration of C5a (100 nM) on intact human PMNs. Such potent C5a antagonists may be useful probes to investigate the role of C5a in host defenses and to develop therapeutic agents for the treatment of many currently intractable inflammatory conditions.

Crystal structure of mammalian purple acid phosphatase

Background: Mammalian purple acid phosphatases are highly conserved binuclear metal-containing enzymes produced by osteoclasts, the cells that resorb bone. The enzyme is a target for drug design because there is strong evidence that it is involved in bone resorption. Results: The 1.55 Angstrom resolution structure of pig purple acid phosphatase has been solved by multiple isomorphous replacement. The enzyme comprises two sandwiched beta sheets flanked by or-helical segments. The molecule shows internal symmetry, with the metal ions bound at the interface between the two halves. Conclusions: Despite less than 15% sequence identity, the protein fold resembles that of the catalytic domain of plant purple acid phosphatase and some serine/threonine protein phosphatases. The active-site regions of the mammalian and plant purple acid phosphatases differ significantly, however. The internal symmetry suggests that the binuclear centre evolved as a result of the combination of mononuclear ancestors. The structure of the mammalian enzyme provides a basis for antiosteoporotic drug design.

Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: Site-directed mutagenesis and kinetic studies

Sulfonation is an important metabolic process involved in the excretion and in some cases activation of various endogenous compounds and xenobiotics. This reaction is catalyzed by a family of enzymes named sulfotransferases. The cytosolic human sulfotransferases SULT1A1 and SULT1A3 have overlapping yet distinct substrate specificities. SULT1A1 favors simple phenolic substrates such as p-nitrophenol, whereas SULT1A3 prefers monoamine substrates such as dopamine. In this study we have used a variety of phenolic substrates to functionally characterize the role of the amino acid at position 146 in SULT1A1 and SULT1A3. First, the mutation A146E in SULT1A1 yielded a SULT1A3-like protein with respect to the Michaelis constant for simple phenols. The mutation E146A in SULT1A3 resulted in a SULT1A1-like protein with respect to the Michaelis constant for both simple phenols and monoamine compounds. When comparing the specificity of SULT1A3 toward tyramine with that for p-ethylphenol (which differs from tyramine in having no amine group on the carbon side chain), we saw a 200-fold preference for tyramine. The kinetic data obtained with the E146A mutant of SULT1A3 for these two substrates clearly showed that this protein preferred substrates without an amine group attached. Second, changing the glutamic acid at position 146 of SULT1A3 to a glutamine, thereby neutralizing the negative charge at this position, resulted in a 360-fold decrease in the specificity constant for dopamine. The results provide strong evidence that residue 146 is crucial in determining the substrate specificity of both SULT1A1 and SULT1A3 and suggest that there is a direct interaction between glutamic acid 146 in SULT1A3 and monoamine substrates.

Crystallization and preliminary X-ray diffraction studies of mammalian purple acid phosphatase

The oxidized form of purple acid phosphatase from pig allantoic fluid has been crystallized in the presence of phosphate using the hanging-drop technique. The crystals belong to the space group P2(1)2(1)2(1) and have unit-cell parameters a = 66.8, b = 70.3, c = 78.7 Angstrom. Diffraction data collected from a cryocooled crystal using a conventional X-ray source extend to 1.55 Angstrom resolution. A knowledge of the three-dimensional structure of mammalian purple acid phosphatase will aid in understanding the substrate specificity of the enzyme and will be important in the rational design of inhibitors, with potential in the treatment of bone diseases.