An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: Relationship to cystatins and the tumor suppressor TIG1

Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 Angstrom resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.

Prediction of protein B-factor profiles

The polypeptide backbones and side chains of proteins are constantly moving due to thermal motion and the kinetic energy of the atoms. The B-factors of protein crystal structures reflect the fluctuation of atoms about their average positions and provide important information about protein dynamics. Computational approaches to predict thermal motion are useful for analyzing the dynamic properties of proteins with unknown structures. In this article, we utilize a novel support vector regression (SVR) approach to predict the B-factor distribution (B-factor profile) of a protein from its sequence. We explore schemes for encoding sequences and various settings for the parameters used in SVR. Based on a large dataset of high-resolution proteins, our method predicts the B-factor distribution with a Pearson correlation coefficient (CC) of 0.53. In addition, our method predicts the B-factor profile with a CC of at least 0.56 for more than half of the proteins. Our method also performs well for classifying residues (rigid vs. flexible). For almost all predicted B-factor thresholds, prediction accuracies (percent of correctly predicted residues) are greater than 70%. These results exceed the best results of other sequence-based prediction methods. (C) 2005 Wiley-Liss, Inc.

A new method for identification of protein (sub)families in a set of proteins based on hydropathy distribution in proteins

Structural similarity among proteins is reflected in the distribution of hydropathicity along the amino acids in the protein sequence. Similarities in the hydropathy distributions are obvious for homologous proteins within a protein family. They also were observed for proteins with related structures, even when sequence similarities were undetectable. Here we present a novel method that employs the hydropathy distribution in proteins for identification of (sub)families in a set of (homologous) proteins. We represent proteins as points in a generalized hydropathy space, represented by vectors of specifically defined features. The features are derived from hydropathy of the individual amino acids. Projection of this space onto principal axes reveals groups of proteins with related hydropathy distributions. The groups identified correspond well to families of structurally and functionally related proteins. We found that this method accurately identifies protein families in a set of proteins, or subfamilies in a set of homologous proteins. Our results show that protein families can be identified by the analysis of hydropathy distribution, without the need for sequence alignment. (C) 2005 Wiley-Liss, Inc.

Expression analysis of a human hepatic cell line in response to palmitate

Saturated fat plays a role in common debilitating diseases such as obesity, type 2 diabetes, and coronary heart disease. It is also clear that certain fatty acids act as regulators of metabolism via both direct and indirect signalling of target tissues. As the molecular mechanisms of saturated fatty acid signalling in the liver are poorly defined, hepatic gene expression analysis was undertaken in a human hepatocyte cell line after incubation with palmitate. Profiling of mRNA expression using cDNA microarray analysis revealed that 162 of approximately 18,000 genes tested were differentially expressed after incubation with palmitate for 48 h. Altered transcription profiles were observed in a wide variety of genes, including genes involved in lipid and cholesterol transport, cholesterol catabolism, cell growth and proliferation, cell signalling, P-oxidation, and oxidative stress response. While palinitate signalling has been examined in pancreatic beta-cells, this is the first report showing that palmitate regulates expression of numerous genes via direct molecular signalling mechanisms in liver cells. (C) 2005 Elsevier Inc. All rights reserved.

Structural plasticity in MS channels

Gating of the mechanosensitive channel MscS involves cooperative action of glycine and alanine residues along the pore-lining transmembrane helix. Opening of the channel is facilitated by an iris-like rotation and tilt of the pore-lining helices. Site-directed mutagenesis indicates that substantial structural plasticity can be tolerated by MscS without impairing its function.

The combined SPE : ToxY-PAM phytotoxicity assay; application and appraisal of a novel biomonitoring tool for the aquatic environment

Mounting concerns regarding the environmental impact of herbicides has meant a growing requirement for accurate, timely information regarding herbicide residue contamination of, in particular, aquatic systems. Conventional methods of detection remain limited in terms of practicality due to high costs of operation and the specialised information that analysis provides. A new phytotoxicity bioassay was trialled for the detection of herbicide residues in filter-purified (Milli-Q) as well as natural waters. The performance of the system, which combines solid-phase extraction (SPE) with the ToxY-PAM dual-channel yield analyser (Heinz Walz GmbH), was tested alongside the traditional method of liquid chromatography-mass spectrometry (LC-MS). The assay methodology was found to be highly sensitive (LOD 0.1 ng L-1 diuron) with good reproducibility. The study showed that the assay protocol is time effective and can be employed for the aquatic screening of herbicide residues in purified as well as natural waters.

Functional characterization and genomic organization of the human Na+-sulfate cotransporter hNaS2 gene (SLC13A4)

Sulfate plays an essential role in human growth and development. Here, we characterized the functional properties of the human Na+-sulfate cotransporter (hNaS2), determined its tissue distribution, and identified its gene (SLC13A4) structure. Expression of hNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by thiosulfate, phosphate, molybdate. selenate and tungstate, but not by oxalate, citrate, succinate, phenol red or DIDS. Transport kinetics of hNaS2 determined a K, for sulfate of 0.38 mM, suggestive of a high affinity sulfate transporter. Na+ kinetics determined a Hill coefficient of 1.6 +/- 0.6, suggesting a Na: SO42- stoichiometry of 2:1. hNaS2 mRNA was highly expressed in placenta and testis, with intermediate levels in brain and lower levels found in the heart, thymus, and liver. The SLC13A4 gene contains 16 exons, spanning over 47 kb in length. Its 5'-flanking region contains CAAT- and GC-box motifs, and a number of putative transcription factor binding sites, including GATA-1, AP-1, and AP-2 consensus sequences. This is the first study to characterize hNaS2 transport kinetics, define its tissue distribution, and resolve its gene (SLC13A4) structure and 5' flanking region. (C) 2004 Elsevier Inc. All rights reserved.

Mechanisms of acetohydroxyacid synthases

Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

Conformational changes involved in MscL channel gating measured using FRET spectroscopy

We demonstrate that fluorescence resonance energy transfer spectroscopy is a powerful tool for in situ structural analysis of multimeric membrane proteins by measuring the conformational changes involved in gating the mechanosensitive ion channel of large conductance. Ensemble analysis is used to analyze the intensity of light emitted by AlexaFluor-labeled cysteine mutants reconstituted into artificial liposomes before and after acceptor photobleaching. The diameter of the protein is found to increase by 16 angstrom upon channel activation.

Effect of osmolytes on the interaction of flavin adenine dinucleotide with muscle glycogen phosphorylase b

The effect of three osmolytes, trimethylamine N-oxide (TMAO), betaine and proline, on the interaction of muscle glycogen phosphorylase b with allosteric inhibitor FAD has been examined. In the absence of osmolyte, the interaction is described by a single intrinsic dissociation constant (17.8 muM) for two equivalent and independent binding sites on the dimeric enzyme. However, the addition of osmolytes gives rise to sigmoidal dependencies of fractional enzyme-site saturation upon free inhibitor concentration. The source of this cooperativity has been shown by difference sedimentation velocity to be an osmolyte-mediated isomerization of phosphorylase b to a smaller dimeric state with decreased affinity for FAD. These results thus have substantiated a previous inference that the tendency for osmolyte-enhanced self-association of dimeric glycogen phosphorylase b in the presence of AMP was being countered by the corresponding effect of molecular crowding on an isomerization of dimer to a smaller, nonassociating state. (C) 2004 Elsevier Ltd. Inc. All rights reserved.

Structure-activity relationships for a new family of sulfonylurea herbicides

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyzes the first common step in branched-chain amino acid biosynthesis. The enzyme is inhibited by several chemical classes of compounds and this inhibition is the basis of action of the sulfonylurea and imidazolinone herbicides. The commercial sulfonylureas contain a pyrimidine or a triazine ring that is substituted at both meta positions, thus obeying the initial rules proposed by Levitt. Here we assess the activity of 69 monosubstituted sulfonylurea analogs and related compounds as inhibitors of pure recombinant Arabidopsis thaliana AHAS and show that disubstitution is not absolutely essential as exemplified by our novel herbicide, monosulfuron (2-nitro-N-(4'-methyl-pyrimidin-2'-yl) phenyl-sulfonylurea), which has a pyrimidine ring with a single meta substituent. A subset of these compounds was tested for herbicidal activity and it was shown that their effect in vivo correlates well with their potency in vitro as AHAS inhibitors. Three-dimensional quantitative structure-activity relationships were developed using comparative molecular field analysis and comparative molecular similarity indices analysis. For the latter, the best result was obtained when steric, electrostatic, hydrophobic and H-bond acceptor factors were taken into consideration. The resulting fields were mapped on to the published crystal structure of the yeast enzyme and it was shown that the steric and hydrophobic fields are in good agreement with sulfonylurea-AHAS interaction geometry.

A quantum yield map for synthetic eumelanin

The quantum yield of synthetic eumelanin is known to be extremely low and it has recently been reported to be dependent on excitation wavelength. In this paper, we present quantum yield as a function of excitation wavelength between 250 and 500 nm, showing it to be a factor of 4 higher at 250 nm than at 500 nm. In addition, we present a definitive map of the steady-state fluorescence as a function of excitation and emission wavelengths, and significantly, a three-dimensional map of the specific quantum yield: the fraction of photons absorbed at each wavelength that are subsequently radiated at each emission wavelength. This map contains clear features, which we attribute to certain structural models, and shows that radiative emission and specific quantum yield are negligible at emission wavelengths outside the range of 585 and 385 nm (2.2 and 3.2 eV), regardless of excitation wavelength. This information is important in the context of understanding melanin biofunctionality, and the quantum molecular biophysics therein. (c) 2005 American Institute of Physics.

Promoter-, cell-, and ligand-specific transactivation responses of the VDRB1 isoform

The vitamin D receptor (VDR) mediates the effects of 1,25(OH)(2)D-3, the active form of vitamin D. The human VDRB1 isoform differs from the originally described VDR by an N-terminal extension of 50 amino acids. Here we investigate cell-, promoter-, and ligand-specific transactivation by the VDRB1 isoform. Transactivation by these isoforms of the cytochrome P450 CYP24 promoter was compared in kidney (HEK293 and COS1), tumor-derived colon (Caco-2, LS174T, and HCT15), and mammary (HS578T and MCF7) cell lines. VDRB1 transactivation in response to 1,25(OH)(2)D-3 was greater in Cost and HCT15 cells (145%), lower in HEK293 and Caco-2 cells (70-85%) and similar in other cell lines tested. By contrast, on the cytochrome P450 CYP3A4 promoter, 1,25(OH)(2)D-3-induced VDRB1 transactivation was significantly lower than VDRA in Caco-2 (68%), but comparable to VDRA in HEK293 and COS1 cells. Ligand-dependence of VDRB1 differential transactivation was investigated using the secondary bile acid lithocholic acid (LCA). On the CYP24 promoter LCA-induced transactivation was similar for both isoforms in COS1, whereas in Caco-2 and HEK293 cells VDRB1 was less active. On the CYP3A4 promoter, LCA activation of VDRB1 was comparable to VDRA in all the cell lines tested. Mutational analysis indicated that both the 1,25(OH)(2)D-3 and LCA-regulated activities of both VDR isoforms required a functional ligand-dependent activation function (AF-2) domain. In gel shift assays VDR:DNA complex formation was stronger in the presence of 1,25(OH)(2)D-3 than with LCA. These results indicate that regulation of VDRB1 transactivation activity is dependent on cellular context, promoter, and the nature of the ligand. (c) 2005 Elsevier Inc. All rights reserved.

Live cell imaging of heavy-ion-induced radiation responses by beamline microscopy

To study the dynamics of protein recruitment to DNA lesions, ion beams can be used to generate extremely localized DNA damage within restricted regions of the nuclei. This inhomogeneous spatial distribution of lesions can be visualized indirectly and rapidly in the form of radiation-induced foci using immunocytochemical detection or GFP-tagged DNA repair proteins. To analyze faster protein translocations and a possible contribution of radiation-induced chromatin movement in DNA damage recognition in live cells, we developed a remote-controlled system to obtain high-resolution fluorescence images of living cells during ion irradiation with a frame rate of the order of seconds. Using scratch replication labeling, only minor chromatin movement at sites of ion traversal was observed within the first few minutes of impact. Furthermore, time-lapse images of the GFP-coupled DNA repair protein aprataxin revealed accumulations within seconds at sites of ion hits, indicating a very fast recruitment to damaged sites. Repositioning of the irradiated cells after fixation allowed the comparison of live cell observation with immunocytochemical staining and retrospective etching of ion tracks. These results demonstrate that heavy-ion radiation-induced changes in sub-nuclear structures can be used to determine the kinetics of early protein recruitment in living cells and that the changes are not dependent on large-scale chromatin movement at short times postirradiation. © 2005 by Radiation Research Society.