SCORPION2: A database for structure-function analysis of scorpion toxins

Scorpion toxins are important experimental tools for characterization of vast array of ion channels and serve as scaffolds for drug design. General public database entries contain limited annotation whereby rich structure-function information from mutation studies is typically not available. SCORPION2 contains more than 800 records of native and mutant toxin sequences enriched with binding affinity and toxicity information, 624 three-dimensional structures and some 500 references. SCORPION2 has a set of search and prediction tools that allow users to extract and perform specific queries: text searches of scorpion toxin records, sequence similarity search, extraction of sequences, visualization of scorpion toxin structures, analysis of toxic activity, and functional annotation of previously uncharacterized scorpion toxins. The SCORPION2 database is available at http://sdmc.i2r.a-star.edu.sg/scorpion/. (c) 2006 Elsevier Ltd. All rights reserved.

Structure/function studies of S100A8/A9

The functional importance of members of the S100 Ca2+-binding protein family is recently emerging. A variety of activities, several of which are apparently opposing, are attributed to S100A8, a protein implicated in embryogenesis, growth, differentiation, and immune and inflammatory processes. Murine (m) S100A8 was initially described as a chemoattractant (CP-10) for myeloid cells. It is coordinately expressed with mS100A9 (MRP14) in neutrophils and the non-covalent heterodimer is presumed to be the functional intracellular species. The extracellular chemotactic activity of mS100A8, however, is not dependent on mS100A9 and occurs at concentrations (10(-13)-10(-11) M) at which the non-covalent heterodimer would probably dissociate. This review focuses on the structure and post-translational modifications of mS100A8/A9 and their effects on function, particularly chemotaxis.

Noninvasive tests for arterial structure, function, and compliance: Do they identify risk or diagnose disease?

Background: Brachial artery reactivity (BAR), carotid intima-media thickness (IMT), and applanation tonometry for evaluation of total arterial compliance may provide information about preclinical vascular disease. We sought to determine whether these tests could be used to identify patients with coronary artery disease (CAD) without being influenced by their ability to identify those at risk ford CAD developing. Methods: We studied 100 patients and compared 3 groups: 35 patients with known CAD; 34 patients with symptoms and risk factors but no CAD identified by stress echocardiography (risk group); and 31 control subjects. BAR and IMT were measured using standard methods, and total arterial compliance was calculated by the pulse-pressure method from simultaneous radial applanation tonometry and pulsed wave Doppler of the left ventricular outflow. Ischemia was identified as a new or worsening wall-motion abnormality induced by stress. Results: In a comparison between the control subjects and patients either at risk for developing CAD or with CAD, the predictors of risk for CAD were: age (P = .01); smoking history (P = .002); hypercholesterolemia (P = .002); and hypertension (P = .004) (model R = 0.82; P = .0001). The independent predictors of CAD were: IMT (P = .001); BAR (P = .04); sex (P = .005); and hypertension (P = .005) (model R = 0.80; P = .0001). Conclusion: IMT, BAR, and traditional cardiovascular risk factors appear to identify patients at risk for CAD developing. However, only IMT was significantly different between patients at risk for developing CAD and those with overt CAD.

Structure-function studies of the plant cyclotides: The role of a circular protein backbone

The traditional idea of proteins as linear chains of amino acids is being challenged with the discovery of miniproteins that contain a circular backbone. The cyclotide family is the largest group of circular proteins and is characterized by an amide-circularized protein backbone and six conserved cysteine residues. These conserved cysteines are paired to form a knotted network of disulfide bonds. The combination of the circular backbone and a cystine knot, known as the cyclic cystine knot (CCK) motif, confers exceptional stability upon the cyclotides. This review discusses the role of the circular backbone based on studies of both the oxidative folding of kalata B1, the prototypical cyclotide, and a comparison of the structure and activity of kalata B1 and its acyclic permutants.

NaSi-1 and Sat-1: structure, function and transcriptional regulation of two genes encoding renal proximal tubular sulfate transporters

Sulfate (SO42-) is an important anion regulating many metabolic and cellular processes. Maintenance Of SO42- homeostasis occurs in the renal proximal tubule via membrane transport proteins. Two SO42- transporters that have been characterized and implicated in regulating serum SO42- levels are: NaSi- 1, a Na+-SO4 (2-) cotransporter located at the brush border membrane and Sat-1, a SO4 (2-) -anion exchanger located on the basolateral membranes of proximal tubular cells. Unlike Sat-1, for which very few studies have looked at regulation of its expression, NaSi- 1 has been shown to be regulated by various hormones and dietary conditions in vivo. To study this further, NaSj- I (SLC13A1) and Sat- I (SLC26A1) gene structures were determined and recent studies have characterized their respective gene promoters. This review presents the current understanding of the transcriptional regulation of NaSj- I and Sat- 1, and describes possible pathogenetic implications which arise as a consequence of altered SO(4)(2-)homeostasis. (c) 2005 Elsevier Ltd. All rights reserved.

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.

Accurate prediction of scorpion toxin functional properties from primary structures

Scorpion toxins are common experimental tools for studies of biochemical and pharmacological properties of ion channels. The number of functionally annotated scorpion toxins is steadily growing, but the number of identified toxin sequences is increasing at much faster pace. With an estimated 100,000 different variants, bioinformatic analysis of scorpion toxins is becoming a necessary tool for their systematic functional analysis. Here, we report a bioinformatics-driven system involving scorpion toxin structural classification, functional annotation, database technology, sequence comparison, nearest neighbour analysis, and decision rules which produces highly accurate predictions of scorpion toxin functional properties. (c) 2005 Elsevier Inc. All rights reserved.

Functional characterisation of an engineered multidomain human P450 E1 by molecular Lego

The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (K (M)=1.84 +/- 0.09 mM and k (cat) of 2.98 +/- 0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (K (M)=0.65 +/- 0.08 mM and k (cat) of 0.95 +/- 0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

Deduction of functional peptide motifs in scorpion toxins

Scorpion toxins are important physiological probes for characterizing ion channels. Molecular databases have limited functional annotation of scorpion toxins. Their function can be inferred by searching for conserved motifs in sequence signature databases that are derived statistically but are not necessarily biologically relevant. Mutation studies provide biological information on residues and positions important for structure-function relationship but are not normally used for extraction of binding motifs. 3D structure analyses also aid in the extraction of peptide motifs in which non-contiguous residues are clustered spatially. Here we present new, functionally relevant peptide motifs for ion channels, derived from the analyses of scorpion toxin native and mutant peptides. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.