MAGIC Tool: integrated microarray data analysis

Summary: Several programs are now available for analyzing thelarge datasets arising from cDNA microarray experiments. Mostprograms are expensive commercial packages or require expensivethird party software. Some are freely available to academicresearchers, but are limited to one operating system. MicroArrayGenome Imaging and Clustering Tool (MAGIC Tool) is an open sourceprogram that works on all major platforms, and takes users ‘fromtiff to gif’. Several unique features of MAGIC Tool areparticularly useful for research and teaching. Availability: http://www.bio.davidson.edu/MAGIC Contact: laheyer{at}davidson.edu     

Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.     

Drosophila, the golden bug, emerges as a tool for human genetics

Drosophila melanogaster is emerging as one of the most effective tools for analyzing the function of human disease genes, including those responsible for developmental and neurological disorders, cancer, cardiovascular disease, metabolic and storage diseases, and genes required for the function of the visual, auditory and immune systems. Flies have several experimental advantages, including their rapid life cycle and the large numbers of individuals that can be generated, which make them ideal for sophisticated genetic screens, and in future should aid the analysis of complex multigenic disorders. The general principles by which D. melanogaster can be used to understand human disease, together with several specific examples, are considered in this review.     

Rapid cryopreservation of five mammalian and one mosquito cell line at −80°C while attached to flasks in a serum free cryopreservative

Cell culturing, and the requisite storage of cell lines at ultra-low temperatures, is used in most laboratories studying or using eukaryotic proteomics, genomics, microarray, and RNA technologies. In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at −80°C while attached to the substratum of 25cm² tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines.     

Recognition of a new ARTC1 peptide ligand uniquely expressed in tumor cells by antigen-specific CD4+ regulatory T cells

CD4(+) regulatory T (Treg) cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Yet very little is known about the natural antigenic ligands that preferentially activate CD4(+) Treg cells. Here we report the establishment of tumor-specific CD4(+) Treg cell clones from tumor-infiltrating lymphocytes (TILs) of cancer patients, and the identification of an Ag recognized by Treg cells (ARTC1) gene encoding a peptide ligand recognized by tumor-specific TIL164 CD4(+) Treg cells. The mutations in a gene encoding an ARTC1 in 164mel tumor cells resulted in the translation of a gene product containing the peptide ligand recognized by CD4(+) Treg cells. ARTC1 peptide-activated CD4(+) Treg cells suppress the physiological function (proliferation and IL-2 secretion) of melanoma-reactive T cells. Furthermore, 164mel tumor cells, but not tumor lysates pulsed on B cells, were capable of activating TIL164 CD4(+) Treg cells. These results suggest that tumor cells may uniquely present an array of peptide ligands that preferentially recruit and activate CD4(+) Treg cells in sites where tumor-specific self-peptide is expressed, leading to the induction of local and tumor-specific immune suppression.     

Bone marrow-derived dendritic cells reverse the anergic state of CD4+CD25+ T cells without reversing their suppressive function

Dendritic cells (DC) are potent inducers of immunity to foreign Ags, but also contribute to self-tolerance by induction of regulatory T cells or deletion/anergy of self-reactive T cells. In this study, we have studied the capacity of DC to activate naturally occurring CD4+CD25+ regulatory T cells as well as the ability of CD4+CD25+ T cells to suppress the DC-mediated activation of CD4+CD25- T cells. Mature bone marrow-derived dendritic cells, but not splenic DC, were able to induce the proliferation of CD4+CD25+ T cells in the presence of a polyclonal stimulus and in the absence of exogenous IL-2. The DC-induced proliferative response of the CD4+CD25+ T cells was partially dependent on IL-2 produced by a small number of contaminating CD25+ effector cells. Because bone marrow-derived dendritic cells induce proliferation of both CD4+CD25+ and CD4+CD25- T cells in vitro, it was impossible to assay the suppressive function of the CD4+CD25+ T cells using [3H]TdR uptake or CFSE dilution. We therefore measured IL-2 production in cocultures of CD4+CD25+ and CD4+CD25- T cells using the IL-2 secretion assay. Surprisingly, CD4+CD25+ T cells markedly suppressed IL-2 secretion by the CD4+CD25- T cells without inhibiting their proliferation. Collectively, these results suggest that Ag presentation by DC can induce the expansion of CD4+CD25+ T cells while simultaneously activating their ability to suppress cytokine secretion by effector T cells.     

Cysteine repeat domains and adjacent sequences determine distinct bone morphogenetic protein modulatory activities of the Drosophila Sog protein

The Drosophila short gastrulation gene (sog) encodes a large extracellular protein (Sog) that inhibits signaling by BMP-related ligands. Sog and its vertebrate counterpart Chordin contain four copies of a cysteine repeat (CR) motif defined by 10 cysteine residues spaced in a fixed pattern and a tryptophan residue situated between the first two cysteines. Here we present a structure-function analysis of the CR repeats in Sog, using a series of deletion and point mutation constructs, as well as constructs in which CR domains have been swapped. This analysis indicates that the CR domains are individually dispensable for Sog function but that they are not interchangeable. These studies reveal three different types of Sog activity: intact Sog, which inhibits signaling mediated by the ligand Glass bottom boat (Gbb), a more broadly active class of BMP antagonist referred to as Supersog, and a newly identified activity, which may promote rather than inhibit BMP signaling. Analysis of the activities of CR swap constructs indicates that the CR domains are required for full activity of the various forms of Sog but that the type of Sog activity is determined primarily by surrounding protein sequences. Cumulatively, our analysis suggests that CR domains interact physically with adjacent protein sequences to create forms of Sog with distinct BMP modulatory activities.     

Thiamin biosynthesis in Bacillus subtilis: structure of the thiazole synthase/sulfur carrier protein complex

Thiazole synthase is the key enzyme involved in the formation of the thiazole moiety of thiamin pyrophosphate. We have determined the structure of this enzyme in complex with ThiS, the sulfur carrier protein, at 3.15 A resolution. Thiazole synthase is a tetramer with 222 symmetry. The monomer is a (betaalpha)(8) barrel with similarities to the aldolase class 1 and flavin mononucleotide dependent oxidoreductase and phosphate binding superfamilies. The sulfur carrier protein (ThiS) is a compact protein with a fold similar to that of ubiquitin. The structure allowed us to model the substrate, deoxy-D-xylulose 5-phosphate (DXP), in the active site. This model identified Glu98 and Asp182 as new active site residues likely to be involved in the catalysis of thiazole formation. The function of these residues was probed by mutagenesis experiments, which confirmed that both residues are essential for thiazole formation and identified Asp182 as the base involved in the deprotonation at C3 of the thiazole synthase DXP imine. Comparison of the ThiS binding surface to the surface of ubiquitin identified a conserved hydrophobic patch of unknown function on ubiquitin that may be involved in complex formation between ubiquitin and one of its binding partners.     

Structural comparison of MTA phosphorylase and MTA/AdoHcy nucleosidase explains substrate preferences and identifies regions exploitable for inhibitor design

The development of new and effective antiprotozoal drugs has been a difficult challenge because of the close similarity of the metabolic pathways between microbial and mammalian systems. 5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is thought to be an ideal target for therapeutic drug design as the enzyme is present in many microbes but not in mammals. MTA/AdoHcy nucleosidase (MTAN) irreversibly depurinates MTA or AdoHcy to form adenine and the corresponding thioribose. The inhibition of MTAN leads to a buildup of toxic byproducts that affect various microbial pathways such as quorum sensing, biological methylation, polyamine biosynthesis, and methionine recycling. The design of nucleosidase-specific inhibitors is complicated by its structural similarity to the human MTA phosphorylase (MTAP). The crystal structures of human MTAP complexed with formycin A and 5'-methylthiotubercidin have been solved to 2.0 and 2.1 A resolution, respectively. Comparisons of the MTAP and MTAN inhibitor complexes reveal size and electrostatic potential differences in the purine, ribose, and 5'-alkylthio binding sites, which account for the substrate specificity and reactions catalyzed. In addition, the differences between the two enzymes have allowed the identification of exploitable regions that can be targeted for the development of high-affinity nucleosidase-specific inhibitors. Sequence alignments of Escherichia coli MTAN, human MTAP, and plant MTA nucleosidases also reveal potential structural changes to the 5'-alkylthio binding site that account for the substrate preference of plant MTA nucleosidases.     

Metabolic biotinylation as a probe of supramolecular structure of the triad junction in skeletal muscle

Excitation-contraction coupling in skeletal muscle involves conformational coupling between dihydropyridine receptors (DHPRs) in the plasma membrane and ryanodine receptors (RyRs) in the sarcoplasmic reticulum. However, it remains uncertain what regions, if any, of the two proteins interact with one another. Toward this end, it would be valuable to know the spatial interrelationships of DHPRs and RyRs within plasma membrane/sarcoplasmic reticulum junctions. Here we describe a new approach based on metabolic incorporation of biotin into targeted sites of the DHPR. To accomplish this, cDNAs were constructed with a biotin acceptor domain (BAD) fused to selected sites of the DHPR, with fluorescent protein (XFP) attached at a second site. All of the BAD-tagged constructs properly targeted to junctions (as indicted by small puncta of XFP) and were functional for excitation-contraction coupling. To determine whether the introduced BAD was biotinylated and accessible to avidin (approximately 60 kDa), myotubes were fixed, permeablized, and exposed to fluorescently labeled avidin. Upon expression in beta1-null or dysgenic (alpha1S-null) myotubes, punctate avidin fluorescence co-localized with the XFP puncta for BAD attached to the beta1a N- or C-terminals, or the alpha1S N-terminal or II-III loop. However, BAD fused to the alpha1S C-terminal was inaccessible to avidin in dysgenic myotubes (containing RyR1). In contrast, this site was accessible to avidin when the identical construct was expressed in dyspedic myotubes lacking RyR1. These results indicate that avidin has access to a number of sites of the DHPR within fully assembled (RyR1-containing) junctions, but not to the alpha1S C-terminal, which appears to be occluded by the presence of RyR1.