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PubMed2000

RNA cleavage by a DNA enzyme with extended chemical functionality.

In vitro selection techniques were applied to the development of a DNA enzyme that contains three catalytically essential imidazole groups and catalyzes the cleavage of RNA substrates. Nucleic acid libraries for selection were constructed by polymerase-catalyzed incorporation of C5-imidazole-functionalized deoxyuridine in place of thymidine. Chemical synthesis was used to define a minimized catalytic domain composed of only 12 residues. The catalytic domain forms a compact hairpin structure that displays the three imidazole-containing residues. The enzyme can be made to cleave RNAs of almost any sequence by simple alteration of the two substrate-recognition domains that surround the catalytic domain. The enzyme operates with multiple turnover in the presence of micromolar concentrations of Zn2+, exhibiting saturation kinetics and a catalytic rate of >1 min-1. The imidazole-containing DNA enzyme, one of the smallest known nucleic acid enzymes, combines the substrate-recognition properties of nucleic acid enzymes and the chemical functionality of protein enzymes in a molecule that is small, yet versatile and catalytically efficient.

Base Composition↗
PubMed2006

Detection of proteins based on amino acid sequences by multiple aptamers against tripeptides.

A number of different ligands have been tested in the course of the development of protein array technology. The most extensively studied example of protein ligands has been based on antibody-antigen interaction. Other examples include protein-protein, protein-nucleic acid, and protein-small molecule interactions. All these ligands can recognize and specifically bind to protein epitopes. In this study, we have developed a novel technology using DNA-based aptamers to detect proteins based on their amino acid sequences. Mouse cathepsin D was used for the proof of principle experiment. Four tripeptides, Leu-Ala-Ser, Asp-Gly-Ile, Gly-Glu-Leu, and Lys-Ala-Ile, were selected based on the published amino acid sequence of mouse cathepsin D. DNA aptamers against the tripeptides were isolated using the systematic evolution of ligands of exponential enrichment method. We have demonstrated that the aptamers specifically interacted with mouse cathepsin D using the structure-switch method. We further performed a proximity-dependent ligation assay to demonstrate that multiple aptamers could specifically detect the protein from cell extracts. In principle, one library containing 8000 aptamers should be enough to detect almost all proteins in the whole proteome in all organisms. This technology could be applied to generate a new generation of protein arrays.

Amino Acid Sequence↗
PubMed1988

The structure of human complement component C7 and the C5b-7 complex.

The molecular architecture of human complement component C7 was elucidated at several structural levels. The complete primary structure of C7 was derived from the cDNA sequence of clones isolated from a human liver library. C7 is a mosaic protein that consists of 821 amino acids. The amino-terminal two-thirds of C7 has 23-30% homology with complement components C8 and C9. In addition, the carboxyl-terminal third contains four cysteine-rich segments that have overlapping internal homology. The protein is a single polypeptide chain with 28 disulfide bonds and is glycosylated at two sites. Virtually all the cysteines are found in small units of 35-77 amino acids that exhibit homology with those of various proteins including the low density lipoprotein receptor, epidermal growth factor precursor, thrombospondin, and blood coagulation factors IX and X. The secondary structural analysis, estimated by circular dichroism, suggested a high content of beta-sheet (38%) and beta-turns (24%). The tertiary structure, visualized by transmission electron microscopy, indicated a flexible elongated molecule with dimensions of 151 X 59 X 43 A. The quaternary structure of the C5b-7 complex bound to lipid vesicles was observed to be in the form of monomers or dimers. The monomer C5b-7 consists of a leaflet and a long flexible stalk, and the dimer has two leaflets linked through a supercoiled stalk. Membrane binding is mediated by the stalk part of the complexes. Using a radioiodinated photoreactive cross-linking reagent bound to the polar head group of phosphatidylethanolamine, the stalk part of the C5b-7 complex could be labeled preferentially, and it was found to consist mainly of C6 and C7. Thus, C7 plays a major role in bringing about the hydrophilic-amphiphilic transition during the formation of the membrane attack complex, and it serves as a membrane anchor for the C5b-7 complex.

Amino Acid Sequence↗
PubMed1997

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants.

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1. 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).

Chloroplasts↗
PubMed2000

Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

Allosteric Regulation↗
PubMed1997

BMP7 null mutation in mice: developmental defects in skeleton, kidney, and eye.

While generating bcl2 alpha transgenic mice, we found some F2 offspring of one of the transgenic lines which were very small and had closed eyes at the time of weaning. These pups died within 1 month after birth. In order to determine the molecular basis of this phenotype, we screened a genomic library of the above transgenic line with a transgene-specific probe and found that the Bmp7 gene, a member of the TGF beta superfamily, was inactivated by insertional mutagenesis due to transgene integration. The Bmp7 homozygous null condition in mice is a postnatal lethal mutation and is associated with various developmental defects: holes in the basisphenoid bone and the xyphoid cartilage, retarded ossification of bones, fused ribs and vertebrae, underdeveloped neural arches of the lumbar and sacral vertebrae, polydactyly of the hind limbs, a kinked tail, a reduced number of nephrons, polycystic kidney, lack of retinal pigmentation, and retarded lens development. These findings indicate that BMP7 is an important signaling molecule for normal development of the mammalian skeleton, kidney, and eye. Academic Press

Abnormalities, Multiple↗
PubMed2002

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis.

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3' end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.

Base Sequence↗
PubMed2003

Aurintricarboxylic acid blocks in vitro and in vivo activity of YopH, an essential virulent factor of Yersinia pestis, the agent of plague.

Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to Bubonic Plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. YopH is an essential virulence factor whose protein-tyrosine phosphatase (PTP) activity is required for Yersinia pathogenicity. Consequently, there is considerable interest in developing potent and selective YopH inhibitors as novel anti-plague agents. We have screened a library of 720 structurally diverse commercially available carboxylic acids and identified 26 YopH inhibitors with IC50 values below 100 mum. The most potent and specific YopH inhibitor is aurintricarboxylic acid (ATA), which exhibits a Ki value of 5 nm for YopH and displays 6-120-fold selectivity in favor of YopH against a panel of mammalian PTPs. To determine whether ATA can block the activity of YopH in a cellular context, we have examined the effect of ATA on T-cell signaling in human Jurkat cells transfected with YopH. We show that YopH severely decreases the T-cell receptor-induced cellular tyrosine phosphorylation, ERK1/2 activity, and interleukin-2 transcriptional activity. We demonstrate that ATA can effectively block the inhibitory activity of YopH and restore normal T-cell function. These results provide a proof-of-concept for the hypothesis that small molecule inhibitors that selectively target YopH may be therapeutically useful. In addition, it is expected that potent and selective YopH inhibitors, such as ATA, should be useful reagents to delineate YopH's cellular targets in plague and other pathogenic conditions caused by Yersinia infection.

Aurintricarboxylic Acid↗
PubMed1995

Predicting ligand binding to proteins by affinity fingerprinting.

BACKGROUND: There are many ways to represent a molecule's properties, including atomic-connectivity drawings, NMR spectra, and molecular orbital models. Prior methods for predicting the biological activity of compounds have largely depended on these physical representations. Measuring a compound's binding potency against a small reference panel of diverse proteins defines a very different representation of the molecule, which we call an affinity fingerprint. Statistical analysis of such fingerprints provides new insights into aspects of binding interactions that are shared among a wide variety of proteins. These analyses facilitate prediction of the binding properties of these compounds assayed against new proteins. RESULTS: Affinity fingerprints are reported for 122 structurally-diverse compounds using a reference panel of eight proteins that collectively are able to generate unique fingerprints for about 75% of the small organic compounds tested. Application of multivariate regression techniques to this database enables the creation of computational surrogates to represent new proteins that are surprisingly effective at predicting binding potencies. We illustrate this for two enzymes with no previously recognizable similarity to each other or to any of the reference proteins. Fitting of analogous computational surrogates to four other proteins confirms the generality of the method; when applied to a fingerprinted library of 5000 compounds, several sub-micromolar hits were correctly predicted. CONCLUSIONS: An affinity fingerprint database, which provides a rich source of data defining operational similarities among proteins, can be used to test theories of cryptic homology unexpected from current understanding of protein structure. Practical applications to drug design include efficient pre-screening of large numbers of compounds against target proteins using fingerprint similarities, supplemented by a small number of empirical measurements, to select promising compounds for further study.

Chromatography, Affinity↗
PubMed2001

Invertebrate connectin spans as much as 3.5 microm in the giant sarcomeres of crayfish claw muscle.

In crayfish claw closer muscle, the giant sarcomeres are 8.3 microm long at rest, four times longer than vertebrate striated muscle sarcomeres, and they are extensible up to 13 microm upon stretch. Invertebrate connectin (I-connectin) is an elastic protein which holds the A band at the center of the sarcomere. The entire sequence of crayfish I-connectin was predicted from cDNA sequences of 53 424 bp (17 352 residues; 1960 kDa). Crayfish I-connectin contains two novel 68- and 71-residue repeats, and also two PEVK domains and one kettin region. Kettin is a small isoform of I-connectin. Immunoblot tests using antibody to the 68-residue repeats revealed the presence of I-connectin also in long sarcomeres of insect leg muscle and barnacle ventral muscle. Immunofluorescence microscopy demonstrated that the two repeats, the long spacer and the two PEVK domains contribute to sarcomere extension. These regions rich in charged amino acids, occupying 63% of the crayfish I-connectin molecule, may allow a span of a 3.5 microm distance as a new class of composite spring.

Amino Acid Sequence↗
PubMed2006

Electrochemical and ligand binding studies of a de novo heme protein.

Heme proteins can perform a variety of electrochemical functions. While natural heme proteins carry out particular functions selected by biological evolution, artificial heme proteins, in principle, can be tailored to suit specified technological applications. Here we describe initial characterization of the electrochemical properties of a de novo heme protein, S824C. Protein S824C is a four-helix bundle derived from a library of sequences that was designed by binary patterning of polar and nonpolar amino acids. Protein S824C was immobilized on a gold electrode and the formal potential of heme-protein complex was studied as a function of pH and ionic strength. The binding of exogenous N-donor ligands to heme/S824C was monitored by measuring shifts in the potential that occurred upon addition of various concentrations of imidazole or pyridine derivatives. The response of heme/S824C to these ligands was then compared to the response of isolated heme (without protein) to the same ligands. The observed shifts in potential depended on both the concentration and the structure of the added ligand. Small changes in structure of the ligand (e.g. pyridine versus 2-amino pyridine) produced significant shifts in the potential of the heme-protein. The observed shifts correlate to the differential binding of the N-donor molecules to the oxidized and reduced states of the heme. Further, it was observed that the electrochemical response of the buried heme in heme/S824C differed significantly from that of isolated heme. These studies demonstrate that the structure of the de novo protein modulates the binding of N-donor ligands to heme.

Binding Sites↗
PubMed2002

Interaction of the developmental regulator SALL1 with UBE2I and SUMO-1.

Mutations in the SALL1 gene on chromosome 16q12.1 cause Townes-Brocks syndrome (TBS). This autosomal dominantly inherited disorder is characterized by typical malformations of the thumbs, the ears, and the anus, and also commonly affects the kidneys and other organ systems. SALL1 has recently been shown to localize to chromocenters and other heterochromatin foci in murine fibroblasts and to interact with the telomere-repeat-binding factor TRF1/PIN2. Here, we show that the ubiquitin-conjugating enzyme 2I (UBE2I), the human homolog of S. cerevisiae UBC9, and the small ubiquitin-like modifier-1 (SUMO-1) interact with SALL1 in the yeast two-hybrid system. The interaction of SALL1 and UBE2I was confirmed in a glutathione S-transferase (GST) pull-down experiment. In an in vitro assay, it could be demonstrated that SALL1 is covalently modified by at least two SUMO-1 molecules in the presence of UBA2/AOS1 and UBE2I. Mutation of lysine 1086 of SALL1 to arginine abrogates SALL1 sumoylation, suggesting the presence of a polymeric SUMO-1 chain in the wild type state.

Abnormalities, Multiple↗
PubMed1990

Pulse-field electrophoresis indicates full-length Mycoplasma chromosomes range widely in size.

Full-size linear chromosomes were prepared from mycoplasmas by using gamma-irradiation to introduce one (on average) double-strand break in their circular chromosomes. Chromosome sizes were estimated by pulsed-field gel electrophoresis (PFGE) from the mobilities of these full-length molecules relative to DNA size references. Sizes estimated for Ureaplasma urealyticum T960 and 16 Mycoplasma species ranged from 684 kbp (M. hominis) to 1315 kbp (M. iowae). Using this sample, we found no correlation between the mobility of the full-size linear chromosomes and their G + C content. Sizes for A. laidlawii and A. hippikon were within the range expected from renaturation kinetics. PFGE size estimates are in good agreement with sizes determined by other methods, including electron microscopy, an ordered clone library, and summation of restriction fragments. Our estimates also agree with those from renaturation kinetics for both the largest and some of the smallest chromosomes, but in the intermediate size range, renaturation kinetics consistently provides lower values than PFGE or electron microscopy. Our PFGE estimates show that mycoplasma chromosomes span a continual range of sizes, with several intermediate values falling between the previously recognized large and small chromosome size clusters.

Acholeplasma↗
PubMed2004

Quality, not quantity: the role of natural products and chemical proteomics in modern drug discovery.

Chemical genetics and reverse chemical genetics parallel classical genetics but target genes at the protein level and have proven useful in recent years for screening combinatorial libraries for compounds of biological interest. However, the performance of combinatorial chemistry in filling pharmaceutical pipelines has been lower than anticipated and the tide may be turning back to Nature in the search for new drug candidates. Even though diversity oriented synthesis is now producing molecules that are natural product-like in terms of size and complexity, these molecules have not evolved to interact with biomolecules. Natural products, on the other hand, have evolved to interact with biomolecules, which is why so many can be found in pharmacopoeias. However, the cellular targets and modes of action of these fascinating compounds are seldom known, hindering the drug development process. This review focuses on the emergence of chemical proteomics and reverse chemical proteomics as tools for the discovery of cellular receptors for natural products, thereby generating protein/ligand pairs that will prove useful in identifying new drug targets and new biologically active small molecule scaffolds. Such a system-wide approach to identifying new drugable targets and their small molecule ligands will help unblock the pharmaceutical product pipelines by speeding the process of target and lead identification.

Animals↗
PubMed1988

A human T cell-specific molecule is a member of a new gene family.

We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.

Amino Acid Sequence↗
PubMed2005

Isolation and cloning of exendin precursor cDNAs from single samples of venom from the Mexican beaded lizard (Heloderma horridum) and the Gila monster (Heloderma suspectum).

Reptile venoms are complex cocktails of bioactive molecules, including peptides. While the drug discovery potential of most species remains unrealized, many are endangered and afforded protection under international treaties. In this study, we describe how potential clinically important bioactive peptides and their corresponding mRNAs can be structurally characterized from single, small samples of reptile venom. The potential type-2 diabetes therapeutics, exendin-3 and exendin-4, from the Mexican beaded lizard (Heloderma horridum) and the Gila monster (Heloderma suspectum), respectively, have been characterized at both protein and nucleic acid levels to illustrate the efficacy of the technique and its contribution to biodiversity conservation.

Animals↗
PubMed1989

Detection of circular forms of eliminated DNA during macronuclear development in E. crassus.

Following their sexual cycle, hypotrichous ciliated protozoa transform a copy of a chromosomal micronucleus into a macronucleus containing small, linear DNA molecules. A frequent event during macronuclear development is the removal of short segments of DNA (internal eliminated sequences: IESs) by a process equivalent to DNA breakage and rejoining. In this study we used a polymerase chain reaction procedure to demonstrate that free circular forms of IESs are present in cells undergoing macronuclear development. Sequencing of the junctions of the free circular IESs suggests that they share 12 nucleotides with the macronuclear DNA molecules that are generated following IES removal. The results provide evidence that IESs are removed by an active DNA breakage and rejoining process, which may involve staggered cuts in the substrate DNA.

Animals↗
PubMed2004

Chemical genetics for therapeutic target mining.

Chemical genetics is an emerging research field that utilises biologically active small molecules to study biological functions of genes and their products. The direct regulation of the protein function by the biologically active small molecules can alternate the gene mutagenesis studies utilised in conventional genetics. Like conventional genetics, chemical genetics can be divided into two concepts - 'forward' and 'reverse' chemical genetics. These approaches of chemical genetics have a tremendous impact on both functional genomics and drug development. This review focuses on the two ways in which chemical genetics can be used for therapeutic target mining and their practical application in drug development, particularly, in angiogenesis-related diseases.

Angiogenesis Inhibitors↗