Biomedical subjects

David S King

Publications and source records attributed to David S King.

At least 19 recordsLinked to original sources

PubMed2006

Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent alpha-factor derivatives.

Much progress defining the order and timing of endocytic internalization events has come as a result of real-time, live-cell fluorescence microscopy. Although the availability of numerous endocytic mutants makes yeast an especially valuable organism for functional analysis of endocytic dynamics, a serious limitation has been the lack of a fluorescent cargo for receptor-mediated endocytosis. We have now synthesized biologically active fluorescent mating-pheromone derivatives and demonstrated that receptor-mediated endocytosis in budding yeast occurs via the clathrin- and actin-mediated endocytosis pathway. We found that endocytic proteins first assemble into patches on the plasma membrane, and then alpha-factor associates with the patches. Internalization occurs next, concomitant with actin assembly at patches. Additionally, endocytic vesicles move toward early endosomes on actin cables. Early endosomes also associate with actin cables, and they actively move toward endocytic sites to capture vesicles being released from the plasma membrane. Thus, endocytic vesicle formation and capture of the newly released vesicles by early endosomes occur in a highly concerted manner, mediated by the actin cytoskeleton.

Actins↗

PubMed2006

Optimization of xenon biosensors for detection of protein interactions.

Hyperpolarized 129Xe NMR spectroscopy can detect the presence of specific low-concentration biomolecular analytes by means of a xenon biosensor that consists of a water-soluble, targeted cryptophane-A cage that encapsulates the xenon. In this work, we use the prototypical biotinylated xenon biosensor to determine the relationship between the molecular composition of the xenon biosensor and the characteristics of protein-bound resonances. The effects of diastereomer overlap, dipole-dipole coupling, chemical-shift anisotropy, xenon exchange, and biosensor conformational exchange on the protein-bound biosensor signal were assessed. It was found that an optimal protein-bound biosensor signal can be obtained by minimizing the number of biosensor diastereomers and using a flexible linker of appropriate length. Both the line width and sensitivity of chemical shift to protein binding of the xenon biosensor were found to be inversely proportional to linker length.

Avidin↗

PubMed2006

Listeria monocytogenes 10403S HtrA is necessary for resistance to cellular stress and virulence.

The HtrA serine protease has been shown to be essential for bacterial virulence and for survival after exposure to many types of environmental and cellular stresses. A Listeria monocytogenes 10403S htrA mutant was found to be sensitive to oxidative and puromycin-induced stress at high temperatures, showed a reduced ability to form biofilms, and was attenuated for virulence in mice.

Animals↗

PubMed2005

Identification and characterization of potent small molecule inhibitor of hemorrhagic fever New World arenaviruses.

Category A arenaviruses as defined by the National Institute of Allergy and Infectious Diseases (NIAID) are human pathogens that could be weaponized by bioterrorists. Many of these deadly viruses require biosafety level-4 (BSL-4) containment for all laboratory work, which limits traditional laboratory high-throughput screening (HTS) for identification of small molecule inhibitors. For those reasons, a related BSL-2 New World arenavirus, Tacaribe virus, 67-78% identical to Junín virus at the amino acid level, was used in a HTS campaign where approximately 400,000 small molecule compounds were screened in a Tacaribe virus-induced cytopathic effect (CPE) assay. Compounds identified in this screen showed antiviral activity and specificity against not only Tacaribe virus, but also the Category A New World arenaviruses (Junín, Machupo, and Guanarito). Drug resistant variants were isolated, suggesting that these compounds act through inhibition of a viral protein, the viral glycoprotein (GP2), and not through cellular toxicity mechanisms. A lead compound, ST-294, has been chosen for drug development. This potent and selective compound, with good bioavailability, demonstrated protective anti-viral efficacy in a Tacaribe mouse challenge model. This series of compounds represent a new class of inhibitors that may warrant further development for potential inclusion in a strategic stockpile.

Amino Acid Sequence↗

PubMed2005

High yield bacterial expression of active c-Abl and c-Src tyrosine kinases.

The Abl and Src tyrosine kinases are key signaling proteins that are of considerable interest as drug targets in cancer and many other diseases. The regulatory mechanisms that control the activity of these proteins are complex, and involve large-scale conformational changes in response to phosphorylation and other modulatory signals. The success of the Abl inhibitor imatinib in the treatment of chronic myelogenous leukemia has shown the potential of kinase inhibitors, but the rise of drug resistance in patients has also shown that drugs with alternative modes of binding to the kinase are needed. The detailed understanding of mechanisms of protein-drug interaction and drug resistance through biophysical methods demands a method for the production of active protein on the milligram scale. We have developed a bacterial expression system for the kinase domains of c-Abl and c-Src, which allows for the quick expression and purification of active wild-type and mutant kinase domains by coexpression with the YopH tyrosine phosphatase. This method makes practical the use of isotopic labeling of c-Abl and c-Src for NMR studies, and is also applicable for constructs containing the SH2 and SH3 domains of the kinases.

Animals↗

PubMed2005

NMR-based biosensing with optimized delivery of polarized 129Xe to solutions.

Laser-enhanced (LE) 129Xe nuclear magnetic resonance (NMR) is an exceptional tool for sensing extremely small physical and chemical changes; however, the difficult mechanics of bringing polarized xenon and samples of interest together have limited applications, particularly to biological molecules. Here we present a method for accomplishing solution 129Xe biosensing based on flow (bubbling) of LE 129Xe gas through a solution in situ in the NMR probe, with pauses for data acquisition. This overcomes fundamental limitations of conventional solution-state LE 129Xe NMR, e.g., the difficulty in transferring hydrophobic xenon into aqueous environments, and the need to handle the sample to refresh LE 129Xe after an observation pulse depletes polarization. With this new method, we gained a factor of >100 in sensitivity due to improved xenon transfer to the solution and the ability to signal average by renewing the polarized xenon. Polarized xenon in biosensors was detected at very low concentrations, </=250 nanomolar, while retaining all the usual information from NMR. This approach can be used to simultaneously detect multiple sensors with different chemical shifts and is also capable of detecting signals from opaque, heterogeneous samples, which is a unique advantage over optical methods. This general approach is adaptable for sensing minute quantities of xenon in heterogeneous in vitro samples, in miniaturized devices and should be applicable to certain in-vivo environments.

Biosensing Techniques↗

PubMed2005

Amino acid substitution and modification resulting from Escherichia coli expression of recombinant Plasmodium falciparum histidine-rich protein II.

The histidine-rich protein II (HRP II) from Plasmodium falciparum is an unusual protein composed of 40% alanine, 36% histidine, and 11% aspartate residues. Expression of HRP II in Escherichia coli results in the isolation of a heterogeneous protein. Mass spectrometry reveals a reduction in mass by multiples of 9 Da from the expected molecular mass that can be attributed to the substitution of glutamine for some histidine residues in the sequence. The extent of the glutamine for histidine substitution can be reduced by slowing the expression rate. Mass spectral analysis of HRP II also revealed alpha-amino methylation of the N-terminal alanine residue of HRP II.

Amino Acid Sequence↗

PubMed2004

Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells.

An orthogonal tryptophanyl-transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNA(Trp) (mutRNA(UCA)(Trp)) pair was generated for use in mammalian cells. The anticodon loop of the Bacillus subtilis tRNA(Trp) was mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the mutRNA(UCA)(Trp) gene was inserted between the 5' and 3' flanking sequences of the tRNA(Trp-1) gene from Arabidopsis to enhance its expression in mammalian cells. In vitro aminoacylation assays and in vivo opal suppression assays showed that B. subtilis TrpRS (BsTrpRS) charges only the cognate mutRNA(UCA)(Trp) and no endogenous mammalian tRNAs. Similarly, the mutRNA(UCA)(Trp) is specifically charged by B. subtilis TrpRS and not by endogenous synthetases in mammalian cells. Site-directed mutagenesis was then used to alter the specificity of BsTrpRS to uniquely charge 5-hydoxy-l-tryptophan. The resulting mutant BsTrpRS-mutRNA(UCA)(Trp) pair allows the efficient and selective incorporation of 5-hydroxy-l-tryptophan into mammalian proteins in response to the codon, TGA. This amino acid can be used as a fluorescence probe and also undergoes electrochemical oxidation in situ to generate an efficient protein crosslinking.

5-Hydroxytryptophan↗

PubMed2004

An expanded genetic code with a functional quadruplet codon.

With few exceptions the genetic codes of all known organisms encode the same 20 amino acids, yet all that is required to add a new building block are a unique tRNA/aminoacyl-tRNA synthetase pair, a source of the amino acid, and a unique codon that specifies the amino acid. For example, the amber nonsense codon, TAG, together with orthogonal Methanococcus jannaschii or Escherichia coli tRNA/synthetase pairs have been used to genetically encode a variety of unnatural amino acids in E. coli and yeast, respectively. However, the availability of noncoding triplet codons ultimately limits the number of amino acids encoded by any organism. Here, we report the design and generation of an orthogonal synthetase/tRNA pair derived from archaeal tRNA(Lys) sequences that efficiently and selectively incorporates an unnatural amino acid into proteins in response to the quadruplet codon, AGGA. Frameshift suppression with L-homoglutamine (hGln) does not significantly affect protein yields or cell growth rates and is mutually orthogonal with amber suppression, permitting the simultaneous incorporation of two unnatural amino acids, hGln and O-methyl-L-tyrosine, at distinct positions within myoglobin. This work suggests that neither the number of available triplet codons nor the translational machinery itself represents a significant barrier to further expansion of the genetic code.

Amino Acids↗

PubMed2003

The role of the protein core in the inhibitory power of the classic serine protease inhibitor, chymotrypsin inhibitor 2.

A synthetic cyclic peptide, reported to be a tight-binding inhibitor of serine proteases, is instead found to be a good substrate, as is the linear peptide of the same sequence. Both of the peptides, designed to mimic the binding loop of chymotrypsin inhibitor 2 (CI2), were cleaved by subtilisin primarily at the CI2 reactive-site Met-59-Glu-60 bond, revealing that the sequence, in the absence of the structural context of the inhibitor, provides sufficient specificity for hydrolysis of this bond. Insights from the crystal structure of the CI2/subtilisin complex, together with biochemical analysis of a CI2 Gly-83 deletion mutant, have allowed us to identify key features that make CI2 an effective inhibitor, while the cyclic and linear peptides are substrates.

Bacillus subtilis↗

PubMed2003

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8.

GPR7 and GPR8 are orphan G protein-coupled receptors that are highly similar to each other. These receptors are expressed predominantly in brain, suggesting roles in central nervous system function. We have purified an endogenous peptide ligand for GPR7 from bovine hypothalamus extracts. This peptide, termed neuropeptide B (NPB), has a C-6-brominated tryptophan residue at the N terminus. It binds and activates human GPR7 or GPR8 with median effective concentrations (EC(50)) of 0.23 nM and 15.8 nM, respectively. In situ hybridization shows distinct localizations of the prepro-NPB mRNA in mouse brain, i.e., in paraventricular hypothalamic nucleus, hippocampus, and several nuclei in midbrain and brainstem. Intracerebroventricular (i.c.v.) injection of NPB in mice induces hyperphagia during the first 2 h, followed by hypophagia. Intracerebroventricular injection of NPB produces analgesia to s.c. formalin injection in rats. Through EST database searches, we identified a putative paralogous peptide. This peptide, termed neuropeptide W (NPW), also has an N-terminal tryptophan residue. Synthetic human NPW binds and activates human GPR7 or GPR8 with EC(50) values of 0.56 nM and 0.51 nM, respectively. The expression of NPW mRNA in mouse brain is confined to specific nuclei in midbrain and brainstem. These findings suggest diverse physiological functions of NPB and NPW in the central nervous system, acting as endogenous ligands on GPR7 andor GPR8.

Amino Acid Sequence↗

PubMed2003

Generation of a bacterium with a 21 amino acid genetic code.

We have generated a completely autonomous bacterium with a 21 amino acid genetic code. This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon. The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli. Denaturing gel electrophoresis and mass spectrometric analysis show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids. This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biological functions.

Amino Acyl-tRNA Synthetases↗

PubMed2003

The relationship between executive function and verbal memory in Parkinson's disease.

A growing body of evidence suggests that the various cognitive symptoms found in Parkinson's disease (PD) are secondary to executive dysfunction. Studies addressing this possibility for memory impairment specifically have not included measures of working memory nor have they ruled-out potential mediating variables such as overall level of cognitive impairment or depression. The purpose of this study was to include measures of these variables in determining the relationship between multiple aspects of executive function and delayed verbal recall in 32 idiopathic PD patients. Results were consistent with the original hypothesis and further suggest that working memory is a key factor in recall memory and may mediate the relationship between other executive measures and recall in PD.

Cognition Disorders↗

PubMed2002

Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform, Pik1.

Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10-192), showed that residues within 80-192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151-199) competed for binding of [(35)S]Pik1-(10-192) to bead-immobilized Frq1, whereas shorter peptides (164-199 and 174-199) did not. Correspondingly, a deletion mutant, Pik1(delta152-191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10-192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110-192), or the 151-199 peptide to Frq1 shows high affinity (K(d) approximately 100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110-192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is not S-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Delta cells. This study defines the segment of Pik1 required for high affinity binding of Frq1.

1-Phosphatidylinositol 4-Kinase↗

PubMed2002

An efficient system for the evolution of aminoacyl-tRNA synthetase specificity.

A variety of strategies to incorporate unnatural amino acids into proteins have been pursued, but all have limitations with respect to technical accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo, provides a potentially powerful approach to the study of protein function and to large-scale production of novel proteins. Here we describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity. Our strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli. A chloramphenicol-resistance (Cm(r)) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-containing protein in E. coli.

Amino Acids↗

PubMed2002

Largest subunits of the human SWI/SNF chromatin-remodeling complex promote transcriptional activation by steroid hormone receptors.

The mammalian SWI/SNF-related complexes facilitate gene transcription by remodeling chromatin using the energy of ATP hydrolysis. The recruitment of these complexes to promoters remains poorly understood and may involve histone modifications or direct interactions with site-specific transcription factors or other cofactors. Here we report the isolation of two related but distinct cDNA clones, hOsa1 and hOsa2, that encode the largest subunits of human SWI/SNF. hOsa1 is identical to previously reported BAF250, and hOsa2 shares a high degree of sequence similarity with hOsa1. Mass spectrometric analysis, and immunoblotting with antibodies specific to hOsa1 or hOsa2 demonstrate the presence of both proteins in SWI/SNF-A but not in the related polybromo-BRG1-associated factors complex purified from HeLa cells. Co-precipitation studies indicate that hOsa1 and hOsa2 associate with BRG1 and hBRM through the C-terminal domain of hOsa. We define multiple domains within hBRM and BRG1 that interact with the hOsa C terminus. In cultured mammalian cells, hOsa1 and hOsa2 stimulate transcription by the glucocorticoid, estrogen, and androgen receptors. The glucocorticoid receptor-mediated activation is not observed with the C-terminal domain or with the hOsa2 polypeptide lacking the ARID DNA binding domain. These results suggest that hOsa1 and hOsa2 participate in promoting transcriptional activation by the steroid hormone receptors.

Amino Acid Sequence↗

PubMed2002

Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli.

We report the selection of a new orthogonal aminoacyl tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-azido-l-phenylalanine, into proteins in response to the amber codon, TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslink interacting proteins.

Azides↗

PubMed2002

Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli.

Benzophenones are among the most useful photocrosslinking agents in biology. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-l-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradiation resulted in efficient crosslinking (>50%) of the protein subunits. This methodology may prove useful for discovering and defining protein interactions in vitro and in vivo.

Amino Acids↗