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Publications (34)

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    [Show abstract] [Hide abstract] ABSTRACT: All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-Type-specific gene regulation.
    Full-text Article · Apr 2016 · Nature Communications
  • [Show abstract] [Hide abstract] ABSTRACT: GPR126 is an orphan heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) that is essential for the development of diverse organs. We found that type IV collagen, a major constituent of the basement membrane, binds to Gpr126 and activates its signaling function. Type IV collagen stimulated the production of cyclic adenosine monophosphate in rodent Schwann cells, which require Gpr126 activity to differentiate, and in human embryonic kidney (HEK) 293 cells expressing exogenous Gpr126. Type IV collagen specifically bound to the extracellular amino-terminal region of Gpr126 containing the CUB (complement, Uegf, Bmp1) and pentraxin domains. Gpr126 derivatives lacking the entire amino-terminal region were constitutively active, suggesting that this region inhibits signaling and that ligand binding relieves this inhibition to stimulate receptor activity. A new zebrafish mutation that truncates Gpr126 after the CUB and pentraxin domains disrupted development of peripheral nerves and the inner ear. Thus, our findings identify type IV collagen as an activating ligand for GPR126, define its mechanism of activation, and highlight a previously unrecognized signaling function of type IV collagen in basement membranes.
    Article · Aug 2014 · Science Signaling
  • [Show abstract] [Hide abstract] ABSTRACT: Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.
    Article · Apr 2014 · Proceedings of the National Academy of Sciences
  • [Show abstract] [Hide abstract] ABSTRACT: Vital to bacterial survival is the faithful propagation of cellular signals, and in Caulobacter crescentus, ChpT is an essential mediator within the cell-cycle circuit. ChpT functions as a histidine-containing phosphotransfer protein (HPt) that shuttles a phosphoryl group from the receiver domain of CckA, the upstream hybrid histidine kinase (HK), to one of two downstream response regulators (CtrA or CpdR) that controls cell-cycle progression. To understand how ChpT interacts with multiple signaling partners, we solved the crystal structure of ChpT at 2.3 Å resolution. ChpT adopts a pseudo-HK architecture but does not bind ATP. We identified two point mutation classes affecting phosphotransfer and cell morphology: one that globally impairs ChpT phosphotransfer, and a second that mediates partner selection. Importantly, a small set of conserved ChpT residues promotes signaling crosstalk and contributes to the branched signaling that activates the master regulator CtrA while inactivating the CtrA degradation signal, CpdR.
    Article · Aug 2013 · Structure
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    [Show abstract] [Hide abstract] ABSTRACT: A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development.
    Full-text Article · Dec 2011 · Journal of Biological Chemistry
  • [Show abstract] [Hide abstract] ABSTRACT: A potent neutralizing antibody to a conserved HCV epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralized most HCV genotypes but having modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants and using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and kappa chain variable (Vk) genes separately and then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wildtype (wt) HC-1. Moreover, propagating 2a HCVcc under the selective pressure of wt HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants, and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development.
    Article · Oct 2011 · Journal of Biological Chemistry
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    [Show abstract] [Hide abstract] ABSTRACT: Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.
    Full-text Article · Aug 2010 · Nature Cell Biology
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    [Show abstract] [Hide abstract] ABSTRACT: A series of novel HIV-1 protease inhibitors based on the (hydroxyethylamino)-sulfonamide isostere incorporating substituted phenyls and benzheterocycle derivatives bearing rich hydrogen bonding acceptors as P(2) ligands were synthesized. Prolonged chain linking the benzhereocycle to the carbonyl group resulted in partial loss of binding affinities. Introduction of a small alkyl substituent with appropriate size to the -CH2- of P(1)-P(2) linkage as a side chain resulted in improved inhibitory potency, and in this study, isopropyl was the best side chain. Replacement of the isobutyl substituent at P(1)'group with phenyl substituent decreased the inhibitory potency. One of the most potent inhibitor, compound 23 showing high affinity to HIV-1 protease with an IC(50) value of 5 nM, also exhibited good anti-SIV activity (EC(50) = 0.8 microM) with low toxicity (TC(50) > 100 microM). The flexible docking of inhibitor 23 to HIV-1 protease active site rationalized the interactions with protease.
    Full-text Article · Aug 2010 · Chemical Biology & Drug Design
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    [Show abstract] [Hide abstract] ABSTRACT: Transient transfection of mammalian cells in suspension culture has recently emerged as a very useful method for production of research-scale quantities of recombinant proteins. The most commonly used cell lines for this purpose are suspension-adapted HEK and CHO cells. We report here that the plasma exposure in mice of an IL-23R extracellular domain Fc fusion protein (IL23R-Fc) differed dramatically depending on whether the protein was prepared by transient transfection of HEK or CHO cells. Specifically, IL23R-Fc expressed using CHO cells had about 30-fold higher in vivo plasma exposure compared to the HEK-expressed protein. In contrast to their differing plasma exposures, the HEK- and CHO-expressed proteins had equivalent in vitro biological activity. Characterization of the CHO- and HEK-expressed IL23R-Fc proteins indicated that the differences in in vivo plasma exposure between them are due to differential glycosylation.
    Full-text Article · May 2010 · Protein Expression and Purification
  • [Show abstract] [Hide abstract] ABSTRACT: Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the world's supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.
    Article · Jan 2009 · Phytochemistry
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    Dataset: Figure S2
    [Show abstract] [Hide abstract] ABSTRACT: Experimental strategy. Embryos at the 2 pronuclei (2PN) or 1-cell stage are collected from wild type matings, and injected with an antisense morpholino oligomer (MO) that has been designed to target a specific gene. MO binds to 5′ UTR or transcription start site and blocks translation by steric hindrance. Microinjected embryos and uninjected control embryos are cultured in vitro and observed for developmental phenotypes such as fragmentation, or arrest at the 2-cell, 4-cell, multicell, or morula stages. Theoretically, this strategy may uncover other phenotypes such as asymmetrical division, but we have not observed them in the genes that we have tested. If a gene-specific MO produces the same phenotype consistently, while the mismatch control MO allows normal development, then we validate knockdown of the gene of interest by immunocytochemistry and/or immunoblotting. Mechanism of gene function is further investigated by obtaining global gene expression profiles from injected and control embryos at the mid-2-cell stage (43 hours post-HCG). Candidate downstream genes are tested for differential expression, and gene function in the early embryo. It is expected that multiple iterations of this strategy to test functions of different transcriptional regulators and their downstream targets will help to deconstruct the gene regulatory network in the mouse embryo at the cusp of embryonic genome activation. (0.77 MB DOC)
    Full-text Dataset · Dec 2008
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    Dataset: Table S5
    [Show abstract] [Hide abstract] ABSTRACT: Genes that have higher expression levels in Ccna2-MO-injected compared to uninjected embryos. (0.03 MB XLS)
    Full-text Dataset · Dec 2008
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    Dataset: Table S4
    [Show abstract] [Hide abstract] ABSTRACT: Genes that have lower expression levels in Oct4-MO-injected compared to uninjected embryos. (0.11 MB XLS)
    Full-text Dataset · Dec 2008
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    Dataset: Table S11
    [Show abstract] [Hide abstract] ABSTRACT: Functional categories that were enriched in upregulated genes in the Oct4 knockdown model. (0.01 MB PDF)
    Full-text Dataset · Dec 2008
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    Dataset: Table S3
    [Show abstract] [Hide abstract] ABSTRACT: Genes that have higher expression levels in Oct4-MO-injected compared to uninjected embryos. (0.05 MB XLS)
    Full-text Dataset · Dec 2008
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    Dataset: Table S2
    [Show abstract] [Hide abstract] ABSTRACT: Summary of the number of embryos tested and the number of experiments performed for each condition. (0.07 MB PDF)
    Full-text Dataset · Dec 2008
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    Dataset: Figure S5
    [Show abstract] [Hide abstract] ABSTRACT: Decreased Oct4 expression at the multicell stage in Oct4-MO-injected embryos. Only 6.4±3.2% of Oct4-MO-injected embryos showed nuclear Oct4 signal, while 88.9±11.1% of Oct4-MM-injected embryos and 82.7±10.9% of uninjected control embryos showed unequivocal nuclear Oct4 expression at the multicell stage; p<0.05. (10.82 MB TIF)
    Full-text Dataset · Dec 2008
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    Dataset: Figure S3
    [Show abstract] [Hide abstract] ABSTRACT: Oct4 expression in the mouse zygote by single embryo RT-PCR. (0.11 MB TIF)
    Full-text Dataset · Dec 2008
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    Dataset: Figure S4
    [Show abstract] [Hide abstract] ABSTRACT: Decreased Oct4 expression was evident by the 4-cell stage in Oct4-MO-injected embryos. Oct4 signal was absent in embryos injected with Oct4-MO, but its nuclear localization was present in uninjected and mismatch controls. Scale bar 40 µm. (10.94 MB TIF)
    Full-text Dataset · Dec 2008
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    Dataset: Figure S7
    [Show abstract] [Hide abstract] ABSTRACT: Confirmation of the requirement of Oct4 in early embryo development by Oct4E4-MO, an antisense morpholino that targets the splice site of exon 4 of Oct4. a, sites targeted by the two morpholinos, Oct4-MO and Oct4E4-MO. Oct4-MO targets the 25 nucleotides starting at the ATG start site, while Oct4E4-MO targets the splice site at the intron (I)-exon (E) boundary of the 4th exon (E4). Removal of E4 is expected to result in a protein product that lacks the DNA-binding and activation domains (1). b, 64.6±19.9% of embryos injected with Oct4E4-MO, while none that were injected with the mismatch control, Oct4E4-MM, arrested at the 2-cell stage. c, Blastocyst development is severely compromised after injection of Oct4E4-MO compared to the mismatch control, Oct4E4-MM. Reference. 1. Morcos PA (2007) Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos. Biochem Biophys Res Commun 358, 521–527. (2.33 MB TIF)
    Full-text Dataset · Dec 2008