Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past ...been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the
Escherichia
coli periplasm. The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs. One
E. coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor. As predicted, the protein lacks binding to C1q and the FcγRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental. In addition, a limited chimpanzee study suggests that the
E. coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.
A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological ...signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.
Careful regulation of the Wnt–Β‐catenin signaling pathway is critical to many aspects of development and cancer. Casein kinase Iε is a Wnt‐activated positive regulator of this pathway. Members of the ...Dishevelled family have been identified as key substrates of casein kinase I (CKI). However, the specific sites phosphorylated in vivo by CKI and their relative importance in the physiologic regulation of these proteins in the canonical Wnt–β‐catenin signaling pathway remain unclear. To address this question, recombinant mouse Dishevelled (mDvl‐1) was phosphorylated by CKIin vitro and phosphorylation sites were identified by MS. CKI phosphorylation of mDvl‐1 at two highly conserved residues, serines 139 and 142, was observed by MS and confirmed by phosphopeptide mapping of in vivo phosphorylated protein. Phosphorylation of these sites is dependent on casein kinase I epsilon activity in vivo. Phenotypic analysis of mutant mDvl‐1 indicates that phosphorylation of these sites stimulates the Dvl‐activated β‐catenin‐dependent Wnt signaling pathway in both cell culture and in Xenopus development. Casein kinase I epsilon is a Wnt‐regulated kinase, and regulated phosphorylation of Dvl allows fine tuning of the Wnt–β‐catenin signaling pathway.
The Wnt/β-catenin signaling pathway is important in both development and cancer. Casein kinase Iϵ (CKIϵ) is a positive regulator of the canonical Wnt pathway. CKIϵ itself can be regulated in vitro by ...inhibitory autophosphorylation, and recent data suggest that in vivo kinase activity can be regulated by extracellular stimuli. We show here that the phosphorylation state and kinase activity of CKIϵ are directly regulated by Wnt signaling. Coexpression of XWnt-8 or addition of soluble Wnt-3a ligand led to a significant and rapid increase in the activity of endogenous CKIϵ. The increase in CKIϵ activity is the result of decreased inhibitory autophosphorylation because it is abolished by preincubation of immunoprecipitated kinase with ATP. Furthermore, mutation of CKIϵ inhibitory autophosphorylation sites creates a kinase termed CKIϵ(MM2) that is significantly more active than CKIϵ and is not activated further upon Wnt stimulation. Autoinhibition of CKIϵ is biologically relevant because CKIϵ(MM2) is more effective than CKIϵ at activating transcription from a Lef1-dependent promoter. Finally, CKIϵ(MM2) expression in Xenopus embryos induces both axis duplication and additional developmental abnormalities. The data suggest that Wnt signaling activates CKIϵ by causing transient dephosphorylation of critical inhibitory sites present in the carboxyl-terminal domain of the kinase. Activation of the Wnt pathway may therefore stimulate a cellular phosphatase to dephosphorylate and activate CKIϵ
Careful regulation of the Wnt-beta-catenin signaling pathway is critical to many aspects of development and cancer. Casein kinase Iepsilon is a Wnt-activated positive regulator of this pathway. ...Members of the Dishevelled family have been identified as key substrates of casein kinase I (CKI). However, the specific sites phosphorylated in vivo by CKI and their relative importance in the physiologic regulation of these proteins in the canonical Wnt-beta-catenin signaling pathway remain unclear. To address this question, recombinant mouse Dishevelled (mDvl-1) was phosphorylated by CKIin vitro and phosphorylation sites were identified by MS. CKI phosphorylation of mDvl-1 at two highly conserved residues, serines 139 and 142, was observed by MS and confirmed by phosphopeptide mapping of in vivo phosphorylated protein. Phosphorylation of these sites is dependent on casein kinase I epsilon activity in vivo . Phenotypic analysis of mutant mDvl-1 indicates that phosphorylation of these sites stimulates the Dvl-activated beta-catenin-dependent Wnt signaling pathway in both cell culture and in Xenopus development. Casein kinase I epsilon is a Wnt-regulated kinase, and regulated phosphorylation of Dvl allows fine tuning of the Wnt-beta-catenin signaling pathway. PUBLICATION ABSTRACT
Careful regulation of the Wnt-Beta-catenin signaling pathway is critical to many aspects of development and cancer. Casein kinase Iepsilon is a Wnt-activated positive regulator of this pathway. ...Members of the Dishevelled family have been identified as key substrates of casein kinase I (CKI). However, the specific sites phosphorylated in vivo by CKI and their relative importance in the physiologic regulation of these proteins in the canonical Wnt-beta-catenin signaling pathway remain unclear. To address this question, recombinant mouse Dishevelled (mDvl-1) was phosphorylated by CKIin vitro and phosphorylation sites were identified by MS. CKI phosphorylation of mDvl-1 at two highly conserved residues, serines 139 and 142, was observed by MS and confirmed by phosphopeptide mapping of in vivo phosphorylated protein. Phosphorylation of these sites is dependent on casein kinase I epsilon activity in vivo. Phenotypic analysis of mutant mDvl-1 indicates that phosphorylation of these sites stimulates the Dvl-activated beta-catenin-dependent Wnt signaling pathway in both cell culture and in Xenopus development. Casein kinase I epsilon is a Wnt-regulated kinase, and regulated phosphorylation of Dvl allows fine tuning of the Wnt-beta-catenin signaling pathway.
The Wnt/β-catenin signaling pathway is important in both development and cancer. Casein kinase Iϵ (CKIϵ) is a positive regulator
of the canonical Wnt pathway. CKIϵ itself can be regulated in ...vitro by inhibitory autophosphorylation, and recent data suggest that in vivo kinase activity can be regulated by extracellular stimuli. We show here that the phosphorylation state and kinase activity
of CKIϵ are directly regulated by Wnt signaling. Coexpression of XWnt-8 or addition of soluble Wnt-3a ligand led to a significant
and rapid increase in the activity of endogenous CKIϵ. The increase in CKIϵ activity is the result of decreased inhibitory
autophosphorylation because it is abolished by preincubation of immunoprecipitated kinase with ATP. Furthermore, mutation
of CKIϵ inhibitory autophosphorylation sites creates a kinase termed CKIϵ(MM2) that is significantly more active than CKIϵ
and is not activated further upon Wnt stimulation. Autoinhibition of CKIϵ is biologically relevant because CKIϵ(MM2) is more
effective than CKIϵ at activating transcription from a Lef1-dependent promoter. Finally, CKIϵ(MM2) expression in Xenopus embryos induces both axis duplication and additional developmental abnormalities. The data suggest that Wnt signaling activates
CKIϵ by causing transient dephosphorylation of critical inhibitory sites present in the carboxyl-terminal domain of the kinase.
Activation of the Wnt pathway may therefore stimulate a cellular phosphatase to dephosphorylate and activate CKIϵ
A
Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by ...chemotherapeutically-abbreviated
D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in
E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in
D. immitis larval and adult parasite extracts.
Numerous regulatory mechanisms are involved in the controlling Wnt signal transduction in development and tumorigenesis. Members of the casein kinase I (CKI) family play a significant role in ...determining the molecular events in response to Wnt hormone. This dissertation primarily describes the crucial role of CKI-specific isoforms in regulating Wnt signal transduction. Investigation of the mechanisms for CKI isoform activation and identification of CKI-specific phosphorylation sites required for Wnt signal transduction are presented. First, canonical Wnt hormones transduce Wnt signal that activates intracellular CKIϵ activity. CKIϵ is inactive in vivo until it is specifically activated by dephosphorylation of its autoinhibitory domain. After the concentration Wnt ligand diminishes, CKIϵ autophosphorylates its carboxy-tail domain, inhibiting its activity, and CKIϵ activity returns to baseline levels. Second, CKIϵ-specific phosphorylation of dishevelled on serine 139 and serine 142 modulates activation of Wnt signal transduction. Mutation of serine 139 and 142 to alanines results in diminished CKIϵ-dependent activation of Wnt signaling in cell culture and Xenopus embryo model systems. These results provide molecular insights into the mechanism by which CKIϵ-regulates the intensity of β-catenin signaling. Third, CKI-specific isoform activities differentially control negative regulatory components of the canonical Wnt pathway. A potential CKIα-specific phosphorylation site was identified on β-catenin which contributes to the negative regulation of Wnt signaling and may contribute to a molecular conformation pool of β-catenin necessary to maintain cellular homeostasis. Potential CKIϵ-specific phosphorylation sites were identified on axin that may regulate cellular distribution and degradation of axin in the cell. Fourth, tools for deciphering the balance between kinases and phosphatases involved in the Wnt pathway were examined. We demonstrate dsRNAi knockout of endogenous PP2A-C subunit leads to hype rphosphorylation of the Wnt substrates Dsh and dysregulation of the Arm destruction complex in genetically engineered S2R+ cells. This dissertation extended our knowledge of how CKI-specific isoforms can differentiate Wnt signal transduction in the cell. Results from these studies specify specific-CKIϵ residues that may be important targets in the Wnt pathway and ultimately drive development of pharmacological agents specifically attenuating dysregulation of the β-catenin accumulation in human cancers.
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