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1.  Fusion of a Short Peptide that Binds Immunoglobulin G to a Recombinant Protein Substantially Increases Its Plasma Half-Life in Mice 
PLoS ONE  2014;9(7):e102566.
We explore a strategy to substantially increase the half-life of recombinant proteins by genetic fusion to FcIII, a 13-mer IgG-Fc domain binding peptide (IgGBP) originally identified by DeLano and co-workers at Genentech [DeLano WL, et al. (2000) Science 287∶1279–1283]. IgGBP fusion increases the in vivo half-life of proteins by enabling the fusion protein to bind serum IgG, a concept originally introduced by DeLano and co-workers in a patent but that to the best of our knowledge has never been pursued in the scientific literature. To further investigate the in vitro and in vivo properties of IgGBP fusion proteins, we fused FcIII to the C-terminus of a model fluorescent protein, monomeric Katushka (mKate). mKate-IgGBP fusions are easily expressed in Escherichia coli and bind specifically to human IgG with an affinity of ∼40 nM and ∼20 nM at pH 7.4 and pH 6, respectively, but not to mouse or rat IgG isotypes. mKate-IgGBP binds the Fc-domain of hIgG1 at a site overlapping the human neonatal Fc receptor (hFcRn) and as a consequence inhibits the binding of hIgG1 to hFcRn in vitro. High affinity binding to human IgG also endows mKate-IgGBP with a long circulation half-life of ∼8 hr in mice, a 75-fold increase compared to unmodified mKate. Thus, IgGBP fusion significantly reduces protein clearance by piggybacking on serum IgG without substantially increasing protein molecular weight due to the small size of the IgGBP. These attractive features could result in protein therapies with reduced dose frequency and improved patient compliance.
PMCID: PMC4109916  PMID: 25057984
2.  Glucose Sensor O-GlcNAcylation Coordinates with Phosphorylation to Regulate Circadian Clock 
Cell metabolism  2013;17(2):291-302.
Post-translational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3β dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3β. Interestingly, OGT activity is regulated by GSK3β, hence OGT and GSK3β exhibit reciprocal regulation. Modulating OGlcNAcylation levels alter circadian period length in both mice and Drosophila, and conversely protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662–S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine tune circadian clock.
PMCID: PMC3597447  PMID: 23395175
3.  All Dact (Dapper/Frodo) scaffold proteins dimerize and exhibit conserved interactions with Vangl, Dvl, and serine/threonine kinases 
BMC Biochemistry  2011;12:33.
The Dact family of scaffold proteins was discovered by virtue of binding to Dvl proteins central to Wnt and Planar Cell Polarity (PCP) signaling. Subsequently Dact proteins have been linked to a growing list of potential partners implicated in β-catenin-dependent and β-catenin-independent forms of Wnt and other signaling. To clarify conserved and non-conserved roles for this protein family, we systematically compared molecular interactions of all three murine Dact paralogs by co-immunoprecipitation of proteins recombinantly expressed in cultured human embryonic kidney cells.
Every Dact paralog readily formed complexes with the Vangl, Dvl, and CK1δ/ε proteins of species ranging from fruit flies to humans, as well as with PKA and PKC. Dact proteins also formed complexes with themselves and with each other; their conserved N-terminal leucine-zipper domains, which have no known binding partners, were necessary and sufficient for this interaction, suggesting that it reflects leucine-zipper-mediated homo- and hetero-dimerization. We also found weaker, though conserved, interactions of all three Dact paralogs with the catenin superfamily member p120ctn. Complex formation with other previously proposed partners including most other catenins, GSK3, LEF/TCF, HDAC1, and TGFβ receptors was paralog-specific, comparatively weak, and/or more sensitive to empirical conditions.
Combined with published functional evidence from targeted knock-out mice, these data support a conserved role for Dact proteins in kinase-regulated biochemistry involving Vangl and Dvl. This strongly suggests that a principal role for all Dact family members is in the PCP pathway or a molecularly related signaling cascade in vertebrates.
PMCID: PMC3141656  PMID: 21718540
4.  Dact1-3 mRNAs exhibit distinct expression domains during tooth development 
Gene expression patterns : GEP  2010;10(2-3):140-143.
Wnt signaling is essential for tooth formation. Dact proteins modulate Wnt signaling by binding to the intracellular protein Dishevelled (Dvl). Comparison of all known mouse Dact genes, Dact1-3, from the morphological initiation of mandibular first molar development after the onset of the root formation using sectional in situ hybridization showed distinct, complementary and overlapping expression patterns for the studied genes. While Dact2 expression was restricted to the dental epithelium including the enamel knot signaling centers and tooth specific preameloblasts, Dact1 and Dact3 showed developmentally regulated expression in the dental mesenchyme. Both mRNAs were first detected in the presumptive dental mesenchyme. After being downregulated from the condensed dental mesenchyme of the bud stage tooth germ, Dact1 was upregulated in the dental follicle masenchyme at the cap stage and subsequently also in the dental papilla at the bell stage where the expression persisted to the postnatal stages. In contrast, Dact3 transcripts persisted throughout the dental mesenchymal tissue components including the tooth-specific cells, preodontoblasts before transcripts were largely downregulated from the tooth germ postnatally. Collectively these results suggest that Dact1 and -3 may contribute to early tooth formation by modulation of Wnt signaling pathways in the mesenchyme, including preodontoblasts, whereas Dact2 may play important signal-modulating roles in the adjacent epithelial cells including the enamel knot signaling centers and preameloblasts. Future loss-of-function studies will help elucidate whether any of these functions are redundant, particularly for Dact1 and Dact3.
PMCID: PMC2849867  PMID: 20170752
5.  Dact1 is a postsynaptic protein required for dendrite, spine, and excitatory synapse development in the mouse forebrain 
Dact1 (Dapper/Frodo), an intracellular phosphoprotein that binds Dishevelled, catenins, and other signaling proteins, is expressed in the developing and mature mammalian central nervous system, but its function there is unknown. Dact1 colocalized with synaptic markers and partitioned to postsynaptic fractions from cultured mouse forebrain neurons. Hippocampal neurons from Dact1 knockout mice had simpler dendritic arbors and fewer spines than hippocampal neurons from wild type littermates. This correlated with reductions in excitatory synapses and miniature excitatory postsynaptic currents, whereas inhibitory synapses were not affected. Loss of Dact1 resulted in a decrease in activated Rac, and recombinant expression of either Dact1 or constitutively active Rac, but not Rho or Cdc42, rescued dendrite and spine phenotypes in Dact1 mutant neurons. Our findings suggest that during neuronal differentiation Dact1 plays a critical role in a molecular pathway promoting Rac activity underlying the elaboration of dendrites and the establishment of spines and excitatory synapses.
PMCID: PMC2848693  PMID: 20335472
Dendritic spine; Synapse; Rac; Dendrite; Forebrain; Hippocampus
6.  Posterior Malformations in Dact1 mutant mice arise through misregulated Vangl2 at the Primitive Streak 
Nature genetics  2009;41(9):977-985.
Mice homozygous for mutations in Dact1 (Dpr/Frodo) phenocopy human malformations involving the spine, genitourinary system, and distal digestive tract. We trace this phenotype to disrupted germ layer morphogenesis at the primitive streak (PS). Remarkably, heterozygous mutation of Vangl2, a transmembrane component of the Planar Cell Polarity (PCP) pathway, rescues recessive Dact1 phenotypes, whereas loss of Dact1 reciprocally rescues semidominant Vangl2 phenotypes. We show that Dact1, an intracellular protein, forms a complex with Vangl2. In Dact1 mutants, Vangl2 is increased at the PS where cells ordinarily undergo an epithelial-mesenchymal transition. This is associated with abnormal E-cadherin distribution and changes in biochemical measures of the PCP pathway. We conclude that Dact1 contributes to morphogenesis at the PS by regulating Vangl2 upstream of cell adhesion and the PCP pathway.
PMCID: PMC2733921  PMID: 19701191
Dact (Dapper Frodo); Vangl2 (Van Gogh Strabismus); PCP; Wnt; spina bifida; caudal regression; OEIS; primitive streak
7.  DACT3 is an epigenetic regulator of Wnt/β-catenin signaling in colorectal cancer and is a therapeutic target of histone modifications 
Cancer cell  2008;13(6):529-541.
Genetic and epigenetic defects in Wnt/β-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/β-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly de-repressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/β-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/β-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/β-catenin signaling in colorectal cancer.
PMCID: PMC2577847  PMID: 18538736
8.  Activating PER Repressor through a DBT-Directed Phosphorylation Switch  
PLoS Biology  2008;6(7):e183.
Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT), a Drosophila ortholog of human casein kinase I (CKI)ɛ/δ. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER). DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The perS mutation, which is associated with short-period (19-h) circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function.
Author Summary
Most proteins involved in circadian transcriptional feedback loops undergo reversible chemical modifications (called phosphorylation) that regulate their activity in a time-of-day–dependent manner. Doubletime (DBT), a Drosophila kinase, phosphorylates the circadian transcriptional repressor PERIOD (PER). Mutations of dbt shorten or lengthen the period of circadian behavioral rhythms, or abolish the rhythms altogether in flies. A mutation of the human ortholog of dbt, casein kinase I (CKI)δ, has been associated with certain forms of a heritable sleep disorder. The disorder may reflect altered activity of a human PER protein, as the syndrome can also be caused by mutation of a CKIɛ/δ phosphorylation site within PER2. In this study, we locate DBT-directed phosphorylation sites in the Drosophila PER protein, including a DBT target region of PER that was previously shown to regulate DBT activity. Two PER domains within this region appear to serve as alternative targets for DBT. Phosphorylation of the upstream domain seems to suppress phosphorylation elsewhere in the region, producing a stable PER protein with little activity as a transcriptional repressor. However, when phosphorylation of the upstream domain is blocked, downstream DBT targets appear to be phosphorylated, producing a highly active, but short-lived repressor. Our results suggest that ordered patterns of DBT-directed phosphorylation contribute to the timing of PER's function and disappearance, and thus influence the pace of the circadian clock.
Two phosphorylation domains inDrosophila PERIOD protein interact in a switch-like fashion with each other and the kinase DOUBLETIME to regulate PER's stability and activity as a transcriptional repressor in the circadian transcriptional feedback loop.
PMCID: PMC2486307  PMID: 18666831

Results 1-8 (8)