XCR2, one of three Xenopus EGF-CFC genes, has a distinct role in the regulation of left-right patterning

Members of the EGF-CFC family facilitate signaling by a subset of TGFbeta superfamily ligands that includes the nodal-related factors and GDF1/VG1. Studies in mouse, zebrafish, and chick point to an essential role for EGF-CFC proteins in the action of nodal/GDF1 signals in the early establishment of the mesendoderm and later visceral left-right patterning. Antisense knockdown of the only known frog EGF-CFC factor (FRL1), however, has argued against an essential role for this factor in nodal/GDF1 signaling. To address this apparent paradox, we have identified two additional Xenopus EGF-CFC family members. The three Xenopus EGF-CFC factors show distinct patterns of expression. We have examined the role of XCR2, the only Xenopus EGF-CFC factor expressed in post-gastrula embryos, in embryogenesis. Antisense morpholino oligonucleotide-mediated depletion of XCR2 disrupts left-right asymmetry of the heart and gut. Although XCR2 is expressed bilaterally at neurula stage, XCR2 is required on the left side, but not the right side, for normal left-right patterning. Left-side expression of XNR1 in the lateral plate mesoderm depends on XCR2, whereas posterior bilateral expression of XNR1 does not, suggesting that distinct mechanisms maintain XNR1 expression in different regions of neurula-tailbud embryos. Ectopic XCR2 on the right side initiates premature right-side expression of XNR1 and XATV, and can reverse visceral patterning. This activity of XCR2 depends on its co-receptor function. These observations indicate that XCR2 has a crucial limiting role in maintaining a bistable asymmetry in nodal family signaling across the left-right axis.     

The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif

Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.     

Human corneal epithelial cells respond to ocular-pathogenic, but not to nonpathogenic-flagellin

In this study, we investigated the expression of TLR5 in human corneal epithelial cells (CEC), and the functional outcome of TLR5 triggering by flagellins of pathogenic- and nonpathogenic bacteria. Flagellins derived from Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescense or Bacillus subtilis were used. The TLR5 protein and TLR5 specific mRNA expression was evident on human CEC. In human corneal epithelium tissues, TLR5 protein was detected at the basal and wing cells of the tissues. Ocular pathogenic bacteria, namely P. aeruginosa and S. marcescense, derived flagellin induced the significantly increased level of gene activation and IL-6 and IL-8 production. In contrast, ocular nonpathogenic S. typhimurium- and B. subtilis-derived flagellin induced neither the gene activation nor the increased production of IL-6 and IL-8 in human CEC. Human CEC would respond only to flagellin derived of ocular pathogenic bacteria, but not to those derived of ocular nonpathogenic bacteria, to generate pro-inflammatory cytokines.     

LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells

LKB1 is a 50 kDa serine/threonine kinase that phosphorylates and activates the catalytic subunit of AMPK at its T-loop residue Thr 172. We prepared adenoviruses expressing the constitutive active (wild-type) form (CA) or dominant negative (kinase inactive, D194A mutant) form (DN) of LKB1 and overexpressed these proteins in cultured myotubes (C2C12 cells) and rat hepatoma cells (FAO cells). When analyzed by immunoblotting with the antibody against Thr172-phosphorylated AMPK, the phosphorylation of AMPK was increased (2.5-fold) and decreased (0.4-fold) in cells expressing CA and DN LKB1, respectively, as compared with Lac-Z expressing control cells. Immunoprecipitation experiments, using isoform-specific antibody, revealed these alterations of AMPK phosphorylation to be attributable to altered phosphorylation of AMPK alpha2, but not alpha1 catalytic subunits, strongly suggesting the alpha2 catalytic subunit to be the major substrate for LKB1 in mammalian cells. In addition, adiponectin or AICAR-stimulated AMPK phosphorylation was inhibited by overexpression of DN LKB1, while phenformin-stimulated phosphorylation was unaffected. These results may explain the difference in AMPK activation mechanisms between AMP and phenformin, and also indicate that AMPK phosphorylation by LKB1 is involved in AMP-stimulated AMPK activation. As a downstream target for AMPK, AICAR-induced glucose uptake and ACCbeta phosphorylation were found to be significantly reduced in DN LKB1 expressing C2C12 cells. The expression of key enzymes for gluconeogenesis, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was also dependent on LKB1 activities in FAO cells. These results demonstrate that LKB1 is a crucial regulator of AMPK activation in muscle and liver cells and, therefore, that LKB1 activity is potentially of importance to our understanding of glucose and lipid metabolism.     

A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.     

Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.     

EpCAM contributes to formation of functional tight junction in the intestinal epithelium by recruiting claudin proteins

Tight junctions (TJs) connect epithelial cells and form a semipermeable barrier that only allows selective passage of ions and solutes across epithelia. Here we show that mice lacking EpCAM, a putative cell adhesion protein frequently overexpressed in human cancers, manifest intestinal barrier defects and die shortly after birth as a result of intestinal erosion. EpCAM was found to be highly expressed in the developing intestinal epithelium of wild-type mice and to localize to cell-cell junctions including TJs. Claudin-7 colocalized with EpCAM at cell-cell junctions, and the two proteins were found to associate with each other. Claudins 2, 3, 7, and 15 were down-regulated in the intestine of EpCAM mutant mice, with claudin-7 being reduced to undetectable levels. TJs in the mutant intestinal epithelium were morphologically abnormal with the network of TJ strands scattered and dispersed. Finally, the barrier function of the intestinal epithelium was impaired in the mutant animals. These results suggest that EpCAM contributes to formation of intestinal barrier by recruiting claudins to cell-cell junctions.