Increase in ADP-ribosyltransferase activity of rat T lymphocyte alloantigen RT6.1 by a single amino acid mutation

AbstractA family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various mammalian cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini. Although the first Glu in the common motif is replaced by Gln (Q207EEVLIP) in rat T lymphocyte alloantigens RT6.1 and RT6.2, the two RT6s appear to have both activities of NAD+ glycohydrolase and ADP-ribosyltransferase to a lesser extent. To investigate the significance of the Glu-rich motif in the two enzyme activities, we produced a mutant RT6.1 (Q207E), in which Gln207 was replaced by Glu, together with wild-type RT6s, in Escherichia coli. Kinetic analysis revealed that there were no marked differences in the Vmax and Km values of NAD+ glycohydrolases among the three recombinant proteins. The recombinant RT6.1 and RT6.2 displayed extremely low auto-ADP-ribosylation, although the latter modification was somewhat higher than the former. In contrast, much greater auto-modification was observed for the Q207E mutant. Moreover, the mutant could effectively ADP-ribosylate agmatine as a substrate. Thus, the single amino acid mutation of RT6.1 caused a marked increase in its ADP-ribosyltransferase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the enzyme activity

The C. elegans Hypodermis Couples Progenitor Cell Quiescence to the Dietary State

SummaryThe nutritional status of an organism can greatly impact the function and behavior of stem and progenitor cells [1]. However, the regulatory circuits that inform these cells about the dietary environment remain to be elucidated. Newly hatched C. elegans larvae (L1s) halt development in “L1 arrest” or “L1 diapause” until ample food is encountered and triggers stem and progenitor cells to exit from quiescence [2]. The insulin/insulin-like growth factor signaling (IIS) pathway plays a key role in this reactivation [3, 4], but its site(s) of action have not been elucidated nor have the nutrient molecule(s) that stimulate the pathway been identified. By tissue-specifically modulating the activity of its components, we demonstrate that the IIS pathway acts in the hypodermis to regulate nutrition-responsive reactivation of neural and mesodermal progenitor cells. We identify ethanol, a likely component of the natural Caenorhabditis habitat, and amino acids as nutrients that synergistically reactivate somatic progenitor cells and upregulate expression of insulin-like genes in starved L1 larvae. The hypodermis likely senses the availability of amino acids because forced activation of the amino-acid-responsive Rag-TORC1 (target of rapamycin complex 1) pathway in this tissue can also release somatic progenitor cell quiescence in the presence of ethanol. Finally, there appears to be crosstalk between the IIS and Rag-TORC1 pathways because constitutive activation of the IIS pathway requires Rag to promote reactivation. This work demonstrates that ethanol and amino acids act as dietary cues via the IIS and Rag-TORC1 pathways in the hypodermis to coordinately control progenitor cell behavior

Guanine nucleotide-binding protein in sea urchin eggs serving as the specific substrate of islet-activating protein, pertussis toxin

AbstractA GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from Lubrol extract of sea urchin egg membranes. The partially purified protein possessed two polypeptides of 39 and 37 kDa; the 39 kDa polypeptide was specifically ADP-ribosylated by IAP and the 37 kDa protein cross-reacted with the antibody prepared against purified βγ-subunits of αβγ-heterotrimeric IAP substrates from rat brain. Incubation of this sea urchin IAP substrate with a non-hydrolyzable GTP analogue resulted in a reduction of the apparent molecular mass on a column of gel filtration as had been the case with purified rat brain IAP substrates, suggesting that the sea urchin IAP substrate was also a heterooligomer dissociable into two polypeptides in the presence of GTP analogues. Thus, the 39 and 37 kDa polypeptides of the sea urchin IAP substrate correspond to the α- and β-subunits, respectively, of mammalian IAP substrates which are involved in the coupling between membrane receptor and effector systems

Evidence for receptor-mediated inhibition of intrinsic activity of GTP-binding protein, Gi1 and Gi2, but not G0 in reconstitution experiments

AbstractThe receptor-mediated inhibition of intrinsic activities of GTP-binding proteins (G-proteins) was studied. Pertussis toxin (IAP)-substrate G-protein, Gi1, Gi2 or G0, was prelabeled with [α-32P]GDP and reconstituted with synaptic membranes of the guinea pig cerebellum in the presence of 0.02% of Chaps. Intrinsic activities of G-proteins were evaluated by the release of [α-32P]GDP in exchange for added GppNHp or GDP in reconstituted preparations. U-50,488H (1 nM-10 μM), a specific ϰ-subtype of opioid receptor agonist, inhibited the [α-32P]GDP release in exchange for added 1 μM GppNHp in Gi1-reconstituted preparations in a concentration-dependent manner. On the other hand, the ϰ-opioid agonist at 10 μM increases the Km values of GppNHp, but not GDP in exchange for [α-32P]GDP release in preparations reconstituted with Gi1 or Gi2, but not with G0. These findings indicate that ϰ-opioid receptor is coupled to inhibition of intrinsic activities of Gi1 and Gi2, but not G0, in guinea pig cerebellar membranes. In addition, it was revealed that the mode of action is mediated by a decrease in affinity of GTP (or its analog) for G-proteins, but not by a change in affinity of GDP

Ependymoma associated protein Zfta is expressed in immature ependymal cells but is not essential for ependymal development in mice

The fusion protein of uncharacterised zinc finger translocation associated (ZFTA) and effector transcription factor of tumorigenic NF‑κB signalling, RELA (ZFTA‑RELA), is expressed in more than two‑thirds of supratentorial ependymoma (ST‑EPN‑RELA), but ZFTA¿s expression profile and functional analysis in multiciliated ependymal (E1) cells have not been examined. Here, we showed the mRNA expression of mouse Zfta peaks on embryonic day (E) 17.5 in the wholemount of the lateral walls of the lateral ventricle. Zfta was expressed in the nuclei of FoxJ1‑positive immature E1 (pre‑E1) cells in E18.5 mouse embryonic brain. Interestingly, the transcription factors promoting ciliogenesis (ciliary TFs) (e.g., multicilin) and ZFTA‑RELA upregulated luciferase activity using a 5′ upstream sequence of ZFTA in cultured cells. Zftatm1/tm1 knock‑in mice did not show developmental defects or abnormal fertility. In the Zftatm1/tm1 E1 cells, morphology, gene expression, ciliary beating frequency and ependymal flow were unaffected. These results suggest that Zfta is expressed in pre‑E1 cells, possibly under the control of ciliary TFs, but is not essential for ependymal development or flow. This study sheds light on the mechanism of the ZFTA‑RELA expression in the pathogenesis of ST‑EPNRELA: Ciliary TFs initiate ZFTA‑RELA expression in pre‑E1 cells, and ZFTA‑RELA enhances its own expression using positive feedback