An electric lobe suppressor for a yeast choline transport mutation belongs to a new family of transporter-like proteins

Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3.5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2–4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNγ-responsive promoters in macrophages

IFNγ, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNγ's functions depend on the activation of STAT1, which stimulates transcription of IFNγ-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbγ or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNγ-inducible genes in macrophages. We found that macrophages from ICSBP−/− mice were defective in inducing some IFNγ-responsive genes, even though they were capable of activating STAT1 in response to IFNγ. Accordingly, IFNγ activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP−/− macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNγ-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNγ treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNγ through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNγ's unique activities in immune cells.

The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein β subunit

Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein α subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Here we report that functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein β subunit (Gβ5L). RGS9 and Gβ5L also form a complex when coexpressed in cell culture. Our data are consistent with the recent observation that several RGS proteins, including RGS9, contain G protein γ-subunit like domain that can mediate their association with Gβ5 (Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G. & Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. USA 95, 13307–13312). We report an example of such a complex whose cellular localization and function are clearly defined.

Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID

Coactivators previously implicated in ligand-dependent activation functions by thyroid hormone receptor (TR) include p300 and CREB-binding protein (CBP), the steroid receptor coactivator-1 (SRC-1)-related family of proteins, and the multicomponent TR-associated protein (TRAP) complex. Here we show that two positive cofactors (PC2 and PC4) derived from the upstream stimulatory activity (USA) cofactor fraction act synergistically to mediate thyroid hormone (T3)-dependent activation either by TR or by a TR-TRAP complex in an in vitro system reconstituted with purified factors and DNA templates. Significantly, the TRAP-mediated enhancement of activation by TR does not require the TATA box-binding protein-associated factors of TFIID. Furthermore, neither the pleiotropic coactivators CBP and p300 nor members of the SRC-1 family were detected in either the TR-TRAP complex or the other components of the in vitro assay system. These results show that activation by TR at the level of naked DNA templates is enhanced by cooperative functions of the TRAP coactivators and the general coactivators PC2 and PC4, and they further indicate a potential functional redundancy between TRAPs and TATA box-binding protein-associated factors in TFIID. In conjunction with earlier studies on other nuclear receptor-interacting cofactors, the present study also suggests a multistep pathway, involving distinct sets of cofactors, for activation of hormone responsive genes.

Cryptocyanin, a crustacean molting protein: Evolutionary link with arthropod hemocyanins and insect hexamerins

Cryptocyanin, a copper-free hexameric protein in crab (Cancer magister) hemolymph, has been characterized and the amino acid sequence has been deduced from its cDNA. It is markedly similar in sequence, size, and structure to hemocyanin, the copper-containing oxygen-transport protein found in many arthropods. Cryptocyanin does not bind oxygen, however, and lacks three of the six highly conserved copper-binding histidine residues of hemocyanin. Cryptocyanin has no phenoloxidase activity, although a phenoloxidase is present in the hemolymph. The concentration of cryptocyanin in the hemolymph is closely coordinated with the molt cycle and reaches levels higher than hemocyanin during premolt. Cryptocyanin resembles insect hexamerins in the lack of copper, molt cycle patterns of biosynthesis, and potential contributions to the new exoskeleton. Phylogenetic analysis of sequence similarities between cryptocyanin and other members of the hemocyanin gene family shows that cryptocyanin is closely associated with crustacean hemocyanins and suggests that cryptocyanin arose as a result of a hemocyanin gene duplication. The presence of both hemocyanin and cryptocyanin in one animal provides an example of how insect hexamerins might have evolved from hemocyanin. Our results suggest that multiple members of the hemocyanin gene family—hemocyanin, cryptocyanin, phenoloxidase, and hexamerins—may participate in two vital functions of molting animals, oxygen binding and molting. Cryptocyanin may provide important molecular data to further investigate evolutionary relationships among all molting animals.

Copper binding to the prion protein: Structural implications of four identical cooperative binding sites

Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd ≈ 6 μM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nɛ2 and Nδ1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP.

Epithelial sodium channel regulated by aldosterone-induced protein sgk

Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na+ absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na+ permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially. Rather, the open probability and/or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine–threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na+ absorption and thus in the control of extracellular fluid volume, blood pressure, and sodium homeostasis.

Solution structure and peptide binding studies of the C-terminal Src homology 3-like domain of the diphtheria toxin repressor protein

The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five β-strands and three helices arranged into a partially orthogonal, two-sheet β-barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in β-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.

Mutations in HYAL1, a member of a tandemly distributed multigene family encoding disparate hyaluronidase activities, cause a newly described lysosomal disorder, mucopolysaccharidosis IX

Hyaluronan (HA), a large glycosaminoglycan abundant in the extracellular matrix, is important in cell migration during embryonic development, cellular proliferation, and differentiation and has a structural role in connective tissues. The turnover of HA requires endoglycosidic breakdown by lysosomal hyaluronidase, and a congenital deficiency of hyaluronidase has been thought to be incompatible with life. However, a patient with a deficiency of serum hyaluronidase, now designated as mucopolysaccharidosis IX, was recently described. This patient had a surprisingly mild clinical phenotype, including notable periarticular soft tissue masses, mild short stature, an absence of neurological or visceral involvement, and histological and ultrastructural evidence of a lysosomal storage disease. To determine the molecular basis of mucopolysaccharidosis IX, we analyzed two candidate genes tandemly distributed on human chromosome 3p21.3 and encoding proteins with homology to a sperm protein with hyaluronidase activity. These genes, HYAL1 and HYAL2, encode two distinct lysosomal hyaluronidases with different substrate specificities. We identified two mutations in the HYAL1 alleles of the patient, a 1412G → A mutation that introduces a nonconservative amino acid substitution (Glu268Lys) in a putative active site residue and a complex intragenic rearrangement, 1361del37ins14, that results in a premature termination codon. We further show that these two hyaluronidase genes, as well as a third recently discovered adjacent hyaluronidase gene, HYAL3, have markedly different tissue expression patterns, consistent with differing roles in HA metabolism. These data provide an explanation for the unexpectedly mild phenotype in mucopolysaccharidosis IX and predict the existence of other hyaluronidase deficiency disorders.

Induction of the Tat-binding protein 1 gene accompanies the disabling of oncogenic erbB receptor tyrosine kinases

Conversion of a malignant phenotype into a more normal one can be accomplished either by down-regulation of erbB family surface receptors or by creating inactive erbB heterodimers on the cell surface. In this report, we report the identification and cloning of differentially expressed genes from antibody-treated vs. untreated fibroblasts transformed by oncogenic p185neu. We repeatedly isolated a 325-bp cDNA fragment that, as determined by Northern analysis, was expressed at higher levels in anti-p185neu-treated tumor cells but not in cells expressing internalization defective p185neu receptors. This cDNA fragment was identical in amino acid sequence to the recently cloned mouse Tat binding protein-1 (mTBP1), which has 98.4% homology to the HIV tat-binding protein-1 (TBP1). TBP1 mRNA levels were found to be elevated on inhibition of the oncogenic phenotype of transformed cells expressing erbB family receptors. TBP1 overexpression diminished cell proliferation, reduced the ability of the parental cells to form colonies in vitro, and almost completely inhibited transforming efficiency in athymic mice when stably expressed in human tumor cells containing erbB family receptors. Collectively, these results suggest that the attenuation of erbB receptor signaling seems to be associated with activation/induction or recovery of a functional tumor suppressor-like gene, TBP1. Disabling erbB tyrosine kinases by antibodies or by trans-inhibition represents an initial step in triggering a TBP1 pathway.

Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene

The Arabidopsis thaliana NPR1 has been shown to be a key regulator of gene expression during the onset of a plant disease-resistance response known as systemic acquired resistance. The npr1 mutant plants fail to respond to systemic acquired resistance-inducing signals such as salicylic acid (SA), or express SA-induced pathogenesis-related (PR) genes. Using NPR1 as bait in a yeast two-hybrid screen, we identified a subclass of transcription factors in the basic leucine zipper protein family (AHBP-1b and TGA6) and showed that they interact specifically in yeast and in vitro with NPR1. Point mutations that abolish the NPR1 function in A. thaliana also impair the interactions between NPR1 and the transcription factors in the yeast two-hybrid assay. Furthermore, a gel mobility shift assay showed that the purified transcription factor protein, AHBP-1b, binds specifically to an SA-responsive promoter element of the A. thaliana PR-1 gene. These data suggest that NPR1 may regulate PR-1 gene expression by interacting with a subclass of basic leucine zipper protein transcription factors.

The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases

The hy1 mutants of Arabidopsis thaliana fail to make the phytochrome-chromophore phytochromobilin and therefore are deficient in a wide range of phytochrome-mediated responses. Because this defect can be rescued by feeding seedlings biliverdin IXα, it is likely that the mutations affect an enzyme that converts heme to this phytochromobilin intermediate. By a combination of positional cloning and candidate-gene isolation, we have identified the HY1 gene and found it to be related to cyanobacterial, algal, and animal heme oxygenases. Three independent alleles of hy1 contain DNA lesions within the HY1 coding region, and a genomic sequence spanning the HY1 locus complements the hy1–1 mutation. HY1 is a member of a gene family and is expressed in a variety of A. thaliana tissues. Based on its homology, we propose that HY1 encodes a higher-plant heme oxygenase, designated AtHO1, responsible for catalyzing the reaction that opens the tetrapyrrole ring of heme to generate biliverdin IXα.

Inhibition of proteasomal degradation by the Gly-Ala repeat of Epstein–Barr virus is influenced by the length of the repeat and the strength of the degradation signal

The Gly-Ala repeat (GAr) of the Epstein–Barr virus nuclear antigen-1 is a transferable element that inhibits in cis ubiquitin/proteasome-dependent proteolysis. We have investigated this inhibitory activity by using green fluorescent protein-based reporters that have been targeted for proteolysis by N end rule or ubiquitin-fusion degradation signals, resulting in various degrees of destabilization. Degradation of the green fluorescent protein substrates was inhibited on insertion of a 25-aa GAr, but strongly destabilized reporters were protected only partially. Protection could be enhanced by increasing the length of the repeat. However, reporters containing the Ub-R and ubiquitin-fusion degradation signals were degraded even in the presence of a 239-aa GAr. In accordance, insertion of a powerful degradation signal relieved the blockade of proteasomal degradation in Epstein–Barr virus nuclear antigen-1. Our findings suggest that the turnover of natural substrates may be finely tuned by GAr-like sequences that counteract targeting signals for proteasomal destruction.

Native protein sequences are close to optimal for their structures

How large is the volume of sequence space that is compatible with a given protein structure? Starting from random sequences, low free energy sequences were generated for 108 protein backbone structures by using a Monte Carlo optimization procedure and a free energy function based primarily on Lennard–Jones packing interactions and the Lazaridis–Karplus implicit solvation model. Remarkably, in the designed sequences 51% of the core residues and 27% of all residues were identical to the amino acids in the corresponding positions in the native sequences. The lowest free energy sequences obtained for ensembles of native-like backbone structures were also similar to the native sequence. Furthermore, both the individual residue frequencies and the covariances between pairs of positions observed in the very large SH3 domain family were recapitulated in core sequences designed for SH3 domain structures. Taken together, these results suggest that the volume of sequence space optimal for a protein structure is surprisingly restricted to a region around the native sequence.

Essential role for p38α mitogen-activated protein kinase in placental angiogenesis

The p38 family of mitogen-activated protein kinases (MAPKs) mediates signaling in response to environmental stresses and inflammatory cytokines, but the requirements for the p38 MAPK pathway in normal mammalian development have not been elucidated. Here, we show that targeted disruption of the p38α MAPK gene results in homozygous embryonic lethality because of severe defects in placental development. Although chorioallantoic placentation is initiated appropriately in p38α null homozygotes, placental defects are manifest at 10.5 days postcoitum as nearly complete loss of the labyrinth layer and significant reduction of the spongiotrophoblast. In particular, p38α mutant placentas display lack of vascularization of the labyrinth layer as well as increased rates of apoptosis, consistent with a defect in placental angiogenesis. Furthermore, p38α mutants display abnormal angiogenesis in the embryo proper as well as in the visceral yolk sac. Thus, our results indicate a requirement for p38α MAPK in diploid trophoblast development and placental vascularization and suggest a more general role for p38 MAPK signaling in embryonic angiogenesis.