ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma,interacts directly with metallothionein 2A and links to apoptosis

Esophageal cancer related gene 2 (ECRG2) is a novel candidate of the tumor suppressor gene identified from human esophagus. To study the biological role of the ECRG2 gene, we performed a GAL4-based yeast two-hybrid screening of a human fetal liver cDNA library. Using the ECRG2 cDNA as bait, we identified nine putative clones as associated proteins. The interaction of ECRG2 and metallothionein 2A (MT2A) was confirmed by glutathione S-transferase pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG2 co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by confocal microscopy. Transfection of ECRG2 gene inhibited cell proliferation and induced apoptosis in esophageal cancer cells. In the co-transfection of ECRG2 and MT2A assays, cell proliferation was inhibited and apoptosis was slightly induced compared with control groups. When we used antisense MT2A to interdict the effect of MT2A, the inhibition of cell proliferation and induction of apoptosis were significantly enhanced. When we used antisense ECRG2 to interdict the effect of ECRG2 in the group of Bel7402 cells co-transfected with ECRG2 and MT2A, the inhibition of cell proliferation and induction of apoptosis disappeared. The results provide evidence for ECRG2 in esophageal cancer cells acting as a bifunctional protein associated with the regulation of cell proliferation and induction of apoptosis. ECRG2 might reduce the function of MT2A on the regulation of cell proliferation and induction of apoptosis. The physical interaction of ECRG2 and MT2A may play an important role in the carcinogenesis of esophageal cancer.     

The physical and functional interaction of NDRG2 with MSP58 in cells

NDRG2, a member of N-Myc downstream regulated gene family, exerts the important functions in cell differentiation and tumor suppression. Although the ectopic expressed Ndrg2 inhibits the proliferation of tumor cells, its intracellular signal transduction pathway is hardly known. Here, we identified MSP58, a 58-kDa microspherule protein, as an interacting partner of human Ndrg2 by using yeast two-hybrid screening. The interaction was confirmed by glutathione S-transferase pull-down assay in vitro and by co-immune-precipitation assay in vivo. The forkhead associated domain of MSP58 is essential for its interaction with Ndrg2. Ndrg2 could co-localize with MSP58 in nuclear of HeLa cell during cell stress. Furthermore, the modulation of Ndrg2 level influences the cell cycle process together with MSP58. In conclusion, the findings offered a novel insight into the physiological roles of Ndrg2.     

Two Mms2 residues cooperatively interact with ubiquitin and are critical for Lys63 polyubiquitination in vitro and in vivo

Recent structural analyses support a model whereby Mms2 interacts with and orientates Ub to promote Ubc13-mediated Lys63 chain formation. However, residues of the hMms2-Ub interface have not been addressed. We found two hMms2 residues to be critical for binding and polyUb conjugation. Surprisingly, while each single mutation reduces the binding affinity, the double mutation causes significant reduction of Ub binding and abolishes polyUb chain formation. Furthermore, the corresponding yeast mms2 double mutant exhibited an additive phenotype that caused a complete loss of MMS2 function. Taken together, this study identifies key residues of the Mms2-Ub interface and provides direct experimental evidence that Mms2 physical association with Ub is correlated with its ability to promote Lys63-linked Ub chain assembly.     

密度感应系统对弗氏志贺菌生长竞争能力的影响

密度感应系统调节细菌应答反应的发生,这些应答反应与细胞密度有关。通过对比大肠杆菌(Escherichia coli)和志贺氏菌(Shigella spp.)的序列发现,志贺菌属密度感应系统操纵子普遍存在丢失或突变。为研究其密度感应系统的功能,文章利用哈氏弧菌(Vibrio harveyi)BB170作为指示菌,检测弗氏志贺菌(Shigella flexneri)密度感应系统信号分子AI-2,证明其可以分泌有活性的AI-2;其次,采用Golden Gate克隆法将大肠杆菌MG1655的密度感应系统基因克隆至弗氏志贺菌301中,获得密度感应系统回复株301。通过菌落计数表明,在混合培养条件下,密度感应系统基因回复株301比野生株301存在生长优势;通过双向电泳初步比较分析表明,密度感应系统基因可以在志贺菌中表达,并鉴定到了其他一些与应激反应相关的差异表达蛋白, 如Hsp60、GroEL、SodB。     

Brain-derived neurotrophic factor promotes interaction of the Nck2 adaptor protein with the TrkB tyrosine kinase receptor

Brain-derived neurotrophic factor (BDNF) binds to and activates the TrkB tyrosine kinase receptor to regulate cell differentiation, survival, and neural plasticity in the nervous system. However, the identities of the downstream signaling proteins involved in this process remain unclear. Using a yeast two-hybrid screen with the intracellular domain (ICD-TrkB) of the TrkB BDNF receptor, we identified the Nck2 adaptor protein as a novel interaction partner of the active form of TrkB. Additionally, we identified three tyrosines in ICD-TrkB (Y694, Y695, and Y771) that are crucial for this interaction. Similar results were obtained for Nck1, an Nck2 homolog. We also found that TrkB could be co-precipitated with GST-Nck2 recombinant protein or anti-Nck antibody in BDNF-activated cortical neurons. These results suggest that BDNF stimulation promotes interaction of Ncks with TrkB in cortical neurons.     

The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity.

It has been suggested that the last seven to nine amino acid residues at the C terminus of the gamma subunit of the ATP synthase act as a spindle for rotation of the gamma subunit with respect to the alpha beta subunits during catalysis (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). To test this hypothesis we selectively deleted C-terminal residues from the chloroplast gamma subunit, two at a time starting at the sixth residue from the end and finishing at the 20th residue from the end. The mutant gamma genes were overexpressed in Escherichia coli and assembled with a native alpha3beta3 complex. All the mutant forms of gamma assembled as effectively as the wild-type gamma. Deletion of the terminal 6 residues of gamma resulted in a significant increase (>50%) in the Ca-dependent ATPase activity when compared with the wild-type assembly. The increased activity persisted even after deletion of the C-terminal 14 residues, well beyond the seven residues proposed to form the spindle. Further deletions resulted in a decreased activity to approximately 19% of that of the wild-type enzyme after deleting all 20 C-terminal residues. The results indicate that the tip of the gammaC terminus is not essential for catalysis and raise questions about the role of the C terminus as a spindle for rotation.     

The proximal N-terminal amino acid residues are required for the coupling activity of the bovine heart mitochondrial factor B

Treatment of the recombinant bovine factor B with trypsin yielded a fragment (amino acid residues 62-175) devoid of coupling activity. Removal of the N-terminal Trp2-Gly3-Trp4 peptide resulted in a significant loss of coupling activity in the FB_ΔW²_−W⁴ deletion mutant. Sucrose density gradient centrifugation demonstrated co-sedimentation of recombinant factor B with the ADP/ATP carrier, which is present in preparations of H⁺-translocating F₀F₁-ATPase, but not in preparations of complex V. The N-terminally truncated factor B mutant FB_ΔW²_−W⁴ did not co-sediment with the ADP/ATP carrier. Recombinant factor B co-sedimented with partially purified membrane sector F₀, extracted from F₁-stripped bovine submitochondrial particles with n-dodecyl-β-d-maltoside. Factor B inhibited the passive proton conductance catalyzed by F₀ reconstituted into asolectin liposomes. A factor B mutant, bearing a photoreactive unnatural amino acid pbenzoyl-l-phenylalanine (pBpa) substituted for Trp2, cross-linked with F₀ subunits e and g as well as the ADP/ATP carrier. These results suggest that the N-terminal domain and, in particular, the proximal N-terminal amino acids are important for the coupling activity and protein-protein interactions of bovine factor B.     

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.     

D77, one benzoic acid derivative, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular LEDGF/p75

Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-factor for integration. Since LEDGF/p75 plays an important role in HIV integration, disruption of the LEDGF/p75 interaction with IN has provided a special interest for anti-HIV agent discovery. In this work, we reported that a benzoic acid derivative, 4-[(5-bromo-4-{[2,4-dioxo-3-(2-oxo-2-phenylethyl)-1,3-thiazolidin-5-ylidene]methyl}-2-ethoxyphenoxy)methyl]benzoic acid (D77) could potently inhibit the IN-LEDGF/p75 interaction and affect the HIV-1 IN nuclear distribution thus exhibiting antiretroviral activity. Molecular docking with site-directed mutagenesis analysis and surface plasmon resonance (SPR) binding assays has clarified possible binding mode of D77 against HIV-1 integrase. As the firstly discovered small molecular compound targeting HIV-1 integrase interaction with LEDGF/p75, D77 might supply useful structural information for further anti-HIV agent discovery.     

TRPM4b channel suppresses store-operated Ca2+ entry by a novel protein-protein interaction with the TRPC3 channel

We identified human TRPC3 protein by yeast two-hybrid screening of a human brain cDNA library with human TRPM4b as a bait. Immunoprecipitation and confocal microscopic analyses confirmed the protein-protein interaction between TRPM4b and TRPC3, and these two TRPs were found to be highly colocalized at the plasma membrane of HEK293T cells. Overexpression of TRPM4b suppressed TRPC3-mediated whole cell currents by more than 90% compared to those in TRPC3-expressed HEK293T cells. Furthermore, HEK293T cells stably overexpressing red fluorescent protein (RFP)-TRPM4b exhibited an almost complete abolition of UTP-induced store-operated Ca²⁺ entry, which is known to take place via endogenous TRPC channels in HEK293T cells. This study is believed to provide the first clear evidence that TRPM4b interacts physically with TRPC3, a member of a different TRP subfamily, and regulates negatively the channel activity, in turn suppressing store-operated Ca²⁺ entry through the TRPC3 channel.     

Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.     

The hydrophobic amino acid residues in the membrane-proximal C tail of the G protein-coupled vasopressin V2 receptor are necessary for transport-competent receptor folding

It is believed that the membrane-proximal C tail of the G protein-coupled receptors forms an additional alpha helix with amphipathic properties (helix 8). It was previously shown for the vasopressin V2 receptor (V2R) that a conserved dileucine motif (L(339), L(340)) in this putative helix 8 is necessary for endoplasmic reticulum (ER) to Golgi transfer of the receptor. Here, we demonstrate that the other hydrophobic residues forming the non-polar side of this helix (F(328), V(332) and L(336)) are also transport-relevant. In contrast, the multiple serine residues contributing to the more hydrophilic side (S(330), S(331), S(333), S(334), S(338)) do not influence receptor trafficking. In addition, we show unambiguously by the use of pharmacological chaperones that the hydrophobic residues of the putative helix 8 do not form a transport signal necessary for receptor sorting into ER to Golgi vesicles. Instead, they are necessary to establish a transport-competent folding state in the early secretory pathway.     

The N-terminal lysine residue-rich domain II and the 340-430 amino acid segment of eukaryotic initiation factor 2-associated glycoprotein p67 are the binding sites for the gamma-subunit of eIF2

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein, p67, plays an important role in protecting eIF2alpha from phosphorylation by eIF2alpha-specific kinases. To understand the molecular details of interaction between p67 and the subunits of eIF2, we applied several biochemical and mutational analyses to identify interacting domains within p67 and eIF2gamma. These studies were combined with functional in vivo and in vitro assays to address the importance of the interactions between p67 and eIF2gamma in eIF2alpha phosphorylation. Studies from yeast two-hybrid assays show that p67 interacts strongly with eIF2gamma, relatively weakly with eIF2alpha, and no interaction with eIF2beta. Further mutational analyses provided evidence that the N-terminal lysine-rich domain II and the 340-430 amino acid segment of p67 interact strongly with the C-terminal 409-472 amino acid segment of eIF2gamma. GST pull-down assays show that the interaction between p67 and eIF2gamma is direct. From co-immunoprecipitation studies, we find that the interaction between p67 and eIF2gamma could not only be detected in mammalian cells growing in growth medium, it could also be detected in transiently transfected cells with expression plasmids encoding p67 and eIF2gamma. However, this interaction could not be detected in p67 mutants lacking lysine-rich domain II and the 340-430 amino acid segment. We also find a very good correlation between p67 binding to eIF2gamma and the protection of eIF2alpha from phosphorylation. Altogether, our data provide genetic evidence for the interaction between p67 and eIF2gamma and that this interaction modulates the phosphorylation of eIF2alpha.     

The binding between p67 and eukaryotic initiation factor 2 plays important roles in the protection of eIF2alpha from phosphorylation by kinases

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2alpha from kinases. Previously, we reported that the D6/2 mutant of p67 has higher levels of protection of eIF2alpha phosphorylation (POEP) activity. In this study, we report that the D6/2 mutant and its double mutants containing second-site alanine substitutions at the five conserved amino acid residues (D251, D262, H331, E364, and E459) show increased POEP activity in serum-starved rat tumor hepatoma cells. Serum-restoration to those cells did not abolish their increased POEP activity except the D6/2+H331A double mutant. The latter mutant shows slight inhibition of POEP activity during serum starvation and this inhibition increased significantly during serum restoration. KRC-7 cells constitutively expressing the D6/2 mutant showed slightly decreased levels of PKR phosphorylation and significantly low level of phosphorylation of ERKs 1 and 2. The D6/2 mutant also showed increased binding with eIF2alpha and eIF2gamma and almost similar binding with ERKs 1 and 2 as compared to wild type p67. Altogether, our data demonstrate that the increased binding of the D6/2 mutant with the subunits of eIF2 may be in part the cause for its high POEP activity.     

The novel function of the Saccharomyces cerevisiaeCBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasiiLSU intron in vivo

In the yeast Saccharomyces cerevisiae, the product of the nuclear gene CBP2 is required exclusively for the splicing of the terminal intron of the mitochondrial cytochrome b gene. The homologous gene from the related yeast, Saccharomyces douglasii, has been shown to be essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome and dispensable in the presence of an intronless mitochondrial genome. The two CBP2 genes are functionally interchangeable although the target intron of the S. cerevisiaeCBP2 gene is absent from the S. douglasii mitochondrial genome. To determine the function of the CBP2 gene in S. douglasii mitochondrial pre-RNA processing we have constructed and analyzed interspecific hybrid strains between the nuclear genome of S. cerevisiae carrying an inactive CBP2 gene and S. douglasii mitochondrial genomes with different intron contents. We have demonstrated that inactivation of the S. cerevisiaeCBP2 gene affects the maturation of the S. douglasii LSU pre-RNA, leading to a respiratory-deficient phenotype in the hybrid strains. We have shown that the CBP2 gene is essential for excision of the S. douglasii LSU intron in vivo and that the gene is dispensable when this intron is deleted or replaced by the S. cerevisiae LSU intron. Received: 1 October 1997 / Accepted: 18 November 1997     

Cysteine residues are critical for chemokine receptor CXCR2 functional properties

We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.     

The regulatory role of spermine and fatty acid in the interaction of AMP deaminase with phosphofructokinase

The role of fatty acid and polyamine in the interaction of AMP deaminase (EC 3.5.4.6)-ammonium system with glycolysis was investigated using permeabilized yeast cells. (1) The addition of fatty acid inhibited the activity of AMP deaminase in situ, resulting in a decrease in the total adenylate pool depletion, and in the recovery of the adenylate energy charge. (2) The addition of fatty acid resulted in an indirect decrease in the activity of phosphofructokinase (EC 2.7.1.11) through a reduced level of ammonium ion; fatty acid itself did not inhibit phosphofructokinase activity in the presence of excess ammonium ion. (3) Spermine protected AMP deaminase from inhibition by fatty acid: the increased ammonium level enhanced phosphofructokinase activity, glycolytic flux and the recovery of the energy charge. In contrast, alkali metals, which are also activators of AMP deaminase had little effect on the inhibition of the enzyme. The inhibition of glycolysis by fatty acid and its reversal by polyamine can be accounted for by the changes in ammonium ion through the action of AMP deaminase-ammonium system, and the physiological relevance is discussed.