bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of the leucocyte NADPH oxidase subunit p47phox by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity
Authors: PARK Hee-Sae , LEE S. Min , LEE Jin Hyup , KIM Yun-Sook , BAE Young-Seuk , PARK Jeen-Woo
Published: August 2015

The leucocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyses the reduction of oxygen to O−2 at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to the appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47phox becomes phosphorylated on several serines and migrates to the plasma membrane. Protein kinase CK2 is an essential serine/threonine kinase present in all eukaryotic organisms. The leucocyte NADPH oxidase subunit p47phox has several putative CK2 phosphorylation sites. In the present study, we report that CK2 is able to catalyse the phosphorylation of p47phoxin vitro. Phosphoamino acid analysis of phosphorylated p47phox by CK2 indicated that the phosphorylation occurs on serine residues. CNBr mapping and phosphorylation of peptides containing the putative site of CK2 indicated that the main phosphorylated residues are Ser-208 and Ser-283 in the Src homology 3 (SH3) domains, and Ser-348 in the C-terminal domain of p47phox. Dependence of phosphorylation on the conformation of p47phox is supported by the finding that p47phox undergoes better phosphorylation by CK2 in the presence of arachidonic acid, a known activator of NADPH oxidase which induces conformational changes in p47phox. In addition, 5,6-dichloro-1-β-o-ribofuranosyl benzimidazole, a CK2 inhibitor, potentiates formyl-Met-Leu-Phe-induced NADPH oxidase activity in DMSO-differentiated HL-60 cells. Taken together, we propose that CK2 is the p47phox kinase, and that phosphorylation of p47phox by CK2 regulates the deactivation of NADPH oxidase.
RhoGDI-binding-defective mutant of Cdc42Hs targets to membranes and activates filopodia formation but does not cycle with the cytosol of mammalian cells
Authors: GIBSON Richard M. , WILSON-DELFOSSE Amy L.
Published: August 2015

We have identified a mutant of the human G-protein Cdc42Hs, R66E, that fails to form a detectable complex with the GDP-dissociation inhibitor RhoGDI in cell-free systems or in intact cells. This point mutant is prenylated, binds guanine nucleotide and interacts with GTPase-activating protein in a manner indistinguishable from wild-type Cdc42Hs. Immunofluorescence localization studies revealed that this RhoGDI-binding-defective mutant is found predominantly in the Golgi apparatus, with a staining pattern similar to that of the wild-type protein. However, unlike wild-type Cdc42Hs, which is distributed in both the microsomal membrane and cytosolic fractions, studies using differential centrifugation show that prenylated R66E Cdc42Hs is found exclusively in association with lipid bilayers. Additionally, whereas the overexpression of RhoGDI results in an apparent translocation of wild-type Cdc42Hs from the Golgi apparatus into the cytosol, identical RhoGDI-overexpression conditions do not alter the Golgi localization of the R66E mutant. Furthermore, overexpression of this RhoGDI-binding-defective mutant of Cdc42Hs seems to activate redistribution of the actin cytoskeleton and filopodia formation in fibroblasts in a manner indistinguishable from the wild-type protein. Taken together, these results suggest that the interaction of Cdc42Hs with RhoGDI is not essential for proper membrane targeting of nascent prenylated Cdc42Hs in mammalian cells; neither is this interaction an essential part of the mechanism by which Cdc42Hs activates filopodia formation. However, it does seem that redistribution of Cdc42Hs to the cytosolic compartment is absolutely dependent on RhoGDI interaction.
Identification of the catalytic nucleophile of the Family 31 α-glucosidase from Aspergillus niger via trapping of a 5-fluoroglycosyl–enzyme intermediate
Authors: LEE Seung Seo , HE Shouming , WITHERS Stephen G.
Published: August 2015

The mechanism-based reagent 5-fluoro-α-d-glucopyranosyl fluoride (5FαGlcF) was used to trap a glycosyl–enzyme intermediate and identify the catalytic nucleophile at the active site of Aspergillus niger α-glucosidase (Family 31). Incubation of the enzyme with 5FαGlcF, followed by peptic proteolysis and comparative liquid chromatography/MS mapping allowed the isolation of a labelled peptide. Fragmentation analysis of this peptide by tandem MS yielded the sequence WYDMSE, with the label located on the aspartic acid residue (D). Comparison with the known protein sequence identified the labelled amino acid as Asp-224 of the P2 subunit.
Cloning and characterization of the mouse polyamine-modulatedfactor-1 (mPMF-1) gene: an alternatively spliced homologue of the human transcription factor
Authors: WANG Yanlin , DEVEREUX Wendy , WOSTER Patrick M. , CASERO Robert A.
Published: August 2015

The natural polyamines and their analogues have been implicated in transcriptional regulation of specific genes. Human polyamine-modulated factor-1 (hPMF-1) was the first polyamine-responsive transcription factor identified. Here the mouse homologue of the hPMF-1 gene is described. Interestingly, the mouse gene (mPMF-1) codes for two alternatively spliced mRNAs. Both of the mouse splice variants, mPMF-1S and mPMF-1L, possess C-terminal coiled-coil domains nearly identical to that found in hPMF-1 and are highly homologous with the human protein. The C-terminal coiled-coil structure is necessary for transcriptional activation. However, the shorter protein, mPMF-1S, does not contain an N-terminal coiled-coil region as do both hPMF-1 and the longer mPMF-1L. mPMF-1L mRNA codes for a protein of 202 amino acids, 37 amino acids longer than the human protein. By contrast, mPMF-1S codes for only 133 amino acids, as a result of two exons being omitted compared with mPMF-1L. Both mouse transcription factors can interact with Nrf-2 (nuclear factor-E2-related factor 2), the normal partner of hPMF-1, substantiating the importance of the C-terminal coiled-coil region responsible for this interaction. Finally, the expression of mPMF-1 is induced when mouse M1 myeloid leukaemia cells are exposed to polyamine analogues, suggesting control similar to that observed for the hPMF-1.
Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase
Authors: SUD'INA Galina F. , BROCK Thomas G. , PUSHKAREVA Marina A. , GALKINA Svetlana I. , TURUTIN Denis V. , PETERS-GOLDEN Marc , ULLRICH Volker
Published: August 2015

Sulphatides are sulphate esters of galactocerebrosides that are present on the surfaces of many cell types and act as specific ligands to selectins. The present study was undertaken to investigate the effect of sulphatides on polymorphonuclear granulocyte (PMN) attachment, spreading and 5-lipoxygenase (5-LO) metabolism. Sulphatides, but not non-sulphated galactocerebrosides, dose-dependently enhanced attachment to collagen, as measured by the myeloperoxidase assay. Studies with blocking antibodies indicated that the increased attachment was mediated by CD11b/CD18 (Mac-1) β2 integrin. Scanning electron microscopy indicated that sulphatides also greatly enhanced the degree of cell spreading. In PMNs treated in suspension, sulphatides had no effect on the ionophore A23187-stimulated release of arachidonic acid and the synthesis of 5-LO metabolites. In contrast, in PMNs attached to collagen, the enzymic conversion of arachidonic acid by 5-LO was inhibited by sulphatides. Inhibition of 5-LO metabolism by sulphatides was observed even in the presence of exogenous substrate, suggesting that sulphatides directly inhibited 5-LO action. Consistent with this, sulphatides interfered with ionophore-induced translocation of the 5-LO to the nuclear envelope. Substances competing with sulphatide binding to cells, like dextran sulphate, or a strong inhibitor of cell spreading, like the actin-polymerizing agent jasplakinolide, prevented the effects of sulphatides on PMN attachment and spreading and leukotriene synthesis. We conclude that shape changes occurring in response to sulphatides specifically impair PMN leukotriene synthesis by inhibiting translocation of 5-LO.
cDNA cloning and promoter analysis of rat caspase-9
Authors: NISHIYAMA Junichiro , YI Xiaolan , VENKATACHALAM Manjeri A. , DONG Zheng
Published: August 2015

Caspase-9 is the apex caspase of the mitochondrial pathway of apoptosis, which plays a critical role in apoptotic initiation and progression. However, gene regulation of caspase-9 is largely unknown. This is in part due to the lack of information on the gene promoter. Here we have cloned the full-length cDNA of rat caspase-9 and have isolated promoter regions of this gene. The rat caspase-9 cDNA of 2058bp predicts a protein of 454 amino acids, which contains a caspase-recruitment domain (‘CARD’) at the N-terminus and enzymic domains at the C-terminus. The enzyme's active site, with a characteristic motif of QACGG, was also identified. Overall, rat and human caspase-9 have 71% identity. With the cDNA sequence, we subsequently isolated the proximal 5′-flanking regions of rat caspase-9 by the procedure of genomic walking. The 2270bp genomic segment is ‘TATA-less’, but contains several GC boxes. Elements binding known transcription factors such as Sp-1, Pit-1, CCAAT-enhancer-binding protein (C/EBP), glucocorticoid receptor and hypoxia-inducible factor 1 (HIF-1) were also identified. When cloned into reporter gene vectors, the genomic segment showed significant promoter activity, indicating that the 5′-flanking regions isolated by genomic walking contain the gene promoter of rat caspase-9. Of significance is that the cloned promoter segments were activated by severe hypoxia, conditions inducing caspase-9 transcription. Thus, the genomic sequences reported here contain not only the basal promoter of rat caspase-9 but also regulatory elements responsive to pathophysiological stimuli including hypoxia.
α-, β- or γ-chain-specific RNA interference of laminin assembly in Drosophila Kc167 cells
Authors: GOTO Akira , AOKI Makoto , ICHIHARA Shigeyuki , KITAGAWA Yasuo
Published: August 2015

Drosophila laminin αβγ trimer assembly in Kc167 cells was perturbed by chain-specific RNA interference (RNAi). The intracellular pool of α and γ chains remained unchanged under β-chain RNAi by lipofection of double-stranded RNA encoding a β-chain partial sequence. This was also the case for the intracellular pool of α and β chains under γ-chain-specific RNAi. Nonetheless, the intracellular pool of β and γ chains increased markedly under α-chain-specific RNAi. Non-reducing SDS/PAGE revealed that some of the increased β and γ chains migrated as disulphide-linked βγ dimers but that the rest migrated as monomers. Since the monomeric β and γ bands detected under α-chain RNAi were denser than the β band under γ-chain RNAi and the γ band under β-chain RNAi, respectively, β and γ also appeared to accumulate by forming βγ dimers without the disulphide linkage. We suggest that interconversion of these βγ dimers is crucial for the replaceable and selective assembly of the α chain for αβγ trimer formation.
Single thyroid hormone receptor monomers are competent for co-activator-mediated transactivation
Authors: QUACK Marcus , CARLBERG Carsten
Published: August 2015

Thyroid hormone receptor (T3R) belongs to the superfamily of nuclear receptors containing highly related transcription factors that transform an incoming signal in the form of a lipophilic hormone into an activation of the basal transcriptional machinery. Like many other nuclear receptors, T3R acts preferentially as a heterodimer with retinoid X receptor (RXR) but it also has the unique property of binding as a monomer to DNA. This study demonstrates that T3R monomers bind preferentially to AGGTCA binding motifs and are able to co-exist with T3R–RXR heterodimers in the presence of limiting amounts of RXR. DNA-bound T3R monomers efficiently contact all three members of the p160 co-activator family, which in turn boost T3R monomer-mediated transactivation. In solution T3R monomers take only one agonistic conformation (c2LPD), whereas bound to DNA they also stabilize, like T3R–RXR heterodimers, a second agonistic conformation (c1LPD). Conformation c2LPD seems to be of lower ligand sensitivity (10nM), whereas, both in T3R–RXR heterodimers and in DNA-bound T3R monomers, c1LPD is already activated at a ligand concentration of 1nM. Taken together, these results suggest that single T3R monomers are fully competent for ligand-induced transactivation and that their role in gene regulation by thyroid hormone might have been underestimated.
Preptin derived from proinsulin-like growth factor II (proIGF-II) is secreted from pancreatic islet β-cells and enhances insulin secretion
Authors: BUCHANAN Christina M. , PHILLIPS Anthony R. J. , COOPER Garth J. S.
Published: August 2015

Pancreatic islet β-cells secrete the hormones insulin, amylin and pancreastatin. To search for further β-cell hormones, we purified peptides from secretory granules isolated from cultured murine βTC6-F7 β-cells. We identified a 34-amino-acid peptide (3948Da), corresponding to Asp69–Leu102 of the proinsulin-like growth factor II E-peptide, which we have termed ‘preptin’. Preptin, is present in islet β-cells and undergoes glucose-mediated co-secretion with insulin. Synthetic preptin increases insulin secretion from glucose-stimulated βTC6-F7 cells in a concentration-dependent and saturable manner. Preptin infusion into the isolated, perfused rat pancreas increases the second phase of glucose-mediated insulin secretion by 30%, while anti-preptin immunoglobulin infusion decreases the first and second phases of insulin secretion by 29 and 26% respectively. These findings suggest that preptin is a physiological amplifier of glucose-mediated insulin secretion.
Transcriptional repressor of vasoactive intestinal peptide receptor mediates repression through interactions with TFIIB and TFIIEβ
Authors: PEI Lin
Published: August 2015

The transcriptional repressor for rat vasoactive-intestinal-polypeptide receptor 1 (VIPR-RP) is a recently characterized transcription factor that belongs to a family of proteins, which include components of the DNA replication factor C complex. In this study, I investigated the mechanisms by which VIPR-RP represses transcription. I show here that transcriptional repression by VIPR-RP is mediated by a histone deacetylase-independent mechanism. I provide evidence that VIPR-RP makes direct physical contacts with two proteins of the basal transcription apparatus, the transcription factors TFIIB and TFIIEβ. The interaction with TFIIB is mediated by the N-terminal 180 amino acids, whereas the interactive domain with TFIIEβ is located between residues 367 and 527 of VIPR-RP. Using gel mobility-shift assays I demonstrated that interaction between VIPR-RP and TFIIB prevents the recruitment of TFIIB into a DNA–TATA-box-binding protein complex. My results indicate that VIPR-RP mediates transcriptional repression through direct interactions with the general transcription machinery.
Comparative analysis of the influence of the high-mobility group box 1 protein on DNA binding and transcriptional activation by the androgen, glucocorticoid, progesterone and mineralocorticoid receptors
Authors: VERRIJDT Guy , HAELENS Annemie , SCHOENMAKERS Erik , ROMBAUTS Wilfried , CLAESSENS Frank
Published: August 2015

We performed a comparative analysis of the effect of high-mobility group box protein 1 (HMGB1) on DNA binding by the DNA-binding domains (DBDs) of the androgen, glucocorticoid, progesterone and mineralocorticoid receptors. The affinity of the DBDs of the different receptors for the tyrosine aminotransferase glucocorticoid response element, a classical high-affinity binding element, was augmented up to 7-fold by HMGB1. We found no major differences in the effects of HMGB1 on DNA binding between the different steroid hormone receptors. In transient transfection assays, however, HMGB1 significantly enhances the activity of the glucocorticoid and progesterone receptors but not the androgen or mineralocorticoid receptor. We also investigated the effect of HMGB1 on the binding of the androgen receptor DBD to a subclass of directly repeated response elements that is recognized exclusively by the androgen receptor and not by the glucocorticoid, progesterone or mineralocorticoid receptor. Surprisingly, a deletion of 26 amino acid residues from the C-terminal extension of the androgen receptor DBD does not influence DNA binding but destroys its sensitivity to HMGB1. Deletion of the corresponding fragment in the DBDs of the glucocorticoid, progesterone and mineralocorticoid receptor destroyed their DNA binding. This 26-residue fragment is therefore essential for the influence of HMGB1 on DNA recognition by all steroid hormone receptors that were tested. However, it is dispensable for DNA binding by the androgen receptor.
Isolation and characterization of a Drosophila homologue of mitogen-activated protein kinase phosphatase-3 which has a high substrate specificity towards extracellular-signal-regulated kinase
Authors: KIM Sun-Hong , KWON Hyung-Bae , KIM Yong-Sik , RYU Ji-Hwan , KIM Kyung-Sub , AHN Yongho , LEE Won-Jae , CHOI Kang-Yell
Published: August 2015

A partial C-terminal cDNA sequence of a novel Drosophila mitogen-activated protein kinase phosphatase (MKP), designated DMKP-3, was identified from an epitope expressed sequence tag database, and the missing N-terminal cDNA fragment was cloned from a Drosophila cDNA library. DMKP-3 is a protein of 411 amino acids, with a calculated molecular mass of 45.8kDa; the deduced amino acid sequence is most similar to that of mammalian MKP-3. Recombinant DMKP-3 produced in Escherichia coli retained intrinsic tyrosine phosphatase activity. In addition, DMKP-3 specifically inhibited extracellular-signal-regulated kinase (ERK) activity, but was without a significant affect on c-Jun N-terminal kinase (JNK) and p38 activities, when it was overexpressed in Schneider cells. DMKP-3 interacted specifically with Drosophila ERK (DERK) via its N-terminal domain. In addition, DMKP-3 specifically inhibited Elk-1-dependent trans-reporter gene expression in mammalian CV1 cells, and dephosphorylated activated mammalian ERK in vitro. DMKP-3 is uniquely localized in the cytoplasm within Schneider cells, and gene expression is tightly regulated during development. Thus DMKP-3 is a Drosophila homologue of mammalian MKP-3, and may play important roles in the regulation of various developmental processes.
Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ibα is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway
Authors: MARSHALL Stuart J. , ASAZUMA Naoki , BEST Denise , WONEROW Peter , SALMON Gary , ANDREWS Robert K. , WATSON Steve P.
Published: August 2015

It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) γ-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR γ-chain, Syk and PLCγ2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCγ2 nor functional responses, such as Ca2+ elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbα in washed platelets does not promote functional responses.
Expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans and some properties of the recombinant enzyme
Authors: ADAMS Benjamin , LOWE David J. , SMITH Andrew T. , SCAZZOCCHIO Claudio , DEMAIS Stephane , BRAY Robert C.
Published: August 2015

Recent crystal structures of xanthine dehydrogenase, xanthine oxidase and related enzymes have paved the way for a detailed structural and functional analysis of these enzymes. One problem encountered when working with these proteins, especially with recombinant protein, is that the preparations tend to be heterogeneous, with only a fraction of the enzyme molecules being active. This is due to the incompleteness of post-translational modification, which for this protein is a complex, and incompletely understood, process involving incorporation of the Mo and Fe/S centres. The enzyme has been expressed previously in both Drosophila and insect cells using baculovirus. The insect cell system has been exploited by Iwasaki et al. [Iwasaki, Okamoto, Nishino, Mizushima and Hori (2000) J. Biochem (Tokyo) 127, 771–778], but, for the rat enzyme, yields a complex mixture of enzyme forms, containing around 10% of functional enzyme. The expression of Drosophila melanogaster xanthine dehydrogenase in Aspergillus nidulans is described. The purified protein has been analysed both functionally and spectroscopically. Its specific activity is indistinguishable from that of the enzyme purified from fruit flies [Doyle, Burke, Chovnick, Dutton, Whittle and Bray (1996) Eur. J. Biochem. 239, 782–795], and it appears to be more active than recombinant xanthine dehydrogenase produced with the baculovirus system. EPR spectra of the recombinant Drosophila enzyme are reported, including parameters for the Fe/S centres. Only a very weak ‘Fe/SIII’ signal (g1,2,3, 2.057, 1.930, 1.858) was observed, in contrast to the strong analogous signal reported for the enzyme from baculovirus. Since this signal appears to be associated with incomplete post-translational modification, this is consistent with relatively more complete cofactor incorporation in the Aspergillus-produced enzyme. Thus we have developed a recombinant expression system for D. melanogaster xanthine dehydrogenase, which can be used for the production of site-specific mutations of this enzyme.
A novel human small subunit of calpains
Authors: SCHÁD Éva , FARKAS Attila , JÉKELY Gáspár , TOMPA Peter , FRIEDRICH Peter
Published: August 2015

Typical calpains are heterodimeric cysteine proteases which have distinct large catalytic subunits (80kDa) but share a common small regulatory subunit (30kDa; css1). Here we report the identification, cloning and characterization of a novel human small subunit (css2) encoded by an intronless gene, capns2, located on chromosome 16. This new protein displays 73% sequence identity within the Ca2+-binding region but lacks two oligo-Gly stretches characteristic of the N-terminal domain of the conventional small subunit. css2 appears to be the functional equivalent of the conventional small subunit in vitro in that it helps the large subunit fold into the active conformation of similar Ca2+ sensitivity when the two proteins are co-expressed in Escherichia coli. The purification of various chimaeric rat 80kDa—human css2 constructs, on the other hand, shows that css2 binds the large subunit much more weakly than css1. Further, it does not undergo the autolytic conversion typical of the classical small subunit. The expression of this protein in vivo, as assessed from its appearance in expressed sequence tag clones, is rather limited, making it an example of a tissue-specific, rather than ubiquitous, small subunit.
Crystal structures of 7-methylguanosine 5′-triphosphate (m7GTP)- and P1-7-methylguanosine-P3-adenosine-5′,5′-triphosphate (m7GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region
Authors: TOMOO Koji , SHEN Xu , OKABE Koumei , NOZOE Yoshiaki , FUKUHARA Shoichi , MORINO Shigenobu , ISHIDA Toshimasa , TANIGUCHI Taizo , HASEGAWA Hiroshi , TERASHIMA Akira , SASAKI Masahiro , KATSUYA Yoshio , KITAMURA Kunihiro , MIYOSHI Hiroshi , ISHIKAWA Masahide , MIURA Kin-ichiro
Published: August 2015

The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5′-triphosphate (m7GTP) and P1-7-methylguanosine-P3-adenosine-5′,5′-triphosphate (m7GpppA)] were determined at 2.0Å resolution (where 1Å = 0.1nm). The flexibility of the C-terminal loop region of eIF4E complexed with m7GTP was significantly reduced when complexed with m7GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein—eIF4E phosphorylation.
Substrate access channel topology in membrane-bound prostacyclin synthase
Authors: DENG Hui , HUANG Aimin , SO Shui-Ping , LIN Yue-Zhen , RUAN Ke-He
Published: August 2015

Results from our molecular-modelling and site-directed-mutagenesis studies of prostaglandin I2 synthase (PGIS) have suggested that the large PGIS cytoplasmic domain is anchored to the endoplasmic reticulum (ER) membrane by the N-terminal segment in a way that orients the substrate access channel opening to face the membrane. To test this hypothesis we have explored the accessibility of the PGIS substrate channel opening to site-specific antibodies. The working three-dimensional PGIS model constructed by protein homology modelling was used to predict surface portions near the substrate access channel opening. Two peptides corresponding to the surface immediately near the opening [residues 66–75 (P66–75) and 95–116 (P95–116)], and two other peptides corresponding to the surface about 10–20Å (1Å = 0.1nm) away from the opening [residues 366–382 (P366–382) and 472–482 (P472–482)] were used to prepare site-specific antibodies. All four antipeptide antibodies specifically recognized the synthetic segments of human PGIS and recombinant PGIS, as shown by binding assays and Western-blot analysis. The site-specific antibodies were used to probe the accessibility of the substrate access channel opening in transiently transfected COS-1 cells expressing recombinant human PGIS, and in spontaneously transformed human endothelial cell line ECV cells expressing endogenous human PGIS. Immunofluorescence staining was performed for cells selectively permeabilized with streptolysin O and for cells whose membranes were permeabilized with detergent. Antibodies to peptides in the immediate vicinity of the substrate channel (P66–75 and P95–116) bound to their targets only after general permeabilization with Triton X-100. In contrast, the two antibodies to peptides further from the channel opening (P366–382 and P472–482) bound to their targets even in cells with intact ER membranes. These observations support our topology model in which the PGIS substrate access channel opening is positioned close to the ER membrane.
Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface
Authors: BALYASNIKOVA Irina V. , KARRAN Eric H. , ALBRECHT Ronald F. , DANILOV Sergei M.
Published: August 2015

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as ‘shedding'. The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20–40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4°C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.
Cinnamomin, a type II ribosome-inactivating protein, is a storage protein in the seed of the camphor tree (Cinnamomum camphora)
Authors: LIU Ren-shui , WEI Guo-qing , YANG Qiang , HE Wen-jun , LIU Wang-Yi
Published: August 2015

Cinnamomin is a novel type II ribosome-inactivating protein (RIP) isolated in our laboratory from the seed of the camphor tree (Cinnamomum camphora). In this paper the physiological role it plays in the plant cell was studied. Northern and Western blotting revealed that cinnamomin was expressed specifically in cotyledons. It accumulated in large amounts simultaneously with other proteins at the post-stages of seed development. Cinnamomin degraded rapidly during the early stages of seed germination. Endopeptidase was proved to play an important role in the degradation of cinnamomin. Western blotting of total proteins from the protein body with antibodies against cinnamomin demonstrated that it only existed in this specific cellular organelle as a storage protein. The similar properties of cinnamomin and other seed storage proteins of dicotyledons were compared. We conclude that cinnamomin is a special storage protein in the seed of C. camphora.
Human insulin-like growth factor II leader 2 mediates internal initiation of translation
Authors: PEDERSEN Susanne K. , CHRISTIANSEN Jan , HANSEN Thomas v.O. , LARSEN Martin R. , NIELSEN Finn C.
Published: August 2015

Insulin-like growth factor II (IGF-II) is a fetal growth factor, which belongs to the family of insulin-like peptides. During fetal life, the IGF-II gene generates three mRNAs with different 5′ untranslated regions (UTRs), but identical coding regions and 3′ UTRs. We have shown previously that IGF-II leader 3 mRNA translation is regulated by a rapamycin-sensitive pathway, whereas leader 4 mRNA is constitutively translated, but so far the significance of leader 2 mRNA has been unclear. Here, we show that leader 2 mRNA is translated efficiently in an eIF4E-independent manner. In a bicistronic vector system, the 411 nt leader 2 was capable of internal initiation via a phylogenetically conserved internal ribosome entry site (IRES), located in the 3′ half of the leader. The IRES is composed of an approx. 120 nt ribosome recruitment element, followed by an 80 nt spacer region, which is scanned by the ribosomal pre-initiation complex. Since cap-dependent translation is down-regulated during cell division, leader 2 might facilitate a continuous IGF-II production in rapidly dividing cells during development.
Investigation of the role of Endo180/urokinase-type plasminogen activator receptor-associated protein as a collagenase 3 (matrix metalloproteinase 13) receptor
Authors: BAILEY Louise , WIENKE Dirk , HOWARD Matthew , KNÄUPER Vera , ISACKE Clare M. , MURPHY Gillian
Published: August 2015

Procollagenase 3 can be activated by interaction with and cleavage by the cell-associated membrane type 1 metalloproteinase (MT1 MMP; MMP 14). It has also been shown to bind to a specific receptor, and is subsequently internalized via the low-density lipoprotein-related receptor by osteoblast cell lines. The receptor was identified as a recycling glycoprotein of the macrophage mannose receptor family, Endo180. In order to ascertain whether there is a relationship between Endo180 binding and procollagenase 3 activation, we have compared procollagenase 3 activation by an HT1080 fibrosarcoma cell line overexpressing MT1 MMP, without and with overexpression of Endo180. No difference in procollagenase 3 activation was observed, and neither was the enzyme bound to the cells or internalized. In contrast, the osteoblast cell lines, MG63 and UMR-106, both bound and internalized procollagenase 3. However, immunolocalization studies showed that the Endo180 abundantly expressed by these cells did not co-localize with the procollagenase 3. In further biochemical studies we confirmed that procollagenase 3 did not bind to Endo180, using both ligand- blotting and immunoprecipitation techniques. We conclude that Endo180 is unlikely to be a receptor for collagenase 3 in relation to either its activation or cell binding and internalization, and that other interaction partners must be sought.
Protein kinase A enhances, whereas glycogen synthase kinase-3β inhibits, the activity of the exon 2-encoded transactivator domain of heterogeneous nuclear ribonucleoprotein D in a hierarchical fashion
Authors: TOLNAY Mate , JUANG Yuang-Taung , TSOKOS George C.
Published: August 2015

Heterogeneous nuclear ribonucleoprotein D (hnRNP D) is implicated in transcriptional regulation. Alternative splicing of exons 2 and 7 generates four isoforms of the protein. We report here that only isoforms that contain the product of exon 2 (amino acids 79–97) were able to transactivate. Moreover, the exon 2-encoded protein domain alone was sufficient to drive transcription. TATA-binding protein and p300 interacted with a synthetic peptide corresponding to exon 2, and both proteins co-precipitated with hnRNP D. Stimulation of protein kinase A (PKA) and protein kinase C (PKC) synergistically induced the transactivating ability of hnRNP D, and the exon 2-encoded domain was sufficient for this inducibility. In kinase assays PKA phosphorylated Ser-87 of hnRNP D, whereas glycogen synthase kinase-3β (GSK-3β) phosphorylated Ser-83, but only if Ser-87 had been pre-phosphorylated by PKA. Phosphorylation of Ser-87 enhanced, whereas phosphorylation of Ser-83 repressed, transactivation. Overexpression of GSK-3β inhibited transactivation by hnRNP D, but stimulation of PKC negated the inhibitory effect of GSK-3β. We suggest that a hierarchical phosphorylation pathway regulates the transactivating ability of hnRNP D: PKA activates hnRNP D, but at the same time renders it sensitive to inhibition by GSK-3β; the latter inhibition can be suspended by inactivating GSK-3β with PKC.
Opposing roles of serine/threonine kinases MEKK1 and LOK in regulating the CD28 responsive element in T-cells
Authors: TAO Li , WADSWORTH Scott , MERCER Jason , MUELLER Cynthia , LYNN Kirsten , SIEKIERKA John , AUGUST Avery
Published: August 2015

T-cell activation requires signals from both the T-cell receptor (TcR) and other co-stimulatory molecules such as CD28. TcR- and CD28-mediated signals are integrated during T-cell activation resulting in the expression of cytokine genes such as interleukin-2 (IL-2). An enhancer element (CD28RE) of the IL-2 gene specifically responsive to CD28 signals has been previously identified and characterized. This response element and an adjacent Activated Protein-1 (nuclear factor-interleukin-2B) site together (RE/AP1) were shown to complex with c-rel, AP-1 and other factors. However, details of the signal transduction pathways leading from CD28 to the composite response element remain poorly understood. We present data showing that overexpression of the serine threonine kinase, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase-1 (MEKK1), but not nuclear factor-κB inducing kinase, or MAP kinase/ERK kinase-1 (MEK1), can significantly increase the level of CD28RE/AP1-driven luciferase (Luc) reporter gene expression in Jurkat E6-1 cells. A MEKK1 dominant negative mutant blocked such activation induced by stimulation with Raji B cells and the superantigen staphylococcus enterotoxin E (SEE), as well as via CD3/CD28. Mutations in either site of the RE/AP1 element abolished MEKK1-induced Luc expression. Calcineurin inhibitors, CsA and FK520, or inhibitors of p38 kinase (SB 203580), or MEK1 (PD 098059), did not affect MEKK1-induced reporter activation. These results directly implicate MEKK1 in the CD28 signalling pathway that activates the CD28 response element. Co-expression of the lymphocyte-oriented kinase (LOK) kinase attenuated Raji/SEE-induced IL-2 production in Jurkat cells, as well as MEKK1 and Raji/SEE-induced reporter gene activation. These data suggest that MEKK1 and LOK may have opposing roles in regulating the CD28RE/AP1 element.
Type IIα phosphatidylinositol phosphate kinase associates with the plasma membrane via interaction with type I isoforms
Authors: HINCHLIFFE Katherine A. , GIUDICI Maria Luisa , LETCHER Andrew J. , IRVINE Robin F.
Published: August 2015

The phosphatidylinositol phosphate kinases (PIPkins) are a family of enzymes involved in regulating levels of several functionally important inositol phospholipids within cells. The PIPkin family is subdivided into three on the basis of substrate specificity, each subtype presumably regulating levels of different subsets of the inositol lipids. The physiological function of the type II isoforms, which exhibit a preference for phosphatidylinositol 5-phosphate, a lipid about which very little is known, is particularly poorly understood. In the present study, we demonstrate interaction between, and co-immunoprecipitation of, type IIα PIPkin with the related, but biochemically and immunologically distinct, type I PIPkin isoforms. Type IIα PIPkin interacts with all three known type I PIPkins (α, β and γ), and in each case co-expression of the type I isoform with type IIα results in recruitment of the latter from the cytosol to the plasma membrane of the cell. This change in subcellular localization could result in improved access of the type IIα PIPkin to its lipid substrates.
Characterization of a novel inhibitor of cytosolic phospholipase A2α, pyrrophenone
Authors: ONO Takashi , YAMADA Katsutoshi , CHIKAZAWA Yukiko , UENO Masahiko , NAKAMOTO Shozo , OKUNO Takayuki , SENO Kaoru
Published: August 2015

Cytosolic phospholipase A2α (cPLA2α), one of the three subtypes of cPLA2 (α, β and γ), is thought to be a rate-limiting enzyme in eicosanoid biosynthesis. We developed a novel and potent cPLA2α inhibitor with an optically active pyrrolidine, termed pyrrophenone, and characterized this compound in detail using enzyme and cellular assay systems. Pyrrophenone, which shows strong inhibition of cPLA2α activity, is one of the most potent cPLA2α inhibitors reported to date. Similar inhibitory potencies for cPLA2α were obtained from three different assays. The inhibitory activity of pyrrophenone is two or three orders of magnitude more potent than arachidonyl trifluoromethyl ketone (AACOCF3) under the same assay conditions. Pyrrophenone shows reversible inhibition of cPLA2α and displays no characteristics of the slow-binding inhibition observed for AACOCF3. Pyrrophenone also inhibited the esterase and lysophospholipase activities of cPLA2α. However, the inhibition by pyrrophenone of 14kDa secretory PLA2s, types IB and IIA, was over two orders of magnitude less potent than that for cPLA2α. Pyrrophenone strongly inhibited arachidonic acid release in calcium ionophore (A23187)-stimulated human monocytic cells (THP-1 cells) in a dose-dependent manner with an IC50 value of 0.024μM, followed by suppression of eicosanoid synthesis, and also showed dose-dependent inhibition for interleukin-1-induced prostaglandin E2 synthesis in human renal mesangial cells with an IC50 value of 0.0081μM. The mechanism of inhibition of eicosanoid synthesis in these cell-based assays was due to inhibition of only one step of arachidonic acid release without any effect on cyclo-oxygenase or lipoxygenase pathways. These results suggest that pyrrophenone could be a potential therapeutic agent for inflammatory diseases.
Expression and characterization of the active molecular forms of choline/ethanolamine kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver
Authors: AOYAMA Chieko , OHTANI Akiko , ISHIDATE Kozo
Published: August 2015

Choline/ethanolamine kinase (ChoK/EtnK) exists as at least three isoforms (α1, α2 and β) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the α (α1 plus α2) or the β isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the α isoform was highest in testis, whereas that for the β isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of α/β hetero-oligomers, with relatively small parts of activity expressed by α/α and β/β homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl4) was shown to induce ChoK activity 2–4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl4 was α, not β. The level of α mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the α isoform. Consequently, the composition of α/α homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl4-induced liver, whereas it was less than 20% in normal uninduced liver.
Association of the 72/74-kDa proteins, members of the heterogeneous nuclear ribonucleoprotein M group, with the pre-mRNA at early stages of spliceosome assembly
Authors: KAFASLA Panayiota , PATRINOU-GEORGOULA Meropi , LEWIS Joe D. , GUIALIS Apostolia
Published: August 2015

We have investigated the role played in precursor mRNA (pre-mRNA) splicing by the protein pair of molecular size 72/74kDa, which are integral components of a discrete subset of heterogeneous nuclear (hn) ribonucleoproteins (RNPs) named large heterogeneous nuclear RNP (LH-nRNP). This 72/74kDa pair of proteins has been shown to belong to the hnRNP M group, and are referred to as 72/74(M). By applying specific immunoprecipitation assays in a consecutive series of splicing reactions in vitro, the antigenic 72/74(M) protein species were found to associate with the pre-mRNA and not the intermediate or final products of splicing. Kinetic studies, combined with isolation of pre-spliceosomal and spliceosomal complexes from the splicing reaction, indicated a loose association of 72/74(M) with both the initially formed H assembly and the first splicing-committed E complex. Stable binding was seen at a later stage of the reaction, well in advance of the appearance of the first intermediate products of RNA splicing. Evidence is provided that supports the almost exclusive association of 72/74(M) with pre-mRNA within the pre-spliceosomal A complex. This dynamic binding appeared to involve pre-mRNA sites similar to those of spliceosomal U1 and U2 small nuclear RNP complexes. Moreover, a preferential binding to a truncated RNA containing the 5′ exon—intron part, rather than the intron—3′ exon part, of pre-mRNA was observed.
Chromatographic assay of glycation adducts in human serum albumin glycated in vitro by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and intrinsic fluorescence
Authors: AHMED Naila , THORNALLEY Paul J.
Published: August 2015

Glycation of proteins leads to the formation of advanced glycation endproducts (AGEs) of diverse molecular structure and biological function. Serum albumin derivatives modified to minimal and high extents by methylglyoxal and glucose in vitro have been used in many studies as model AGE proteins. The early and advanced glycation adduct contents of these proteins were investigated using the 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate (AQC) chromatographic assay of enzymic hydrolysates. AGEs derived from methylglyoxal, glyoxal and 3-deoxyglucosone, the hydroimidazolones Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), Nδ-(5-hydro-4-imidazolon-2-yl)ornithine (G-H1) and Nδ-[5-(2,3,4-trihydroxybutyl)-5-hydro-4-imidazolon-2-yl]ornithine (3DG-H1), bis(lysyl)imidazolium cross-links methylglyoxal-derived lysine dimer (MOLD), glyoxal-derived lysine dimer (GOLD), 3-deoxyglucosone-derived lysine dimer (DOLD), monolysyl adducts N∊-(1-carboxyethyl)lysine (CEL), N∊-carboxymethyl-lysine (CML) and pyrraline, other AGEs, Nδ-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)ornithine (THP), argpyrimidine and pentosidine, and fructosyl-lysine were determined. AGEs with intrinsic fluorescence (argpyrimidine and pentosidine) were assayed without derivatization. Human serum albumin (HSA) glycated minimally by methylglyoxal in vitro contained mainly MG-H1 with minor amounts of THP and argpyrimidine. Similar AGEs were found in prothrombin glycated minimally by methylglyoxal and in Nα-t-butyloxycarbonyl-arginine incubated with methylglyoxal. HSA glycated highly by methylglyoxal contained mainly argpyrimidine, MG-H1 and THP, with minor amounts of CEL and MOLD. HSA glycated minimally by glucose in vitro contained mainly fructosyl-lysine and CML, with minor amounts of THP, MG-H1, G-H1, 3DG-H1, argpyrimidine and DOLD. HSA glycated highly by glucose contained these AGEs and pyrraline, and very high amounts (≈8mol/mol of protein) of fructosyl-lysine. Most AGEs in albumin glycated minimally by methylglyoxal and glucose were identified. Significant proportions of arginine and lysine-derived AGEs in albumin modified highly by methylglyoxal, and lysine-derived AGEs in albumin modified highly by glucose, remain to be identified.
Protein kinase CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) induces apoptosis and caspase-dependent degradation of haematopoietic lineage cell-specific protein 1 (HS1) in Jurkat cells
Authors: RUZZENE Maria , PENZO Daniele , PINNA Lorenzo A.
Published: August 2015

Incubation of Jurkat cells with 4,5,6,7-tetrabromobenzotriazole (TBB), a specific inhibitor of protein kinase CK2, induces dose-and time-dependent apoptosis as judged by several criteria. TBB-promoted apoptosis is preceded by inhibition of Ser/Thr phosphorylation of haematopoietic lineage cell-specific protein 1 (HS1) and is accompanied by caspase-dependent fragmentation of the same protein. Both effects are also observable if apoptosis is promoted by anti-Fas antibodies and by etoposide. Moreover, in vitro experiments show that HS1, once phosphorylated by CK2, becomes refractory to cleavage by caspase-3. These findings, in conjunction with similar data in the literature concerning two other CK2 protein substrates, Bid and Max, suggest that CK2 may play a general anti-apoptotic role through the generation of phosphorylated sites conferring resistance to caspase cleavage.