bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of cleavage of membrane proteins by calmodulin inhibitors
Authors: DÍAZ-RODRÍGUEZ Elena , ESPARÍS-OGANDO Azucena , MONTERO Juan Carlos , YUSTE Laura , PANDIELLA Atanasio
Published: August 2015

The ectodomain of several membrane-bound proteins can be shed by proteolytic cleavage. The activity of the proteases involved in shedding is highly regulated by several intracellular second messenger pathways, such as protein kinase C (PKC) and intracellular Ca2+. Recently, the shedding of the adhesion molecule L-selectin has been shown to be regulated by the interaction of calmodulin (CaM) with the cytosolic tail of L-selectin. Prevention of CaM-L-selectin interaction by CaM inhibitors or mutation of a CaM binding site in L-selectin induced L-selectin ectodomain shedding. Whether this action of CaM inhibitors also affects other membrane-bound proteins is not known. In the present paper we show that CaM inhibitors also stimulate the cleavage of several other transmembrane proteins, such as the membrane-bound growth factor precursors pro-transforming growth factor-α and pro-neuregulin-α2c, the receptor tyrosine kinase, TrkA, and the β-amyloid precursor protein. Cleavage induced by CaM inhibitors was a rapid event, and resulted from the activation of a mechanism that was independent of PKC or intracellular Ca2+ increases, but was highly sensitive to hydroxamic acid-based metalloprotease inhibitors. Mutational analysis of the intracellular domain of the TrkA receptor indicated that CaM inhibitors may stimulate membrane-protein ectodomain cleavage by mechanisms independent of CaM-substrate interaction.
Protein phosphatase 2A is associated in an inactive state with microtubules through 2A1-specific interaction with tubulin
Authors: HIRAGA Akira , TAMURA Shinri
Published: August 2015

Protein phosphatase (PP) 2A1, a trimer composed of A-, B- and C-subunits in the PP2A family, has been regarded as a principal form localizing at microtubules (MT), but PP2A2, the dimer of A- and C-subunits, has not. Substantiating the claim, the present work shows that the PP2A1 but not PP2A2, both isolated from bovine extract, largely associated with the purified preparation of MT. Furthermore, PP2A1 was found to bind purifiedtubulin polymerized by taxol. The presence of MT associated proteins with purified tubulin hardly affected the binding of PP2A1 to the tubulin. In addition, PP2A1 activity towards glycogen phosphorylase, a probably unphysiological but good substrate, was similarly inhibited by MT proteins and purifiedtubulin, which accounts for ≥ 85% of MT proteins, with their IC50 of about 0.15 mg/ml. In contrast, the inhibition of PP2A2 was about 40% with 1 mg/ml MT proteins and 20% with 0.8 mg/ml tubulin, consistent with its weak association with MT. Therefore, the association with and resultant inhibition by MT proteins of PP2A1 is largely effected by the binding of PP2A1 to tubulin molecule. Moreover, PP2A1 isolated from MT has higher affinity for polymerized MT proteins than has PP2A1 from the postmicrotubule supernatant. The MT PP2A1 has also higher sensitivity to the inhibition by tubulin and MT proteins than has the supernatant PP2A1 (IC50: 0.1-0.2 mg/ml vs. 0.3-0.6 mg/ml), demonstrating the importance of its association with polymerized tubulin.
Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts
Authors: BOEHM Mark K. , CORPER Adam L. , WAN Tommy , SOHI Maninder K. , SUTTON Brian J. , THORNTON Jeremy D. , KEEP Patricia A. , CHESTER Kerry A. , BEGENT Richard H. J. , PERKINS Stephen J.
Published: August 2015

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly4Ser)3 linker to a variable light-chain (VL) domain (ĸ chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 Å resolution by molecular replacement with an Rcryst of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a β-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the β-sheet of the VL domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.
Characterization of MAX.3 antigen, a glycoprotein expressed on mature macrophages, dendritic cells and blood platelets: identity with CD84
Authors: KRAUSE Stefan W. , REHLI Michael , HEINZ Sven , EBNER Reinhard , ANDREESEN Reinhard
Published: August 2015

MAX.3 is a monoclonal antibody that preferentially reacts with mature macrophages (MAC), monocyte-derived dendritic cells, megakaryocytes and platelets. In this study, we describe the characterization, purification and identification of the MAX.3 antigen. Immunoprecipitation and SDS/PAGE revealed different molecular masses of MAX.3 antigen in MAC (60-90 kDa) and platelets (58-64 kDa), whereas a similar size (45 kDa) was observed in both cell types after digestion with N-glycosidase F. Lectin affinity and sequential treatment with different glycosidases suggests complex type glycosylation of MAX.3 antigen in MAC and hybrid type glycosylation in platelets. Amino acid sequencing led to the identification of a corresponding cDNA clone and showed its identity to the sequence of the CD84 antigen, a member of the CD2 family of cell surface molecules. MAX.3/CD84 was further studied by immunohistochemistry and a variable expression was found on tissue MAC, confirming this antigen to be mainly a marker for MAC in situ.
Analysis of the cellular functions of PTEN using catalytic domain and C-terminal mutations: differential effects of C-terminal deletion on signalling pathways downstream of phosphoinositide 3-kinase
Authors: LESLIE Nick R. , GRAY Alex , PASS Ian , ORCHISTON Elaine A. , DOWNES C. Peter
Published: August 2015

The tumour suppressor protein, PTEN (phosphatase and tensin homolog deleted on chromosome 10), is a phosphatase that can dephosphorylate tyrosine-containing peptides, Shc, focal adhesion kinase and phosphoinositide substrates. In cellular assays, PTEN has been shown to antagonize the PI-3K-dependent activation of protein kinase B (PKB) and to inhibit cell spreading and motility. It is currently unclear, however, whether PTEN accomplishes these effects through its lipid- or protein-phosphatase activity, although strong evidence has demonstrated the importance of the latter for tumour suppression by PTEN. By using a PTEN G129E (Gly129 → Glu) mutant that has lost its lipid phosphatase activity, while retaining protein phosphatase activity, we demonstrated a requirement for the lipid phosphatase activity of PTEN in the regulation of PKB activity, cell viability and membrane ruffling. We also made a small C-terminal deletion of PTEN, removing a putative PDZ (PSD95, Dlg and ZO1)-binding motif, with no detectable effect on the phosphatase activity of the protein expressed in HEK293 cells (human embryonic kidney 293 cells) assayed in vitro. Surprisingly, expression of this mutant revealed differential requirements for the C-terminus in the different functional assays. Wild-type and C-terminally deleted PTEN appeared to be equally active in down-regulating PKB activity, but this mutant enzyme had no effect on platelet-derived growth factor (PDGF)-induced membrane ruffling and was only partially active in a cell viability assay. These results stress the importance of the lipid phosphatase activity of PTEN in the regulation of several signalling pathways. They also identify a mutation, similar to mutations that occur in some human tumours, which removes the effect of PTEN on membrane ruffling but not that on PKB.
Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substrates
Authors: PORTARO Fernada C. Vieira , SANTOS Ana Beatriz F. , CEZARI Maria Helena S. , JULIANO Maria Aparecida , JULIANO Luiz , CARMONA Euridice
Published: August 2015

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S4 and S3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S2ʹ and S3ʹ. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P4, P3, P2ʹ and P3ʹ were made. The S4 to S2ʹ subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S3ʹ, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S4, S3, S2ʹ and S3ʹ of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P2ʹ respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P3ʹ. Basic residues at P3 and P4 were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P2ʹ (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His111 (H111A) and His110 (H110A) of cathepsin B led to an increase in kcat values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.
Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin
Authors: OLEINIKOV Andrew V. , ZHAO Jun , MAKKER Sudesh P.
Published: August 2015

gp600/megalin, an endocytic receptor, belongs to the low-density lipoprotein receptor family. It is most abundant in the renal proximal tubular cells, where it is implicated in the reabsorption of a number of molecules filtered through the glomerulus. The cytoplasmic tail (CT) of gp600/megalin contains a number of sequence similarities, which indicate that gp600/megalin might be involved in signal transduction. To find intracellular proteins that would interact with the gp600/megalin CT, a human kidney cDNA library was screened by using the yeast two-hybrid system. The phosphotyrosine interaction domain (PID) of the Disabled protein 2 (Dab2), a mammalian structural analogue of Drosophila Disabled, was found to bind to the gp600/megalin CT in this system. The interaction between these two proteins was confirmed by a binding assay in vitro and by the co-immunoprecipitation of both proteins from renal cell lysates. The gp600/megalin CT contains three ΨXNPXY motifs (in which Ψ represents a hydrophobic residue) that are potentially able to interact with PID. Analysis of the CT deletion and point-mutation variants of gp600/megalin by the two-hybrid system revealed that the third ΨXNPXY motif is most probably involved in this interaction. Dab2 is a mitogen-responsive phosphoprotein thought to be an adaptor molecule involved in signal transduction, and a suggested negative regulator of cell growth. Dab2 is the first intracellular ligand identified for gp600/megalin; gp600/megalin is the first known transmembrane receptor that interacts with the cytosolic protein Dab2. We speculate that their interaction might involve gp600/megalin in signal transduction pathways or might mediate the intracellular trafficking of this receptor.
Isolation and characterization of protoporphyrin glycoconjugates from rat Harderian gland by HPLC, capillary electrophoresis and HPLC/electrospray ionization MS
Authors: LIM Chang Kee , RAZZAQUE M. Aaqib , LUO Jinli , FARMER Peter B.
Published: August 2015

It has been widely reported that the Harderian gland, present in most vertebrates, accumulates high levels of porphyrins, particularly protoporphyrin. The present study describes the extraction, identification and characterization of a group of hitherto unreported protoporphyrin glycoconjugates in the rat Harderian gland using HPLC, capillary electrophoresis, on-line HPLC/electrospray ionization MS and tandem MS. The major glycoconjugate was identified as protoporphyrin-1-O-acyl β-xyloside with a smaller amount of protoporphyrin-1-O-acyl β-glucoside also detected. In the Harderian glands studied, 50-70% of the porphyrins present were in the form of protoporphyrin glycoconjugates. This is the first reported occurrence of glycoconjugates of porphyrins in Nature and suggests that previous studies have wrongly identified the major porphyrin in the Harderian gland as the unconjugated protoporphyrin.
Zebrafish ftz-f1 gene has two promoters, is alternatively spliced, and is expressed in digestive organs
Authors: LIN Wen-wen , WANG Hsin-Wei , SUM Cristina , LIU Dong , HEW Choy L. , CHUNG Bon-chu
Published: August 2015

Fushi-tarazu Factor-1 (FTZ-F1) is a family of nuclear receptors involved in various developmental processes. We have cloned a zebrafish FTZ-F1 gene, termed ff1, which belongs to the fetoprotein transcription factor/liver receptor homologue-1 (FTF/LRH-1) subgroup of the FTZ-F1 family. Four transcripts arise as a result of differential promoter usage and alternative splicing at the 3ʹ-most exons. The longer transcript, form A, encodes a transcriptional activator. The shorter transcript, form B, lacks the activation domain, and hence could not activate transcription. The difference in promoter usage generates FF1 proteins with different N-terminal sequences. All four transcripts appear to be expressed in most of the adult tissues, whereas, during embryo development, the IIA form is the predominant transcript. Reverse transcriptase-PCR and in situ hybridization experiments showed that the ff1 transcript is expressed in the hypothalamus, spinal cord, mandibular arch and digestive organs, including pancreas, liver, and intestine. The expression of ff1 in the digestive organs implies its function in gut development.
Atomic force microscopy of gastric mucin and chitosan mucoadhesive systems
Authors: DEACON Matthew P. , McGURK Simon , ROBERTS Clive J. , WILLIAMS Phillip M. , TENDLER Saul J. B. , DAVIES Martyn C. , DAVIS S. S. (Bob) , HARDING Stephen E.
Published: August 2015

Atomic force microscopy has been utilized to probe, at a molecular level, the interaction between purified pig gastric mucin (PGM) and a mucoadhesive cationic polymer, chitosan (sea cure 210+), with a low degree (approx. 11%) of acetylation. Images were produced detailing the structures of both PGM and chitosan in 0.1 M acetate buffer (pH 4.5), followed by the complex of the two structures in the same buffer. PGM in 0.1 M acetate buffer revealed long linear filamentous structures, consistent with earlier electron microscopy and scanning tunnelling micoscopy studies. The chitosan molecules also adopted a linear conformation in the same buffer, although with a smaller average length and diameter. They appeared to adopt a stiff-coil conformation consistent with earlier hydrodynamic measurements. The complexes formed after mixing PGM and chitosan together revealed large aggregates. In 0.1 M ionic strength buffer they were of the order of 0.7 μm in diameter, consistent with previous electron microscopy studies. The effect of ionic strength of the buffer on the structure of the complex was also studied and, together with molecular hydrodynamic data, demonstrates that the interaction is principally electrostatic in nature.
HSP43, a small heat-shock protein localized to specific cells of the vulva and spermatheca in the nematode Caenorhabditis elegans
Authors: DING Lily , CANDIDO E. M. Peter
Published: August 2015

Heat-shock protein 43 (HSP43) of Caenorhabditis elegans is prominently expressed in the utse cell, which attaches the uterus to the hypodermis, the uv1 cells joining the vulva and the uterus, the spermathecal valve and junctions between cells of the spermathecal cage. In body-wall muscle, HSP43 forms a punctate pattern of circumferential lines, probably corresponding to regions where the hypodermis contacts the muscle cells.
The specificity of the protein kinase C α, βII and γ isoforms as assessed by an unnatural alcohol-appended peptide library
Authors: YAN Xiongwei , CURLEY Kieran , LAWRENCE David S.
Published: August 2015

Previous studies using conventional peptide-based libraries have demonstrated that homologous protein-processing enzymes [e.g. the α, βII and γ isoforms of protein kinase (PKC)] typically display identical amino acid consensus sequences. These observations have hampered the acquisition of selective synthetic substrates for the individual members of these enzyme families. We describe here a parallel synthesis strategy, readily adaptable to the preparation of large libraries, that has led to the emergence of the first examples of selective substrates for the conventional PKC isoforms. In addition, we have found that a wide variety of structurally diverse N-appended alcohol-containing residues, including tyrosine, serve as substrates for the PKC α, βII and γ isoforms. This broad active-site substrate specificity with respect to both natural and unnatural residues may prove to be especially applicable to the construction of transition-state analogues and suicide substrates, species that often require the presence of structurally elaborate functionality.
Regulation of interleukin-6 gene expression by pro-inflammatory cytokines in a colon cancer cell line
Authors: LEGRAND-POELS Sylvie , SCHOONBROODT Sonia , PIETTE Jacques
Published: August 2015

The two carcinoma cell lines HeLa and HTM-29 show different behaviour in terms of interleukin-6 (IL-6) production. Analyses of secreted IL-6 by ELISA and of IL-6 mRNA by reverse transcription–PCR revealed that, whereas HeLa cells produced high levels of IL-6 in response to tumour necrosis factor-α (TNF-α) and IL-1β, the HTM-29 cell line failed to produce both IL-6 protein and mRNA. Nevertheless, the transcription factors nuclear factor-κB (NF-κB) and NF-IL6, the main factors involved in IL-6 gene transcriptional activation by cytokines, were activated in both cell lines after treatment with TNF-α or IL-1β. In order to verify that the lack of IL-6 expression in HTM-29 cells was not due to an endogenous IL-6 gene deficiency or to IL-6 mRNA instability, we carried out transient transfection assays with an IL-6 promoter–reporter construct. Strong activation of the IL-6 promoter by cytokines could be observed in HeLa cells, whereas no induction could be detected in cytokine-treated HTM-29 cells. These cytokines induced a very strong stimulation of NF-κB-mediated transcription in HeLa cells transfected with a κB luceriferase reporter construct, whereas no induction could be detected in cytokine-stimulated HTM-29 cells. Thus IL-6 promoter repression in HTM-29 cells probably results from a failure of cytokine-activated NF-κB to exert its transactivating activities. Western blotting experiments demonstrated that the lack of NF-κB-mediated transcription was not due to increased expression of IκB (inhibitor of NF-κB) proteins in HTM-29 cells. Co-transfection experiments with the κB Luc reporter construct and the CBP [CREB (cAMP response element binding protein) binding protein] expression vector showed that the impairment in NF-κB-dependent transcription did not result from a deficiency in the co-activator CBP. Interestingly, both NF-κB-mediated transcription and IL-6 promoter activation could be restored in HTM-29 cells by transfection with RelA. Furthermore, CBP could have a significant synergistic effect on exogenous RelA-mediated transcription. Since sequencing of the endogenous relA gene did not reveal any mutation, it is likely that repression of NF-κB-mediated transcription results from negative cross-talk between NF-κB and another nuclear factor specifically expressed or regulated by TNF-α in HTM-29 cells.
Peroxisome-proliferator-activated receptor δ mediates the effects of long-chain fatty acids on post-confluent cell proliferation
Authors: JEHL-PIETRI Chantal , BASTIE Claire , GILLOT Isabelle , LUQUET Serge , GRIMALDI Paul A.
Published: August 2015

Nutritional long-chain fatty acids control adipose tissue mass by regulating the number and the size of adipocytes. It is now established that peroxisome-proliferator-activated receptors (PPARs) play crucial functions in the control of gene expression and the level of cell differentiation. PPARγ, which is activated by specific prostanoids, is a key factor in activating terminal differentiation and adipogenesis. We have recently demonstrated that PPARδ, once activated by fatty acids, drives the expression of a limited set of genes, including that encoding PPARγ, thereby inducing adipose differentiation. Thus far, the mechanism of action of fatty acids in the control of preadipocyte proliferation has remained unknown. We show here that PPARδ is directly implicated in fatty acid-induced cell proliferation. Ectopic expression of PPARδ renders 3T3C2 cells capable of responding to treatment with long-chain fatty acids by a resumption of mitosis, and this effect is limited to a few days after confluence. This response is restricted to PPARδ activators and, for fatty acids, takes place within the range of concentrations found to trigger differentiation of preadipocytes both in vitro and in vivo. Furthermore, the use of a mutated inactive PPARδ demonstrated that transcriptional activity of the nuclear receptor is required to mediate fatty acid-induced proliferation. These data demonstrate that PPARδ, as a transcription factor, is directly implicated in fatty acid-induced proliferation, and this could explain the hyperplastic development of adipose tissue that occurs in high-fat-fed animals.
Identification of domains responsible for signal recognition and transduction within the QUTR transcription repressor protein
Authors: LEVETT Lisa J. , SI-HOE Shi Ming , LIDDLE Stuart , WHEELER Kerry , SMITH Deborah , LAMB Heather K. , NEWTON Giles H. , COGGINS John R. , HAWKINS Alastair R.
Published: August 2015

QUTR (qutR-encoded transcription-repressing protein) is a multi-domain repressor protein active in the signal-transduction pathway that regulates transcription of the quinic acid utilization (qut) gene cluster in Aspergillus nidulans. In the presence of quinate, production of mRNA from the eight genes of the qut pathway is stimulated by the activator protein QUTA (qutA-encoded transcription-activating protein). Mutations in the qutR gene alter QUTR function such that the transcription of the qut gene cluster is permanently on (constitutive phenotype) or is insensitive to the presence of quinate (super-repressed phenotype). These mutant phenotypes imply that the QUTR protein plays a key role in signal recognition and transduction, and we have used deletion analysis to determine which regions of the QUTR protein are involved in these functions. We show that the QUTR protein recognizes and binds to the QUTA protein in vitro and that the N-terminal 88 amino acids of QUTR are sufficient to inactivate QUTA function in vivo. Deletion analysis and domain-swap experiments imply that the two C-terminal domains of QUTR are mainly involved in signal recognition.
Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration
Authors: LEE Sang Hyoung , JOHNSON J. David , WALSH Michael P. , VAN LIEROP Jacquelyn E. , SUTHERLAND Cindy , XU Ande , SNEDDEN Wayne A. , KOSK-KOSICKA Danuta , FROMM Hillel , NARAYANAN Njanoor , CHO Moo Je
Published: August 2015

Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca2+ dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (Kact 1.8 and 1.7nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher Kact than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca2+-ATPases. The plant Ca2+-ATPase was activated maximally by both isoforms, while the erythrocyte Ca2+-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in Kact and an approx. 25% reduction in Vmax. Importantly, SCaM isoforms showed a distinct Ca2+ concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca2+] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca2+] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca2+ transients.
Peptide YY Y1 receptor activates mitogen-activated protein kinase and proliferation in gut epithelial cells via the epidermal growth factor receptor
Authors: MANNON Peter J. , MELE Jennifer M.
Published: August 2015

The G-protein-coupled peptide YY (PYY)/neuropeptide Y Y1 receptor (Y1R) subtype is highly expressed in the proliferative zone of human colonic crypt epithelial cells but biochemical and biological support for growth effects have been lacking. Using a model gut epithelial cell system, we have stably expressed the human Y1R in IEC-6 cells and show that the Y1R does couple to mitogen-activated protein kinase (MAPK) phosphorylation and cell growth. This pathway uses pertussis-toxin-sensitive G-proteins and βγ subunits, inhibited by co-transfected α-transducin. The Src-family tyrosine kinase inhibitor PP1, as well as specific inhibition of the epidermal growth factor receptor tyrosine kinase (EGFR TK) by PD153035, also blocks PYY stimulation of MAPK. This pathway further requires protein kinase C with EGFR TK inhibition blocking PYY-induced protein kinase Cε (PKCε) translocation to the cell membrane. Finally, we show that PYY stimulates growth in Y1R-expressing gut epithelial cells that is dependent on EGFR TK activity. These results demonstrate a novel pathway involving Gi/Go protein, EGFR and PKC to activate MAPK. Further, they support a role for PYY and the Y1R in regulating growth in human colonic epithelium.
Definition of a minimal munc18c domain that interacts with syntaxin 4
Authors: GRUSOVIN Julian , STOICHEVSKA Violet , GOUGH Keith H. , NUNAN Katrina , WARD Colin W. , MACAULAY S. Lance
Published: August 2015

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.
Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids
Authors: LEONARD Amanda E. , BOBIK Emil G. , DORADO Joseph , KROEGER Paul E. , CHUANG Lu-Te , THURMOND Jennifer M. , PARKER-BARNES Jennifer M. , DAS Tapas , HUANG Yung-Sheng , MUKERJI Pradip
Published: August 2015

The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiaeELO2 gene, a consensus cDNA sequence was identified from the LifeSeq® database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5´ and 3´ ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of γ-linolenic acid (C18:3, n-6) into dihomo-γ-linolenic acid (C20:3, n-6), arachidonic acid (C20:4, n-6) into adrenic acid (C22:4, n-6), stearidonic acid (C18:4, n-3) into eicosatetraenoic acid (C20:4, n-3), eicosapentaenoic acid (C20:5, n-3) into ω3-docosapentaenoic acid (C22:5, n-3) and α-linolenic acid (C18:3, n-3) into ω3-eicosatrienoic acid (C20:3, n-3). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.
Functional and molecular modelling studies of two hereditary fructose intolerance-causing mutations at arginine 303 in human liver aldolase
Authors: SANTAMARIA Rita , ESPOSITO Gabriella , VITAGLIANO Luigi , RACE Vincenza , PAGLIONICO Immacolata , ZANCAN Lucia , ZAGARI Adriana , SALVATORE Francesco
Published: August 2015

We have identified a novel hereditary fructose intolerance mutation in the aldolase B gene (i.e. liver aldolase) that causes an arginine-to-glutamine substitution at residue 303 (Arg303 → Gln). We previously described another mutation (Arg303 → Trp) at the same residue. We have expressed the wild-type protein and the two mutated proteins and characterized their kinetic properties. The catalytic efficiency of protein Gln303 is approx. 1/100 that of the wild-type for substrates fructose 1,6-bisphosphate and fructose 1-phosphate. The Trp303 enzyme has a catalytic efficiency approx. 1/4800 that of the wild-type for fructose 1,6-bisphosphate; no activity was detected with fructose 1-phosphate. The mutation Arg303 → Trp thus substitution impairs enzyme activity more than Arg303 → Gln. Three-dimensional models of wild-type, Trp303 and Gln303 aldolase B generated by homology-modelling techniques suggest that, because of its larger size, tryptophan exerts a greater deranging effect than glutamine on the enzyme's three-dimensional structure. Our results show that the Arg303 → Gln substitution is a novel mutation causing hereditary fructose intolerance and provide a functional demonstration that Arg303, a conserved residue in all vertebrate aldolases, has a dominant role in substrate binding during enzyme catalysis.
The β-subunit of the hepatocyte growth factor/scatter factor (HGF/SF) receptor phosphorylates and associates with CrkII: expression of CrkII enhances HGF/SF-induced mitogenesis
Authors: RIORDAN Stephen M. , LIDDER Sukhwinderjit , WILLIAMS Roger , SKOUTERIS George G.
Published: August 2015

CrkII, a 40kDa adaptor possessing a Src homology (SH)2 domain followed by two SH3 domains, although not endowed with catalytic activity, participates in intracellular signalling, presumably by activating the Ras pathway. CrkII was found to be phosphorylated in response to hepatocyte growth factor/scatter factor (HGF/SF) and to associate with the beta-subunit of the HGF receptor (MET). CrkII associated with p145βMET via its SH2 domain. Growth-factor-receptor-bound protein 2 (Grb2) co-immunoprecipitated with CrkII species. By transient transfection of A431 human epidermoid carcinoma cells with wild-type and dominant-negative Grb2 expression constructs lacking either the SH2 or SH3 domains, we have concluded that Grb2 does not contribute to the ‘presentation’ of CrkII to p145βMET. Overexpression of wild-type CrkII in A431 cells enhanced HGF/SF-induced proliferation, while a CrkII dominant-negative mutant lacking the SH2 domain prevented a similar proliferating response to HGF/SF. The effect of CrkII on HGF/SF-induced proliferation was also abolished in cells co-expressing CrkII and Son-of-sevenless lacking the guanine exchange domain, suggesting that CrkII is likely to induce cell proliferation partly via the Ras/mitogen-activated protein kinase route.
Characterization of a calcium-regulation domain of the skeletal-muscle ryanodine receptor
Authors: HAYEK Salim M. , ZHU Xinsheng , BHAT Manjunatha B. , ZHAO Jiying , TAKESHIMA Hiroshi , VALDIVIA Hector H. , MA Jianjie
Published: August 2015

A negatively charged region of the N-terminal portion of the skeletal ryanodine receptor (RyR), located between residues 1872–1923, is involved in Ca 2+-dependent regulation of the Ca2+-release channel. This region is divergent between the skeletal (RyR1) and cardiac (RyR2) isoforms of the channel, and is known as D3. Ca2+ exerts important regulatory functions on the RyR, being involved in both activation and inactivation functions of the channel, i.e. the effects occurring at micromolar and millimolar Ca2+ concentrations respectively. To characterize the role of D3 in the Ca2+-dependent regulation of the Ca2+-release channel, we studied the functional consequences of deleting the D3 region from RyR1 (∆D3-RyR1) using a heterologous expression system, [3H]ryanodine binding assays and single-channel recordings in lipid bilayers. Deletion of the D3 region selectively affected Ca2+-dependent regulation of RyR1, but did not alter [3H]ryanodine binding or the effect of other modulators on the RyR. Compared with full-length RyR1 (wt-RyR1), the Ca2+-dependence curve of ∆D3-RyR1 is broader, reflecting increased sensitivity to Ca2+ activation and decreased sensitivity to Ca2+ inactivation. In addition, ∆D3-RyR1 was more resistant to inhibition by Mg2+. Comparison of the effect of caffeine on wt-RyR1 and ∆D3-RyR1 suggested that D3 is an important region of RyR that participates in Ca2+-dependent activation and inactivation of the Ca2+-release channel.
Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association
Authors: YIP-SCHNEIDER Michele T. , MIAO Wenyan , LIN Amy , BARNARD Darlene S. , TZIVION Guri , MARSHALL Mark S.
Published: August 2015

The Raf-1 kinase domain is kept in an inactive state by the N-terminal regulatory domain. Activation of the kinase domain occurs following release from the N-terminal repression and possible catalytic upregulation. To distinguish the regulatory mechanisms that directly influence the catalytic activity of the enzyme from those which act through the inhibitory domain, the catalytic domain of Raf-1 (CR3) was expressed in COS-7 cells. The role of phosphorylation in the direct regulation of this domain was determined by substituting non-phosphorylatable amino acids for known serine and tyrosine phosphorylation sites. The intrinsic activity of each mutant protein was determined as well as stimulation by v-Src and phorbol esters. Both v-Src and phorbol esters were potent activators of CR3, requiring the serine 338/339 (p21-activated protein kinase, Pak) and tyrosine 340/341 (Src) phosphorylation sites for full stimulation of CR3. In contrast, loss of the serine 497/499 protein kinase C phosphorylation sites had little effect on CR3 activation by either v-Src or phorbol esters. Loss of serine 621, a 14-3-3 adaptor-protein-binding site, prevented activation of CR3 by v-Src or phorbol esters and partially decreased the high basal activity of the kinase fragment. When co-expressed in COS-7 cells, 14-3-3 associated strongly with full-length Raf-1, weakly with wild-type CR3 and not at all with the A621 and D621 CR3 mutants. The role of 14-3-3 in maintaining the activity of the catalytic domain of Raf-1 was investigated further by performing peptide-competition studies with wild-type CR3, wild-type CR3 and v-Src or constitutively active CR3 (CR3[YY340/341DD]). In each case, incubation of the proteins with a phosphoserine-621 Raf-1 peptide, which we show displaced Raf-1 and CR3[YY340/341DD] from 14-3-3, was found to substantially reduce catalytic activity. Taken together, our results support a model of Raf regulation in which the activity of the Raf-1 catalytic domain is directly upregulated by phosphorylation, following relief of inhibition by the N-terminal regulatory domain upon Ras-GTP binding. Moreover, the presence of serine 621 in the free catalytic fragment is required for full CR3 activation by stimulatory factors, and the continuous presence of 14-3-3 at this site is necessary for retaining activity once the kinase is activated.
Kinetic alteration of a human dihydrodiol/3α-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine
Authors: OHTA Tatuya , ISHIKURA Syuhei , SHINTANI Syunichi , USAMI Noriyuki , HARA Akira
Published: August 2015

Human dihydrodiol dehydrogenase with 3α-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1Ő1C4) that belong to the aldoŐketo reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1Ő1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the Km for NADP+ and differently influenced the Km and kcat for substrates depending on their structures. The kinetic constants for bile acids with a 12α-hydroxy group were decreased 1.5Ő7-fold and those for the other substrates were increased 1.3Ő9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17β-oestradiol, phenolphthalein and flufenamic acid and 3,5,3´,5´-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the Km for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators.
Control of pancreatic bile-salt-dependent-lipase secretion by the glucose-regulated protein of 94kDa (Grp94)
Authors: NGANGA Alain , BRUNEAU Nadine , SBARRA Veronique , LOMBARDO Dominique , LE PETIT-THEVENIN Josette
Published: August 2015

Bile-salt-dependent lipase (BSDL; EC 3.1.1.13) is an enzyme expressed by the pancreatic acinar cell and secreted as a component of the pancreatic juice. During its route towards secretion, BSDL is associated with intracellular membranes by means of a multiprotein folding complex, which includes the glucose-regulated protein of 94kDa (Grp94). We have postulated that the association of BSDL with membranes is required for the complete O-glycosylation of the protein, which diverts BSDL from a degradation route and consequently allows its secretion. To further characterize the role of Grp94 in BSDL secretion, we have studied the effect of a ribozyme specifically targeted to Grp94 mRNA. This ribozyme has been transfected into AR4-2J cells, and we have shown that a decrease in Grp94 expression leads to a concomitant decrease in BSDL secretion and expression. Geldanamycin (GA), which alters Grp94 functions, also affects the release of BSDL into the culture medium of AR4-2J cells. BSDL expressed in GA-treated AR4-2J cells is unstable. Furthermore, under conditions that decrease the level of BSDL secretion, no intracellular accumulation of the enzyme was observed, suggesting that BSDL that cannot associate with (or be structured by) Grp94 could be rapidly degraded. We have further shown that this degradation probably occurs via the ubiquitin-dependent pathway. Altogether, these results indicate that Grp94 has a pivotal role in BSDL folding and in the sorting of this pancreatic enzyme.
Coexpression of α and β subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen
Authors: WAGNER Klaus , PÖSCHL Ernst , TURNAY Javier , BAIK Jeong-mi , PIHLAJANIEMI Taina , FRISCHHOLZ Svenja , VON DER MARK Klaus
Published: August 2015

We have reported previously on the expression of recombinant human type X collagen (hrColX) in HEK 293 and HT 1080 cells by using the eukaryotic expression vector pCMVsis (in which CMV stands for cytomegalovirus). Several stably transfected clones secreted full-length triple-helical hrColX molecules in large amounts, but the secreted collagen was underhydroxylated, with a hydroxyproline-to-proline ratio of 0.25 and a melting temperature (Tm) of 31°C. By comparison, native chicken type X procollagen has a Tm of 46°C. To stabilize the triple helix of hrColX, an hrColX-expressing clone (A6/16) was co-transfected with both α and β subunits of human prolyl 4-hydroxylase. Clones were selected that secreted proα1(X) collagen chains with an apparent molecular mass of 75kDa and an increased hydroxyproline-to-proline ratio of close to 0.5. As a result of enhanced prolyl hydroxylation, the Tm of the hrColX was increased to 41°C as measured by CD analysis at various temperatures. The CD spectra indicated a minimum ellipticity at 198nm and a peak at 225nm at 20°C, confirming the presence of a triple helix. The same Tm of 41°C was measured for the triple-helical core fragments of hrColX of 60Ő65kDa that were retained after brief digestion with chymotrypsin/trypsin at increasing temperatures. This shows that the human cell line HEK-293 is suitable for the simultaneous expression of three genes and the stable production of substantial amounts of recombinant, fully hydroxylated type X collagen over several years.
An uncoupling protein homologue putatively involved in facultative muscle thermogenesis in birds
Authors: RAIMBAULT Serge , DRIDI Sami , DENJEAN Frédérique , LACHUER Joël , COUPLAN Elodie , BOUILLAUD Frédéric , BORDAS André , DUCHAMP Claude , TAOUIS Mohamed , RICQUIER Daniel
Published: August 2015

The cDNA of an uncoupling protein (UCP) homologue was obtained by screening a chicken skeletal-muscle library. The predicted 307-amino-acid sequence of avian UCP (avUCP) is 55, 70, 70 and 46% identical with mammalian UCP1, UCP2 and UCP3 and plant UCP respectively. avUCP mRNA expression is restricted to skeletal muscle and its abundance was increased 1.3-fold in a chicken line showing diet-induced thermogenesis, and 3.6- and 2.6-fold in cold-acclimated and glucagon-treated ducklings developing muscle non-shivering thermogenesis respectively. The present data support the implication of avUCP in avian energy expenditure.
Tunicamycin induces ubiquitination and degradation of apolipoprotein B in HepG2 cells
Authors: LIAO Wei , CHAN Lawrence
Published: August 2015

Apolipoprotein (apo) B-100 is an essential component of atherogenic plasma lipoproteins. Previous studies have demonstrated that the production of apoB-100 is regulated largely by intracellular degradation at both the co-translational and post-translational levels and that proteasome-mediated and non-proteasome-mediated pathways are involved in this process. ApoB-100 is a glycoprotein. The present study was undertaken to address the question of whether the inhibition of N-linked glycosylation with tunicamycin would interfere with apoB-100 production. We demonstrated that the treatment of HepG2 cells with tunicamycin decreased the net production of apoB-100 by enhancing co-translational degradation of the protein. This effect of tunicamycin was partly prevented by lactacystin, a specific proteasome inhibitor. Because lactacystin only partly reversed the effects of tunicamycin on apoB biogenesis, tunicamycin seemed also to induce apoB co-translational degradation in HepG2 cells by one or more non-proteasomal pathways. Furthermore, tunicamycin increased apoB ubiquitination approx. 4-fold. The proportion of the newly synthesized apoB-100 that was secreted and incorporated into the nascent lipoprotein particles was unaffected by tunicamycin. Thus the tunicamycin-mediated inhibition of N-linked glycosylation interferes with the production of apoB-100 that is mediated by both proteasomal and non-proteasomal pathways.
Implication of the proprotein convertases furin, PC5 and PC7 in the cleavage of surface glycoproteins of Hong Kong, Ebola and respiratory syncytial viruses: a comparative analysis with fluorogenic peptides
Authors: BASAK Ajoy , ZHONG Mei , MUNZER Jon S. , CHRÉTIEN Michel , SEIDAH Nabil G.
Published: August 2015

Fluorogenic peptides encompassing the processing sites of envelope glycoproteins of the infectious influenza A Hong Kong virus (HKV), Ebola virus (EBOV) and respiratory syncytial virus (RSV) were tested for cleavage by soluble recombinants of the proprotein convertases furin, PC5 and PC7. Kinetic studies with these intramolecularly quenched fluorogenic peptides revealed selective cleavages at the physiological dibasic sites. The HKV peptide is cleaved by both furin and PC5 with similar efficacy; in comparison, PC7 cleaves this substrate poorly. In contrast with the basic tetrapeptide insertion within the haemagglutinin sequence of HKV, two other dipeptide insertions revealed a poorer cleavage with a similar rank order of potency. These results demonstrate that the N-terminal RERR insertion to the wild-type avian RKKR↓ sequence is functionally significant, and suggest that the approx. 5-fold increase in cleavage efficacy contributes to the high infectivity of the H5N1 virus subtype. With regard to RSV peptide processing, PC7 is twice as effective as PC5 and furin. The EBOV peptide was processed with similar efficiency by the three enzymes. Our observations that all of these cleavages can be effectively inhibited by a plant andrographolide derivative at 250µM or less might aid in the design of potent convertase inhibitors as alternative antiviral therapies.