http://www.biochemj.org Biochemical Journal RSS feed -- BJ Cell Immediate Publications 0264-6021 1470-8728 Biochemical Journal http://www.biochemj.org/images/BJ_Name.gif http://www.biochemj.org http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111912 I_Ks channel function. In this study we attempted to rescue the function of these mutants, in human embryonic kidney-293 cells, by promoting readthrough of their premature termination codons (PTCs) using the pharmacological agents G-418, gentamicin and PTC124. Gentamicin and G-418, acted to promote full-length channel protein expression from R518X at 100 µM and from Q530X at 1 mM. In contrast, PTC124 did not, at any dose tested, induce readthrough of either mutant. G-418 (1 mM) treatment also acted to significantly (P<0.05) increase current density and peak-tail current density, at +80 mV, for R518X, but not Q530X, to 58±11% and 82±17% of the wild-type level respectively. However, the biophysical properties of the currents produced, from R518X, while similar were not identical to wild-type as the voltage dependence of activation was significantly (P<0.05) shifted by +25 mV. Overall, these findings indicate that although functional rescue of LQT1 nonsense mutations is possible it is dependent on the degree of readthrough achieved and the effect on channel function of the amino acid substituted for the PTC. Such considerations will determine the success of future therapies.]]> S C Harmer, J S Mohal, D Kemp, A Tinker 2012-02-06T15:18:15Z doi:10.1042/BJ20111912 Portland Press Limited 2012-02-06 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111645 H Li, J Liu 2012-01-31T12:11:41Z doi:10.1042/BJ20111645 Portland Press Limited 2012-01-31 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111627 Ser15phosphorylation induces dissociation of p53-RPA70 complex in hypoxia. DNA-PK is shown to induce p53^Ser15phosphorylation thus enhancing RPA70-mediated NER/NHEJ-repair. Further, RPA70 gene silencing induces significant increase in the cellular apoptosis in the resistant hypoxic cancer cells. We have thus elucidated a novel pathway showing how DNA-PK-mediated p53^Ser15 phosphorylation dissociates p53-RPA70 complex in enhancing NER/NHEJ-repair that causes resistance to apoptosis, in hypoxic cancer cells. This novel finding may open new strategies in developing cancer therapeutics based upon regulation of RPA70-mediated NER/NHEJ-repair.]]> E Madan, R Gogna, U Pati 2012-01-30T13:47:24Z doi:10.1042/BJ20111627 Portland Press Limited 2012-01-30 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110348 FBLN5^+/+ MEFs), but has no effect on fibulin-5-deficient FBLN5^-/- MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (FBLN5^RGE/RGEMEFs) do not migrate in response to uPA. Moreover, a blocking anti-human β1 antibody inhibited the migration of pulmonary arterial smooth muscle cells (PASMCs) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal calcium-binding epidermal growth factor-like (cbEGF) domain, leading to loss of b1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from b1 integrin and thereby unblocks integrin’s capacity to facilitate cell motility.]]> A Kapustin, V Stepanova, N Aniol, D B. Cines, A Poliakov, S Yarovoi, T Lebedeva, R Wait, G Ryzhakov, Y Parfyonova, Y Gursky, H Yanagisawa, M Minashkin, R Beabealashvilli, A Vorotnikov, A Bobik, V Tkachuk 2012-01-26T15:50:10Z doi:10.1042/BJ20110348 Portland Press Limited 2012-01-26 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111839 in vitro kinase reaction. The phosphorylation sites were identified by mass spectrometry and the major phosphorylation site was found to be at the highly conserved S1366 residue. A phospho-specific antibody was generated that can specifically recognize ROCKII S1366 phosphorylation. We found that the extent of S1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that S1366 phosphorylation reflects its kinase activity. In addition, ROCKII S1366 phosphorylation could be detected in human breast tumors by immunohistochemical staining. Our study provides a new approach for revealing ROCKII activation status by directly probing ROCKII S1366 phosphorylation in cells or tissues.]]> H Chuang, C Yang, Y Tsay, C Hsu, L Tseng, Z Chang, H Lee 2012-01-24T14:38:48Z doi:10.1042/BJ20111839 Portland Press Limited 2012-01-24 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112061 Agrocybe aegerita (designated AAL-2) by affinity chromatography with N-acetylglucosamine (GlcNAc) coupled Sepharose 6B after (NH₄)₂SO₄ precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by mass spectrometry against the translated transcriptome of A. aegerita. The molecular weight of AAL-2 was calculated to be 43.175 kDa from mass spectrometry (MS), which was consistent with the data calculated from the amino acid sequence. To analyze the sugar binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed and the result showed that AAL-2 bound with high selectivity to terminal, nonreducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal, nonreducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as wheat germ agglutinin (WGA) and Griffonia simplicifolia lectin-II (GSL-II). Isothermal titration calorimetry (ITC) further showed that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the current study, we also evaluated the antitumor activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumor growth and prolongation of survival time of tumor bearing mice in vivo.]]> S Jiang, Y Chen, M Wang, Y Yin, Y Pan, B Gu, G Yu, Y Li, B Wong, Y Liang, h sun 2012-01-23T15:09:25Z doi:10.1042/BJ20112061 Portland Press Limited 2012-01-23 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111236 in-vitro, an effect reversed by PKR-inhibitor 2-aminopurine, suggests a direct effect of the drug in the kinase activation. The results identify PKR as a novel target of indomethacin, opening new scenarios on the molecular mechanisms underlying the pleiotropic activity of this traditional NSAID.]]> C Brunelli, C Amici, M Angelini, C Fracassi, G Belardo, M Santoro 2012-01-23T14:57:01Z doi:10.1042/BJ20111236 Portland Press Limited 2012-01-23 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111861 via decreased HNF binding to its cognate promoter element.]]> R H. Jenkins, J Martin, A O. Phillips, T Bowen, D J. Fraser 2012-01-23T12:05:33Z doi:10.1042/BJ20111861 Portland Press Limited 2012-01-23 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111837 O-gallate (EGCG), the major polyphenol of green tea, has cancer chemopreventive and chemotherapeutic activities. EGCG selectively inhibits cell growth and induces apoptosis in cancer cells without adversely affecting normal cells; however, the underlying molecular mechanism in vivo is unclear. In this study, we show that EGCG-induced apoptotic activity is attributed to a lipid raft clustering mediated through 67-kDa laminin receptor (67LR) that is significantly elevated in multiple myeloma (MM) cells relative to normal peripheral blood mononuclear cells, and that acid sphingomyelinase (aSMase) is critical for the lipid raft clustering and the apoptotic cell death induced by EGCG. We also found that EGCG induces aSMase translocation to the plasma membrane and protein kinase C delta (PKCδ) phosphorylation at Ser⁶⁶⁴, which was necessary for aSMase/ceramide signaling, via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell death pathway triggered by EGCG for the specific killing of MM cells.]]> S Tsukamoto, K Hirotsu, M Kumazoe, Y Goto, K Sugihara, T Suda, Y Tsurudome, T Suzuki, S Yamashita, Y Kim, Y Huang, K Yamada, H Tachibana 2012-01-19T11:59:48Z doi:10.1042/BJ20111837 Portland Press Limited 2012-01-19 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111351 A Hafner, N Obermajer, J Kos 2012-01-19T11:42:24Z doi:10.1042/BJ20111351 Portland Press Limited 2012-01-19 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111996 D A. Carrino, S Mesiano, N M. Barker, W W. Hurd, A I. Caplan 2012-01-19T11:22:29Z doi:10.1042/BJ20111996 Portland Press Limited 2012-01-19 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110554 golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, neuroendocrine long coiled-coil protein 1 (NECC1). Our studies show that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K⁺-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture but enhanced K⁺-stimulated hormone secretion in PC12 cells. In all, our results identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells.]]> D Cruz-Garcia, A Diaz-Ruiz, Y Rabanal-Ruiz, J R. Peinado, F Gracia-Navarro, J P. Castaño, M Montero-Hadjadje, M Tonon, H Vaudry, Y Anouar, R Vazquez-Martinez, M M Malagon 2012-01-18T12:34:00Z doi:10.1042/BJ20110554 Portland Press Limited 2012-01-18 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111589 2) resides in raft domains and DHA can alter the size of rafts, we hypothesized that PI(4,5)P₂ and downstream actin remodeling are perturbed by the incorporation of n-3 PUFA into membranes, resulting in suppressed T cell activation. CD4⁺ T cells isolated from Fat-1 transgenic mice (membranes enriched in n-3 PUFA) exhibited a 50% decrease in PI(4,5)P₂. Upon activation by plate bound anti-CD3/anti-CD28 or PMA/ionomycin, Fat-1 CD4⁺ T cells failed to metabolize PI(4,5)P₂. Furthermore, actin remodeling, failed to initiate in Fat-1 CD4⁺ T cells upon stimulation; however, the defect was reversed by incubation with exogenous PI(4,5)P₂. When Fat-1 CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 coated beads, the Wiskott-Aldrich syndrome protein (WASP) failed to translocate to the immunological synapse. The suppressive phenotype, consisting of defects in PI(4,5)P₂ metabolism and actin remodeling, were recapitulated in CD4⁺ T cells isolated from mice fed a 4% DHA triglyceride-enriched diet. Collectively, these data demonstrate that DHA alters PI(4,5)P₂ in CD4⁺ T cells, thereby suppressing the recruitment of WASP to the IS, and impairing actin remodeling in CD4⁺ T cells.]]> T Y Hou, J M Monk, Y Fan, R Barhoumi, Y Q Chen, G M Rivera, D N McMurray, R S Chapkin 2012-01-18T11:24:15Z doi:10.1042/BJ20111589 Portland Press Limited 2012-01-18 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111255 2+ with a K_d = 1.6 x 10^-6 M for trout hair cell proteins and a K_d = 2.7 x 10^-7 M for OC proteins at 68 µM Ca²⁺. Pull-down assays indicated that the binding to OC CNGA3 was attributable to EMILIN1 intracellular sequence that follows a predicted transmembrane domain in the carboxy terminus. Saccular hair cells also express transcript for phosphodiesterase 6C (PDE6C), which in cone photoreceptors regulates degradation of cGMP used to gate CNGA3 in phototransduction. Taken together, the evidence supports the existence in saccular hair cells of a molecular pathway linking CNGA3, its binding partner EMILIN1 (and beta1-integrin) and cGMP-specific PDE6C, which is potentially replicated in cochlear outer hair cells, given stereociliary immunolocalizations of CNGA3, EMILIN1 and PDE6C.]]> D Selvakumar, M J Drescher, J R Dowdall, K M Khan, J S Hatfield, N A Ramakrishnan, D G Drescher 2012-01-17T12:23:32Z doi:10.1042/BJ20111255 Portland Press Limited 2012-01-17 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111889 2-producing enzyme and a lipid-transporter. L-PGDS is a member of the lipocalin superfamily and can bind a wide variety of lipophilic molecules. Here we show the protective effect of L-PGDS on H₂O₂-induced apoptosis in neuroblastoma cell line SH-SY5Y. L-PGDS expression was increased in H₂O₂-treated neuronal cells, and the L-PGDS level was highly associated with H₂O₂-induced apoptosis, indicating that L-PGDS protected the neuronal cells against H₂O₂-mediated cell death. Cell viability assay revealed that L-PGDS protected against H₂O₂-induced cell death in a concentration-dependent manner. Further, the titration of free-thiols in H₂O₂-treated L-PGDS revealed that H₂O₂ reacted with the thiol of Cys65 of L-PGDS. The MALDI-TOF MS spectrum of H₂O₂-treated L-PGDS showed a 32-Da increase in the mass relative to that of the untreated protein, showing that the thiol was oxidized to sulfinic acid. The binding affinities of oxidized L-PGDS for lipophilic molecules were comparable to those of untreated L-PGDS. Taken together, these results demonstrate that L-PGDS protected against neuronal cell death by scavenging reactive oxygen species without losing its ligand-binding function. The novel function of L-PGDS could be useful for the suppression of oxidative stress-mediated neurodegenerative diseases.]]> A Fukuhara, M Yamada, K Fujimori, Y Miyamoto, T Kusumoto, H Nakajima, T Inui 2012-01-16T14:00:57Z doi:10.1042/BJ20111889 Portland Press Limited 2012-01-16 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112078 3 and Gbg subunits and inhibited by PKA. Here, we show that Protein Phosphatase 1a (PP1a) binds P‑Rex1 through an RVxF-type docking motif. PP1a activates P‑Rex1 directly in vitro, both independently of and additively to PIP₃ and Gbg. PP1a also substantially activates P‑Rex1 in vivo, both in basal and PDGF- or LPA-stimulated cells. The phosphatase activity of PP1a is required for P‑Rex1 activation. PP1b, a close homologue of PP1a, is also able to activate P‑Rex1, but less effectively. PP1a stimulates P-Rex1-mediated, Rac-dependent changes in endothelial cell morphology. Mass spectrometric analysis of wild-type P‑Rex1 and a PP1a-binding deficient mutant revealed that endogenous PP1a dephosphorylates P‑Rex1 on at least three residues, S834, S1001 and S1165. Site-directed mutagenesis of S1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1a, confirming S1165 as a dephosphorylation site important in regulating P‑Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P‑Rex1 through PP1a-dependent dephosphorylation.]]> M A Barber, A Hendrickx, M Beullens, H Ceulemans, D Oxley, S Thelen, M Thelen, M Bollen, H C E Welch 2012-01-16T12:34:24Z doi:10.1042/BJ20112078 Portland Press Limited 2012-01-16 BJ Signal http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111057 In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulates both the K_M and the V_M of PARP-1. In addition, we showed that upon treatment by lithium, a very potent inhibitor of the enzyme responsible of pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity.]]> E Toledano, V Ogryzko, A Danchin, D Ladant, U Mechold 2012-01-12T14:34:48Z doi:10.1042/BJ20111057 Portland Press Limited 2012-01-12 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110868 I Boo, K teWierek, F Douam, D LAVILLETTE, P Poumbourios, H Edelgard Drummer 2012-01-12T14:09:38Z doi:10.1042/BJ20110868 Portland Press Limited 2012-01-12 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111994 S A. Maroney, P E. Ellery, J P. Wood, J P. Ferrel, C E. Bonesho, A E. Mast 2012-01-12T13:53:45Z doi:10.1042/BJ20111994 Portland Press Limited 2012-01-12 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112044 2+ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. Here we used in vitro lipid mixing assays to investigate the Ca²⁺-dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS in the vesicle was varied. Syt1 stimulated lipid mixing by factors of 3-10 without Ca²⁺. However, with Ca²⁺ there was additional factors of 2-5 enhancement. But the Ca²⁺-dependent stimulation was observed only when there was excess PS on the t-SNARE side, and if there was equal or more PS on v-SNARE side, the Ca²⁺-dependent stimulation was not observed. We found that Ca²⁺ in a few ten µM level was sufficient to give rise to the maximal enhancement. The single vesicle fusion assay indicates that the Ca²⁺-dependent enhancement was mainly on docking while its effect on lipid mixing was small. Thus, for Syt1 to function as a Ca²⁺ sensor, a charge asymmetry appears to be important and it may play a role in steering Syt1 to make productive trans binding to the plasma membrane.]]> Y Lai, Y Shin 2012-01-09T15:31:00Z doi:10.1042/BJ20112044 Portland Press Limited 2012-01-09 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111420 + are synthesized outside the peroxisomes and therefore must be transported into the peroxisomal matrix where they are required for important processes. In this work we have functionally identified and characterized SLC25A17, which is the only member of the mitochondrial carrier family that has previously been shown to be localized in the peroxisomal membrane. Herein, recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN, AMP and to a lesser extent of NAD⁺, adenosine 3’,5’-diphosphate (PAP) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and inhibited by pyridoxal-5’-phosphate and other mitochondrial carrier inhibitors. It was expressed to various degrees in all the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD⁺ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP. This is the first report describing the identification and characterization of a transporter for multiple free cofactors in peroxisomes.]]> G Agrimi, A Russo, P Scarcia, F Palmieri 2011-12-21T12:59:00Z doi:10.1042/BJ20111420 Portland Press Limited 2011-12-21 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110687 C Stroupe 2011-12-16T11:28:00Z doi:10.1042/BJ20110687 Portland Press Limited 2011-12-16 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111360 Drosophila melanogaster crammer is a novel cathepsin inhibitor that is involved in long-term memory (LTM) formation. The mechanism by which the inhibitory activity is regulated remains unclear. Here we have shown that the oligomeric state of crammer is pH dependent. At neutral pH, crammer is predominantly dimeric in vitro as a result of disulfide bond formation, and is monomeric at acidic pH. Our inhibition assay shows that monomeric crammer, not disulfide bonded dimer, is a strong competitive inhibitor of cathepsin L. Crammer is a monomeric molten globule in acidic solution, a condition that is similar to the environment in the lysosome where crammer is likely located. Upon binding to cathepsin L, however, crammer undergoes a molten globule-to-ordered structural transition. Using high-resolution NMR spectroscopy, we have shown that a cysteine-to-serine point mutation at position 72 (C72S) renders crammer monomeric at pH 6.0 and that the structure of the C72S variant highly resembles that of wild-type crammer in complex with cathepsin L at pH 4.0. We have determined the first solution structure of propeptide-like protease inhibitor in its active form and examined in detail using a variety of spectroscopic methods the folding properties of crammer in order to delineate its biomolecular recognition of cathepsin.]]> T Tseng, C Cheng, D Chen, M Shih, Y Liu, S Danny Hsu, P Lyu 2011-12-09T14:32:00Z doi:10.1042/BJ20111360 Portland Press Limited 2011-12-09 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111661 42-EGFP fusion protein in the HEK cell line, T-REx293, and use this to identify both macromolecules and small molecules that reduced aggregation and associated cell toxicity. Thus, a molecular shield protein, AavLEA1, which limits aggregation of proteins with expanded polyglutamine (polyQ) repeats, is also effective against Aβ₄₂-EGFP when co-expressed in T-REx293 cells. A screen of polysaccharide and small organic molecules from medicinal plants and fungi reveals one candidate in each category, PS5 and ganoderic acid DM, respectively, with activity against Aβ peptide. Both PS5 and ganoderic acid DM probably promote Aβ aggregate clearance indirectly through the proteasome. The model is therefore of value to study the effects of intracellular Aβ peptide on cell physiology, and to identify reagents that counteract those effects.]]> S Chakrabortee, Y Liu, L Zhang, H R Matthews, H Zhang, N Pan, C Cheng, S Guan, D Guo, Z Huang, Y Zheng, A Tunnacliffe 2011-12-09T14:08:00Z doi:10.1042/BJ20111661 Portland Press Limited 2011-12-09 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111678 Pdx1, Ngn3 and MafA have been reported to bring about the transdifferentiation of pancreatic exocrine cells to beta cells in vivo (Zhou et al. (2008) Nature 455, 627-630), We have investigated the mechanism of this process using a standard in vitro model of pancreatic exocrine cells, the rat AR42j-B13 cell line. We constructed a new adenoviral vector encoding all three genes, called Ad-PNM. When introduced into AR42j-B13 cells, Ad-PNM causes a rapid change to a flattened morphology, and a cessation of cell division. The expression of exocrine markers is suppressed. Both insulin genes are upregulated as well as a number of transcription factors normally characteristic of beta cells. At the chromatin level, histone tail modifications of the Pdx1, Ins1 and Ins2 gene promoters are shifted in a direction associated with gene activity, and the level of DNA CpG methylation is reduced at the Ins1 promoter. The transformed cells secrete insulin and are capable of relieving diabetes in streptozotocin-treated NOD-SCID mice. However the transformation is not complete. The cells lack expression of several genes important for beta cell function and they do not show glucose-sensitive insulin secretion. We conclude that, for this exocrine cell model, although the transformation is dramatic, the reprogramming is not complete and lacks critical aspects of the beta cell phenotype.]]> E Akinci, A Banga, L V Greder, J R Dutton, J M.W. Slack 2011-12-09T12:11:00Z doi:10.1042/BJ20111678 Portland Press Limited 2011-12-09 BJ Disease http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111831 S Jayaraman, G Cavigiolio, O Gursky 2011-12-08T11:26:00Z doi:10.1042/BJ20111831 Portland Press Limited 2011-12-08 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111745 A K Panigrahi, N Zhang, S K Otta, D Pati 2011-12-07T12:10:00Z doi:10.1042/BJ20111745 Portland Press Limited 2011-12-07 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111653 th position. Arg14Ala SARM mutant also showed reduced apoptotic potential when compared to the wild type. Taken together S27, which is a bona fide signal sequence that targets SARM to the mitochondria, explains the proapoptotic activity of SARM.]]> P Panneerselvam, L Pradeepkumar Singh, B Ho, J Chen, J L Ding 2011-12-07T11:51:00Z doi:10.1042/BJ20111653 Portland Press Limited 2011-12-07 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111398 2, differing in their rate of digoxin release in response to Na and ATP. Treating cells with angiotensin II (Ang II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. We test if Ang II regulates release by increasing phosphorylation at Ser-11/Ser-18 and Ser-938. Opossum kidney cells co-expressing the AT_1a receptor and either alpha-1.wild-type, alpha-1.S11A/S18A or alpha-1.S938A were treated ± 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na,K-ATPase was bound to digoxin-affinity columns in the presence of Na, ATP, and Mg. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Na,K-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in alpha-1.wild-type and alpha-1.S938A, but not alpha-1.S11A/S18A cells. Another 50% of bound Na,K-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in alpha-1.wild-type, but not a-1.S938A cells. Thus, Ang II changes the conformation of two forms of EP₂ via differential phosphorylation.]]> K J. Massey, Q Li, N F. Rossi, R R. Mattingly, D R. Yingst 2011-12-07T09:52:00Z doi:10.1042/BJ20111398 Portland Press Limited 2011-12-07 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111220 Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. Eeyarestatin I (ES_I) is a novel modulator of endoplasmic reticulum (ER) function. Here, we show that in addition to acutely inhibiting ERAD, ES_I causes production of mislocalised polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest these nontranslocated polypeptides promote activation of the unfolded protein response (UPR), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These data suggest that the accumulation of nontranslocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation.]]> C McKibbin, A Mares, M Piacenti, H Williams, P Roboti, M Puumalainen, A C Callan, K Lesiak-Mieczkowska, S Linder, H Harant, S High, S Flitsch, R C Whitehead, E Swanton 2011-12-06T14:06:00Z doi:10.1042/BJ20111220 Portland Press Limited 2011-12-06 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111363 E Merklinger, Y Gofman, A Kedrov, A J.M. Driessen, N Ben-Tal, Y Shai, D Rapaport 2011-12-05T14:30:00Z doi:10.1042/BJ20111363 Portland Press Limited 2011-12-05 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111618 Lactococcus lactis cannot synthesize heme, but when supplied with heme, expresses a cytochrome bd oxidase. Aside of the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in heme metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is know for hemW. HemW-like proteins occur in bacteria, plants, and animals, and are usually annotated as coproporphyrinogen III dehydrogenases. However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of coproporphyrinogen III dehydrogenase activity in vivo and in vitro. Recombinantly produced, purified HemW contained an iron-sulfur cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound heme b in vitro, with a stoichiometry of one heme per monomer and a K_D of 8 µM. In vivo, HemW occurred as a heme free, cytosolic form, as well as a heme-containing, membrane-associated form. Addition of L. lactis membranes to heme-containing HemW triggered the release of heme from HemW in vitro. Based these findings, we propose a role of HemW in heme trafficking. HemW-like proteins form a distinct phylogenetic clade which has not previously been recognized.]]> H K Abicht, J Martinez, G Layer, D Jahn, M Solioz 2011-12-05T14:11:00Z doi:10.1042/BJ20111618 Portland Press Limited 2011-12-05 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110605 Slc28a2) is the purine-preferring concentrative nucleoside transporter. It is expressed in both non polarized and polarized cells, where it is localized in the brush border membrane. Since no information about the domains implicated in the plasma membrane sorting of rCNT2 is available, this work aims at identifying structural and functional requirements for rCNT2 trafficking. The comprehensive topological mapping of the intracellular N terminal tail revealed two main features: 1) a glutamate enriched region (NPGLELME) between residues 21 and 28 seems to be implicated in the stabilization of rCNT2 in the cell surface, since mutagenesis of these conserved glutamates resulted in enhanced endocytosis. 2) Mutation of a potential Casein Kinase 2 (CK2) domain led to a loss of brush border-specific sorting. Although the shortest proteins assayed (-74AA, -48AA and -37AA) were accumulated intracellularly and lost their brush border membrane preference, they were still functional. A deeper analysis of CK2 implication in CNT2 trafficking, using a CK2 specific inhibitor (DMAT) and other complementary mutations mimicking the negative charge provided by phosphorylation (S46D and S46E), demonstrated an effect of this kinase on CNT2 activity. In summary, rCNT2 N-terminal tail contains dual sorting signals. An acidic region is responsible for its proper stabilization at the plasma membrane whereas the putative CK2 domain (Ser⁴⁶) is implicated in the apical sorting of the transporter.]]> I Pinilla-Macua, F Casado, M Pastor-Anglada 2011-12-02T15:33:00Z doi:10.1042/BJ20110605 Portland Press Limited 2011-12-02 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110247 +/Cl^-/glycine cotransport with a 2:1:1 stoichiometry. By contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na⁺. Using Molecular Interaction Potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na⁺ interactions. Replacement of D471 in this region reduced Na⁺ affinity and Na⁺ cooperativity of transport, an effect not produced in the homologous position (D295) in GLYT1. Unlike the GLYT1D295 mutation, this D471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [³H]glycine accumulation. The homologous D471 and D295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na⁺ and Li⁺-induced conformational changes. While these two cations had opposite effects on GLYT1 they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. Based on these findings, we propose a role for D471 in controlling cation access to GLYT2 Na⁺ sites, ion coupling during transport and the subsequent conformational changes.]]> G Pérez-Siles, E Nunez, A Morreale, E Jiménez, A Leo-Macías, G Pita, F Cherubino, R Sangaletti, E Bossi, Á R. Ortíz, C Aragon, B Lopez-Corcuera 2011-12-01T15:15:00Z doi:10.1042/BJ20110247 Portland Press Limited 2011-12-01 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111461 trans-Golgi membranes come in close apposition with endoplasmic reticulum (ER) membranes to form ER-trans-Golgi contact sites, which may facilitate transfer of newly synthesized ceramide from the ER to sphingomyelin (SM) synthase at the trans-Golgi via ceramide transfer protein (CERT). CERT interacts with both ER and Golgi membranes, and together with Golgi morphology contributes to efficient SM synthesis. Here, we show that Golgi disassembly during proapoptotic stress induced by tumor necrosis factor (TNFa) and anisomycin results in decreased levels of CERT at the Golgi region. This is accompanied by a caspase-dependent loss of full-length CERT and reduction in de novo SM synthesis. In vitro, CERT is cleaved by caspases-2, -3 and -9. Truncated versions of CERT corresponding to fragments generated by caspase-2 cleavage at Asp²¹³ were mislocalized and did not promote efficient de novo SM synthesis. Thus, it is likely that during cellular stress, disassembly of Golgi structure together with inactivation of CERT by caspases causes a reduction in ceramide trafficking and SM synthesis, and could contribute to the cellular response to proapoptotic stress.]]> S Chandran, C E. Machamer 2011-12-01T11:55:00Z doi:10.1042/BJ20111461 Portland Press Limited 2011-12-01 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110535 in vitro the pGlu formation on MCPs by QC using mass spectrometry. A potent QC inhibitor, PBD150, significantly reduced the N-terminal uncyclized MCPs precursor (preMCPs)-stimulated monocyte migration, whereas pGlu-containing MCPs (pMCPs)-induced cell migration was unaffected. QC siRNA revealed a similar inhibitory effect. Lastly, we demonstrated that inhibiting QC can attenuate cell migration by LPS. These results strongly suggest that QC-catalyzed N-terminal pGlu formation of MCPs is required for monocyte migration, and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a drug target for some inflammatory disorders.]]> Y Chen, K Huang, W Kuo, Y Lo, Y Lee, A H.-J. Wang 2011-11-08T11:22:00Z doi:10.1042/BJ20110535 Portland Press Limited 2011-11-08 BJ ChemBio