MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'BJ Cell' source. Mon, 06 Feb 2012 13:31:35 +0100 http://www.medworm.com/index.php?rid=5664016&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111912 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 w... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5664016 Mon, 06 Feb 2012 05:00:00 +0100 5664016 http://www.medworm.com/index.php?rid=5656179&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111645 In this study, we have identified a novel histone nuclear factor P (HINFP) recognition motif residing between HNF1 motif and SRE that is essential for basal and sterol-regulated transcriptions of the PCSK9 promoter. Mutation of this motif lowers the basal promoter activity and abolishes the sterol-mediated repression as well as the SREBP2-induced activation of the PCSK9 promoter. We further show that the activity of SREBP2 in stimulating PCSK9 promoter activity is greatly enhanced by HINFP. Additional experiments suggest that HINFP and its cofactor NPAT form a functional complex, and NPAT may subsequently recruit HAT cofactor TRRAP to facilitate the histone H4 acetylation of the PCSK9 promoter. Knockdown of HINFP, NPAT or TRRAP each markedly reduces the amount of acetylated histone H4 on t... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5656179 Tue, 31 Jan 2012 05:00:00 +0100 5656179 http://www.medworm.com/index.php?rid=5643646&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111627 Cellular stressors are known to inhibit p53-RPA70 complex and RPA70 increases cellular DNA repair in cancer cells. We hypothesized that regulation of RPA70-mediated DNA repair might be responsible for inhibition of apoptosis in hypoxic tumors. We have shown that, in cancer cells, hypoxia disrupts p53-RPA70 complex thereby enhancing RPA70-mediated NER/NHEJ-repair. In normal cells, RPA70 binds to p53 N-terminus (NTD) whereas this binding is disrupted in hypoxia. Phosphorylation of p53-NTD is a crucial event in dissociating both NTD-RPA70 and p53-RPA70 complexes. Serial mutations at serine and threonine residues in NTD confirm that p53Ser15phosphorylation induces dissociation of p53-RPA70 complex in hypoxia. DNA-PK is shown to induce p53Ser15phosphorylation thus enhancing RPA70-mediated NER/N... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5643646 Mon, 30 Jan 2012 05:00:00 +0100 5643646 http://www.medworm.com/index.php?rid=5635192&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110348 We report uPA-mediated cell migration requires its interaction with fibulin-5. uPA stimulates migration of wild-type mouse embryonic fibroblasts (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 (FBLN5RGE/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... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5635192 Thu, 26 Jan 2012 05:00:00 +0100 5635192 http://www.medworm.com/index.php?rid=5624466&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111839 In this study, we show autophosphorylation of ROCKII in an 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 S1... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5624466 Tue, 24 Jan 2012 05:00:00 +0100 5624466 http://www.medworm.com/index.php?rid=5624469&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111861 MicroRNA-192 (miR-192) plays key roles in renal pathological and physiological responses, by repressing targets including Zeb1, Zeb2, and Wnk1. Here, we have studied regulation of miR-192 expression. We found that Transforming Growth Factor Beta-1 (TGF-b1) down-regulates miR-192 and miR-194, co-transcribed in the shared precursor pri-miR-192/194. Luciferase reporter analysis showed constitutive promoter activity within nucleotides +21 to -223. We identified Hepatocyte Nuclear Factor and p53 binding sites within this region that were required for constitutive promoter activity, which was decreased by TGF-b1 via an Alk5-dependent mechanism. TGF-b1-treatment decreased HNF binding to the miR-194-2/192 promoter, while knockdown of HNF-1 inhibited mature miR-192 and -194 expression. miR-1... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5624469 Mon, 23 Jan 2012 05:00:00 +0100 5624469 http://www.medworm.com/index.php?rid=5624468&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111236 The non-steroidal anti-inflammatory drug (NSAID) indomethacin, a cyclooxygenase-1 and -2 inhibitor with anti-inflammatory and analgesic properties, is known to possess anticancer activity against colorectal cancer (CRC) and other malignancies in humans; however, the mechanism underlying the anticancer action remains elusive. Herein we show that indomethacin selectively activates the double-stranded RNA (dsRNA)-dependent protein kinase PKR in a cyclooxygenase-independent manner, causing rapid phosphorylation of the alpha-subunit of eukaryotic translation initiation-factor 2 (eIF2alpha) and inhibiting protein synthesis in colorectal carcinoma and other types of cancer cells. The PKR-mediated translational block was followed by inhibition of CRC cell proliferation and apoptosis induction. Ind... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5624468 Mon, 23 Jan 2012 05:00:00 +0100 5624468 http://www.medworm.com/index.php?rid=5624467&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20112061 A novel lectin was isolated from the mushroom 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 re... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5624467 Mon, 23 Jan 2012 05:00:00 +0100 5624467 http://www.medworm.com/index.php?rid=5616836&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111996 Fibrosis is the formation of excess and abnormal fibrous connective tissue as a result of either a reparative or reactive process. A defining feature of connective tissue is its extracellular matrix, which provides structural support and also influences cellular activity. Two common human conditions that result from fibrosis are uterine fibroids (leiomyomas) and keloid scars. Because these conditions share a number of similarities and because their growth is due primarily to excessive extracellular matrix deposition, we compared the proteoglycans of uterine fibroids and keloid scars to corresponding normal tissues. Our analysis indicates that uterine fibroids and keloid scars contain higher amounts of proteoglycans relative to normal myometrium and normal adult skin, respectively. Proteogl... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5616836 Thu, 19 Jan 2012 05:00:00 +0100 5616836 http://www.medworm.com/index.php?rid=5616835&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111351 In this study, we investigated the involvement of γ-enolase in PI 3-K/Akt (phosphatidylinositol 3-kinase/Akt) and MAPK/ERK (mitogen-activated protein kinase/ extracellular-signal-regulated kinase) signaling, the two pathways triggered predominantly by neurotrophic factors. While the PI 3-K/Akt pathway, rather than the MAPK/ERK pathway, is involved in γ-enolase-enhanced cell survival, γ-enolase-stimulated neurite outgrowth requires both pathways, i.e. the activation of both PI 3-K and ERK1/2, leading to subsequent expression of growth cone-specific GAP-43 protein. MEK (mitogen-activated protein kinase kinase) and PI 3-K inhibition blocked or attenuated the neurite outgrowth associated with dynamic remodeling of the actin-based cytoskeleton. We show that γ-enolase... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5616835 Thu, 19 Jan 2012 05:00:00 +0100 5616835 http://www.medworm.com/index.php?rid=5616834&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111837 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 ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5616834 Thu, 19 Jan 2012 05:00:00 +0100 5616834 http://www.medworm.com/index.php?rid=5603661&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111589 n-3 polyunsaturated fatty acids (PUFA), i.e. docosahexaenoic acid (DHA), found in fish oil, exhibit anti-inflammatory properties; however, the molecular mechanisms remain unclear. Since phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) 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... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5603661 Wed, 18 Jan 2012 05:00:00 +0100 5603661 http://www.medworm.com/index.php?rid=5603660&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110554 Golgi-associated long coiled-coil proteins, often referred to as 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 a... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5603660 Wed, 18 Jan 2012 05:00:00 +0100 5603660 http://www.medworm.com/index.php?rid=5603662&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111255 The molecular characteristics of cyclic nucleotide-gated (CNG) channels in auditory/vestibular hair cells are largely unknown, unlike those of CNG channels mediating sensory transduction in vision and olfaction. Here, we report full-length sequence for three CNGA3 variants in a hair cell preparation from the trout saccule with high identity to CNGA3 in olfactory receptor neurons/cone photoreceptors. A custom antibody targeting amino terminus sequence immunolocalized CNGA3 to the stereocilia and subcuticular plate region of saccular hair cells. The cytoplasmic carboxyl terminus of CNGA3 was found by yeast two-hybrid analysis to bind the carboxy terminus of elastin microfibril interface-located protein 1 (EMILIN1) in both the vestibular hair cell model and rat organ of Corti (OC). Specific b... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5603662 Tue, 17 Jan 2012 05:00:00 +0100 5603662 http://www.medworm.com/index.php?rid=5603664&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20112078 P‑Rex1 is a guanine-nucleotide exchange factor (GEF) for the small G protein Rac that is activated by PIP3 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 def... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5603664 Mon, 16 Jan 2012 05:00:00 +0100 5603664 http://www.medworm.com/index.php?rid=5603663&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111889 Lipocalin-type prostaglandin D synthase (L-PGDS) is a dual functional protein, acting as a PGD2-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... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5603663 Mon, 16 Jan 2012 05:00:00 +0100 5603663 http://www.medworm.com/index.php?rid=5592673&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111994 Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that prevents intravascular coagulation through inhibition of factor Xa (fXa) and the tissue factor (TF)-factor VIIa complex (TF-fVIIa). Localization of TFPI within caveolae enhances its anticoagulant activity. To further define how caveolae contribute to TFPI anticoagulant activity, CHO cells were co-transfected with TF and membrane associated TFPI targeted to either caveolae (TFPI-GPI) or to bulk plasma membrane (TFPI-TM). Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent insoluble membrane fractions, while TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However,... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592673 Thu, 12 Jan 2012 05:00:00 +0100 5592673 http://www.medworm.com/index.php?rid=5592672&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110868 We examined the functional roles of conserved histidines located within E1 and E2. The E1 mutations, H222A/R, H298R, and H352A, disrupted E1-E2 heterodimerization and reduced virus entry. Five of 6 histidines located within the E2 receptor-binding domain (RBD) were important for the E2 fold, their substitution with Arg or Ala causing aberrant heterodimerization and/or CD81 binding. Distinct roles in E1/E2 heterodimerization and in virus entry were identified for His-691 and His-693, respectively, within the membrane-proximal stem region. Viral entry and cell-cell fusion at neutral and low pH were enhanced with H445R, indicating that the protonation state of His-445 is a key regulator of HCV fusion. However, H445R did not overcome the block to virus entry induced by bafilomycin A1 indicatin... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592672 Thu, 12 Jan 2012 05:00:00 +0100 5592672 http://www.medworm.com/index.php?rid=5592671&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111057 We report here a new target of pAp, Poly(ADP-ribose) Polymerase 1 (PARP-1), a key enzyme in the detection of DNA single strand breaks. We show that pAp can interact with PARP-1 and inhibit its poly(ADP-ribosyl)ation activity. 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 KM and the VM 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 re... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592671 Thu, 12 Jan 2012 05:00:00 +0100 5592671 http://www.medworm.com/index.php?rid=5592674&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20112044 Synaptotagmin 1 (Syt1) is a major Ca2+ 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 te... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592674 Mon, 09 Jan 2012 05:00:00 +0100 5592674 http://www.medworm.com/index.php?rid=5592675&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111420 The essential cofactors coenzyme A (CoA), FAD and NAD+ 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 pyr... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592675 Wed, 21 Dec 2011 05:00:00 +0100 5592675 http://www.medworm.com/index.php?rid=5532954&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111420 The essential cofactors coenzyme A (CoA), FAD and NAD+ 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 in... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5532954 Wed, 21 Dec 2011 05:00:00 +0100 5532954 http://www.medworm.com/index.php?rid=5512091&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110687 A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE proteins, Sec18p and Sec17p (yeast NSF[1] and a-SNAP), and the HOPS/Class C Vps complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for lipid mixing of proteoliposomes lacking cardiolipin. Omission of other lipids with negatively charged and/or small headgroups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for cardiolipin synthase (CRD1) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5512091 Fri, 16 Dec 2011 05:00:00 +0100 5512091 http://www.medworm.com/index.php?rid=5492379&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111678 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... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5492379 Fri, 09 Dec 2011 05:00:00 +0100 5492379 http://www.medworm.com/index.php?rid=5492378&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111661 Aβ peptide has a central role in Alzheimer’s disease (AD) where neuronal toxicity is linked to its extracellular and intracellular accumulation as oligomeric species. Searching for molecules that attenuate Aβ aggregation could uncover novel therapies for AD, but most studies in mammalian cells have inferred aggregation indirectly by assessing levels of secreted Aβ peptide. Here we establish a mammalian cell system for the direct visualisation of β-amyloid formation by expression of an Aβ42-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 expand... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5492378 Fri, 09 Dec 2011 05:00:00 +0100 5492378 http://www.medworm.com/index.php?rid=5492377&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111360 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-res... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5492377 Fri, 09 Dec 2011 05:00:00 +0100 5492377 http://www.medworm.com/index.php?rid=5492380&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111831 High-density lipoproteins (HDL) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apolipoprotein A-I (apoA-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed protein:lipid weight ratio 3:1, with apoA-I, phosphatidylcholine and cholesterol ester at approximate molar ratios of 1:8:1. Compared to lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5492380 Thu, 08 Dec 2011 05:00:00 +0100 5492380 http://www.medworm.com/index.php?rid=5482757&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111398 Kidney plasma membranes, which contain a single a-1 isoform of Na,K-ATPase, simultaneously contain two sub-conformations of EP2, 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 AT1a 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% ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5482757 Wed, 07 Dec 2011 05:00:00 +0100 5482757 http://www.medworm.com/index.php?rid=5482756&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111653 The fifth and the most well conserved member of the TLR adaptor, SARM, has been reported to be an important mediator of apoptosis. However, the exact cellular localization of SARM with respect to its role is unclear. Here we show that SARM specifically co-localizes with the mitochondria. Endogenous SARM is mainly found in the mitochondria. We demonstrated that the N terminal 27 amino acids (S27) of SARM, which is hydrophobic and polybasic, acts as a mitochondria-targeting signal sequence, associating SARM to the mitochondria. The S27 peptide has an inherent ability to bind to lipids and mitochondria. This sequence effectively translocates the soluble EGFP reporter into the mitochondria. Positioning S27 downstream of the EGFP abrogates its mitochondria-targeting ability. Transmission electr... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5482756 Wed, 07 Dec 2011 05:00:00 +0100 5482756 http://www.medworm.com/index.php?rid=5482755&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111745 The cohesin complex holds the sister chromatids together from S phase until the metaphase-to-anaphase transition and ensures both their proper cohesion and timely separation. In addition to its canonical function in chromosomal segregation, cohesion has been suggested by several lines of investigation in recent years to play additional roles in apoptosis, DNA damage response, transcriptional regulation, and hematopoiesis. To better understand the basis of the disparate cellular functions of cohesin in these various processes, we have characterized a comprehensive protein interactome of cohesin-RAD21 by using three independent approaches: yeast 2-hybrid (Y2H) screening, immunoprecipitation-coupled-mass spectrometry (IP-M) of cytoplasmic and nuclear extracts from MOLT-4 T lymphocytes in the ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5482755 Wed, 07 Dec 2011 05:00:00 +0100 5482755 http://www.medworm.com/index.php?rid=5482758&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111220 Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. Eeyarestatin I (ESI) is a novel modulator of endoplasmic reticulum (ER) function. Here, we show that in addition to acutely inhibiting ERAD, ESI 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. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=5482758 Tue, 06 Dec 2011 05:00:00 +0100 5482758 http://www.medworm.com/index.php?rid=5592676&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111618 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 ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592676 Mon, 05 Dec 2011 05:00:00 +0100 5592676 http://www.medworm.com/index.php?rid=5482760&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111618 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 ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5482760 Mon, 05 Dec 2011 05:00:00 +0100 5482760 http://www.medworm.com/index.php?rid=5482759&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111363 The mitochondrial outer membrane (OM) contains signal-anchored proteins that bear at their N-terminus a single hydrophobic segment that serves as both a mitochondrial targeting signal and an anchor at the membrane. These proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus and have to be imported into the organelle. Currently, the mechanisms by which they are targeted to and inserted into the OM are unclear. To shed light on these issues, we employed a recombinant version of the signal-anchored protein OM45 and a synthetic peptide corresponding to its signal-anchor segment. Both forms associated with isolated mitochondria independently of cytosolic factors. Interaction with mitochondria was diminished when a mutated form of the signal-anchor was employed. ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5482759 Mon, 05 Dec 2011 05:00:00 +0100 5482759 http://www.medworm.com/index.php?rid=5473454&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110605 rCNT2 (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 borde... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5473454 Fri, 02 Dec 2011 05:00:00 +0100 5473454 http://www.medworm.com/index.php?rid=5592678&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111461 The mammalian Golgi apparatus is composed of multiple stacks of cisternal membranes organized laterally into a polarized ribbon. Furthermore, 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 ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592678 Thu, 01 Dec 2011 05:00:00 +0100 5592678 http://www.medworm.com/index.php?rid=5592677&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110247 Synaptic glycine levels are controlled by glycine transporters (GLYTs). GLYT1 is the main regulator of synaptic glycine concentrations, which catalyzes Na+/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 ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592677 Thu, 01 Dec 2011 05:00:00 +0100 5592677 http://www.medworm.com/index.php?rid=5464992&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111461 The mammalian Golgi apparatus is composed of multiple stacks of cisternal membranes organized laterally into a polarized ribbon. Furthermore, 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 ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5464992 Thu, 01 Dec 2011 05:00:00 +0100 5464992 http://www.medworm.com/index.php?rid=5464991&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110247 Synaptic glycine levels are controlled by glycine transporters (GLYTs). GLYT1 is the main regulator of synaptic glycine concentrations, which catalyzes Na+/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 GL... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5464991 Thu, 01 Dec 2011 05:00:00 +0100 5464991 http://www.medworm.com/index.php?rid=5592679&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111256 This study provides support for BAD-Ser170 phosphorylation playing a key role not only in regulating BAD’s pro-apoptotic activity, but also its role in cell proliferation. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592679 Tue, 22 Nov 2011 05:00:00 +0100 5592679 http://www.medworm.com/index.php?rid=5437105&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111256 This study provides support for BAD-Ser170 phosphorylation playing a key role not only in regulating BAD’s pro-apoptotic activity, but also its role in cell proliferation. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=5437105 Tue, 22 Nov 2011 05:00:00 +0100 5437105 http://www.medworm.com/index.php?rid=5592680&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110679 This study focuses on the effect of mhtt on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing GABA. In this study, we report that the subcellular distribution of GAD is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated with Golgi membrane in cells expressing normal htt. However, it diffuses in the cytosol of cells expressing mhtt. As a result, vesicle-associated GAD65 trafficking is impaired. Since palmitoylation of GAD65 is required for GAD65 trafficking, we then demonstrate that palmitoylation of GAD65 is reduced in the HD model. Furthermore, overexpression of huntingtin-interacting protein 14, the enzyme responsible for palmitoylating GAD65 in... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592680 Mon, 21 Nov 2011 05:00:00 +0100 5592680 http://www.medworm.com/index.php?rid=5437106&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110679 This study focuses on the effect of mhtt on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing GABA. In this study, we report that the subcellular distribution of GAD is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated with Golgi membrane in cells expressing normal htt. However, it diffuses in the cytosol of cells expressing mhtt. As a result, vesicle-associated GAD65 trafficking is impaired. Since palmitoylation of GAD65 is required for GAD65 trafficking, we then demonstrate that palmitoylation of GAD65 is reduced in the HD model. Furthermore, overexpression of huntingtin-interacting protein 14, the enzyme responsible for palmitoylating GAD65 in... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5437106 Mon, 21 Nov 2011 05:00:00 +0100 5437106 http://www.medworm.com/index.php?rid=5592681&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111030 Nemaline myopathy (NM) is a rare, genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding beta-tropomyosin (Tm). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium sensitive manner. We have studied the pathogenetic mechanisms underlying six disease causing mutations in Tm. We show that five of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, while two show defective Ca2+ activation of contractility. We have also mapped the amino acids ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592681 Wed, 16 Nov 2011 05:00:00 +0100 5592681 http://www.medworm.com/index.php?rid=5417162&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111030 Nemaline myopathy (NM) is a rare, genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding beta-tropomyosin (Tm). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium sensitive manner. We have studied the pathogenetic mechanisms underlying six disease causing mutations in Tm. We show that five of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, while two show defective Ca2+ activation of contractility. We have also mapped the amino... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5417162 Wed, 16 Nov 2011 05:00:00 +0100 5417162 http://www.medworm.com/index.php?rid=5417163&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111230 Transcriptional regulation requires coordinated action of transcription factors, co-activator complexes and general transcription factors to access specific loci in the dense chromatin structure. Here we demonstrate that the transcriptional co-regulator Stromelysin-1 PDGF responsive element binding protein (SPBP) contains two independent chromatin binding domains, the SPBP(1551-1666) region and the C-terminal extended PHD (ePHD/ADD) domain. The region 1551-1666 is a novel core nucleosome interaction domain located adjacent to the AT-hook motif in the DNA binding domain. This novel nucleosome binding region is critically important for proper localisation of SPBP in the cell nucleus. The ePHD/ADD domain associates with nucleosomes in a histone tail dependent manner, and has significant impac... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5417163 Mon, 14 Nov 2011 05:00:00 +0100 5417163 http://www.medworm.com/index.php?rid=5404624&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111029 This study provides intriguing insights into the molecular details of LOX-1 recognition of OxLDL. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=5404624 Fri, 11 Nov 2011 05:00:00 +0100 5404624 http://www.medworm.com/index.php?rid=5396482&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111761 The retromer complex is a conserved endosomal protein sorting complex that sorts membrane proteins into nascent endosomal tubules. The recognition of membrane proteins is mediated by the cargo-selective retromer complex, a stable trimer of the VPS35, VPS29 and VPS26 proteins. We have recently reported that the cargo-selective retromer complex associates with the WASH complex, a multimeric protein complex that regulates tubule dynamics at endosomes. Here we show that the retromer-WASH complex interaction occurs through the long unstructured “tail” domain of the WASH complex-Fam21 protein binding to VPS35, an interaction that is necessary and sufficient to target the WASH complex to endosomes. The Fam21-tail also binds to FKBP15, a protein associated with ulcerative colitis, to... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5396482 Wed, 09 Nov 2011 05:00:00 +0100 5396482 http://www.medworm.com/index.php?rid=5396483&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110535 This study examines the role of QC-mediated pGlu formation on monocyte chemoattractant proteins (MCPs) in inflammation. We demonstrated 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 i... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5396483 Tue, 08 Nov 2011 05:00:00 +0100 5396483 http://www.medworm.com/index.php?rid=5374767&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111020 The PDGF family members are potent mitogens for cells of mesenchymal origin and serve as important regulators of cell migration, survival, apoptosis, and transformation. Tumor-derived PDGF ligands are thought to function in both autocrine and paracrine manners, activating receptors on tumor and surrounding stromal cells. PDGF-C and -D are secreted as latent dimers, unlike PDGF-A and -B. Cleavage of the CUB domain from the PDGF-C and -D dimers is required for their biological activity. At present, little is known about the proteolytic processing of PDGF-C, the rate-limiting step in the regulation of PDGF-C activity. Here we show that the breast carcinoma cell line, MCF7, engineered to overexpress PDGF-C, produces proteases capable of cleaving PDGF-C to its active form. Increased PDGF-C expr... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5374767 Fri, 28 Oct 2011 04:00:00 +0100 5374767 http://www.medworm.com/index.php?rid=5592682&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111358 In this study, we identified ubiquitin-specific protease USP4 as a novel deubiquitinase targeting TRAF2 and TRAF6 for deubiquitination. We found that USP4 specifically interacts with TRAF2 and TRAF6, but not TRAF3. Moreover, USP4 associates with TRAF6 both in vitro and in vivo independent of its deubiquitinase activity. The USP domain is responsible for USP4 to interact with TRAF6. Ectopic expression of USP4 inhibits the TRAF2 and TRAF6-stimulated NF-κB reporter gene and negatively regulates the TNFa-induced IkBa degradation and NF-kB activation. Knockdown of USP4 significantly increased TNFa-induced cytokine expression. Furthermore, we found that USP4 deubiquitinates both TRAF2 and TRAF6 in vivo and in vitro in a deubiquitinase activity-dependent manner. Importantly, our data showe... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592682 Thu, 27 Oct 2011 04:00:00 +0100 5592682 http://www.medworm.com/index.php?rid=5355101&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111358 In this study, we identified ubiquitin-specific protease USP4 as a novel deubiquitinase targeting TRAF2 and TRAF6 for deubiquitination. We found that USP4 specifically interacts with TRAF2 and TRAF6, but not TRAF3. Moreover, USP4 associates with TRAF6 both in vitro and in vivo independent of its deubiquitinase activity. The USP domain is responsible for USP4 to interact with TRAF6. Ectopic expression of USP4 inhibits the TRAF2 and TRAF6-stimulated NF-κB reporter gene and negatively regulates the TNFa-induced IkBa degradation and NF-kB activation. Knockdown of USP4 significantly increased TNFa-induced cytokine expression. Furthermore, we found that USP4 deubiquitinates both TRAF2 and TRAF6 in vivo and in vitro in a deubiquitinase activity-dependent manner. Importantly, our data showe... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5355101 Thu, 27 Oct 2011 04:00:00 +0100 5355101 http://www.medworm.com/index.php?rid=5592683&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111234 In conclusion, NTCP adopts a dimeric structure in which individual subunits are functional. Bile salt uptake is influenced by hetero-dimerization when this impairs NTCP plasma membrane trafficking. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592683 Wed, 26 Oct 2011 04:00:00 +0100 5592683 http://www.medworm.com/index.php?rid=5355103&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111252 Different types of nanoparticles (NPs) are currently under development for diagnostic and therapeutic applications in the biomedical field. Yet our knowledge about their possible effects and fate in living cells is still limited. Here we examined the cellular response of human and rat brain-derived endothelial cells to NPs of different size and structure: uncoated and oleic acid-coated iron oxide NPs (8-9 nm core), fluorescent 25 and 50 nm silica NPs, titanium dioxide NPs (21 nm mean core diameter) and PLGA-PEO polymeric NPs (150 nm). We evaluated their uptake by the cells, and their localization, generation of oxidative stress and DNA-damaging effects in exposed cells. We show that NPs are internalized by human brain-derived endothelial cells, however the extent of their intracellular upt... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5355103 Wed, 26 Oct 2011 04:00:00 +0100 5355103 http://www.medworm.com/index.php?rid=5355102&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111234 In conclusion, NTCP adopts a dimeric structure in which individual subunits are functional. Bile salt uptake is influenced by hetero-dimerization when this impairs NTCP plasma membrane trafficking. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=5355102 Wed, 26 Oct 2011 04:00:00 +0100 5355102 http://www.medworm.com/index.php?rid=5592685&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111604 Downregulation of Kv4.3 K+ channels commonly occurs in multiple diseases, but the understandings of the regulation of Kv4.3 K+ channels and the role of Kv4.3 K+ channels in pathological conditions are limited. HEK293T cells are derived from HEK293 cells which are transformed by expression of the large T antigen. Here, by comparing the common couple HEK293 and HEK293T cells, we find that HEK293T cells express more Kv4.3 K+ channels and transcription factor Sp1 than HEK293 cells. Inhibition of Sp1 with Sp1 decoy oligonucleotide reduces Kv4.3 K+ channel expression in HEK293T cells. Transfection of pN3-Sp1FL vector increases Sp1 protein expression and results in increased Kv4.3 K+ expression in HEK293 cells. Since the ultimate determinant of the phenotype difference between HEK293 and HEK293T ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592685 Tue, 25 Oct 2011 04:00:00 +0100 5592685 http://www.medworm.com/index.php?rid=5592684&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111454 The opportunistic fungus Candida albicans causes oral thrush and vaginal candidiasis, as well as candidemia in immunocompromised patients including those undergoing cancer chemotherapy, organ transplant and those with AIDS. We previously found that the antimicrobial peptides (AMPs) LL37 and hBD-3 inhibited C. albicans viability and its adhesion to plastic. For this study, the mechanism by which LL37 and hBD-3 reduced C. albicans adhesion was investigated. After AMP treatment, C. albicans adhesion to plastic was reduced by up to ~60% and was dose-dependent. Our previous study indicated that LL37 might interact with the cell-wall β-1,3-exoglucanase Xog1p, which is involved in cell-wall β-glucan metabolism, and consequently the binding of LL37 or hBD-3 to Xog1p might cause the d... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592684 Tue, 25 Oct 2011 04:00:00 +0100 5592684 http://www.medworm.com/index.php?rid=5355105&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111604 Downregulation of Kv4.3 K+ channels commonly occurs in multiple diseases, but the understandings of the regulation of Kv4.3 K+ channels and the role of Kv4.3 K+ channels in pathological conditions are limited. HEK293T cells are derived from HEK293 cells which are transformed by expression of the large T antigen. Here, by comparing the common couple HEK293 and HEK293T cells, we find that HEK293T cells express more Kv4.3 K+ channels and transcription factor Sp1 than HEK293 cells. Inhibition of Sp1 with Sp1 decoy oligonucleotide reduces Kv4.3 K+ channel expression in HEK293T cells. Transfection of pN3-Sp1FL vector increases Sp1 protein expression and results in increased Kv4.3 K+ expression in HEK293 cells. Since the ultimate determinant of the phenot... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5355105 Tue, 25 Oct 2011 04:00:00 +0100 5355105 http://www.medworm.com/index.php?rid=5355104&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111454 The opportunistic fungus Candida albicans causes oral thrush and vaginal candidiasis, as well as candidemia in immunocompromised patients including those undergoing cancer chemotherapy, organ transplant and those with AIDS. We previously found that the antimicrobial peptides (AMPs) LL37 and hBD-3 inhibited C. albicans viability and its adhesion to plastic. For this study, the mechanism by which LL37 and hBD-3 reduced C. albicans adhesion was investigated. After AMP treatment, C. albicans adhesion to plastic was reduced by up to ~60% and was dose-dependent. Our previous study indicated that LL37 might interact with the cell-wall β-1,3-exoglucanase Xog1p, which is involved in cell-wall β-glucan metabolism, and consequently the binding of LL37 or hBD-3 to Xog1p might cause the d... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5355104 Tue, 25 Oct 2011 04:00:00 +0100 5355104 http://www.medworm.com/index.php?rid=5343379&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111682 Tumor endothelial marker 5 (TEM5/GPR124) is an adhesion G protein-coupled receptor containing a cryptic RGD motif in its extracellular domain. TEM5 is expressed in endothelial cells and pericytes during angiogenesis. Here, we report that thrombin mediates shedding of an N-terminal TEM5 fragment of 60 kDa (N60) containing the RGD motif in an open conformation. Thrombin directly cleaved recombinant soluble TEM5 (rsTEM5) five and 34 residues downstream of the RGD motif resulting in formation of N60 and its C-terminal counterpart (C50). Interestingly, N60 derived from thrombin cleavage of rsTEM5 was covalently linked to C50 by disulfide bonds, whereas N60 shed from thrombin-treated cells was not associated with its membrane-bound C-terminal counterpart. Inhibition of the reducing function of c... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5343379 Thu, 20 Oct 2011 04:00:00 +0100 5343379 http://www.medworm.com/index.php?rid=5343378&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110951 Mena/VASP proteins are the homologue of Drosophila Enabled (Ena). In Drosophila, Ena is a substrate of the tyrosine kinase, Drosophila Abl (DAbl). However, the link between Abl and the Mena/VASP family is not fully understood in mammals. We previously reported that Abi-1 promotes phosphorylation of Mena and BCAP by bridging the interaction between c-Abl and the substrate. Here we identified VASP, another member of the Mena/VASP family, as an Abi-1-bridged substrate of Abl. VASP is phosphorylated by Abl when Abi-1 is coexpressed. We also found that VASP interacted with Abi-1 both in vitro and in vivo. VASP was tyrosine-phosphorylated in Bcr-Abl-positive leukemic cells in an Abi-1-dependent manner. Coexpression of c-Abl and Abi-1 or the phosphomimetic Y39D mutation in VASP resulted in less a... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5343378 Thu, 20 Oct 2011 04:00:00 +0100 5343378 http://www.medworm.com/index.php?rid=5592686&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111277 A yeast two-hybrid screen performed to identify binding partners of the calcium-sensing receptor (CaR) intracellular tail, identified the adaptor protein, 14-3-3theta, as a novel binding partner that bound to the proximal membrane region important for CaR expression and signaling. 14-3-3theta directly interacted with the CaR tail in pulldown studies and FLAG-tagged CaR co-immunoprecipitated with EGFP-tagged 14-3-3theta when co-expressed in HEK293 or COS-1 cells. The interaction between the CaR and 14-3-3theta did not require a putative binding site in the membrane proximal region of the CaR tail and was independent of PKC phosphorylation. Confocal microscopy demonstrated colocalization of the CaR and EGFP-14-3-3theta in the endoplasmic reticulum of HEK293 cells that stably expressed the Ca... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5592686 Wed, 19 Oct 2011 04:00:00 +0100 5592686 http://www.medworm.com/index.php?rid=5330238&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111277 A yeast two-hybrid screen performed to identify binding partners of the calcium-sensing receptor (CaR) intracellular tail, identified the adaptor protein, 14-3-3theta, as a novel binding partner that bound to the proximal membrane region important for CaR expression and signaling. 14-3-3theta directly interacted with the CaR tail in pulldown studies and FLAG-tagged CaR co-immunoprecipitated with EGFP-tagged 14-3-3theta when co-expressed in HEK293 or COS-1 cells. The interaction between the CaR and 14-3-3theta did not require a putative binding site in the membrane proximal region of the CaR tail and was independent of PKC phosphorylation. Confocal microscopy demonstrated colocalization of the CaR and EGFP-14-3-3theta in the endoplasmic reticulum of HEK293 cells that stably expressed the Ca... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5330238 Wed, 19 Oct 2011 04:00:00 +0100 5330238 http://www.medworm.com/index.php?rid=5330240&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111111 Rho GTPases regulate the assembly of cellular actin structures and are activated by guanine nucleotide exchange factors (GEFs) and rendered inactive by GTPase activating proteins (GAPs). Using the Rho GTPases Cdc42, Rac1, and RhoA, and the GTPase binding portions of the effector proteins p21 Activated Kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio, and Asef) and GAPs (p190, CdGAP, and PARG). The assay detected activities associated with purifie... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5330240 Mon, 17 Oct 2011 04:00:00 +0100 5330240 http://www.medworm.com/index.php?rid=5330239&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111671 Ubiquitin and ubiquitin-like modifiers (UBLs) are small proteins that covalently modify other proteins to alter their properties or behaviors. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 is the UBL most closely related to ubiquitin, and its best-studied role is the activation of Cullin-Ring ubiquitin Ligases (CRLs) by its conjugation to a conserved C-terminal lysine on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. Here we now report a new mode of NEDD8 conjugation (neddylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical neddylation is independent... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5330239 Mon, 17 Oct 2011 04:00:00 +0100 5330239 http://www.medworm.com/index.php?rid=5321231&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111457 In the present report we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its NH2-terminal encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, while its COOH-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes where it appears in the post-microsomal supernatant, however in skeletal muscle SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS 7C subfamily. Retinol is the substrate of SRP-35 since its transient over-expression leads to an incr... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5321231 Fri, 14 Oct 2011 04:00:00 +0100 5321231 http://www.medworm.com/index.php?rid=5321233&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111004 Mutations that perturb the function of photoreceptor cyclic nucleotide-gated (CNG) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the endoplasmic reticulum (ER) is known to cause ER stress and trigger the unfolded protein response (UPR), an evolutionarily conserved cellular program that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H an... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5321233 Thu, 13 Oct 2011 04:00:00 +0100 5321233 http://www.medworm.com/index.php?rid=5321232&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111566 The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the apical membrane of epithelial cells. Cell surface CFTR levels are regulated by endocytosis and recycling. A number of adaptor proteins including AP-2 (μ2) and Dab2 have been proposed to modulate CFTR internalization. We used small interfering RNA-mediated (siRNA) silencing of these adaptors to test their roles in the regulation of CFTR cell surface trafficking and stability in human airway epithelial cells. The results indicated that m2 and Dab2 performed partially overlapping, but divergent functions. While m2 depletion dramatically decreased CFTR endocytosis with little effect on CFTR protein half-life, Dab2 depletion increased CFTR half-life ~3-fold in addition to inhibiting CFTR endocytosis. Furth... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5321232 Thu, 13 Oct 2011 04:00:00 +0100 5321232 http://www.medworm.com/index.php?rid=5292616&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111377 The metabolism under hypoxia is significantly different from that under normoxia. It has been well elucidated that hypoxia inducible factor 1 (HIF1) plays a central role in regulating glucose metabolism under hypoxia. However, the role of HIF1 in lipid metabolism has not been well addressed yet. Here we demonstrated that HIF1 promotes low density lipoprotein (LDL) and very low density lipoprotein (VLDL) uptake through regulating very low density lipoprotein receptor gene (VLDLR) expression under hypoxia. Increased VLDLR mRNA and protein levels were observed under hypoxic or DFO treatment in MCF7, HepG2, and HeLa cells. By dual luciferase reporter assay and ChIP assay we confirmed a functional HRE which is localized at +405 in the exon1 of VLDLR gene. Knockdown of HIF1α and VL... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5292616 Tue, 04 Oct 2011 04:00:00 +0100 5292616 http://www.medworm.com/index.php?rid=5281215&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110558 Fast Ca2+-dependent inactivation (FCDI) is a mechanism that limits Ca2+ entry through Ca2+ channels, including Ca2+ release-activated Ca2+ (CRAC) channels. This phenomenon occurs when the Ca2+ concentration rises beyond a certain level in the vicinity of the intracellular mouth of the channel pore. In CRAC channels, several regions of the pore-forming protein Orai1, and STIM1, the SR/ER Ca2+ sensor that communicates the Ca2+ load of the intracellular stores to Orai1, have been shown to regulate fast Ca2+ dependent inactivation. Although significant advances in unravelling the mechanisms of CRAC channel gating have occurred, the mechanisms regulating fast Ca2+ dependent inactivation in this channel are not well understood... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5281215 Mon, 03 Oct 2011 04:00:00 +0100 5281215 http://www.medworm.com/index.php?rid=5281214&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111379 In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell-matrix interface. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=5281214 Mon, 03 Oct 2011 04:00:00 +0100 5281214 http://www.medworm.com/index.php?rid=5281216&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111385 In this study, we report the 1.9 Å X-ray structure of the Cwc2 core domain which is both necessary and sufficient for RNA binding. The Cwc2 core domain contains two subdomains, a CCCH-type zinc finger (ZnF) and an RNA recognition motif (RRM). Unexpectedly, the ZnF domain and the RRM motif form a single folding unit, glued together by extensive hydrophobic interactions and hydrogen bonds. Structure-guided mutational analysis revealed that the intervening loop (known as RB loop) between ZnF and RRM plays an essential role in RNA binding. In addition, a number of highly conserved, positively charged residues on the b-strands of RRM make important contribution to RNA binding. Intriguingly, these residues and a portion of the RB loop constitute an extended, basic surface strip that encir... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5281216 Fri, 30 Sep 2011 04:00:00 +0100 5281216 http://www.medworm.com/index.php?rid=5268373&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110749 Ab42 plays a central role in Alzheimer’s disease and is known to have a detrimental effect on neuronal cell function and survival when assembled into an oligomeric form. We show that administration of freshly prepared Ab42 oligomers to a neuroblastoma cell line results in a reduction in survival and that Ab42 enters the cells prior to cell death. Immunoconfocal and immunogold electron microscopy reveal the path of the Ab42 with time, through the endosomal system and shows that it accumulates in lysosomes. 24-hour incubation with Ab results in cells that have damaged lysosomes show signs of enzyme leakage, accumulate autophagic vacuoles and exhibit severely disrupted nuclei. Endogenous Ab is evident in the cells and our results suggest that the addition of Ab oligomers disrupts a cru... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5268373 Thu, 29 Sep 2011 04:00:00 +0100 5268373 http://www.medworm.com/index.php?rid=5268374&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110870 ERK1/2 MAP kinases are tightly regulated by the cellular microenvironment in which they operate. Mxi2 is a p38a splice isoform capable of binding to ERK1/2 and ensuing their translocation to the nucleus. Therein, Mxi2 sustains ERK1/2 phosphorylation levels and as a consequence ERK1/2 nuclear signals are enhanced. However, the molecular mechanisms underlying this process are still unclear. Herein, we show that Mxi2 prevents nuclear but not cytoplasmic phosphatases from binding to and dephosphorylating ERK1/2, disclosing an unprecedented mechanism for the spatial regulation of ERK1/2 activation. We also demonstrate that the kinetics of ERK1/2 extranuclear signals can be significantly altered by artificially tethering Mxi2 to the cytoplasm. In this case, Mxi2 abolishes ERK1/2 inactivation by ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5268374 Tue, 27 Sep 2011 04:00:00 +0100 5268374 http://www.medworm.com/index.php?rid=5257056&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111148 Hemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE, transferrin receptor 2, ferroportin, hepcidin, and hemojuvelin (HJV). Recent studies have established the expression of all the five genes in retina, indicating their importance in retinal iron homeostasis. We previously demonstrated that Hjv is expressed in retinal pigment epithelium (RPE), outer and inner nuclear layers, and ganglion cell layer. Here we report on the consequences of Hjv deletion on the retina in mice. Hjv-null mice at ³18 months of age had increased iron accumulation in retina with marked morphological damage compared to age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5257056 Mon, 26 Sep 2011 04:00:00 +0100 5257056 http://www.medworm.com/index.php?rid=5257055&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110833 Signal transduction networks in mammalian cells, comprised of a limited set of interacting biochemical pathways, are accessed by various growth factor and cytokine receptors to elicit distinct cell responses. This raises the question as to how specificity of the stimulus-response relationship is encoded at the molecular level. It has been proposed that specificity arises not simply from the activation of unique signalling pathways but also from quantitative differences in the activation and regulation of shared, receptor-proximal signalling proteins. To address such hypotheses, data sets with greater precision and coverage of experimental conditions will need to be acquired, and rigorous frameworks that codify and parameterise the inherently nonlinear relationships among signalling activit... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5257055 Mon, 26 Sep 2011 04:00:00 +0100 5257055 http://www.medworm.com/index.php?rid=5257054&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110533 Tissue factor pathway inhibitor-2 (TFPI-2) has recently been recognized as a new tumor suppressor gene. Low expression of this protein in several types of cancers allows for enhanced tumor growth, invasion and metastasis. To investigate the molecular mechanism responsible for the tumor-suppressor effects of TFPI-2, we performed yeast two-hybrid analysis and identified prosaposin (PSAP) as a TFPI-2-interacting partner. This interaction was confirmed by co-immunoprecipitation and immunofluorescence. The region of TFPI-2 that interacts with PSAP is located in the second Kunitz-type domain (KD2). Further study showed that PSAP does not affect TFPI-2’s function as serine proteinase inhibitor but that TFPI-2 could inhibit the invasion-promoting effects of PSAP in human HT1080 fibrosarcoma... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5257054 Mon, 26 Sep 2011 04:00:00 +0100 5257054 http://www.medworm.com/index.php?rid=5246005&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111424 Beclin 1, a subunit of class III phosphatidylinositol 3-kinase complex, is a tumor suppressor with a central role in endocytic trafficking, cytokinesis, and the cross-regulation between autophagy and apoptosis. Interestingly, not only reduced expression but also overexpression of Beclin 1 is correlated with cancer development and metastasis. Thus, it seems necessary for the cell to balance the protein levels of Beclin 1. Here, we describe a regulatory link between Beclin 1 and the ubiquitin ligase Nedd4. We establish Nedd4 as a novel binding partner of Beclin 1 and demonstrate that Nedd4 polyubiquitinates Beclin 1 with K11- and K63-linked chains. Importantly, Nedd4 expression controls the stability of Beclin 1, and depletion of the Beclin 1-interacting protein VPS34 causes Nedd4-mediated p... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5246005 Thu, 22 Sep 2011 04:00:00 +0100 5246005 http://www.medworm.com/index.php?rid=5233091&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111117 Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. Botulinum neurotoxin type A (/A) is a protease that binds to peripheral nerve terminals, gets endocytosed and causes prolonged blockade of transmitter release by cleaving synaptosomal-associated protein of Mr = 25k (SNAP-25). Retrograde transport of the toxin has been suggested, but not of the transient muscle relaxant, type E. Herein, dispersal of these proteases in compartmented cultures of rat sympathetic neurons was examined after focal application of /A or /E to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. /E yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5233091 Mon, 19 Sep 2011 04:00:00 +0100 5233091 http://www.medworm.com/index.php?rid=5233092&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110855 Heat shock protein 90 (Hsp90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and cochaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are getting increasingly noted to be involved in this process. Recent literatures have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. Here, we systematically investigated the impact of phosphorylation of a single Threonine 90 (T90) residue of Hsp90α (pT90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5233092 Thu, 15 Sep 2011 04:00:00 +0100 5233092 http://www.medworm.com/index.php?rid=5203886&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111243 Cell-to-cell fusion plays an important role in normal physiology and in different pathological conditions. Early fusion stages mediated by specialized proteins and yielding fusion pores are followed by a pore expansion stage that is dependent on cell metabolism and yet unidentified machinery. Because of a similarity of membrane bending in the fusion pore rim and in highly curved intracellular membrane compartments, here we explored whether changes in the activity of the proteins that generate these compartments affect cell fusion initiated by protein fusogens of influenza virus and baculovirus. We raised the intracellular concentration of curvature generating proteins in cells by either expressing or microinjecting the ENTH domain of Epsin or by expressing GRAF1 BAR domain or FCHo2 F-BAR d... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5203886 Wed, 07 Sep 2011 04:00:00 +0100 5203886 http://www.medworm.com/index.php?rid=5203887&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111147 The T-type calcium channel Cav3.2 is expressed in nociceptive and mechanosensitive sensory neurons. The mechanosensitive D-hair neurons, which innervate hair follicles, are characterized by a large-amplitude Cav3.2 T-current involved in the amplification of slow moving stimuli. The molecules and signaling pathways that regulate T-current expression in mechanoreceptors are unknown. We investigated in vitro the effects of neurotrophin 4, NT-4, on Cav3.2 T-current expression in D-hair neurons. Interruption of NT-4 supply with peripheral nerve axotomy induced a non-transcriptional decrease in the T-current amplitude of fluoro-gold labeled axotomized sensory neurons. The T-current amplitude was restored with NT-4 incubation. Deletion of NT-4 through genetic ablation resulted in a similar select... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5203887 Mon, 05 Sep 2011 04:00:00 +0100 5203887 http://www.medworm.com/index.php?rid=5190978&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111300 The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export recepor CRM1 binds to specific amino acid motifs termed nuclear export sequences (NESs). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These data allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified here reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor leptomycin B (LMB) on the localization of YFP- or GFP-tagged versions o... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5190978 Thu, 01 Sep 2011 23:00:00 +0100 5190978 http://www.medworm.com/index.php?rid=5190979&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110739 Upon environmental insults, stress granules (SGs) aid cell survival by serving as sites of translational silencing. RNA helicase DDX3 was reported to associate with SG. However, its role in SG physiology remains undefined. We have previously demonstrated that DDX3 acts as an eIF4E inhibitory protein to suppress translation. Here, we indentified the SG marker poly(A)-binding protein 1 (PABP1) as another direct interaction partner of DDX3. We established various stimuli as novel stressors that direct DDX3 with eIF4E and PABP1 into SGs, but not to processing bodies. Interestingly, downregulation of DDX3 interfered with SG assembly, led to nuclear accumulation of PABP1 and reduced cell viability following stress. Conversely, supplementation with a shRNA-resistant DDX3 restored SG formation, th... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5190979 Wed, 31 Aug 2011 23:00:00 +0100 5190979 http://www.medworm.com/index.php?rid=5168720&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110910 The critical involvement of transforming growth factor-β1 (TGF-β1) in diabetic nephropathy (DN) is well established, however the role of connective tissue growth factor (CTGF) in regulating the complex interplay of TGF-β1 signalling networks is poorly understood. The purpose of this study was to investigate co-operative signaling between CTGF and TGF-β1 and its physiological significance. CTGF was determined to bind directly to TβRIII and antagonize TGF-β1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-β1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-β1 signalling and expression of a unique profile of differentially regulated genes wa... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5168720 Thu, 25 Aug 2011 23:00:00 +0100 5168720 http://www.medworm.com/index.php?rid=5168719&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110530 Invasive migration of carcinoma cells is a pre-requisite for the metastatic dissemination of solid tumors. Numerous mechanisms control the ability of cancer cells to acquire a motile and invasive phenotype and subsequently degrade and invade the basement membrane. Several genes that are upregulated in breast carcinoma are responsible for mediating the metastatic cascade. Recent studies have revealed that the nuclear factor of activated T cells (NFAT) is a transcription factor that is highly expressed in aggressive breast cancer cells and tissues, and mediates invasion through transcriptional induction of pro-invasion and migration genes. Here we demonstrate that NFAT promotes breast carcinoma invasion through induction of glypican-6 (GPC6), a cell surface glycoprotein. NFAT transcriptional... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5168719 Thu, 25 Aug 2011 23:00:00 +0100 5168719 http://www.medworm.com/index.php?rid=5168721&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111281 Xenotoxic damage in inflammatory diseases, including inflammatory bowel disease (IBD), is importantly compounded by reduced activity of the xenobiotic transporter ABCG2 during the inflammatory state. An association between activation of the unfolded protein response pathway and inflammation prompted us to investigate the possibility that reduced ABCG2 activity is causally linked to this response. To this end we correlated expression of ABCG2 and the unfolded protein response marker GRP78 in colon biopsies from healthy individuals (n=9), and patients with inactive (n=67), or active (n=55) IBD, ischemic colitis (n=10), or infectious colitis (n=14). In addition, tissue-specimens throughout the small bowel from healthy individuals (n=27), and from patients with inactive (n=9) or active (n=25) ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5168721 Wed, 24 Aug 2011 23:00:00 +0100 5168721 http://www.medworm.com/index.php?rid=5155304&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110438 In this study, we generated and evaluated a panel of neutralizing nanobodies targeting the leptin receptor (LR). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2, Ig-like and FNIII domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic neuropeptide Y expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All these characteristics resemble the phenotyp... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5155304 Thu, 18 Aug 2011 23:00:00 +0100 5155304 http://www.medworm.com/index.php?rid=5137670&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110676 In erythrocytes, the 80kDa isoform of protein 4.1R (4.1R80) binds to the cytoplasmic tail of transmembrane proteins band 3 and glycophorin C (GPC), and to the membrane-associated protein p55 through the N-, a-, and C-lobes of its N-terminal 30kDa FERM domain (R30), respectively. We have previously shown that R30 binds to calmodulin (CaM) in a Ca2+- independent manner, the dissociation constant (K(D)) for R30-CaM binding being very similar (in the submicromolar range) in the presence or absence of Ca2+. In the present study, we investigated the consequences of CaM binding on R30 structural stability using resonant mirror detection and Fourier transform-infrared (FT-IR) spectroscopy. After a 30 minute incubation above 40°C, R30 could no longer bind to band 3 nor to GPC. ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5137670 Tue, 16 Aug 2011 23:00:00 +0100 5137670 http://www.medworm.com/index.php?rid=5137672&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110424 Homo-dimerization of the membrane-bound collagenase MT1-MMP is crucial for its collagenolytic activity. However, it has not been clear if this dimerization is regulated during cellular invasion into 3D collagen matrices. To address this question, we established a fluorescence resonance energy transfer system to detect MT1-MMP dimerization and analysed the process in cells invading through 3D collagen. Our data indicates that dimerization occurrs dynamically and constantly at the leading edge of migrating cells but not the trailing edge. We found that polarised dimerization was not due to ECM attachment, but was rather controlled by reorganization of the actin cytoskeleton by the small GTPases, Cdc42 and Rac1. Our data indicate that cell surface collagenolytic activity is regulated co-ordin... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5137672 Mon, 15 Aug 2011 23:00:00 +0100 5137672 http://www.medworm.com/index.php?rid=5137671&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102148 In this study we investigated mechanisms whereby adiponectin dampens leptin signaling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad-/-) and wild-type mice with leptin and/or carbon tetrachloride (CCl4), or saline. We analyzed Jak2 and Ob-Rb phosphorylation, and PTP1B expression and activity. We also explored potential mechanisms through which adiponectin regulates SOCS-3/Ob-Rb association. Adiponectin inhibited leptin-stimulated Jak2 activation and Ob-Rb phosphorylation in HSCs, while both were increased in Ad-/- mice. Adiponectin stimulated PTP1B expression and activity, in vitro, while PTP1B expression was lower in Ad-/-mice than in wild-type mice. Adiponectin also... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5137671 Mon, 15 Aug 2011 23:00:00 +0100 5137671 http://www.medworm.com/index.php?rid=5116895&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110167 Heparanase is involved in the cleavage of the heparan sulfate (HS) chain of HS proteoglycans (HSPGs) and hence participates in remodeling of the extracellular matrix (ECM) and basement membrane (BM). Here we showed nerve growth factor (NGF) promoted nuclear enrichment of early growth response 1 (EGR1), a transcription factor for heparanase and markedly induced heparanase expression in rat adrenal pheochromocytoma (PC12) cells. K252a, an antagonist of NGF receptor TrkA (tyrosine kinase receptor A), decreased heparanase protein expression induced by NGF in PC12 cells. Suramin, a heparanase inhibitor, decreased heparanase in PC12 cells and blocked NGF-induced PC12 neuritogenesis. Stable overexpression of heparanase activated p38 by phosphorylation and enhanced the neurite outgrowth induced by... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5116895 Wed, 10 Aug 2011 23:00:00 +0100 5116895 http://www.medworm.com/index.php?rid=5116897&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110641 Pyrophosphate (PPi) is a critical element of cellular metabolism as both an energy donor and as an allosteric regulator of several metabolic pathways. The apicomplexan parasite, Toxoplasma gondii, uses PPi in place of ATP as an energy donor in at least two reactions: the glycolytic PPi-dependent phosphofructokinase (PFK), and the proton translocating vacuolar pyrophosphatase (V-H+-PPase). In the present work, we report the cloning, expression, and characterization of a cytosolic pyrophosphatase from T. gondii (TgPPase). Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a family I soluble PPase. Overexpression of the enzyme in extracellular tachyzoites led to a 6-fold decrease in the cytosolic concentration of PPi relative to RH wild type tachyzo... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5116897 Tue, 09 Aug 2011 23:00:00 +0100 5116897 http://www.medworm.com/index.php?rid=5116896&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110876 The action of multidrug efflux pumps in multidrug resistance (MDR) acquisition has been proposed to partially depend on the transport of physiological substrates which may indirectly affect drug partition and transport across cell membranes. In this work, PDR18 gene (ORF YNR070w), encoding a putative pleiotropic drug resistance (PDR) transporter of the ATP-binding cassette superfamily, was found to mediate plasma membrane sterol incorporation in yeast. Pdr18 physiological role is demonstrated to affect plasma membrane potential and is proposed to underlie its action as a MDR determinant, conferring resistance to the herbicide 2,4-D. The action of Pdr18 in yeast tolerance to 2,4-D, which was found to contribute to reduce [14C]-2,4-D intracellular accumulation, may be indirect, given the ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5116896 Tue, 09 Aug 2011 23:00:00 +0100 5116896 http://www.medworm.com/index.php?rid=5102652&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101894 In this study we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3 binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser555 in vitro. Mutation of this residue to Ala abrogated 14-3-3 binding to ULK1 and in vivo phosphorylation of ULK1 was blocked by a dominant negative AMPK mutant. We next identified a high stringency Akt site in ULK1 at Ser774 and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase activation loop of ULK1 contains a consensus phosphorylation site at Thr180 that is required for ULK1 autophosphorylation activity. Collectively, our data suggest that ULK1 may act a... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5102652 Thu, 04 Aug 2011 23:00:00 +0100 5102652 http://www.medworm.com/index.php?rid=5068466&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110610 Geranylgeranoic acid (GGA) is a natural polyprenoic acid derivatives of which have been shown to prevent second primary hepatoma. GGA induces mitochondriamediated programmed cell death (PCD), which may be relevant to cancer prevention. To gain further insights into GGA-induced PCD, autophagy processes were examined in human hepatoma-derived HuH-7 cells. Treatment of HuH-7/GFP-LC3 cells with GGA induced green fluorescent puncta in the cytoplasm within 30 min and their massive accumulation at 24 h. After 15 min of GGA treatment, a burst of mitochondrial superoxide production occurred and LC3β-I was appreciably converted to LC3β-II. GGA-induced early stages of autophagy were unequivocally confirmed by electron-microscopic observation of early/initial autophagic vacuoles... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5068466 Mon, 25 Jul 2011 23:00:00 +0100 5068466 http://www.medworm.com/index.php?rid=5048039&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110584 The alpha2beta1 integrin antagonist rhodocetin from Calloselasma rhodostoma is a heterotetrameric C-type lectin-related protein (CLRP) consisting of four distinct chains, alpha, beta, gamma, and delta. Via their characteristic domain-swapping loops, the individual chains form two subunits, alpha beta and gamma delta. To distinguish the four chains which share similar molecular masses and high sequence homologies, we generated 11 monoclonal antibodies (mAbs) with different epitope specificities. Four groups of distinct mAbs were generated: the first targeted the rhodocetin beta chain, the second group bound to the alpha beta subunit mostly in a conformation-dependent manner, and the third group recognized the gamma delta subunit only when separated from the alpha beta subunit, while a fourt... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5048039 Wed, 20 Jul 2011 23:00:00 +0100 5048039 http://www.medworm.com/index.php?rid=5048038&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110240 Striatal-enriched phosphatase (STEP) is a non-receptor tyrosine phosphatase that is specifically expressed in neurons of the central nervous system. STEP regulates the activity of several effector molecules involved in synaptic plasticity and neuronal cell survival, including mitogen-activated protein kinases (MAPKs), Src family kinases and N-methyl-D-aspartic acid (NMDA) receptors. The critical role of STEP in regulating these effectors requires that its activity be tightly regulated. Previous studies demonstrated that the activity of STEP is regulated through reversible phosphorylation of a serine residue within the kinase interacting motif (KIM), by cAMP-dependent protein kinase A (PKA). Here we show that STEP is endogenously phosphorylated at two additional sites located within the kin... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5048038 Wed, 20 Jul 2011 23:00:00 +0100 5048038 http://www.medworm.com/index.php?rid=5048037&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111036 Mitochondria transport and utilize iron for the synthesis of heme and Fe-S clusters. Although many proteins are known to be involved in these processes, additional proteins are likely to participate. To test this hypothesis, we used a genetic screen looking for yeast mutants that are synthetically lethal with the mitochondrial iron carriers, Mrs3 and Mrs4. Several genes were identified including an isolate mutated for Yfh1, the yeast frataxin homolog. All such triple mutants were complemented by increased expression of Rim2, another mitochondrial carrier protein. Rim2 overexpression was able to enhance heme and Fe-S cluster synthesis in wild-type or ∆mrs3/∆mrs4 backgrounds. Conversely Rim2 depletion impaired heme and Fe-S cluster synthesis in wild-type or ∆mrs3/&#x2206... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5048037 Wed, 20 Jul 2011 23:00:00 +0100 5048037 http://www.medworm.com/index.php?rid=5048036&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110318 In this study, we performed a mutagenic analysis of Cfr to show that two distinct regions contribute to its distribution and stability. First, the C-terminal region retains Cfr in the Golgi apparatus. Second, the Cfr repeats in the extracellular juxtamembrane region destabilizes Cfr passed through the Golgi apparatus. This destabilization does not depend on the cleavage and secretion of the extracellular domain of Cfr. Furthermore, we found that Cfr with a glycosylphosphatidylinositol (GPI)-anchor was predominantly expressed on the cell surface in Ba/F3 cells and affected Fgf18 signaling similarly to the full-length Cfr, indicating that the interaction of Cfr with Fgfs on the cell surface is important for its function in Fgf signaling. These results suggest that the expression of Cfr in th... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5048036 Wed, 20 Jul 2011 23:00:00 +0100 5048036 http://www.medworm.com/index.php?rid=5048042&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110173 PI(3,4,5)P3 and PI(3,4)P2 are major signalling molecules in mammalian cell biology. PI(3,4)P2 can be produced by PI 3-kinases but also by PI 5-phosphatases including SHIP2. Proteomic studies in human cells revealed that SHIP2 can be phosphorylated at more than 20 sites but their individual function is unknown. In a model of PTEN-null astrocytoma cells, lowering SHIP2 expression leads to increased PI(3,4,5)P3 levels and Akt phosphorylation. Mass spec analysis identified SHIP2 phosphosites on S132, T1254 and S1258; phosphotyrosine containing sites were undetectable. By immunostaining, total SHIP2 concentrated in the perinuclear area and in the nucleus, while phospho SHIP2 S132 was in the cytoplasm, the nucleus and nuclear speckles depending on the cell cycle. SHIP2 phosphorylated on S132 dem... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5048042 Mon, 18 Jul 2011 23:00:00 +0100 5048042 http://www.medworm.com/index.php?rid=5048041&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110392 SVCT2 is the major transporter mediating vitamin C uptake in most organs. Its expression is driven by two promoters (CpG-poor exon 1a promoter and CpG-rich exon 1b promoter). In this work we mapped discrete elements within the proximal CpG-poor promoter responsible for the exon 1a transcription. We identified two E boxes for USF binding and one Y box for NF-Y binding. We further show that NF-Y and USF bind to the exon 1a promoter in a cooperative manner, amplifying the binding of each to the promoter, and is absolutely required for the full activity of the exon 1a promoter. The analysis of the CpG site located at the upstream USF binding site in the promoter showed a strong correlation between expression and demethylation. It was also shown that the exon 1a transcription was induced in cel... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5048041 Mon, 18 Jul 2011 23:00:00 +0100 5048041 http://www.medworm.com/index.php?rid=5048040&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111006 In this study, we demonstrated that these miR-138 induced changes were accompanied by marked reduction in E-cadherin (E-cad) expression and enhanced Vimentin (Vim) expression, characteristics of epithelial-to-mesenchymal transition (EMT). Based on a combined experimental and bioinformatics analysis, we identified a number of miR-138 target genes that are associated with EMT, including Vim, Zinc finger E-box-binding homeobox 2 (ZEB2), and Enhancer of Zeste homolog 2 (EZH2). Direct targeting of miR-138 to specific sequences located in the mRNAs of Vim, ZEB2 and EZH2 genes was confirmed using luciferase reporter gene assays. Our functional analyses (knock-in and knockdown) demonstrated that miR-138 regulates the EMT via 3 distinct pathways: 1) direct targeting Vim mRNA and controlling the exp... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5048040 Mon, 18 Jul 2011 23:00:00 +0100 5048040 http://www.medworm.com/index.php?rid=5048043&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110953 The XY-linker region of somatic cell PLC-b, -g, -d and -e isoforms confers potent catalytic inhibition suggesting a common auto-regulatory role. Surprisingly, the sperm PLCz XY-linker does not mediate auto-inhibition. Unlike for somatic PLCs, absence of the PLCz XY-linker significantly diminishes both in vitro PIP2 hydrolysis and in vivo Ca2+ oscillation-inducing activity, revealing evidence for a novel PLCz enzymatic mechanism. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=5048043 Sun, 17 Jul 2011 23:00:00 +0100 5048043 http://www.medworm.com/index.php?rid=5035323&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20111010 EHDs, EH domain containing proteins, participate in different stages of endocytosis. EHD2 is a plasma membrane associated, EHD which regulates trafficking from the plasma membrane [1]and recycling [2]. EHD2 has a role in nucleotide dependent membrane remodeling and its ATP binding domain is involved in dimerization, which creates a membrane binding region. Nucleotide binding is important for association of EHD2 with the plasma membrane, since a nucleotide free mutant (EHD2 T72A) failed to do so [3]. To elucidate the possible function of EHD2 during endocytic trafficking we attempted to unravel proteins that interact with EHD2, using the yeast two-hybrid system. A novel interaction was found between EHD2 and Nek3, a serine/threonine kinase. EHD2 was also found in association with Vav1, a... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5035323 Thu, 14 Jul 2011 23:00:00 +0100 5035323 http://www.medworm.com/index.php?rid=5006152&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110803 Our recent studies have been aimed at understanding the mechanisms regulating apical protein sorting in polarized epithelial cells. In particular, we have been investigating how lipid rafts serve to sort apical proteins in the biosynthetic pathway. The recent findings that lipid domains are too small or transient to host apically-destined cargo have led to newer versions of the hypothesis that invoke proteins required for lipid domain coalescence and stabilization. Myelin and lymphocyte (MAL) and its highly conserved family member, MAL2, have emerged as possible regulators of this process in the direct and indirect apical trafficking pathways, respectively. To test this possibility, we took a biochemical approach. We determined that MAL, but not MAL2, self-associates, forms higher order, c... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5006152 Wed, 06 Jul 2011 23:00:00 +0100 5006152 http://www.medworm.com/index.php?rid=5006151&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110728 The PEA3 group members (ERM, ER81 and PEA3) of the Ets transcription factor family are involved in migration and dissemination processes during organogenesis and cancer development. In the present study, we report that the heterogeneous nuclear ribonucleoprotein (hnRNP)-like protein CoAA interacts with the PEA3 group members and modulates their transcriptional activity. We also demonstrate that the CoAA YQ domain, containing tyrosine/glutamine-rich hexapeptide repeats, is necessary for the interaction, whereas the two N-terminal RNA recognition motifs (RRM) of CoAA are required to enhance transcriptional activity. Finally, we show that CoAA is involved in the migration-enhancing action of PEA3 on MCF7 human cancer cells, suggesting that CoAA might be an important regulator of PEA3 group me... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5006151 Wed, 06 Jul 2011 23:00:00 +0100 5006151 http://www.medworm.com/index.php?rid=5006153&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110905 Glutamate transport is coupled to the co-transport of 3 Na+ and 1 H+ followed by the counter-transport of 1 K+. In addition, glutamate and Na+ binding to glutamate transporters generates an uncoupled anion conductance. The human glial glutamate transporter, EAAT1 also allows significant passive and active water transport, which suggests that water permeation through glutamate transporters may play an important role in glial cell homeostasis. Urea also permeates EAAT1 and has been used to characterize the permeation properties of the transporter. We have previously identified a series of mutations that differentially affect either the glutamate transport process or the substrate-activated channel function of EAAT1. The water and urea permeation properties of wild... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5006153 Tue, 05 Jul 2011 23:00:00 +0100 5006153 http://www.medworm.com/index.php?rid=5006154&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110582 In this study, we elucidated the function of l(2)01810 during megamitochondrial formation. The overexpression of l(2)01810 and the inhibition of glutamine synthesis showed that l(2)01810 is involved in the accumulation of glutamate. l(2)01810 was predicted to contain transmembrane domains and was found to be localized to the plasma membrane. By using 14C-labeled glutamate, l(2)01810 was confirmed to uptake glutamate into Drosophila cells with high affinity (Km = 69.4 μM). l(2)01810 uptakes glutamate in a Na+-independent manner. Interestingly, however, this uptake was not inhibited by cystine that is a competitive inhibitor of Na+-independent glutamate transporter, but by aspartate. A signal peptide consisting of 34 amino acid residues targeting to endoplasmic reticulum... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5006154 Mon, 04 Jul 2011 23:00:00 +0100 5006154 http://www.medworm.com/index.php?rid=4997064&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110767 Periplasmic peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the cis/trans isomerization of peptidyl-prolyl bonds, which is a rate-limiting step during protein folding. We demonstrate that the surA, ppiA, ppiD fkpA and fklB alleles each encode a periplasmic PPIase in the bacterial pathogen Yersinia pseudotuberculosis. Of these, four were purified to homogeneity. Purified SurA, FkpA and FklB, but not PpiD, displayed detectable PPIase activity in vitro. Significantly, only Y. pseudotuberculosis lacking surA caused drastic alterations to the outer membrane protein profile and fatty acid composition. They also exhibited aberrant cellular morphology, leaking LPS into the extracellular environment. The SurA PPIase is therefore most critical for maintaining Y. pseudotuberculosis envelope i... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4997064 Sun, 03 Jul 2011 23:00:00 +0100 4997064 http://www.medworm.com/index.php?rid=4997063&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110544 The hOCTN1 transporter over-expressed in E. coli and purified by Ni-chelating chromatography has been reconstituted in liposomes by detergent removal with a batch-wise procedure. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the time of incubation with Amberlite XAD-4 resin. Time dependent [14C]tetraethylammonium, [3H]carnitine or [3H]ergothioneine uptake was measured in proteoliposomes with activities ratio of 8.0 : 1.3 : 1.0, respectively. Optimal activity was found at pH 8.0. The transport depended on intraliposomal ATP. [14C]tetraethylammonium transport was inhibited by several compounds; the most effective were acetylcholine and γ-butyrobetaine, followed by acetylcarnitine and tetramethylammonium. Reagents such ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4997063 Sun, 03 Jul 2011 23:00:00 +0100 4997063 http://www.medworm.com/index.php?rid=4997062&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110759 We report here that targeted inactivation of Slc7a8 leads to increased urinary loss of small neutral amino acids. Development and growth of Slc7a8-/- mice appears normal suggesting functional compensation of neutral amino acid transport by alternative transporters in kidney, intestine, and placenta. Movement coordination is slightly impaired in mutant mice, although cerebellar development and structure remained inconspicuous. Circulating thyroid hormones, thyrotropin, and thyroid hormone-responsive genes remained unchanged in Slc7a8-/- mice, possibly because of functional compensation by the thyroid hormone transporter Mct8, which is co-expressed in many cell types. The reason for the mild neurological phenotype remains unresolved. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=4997062 Sun, 03 Jul 2011 23:00:00 +0100 4997062 http://www.medworm.com/index.php?rid=4974496&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101475 The factors that influence preadipocyte determination remain poorly understood. Here, we report that CREBL2, a novel bZIP_1 protein, is up-regulated during MDI-induced preadipocyte differentiation. During both over-expression and under physiological conditions, CREBL2 interacted and was entirely co-localized with CREB. Over-expression of CREBL2 was sufficient to promote adipogenesis via up-regulating the expression of PPARγ and C/EBPα and accelerate lipogenesis accompanied with increased GLUT1 and GLUT4. CREBL2 knockdown restrained adipogenic conversion and lipogenesis. Additionally, depletion of CREB could completely block the effects of over-expressed CREBL2 while an increased in CREB couldn’t drive adipogenesis in the absence of CREBL2 indicating that the roles of C... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4974496 Mon, 27 Jun 2011 23:00:00 +0100 4974496 http://www.medworm.com/index.php?rid=4974495&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110649 We report that the C-terminus contains two tandem phosphorylated binding sites (Ser274 and Ser299) for 14-3-3. The C-terminus also contains PEST sequences which are shielded by 14-3-3 binding. Like many proteins containing PEST sequences, the levels of RdgBβ are regulated by proteolysis. RdgBb is degraded with a half-life of 4hrs following ubiquitination via the proteasome. A mutant RdgBβ which is unable to bind 14-3-3 is degraded even faster with a half-life of 2hrs. In vitro, RdgBβ is 100-fold less active than PITPα for PI transfer and RdgBβ proteins (wild-type and a mutant that cannot bind 14-3-3) either expressed in COS-7 cells or endogenous proteins from heart cytosol do not exhibit transfer activity. When cells are treated with PMA, the PITP domain ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4974495 Mon, 27 Jun 2011 23:00:00 +0100 4974495 http://www.medworm.com/index.php?rid=4974498&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102155 Gel-based analysis of thylakoid membrane protein complexes represents a valuable tool to monitor the dynamics of the photosynthetic machinery. The native polyacrylamide gel electrophoresis (native-PAGE) preserves the components and often also the conformation of the protein complexes, thus enabling the analysis of their subunit composition. Nevertheless, the literature and practical experimentation in the field sometimes raise confusion due to a great variety of native-PAGE and thylakoid solubilization systems. Here, we describe optimized methods for separation of higher plant thylakoid membrane protein complexes by native-PAGE addressing particularly (i) the use of detergent and (ii) solubilization buffer as well as (iii) the gel electrophoresis method. Special attention is paid on separa... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4974498 Sun, 26 Jun 2011 23:00:00 +0100 4974498 http://www.medworm.com/index.php?rid=4974497&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110799 We report that Dock is required for effective IR dephosphorylation and inactivation by PTP61F in vitro and in vivo. Furthermore, we demonstrate that Nck interacts with PTP1B and that the Nck/PTP1B complex inducibly associates with the IR for the attenuation of IR activation in mammalian cells. Our studies reveal for the first time that the adaptor protein Dock/Nck attenuates insulin signaling by recruiting PTP61F/PTP1B to its substrate the IR. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=4974497 Sun, 26 Jun 2011 23:00:00 +0100 4974497 http://www.medworm.com/index.php?rid=4967362&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101700 It is clear that RhoA activates the diaphanous-related formin (DRF), mDia2, by disrupting the molecular interaction between the diaphanous autoregulatory domain (DAD) and the diaphanous inhibitory domain (DID). Previous studies indicate that a basic motif within the DAD contributes to mDia2 auto-inhibition, and data presented herein suggest these residues likely bind a conserved acidic region within the DID. Furthermore, we demonstrate that mDia2 is phosphorylated by Rho-kinase (ROCK) at two conserved residues (T1061 and S1070) just C-terminal to the DAD basic region. Phosphomimetic mutations to these residues in the context of the full-length molecule enhanced mDia2 activity as measured by increased actin polymerization, SRF-dependent smooth muscle-specific gene transcription, and nuclear... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4967362 Thu, 23 Jun 2011 23:00:00 +0100 4967362 http://www.medworm.com/index.php?rid=5048044&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110807 SLC41A1 (solute carrier family 41, member 1) is a recently described vertebrate member of the MgtE family of Mg2+ transporters. Although MgtE transporters are found in both prokaryotic and eukaryotic organisms, and are highly conserved, little is known about the regulation of their Mg2+ transport function. Here, we show that endogenous SLC41A1 transporter expression is post-transcriptionally regulated by extracellular Mg2+ in TRPM7-deficient cells, suggesting that SLC41A1 transporters underlie a novel plasma membrane Mg2+ transport function. Consistent with this conclusion, structure/function analyses of heterologous SLC41A1 transporter expression demonstrate that SLC41A1 transporters exhibit the same plasma membrane orientation as homologous bacterial MgtE proteins, are capable of complem... BJ Cell journals http://www.medworm.com/rss/comments.php?id=5048044 Tue, 21 Jun 2011 23:00:00 +0100 5048044 http://www.medworm.com/index.php?rid=4959164&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110166 “Disrupted in renal carcinoma 2” (DIRC2) has been initially identified as a breakpoint spanning gene in a chromosomal translocation putatively associated with the development of renal cancer. The DIRC2 protein belongs to the major facilitator superfamily (MFS) and has been previously detected by organellar proteomics as a tentative constituent of lysosomal membranes. In the present study, lysosomal residence of overexpressed as well as endogenous DIRC2 was shown by several approaches. DIRC2 is proteolytically processed into a N-glycosylated N-terminal and a non-glycosylated C-terminal fragment, respectively. Proteolytic cleavage occurs in lysosomal compartments and critically depends on the activity of cathepsin L which was found to be indispensable for this process in murine... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4959164 Tue, 21 Jun 2011 23:00:00 +0100 4959164 http://www.medworm.com/index.php?rid=4959163&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110807 SLC41A1 (solute carrier family 41, member 1) is a recently described vertebrate member of the MgtE family of Mg2+ transporters. Although MgtE transporters are found in both prokaryotic and eukaryotic organisms, and are highly conserved, little is known about the regulation of their Mg2+ transport function. Here, we show that endogenous SLC41A1 transporter expression is post-transcriptionally regulated by extracellular Mg2+ in TRPM7-deficient cells, suggesting that SLC41A1 transporters underlie a novel plasma membrane Mg2+ transport function. Consistent with this conclusion, structure/function analyses of heterologous SLC41A1 transporter expression demonstrate that SLC41A1 transporters exhibit the same plasma membrane orientation as homologous bacterial MgtE prot... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4959163 Tue, 21 Jun 2011 23:00:00 +0100 4959163 http://www.medworm.com/index.php?rid=4959162&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110274 Lung cell migration is a crucial step for re-epithelialization that in turn is essential for remodeling and repair after lung injury. We hypothesize that secreted autotaxin (ATX), which exhibits lysophospholipase D (lysoPLD) activity, stimulates lung epithelial cell migration through lysophosphatidic acid (LPA) generation-dependent and -independent pathways. Release of endogenous ATX protein and activity was detected in lung epithelial cell culture medium. ATX with V5 tag (ATX-V5) overexpressed conditional medium had higher LPA levels compared to control medium and stimulated cell migration through Gαi-coupled LPA receptors, cytoskeleton rearrangement, phosphorylation of PKCδ and cortactin at the leading edge of migrating cells. Inhibition of PKCδ attenuated ATX-V5 ove... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4959162 Tue, 21 Jun 2011 23:00:00 +0100 4959162 http://www.medworm.com/index.php?rid=4951171&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110577 TIR domain containing proteins play a crucial role in innate immunity in eukaryotes. Brucella is a highly infectious intracellular bacterium that encodes a TIR domain protein (TcpB) to subvert host innate immune responses to establish a beneficial niche for pathogenesis. TcpB inhibits NF-κB activation and pro-inflammatory cytokine secretions mediated by Toll like receptor (TLR) 2 & 4. Here, we demonstrate that TcpB modulates microtubule dynamics by acting as a stabilization factor. TcpB increased the rate of nucleation as well as the polymerization phases of microtubule formation similar to paclitaxel. TcpB could efficiently inhibit nocodazole or cold induced microtubule disassembly. Microtubule stabilization by TcpB is attributed to the BB-loop region of the TIR domain and a point ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4951171 Sun, 19 Jun 2011 23:00:00 +0100 4951171 http://www.medworm.com/index.php?rid=4931917&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110218 In this report, we used immunofluorescence microscopy, transmission and scanning electron microscopy to explore the underlying mechanisms. Our findings indicated that both C. neoformans and gp41-I90 up-regulated ICAM-1 on the HBMEC and elicited membrane ruffling on the surface of HBMEC. HIV-1 gp41 ectodomain could also induce CD44 and β-actin redistribution to the membrane lipid rafts, but it could not enhance PKCα phosphorylation like C. neoformans. Instead, the gp41-I90 was able to induce syncytium formation on HBMEC. Our results suggested that the HIV-1 gp41 enhanced C. neoformans binding to HBMEC via gp41 core domain-induced membrane activities, revealing a potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients. (Sourc... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4931917 Sun, 12 Jun 2011 23:00:00 +0100 4931917 http://www.medworm.com/index.php?rid=4919880&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110694 Heparan sulfate has been shown to be an important mediator of Plasmodium sporozoite homing and invasion of the liver, but the role of this glycosaminoglycan in mosquito vector host-sporozoite interactions is unknown. We have biochemically characterized the function of Anopheles gambiae peptide-O-xylosyltransferase (AgOXT1) and confirmed that AgOXT1 can modify peptides representing model heparan and chondroitin sulfate proteoglycans in vitro. Moreover, we also demonstrated that the mosquito salivary gland basal lamina proteoglycans are modified by heparan sulfate. We used RNAi-mediated knockdown of heparan sulfate biosynthesis in An. gambiae salivary glands to determine if Plasmodium falciparum sporozoites that are released from mosquito midgut oocysts use salivary gland heparan sulfate as ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4919880 Thu, 09 Jun 2011 23:00:00 +0100 4919880 http://www.medworm.com/index.php?rid=4919881&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101316 Tropomyosin (Tm) is an evolutionarily conserved a-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilising actin-filaments and regulate myosin function. The fission yeast, Schizosaccharomyces pombe, possesses a single essential Tm, Cdc8, which can be acetylated on its amino terminal methionine to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with amino terminal substitutions to explore how stability of the Cdc8-polymer overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we hav... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4919881 Wed, 08 Jun 2011 23:00:00 +0100 4919881 http://www.medworm.com/index.php?rid=4910202&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110683 The surface of the protozoan Trypanosoma cruzi is covered by a dense coat of mucin-type glycoconjugates, which have a pivotal contribution to parasite protection and host immune evasion. Their importance is further underscored by the presence of <1,000 mucin-like genes in the parasite genome. Here we demonstrate that one such group of genes termed TcSMUG L, codes for previously unrecognized mucin-type glycoconjugates anchored to, and secreted from the surface of insect-dwelling epimastigotes. These features are supported by the in vivo tracing and characterization of endogenous TcSMUG L products and recombinant, tagged-molecules expressed by transfected parasites. Besides displaying substantial homology to TcSMUG S products, which provide the scaffold for the major Gp35/50 mucins also p... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4910202 Tue, 07 Jun 2011 23:00:00 +0100 4910202 http://www.medworm.com/index.php?rid=4900974&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110257 The SOUL protein is known to induce apoptosis by provoking the mitochondrial permeability transition and a sequence homologous to the Bcl-2 homology 3 (BH3) domains has been recently identified in it thus making it a potential new member of the BH3-only protein family. Here we present NMR, SPR and crystallographic evidence that a peptide spanning SOUL residues 147 – 172 interacts with the anti-apoptotic protein Bcl-xL. We have crystallized SOUL alone and the complex of its BH3 domain peptide with Bcl-xL and solved their three-dimensional structures. The SOUL monomer is a single domain organized as a distorted beta barrel with eight anti-parallel strands and two alpha helices. The BH3 domain extends across 15 residues at the end of the second helix and 8 amino acids in the chain foll... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4900974 Sun, 05 Jun 2011 23:00:00 +0100 4900974 http://www.medworm.com/index.php?rid=4891402&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110129 Tight regulation of serine proteases is essential for their physiological functions and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of urokinase-type plasminogen activator (uPA) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA’s activity. We have now characterised the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4891402 Wed, 01 Jun 2011 23:00:00 +0100 4891402 http://www.medworm.com/index.php?rid=4891401&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101484 The nuclear protein PARP-1 is activated in cardiomyocytes exposed to hypoxia causing DNA breaks. Unlike this stress-induced PARP-1 activation, our results provide evidence for Ca2+ induced PARP-1 activation in contracting newborns' cardiomyocytes treated with growth factors and hormons that increased their contraction rate, induced intracellular Ca2+ mobilization and its rhythmical and transient translocation into the nucleus. Furthermore, activated PARP-1 up-regulated the activity of phosphorylated ERK in the nucleus, promoting expression of Elk1 target gene, c-fos. Up-regulation of transcription factor c-Fos\GATA-4 promoted ANF expression. Given that expression of atrial naturetic factor (ANF) is known to be implicated in morphological changes, growth and development of car... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4891401 Wed, 01 Jun 2011 23:00:00 +0100 4891401 http://www.medworm.com/index.php?rid=4882604&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101958 hPAR2, is a member of the novel family of proteolytically-activated G-protein coupled receptors (GPCR) termed Proteinase-Activated Receptors (PAR). Previous pharmacological studies have found that activation of hPAR2 by mast cell tryptase, can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR2 that can regulate function, we have explored the functional role of the intracellular cysteine, C361. We demonstrated, using autoradiography, that C361 is the primary palmitoylation site of hPAR2. The hPAR2C361A mutant cell line displayed greater cell surface expression compared to the wt-hPAR2 expressing cell line. The hPAR2C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both tryps... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4882604 Mon, 30 May 2011 23:00:00 +0100 4882604 http://www.medworm.com/index.php?rid=4870561&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110447 Vacuolar H+-ATPases (V-ATPases) are a specific class of multisubunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localises with the V-ATPase occurs in plants and many protists: the single-subunit H+-translocating inorganic pyrophosphatase (H+-PPase). Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. Here, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1 that is preferentially targeted to internal membranes of yeast alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4870561 Wed, 25 May 2011 23:00:00 +0100 4870561 http://www.medworm.com/index.php?rid=4860991&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101381 In conclusion, the EWI-2 cytoplasmic tail actively engages with cell membrane via PIP binding and palmitoylation, which play differential roles in EWI-2 functions. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=4860991 Tue, 24 May 2011 23:00:00 +0100 4860991 http://www.medworm.com/index.php?rid=4860990&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110448 Chloroplast precursor proteins encoded in the nucleus depend on their targeting sequences for delivery to chloroplasts. There exist different routes to the chloroplast outer envelope, but a common theme is the involvement of molecular chaperones. Hsp90 delivers precursors via its receptor Toc64, which transfers precursors to the core translocase in the outer envelope. Here, we identify an uncharacterised protein in Arabidopsis thaliana, OEP61, which shares common features with Toc64, and potentially provides an alternative route to chloroplasts. Sequence analysis indicates that OEP61 possesses a clamp-type tetratricopeptide repeat (TPR) domain capable of binding molecular chaperones, and a C-terminal transmembrane domain. Phylogenetic comparisons show sequence similarities between the TPR ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4860990 Tue, 24 May 2011 23:00:00 +0100 4860990 http://www.medworm.com/index.php?rid=4860994&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101666 During αMβ2-mediated phagocytosis, the small GTPase Rap1 activates the β2 integrin by binding to a region between residues 732 – 761. Using COS-7 cells transfected with αMβ2, we show that αMβ2 activation by the phorbol ester, PMA, involves serine residue 756 of β2. This residue is critical for the local positioning of talin, and biochemically interacts with Rap1. Using the calmodulin antagonist W7, we found Rap1 recruitment and inside-out activation of αMβ2 to be affected. We also report a role for calcium/calmodulin kinase II (CamKII) in the activation of Rap1 during integrin activation. These results demonstrate a distinct, physiological role for the serine residue 756 of β2 integrin, in conjunction with ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4860994 Mon, 23 May 2011 23:00:00 +0100 4860994 http://www.medworm.com/index.php?rid=4860993&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110373 Obesity is associated with induction of endoplasmic reticulum (ER)-stress response signalling and insulin resistance. Protein tyrosine phosphatase (PTP)-1B is a major regulator of adiposity and insulin sensitivity. The aim of this study was to investigate the role of liver-PTP1B in chronically- (high-fat diet) and pharmacologically-induced (tunicamycin, thapsigargin) ER-stress response signalling in vitro and in vivo. We assessed the effects of ER-stress response induction on hepatic PTP1B expression, and consequences of hepatic-PTP1B deficiency, in cells and mouse liver, on components of ER-stress response signalling. We found that PTP1B protein and mRNA expression levels were up-regulated in response to acute and/or chronic ER-stress, in vitro and in vivo. Silencing PTP1B in hepati... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4860993 Mon, 23 May 2011 23:00:00 +0100 4860993 http://www.medworm.com/index.php?rid=4860992&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110427 In conclusion, MRP1/Mrp1 activity is not correlated with the level of free cholesterol or with localization in cholesterol-dependent lipid rafts. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=4860992 Mon, 23 May 2011 23:00:00 +0100 4860992 http://www.medworm.com/index.php?rid=4839437&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110706 The influenza virus transmembrane protein M2 is a proton channel, but also plays a role in the scission of nascent virus particles from the plasma membrane. An amphiphilic helix in the cytoplasmic tail (CT) of M2 is supposed to insert into the lipid bilayer thereby inducing curvature. Palmitoylation of the helix and binding to cholesterol via putative CRAC (cholesterol recognition/interaction amino acid consensus) motifs are believed to target M2 to the edge of rafts, the viral budding site. We tested preconditions of this model, i.e. that the CT interacts with membranes and that acylation and cholesterol binding affect targeting of M2. M2-CT, purified as a glutathione-S-transferase fusion protein, associated with [³H]-photocholesterol and with liposomes. Mutation of tyrosines in t... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4839437 Wed, 18 May 2011 23:00:00 +0100 4839437 http://www.medworm.com/index.php?rid=4839439&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110186 During quality control in the endoplasmic reticulum (ER), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins escaping quality control in the endoplasmic reticulum, facilitating secretion of aberrantly folded as well as normal glycoproteins. Here, we employ free oligosaccharides (FOS) released from degrading glycoproteins as biomarkers of ERAD, allowing us to gain a global rather than single protein-centered view on ER-associated degradation. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannos... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4839439 Tue, 17 May 2011 23:00:00 +0100 4839439 http://www.medworm.com/index.php?rid=4839438&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110111 In this study, we found that AKR1B10 protein is secreted from normal intestinal epithelium and cultured cancer cells, as detected by newly developed sandwich enzyme-linked immunosorbent assay and Western blot. AKR1B10’s secretion was not affected by protein synthesis inhibitor cycloheximide and classical protein secretion pathway inhibitor brefeldin A, but stimulated by temperature, ATP, Ca2+ and Ca2+ carrier ionomycin, lysosomotropic NH4Cl, G-protein activator GTPγS, and G-protein coupling receptor fMLP. ADP-ribosylation factor inhibitor Exo-1 and phospholipase C inhibitor U73122 inhibited the secretion of AKR1B10. In cultured cells, AKR1B10 is present in lysosomes and secreted together with Cathepsin D, a lysosomal marker. In the intestine, AKR1B10 is specific... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4839438 Tue, 17 May 2011 23:00:00 +0100 4839438 http://www.medworm.com/index.php?rid=4839440&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101500 Endothelial-mesenchymal transition (EndMT) is a critical process of cardiac development and disease progression. However, little is know about the signaling mechanisms that cause endothelial cells to transform into mesenchymal cells. Here we show that transforming growth factor-beta2 (TGF-beta2) stimulates EndMT through Smad, MEK, PI3K, and p38 MAPK signaling pathways. Inhibitors of these pathways prevent TGF-beta2-induced EndMT. Furthermore, we show that all of these pathways are essential for increasing expression of the cell adhesion suppressing transcription factor Snail. Inhibition of Snail with siRNA prevents TGF-beta2-induced EndMT. However, over-expression of Snail is not sufficient to cause EndMT. Chemical inhibition of GSK-3beta allows EndMT to be induced by Snail over-expression... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4839440 Mon, 16 May 2011 23:00:00 +0100 4839440 http://www.medworm.com/index.php?rid=4827460&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101633 AF4 belongs to a family of proteins implicated in childhood lymphoblastic leukaemia, FRAXE mental retardation and ataxia. AF4 is a transcriptional activator that is involved in transcriptional elongation. Although AF4 has been implicated in MLL-related leukaemogenesis, AF4-dependent physiological mechanisms are not clearly defined. Proteins that interact with AF4 may play important roles also in mediating oncogenesis, and are potential targets for novel therapies. Using a functional proteomic approach involving tandem mass spectrometry and bioinformatics, we identified 51 AF4-interacting proteins of various Gene Ontology Categories. About 60% participate in transcription regulatory mechanisms, including the Mediator complex in eukaryotic cells. Here we report the first extensive proteomic ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4827460 Sun, 15 May 2011 23:00:00 +0100 4827460 http://www.medworm.com/index.php?rid=4819469&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101795 Recent studies suggest that cholesterol binding is widespread among G protein coupled receptors (GPCRs). Here, we analyzed putative cholesterol-induced changes in the oxytocin receptor, a prototype of cholesterol-interacting GPCRs. For this purpose, we have created recombinant oxytocin receptors that are able to bind two small-sized fluorescence-labeled ligands simultaneously. An oxytocin receptor antagonist was chosen as one of the ligands. To create a second ligand binding site, a small-sized α-bungarotoxin binding site was inserted at the N-terminus or within the third extracellular loop of the oxytocin receptor. All receptor constructs were functionally active and bound both ligands with high-affinity in the nanomolar range. Measurements of the quenching behavior, fluorescence a... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4819469 Wed, 11 May 2011 23:00:00 +0100 4819469 http://www.medworm.com/index.php?rid=4819468&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110210 Cytotoxic lymphocyte protease granzyme M (GrM) is a potent inducer of tumor cell death and a key regulator of inflammation. Although human GrM (hGrM) and mouse GrM (mGrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. Here, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM preferred to cleave after a Met residue, whereas hGrM clearly favors a Leu at the P1 position. The kinetic optimal nonprime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and nonprime consensus cleavage motif with Leu and Met being major P1 determi... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4819468 Wed, 11 May 2011 23:00:00 +0100 4819468 http://www.medworm.com/index.php?rid=4819467&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110569 Arachidonic acid (AA), which possesses both neurotoxic and neurotrophic activities, has been implicated as a messenger in both physiological and pathophysiological processes. In the present study, we investigated the effects of both extracellular and intracellular application of AA on the activity of voltage-gated Na+ channels (NaV) in rat cerebellar granule cells. The extracellular application of AA inhibited the resultant NaV current (INa), wherein the current-voltage curve shifted to a negative voltage direction. Because this effect could be reproduced by treating the granule cells with eicosatetraynoic acid (ETYA), or a membrane impermeable analogue of AA, arachidonoyl coenzyme A (arachidonoyl-CoA), we inferred that AA itself exerted the observed modulatory effects on INa. In co... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4819467 Wed, 11 May 2011 23:00:00 +0100 4819467 http://www.medworm.com/index.php?rid=4801521&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20102059 Gap junctions are specialized cell-cell contacts that provide direct intercellular communication between eukaryotic cells. The tyrosine-sorting signal (YXXØ), present on amino acids 286-289 of Cx43, has been implicated in the internalization of the protein. In recent years, ubiquitination of Cx43 has also been proposed to regulate gap junction intercellular communication, however, the underlying mechanism and molecular players involved remain elusive. Here we demonstrate that ubiquitinated Cx43 is internalized through a mechanism that is independent on the YXXØ signal. Indeed, expression of a Cx43-Ub chimera was shown to drive the internalization of a mutant Cx43 in which the YXXØ-motif was eliminated. Immunofluorescence, cycloheximide-chase and cell surface protein bi... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4801521 Sun, 08 May 2011 23:00:00 +0100 4801521 http://www.medworm.com/index.php?rid=4794137&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110226 Granzyme-mediated cell death is the main pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. A major player in this process is granzyme B (GrB), which triggers apoptosis in both caspase-dependent and caspase-independent manners. A caspase-independent substrate of GrB is the highly conserved transmembrane receptor Notch1. The GrB cleavage sites in Notch1 and functional consequences of Notch1 cleavage by GrB were unknown. We confirmed that Notch1 is a direct and caspase-independent substrate of GrB. We demonstrate that GrB cleaved the intracellular Notch1 domain at least at two distinct aspartic acids D1860 and D1961. Granzyme B cleavage of Notch1 can occur in all subcellular compartments, during maturation of the receptor, at the membrane, and in the nucleus. GrB also ... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4794137 Thu, 05 May 2011 23:00:00 +0100 4794137 http://www.medworm.com/index.php?rid=4788011&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110587 This study demonstrates that PKCzeta phosphorylates occludin on specific Thr residues and promotes assembly of epithelial tight junctions. (Source: BJ Cell) BJ Cell journals http://www.medworm.com/rss/comments.php?id=4788011 Wed, 04 May 2011 23:00:00 +0100 4788011 http://www.medworm.com/index.php?rid=4788013&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110435 Cell migration is a highly controlled, essential cellular process often dys-regulated in tumour cells, dynamically controlled by the architecture of the cell. Studies involving cellular fractionation and microarray profiling have previously identified functionally distinct mRNA populations specific to cellular organelles and architectural compartments. However, the interaction between the translational machinery per se and cellular structures is relatively unexplored. To help understand the role for the compartmentalisation and localised protein synthesis in cell migration, we have used scanning confocal microscopy, immunofluorescence and a novel ribopuromycylation method to visualise translating ribosomes. Here we show that initiation factors (eIFs) localise to the leading edge of migrati... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4788013 Tue, 03 May 2011 23:00:00 +0100 4788013 http://www.medworm.com/index.php?rid=4788012&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20101388 In this study, we tested whether and how estrogen-ER signaling regulates KLF5 protein. We found that estrogen caused the degradation of KLF5 protein, and the degradation was sensitive to proteasome inhibitors but not other inhibitors. The estrogen-inducible E3 ligase EFP was identified as a key player in estrogen-mediated degradation of KLF5, as knockdown and over-expression of EFP increased and decreased KLF5 protein levels respectively, and the decrease continued even when protein synthesis was blocked. EFP-mediated degradation impaired the function of KLF5 in gene transcription. While EFP interacted with KLF5 at their unubiquitinated forms, overexpression of EFP appeared to prevent the ubiquitination of KLF5 while heavily ubiquitinated itself. Furthermore, ubiquitination of EFP interrup... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4788012 Tue, 03 May 2011 23:00:00 +0100 4788012 http://www.medworm.com/index.php?rid=4731873&cid=s_37615_60_f&fid=37615&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20110361 The switch of human melanoma cell phenotype from non to highly tumorigenic and metastatic is triggered by the increase of procathepsin L secretion which modifies tumor microenvironment. Our aim was to identify components involved in the regulation of procathepsin L secretion in melanoma cells. We focused on Rab family members, i.e. Rab3A, Rab4A, Rab4B, Rab5A, Rab8A, Rab11A, Rab27A and Rab33A, involved in distinct regulatory pathways. From analysis of mRNA and protein expression of these Rab components and their knock down by specific siRNAs emerged that Rab4A protein is involved in the regulation of procathepsin L secretion. This result was strengthened as procathepsin L secretion was either inhibited by expression of Rab4A dominant negative mutant or increased by overexpression of the Rab... BJ Cell journals http://www.medworm.com/rss/comments.php?id=4731873 Sun, 17 Apr 2011 23:00:00 +0100 4731873