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By admin, on May 15th, 2012
J Biol Chem. 2012 May 10;
Lopez Sambrooks C, Carpio MA, Hallak ME
Post-translational modifications of proteins are important for the regulation of cell fate and functions; one of these post-translational modifications is arginylation. We have previously established that calreticulin (CRT), an endoplasmic reticulum (ER) resident, is also one of the arginylated substrates found in the cytoplasm. In the present study, we describe that arginylated CRT (R-CRT) binds to the cell membrane and identified its role as a pre-apoptotic signal. We also show that cells lacking arginyl-tRNA protein transferase (ATE1-/- cells) are less susceptible to apoptosis than WT cells. Under these conditions R-CRT is present on the cell membrane, but at early stages is differently localized in stress granules (SGs). Moreover, cells induced to undergo apoptosis by arsenite show increased R-CRT on their cell surface. Exogenously applied R-CRT binds to the cell membrane and is able both to increase the number of cells undergoing apoptosis in WT cells and overcome apoptosis resistance in ATE1-/- cells that express R-CRT on the cell surface. Thus, these results demonstrate the importance of surface R-CRT in the apoptotic response of cells, implying that post-translational arginylation of CRT can regulate its intracellular localization, cell function and survival.
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Arginylated calreticulin at the plasma membrane increases the susceptibility of cells to apoptosis.
By admin, on May 10th, 2012
Dalton Trans. 2012 Jun 7; 41(21): 6419-30
Winter PW, Al-Qatati A, Wolf-Ringwall AL, Schoeberl S, Chatterjee PB, Barisas BG, Roess DA, Crans DC
The effects of treatment with bis(maltolato)oxovanadium(iv) (BMOV) on protein localization in membrane microdomains were investigated by comparing the effects of insulin and treatment with BMOV on the lateral motions and compartmentalization of individual insulin receptors (IR). In addition, effects of insulin and BMOV on the association of IR, phosphorylated IR (pIR) and phosphorylated insulin receptor substrate-1 (pIRS-1) with chemically-isolated plasma membrane microdomains on rat basophilic leukemia (RBL-2H3) cells were evaluated. Single particle tracking experiments indicate that individual quantum dot-labeled IR on RBL-2H3 cells exhibit relatively unrestricted lateral diffusion of approximately 1 × 10(-10) cm(2) s(-1) and are confined in approximately 475 nm diameter cell-surface membrane compartments. After treating of RBL-2H3 cells with 10 μM BMOV, IR lateral diffusion and the size of IR-containing membrane compartments is significantly reduced to 6 × 10(-11) cm(2) s(-1) and approximately 400 nm, respectively. BMOV treatment also increases the association of IR with low-density, detergent-resistant membrane fragments isolated using isopycnic sucrose-gradient centrifugation from 2.4% for untreated cells to 25.8% for cells treated with 10 μM BMOV. Additionally, confocal fluorescence microscopic imaging of live RBL-2H3 cells labeled with the phase sensitive aminonaphthylethenylpyridinium-based dye, Di-4-ANEPPDHQ, indicates that BMOV treatment, but not insulin treatment, decreases cell-surface plasma membrane lipid order while fluorescence correlation spectroscopy measurements suggest that BMOV treatment does not affect IR surface-density or insulin binding affinity. Finally, model studies using microemulsions of cetyltrimethylammonium bromide (CTAB) micelles and (1)H NMR spectroscopy show that an oxidized form of BMOV readily localizes near the CTAB head-groups at the lipid-water interface. These observations were supported by IR spectroscopic studies using microemulsions of CTAB reverse micelles showing that both BMOV and oxidized BMOV are associated with the water pool. This conclusion is based on changes in (1)H NMR chemical shifts observed for the complex, oxidized BMOV. Moreover, these shifts appeared to be informative about the location of the complex. No differences were observed in the OD absorption peak positions for the CTAB reverse micelles prepared in the presence and absence of BMOV, oxidized BMOV or maltol. Combined, these results suggest that activation of IR signaling by both insulin and BMOV treatment involves increased association of IR with specialized, nanoscale membrane microdomains. The observed insulin-like activity of BMOV or decomposition products of BMOV may be due to changes in cell-surface membrane lipid order rather than due to direct interactions with IR.
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The anti-diabetic bis(maltolato)oxovanadium(iv) decreases lipid order while increasing insulin receptor localization in membrane microdomains.
By admin, on May 5th, 2012
Oncol Rep. 2012 Apr 30;
Solár P, Hrčková G, Varinská L, Solárová Z, Kriška J, Uhrínová I, Kello M, Mojžiš J, Fedoročko P, Sytkowski AJ
Erythropoietin (Epo) is a critical regulator of erythroid cell proliferation, differentiation and apoptosis. In the form of a recombinant protein, it is widely used to treat various forms of anemia, including that associated with cancer and with the myelosuppressive effects of chemotherapy. Studies of ovarian cancer cell lines have demonstrated the presence of the Epo receptor (EpoR), but there are disagreements regarding its localization and functionality in these cells. Using fluorescence microscopy, we were not able to identify the EpoR on the surface of A2780 cells, in contrast to the positive control K562 cells. Flow cytometry did reveal a weak surface EpoR signal in A2780 cells. Interestingly, most of the EpoR in A2780 cells was found in the cytoplasm, more abundantly as an intracellular membrane-associated protein than a soluble one. Silencing EpoR expression by lentiviral-mediated shRNA resulted in reduced A2780 prolife-ration as well as reduction in Epo-induced phosphorylation of Erk1/2. Our findings provide important insights into the biology of the EpoR in ovarian cancer cells.
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Location and the functionality of erythropoietin receptor(s) in A2780 cells.
By admin, on May 3rd, 2012
J Biol Chem . 2012 Apr 30; Becker D, Richter J, Tocilescu MA, Przedborski S, Voos W The Parkinson`s disease-associated kinase Pink1 is targeted to mitochondria where it is thought to regulate mitochondrial quality control by promoting the selective autophagic removal of dysfunctional mitochondria. Nevertheless, the targeting mode of Pink1 and its submitochondrial localization are still not conclusively resolved
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Pink1 and its {Delta}ψ-dependent cleavage product both localize to the outer mitochondrial membrane by a unique targeting mode.
By admin, on May 2nd, 2012
BMC Bioinformatics. 2012 Apr 27; 13(1): 63
E-Komon T, Burchmore RJ, Herzyk P, Davies RL
ABSTRACT: BACKGROUND: Outer membrane proteins (OMPs) of Pasteurella multocida have various functions related to virulence and pathogenesis and represent important targets for vaccine development. Various bioinformatic algorithms can predict outer membrane localization and discriminate OMPs by structure or function. The designation of a confident prediction framework by integrating different predictors followed by consensus prediction, results integration and manual confirmation will improve the prediction of the outer membrane proteome. RESULTS: In the present study, we used 10 different predictors classified into three groups (subcellular localization, transmembrane beta-barrel protein and lipoprotein predictors) to identify putative OMPs from two available P. multocida genomes: those of avian strain Pm70 and porcine non-toxigenic strain 3480. Predicted proteins in each group were filtered by optimized criteria for consensus prediction: at least two positive predictions for the subcellular localization predictors, three for the transmembrane beta-barrel protein predictors and one for the lipoprotein predictors. The consensus predicted proteins were integrated from each group into a single list of proteins. We further incorporated a manual confirmation step including a public database search against PubMed and sequence analyses, e.g. sequence and structural homology, conserved motifs/domains, functional prediction, and protein-protein interactions to enhance the confidence of prediction. As a result, we were able to confidently predict 98 putative OMPs from the avian strain genome and 107 OMPs from the porcine strain genome with 83% overlap between the two genomes. CONCLUSIONS: The bioinformatic framework developed in this study has increased the number of putative OMPs identified in P. multocida and allowed these OMPs to be identified with a higher degree of confidence. Our approach can be applied to investigate the outer membrane proteomes of other Gram-negative bacteria.
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Predicting the outer membrane proteome of Pasteurella multocida based on consensus prediction enhanced by results integration and manual confirmation.
By admin, on April 26th, 2012
Biochem J. 2012 Apr 24;
Jiang L, Alber J, Wang J, Du W, Yang X, Li X, Sanglard D, Geyer J
Candida albicans RCH1 encodes a protein of 10-transmembrane domains, homologous to human SLC10A7, and localizes in the plasma membrane. Deletion of RCH1 confers hypersensitivity to high concentrations of extracellular calcium and tolerance to azoles and lithium, which phenocopies the deletion of CaPMC1 encoding the vacuolar calcium pump. Additive to CaPMC1 mutation, lack of RCH1 alone shows an increase in calcium sensitivity, calcium uptake and cytosolic calcium level. The calcium hypersensitivity is abolished by cyclosporin A and magnesium. In addition, deletion of RCH1 elevates the expression of CaUTR2, a downstream target of the calcium/calcineurin signaling. Mutational and functional analysis indicates that the Rch1p TM8 domain, but not the TM9 and TM10 domains, are required for its protein stability, cellular functions and subcellular localization. Therefore, Rch1p is a novel regulator of cytosolic calcium homeostasis, which expands the functional spectrum of the vertebrate SLC10 family.
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The Candida albicans plasma membrane protein Rch1p, a member of the vertebrate SLC10 carrier family, is a novel regulator of cytosolic Ca2+ homoeostasis.
By admin, on April 22nd, 2012
J Bacteriol. 2012 Apr 13;
Durand-Heredia J, Rivkin E, Fan G, Morales J, Janakiraman A
The tubulin-homolog, FtsZ, forms a polymeric membrane-associated ring structure (Z-ring) at midcell that establishes the site of division and provides an essential framework for the localization of a multi-protein molecular machine that promotes division in E. coli. A number of regulatory proteins interact with FtsZ and modulate FtsZ-assembly/disassembly processes ensuring the spatiotemporal integrity of cytokinesis. The Z-associated proteins (ZapA, ZapB and ZapC) belong to a group of FtsZ regulatory proteins that exhibit functionally redundant roles in stabilizing FtsZ-ring assembly by binding and bundling polymeric FtsZ at midcell. In this study, we report the identification of ZapD (YacF) as a member of the E. coli midcell division machinery. Genetics and cell biological evidence indicate that ZapD requires FtsZ but not other downstream division proteins for localizing to midcell where it promotes FtsZ-ring assembly via molecular mechanisms that overlap with ZapA. Biochemical evidence indicates that ZapD directly interacts with FtsZ and promotes bundling of FtsZ protofilaments. Similar to ZapA, ZapB and ZapC, ZapD is dispensable for division and therefore belongs to the growing group of FtsZ-associated proteins in E. coli that aid in the overall fitness of the division process.
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Identification of ZapD as a cell division factor that promotes the assembly of FtsZ in Escherichia coli.
By admin, on April 21st, 2012
Plant Cell . 2012 Apr 18; Lung SC, Chuong SD Although Toc159 is known to be one of the key GTPase receptors for selective recognition of chloroplast preproteins, the mechanism for its targeting to the chloroplast surface remains unclear. To compare the targeting of these GTPase receptors, we identified two Toc159 isoforms and a Toc34 from Bienertia sinuspersici, a single-cell C(4) species with dimorphic chloroplasts in individual chlorenchyma cells.
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A Transit Peptide-Like Sorting Signal at the C Terminus Directs the Bienertia sinuspersici Preprotein Receptor Toc159 to the Chloroplast Outer Membrane.
By admin, on April 19th, 2012
By admin, on April 19th, 2012
By admin, on April 17th, 2012
Mol Biosyst . 2012 Apr 11; Lütticke C, Hauske P, Lewandrowski U, Sickmann A, Kaiser M, Ehrmann M YggG is a conserved lipoprotein localized to the outer membrane of Gram negative bacteria. Even though the expressed open reading frame has been identified previously, the Escherichia coli protein remained uncharacterized.
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E. coli LoiP (YggG), a metalloprotease hydrolyzing Phe-Phe bonds.
By admin, on April 13th, 2012
PLoS One. 2012; 7(4): e35163
Pryor S, McCaffrey G, Young LR, Grimes ML
Membrane protein sorting is mediated by interactions between proteins and lipids. One mechanism that contributes to sorting involves patches of lipids, termed lipid rafts, which are different from their surroundings in lipid and protein composition. Although the nerve growth factor (NGF) receptors, TrkA and p75(NTR) collaborate with each other at the plasma membrane to bind NGF, these two receptors are endocytosed separately and activate different cellular responses. We hypothesized that receptor localization in membrane rafts may play a role in endocytic sorting. TrkA and p75(NTR) both reside in detergent-resistant membranes (DRMs), yet they responded differently to a variety of conditions. The ganglioside, GM1, caused increased association of NGF, TrkA, and microtubules with DRMs, but a decrease in p75(NTR). When microtubules were induced to polymerize and attach to DRMs by in vitro reactions, TrkA, but not p75(NTR), was bound to microtubules in DRMs and in a detergent-resistant endosomal fraction. NGF enhanced the interaction between TrkA and microtubules in DRMs, yet tyrosine phosphorylated TrkA was entirely absent in DRMs under conditions where activated TrkA was detected in detergent-sensitive membranes and endosomes. These data indicate that TrkA and p75(NTR) partition into membrane rafts by different mechanisms, and that the fraction of TrkA that associates with DRMs is internalized but does not directly form signaling endosomes. Rather, by attracting microtubules to lipid rafts, TrkA may mediate other processes such as axon guidance.
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NGF Causes TrkA to Specifically Attract Microtubules to Lipid Rafts.
By admin, on April 7th, 2012
J Cell Sci. 2012 Mar 30;
Tran KD, Rodriguez-Contreras D, Shinde U, Landfear SM
Many of the cilia and flagella specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, while two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1 that mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region may present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein binding partners that mediate flagellar targeting of membrane associated proteins.
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Both sequence and context are important for flagellar targeting of a glucose transporter.
By admin, on April 7th, 2012
J Cell Sci. 2012 Mar 30;
Tran KD, Rodriguez-Contreras D, Shinde U, Landfear SM
Many of the cilia and flagella specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, while two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1 that mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region may present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein binding partners that mediate flagellar targeting of membrane associated proteins.
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Both sequence and context are important for flagellar targeting of a glucose transporter.
By admin, on April 5th, 2012
Curr Protoc Neurosci. 2012 Apr; Chapter 2: Unit2.7
Digiovanni J, Sun T, Sheng ZH
Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome-enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures. Curr. Protoc. Neurosci. 59:2.7.1-2.7.22. © 2012 by John Wiley & Sons, Inc.
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Characterizing synaptic vesicle proteins using synaptosomal fractions and cultured hippocampal neurons.
By admin, on April 4th, 2012
PLoS One. 2012; 7(3): e34004
Yuan G, Wang C, Ma C, Chen N, Tian Q, Zhang T, Fu W
The Wnt/β-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/β-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of β-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of β-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated β-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3β and β-catenin. DACT1 stabilizes β-catenin via DACT1-induced effects on GSK-3β and directly interacts with β-catenin proteins. The level of phosphorylated GSK-3β at Ser9 is significantly increased following the elevated expression of DACT1. DACT1 mediates the subcellular localization of β-catenin via increasing the level of phosphorylated GSK-3β at Ser9 to inhibit the activity of GSK-3β. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the β-catenin-dependent pathway.
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Oncogenic Function of DACT1 in Colon Cancer through the Regulation of β-catenin.
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