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Query: UNIPROT:P06889 (Mol)
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Peroxisomes are organelles found in all eukaryotic cells. Peroxisomes import integral membrane proteins post-translationally, and PEX19 is a predominantly cytosolic, farnesylated protein of mammalian and yeast cells that binds multiple peroxisome membrane proteins and is required for their correct targeting/insertion to the peroxisome membrane. We report the characterisation of the Arabidopsisthaliana homologue of PEX19 which is a predominantly cytosolic protein. AtPEX19 is encoded by two genes (designated AtPEX19-1 and AtPEX19-2) that are expressed in all tissues and at all developmental stages of the plant. Quantitative real time PCR shows that AtPEX19-1 and AtPEX19-2 have distinct expression profiles. Using in vitro translation and co-immunoprecipitation AtPEX19-1 was shown to bind to the Arabidopsis peroxisomal membrane protein PEX10. Additionally, bacterially expressed recombinant AtPEX19-1 was able to bind a fusion protein consisting of the C-terminus of PEX10 and glutathione S-transferase in pull-down assays, thereby demonstrating that non-farnesylated AtPEX19 can interact with the C-terminus of AtPEX10. Purified recombinant AtPEX19-1 was analysed by gel filtration chromatography and was found to have a molecular weight consistent with it forming a dimer and a dimer was detected in Arabidopsis cell extracts that was slightly destabilised in the presence of DTT. Moreover, cross-linking studies of native AtPEX19 suggest that in vivo it is the dimeric species of the protein that preferentially forms complexes with other proteins.
Mol Membr Biol
PMID:Arabidopsis PEX19 is a dimeric protein that binds the peroxin PEX10. 1692 26

A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at 60-65 ( composite function)C and retained 100% activity after incubation at 60 ( composite function)C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation at 70 ( composite function)C and 23 min incubation at 80 ( composite function)C. Catalytic function of lipase was activated by Mg(++) (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA, DEPC, betaME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K ( m ) and V (max) of 0.5 mM and 0.139 microM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active site and antigen-binding site of enzyme do not overlap.
Mol Cell Biochem 2006 Oct
PMID:A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: purification and characterization. 1692 23

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl peptidase type-1 (PAP1). The cloned nucleotide sequence has an ORF coding for a primary sequence of 209 amino acid residues, which displays 98% identity with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary between the two sequences. The recombinant bovine PAP1 with a C-terminal His(6) tag (rBtaPAP1(6H)) was expressed in Escherichia coli XL10-Gold cells and purified by immobilised nickel ion affinity chromatography resulting in a yield of 2.6 mg of PAP1 per litre of culture. Purified rBtaPAP1(6H) had a specific activity of 3633 units mg(-1). SDS-PAGE revealed a band for bovine PAP1 with a molecular weight of approximately 24 kDa, which is in good agreement with previously reported data on PAP1. The K (m) and k (cat) values obtained for rBtaPAP1(6H) were 59 muM and 3.5 s(-1), respectively. The optimum pH for activity was 9.0-9.5 and the optimum temperature was 37 degrees C. rBtaPAP1(6H) was found to have an absolute requirement for the thiol-reducing agent DTT, consistent with the expected property of a cysteine protease. Kinetic studies using the peptides pGlu-His-Pro-NH(2) (TRH), pGlu-Ala and pGlu-Val revealed K (i) values of 44.1, 141 and 652.17 microM, respectively. The lowest K (i), observed for Thyrotropin-releasing Hormone (TRH), indicates that rBtaPAP1(6H) has a higher affinity for tripeptides over dipeptides.
Mol Cell Biochem 2007 Mar
PMID:Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli: purification, biochemical and kinetic characterisation. 1711 51

We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.
Mol Cell Biochem 2007 Apr
PMID:Direct evidence that two cysteines in the dopamine transporter form a disulfide bond. 1713 Oct 45

O-GlcNAcylation on serine and threonine side chains of nuclear and cytoplasmic proteins is dynamically regulated in response to various environmental and biological stimuli. O-GlcNAcylation is remarkably similar to O-phosphorylation and appears to have a dynamic interplay with O-phosphate in cellular regulation. A systematic glycoproteomics analysis of the affects of inhibiting specific kinases on O-GlcNAcylation should help reveal both the global and specific dynamic relationships between these two abundant post-translational modifications. Here we report the O-GlcNAc perturbations in response to inhibition of glycogen synthase kinase-3 (GSK-3), a pivotal kinase involved in many signaling pathways. By combining immunoaffinity chromatography and SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative mass spectrometry, we identified 45 potentially O-GlcNAcylated proteins. Quantitative measurements indicated that at least 10 proteins had an apparent increase of O-GlcNAcylation upon GSK-3 inhibition by lithium, whereas surprisingly 19 other proteins showed decreases. O-GlcNAcylation changes on a subset of the proteins were confirmed by follow-up experiments. By combining a new O-GlcNAc peptide enrichment method and beta-elimination followed by Michael addition with DTT, we also mapped the O-GlcNAc site (Ser-55) of vimentin, which showed an apparent increase of O-GlcNAcylation upon GSK-3 inhibition. Based on the MS data, we further investigated potential roles of O-GlcNAc on host cell factor-1, a transcription co-activator, and showed that dynamic regulation of O-GlcNAcylation on host cell factor-1 influenced its subcellular distribution. Taken together, these data indicated the complex interplay between phosphorylation and O-GlcNAcylation that occurs within signaling networks.
Mol Cell Proteomics 2007 Aug
PMID:Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation. 1750 70

Plasminogen activator inhibitor (PAI-1) is an anticancer agent that inhibits plasmin driven proteolysis, limiting angiogenesis and metastasis. In low concentrations it could induce cancer cell motility by interacting with urokinase (uPA), its receptor (uPAR), vitronectin and integrins. Active PAI-1 binds to uPA forming a complex with uPAR, while the latent form of PAI-1 does not. PAI-1 is found in both forms in the circulation. It is not clear which form acts as an anticancer agent and how it interacts with malignant cells. To investigate how these forms reduce angiogenesis or metastasis, we have created PAI-1 cysteine mutants in the active conformation (VLHL PAI-1) with an extended half-life that reaches approximately 700 h and its R369A mutant, which has an active conformation but cannot bind to uPA (VLHLNS PAI-1). Both VLHL PAI-1s convert into the latent form when treated with a reducing agent (DTT) that breaks disulfide bridges. Unexpectedly, during routine investigation of LnCAP cell proliferation, we have found that cells detach from the culture vessels regardless of PAI-1 conformation or activity. Further investigation showed that treatment of cancer cells with VLHL PAI-1 downregulated nucleophosmin, while all forms of PAI-1 downregulated fortilin. These two proteins are implicated in important cellular processes (cell growth, cell cycle, malignant transformation). This suggests that PAI-1, in addition to its well-known anticancer properties, plays an important role in cell signaling. We hope that by exploring PAI-1's structure and function we might be able to understand and separate the different effects of PAI-1 on cancer cells and develop more effective therapeutic strategies in cancer treatment.
Int J Mol Med 2007 Jul
PMID:PAI-1 induces cell detachment, downregulates nucleophosmin (B23) and fortilin (TCTP) in LnCAP prostate cancer cells. 1754 83

Phosphorylation appears to be one of the modulators of chaperone functions of small heat shock proteins. However, the role of phosphorylation is not completely understood. We have investigated the structural and functional consequences of a phosphorylation-mimicking mutation in alpha B-crystallin, a small heat shock protein with chaperone activity. We have used a phosphorylation-mimicking mutant, 3D alpha B-crystallin, in which all the three phosphorylatable serine residues are replaced with aspartic acid. 3D alpha B-Crystallin showed enhanced chaperone-like activity towards DTT-induced aggregation of insulin, heat-induced aggregation of citrate synthase and SDS-induced amyloid fibril formation of alpha-synuclein. Fluorescence and circular dichroism spectroscopic studies showed that 3D alpha B-crystallin exhibits lower stability towards urea-induced denaturation compared to alpha B-crystallin. Subunit exchange studies using fluorescence resonance energy transfer showed that 3D alpha B-crystallin exhibits an observable increase in subunit exchange compared to alpha B-crystallin. Since only part of alpha B-crystallin is phosphorylated in vivo, our subunit exchange studies indicate that formation of mixed oligomers between the unphosphorylated and phosphorylated subunits are likely to play a role in vivo. Our study shows that mixed-oligomer formation modulates the chaperone-like activity. We propose that the degree of phosphorylation of the alpha B-crystallin oligomers and temperature are key modulators to achieve a wide range of chaperone capabilities of the small heat shock protein, alpha -crystallin.
J Mol Biol 2008 Jan 25
PMID:Effect of phosphorylation on alpha B-crystallin: differences in stability, subunit exchange and chaperone activity of homo and mixed oligomers of alpha B-crystallin and its phosphorylation-mimicking mutant. 1806 12

The human papillomavirus (HPV) DNA replication origin (ori) shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundreds of base pairs. The HPV type 11 ori spans 103 bp and contains three palindromic E2 binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 ori binding protein. These sites are separated by 64 and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 DNA ori bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound, indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies on the dependence of looping on salt, ATP, and DTT using full-length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N-terminal domain and factors that may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.
J Mol Biol 2008 Jan 25
PMID:Remodeling of the human papillomavirus type 11 replication origin into discrete nucleoprotein particles and looped structures by the E2 protein. 1806 22

A cDNA encoding glutamine synthetase, one of the enzymes of the GS/GOGAT pathway, was cloned from Camellia sinensis (CsGS). The isolated cDNA consists of 1,071 nucleotides encoding a polypeptide of 356 amino acids with an estimated isoelectric point of 6.13. The recombinant protein purified from Escherichia coli using Ni-NTA affinity chromatography showed molecular mass of 39.2 kDa. The purified protein was confirmed by blotting with anti-His antibodies. Catalytic parameters of the protein were determined using glutamate and ATP as substrates. The observed Km was 9 mM and Vmax was 93 U/mg protein with glutamate as substrate, while with ATP Km and Vmax values were 6 mM and 70 U/mg protein, respectively. Purified enzyme showed pH optima at 8. Cations were found to be showing enhancing effect on the activity of GS enzyme and Mg2+ ion exhibited maximum enhancing effect among the various ions used in this study. This enzyme activity increased by 25% in presence of DTT and decreased by 18% when incubated with PMSF. Transcript analysis in tea bud, youngest leaf, showed that CsGS gene expression is stimulated in response to abscisic acid (ABA), salicylic acid (SA), and hydrogen peroxide (H2O2), while gibberellic acid (GA3) has no influence on its expression levels.
Mol Biotechnol 2008 May
PMID:Cloning and characterization of a cytosolic glutamine synthetase from Camellia sinensis (L.) O. Kuntze that is upregulated by ABA, SA, and H2O2. 1807 44

gamma-Hemolysins are pore-forming toxins which develop from water-soluble monomers by combining two different 'albeit homologous' proteins. They form oligomeric pores in both cell and model membranes by undergoing a still poorly understood conformational rearrangement in the stem region. The stem is formed by three beta-strands, folded onto the core of the soluble protein and completely extended in the pore. We propose a new model to explain such a process. Seven double-cysteine mutants were developed by inserting one cysteine on the stretch that links the beta-hairpin to the core of the protein and another on different positions along the beta-strands. The membrane bound protein was blocked in a non-lytic state by S-S bond formation. Six mutants were oxidized as inactive intermediates, but became active after adding DTT. These results demonstrate that the stem extension can be temporarily frozen and that the beta-barrel formation occurs by beta-strand concerted step-by-step sliding.
Cell Mol Life Sci 2008 Jan
PMID:A molecular pin to study the dynamics of beta-barrel formation in pore-forming toxins on erythrocytes: a sliding model. 1808 87


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